CN116963749A

CN116963749A - Enriched bioactive renal cell populations, their characteristics and uses thereof

Info

Publication number: CN116963749A
Application number: CN202180089551.8A
Authority: CN
Inventors: 蒂莫西·A·伯特伦; 迪帕克·简
Original assignee: Di PakeJian; Di MoxiABotelun
Current assignee: Di PakeJian; Di MoxiABotelun
Priority date: 2020-11-13
Filing date: 2021-11-12
Publication date: 2023-10-27

Abstract

Methods of identifying enriched heterogeneous kidney cell populations having therapeutic potential, and uses thereof.

Description

Enriched bioactive renal cell populations, their characteristics and uses thereof

Background

Chronic Kidney Disease (CKD) is characterized by progressive kidney disease that deteriorates if not subjected to therapeutic intervention; end Stage Renal Disease (ESRD) may be reached by the end patient. Prevalence data from the united states to europe shows that about 10% of the general population suffers from stage 1-3 CKD (ERA, 2009;USRDS,2011;Jha et al.Chronic kidney disease:global dimension and perspectives.Lancet.2013;382:260-72). Worldwide, the incidence and prevalence of CKD and ESRD continue to increase, while the outcome of treatment remains poor (Shaw et al global estimates of the prevalence of diabetes for2010and 2030.Diabetes Res Clin Pract.2010;87:4-14). In 1996 to 2006, the prevalence of chronic kidney disease increased by more than 33% in the united states alone (u.s. Renal Data system. Costs of CKD and ESRD.Minneapolis, MN, 2007). The increasing incidence of CKD poses a significant threat to public health, with only an increasing impact being expected.

The biggest cause of ESRD is diabetes (Postma and de Zeeuw, 2009), while the incidence of CKD continues to increase, mainly due to the increased incidence of type 2 diabetes (Postma and de Zeeuw, 2009). CKD is often accompanied by adverse consequences (Khan et al 2002;Stenvinkel P.Chronic kidney disease-apublic health priority and harbinger of premature cardiovascular disease J Intern med.2010; 268:456-67) due to potential complications and/or risk factors, including hypertension and renal vascular disease. Due to severe complications, the likelihood of premature death in CKD patients is 5-11 times higher than the likelihood of surviving and progressing to ESRD (Collins et al, 2003; smith et al, 2004). For survival, ESRD patients require kidney replacement therapy (dialysis or transplantation). Currently, over 50 tens of thousands of people in the United states need dialysis or kidney transplantation, accounting for over 220 million dollars (6% of the total medical insurance budget) in annual medical insurance costs (Annual Report of the U.S. Organ Procurement and Transplantation Network and the Scientific Registry of Transplant Recipients: transplant Data 1998-2007.Rockville, MD: HHS/HRSA/HSB/DOT, 2008). Kidney transplantation is the ultimate therapeutic standard for CKD, providing better long-term survival (and cost-effectiveness) than dialysis; however, organs are still in long-term shortage. Despite the increasing number of cadaver and living kidney donors, the annual transplant rate per 100 dialysis patients in the united states is actually decreasing. Prevention or delay of adverse consequences of CKD by intervention early in the disease is a major strategy for CKD treatment. Unfortunately, early treatments to prevent disease progression have not been successful.

New therapeutic paradigms involving tissue engineering and cell-based applications can provide significant and durable enhancement of kidney function, slow disease progression, and improve quality of life for this patient population. These next generation regenerative medicine techniques provide isolated kidney cells as a therapeutic option for CKD (Presnell et al wo/2010/056328 and Ilagan et al pct/US 2011/036347). Injection of these bioactive kidney cells into the kidneys of CKD animal models can result in significant improvements in animal survival and kidney function.

There is a need in the art to identify cells that have therapeutic potential for the treatment of kidney disease based on their regenerative or nephrogenic capacity and to ensure that the kidney cell-based treatment reaches the desired level of efficacy.

Summary of The Invention

The present disclosure describes methods for identifying whether an enriched heterogeneous kidney cell population has therapeutic potential. In this method, it is determined whether cells of the enriched heterogeneous kidney cell population express at least one nephrogenic marker. An enriched heterogeneous kidney cell population is identified as having therapeutic potential if the cells of the enriched heterogeneous kidney cell population express at least one nephrogenic marker. The at least one nephrogenic marker comprises one or more of SIX2, OSR1, LHX1, RET, and FGF 8.

The present disclosure also describes further methods of identifying whether the enriched heterogeneous kidney cell population has therapeutic potential. In this method, the expression level of one or more of the RHAMM, C2, C3, C4, fibrinogen, clotting factor XIII, TEK, KDR, notch1, notch3, timp3, vwf, adam15, gas6, igfbp1 and Tm4sf4 genes in the cells of the enriched heterogeneous kidney cell population is determined. An enriched heterogeneous kidney cell population is identified as having therapeutic potential if the expression level of one or more genes in the cells of the enriched heterogeneous kidney cell population is increased relative to the expression level of one or more genes in the cells of a control kidney cell population.

The present disclosure describes yet another method for identifying enriched heterogeneous populations of kidney cells as having therapeutic potential. In this method, it is determined whether cells of the enriched heterogeneous kidney cell population express SIX2, OSR1, RET and podin. If the cells of the enriched heterogeneous kidney cell population are assayed for expression of SIX2, OSR1, RET and podin, the enriched heterogeneous kidney cell population is identified as having therapeutic potential.

The present disclosure describes yet another method for identifying enriched heterogeneous populations of kidney cells as having therapeutic potential. In this method, it is determined whether cells of the enriched heterogeneous kidney cell population express the nephropathy-free protein, podoprotein and LHX1. An enriched heterogeneous kidney cell population is identified as having therapeutic potential if the cells of the enriched heterogeneous kidney cell population are assayed for the expression of kidney disease proteins, podin, and LHX1.

Drawings

FIG. 1A provides a table describing the detected cell markers expressed by cells of a selected kidney cell population (e.g., an enriched heterogeneous kidney cell population), the cellular sources of these markers, and the structures in the kidney in which cells expressing these markers may be involved in development.

Fig. 1B provides a graph showing the percentage of cells determined by Fluorescence Activated Cell Sorting (FACS) to be a selected kidney cell population (e.g., enriched heterogeneous kidney cell population) expressing the markers described in fig. 1A. Average percentages and 95% ci are shown.

FIG. 2 provides a scatter plot of the Kyoto gene and genomic encyclopedia (Kyoto Encyclopedia of Genes and Genomes, KEGG) pathways enriched for the Differential Expressed Gene (DEG) set. The ordinate represents the pathway name, and the abscissa represents the enrichment coefficient. The size and color of each dot represent the number of differential genes in the pathway and the range of different Q values, respectively. TGF-beta signaling pathways, salmonella infection, rheumatoid arthritis, pyrimidine metabolism, proteoglycans in cancer, pathways in cancer, other types of O-glycan biosynthesis, legionella disease, hippo signaling pathways, HTLV-1 infection, foxO signaling pathways, drug metabolism-other enzymes and dark spots of the cell cycle are all in a higher Q range, approaching 1.00. The AGE-RAGE signaling pathway and the amebiasis spots in diabetic complications are both in a lower Q range, approaching 0.00. Points of protein digestion and absorption, small cell lung cancer, ECM-receptor interactions, axonal guidance, and arginine and proline metabolism are within the low-to-medium Q range.

FIG. 3 provides a KEGG pathway annotation diagram for the DEG set.

Fig. 4 shows a violin box plot of the average methylation level distribution of the Differential Methylation Region (DMR). Hyperdmr refers to the dmr with higher methylation in the sample and hypo-dmr refers to the dmr with lower methylation in the sample.

FIGS. 5A-H provide scatter plots of the following gene expression from FACS analysis of cells: (A) SIX2, (B) OSR1, (C) LHX1, (D) RET, (E) nephritic protein, (F) podoprotein, (G) FGF8, and (H) RACK-1. (A) The top scatter plot of each of- (H) used isotype control antibodies that gated negative cells. (A) Bottom scatter plots of each of- (H) used antigen-specific antibodies to detect positive cells, gated away from negative cells. Positive cell populations are located to the right of the vertical axis or above the horizontal axis.

Detailed Description

Kidneys are a complex organ composed of many different cell types, including podocytes, mesangial cells, endothelial cells, fibroblasts, epithelial cells, and numerous populations of stem and progenitor cells organized in discrete, specialized functional units or nephrons in the renal parenchyma for selective filtration of electrolytes in the vasculature. This complexity of the kidneys makes it extremely difficult to create a solid kidney organ replacement structure.

A complex process of mammalian kidney development begins in the mesodermal region known as the mesoderm. The anterior kidney or "first kidney" represents the initial step in the kidney developmental lineage specification produced by the mesendoderm. The anterior kidney is a small, hollow epithelial tubular cytoball connected to the anterior renal tube. The anterior renal tube then extends caudally, while the anterior kidney itself degenerates. The mesendoderm now forms the second kidney or mesokidney, also known as the mesorenal tube. In females this phenomenon subsides, but in males it eventually becomes the epididymis, or connective tissue between the testes and the bladder. The middle kidney consists of about 30 tubular ducts. The lateral end of the middle tubular is fused with the middle tubular, opening the passage from the excretory unit to the cloaca. The cloaca eventually becomes the bladder and rectum. Finally, the final kidney or postrenal interstitium develops from ureteral buds that germinate from the renal tubular and branch extensively, with each newly grown tip acquiring a cap-like aggregate of postrenal embryo tissue, giving the postrenal lobal appearance.

Bidirectional signaling between ureteral buds and the postrenal interstitium is ultimately responsible for mediating an important event of nephrogenesis. Ureteral buds are branches of the renal tube at E10.5, signaling the surrounding postrenal interstitium, inducing postrenal interstitium cells to aggregate around the tip of the invasive ureteral bud. These mesenchymal aggregates then undergo a mesenchymal-epithelial transition, forming primitive epithelial vesicles known as kidney vesicles. Continued branching of the ureteral bud leads to development of the vasculature and renal pelvis components. At the same time, the kidney vesicles undergo a series of systemic morphological changes, eventually fusing with the ureteric bud epithelium to form a continuous epithelial tubule, the S-shaped body. Infiltration of the S-shaped body by endothelial cells results in the formation of glomerular vasculature. Ureteral bud epithelium continues branch morphogenesis in response to signaling from the proximal postrenal interstitium, which in turn leads to the induction of new postrenal interstitium aggregates and persistent nephrogenic events at the tips of ureteral buds. The iterative process of ureteric bud branching morphogenesis and additional mesenchymal aggregate induction continues along the radial axis of the developing kidney, with the youngest nephron induced to the periphery.

The molecular genetics of the branching morphogenesis and concomitant nephrogenesis of the developing kidneys is complex. Briefly, the induction of ureteral buds is triggered by up-regulation of secreted growth factor GDNF by its receptor RET. RET expression is highest along the anterior renal tube at the ureteral bud formation site. Knock-out mutations in GDNF or RET are embryonic lethal and are associated with failure of ureters to bud and subsequent termination of kidney and ureter formation. Upregulation of GDNF expression is achieved by the action of transcription factors (including PAX2, SIX1, 2, 4).

The conversion of postrenal interstitial aggregates into kidney vesicles is primarily controlled by WNT family proteins (including WNT9b and WNT 4). Thus, knockdown of WNT4 died within 24 hours after birth; kidneys are small and abnormal, consisting of undifferentiated postrenal interstitium. Other growth factors that regulate the branching and nephrogenesis aspects of ureteral buds include TGF-beta, FGF2, FGF7, LIF, and LIM1. Multiple interacting signaling pathways are also involved in the regulation of both ureteral bud branching and nephrogenesis. These include the classical WNT/β -catenin pathway, the sonic hedgehog pathway, BMP and FGF members of the TGF- β superfamily signaling pathway.

The localization of the mesendoderm along the anterior/posterior axis is marked by the expression of certain key transcription factors, including PAX2, PAX8, OSR1 and WT 1. OSR1 originally designated the mesendoderm from paraxial and lateral plate fate, but it is critical for the specialization and survival of the hat mesenchyme. OSR1 expression became limited to only the cap mesenchymal, and OSR1 deleted mice failed to show expression of key cap mesenchymal genes (PAX 2, SIX2, GDNF, EYA1, and SALL 1). In contrast, although OSR1 is expressed in the postrenal mesenchyme prior to isolation of these lineages, the formation of foxd1+ matrix compartments does not require OSR1.

The WNT9b gene was expressed in epithelial Wolffian tubes prior to post-induction kidney development. WNT9b continues to express in ureteral buds, with expression being more pronounced in the stem region than in the tip. Expression of WNT9b in mouse manifolds was maintained until adulthood. WNT9 b-mediated induction of hat mesenchymal tissue initiates expression of WNT4, fibroblast growth factor 8 (FGF 8), paired box 8 (PAX 8), and LIM homologous box protein 1 (LHX 1) encoding genes. These genes cannot be expressed in the hat mesenchyme of the embryonic kidney deficient in WNT9b and no nephron is formed, so WNT9b knockout mice die soon after birth.

The observed expression of multiple markers is generally associated with the earliest signaling event in the embryogenesis process, and may indicate that the enriched heterogeneous kidney cell population (after isolation from the kidney and expansion after the isolation step) may have dedifferentiated and acquired more kidney progenitor-like properties. Thus, the introduction of an enriched heterogeneous population of renal cells with these kidney progenitor-like properties into diseased renal parenchyma may trigger the occurrence of a critical signaling cascade that normally mediates nephrogenesis, but is interpreted as regeneration in the context of adult renal parenchyma.

Described herein are methods and uses for identifying enriched heterogeneous kidney cell populations as having therapeutic potential, as well as heterogeneous kidney cell populations having therapeutic potential.

In a method of identifying an enriched heterogeneous population of kidney cells as having therapeutic potential, the therapeutic potential of the enriched heterogeneous population of kidney cells may be treating kidney disease, a defect in tubular transport, or a defect in glomerular filtration.

If the enriched heterogeneous population of kidney cells is identified as having the potential to treat kidney disease, the kidney disease may be associated with any stage or degree of acute or chronic kidney failure that results in the inability of the kidney to perform the following functions: blood filters and eliminates excess liquids, electrolytes, and waste materials from the blood. Kidney diseases may include endocrine dysfunctions, such as anemia, e.g. erythropoietin deficiency, and mineral imbalances, e.g. vitamin D deficiency. Kidney disease may originate in the kidney or may be secondary to another disease, such as heart failure, hypertension, diabetes, autoimmune disease, or liver disease. Alternatively, kidney disease may occur after acute kidney injury, or may be the result of kidney and/or urinary tract abnormalities.

If the enriched heterogeneous kidney cell population is identified as having therapeutic potential, the enriched heterogeneous kidney cell population can restore kidney function, stabilize kidney function, improve kidney function, reduce kidney fibrosis, reduce kidney inflammation, induce tubule formation in the kidney, induce nephrogenesis in the kidney, induce glomerulogenesis in the kidney, or have regenerative effects in the kidney in a patient in need of such treatment. If the enriched heterogeneous kidney cell population is identified as having therapeutic potential, the enriched heterogeneous kidney cell population can restore mineral balance or alleviate anemia in a patient in need of such treatment. If the enriched heterogeneous population of kidney cells is identified as having therapeutic potential, it may delay or prevent the need for dialysis in a patient in need of kidney disease treatment, or it may delay or prevent the need for performing kidney transplantation.

In a method of identifying an enriched heterogeneous kidney cell population as having therapeutic potential, it can be determined whether cells of the enriched heterogeneous kidney cell population express at least one nephrogenic marker. The at least one nephrogenic marker whose expression is determined in the method may be any of SIX homology box 2 (SIX 2), odd-skip-related 1 (odd-skip-related 1; OSR 1), LIM homology box 1 (LHX 1), transfection phase rearrangement (rearragnged during transfection; RET) or fibroblast growth factor 8 (FGF 8). The at least one nephrogenic marker whose expression is determined in the method may be or may include any one, any two, any three, any four or all of SIX2, OSR1, LHX1, RET and FGF 8.

In these methods of identifying an enriched heterogeneous population of kidney cells as having therapeutic potential, determining the expression of at least one nephrogenic marker may be or may include determining the expression of any two of the nephrogenic markers SIX2, OSR1, LHX1, RET, or FGF8. If expression of any two of these nephrogenic markers is measured, the two nephrogenic markers may be or may include SIX2 and OSR1, or SIX2 and LHX1, or SIX2 and RET, or SIX2 and FGF8, or OSR1 and LHX1, or OSR1 and RET, or OSR1 and FGF8, or LHX1 and RET, or LHX1 and FGF8, or RET and FGF8.

In a method of identifying an enriched heterogeneous population of kidney cells as having therapeutic potential, determining the expression of at least one nephrogenic marker may be or may include determining the expression of any three of the nephrogenic markers SIX2, OSR1, LHX1, RET or FGF8. If expression of any three of these kidney generators is measured, these three kidney generation markers may be or may include SIX2, OSR1 and LHX1, or SIX2, OSR1 and RET, or SIX2, OSR1 and FGF8, or SIX2, LHX1 and RET, or SIX2, LHX1 and FGF8, or SIX2, RET and FGF8, or OSR1, LHX1 and RET, or OSR1, LHX1 and FGF8, or OSR1, RET and FGF8, or LHX1, RET and FGF8.

In a method of identifying an enriched heterogeneous kidney cell population as having therapeutic potential, determining the expression of at least one nephrogenic marker may be or may include determining the expression of any four of the nephrogenic markers SIX2, OSR1, LHX1, RET or FGF 8. If expression of any four of these nephrogenic markers is assayed, then the four nephrogenic markers may be or may include SIX2, OSR1, LHX1 and RET, or SIX2, OSR1, LHX1 and FGF8, or SIX2, LHX1, RET and FGF8, or any of SIX2, OSR1, RET and FGF8, or OSR1, LHX1, RET and FGF 8.

In a method of identifying an enriched heterogeneous population of kidney cells as having therapeutic potential, the determination of the expression of at least one nephrogenic marker may be or may include the determination of the expression of each of the nephrogenic markers SIX2, OSR1, LHX1, RET or FGF 8.

In a method of identifying an enriched heterogeneous kidney cell population as having therapeutic potential, determining that cells of the heterogeneous enriched kidney cell population express at least one (e.g., any one, or any two, or any three, or any four or all five) nephrogenic marker can identify the enriched heterogeneous kidney cell population as having therapeutic potential.

In the method of identifying an enriched heterogeneous kidney cell population as having therapeutic potential, determining expression of the at least one nephrogenic marker may further comprise determining the percentage of cells in the enriched heterogeneous kidney cell population that express the at least one nephrogenic marker. If the percentage of cells expressing at least one nephrogenic marker in the enriched heterogeneous population of renal cells is determined, the percentage of cells expressing any one, any two, any three, any four, or all five of the nephrogenic markers SIX2, OSR1, LHX1, RET, and FGF8 can be determined. If the percentage of cells expressing at least one nephrogenic marker in the enriched heterogeneous kidney cell population is determined, the enriched heterogeneous kidney cell population can be identified as having therapeutic potential if the enriched heterogeneous kidney cell population expresses any one, any two, any three, any four, or all five of the nephrogenic markers SIX2, OSR1, LHX1, RET, and FGF8 for about a certain or a particular percentage.

An enriched heterogeneous kidney cell population can be identified as having therapeutic potential if the percentage of cells expressing SIX2 in the enriched heterogeneous kidney cell population is determined in the method and at least about 0.02% of the cells in the enriched heterogeneous kidney cell population are determined to express SIX 2. Alternatively, an enriched heterogeneous kidney cell population can be identified as having therapeutic potential if the percentage of cells expressing SIX2 in the enriched heterogeneous kidney cell population is determined to be at least about 0.04%, or at least about 0.1%, or at least about 0.5%, or at least about 1.0%, or at least about 1.5%, or at least about 2.0%, or at least about 2.5%, or at least about 3.0%, or at least about 3.5%, or at least about 4.0%, or at least about 4.5%, or at least about 5.0%, or at least about 5.5%. An enriched heterogeneous population of kidney cells can be identified as having therapeutic potential if the percentage of cells expressing SIX2 in the enriched heterogeneous population of kidney cells is determined to be greater than 0% and up to about 15.0%, greater than 0% and up to about 10.0%, or greater than 0% and up to about 9.5%, or greater than 0% and up to about 9.0%, or greater than 0% and up to about 8.5%, or greater than 0% and up to about 8.0%, or greater than 0% and up to about 7.5%, or greater than 0% and up to about 7.0%, or greater than 0% and up to about 6.5%, or greater than 0% and up to about 6.0%. In addition, a heterogeneous population of cells having a potential to be enriched between about 0.02% and about 15.0%, or between about 0.02% and about 10.0%, or between about 0.02% and about 9.0%, or between about 0.02% and about 8.0%, or between about 0.02% and about 7.0%, or between about 0.02% and about 6.0%, or between about 0.04% and about 15.0%, or between about 0.04% and about 10.0%, or between about 0.04% and about 9.0%, or between about 0.04% and about 8.0%, or between about 0.04% and about 7.0%, or between about 0.04% and about 6.0%, or between about 1.0% and about 15.0%, or between about 1.0% and about 10.0%, or between about 1.0% and about 9.0%, or between about 1.04% and about 9.0%, or between about 0.0% and about 8.0%, or between about 1.0% and about 7.0% can be identified as having a potential to enrich.

If the percentage of cells expressing OSR1 in the enriched heterogeneous kidney cell population is determined in the method and at least about 30% of the cells in the enriched heterogeneous kidney cell population are determined to express OSR1, the enriched heterogeneous kidney cell population can be identified as having therapeutic potential. Alternatively, an enriched heterogeneous population of kidney cells may be identified as having therapeutic potential if the percentage of cells expressing OSR1 in the enriched heterogeneous population of kidney cells is determined to be at least about 35%, or at least about 36%, or at least about 37%, or at least about 38%, or at least about 39%, or at least about 40%, or at least about 41%, or at least about 42%, or at least about 43%, or at least about 44%, or at least about 45%, or at least about 50%. An enriched heterogeneous kidney cell population may be identified as having therapeutic potential if the percentage of cells expressing OSR1 in the enriched heterogeneous kidney cell population is determined to be greater than 0% and up to about 90%, or greater than 0% and up to about 88%, or greater than 0% and up to about 86%, or greater than 0% and up to about 84%, or greater than 0% and up to about 82%, or greater than 0% and up to about 80%, or greater than 0% and up to about 75%, or greater than 0% and up to about 70%. In addition, if the percentage of cells expressing OSR1 in the enriched heterogeneous population of kidney cells is determined to have an enrichment potential of between about 30% and about 90%, or between about 30% and about 88%, or between about 30% and about 86%, or between about 30% and about 84%, or between about 30% and about 82%, or between about 30% and about 80%, or between 34% and about 90%, or between about 34% and about 88%, or between about 34% and about 86%, or between about 34% and about 84%, or between about 34% and about 82%, or between about 34% and about 80%, or between about 36% and about 90%, or between about 36% and about 88%, or between about 36% and about 86%, or between about 36% and about 84%, or between about 36% and about 82%, or between about 36% and about 80%, or between about 40% and about 90%, or between about 40% and about 88%, or between about 45% and about 85%, or between about 60% and about 90%, or between about 85%, or between about 60% and about 90%.

A heterogeneous enriched kidney cell population can be identified as having regenerative potential if the percentage of cells expressing LHX1 in the enriched heterogeneous kidney cell population is determined in the method and at least about 5% of the cells in the enriched heterogeneous kidney cell population are determined to express LHX 1. Alternatively, a heterogeneous population of kidney cells can be identified as having therapeutic potential if the percentage of cells expressing LHX1 in the enriched heterogeneous population of kidney cells is determined to be at least about 6%, or at least about 7%, or at least about 8%, or at least about 9%, or at least about 10. Enriched heterogeneous renal cell populations can be identified as having therapeutic potential if the percentage of cells expressing LHX1 in the enriched heterogeneous renal cell population is determined to be greater than 0% and up to 75%, or greater than 0% and up to 70%, or greater than 0% and up to about 65%, or greater than 0% and up to about 64%, or greater than 0% and up to about 63%, or greater than 0% and up to about 62%, or greater than 0% and up to about 61%, or greater than 0% and up to about 60%, or greater than 0% and up to about 59%, or greater than 0% and up to about 58%, or greater than 0% and up to about 57%, or greater than 0% and up to about 56%, or greater than 0% and up to about 55%. Further, if the percentage of cells expressing LHX1 in the enriched heterogeneous population of renal cells is determined to be between about 6% and about 60%, or between about 6% and about 59%, or between about 6% and about 58%, or between about 6% and about 57%, or between about 6% and about 56%, or between about 6% and about 55%, or between about 6% and about 54%, or between about 8% and about 60%, or between about 8% and about 59%, or between about 8% and about 58%, or between about 8% and about 57%, or between about 8% and about 56%, or between about 8% and about 55%, or between about 8% and about 54%, or between about 10% and about 60%, or between about 10% and about 59%, or between about 10% and about 58%, or between about 10% and about 57%, or between about 10% and about 56%, or between about 8% and about 60%, or between about 22% and about 16% or between about 16% and about 50%, or between about 22% and about 16% or between about 22% and about 50%, or between about 16% and about 50% or between about 8% and about 55%, or between about 58%, or between about 8% and about 58%, or between about 10% and about 58%. The enriched heterogeneous population of kidney cells can be identified as having regenerative potential.

An enriched heterogeneous kidney cell population can be identified as having therapeutic potential if the percentage of RET expressing cells in the enriched heterogeneous kidney cell population is determined in the method and at least about 45% of the cells in the enriched heterogeneous kidney cell population are determined to express RET. Alternatively, an enriched heterogeneous population of kidney cells can be identified as having therapeutic potential if the percentage of RET expressing cells in the enriched heterogeneous population of kidney cells is determined to be at least about 22%, at least about 24%, at least about 26%, at least about 28%, at least about 30%, at least about 32%, at least about 34%, at least about 36%, at least about 38%, at least about 40%, at least about 42%, at least about 44%, at least about 46%, or at least about 47%, or at least about 48%, or at least about 49%, or at least about 50%, or at least about 51%, or at least about 52%, or at least about 53%, or at least about 54%. An enriched heterogeneous kidney cell population can be identified as having therapeutic potential if the percentage of RET expressing cells in the enriched heterogeneous kidney cell population is determined to be greater than 0% and up to about 95%, or greater than 0% and up to about 94%, or greater than 0% and up to about 93%, or greater than 0% and up to about 92%, or greater than 0% and up to about 91%, or greater than 0% and up to about 90%, or greater than 0% and up to about 89%, or greater than 0% and up to about 88%, or greater than 0% and up to about 87%, or greater than 0% and up to about 86%, or greater than 0% and up to about 85%. Further, if the percentage of cells expressing RET in the enriched heterogeneous renal cell population is determined to have an enriched potential of between about 45% and about 95%, or between about 45% and about 94%, or between about 45% and about 93%, or between about 45% and about 92%, or between about 45% and about 91%, or between about 45% and about 90%, or between about 45% and about 89%, or between about 45% and about 88%, or between about 47% and about 95%, or between about 47% and about 94%, or between about 47% and about 93%, or between about 47% and about 92%, or between about 47% and about 91%, or between about 47% and about 90%, or between about 47% and about 89%, or between about 47% and about 88%, or between about 49% and about 95%, or between about 49% and about 94%, or between about 49% and about 93%, or between about 49% and about 92%, or between about 45% and about 90%, or between about 40% and about 25%, or between about 40% and about 25% or between about 40% and about 20% or between about 25% and about 20% or between about 40% and about 25% or between about 40% and about 40% or between about 45%.

An enriched heterogeneous kidney cell population can be identified as having therapeutic potential if the percentage of cells expressing FGF8 in the enriched heterogeneous kidney cell population is determined in the method and at least about 0.2% of the cells in the enriched heterogeneous kidney cell population are determined to express FGF 8. Alternatively, an enriched heterogeneous kidney cell population can be identified as having therapeutic potential if the percentage of cells expressing FGF8 in the enriched heterogeneous kidney cell population is determined to be at least about 0.25%, or at least about 0.3%, or at least about 0.35%, or at least about 0.4%, or at least about 0.45%, or at least about 0.48%, or at least about 0.5%, or at least about 0.55%, or at least about 0.6%, or at least about 0.8%, or at least about 1.0%, or at least about 1.5%, or at least about 2.0%, or at least about 2.5%. Enriched heterogeneous populations of kidney cells can be identified as having therapeutic potential if the percentage of cells expressing FGF8 in the enriched heterogeneous population of kidney cells is determined to be greater than 0% and up to about 65%, or greater than 0% and up to about 64%, or greater than 0% and up to about 63%, or greater than 0% and up to about 62%, or greater than 0% and up to about 61%, or greater than 0% and up to about 60%, or greater than 0% and up to about 59%, or greater than 0% and up to about 58%, or greater than 0% and up to about 57%, or greater than 0% and up to about 56%, or greater than 0% and up to about 55%. In addition, in the case of the optical fiber, if the percentage of cells expressing FGF8 in the enriched heterogeneous kidney cell population is determined to be between about 0.3% and about 64%, or between about 0.3% and about 63%, or between about 0.3% and about 62%, or between about 0.3% and about 61%, or between about 0.3% and about 60%, or between about 0.3% and about 59%, or between about 0.3% and about 58%, or between about 0.3% and about 57%, or between about 0.4% and about 64%, or between about 0.4% and about 63%, or between about 0.4% and about 62%, or between about 0.4% and about 61%, or between about 0.4% and about 60%, or between about 0.4% and about 59%, or between about 0.4% and about 58%, or between about 0.4% and about 57%, or between about 0.5% and about 63%, or between about 0.4% and about 60%. Or between about 0.5% and about 62%, or between about 0.5% and about 61%, or between about 0.5% and about 60%, or between about 0.5% and about 59%, or between about 0.5% and about 58%, or between about 0.5% and about 57%, or between about 1% and about 64%, or between about 1% and about 54%, or between about 1% and about 44%, or between about 1% and about 34%, or between about 1% and about 24%, or between about 2% and about 64%, or between about 2% and about 54%, or between about 2% and about 24%, or between about 3% and about 64%, or between about 3% and about 54%, the enriched heterogeneous kidney cell population can be identified as having therapeutic potential.

The method of identifying an enriched heterogeneous population of kidney cells as having therapeutic potential may include the step of determining the percentage of cells in the heterogeneous population of kidney cells that express a combination of any two, any three, any four, or all five of the nephrogenic markers SIX2, OSR1, LHX1, RET, and FGF8. For example, an enriched heterogeneous population of kidney cells can be identified as having therapeutic potential if a combination of any two, any three, any four, or all five of the following are determined: more than 0% and up to about 6% of the cells of the heterogeneous kidney cell population express SIX2, at least about 36% of the cells of the heterogeneous kidney cell population express OSR1, at least about 8% of the cells of the enriched heterogeneous kidney cell population express LHX1, at least about 49% of the cells of the enriched heterogeneous kidney cell population express RET, and/or more than 0% and up to about 59% of the cells of the enriched heterogeneous kidney cell population express FGF8. In another example, an enriched heterogeneous population of kidney cells can be identified as having therapeutic potential if any two, any three, any four, or all five of the following are determined: at least about 0.04% of the cells of the enriched heterogeneous kidney cell population express SIX2, greater than 0% and up to about 85% of the cells of the enriched heterogeneous kidney cell population express OSR1, greater than 0% and up to about 58% of the cells of the enriched heterogeneous kidney cell population express LHX1, greater than 0% and up to about 90% of the cells of the enriched heterogeneous kidney cell population express RET, and/or at least about 0.48% of the cells of the enriched heterogeneous kidney cell population express FGF8. Enriched heterogeneous populations of kidney cells can be further identified as having therapeutic potential if any two, any three, any four, or all five of the following are determined: between about 0.04% and about 6.0% of the cells of the enriched heterogeneous kidney cell population express SIX2, between about 36% and about 85% of the cells of the enriched heterogeneous kidney cell population express OSR1, between about 8% and about 58% of the cells of the enriched heterogeneous kidney cell population express LHX1, between about 49% and about 90% of the cells of the enriched heterogeneous kidney cell population express RET and between about 0.48% and/or about 59% of the cells express FGF8. Any two, any three, any four or all five combinations of nephrogenic markers whose expression can be determined in these percentages can be any of the following combinations: SIX2 and OSR1; or SIX2 and LHX1; or SIX2 and RET; or SIX2 and FGF8; or OSR1 and LHX1; or OSR1 and RET; or OSR1 and FGF8; or LHX1 and RET; or LHX1 and FGF8; or RET and FGF8; or SIX2, OSR1 and LHX1; or SIX2, OSR1 and RET; or SIX2, OSR1 and FGF8; or SIX2, LHX1 and RET; or SIX2, LHX1 and FGF8; or SIX2, RET and FGF8; or OSR1, LHX1 and RET; or OSR1, LHX1 and FGF8; or OSR1, RET and FGF8; or LHX1, RET and FGF8; or SIX2, OSR1, LHX1 and RET; SIX2, OSR1, LHX1 and FGF8; SIX2, LHX1, RET and FGF8; SIX2, OSR1, RET and FGF8; OSR1, LHX1, RET and FGF8; or SIX2, OSR1, LHX1, RET and FGF8.

In a method of identifying an enriched heterogeneous population of kidney cells as having therapeutic potential, the enriched heterogeneous population of kidney cells can be selectively identified as having therapeutic potential if any two, any three, any four, or all five of the following are determined: more than 0% and up to about 10% of the cells of the heterogeneous kidney cell population express SIX2, at least about 30% of the cells of the heterogeneous kidney cell population express OSR1, at least about 5% of the cells of the enriched heterogeneous kidney cell population express LHX1, at least about 40% of the cells of the enriched heterogeneous kidney cell population express RET, and/or more than 0% and up to about 60% of the cells of the enriched heterogeneous kidney cell population express FGF8. In another example, an enriched heterogeneous population of kidney cells can be identified as having therapeutic potential if any two, any three, any four, or all five of the following are determined: at least about 0.02% of the cells of the enriched heterogeneous kidney cell population express SIX2, greater than 0% and up to about 90% of the cells of the enriched heterogeneous kidney cell population express OSR1, greater than 0% and up to about 65% of the cells of the enriched heterogeneous kidney cell population express LHX1, greater than 0% and up to about 95% of the cells of the enriched heterogeneous kidney cell population express RET, and/or at least about 0.4% of the cells of the enriched heterogeneous kidney cell population express FGF8. Furthermore, an enriched heterogeneous population of kidney cells can be identified as having therapeutic potential if any two, any three, any four, or all five of the following are determined: between about 0.02% and about 10.0% of the cells of the enriched heterogeneous kidney cell population express SIX2, between about 30% and about 90% of the cells of the enriched heterogeneous kidney cell population express OSR1, between about 5% and about 65% of the cells of the enriched heterogeneous kidney cell population express LHX1, between about 40% and about 95% of the cells of the enriched heterogeneous kidney cell population express RET and/or between about 0.4% and about 60% of the cells express FGF8. Any two, any three, any four or all five combinations of nephrogenic markers whose expression can be determined in these percentages can be any of the following combinations: SIX2 and OSR1; or SIX2 and LHX1; or SIX2 and RET; or SIX2 and FGF8; or OSR1 and LHX1; or OSR1 and RET; or OSR1 and FGF8; or LHX1 and RET; or LHX1 and FGF8; or RET and FGF8; or SIX2, OSR1 and LHX1; or SIX2, OSR1 and RET; or SIX2, OSR1 and FGF8; or SIX2, LHX1 and RET; or SIX2, LHX1 and FGF8; or SIX2, RET and FGF8; or OSR1, LHX1 and RET; or OSR1, LHX1 and FGF8; or OSR1, RET and FGF8; or LHX1, RET and FGF8; or SIX2, OSR1, LHX1 and RET; SIX2, OSR1, LHX1 and FGF8; SIX2, LHX1, RET and FGF8; SIX2, OSR1, RET and FGF8; OSR1, LHX1, RET and FGF8; or SIX2, OSR1, LHX1, RET and FGF8.

In a method of identifying an enriched heterogeneous population of kidney cells as having therapeutic potential, the enriched heterogeneous population of kidney cells can be identified as having therapeutic potential if any two, any three, any four, or all five of the following are determined: more than 0% and up to about 3% of the cells of the heterogeneous kidney cell population express SIX2, at least about 60% of the cells of the heterogeneous kidney cell population express OSR1, at least about 25% of the cells of the enriched heterogeneous kidney cell population express LHX1, at least about 65% of the cells of the enriched heterogeneous kidney cell population express RET, and/or more than 0% and up to about 35% of the cells of the enriched heterogeneous kidney cell population express FGF8. In another example, an enriched heterogeneous population of kidney cells can be identified as having therapeutic potential if any two, any three, any four, or all five of the following are determined: at least about 0.5% of the cells of the enriched heterogeneous kidney cell population express SIX2, greater than 0% and up to about 80% of the cells of the enriched heterogeneous kidney cell population express OSR1, greater than 0% and up to about 55% of the cells of the enriched heterogeneous kidney cell population express LHX1, greater than 0% and up to about 90% of the cells of the enriched heterogeneous kidney cell population express RET, and/or at least about 5% of the cells of the enriched heterogeneous kidney cell population express FGF8. Furthermore, an enriched heterogeneous population of kidney cells can be identified as having therapeutic potential if any two, any three, any four, or all five of the following are determined: between about 0.5% and about 3.0% of the cells of the enriched heterogeneous kidney cell population express SIX2, between about 60% and about 80% of the cells of the enriched heterogeneous kidney cell population express OSR1, between about 25% and about 55% of the cells of the enriched heterogeneous kidney cell population express LHX1, between about 65% and about 90% of the cells of the enriched heterogeneous kidney cell population express RET and/or between about 5% and about 35% of the cells express FGF8. Any two, any three, any four or all five combinations of nephrogenic markers whose expression can be determined in these percentages can be any of the following combinations: SIX2 and OSR1; or SIX2 and LHX1; or SIX2 and RET; or SIX2 and FGF8; or OSR1 and LHX1; or OSR1 and RET; or OSR1 and FGF8; or LHX1 and RET; or LHX1 and FGF8; or RET and FGF8; or SIX2, OSR1 and LHX1; or SIX2, OSR1 and RET; or SIX2, OSR1 and FGF8; or SIX2, LHX1 and RET; or SIX2, LHX1 and FGF8; or SIX2, RET and FGF8; or OSR1, LHX1 and RET; or OSR1, LHX1 and FGF8; or OSR1, RET and FGF8; or LHX1, RET and FGF8; or SIX2, OSR1, LHX1 and RET; SIX2, OSR1, LHX1 and FGF8; SIX2, LHX1, RET and FGF8; SIX2, OSR1, RET and FGF8; OSR1, LHX1, RET and FGF8; or SIX2, OSR1, LHX1, RET and FGF8.

In a method of identifying an enriched heterogeneous population of kidney cells as having therapeutic potential, the enriched heterogeneous population of kidney cells can be identified as having therapeutic potential if any two, any three, any four, or all five of the following are determined: more than 0% and up to about 10% of the cells of the heterogeneous kidney cell population express SIX2, at least about 35% of the cells of the heterogeneous kidney cell population express OSR1, at least about 8% of the cells of the enriched heterogeneous kidney cell population express LHX1, at least about 20% of the cells of the enriched heterogeneous kidney cell population express RET, and/or more than 0% and up to about 60% of the cells of the enriched heterogeneous kidney cell population express FGF8. In another example, an enriched heterogeneous population of kidney cells can be identified as having therapeutic potential if any two, any three, any four, or all five of the following are determined: at least about 0.2% of the cells of the enriched heterogeneous kidney cell population express SIX2, greater than 0% and up to about 85% of the cells of the enriched heterogeneous kidney cell population express OSR1, greater than 0% and up to about 65% of the cells of the enriched heterogeneous kidney cell population express LHX1, greater than 0% and up to about 90% of the cells of the enriched heterogeneous kidney cell population express RET, and/or at least about 0.5% of the cells of the enriched heterogeneous kidney cell population express FGF8. Furthermore, an enriched heterogeneous population of kidney cells can be identified as having therapeutic potential if any two, any three, any four, or all five of the following are determined: between about 0.2% and about 10.0% of the cells of the enriched heterogeneous kidney cell population express SIX2, between about 35% and about 85% of the cells of the enriched heterogeneous kidney cell population express OSR1, between about 8% and about 65% of the cells of the enriched heterogeneous kidney cell population express LHX1, between about 20% and about 90% of the cells of the enriched heterogeneous kidney cell population express RET and/or between about 0.5% and about 60% of the cells express FGF8. Any two, any three, any four or all five combinations of nephrogenic markers whose expression can be determined in these percentages can be any of the following combinations: SIX2 and OSR1; or SIX2 and LHX1; or SIX2 and RET; or SIX2 and FGF8; or OSR1 and LHX1; or OSR1 and RET; or OSR1 and FGF8; or LHX1 and RET; or LHX1 and FGF8; or RET and FGF8; or SIX2, OSR1 and LHX1; or SIX2, OSR1 and RET; or SIX2, OSR1 and FGF8; or SIX2, LHX1 and RET; or SIX2, LHX1 and FGF8; or SIX2, RET and FGF8; or OSR1, LHX1 and RET; or OSR1, LHX1 and FGF8; or OSR1, RET and FGF8; or LHX1, RET and FGF8; or SIX2, OSR1, LHX1 and RET; SIX2, OSR1, LHX1 and FGF8; SIX2, LHX1, RET and FGF8; SIX2, OSR1, RET and FGF8; OSR1, LHX1, RET and FGF8; or SIX2, OSR1, LHX1, RET and FGF8.

It will be appreciated that if the percentage of cells expressing a certain marker is provided as a percentage of a particular number of "about", e.g., about 5%, then the percentage of cells need not be exactly that particular number, e.g., exactly 5%. Conversely, it should be understood that if the percentage of cells expressing a certain marker is provided as "about" a particular number, e.g., about 5%, the percentage of cells expressing a certain marker may be in the range of up to 10% of the particular number, e.g., between 4.5% and 5.5%.

In a method of identifying an enriched heterogeneous kidney cell population as having therapeutic potential, it can be determined whether the cells of the enriched heterogeneous kidney cell population express one or more additional markers, i.e., markers other than the nephrogenic markers SIX2, OSR1, LHX1, RET and FGF 8. The one or more additional markers may be or may include one or more of podocyte markers, epithelial markers, developmental markers, or mirnas. If the one or more additional markers comprise podocyte markers, the one or more additional markers may comprise one or more kidney disease proteins, epiCAM, NPHS2 (encoding podoprotein), podoprotein, wilms tumor protein (WT 1), or podocyte marker proteins. If the one or more additional markers comprise an epithelial cell marker, the one or more additional markers may comprise one or more of E-cadherin, N-cadherin, cubulin/megalin, vitamin D-25 hydroxylase (CYP 2R 1), gamma-glutamyl transferase 1 (GGT 1), liver-enriched transcription protein (LAP), cytokeratin (CK) 18, aquaporin (AQP) 2, kidney injury molecule (KIM 1), erythropoietin (EPO), kinase insertion domain receptor (KDR), epithelial Cell Adhesion Molecule (ECAM), or AQP 1. If the one or more additional markers comprise a developmental marker, the developmental marker may comprise one or more of neutrophil gelatinase-associated lipocalin (NGAL), sonic hedgehog (SHH), NOTCH, C-X-C motif chemokine receptor 4 (CXCR 4), lunatic Fringe (LFNG), or IL-11. The one or more additional markers may be or may include receptors for activated C kinase 1 (RACK-1). If the one or more additional markers comprise mirnas, the mirnas can be one or more of miR22, miR181, or miR 145.

If the one or more additional markers comprise a podocyte marker, the podocyte marker may be a kidney disease protein. In this method, an enriched heterogeneous kidney cell population can be identified as having therapeutic potential if the cells of the enriched heterogeneous kidney cell population are further assayed for the expression of a kidney disease protein. If one or more additional markers are or include a kidney disease protein, the percentage of cells expressing the kidney disease protein in the enriched heterogeneous kidney cell population can be determined. If the percentage of cells expressing a kidney disease protein in the enriched heterogeneous kidney cell population is determined, the enriched heterogeneous kidney cell population can be identified as having therapeutic potential if at least about 4%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% of the cells of the heterogeneous kidney cell population express a kidney disease protein. If the percentage of cells expressing a kidney disease protein in the enriched heterogeneous kidney cell population is determined, the enriched heterogeneous kidney cell population may be identified as having therapeutic potential if the cells of the kidney disease cell population express a kidney disease protein between about 4% and about 95%, or between about 10% and about 95%, or between about 15% and about 95%, or between about 20% and about 95%, or between about 25% and about 95%, or between about 30% and about 95%, or between 35% and about 95%, or between about 40% and about 95%, or between about 45% and 95%, or between about 50% and about 95%, or between about 55% and about 95%, or between about 60% and about 95%, or between about 65% and about 95%, or between 70% and about 95%, or between about 75% and about 95%, or between about 80% and about 95%, or between about 85% and about 95%.

If the one or more additional markers are or include podocyte markers, the podocyte markers may be podoprotein. In this method, the enriched heterogeneous kidney cell population can be identified as having therapeutic potential if the cells of the enriched heterogeneous kidney cell population are further assayed for the expression of podin. If the one or more additional markers are or include podoprotein, the percentage of podoprotein expressing cells in the enriched heterogeneous kidney cell population can be determined. If the percentage of cells expressing podoprotein in the enriched heterogeneous kidney cell population is determined, the enriched heterogeneous kidney cell population can be identified as having therapeutic potential if at least about 80%, at least about 82%, at least about 84%, at least about 86%, at least about 88%, at least about 90%, at least about 92%, at least about 94%, at least about 96%, or at least about 98% of the cells of the heterogeneous kidney cell population express podoprotein.

If the one or more additional markers comprise podocyte markers, then the podocyte markers may comprise both kidney disease proteins and podoproteins. In this method, an enriched heterogeneous kidney cell population can be identified as having therapeutic potential if the cells of the enriched heterogeneous kidney cell population are further assayed for the expression of kidney disease proteins and podoproteins. If the one or more additional markers include a kidney disease protein and a podoprotein, the percentage of cells expressing the kidney disease protein and podoprotein in the enriched heterogeneous kidney cell population can be determined. If the percentage of cells expressing kidney disease protein and podoprotein in the enriched heterogeneous kidney cell population is determined, then if at least 4% of the cells express kidney disease protein and at least about 90% of the cells express podoprotein, or if at least 8% of the cells express kidney disease protein and at least about 90% of the cells express podoprotein, or if at least 10% of the cells express kidney disease protein and at least about 90% of the cells express podoprotein, or if at least 12% of the cells express kidney disease protein and at least about 90% of the cells express podoprotein, or if at least 20% of the cells express kidney disease protein and at least about 90% of the cells express podoprotein, or if at least 25% of the cells express kidney disease protein and at least about 90% of the cells express podoprotein, or if at least 30% of the cells express a kidney disease protein and at least about 90% of the cells express a podin protein, or if at least 35% of the cells express a kidney disease protein and at least about 90% of the cells express a podin protein, or if at least 40% of the cells express a kidney disease protein and at least about 90% of the cells express a podin protein, or if at least 45% of the cells express a kidney disease protein and at least about 90% of the cells express a podin protein, or if at least 55% of the cells express a kidney disease protein and at least about 90% of the cells express a podin protein, or if at least 60% of the cells express a kidney disease protein and at least about 90% of the cells express a podin protein, or if at least 65% of the cells express a kidney disease protein and at least about 90% of the cells express a podin protein, or if at least 70% of the cells express a kidney disease protein and at least about 90% of the cells express a podoprotein, or if at least 75% of the cells express a kidney disease protein and at least about 90% of the cells express a podoprotein, the enriched heterogeneous population of kidney cells can be identified as having therapeutic potential.

If one or more additional markers is or includes RACK-1, an enriched heterogeneous kidney cell population can be identified as having therapeutic potential if the cells of the population express RACK-1. If one or more additional markers is or includes RACK-1, the percentage of cells expressing RACK-1 in the enriched heterogeneous kidney cell population can be determined. If the percentage of cells expressing RACK-1 in the enriched heterogeneous kidney cell population is determined, the enriched heterogeneous kidney cell population can be identified as having therapeutic potential if at least about 80%, at least about 82%, at least about 84%, at least about 86%, at least about 88%, at least about 90%, at least about 92%, at least about 94%, at least about 96%, or at least about 98% of the cells of the heterogeneous kidney cell population express RACK-1.

If the one or more additional markers comprise an epithelial cell marker, the epithelial cell marker may be CYP2R1. In this method, an enriched heterogeneous kidney cell population can be identified as having therapeutic potential if the cells of the enriched heterogeneous kidney cell population are further assayed for CYP2R1 expression. If one or more additional markers is or includes CYP2R1, the percentage of cells expressing CYP2R1 in the enriched heterogeneous kidney cell population can be determined. If the percentage of cells expressing CYP2R1 in the enriched heterogeneous kidney cell population is determined, the enriched heterogeneous kidney cell population can be identified as having therapeutic potential if the cells of the heterogeneous kidney cell population express CYP2R1 between about 75% and about 100%, or between about 80% and about 100%, or between about 85% and about 100%, or between about 86% and about 100%, or between about 87% and about 100%, or between about 88% and about 100%, or between about 75% and about 97%, or between about 80% and about 97%, or between about 85% and about 97%, or between about 86% and about 97%, or between about 87% and about 97%.

If the one or more additional markers comprise a developmental marker, the developmental marker may be CXCR4. In this method, an enriched heterogeneous kidney cell population can be identified as having therapeutic potential if the cells of the enriched heterogeneous kidney cell population are further assayed for CXCR4 expression. If one or more additional markers is or includes CXCR4, the percentage of cells expressing CXCR4 in the enriched heterogeneous kidney cell population can be determined. If the percentage of cells expressing CXCR4 in an enriched heterogeneous kidney cell population is determined, then the enriched heterogeneous kidney cell population can be identified as having therapeutic potential if greater than about 15%, or greater than about 16%, or greater than about 17%, or greater than about 18%, or greater than about 19%, or greater than about 20%, or greater than about 21%, or greater than about 22%, or greater than about 23%, or greater than about 24%, or greater than about 25% of the cells of the heterogeneous kidney cell population express CXCR4.

One or more additional markers may include a combination of one or more of Bone Morphogenic Protein (BMP) 4, BMP7, glial cell-derived neurotrophic factor (GDNF), homeobox protein HOX11, eye deletion homolog 1 (EYA 1), SAL1, and SIX4 (SIX homolog box 4). One or more additional markers may include a combination of one or more of a pairing box 2 (PAX 2), a Cbp/P300-interacting transactivator with a Glu/Asp-rich carboxy-terminal domain 1 (CITED 1), a Fibroblast Growth Factor Receptor (FGFR) 1, FGF7, FGF10, a homeobox protein HOX10, a encapsulating/Tcf 21 (POD 1) POD1, and mucin 1 (MUC 1). One or more additional markers may include a hyaluronic acid-mediated motility Receptor (RHAMM), complement component C2, complement component C3, complement component C4, fibrinogen, factor XIII, TEK tyrosine kinase, KDR, notch1, notch3, timp3, von Willebrand (von Willebrand) factor (VWF), adam15, growth arrest-specific 6 (Gas 6), insulin-like growth factor binding protein (Igfbp) 1, or a combination of one or more of transmembrane 4 superfamily member 4 (Tm 4sf 4). The one or more additional markers may comprise RHAMM.

One or more additional markers may include CDK2A, interleukin 11 (IL 11), transcription Growth Factor (TGF) beta 2, fibronectin (FN) 1, cysteine-rich secreted protein LCCL domain 2 (CRISPLD 2), collagen type 1 alpha 1 chain (COL 1A 1), lysyl Oxidase (LOX), run-related transcription factor 2 (RUNX 2), lunatic Fringe (LFN), brain-derived neurotrophic factor (BDNF), claudin (CLDN) 3, uridine phosphorylase (UPP) 1, kruppel-like factor (KLF) 14, glycosyltransferase-like (LTL) 1B, mannosidase alpha 1C class member 1 (MAN 1C 1) polypeptide N-acetylgalactosamine transferase 9 (GALNT 9), aquaporin (AQP 1), solute carrier family 47 member 1 (SLC 47 A1), WNK lysine deficiency protein kinase (WNK) 2, calcium sensitive receptor (CASR), retinoic acid induction 2 (RAI 2), plasma membrane vesicle associated protein (PLVAP), SHISA family member (shasa) 3, prostaglandins-regulated mucin-like protein 1 (PARM 1), FGF11, fork-box E1 (FOXE 1), WNT family member (WNT) 5A, WNT10A, TGF beta 1, and insulin-like growth factor binding protein (IGFBP) 3. The one or more additional markers may include any one or more of IL11, tgfβ2, CRISPLD2, LOX, LNFG, BDNF, WNT5A, or IGFBP 3.

In some methods of identifying enriched heterogeneous kidney cell populations as having therapeutic potential, it can be determined whether the cells of the enriched heterogeneous kidney cell populations express SIX2, OSR1, RET, and podin. In such methods, an enriched heterogeneous kidney cell population can be identified as having therapeutic potential if the cells of the enriched heterogeneous kidney cell population are assayed for expression of SIX2, OSR1, RET, and podin. Determining that the cells of the enriched heterogeneous kidney cell population express SIX2, OSR, RET, and podoprotein can include determining the percentage of cells of the enriched heterogeneous kidney cell population that express SIX2, OSR, RET, and podoprotein.

If determining that the cells of the enriched heterogeneous kidney cell population express SIX2, OSR, RET, and podoprotein comprises determining the percentage of cells of the enriched heterogeneous kidney cell population that express SIX2, OSR, RET, and podoprotein, the following percentages are expressed in the cells of the population: (i) SIX2 may be greater than 0% and up to 15.0%, or greater than 0% and up to about 10.0%, or greater than 0% and up to about 9.5%, or greater than 0% and up to about 9.0%, or greater than 0% and up to about 8.5%, or greater than 0% and up to about 8.0%, or greater than 0% and up to about 7.5%, or greater than 0% and up to about 7.0%, or greater than 0% and up to about 6.5%, or greater than 0% and up to about 6.0%, or between about 0.02% and about 15%, or between about 0.02% and about 10.0%, or between about 0.02% and about 9.0%, or or between about 0.02% and about 8.0%, or between about 0.02% and about 7.0%, or between about 0.02% and about 6.0%, or between about 0.04% and about 15%, or between about 0.04% and about 10.0%, or between about 0.04% and about 9.0%, or between about 0.04% and about 8.0%, or between about 0.04% and about 7.0%, or between about 0.04% and about 6.0%, or between about 1.0% and about 15.0%, or between about 1.0% and about 10.0%, or between about 1.0% and about 9.0%, or between about 1.0% and about 8.0%, or between about 1.0% and about 7.0%, or between about 1.0% and about 6.0%. (ii) The OSR may be greater than 0% and up to about 90%, or greater than 0% and up to about 88%, or greater than 0% and up to about 86%, or greater than 0% and up to about 84%, or greater than 0% and up to about 82%, or greater than 0% and up to about 80%, or greater than 0% and up to about 75%, or greater than 0% and up to about 70%, between about 30% and about 90%, or between about 30% and about 88%, or between about 30% and about 86%, or between about 30% and about 84%, or between about 30% and about 82%, or between about 30% and about 80%, or between 34% and 90%, or between about 34% and about 88%, or between about 34% and about 86%, or between about 34% and about 84%, or between about 34% and about 82%, or or between about 34% and about 80%, or between about 36% and about 90%, or between about 36% and about 88%, or between about 36% and about 86%, or between about 36% and about 84%, or between about 36% and about 82%, or between about 36% and about 80%, or between about 40% and about 90%, or between about 40% and about 85%, or between about 45% and about 90%, or between about 45% and about 85%, or between about 50% and about 90%, or between about 50% and about 85%, or between about 55% and about 90%, or between about 55% and about 85%, or between about 60% and about 90%, or between about 60% and about 85%, or between about 65% and about 90%, or between about 65% and about 85%, or between about 70% and about 90%, or between about 70% and about 85%. (iii) The RET may be greater than 0% and up to about 94%, or greater than 0% and up to about 93%, or greater than 0% and up to about 92%, or greater than 0% and up to about 91%, or greater than 0% and up to about 90%, or greater than 0% and up to about 89%, or greater than 0% and up to about 88%, or greater than 0% and up to about 87%, or greater than 0% and up to about 86%, or greater than 0% and up to about 85%, or between about 45% and about 95%, or between about 45% and about 94%, or between about 45% and about 93%, or between about 45% and about 92%, or between about 45% and about 91%, or between about 45% and about 90%, or between about 45% and about 89%, or between about 45% and about 88%, or between about 47% and about 95%. Or between about 47% and about 93%, or between about 47% and about 92%, or between about 47% and about 91%, or between about 47% and about 90%, or between about 47% and about 89%, or between about 47% and about 88%, or between about 49% and about 95%, or between about 49% and about 94%, or between about 49% and about 93%, or between about 49% and about 92%, or between about 49% and about 91%, or between about 49% and about 90%, or between about 49% and about 89%, or between about 20% and about 60%, or between about 20% and about 50%, or between about 20% and about 45%, or between about 20% and about 40%, or between about 25% and about 60% Or between about 25% and about 55%, or between about 25% and about 50%, or between about 25% and about 45%, or between about 25% and about 40%; (iv) The podoprotein can be at least about 80%, at least about 82%, at least about 84%, at least about 86%, at least about 88%, at least about 90%, at least about 92%, at least about 94%, at least about 96%, or at least about 98%.

In a method of identifying an enriched heterogeneous kidney cell population as having therapeutic potential, wherein determining whether the cells of the enriched heterogeneous kidney cell population express SIX2, OSR1, RET and podin, it can be further determined whether the cells of the enriched heterogeneous kidney cell population express one or more of LHX1, FGF8, RACK-1 and kidney disease protein. In these methods, an enriched heterogeneous kidney cell population can be identified as having therapeutic potential if it is determined that the cells of the enriched heterogeneous kidney cell population express SIX2, OSR, RET, and podin, and further the cells of the enriched heterogeneous population express one or more of LHX1, FGF8, RACK-1, and kidney disease protein.

In these methods, determining whether the cells of the enriched heterogeneous kidney cell population express SIX2, OSR1, RET, and podin, and further determining whether the cells of the enriched heterogeneous kidney cell population express one or more of LHX1, FGF8, RACK-1, and kidney disease protein, can be determining whether the cells of the enriched heterogeneous kidney cell population express any of: SIX2, OSR1, RET, podin, and LHX1; or SIX2, OSR1, RET, podin, and FGF8; or SIX2, OSR1, RET, podin and RACK-1; or SIX2, OSR1, RET, podoprotein, and nephritic protein; or SIX2, OSR1, RET, podin, LHX1, and FGF8; or SIX2, OSR1, RET, podin, LHX1 and RACK-1; or SIX2, OSR1, RET, podin, LHX1, and nephritic proteins; or SIX2, OSR1, RET, podin, FGF8, and RACK-1; or SIX2, OSR1, RET, podin, FGF8, and kidney disease proteins; or SIX2, OSR1, RET, podoprotein, RACK-1 and kidney disease proteins; or SIX2, OSR1, RET, podin, LHX1, FGF8 and RACK-1; or SIX2, OSR1, RET, podin, LHX1, FGF8, and kidney disease proteins; or SIX2, OSR1, RET, podin, LHX, RACK-1 and kidney disease proteins; or SIX2, OSR1, RET, podin, FGF8, RACK-1, and kidney disease proteins; or SIX2, OSR1, RET, podin, LHX, FGF8, RACK-1 and kidney disease proteins.

Determining whether the cells of the enriched heterogeneous kidney cell population express SIX2, OSR1, RET and podin, and whether the cells further express one or more of LHX1, FGF8, RACK-1 and kidney disease protein, can be determining the percentage of cells that express SIX2, OSR1, RET, podin and further express one or more of LHX1, FGF8, RACK-1 and kidney disease protein. If the cells of the enriched heterogeneous kidney cell population are assayed for expression of SIX2, OSR, RET and podin, and further for expression of one or more of LHX1, FGF8, RACK-1, the following percentages are expressed in the cells of the population: (i) SIX2 may be greater than 0% and up to 15%, or may be greater than 0% and up to about 10.0%, or greater than 0% and up to about 9.5%, or greater than 0% and up to about 9.0%, or greater than 0% and up to about 8.5%, or greater than 0% and up to about 8.0%, or greater than 0% and up to about 7.5%, or greater than 0% and up to about 7.0%, or greater than 0% and up to about 6.5%, or greater than 0% and up to about 6.0%, or between about 0.02% and about 15.0%, or between about 0.02% and about 10.0%, or between about 0.02% and about 9.0%, or or between about 0.02% and about 8.0%, or between about 0.02% and about 7.0%, or between about 0.02% and about 6.0%, or between about 0.04% and about 15%, or between about 0.04% and about 10.0%, or between about 0.04% and about 9.0%, or between about 0.04% and about 8.0%, or between about 0.04% and about 7.0%, or between about 0.04% and about 6.0%, or between about 1.0% and about 15.0%, or between about 1.0% and about 10.0%, or between about 1.0% and about 9.0%, or between about 1.0% and about 8.0%, or between about 1.0% and about 7.0%, or between about 1.0% and about 6.0%. (ii) OSRl may be greater than 0% and up to about 90%, or greater than 0% and up to about 88%, or greater than 0% and up to about 86%, or greater than 0% and up to about 84%, or greater than 0% and up to about 82%, or greater than 0% and up to about 80%, or greater than 0% and up to about 75% or greater than 0% and up to about 70%, between about 30% and about 90%, or between about 30% and about 88%, or between about 30% and about 86%, or between about 30% and 84%, or between about 30% and about 82%, or between about 30% and about 80%, or between 34% and 90%, or between about 34% and about 88%, or between about 34% and about 86%, or between about 34% and about 84%, or between about 34% and about 82%, or between about 82% and about 34% and about 86%. Or between about 34% and about 80%, or between about 36% and about 90%, or between about 36% and about 88%, or between about 36% and about 86%, or between about 36% and about 84%, or between about 36% and about 82%, or between about 36% and about 80%, or between about 40% and about 90%, or between about 40% and about 85%, or between about 45% and about 90%, or between about 45% and about 85%, or between about 50% and about 90%, or between about 50% and about 85%, or between about 55% and about 90%, or between about 55% and about 85%, or between about 60% and about 90%, or between about 60% and about 85%, or between about 65% and about 90%, or between about 65% and about 85%, or between about 70% and about 90%, or between about 70% and about 85%. (iii) The RET may be greater than 0% and up to about 94%, or greater than 0% and up to about 93%, or greater than 0% and up to about 92%, or greater than 0% and up to about 91%, or greater than 0% and up to about 90%, or greater than 0% and up to about 89%, or greater than 0% and up to about 88%, or greater than 0% and up to about 87%, or greater than 0% and up to about 86%, or greater than 0% and up to about 85%, or between about 45% and about 95%, or between about 45% and about 94%, or between about 45% and about 93%, or between about 45% and about 92%, or between about 45% and about 91%, or between about 45% and about 90%, or between about 45% and about 89%, or between about 45% and about 88%, or between about 47% and about 95%. Or between about 47% and about 93%, or between about 47% and about 92%, or between about 47% and about 91%, or between about 47% and about 90%, or between about 47% and about 89%, or between about 47% and about 88%, or between about 49% and about 95%, or between about 49% and about 94%, or between about 49% and about 93%, or between about 49% and about 92%, or between about 49% and about 91%, or between about 49% and about 90%, or between about 49% and about 89%, or between about 20% and about 60%, or between about 20% and about 50%, or between about 20% and about 45%, or between about 20% and about 40%, or between about 25% and about 60% Or between about 25% and about 55%, or between about 25% and about 50%, or between about 25% and about 45%, or between about 25% and about 40%; (iv) The podoprotein can be at least about 80%, at least about 82%, at least about 84%, at least about 86%, at least about 88%, at least about 90%, at least about 92%, at least about 94%, at least about 96%, or at least about 98%; and optionally, the presence of a metal salt, (v) LHXL may be at least about 5%, at least about 6%, or at least about 7%, or at least about 8%, or at least about 9%, or at least about 10%, greater than 0% and up to about 80%, or greater than 0% and up to about 70%, or greater than 0% and up to about 65%, or greater than 0% and up to about 64%, or greater than 0% and up to about 63%, or greater than 0% and up to about 62%, or greater than 0% and up to about 61%, or greater than 0% and up to about 60%, or greater than 0% and up to about 59%, or greater than 0% and up to about 58%, or greater than 0% and up to about 57%, or greater than 0% and up to about 56%, or greater than 0% and up to about 55%, between about 6% and about 60%, or greater than 0% and up to about 61%, or greater than 0% and up to about 58%, or greater than 0% and up to about 57%, or greater than 0% and up to about 55%, or up to about 55%, and up to about 55%, or about or between about 6% and about 59%, or between about 6% and about 58%, or between about 6% and about 57%, or between about 6% and about 56%, or between about 6% and about 55%, or between about 6% and about 54%, or between about 8% and about 60%, or between about 8% and about 59%, or between about 8% and about 58%, or between about 8% and about 56%, or between about 8% and about 55%, or between about 8% and about 54%, or between about 10% and about 60%, or between about 10% and about 58%, or between about 10% and about 57%, or between about 10% and about 54%, or between about 16% and about 80% Or between about 16% and about 70%, or between about 16% and about 60%, or between about 16% and about 58%, or between about 16% and about 56%, or between about 16% and about 54%, or between about 16% and about 52%, or between about 16% and about 50%, or between 22% and about 60%, or between about 22% and about 58%, or between about 22% and about 56%, or between about 22% and about 54%, or between about 22% and about 52%, or between about 22% and about 50%, and optionally (vi) FGF8 can be at least about 0.2%, at least about 0.45%, or at least about 0.48%, or at least about 0.5%, or at least about 0.55%, or at least about 0.6%, or at least about 0.8%, or at least about 1.0%, or at least about 1.5%, or at least about 2.0%, or about 2.5%, greater than 0% and up to about 65%, or greater than 0% and up to about 64%, or greater than 0% and up to about 63%, or greater than 0% and up to about 62%, or greater than 0% and up to about 61%, or greater than 0% and up to about 60%, or greater than 60%, or up to about 60%, of FGF 8. Or greater than 0% and up to about 59%, or up to about 58%, or greater than 0% and up to about 57%, or greater than 0% and up to about 56%, or greater than 0% and up to about 55%, or between about 0.3% and about 64%, or between about 0.3% and about 63%, or between about 0.3% and about 62%, or between about 0.3% and about 61%, or between about 0.3% and about 60%, or between about 0.3% and about 59%, or between about 0.3% and about 58%, or between about 0.3% and about 57%, or between about 0.4% and about 64%, or between about 0.4% and about 63%, or between about, or between about 0.4% and about 62%, or between about 0.4% and about 61%, or between about 0.4% and about 60%, or between about 0.4% and about 59%, or between about 0.4% and about 58%, or between about 0.4% and about 57%, or between about 0.5% and about 64%, or between about 0.5% and about 63%, or between about 0.5% and about 62%, or between about 0.5% and about 61%, or between about 0.5% and about 60%, or between about 0.5% and about 59%, or between about 0.5% and about 58%, or between about 0.5% and about 57%, or between about 1% and about 64%, or between about 1% and about 54%, or between about 44%, or between about 1% and about 34%, or between about 24% and about 24%, or between about 2% and about 64%, or between about 2% and about 44%, or between about 2% and about 34%, or between about 3% and about 24%, or between about 24% and about 54%, or between about 2% and about 44%, or between about 2% and about 34%, or between about 3% and about 59%, or between about 2% and about 24% and about 34%; and optionally, (vii) RACK-1 may be at least about 80%, at least about 82%, at least about 84%, at least about 86%, at least about 88%, at least about 90%, at least about 92%, at least about 94%, at least about 96%, or at least about 98%; and optionally, (viii) the nephron may be at least about 4%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, from about 4% to about 95%, or between about 10% and about 95%, or between about 15% and about 95%, or between about 20% and about 95%, or between about 25% and about 95%, and between about 30% and about 95%, or between 35% and about 95%, or between about 40% and about 95%, or between about 45% and 95%, or between about 50% and about 95%, or between about 55% and about 95%, or between about 60% and about 95%, or between about 65% and about 95%, or between about 70% and about 95%, or between about 75% and about 95%, or between about 95% and about 80%.

In some other methods of identifying enriched heterogeneous kidney cell populations as having therapeutic potential, it can be determined whether cells of the enriched heterogeneous kidney cell populations express kidney disease proteins, podin, and LHX1. In such methods, an enriched heterogeneous kidney cell population can be identified as having therapeutic potential if the cells of the enriched heterogeneous kidney cell population are assayed for the expression of kidney disease proteins, podin, and LHX1.

Determining that cells of the enriched heterogeneous kidney cell population express a kidney disease protein, a podoprotein, and LHX1 can include determining the percentage of cells of the enriched heterogeneous kidney cell population that express a kidney disease protein, podoprotein, and LHX1. If the assay comprises determining the percentage of cells expressing kidney disease protein, podoprotein and LHX1 in the enriched heterogeneous kidney cell population, then to identify the enriched heterogeneous kidney cell population as having therapeutic potential, the following percentages are expressed in the cells of the cell population: (i) The kidney disease protein may be at least about 70%, at least about 72%, at least about 75%, at least about 77%, at least about 80%, at least about 82%, at least about 85%, at least about 87%, at least about 90%, at least about 92%, at least about 95%, or at least about 97%; (ii) The podoprotein can be at least about 80%, at least about 82%, at least about 84%, at least about 86%, at least about 88%, at least about 90%, at least about 92%, at least about 94%, at least about 96%, or at least about 98%; (iii) LHXl may be at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, between about 15% and about 80%, between about 20% and about 80%, between about 15% and about 75%, between about 15% and about 70%, or between about 20% and about 80%.

In a method of identifying an enriched heterogeneous kidney cell population as having therapeutic potential, wherein determining whether the cells of the enriched heterogeneous kidney cell population express a kidney disease protein, a podin, and LHX1, it can be further determined whether the cells of the enriched heterogeneous kidney cell population express one or more of the nephrogenic markers SIX2, OSR1, RET, or FGF8. In these methods, an enriched heterogeneous kidney cell population can be identified as having therapeutic potential if it is determined that the cells of the enriched heterogeneous kidney cell population express kidney disease protein, podin, and LHX1, and further the cells of the enriched heterogeneous population express one or more of SIX2, OSR1, RET, or FGF8.

In these methods, determining whether the cells of the enriched heterogeneous kidney cell population express kidney disease protein, podin, and LHX1, and further express one or more of SIX2, OSR1, RET, or FGF8, can be determining whether the cells of the enriched heterogeneous kidney cell population express any of: kidney disease proteins, podoprotein, LHX1 and SIX2; kidney disease proteins, podoprotein, LHX1 and OSR1, kidney disease proteins, podoprotein, LHX1 and RET; kidney disease proteins, podoproteins, LHX1 and FGF8; kidney disease proteins, podoprotein, LHX1, SIX2 and OSR1; kidney disease proteins, podoprotein, LHX1, SIX2 and RET; kidney disease proteins, podoproteins, LHX1, SIX2 and FGF8; kidney disease proteins, podoprotein, LHX1, OSR1 and RET; kidney disease proteins, podoproteins, LHX1, ORS1 and FGF8; kidney disease proteins, podoproteins, LHX1, RET and FGF8; kidney disease proteins, podoprotein, LHX1, SIX2, OSR1 and RET; kidney disease proteins, podoproteins, LHX1, SIX2, RET and FGF8; kidney disease proteins, podoproteins, LHX1, OSR1, RET and FGF8; kidney disease proteins, podoproteins, LHX1, SIX2, OSR1 and FGF8; or kidney disease proteins, podoproteins, LHX1, SIX2, OSR1, RET and FGF8.

Determining whether the cells of the enriched heterogeneous kidney cell population express kidney disease protein, podin, and LHX1, and further express one or more of SIX2, OSR1, RET, and FGF8, can be determining the percentage of cells that express kidney disease protein, podin, and LHX1, and further express one or more of SIX2, OSR1, RET, and FGF 8. If the percentage of cells in the population that express kidney disease protein, podin and LHX1 and one or more of SIX2, OSR1, RET and FGF8 are determined, then to identify the enriched heterogeneous kidney cell population as having therapeutic potential, the following percentages of cells are expressed: (i) The kidney disease protein may be at least about 70%, at least about 72%, at least about 75%, at least about 77%, at least about 80%, at least about 82%, at least about 85%, at least about 87%, at least about 90%, at least about 92%, at least about 95%, or at least about 97%; (ii) The podoprotein can be at least about 80%, at least about 82%, at least about 84%, at least about 86%, at least about 88%, at least about 90%, at least about 92%, at least about 94%, at least about 96%, or at least about 98%; (iii) LHXl may be at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, between about 15% and about 80%, between about 20% and about 80%, between about 15% and about 75%, between about 15% and about 70%, or between about 20% and about 80%; and optionally, the presence of a metal salt, (iv) SIX2 may be greater than 0% and up to about 15.0%, or greater than 0% and up to about 13%, or greater than 0% and up to about 11%, or greater than 0% and up to about 9%, or greater than 0% and up to about 7%, or greater than 0% and up to about 5%, or greater than 0% and up to about 3%, or between about 0.02% and about 15%, or between about 0.02% and about 13%, or between about 0.02% and about 11%, or between about 0.02% and about 9%, or between about 0.02% and about 7%, or between about 0.02% and about 5%, or between about 0.02% and about 3%, or between about 0.04% and about 15%, or between about 0.04% and about 13%, or between about 0.04% and about 11%, or between about 0.04% and about 9%, or about 1.04% and about 1%, or about 1.02% and about 1% or about 1, about 1% and about 5%, or between about 0.02% and about 3%, or between about 1.04% and about 1, about 1% and about 3%, or about 1.04% and about 1.11%; and optionally, the presence of a metal salt, (v) OSRl may be greater than 0% and up to about 90%, or greater than 0% and up to about 88%, or greater than 0% and up to about 86%, or greater than 0% and up to about 84%, or greater than 0% and up to about 82%, or greater than 0% and up to about 80%, or greater than 0% and up to about 75%, or greater than 0% and up to about 70%, between about 30% and about 90%, or between about 30% and about 88%, or between about 30% and about 86%, or between about 30% and about 84%, or between about 30% and about 82%, or between about 30% and about 80%, or between 34% and about 90%, or between about 34% and about 88%, or between about 34% and about 86%, or between about 34% and about 84%, or between about 34% and about 82%. Or between about 34% and about 80%, or between about 36% and about 90%, or between about 36% and about 88%, or between about 36% and about 86%, or between about 36% and about 84%, or between about 36% and about 82%, or between about 36% and about 80%, or between about 40% and about 90%, or between about 40% and about 85%, or between about 45% and about 90%, or between about 45% and about 85%, or between about 50% and about 90%, or between about 50% and about 85%, or between about 55% and about 90%, or between about 55% and about 85%, or between about 60% and about 90%, or between about 60% and about 85%, or between about 65% and about 90%, or between about 65% and about 85%, or between about 70% and about 90%, or between about 70% and about 85%. And optionally, the presence of a metal salt, (vi) The RET may be greater than 0% and up to about 94%, or greater than 0% and up to about 93%, or greater than 0% and up to about 92%, or greater than 0% and up to about 91%, or greater than 0% and up to about 90%, or greater than 0% and up to about 89%, or greater than 0% and up to about 88%, or greater than 0% and up to about 87%, or greater than 0% and up to about 86%, or greater than 0% and up to about 85%, or between about 45% and about 95%, or between about 45% and about 94%, or between about 45% and about 93%, or between about 45% and about 92%, or between about 45% and about 91%, or between about 45% and about 90%, or between about 45% and about 89%, or between about 45% and about 88%, or between about 47% and about 95%. Or between about 47% and about 93%, or between about 47% and about 92%, or between about 47% and about 91%, or between about 47% and about 90%, or between about 47% and about 89%, or between about 47% and about 88%, or between about 49% and about 95%, or between about 49% and about 94%, or between about 49% and about 93%, or between about 49% and about 92%, or between about 49% and about 91%, or between about 49% and about 90%, or between about 49% and about 89%, or between about 20% and about 60%, or between about 20% and about 50%, or between about 20% and about 45%, or between about 20% and about 40%, or between about 25% and about 60% Or between about 25% and about 55%, or between about 25% and about 50%, or between about 25% and about 45%, or between about 25% and about 40%; and optionally, the presence of a metal salt, (vii) FGF8 can be at least about 0.2%, at least about 0.45%, or at least about 0.48%, or at least about 0.5%, or at least about 0.55%, or at least about 0.6%, or at least about 0.8%, or at least about 1.0%, or at least about 1.5%, or at least about 2.0%, or at least about 2.5%, greater than 0% and up to about 65%, or greater than 0% and up to about 64%, or greater than 0% and up to about 63%, or greater than 0% and up to about 62%, or greater than 0% and up to about 61%, or greater than 0% and up to about 60%, or greater than 0% and up to about 59%, or up to about 58%, or greater than 0% and up to about 57%, or greater than 0% and up to about 56%, or greater than 0% and up to about 55%, between about 0.3% and about 64%, or between about 3% and about 63%. Or between about 0.3% and about 62%, or between about 0.3% and about 61%, or between about 0.3% and about 60%, or between about 0.3% and about 59%, or between about 0.3% and about 58%, or between about 0.3% and about 64%, or between about 0.4% and about 63%, or between about 0.4% and about 62%, or between about 0.4% and about 61%, or between about 0.4% and about 60%, or between about 0.4% and about 59%, or between about 0.4% and about 58%, or between about 0.4% and about 57%, or between about 0.5% and about 64%, or between about 0.5% and about 63%, or between about 0.5% and about 62%, or between about 0.5% and about 61%, or between about 0.4% and about 60%, or between about 0.5% and about 59%, or between about 0.5% and about 58%, or between about 0.5% and about 59% Or between about 0.5% and about 57%, or between about 1% and about 64%, or between about 1% and about 54%, or between about 1% and about 44%, or between about 1% and about 34%, or between 1% and about 24%, or between about 2% and about 64%, or between about 2% and about 54%, or between about 2% and about 44%, or between about 2% and about 34%, or between 2% and about 24%, or between about 3% and about 64%, or between about 3% and about 54%, or between about 3% and about 44%, or between about 3% and about 34%, or between 3% and about 24%.

In a method of identifying an enriched heterogeneous kidney cell population as having therapeutic potential, wherein it is determined whether the cells of the enriched heterogeneous kidney cell population express kidney disease protein, podin and LHX1, it may be further determined whether the cells of the enriched heterogeneous kidney cell population express RACK1, in addition to or in lieu of determining whether the cells express one or more of the nephrogenic markers SIX2, OSR1, RET or FGF 8. In these methods, an enriched heterogeneous kidney cell population can be identified as having therapeutic potential if the cells of the heterogeneous kidney cell population express kidney disease protein, podin, and LHX1, and further express RACK-1.

In these methods, determining whether the cells of the enriched heterogeneous kidney cell population express RACK1 may be determining the percentage of cells in the enriched heterogeneous kidney cell population that express RACK1, in addition to the percentage of cells that express kidney disease protein, podin, and LHX1, and optionally the percentage of cells that express one or more of SIX1, OSR1, RET, and FGF 8. If the percentage of cells expressing RACK1 in the enriched heterogeneous kidney cell population is determined as part of the method, the percentage of enriched kidney cell population identified as having therapeutic potential may be at least about 80%, at least about 82%, at least about 84%, at least about 86%, at least about 88%, at least about 90%, at least about 92%, at least about 94%, at least about 96%, or at least about 98%.

In yet another method of identifying an enriched heterogeneous kidney cell population as having therapeutic potential, it can be determined whether cells of the enriched heterogeneous kidney cell population express gamma-glutamyl transpeptidase (GGT) -1, CK18 and podin. In such methods, an enriched heterogeneous kidney cell population can be identified as having therapeutic potential if the cells of the enriched heterogeneous kidney cell population are assayed for the expression of GGT-1, CK18, and podin. Determining that the cells of the enriched heterogeneous kidney cell population express GGT-1CK18 and podoprotein can include determining the percentage of cells of the enriched heterogeneous kidney cell population that express GGT-1, CK18 and podoprotein. If the cells of the enriched heterogeneous kidney cell population are assayed for the expression of GGT-1, CK18 and podin, the enriched heterogeneous kidney cell population can be identified as having therapeutic potential if: (i) at least 4.5% or at least 10% or at least 18% of the cells of the population express GGT-1, (ii) at least 80% of the cells of the population express CK18, and (iii) at least about 80%, at least about 82%, at least about 84%, at least about 86%, at least about 88%, at least about 90%, at least about 92%, at least about 94%, at least about 96%, or at least about 98% of the cells of the population express podin. In these methods, VEGF and/or KIM-1 secreted by cells of the population in the cell culture medium can further identify whether the cells have therapeutic potential.

In some methods of identifying enriched heterogeneous kidney cell populations as having therapeutic potential, it is not necessary to determine whether cells of the enriched heterogeneous kidney cell populations express at least one nephrogenic marker. In these alternative methods of identifying enriched heterogeneous kidney cell populations as having therapeutic potential, cells can be identified as having therapeutic potential by determining the expression level of one or more of the RHAMM, C2, C3, C4, fibrinogen, clotting factor XIII, TEK, KDR, notch1, notch3, timp3, vwf, adam15, gas6, igfbp1 or Tm4sf4 genes. In these alternative methods, the one or more genes whose expression levels can be determined may be RHAMM. In these alternative methods, an enriched heterogeneous kidney cell population can be identified as having therapeutic potential if the determined expression level of the one or more genes of the cells of the enriched heterogeneous kidney cell population is increased relative to the expression of the one or more genes of the cells of the control kidney cell population.

In addition, in any method that identifies an enriched heterogeneous kidney cell population as having therapeutic potential, the enriched heterogeneous kidney cell population can be further subjected to transcriptional or transcriptomic profiling. An enriched heterogeneous kidney cell population can be identified as having therapeutic potential if it is subjected to transcriptional or transcriptomic profiling if the complement or complement cascade, vascular development, vascular morphogenesis, vasculature development or response to injury associated transcriptional pathways or features are measured and/or the transcriptional pathways or features associated with extracellular matrix-receptor interactions are down-regulated.

Markers such as SIX2, OSR1, RET, LHX1, FGF8, kidney disease protein, podoprotein, and RACK-1, as discussed herein, useful for identifying enriched heterogeneous populations of kidney cells, and methods for identifying whether a patient needs treatment with enriched heterogeneous populations of kidney cells, such as a patient in need of treatment for kidney disease, a defect in tubular transport, or a defect in glomerular filtration, would be intermediate to high responders or low responders to such treatment. In such methods, if a patient is identified as a moderate to high responder, the patient may be identified as a patient whose response to treatment may be an estimated increase in glomerular filtration rate (eGFR). If a patient is identified as a low responder, the patient may be identified as a patient whose response to treatment may be an improvement in the slope of the gfr but no increase in the gfr. To identify whether the patient is likely to be a moderate to high responder or a low responder, the percentage of cells in the enriched heterogeneous kidney cell population that express one or more markers (e.g., SIX2, OSR1, RET, LHX1, FGF8, kidney disease protein, podoprotein, and RACK-1) can be determined. For example, to identify whether a patient is a moderate to high responder or a low responder, the percentage of LHX1 expressing cells in the enriched heterogeneous kidney cell population can be determined. Patients can be identified as moderate to high responders if the percentage of cells expressing LHX1 in the heterogeneous kidney cell population is determined to be at least 50%. If the percentage of cells expressing LHX1 in the heterogeneous kidney cell population is determined to be less than 50%, the patient may be identified as a low responder. As another example, to identify whether a patient is a moderate to high responder or a low responder, the percentage of cells expressing SIX2 in the enriched heterogeneous kidney cell population can be determined. Patients can be identified as moderate to high responders if the percentage of cells expressing SIX2 in the heterogeneous kidney cell population is determined to be at least 3.5%. If the percentage of cells expressing SIX2 in the heterogeneous kidney cell population is determined to be less than 3.5% but greater than 0%, the patient can be identified as a low responder. The percentage of both LHX1 and SIX2 can be determined to identify the patient as a moderate to high responder or a low responder.

In any method of identifying an enriched heterogeneous kidney cell population as having therapeutic potential, determining whether the cells of the enriched heterogeneous kidney cell population express one or more, for example, nephrogenic markers and/or further markers (or determining the expression level of one or more genes) can be determining whether the cells of the enriched heterogeneous kidney cell population express a marker in the form of a nucleic acid (e.g., mRNA or miRNA) or a polypeptide (or can be determining the expression level of one or more genes). The marker (or gene whose expression or expression level is determined) may be membrane-bound or membrane-associated, it may be intracellular or it may be secreted from the cell. The expression of one or more, e.g., nephrogenic markers and/or further markers (or the expression level of a gene) by the cells of the enriched heterogeneous kidney cell population can be determined via any assay suitable for detecting the presence of the marker (or the expressed water of the gene). Many such assays are known in the art. For example, if the markers are determined as polypeptides (or the expression levels and expression of genes are determined), they can be determined by assays such as Western blotting, fluorescence Activated Cell Sorting (FACS), enzyme-linked immunosorbent assay (ELISA), and the like. If the markers are determined in nucleic acid form (or the level of expression and expression of the gene is determined), they can be determined by assays such as Southern blotting, polymerase Chain Reaction (PCR) or reverse transcriptase PCR, serial Analysis of Gene Expression (SAGE), mass ARRAY or Fluorescence In Situ Hybridization (FISH). Whether or not the assay determines whether or not cells of the enriched heterogeneous kidney cell population express nephrogenesis and/or further markers (or determine the expression level of one or more genes), the assay may include labeled detection reagents for determining the presence of markers (or genes whose expression levels have been determined) and/or the percentage of cells expressing markers (or genes). The labeled detection reagent may comprise (i) a label (or gene expression product) directly Or an indirectly complexed moiety and (ii) a detection moiety. Non-limiting detection moieties include radioisotopes (e.g ³⁵ S、 ¹⁴ C、 ¹²⁵ I、 ³ H and ¹³¹ i) Colloidal gold particles, fluorescent labels (e.g., texas red, rhodamine, fluorescein, dansyl, lissamine, phycoerythrin, phycocyanin, spectra ORANGE, spectra GREEN 1), and enzyme substrates (e.g., firefly luciferase, bacterial luciferase, luciferin, horseradish peroxidase, alkaline phosphatase, or beta-galactosidase).

The enriched heterogeneous population of kidney cells that can be identified as having therapeutic potential in any method can be enriched for one or more kidney cell types, such as kidney epithelial cells, tubular epithelial cells, or proximal tubular cells. Enrichment of these one or more kidney cell types by the enriched heterogeneous kidney cell population may refer to an enriched heterogeneous kidney cell population having a greater percentage of one or more kidney cell types than a patient's kidney tissue, a patient's kidney biopsy, or an in vitro culture of cells established from a patient's kidney tissue or kidney biopsy (which are collectively referred to as "starting kidney cell population"). If an in vitro culture of cells is established from patient's kidney tissue or patient's kidney biopsy, the starting kidney cell population may be a kidney cell preparation comprising dissociated cells of the kidney tissue or kidney biopsy (e.g., cells dissociated from the kidney tissue or kidney biopsy via shredding and/or enzymatic digestion), which may or may not have been treated to remove red blood cells and debris. An example of an enriched heterogeneous kidney cell population is a selected kidney cell population (SRC) as described in the examples herein.

The enriched heterogeneous kidney cell population can be enriched for one or more kidney cell types because it is prepared from the starting kidney cell population (e.g., kidney tissue of a patient, kidney biopsy of a patient, or an in vitro culture of cells established from kidney tissue or kidney biopsy of a patient) via a method that includes an isolation step. The step of isolating may be a step of isolating cells of the starting renal cell population that are passaged no more than once, twice, or three times based on the buoyancy density. If the separation step is a step of separating cells based on their buoyant density, the separation step may utilize a single or multiple step continuous or discontinuous density gradient using a density gradient medium such as glycerol, glucose OptiPrep, percoll or Ficoll-Paque. The use of such density gradient media in this manner can result in the separation of cells of the starting renal cell population (or starting renal cell population that has been passaged up to once, twice, or three times) into one or more distinguishable fractions, wherein cells of the enriched heterogeneous renal cell population can be clearly identified and isolated. Distinguishable fractions can be those in which the buoyant density of cells in the fraction is greater than about 1.045g/mL, or greater than or equal to 1.045 g/mL. Distinguishable fractions can be those in which the buoyant density of cells in the fraction is greater than about 1.04g/mL, or greater than or equal to 1.04g/mL, or greater than about 1.0419g/mL, or greater than 1.0419g/mL, or greater than or equal to 1.0419 g/mL. Distinguishable fractions may be those in which the buoyancy density is between about 1.045g/mL and about 1.091g/mL, or between about 1.045g/mL and about 1.052 g/mL. Alternatively, the isolating step may be a step of isolating the cells of the starting renal cell population (or cells of the starting renal cell population that have been passaged no more than once, twice, or three times) based on whether they express a specific marker on their surface. If the separation step separates cells based on the expression of their specific cell surface markers, the separation step may be a step utilizing flow cytometry. Flow cytometry can sort cells from a starting renal cell population (or a starting renal cell population that has been passaged up to once, twice, or three times) if they express a specific surface marker, such as a kidney disease protein (which is characteristic of, for example, renal epithelial cells, tubular epithelial cells, or proximal tubular cells), thereby forming an isolated enriched heterogeneous renal cell population.

The enriched heterogeneous kidney cell population that has been prepared from the starting kidney cell population (or starting kidney cell population that has been passaged up to once, twice, or three times) can be cultured under hypoxic conditions prior to the isolation step. If the cells are cultured under hypoxic conditions prior to the isolation step, the cells can be cultured under conditions of oxygen levels below about 20%, or below about 15%, or below about 10%, or below about 9%, or below about 8%, or below about 7%, or below about 6%, or below about 5% oxygen, or below about 4% oxygen, or below about 3% oxygen, or below about 2% oxygen. If the cells are cultured under hypoxic conditions, the cells can be cultured under hypoxic conditions for at least 6 hours, at least 8 hours, at least 10 hours, at least 12 hours, at least 14 hours, at least 16 hours, at least 20 hours, at least 24 hours, at least 30 hours, at least 36 hours, at least 42 hours, or at least 48 hours.

In general, a preparation of enriched heterogeneous kidney cell populations can be derived from any starting cell population, for example, an in vitro culture of cells established from a patient's kidney tissue or a patient's kidney biopsy. If the enriched heterogeneous population of kidney cells is prepared from an in vitro culture of cells established from a kidney tissue or kidney biopsy of a patient, the cells of the in vitro culture may be expanded by passaging up to once, or up to two or at least three times. Alternatively, if desired, cells of an in vitro culture of cells established from kidney tissue or kidney biopsy may be cryopreserved and then expanded by passaging up to once, or up to two or up to three times. Once the cells are expanded, the expanded cells may be cryopreserved. The expanded cells, whether or not cryopreserved, may then undergo a separation step, or may undergo hypoxic culture conditions, followed by a separation step. The enriched heterogeneous kidney cell population can be isolated by performing a separation step. Once the enriched population of kidney cells is isolated, it can be frozen and/or analyzed prior to use as a therapeutic agent.

In addition, one or more control kidney cell populations can be generated when preparing an enriched heterogeneous kidney cell population from the starting kidney cell population. The control kidney cell population can be any kidney cell population produced by subjecting the starting kidney cell population to one or more steps or conditions in the preparation of an enriched heterogeneous kidney cell population prior to the isolation step. The control kidney cell population may be a kidney cell population generated from the starting kidney cell population after the expansion step of the starting kidney cell population, for example by passaging the starting kidney cell population up to one, two or three times prior to the expansion step. Alternatively, the control kidney cell population may be a kidney cell population produced from a starting kidney cell population after culturing the starting kidney cell population under hypoxic conditions prior to the isolating step. In another example, the control kidney cell population can be a kidney cell population that was generated from the starting kidney cell population as the starting kidney cell population has been passaged up to once, twice, or three times and cultured under hypoxic conditions prior to performing the isolation step. In yet another example, the control kidney cell population may be a kidney cell population that has been generated from the starting kidney cell population prior to performing the isolation step as the starting kidney cell population has been expanded by passaging up to once, twice or three times and/or has been cultured under hypoxic conditions and/or has been cryopreserved. The control kidney cell population may be a kidney cell population generated from an initial kidney cell population that has undergone a complete set of steps or conditions in preparation for the isolation step, but has not undergone the isolation step.

Such a control kidney cell population can be used in the methods provided herein, wherein an enriched heterogeneous kidney cell population can be identified as having therapeutic potential if the expression level of one or more genes (e.g., RHAMM, C2, C3, C4, fibrinogen, clotting factor XIII, TEK, KDR, notch1, notch3, timp3, vwf, adam15, gas6, igfbp1, or Tm4sf 4) of the cells of the enriched kidney cell population is increased relative to the expression level of one or more genes of the cells of the control kidney cell population. The increase in the expression level of one or more genes may be at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% increase. In such methods, the one or more genes may be or may include RHAMM, and the increase in RHAMM expression level in the enriched heterogeneous kidney cell population relative to the control population may be at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% increase. In such methods, the increase in expression level may be an increase in the percentage of cells expressing the one or more genes, or may be an increase in the total amount of expression of the one or more genes (adjusted according to the number of cells).

If the enriched heterogeneous population of kidney cells is identified as having therapeutic potential according to any of the methods disclosed herein, it may be included in a pharmaceutical composition, or administered in a method of treating kidney disease in a patient in need thereof, and/or for the manufacture of a medicament for treating kidney disease. If the enriched heterogeneous kidney cell population is identified as having therapeutic potential and is contained in a pharmaceutical composition, it can be formulated as a hydrogel composition or a liquid composition. Which may or may not comprise hyaluronic acid.

If the pharmaceutical composition is formulated as a hydrogel composition, the cells of the enriched heterogeneous kidney cell composition can be combined with a temperature sensitive cell stabilizing biomaterial that maintains a gel state at about 8 ℃ or less, maintains a substantially liquid state at about ambient temperature or more, and is in a solid-liquid transition state between about 8 ℃ and about ambient temperature or more. The temperature sensitive cell stabilizing biomaterial included in the hydrogel composition may be one or more of a recombinant-derived extracellular matrix protein, an extracellular matrix derived from the kidney or another tissue or organ, or gelatin. If the temperature sensitive cell stabilizing biomaterial comprises gelatin, the gelatin may be derived from type I alpha I collagen, such as porcine type I alpha I collagen or recombinant human type I alpha I collagen. If the temperature sensitive cell stabilizing biological material comprises gelatin, the gelatin may be present in the pharmaceutical composition from about 0.5% (w/v) to about 1% (w/v) or from about 0.8% (w/v) to about 0.9% (w/v) or about 0.88% (w/v). The cells of the enriched heterogeneous kidney cell population can be dispersed throughout the biological material or substantially uniformly distributed throughout the biological material.

If the pharmaceutical composition is formulated as a liquid composition, the enriched heterogeneous kidney cell population can be combined with any suitable liquid (e.g., suitable cell storage or culture medium, saline, or a combination thereof) for immediate use or frozen storage until use.

If the enriched heterogeneous kidney cell population is identified as having therapeutic potential, the enriched heterogeneous kidney cell population or a pharmaceutical composition comprising the enriched heterogeneous kidney cell population can be administered to a patient in a method of treating kidney disease. If the enriched heterogeneous kidney cell population is identified as having therapeutic potential, the enriched heterogeneous kidney cell population or a pharmaceutical composition comprising the enriched heterogeneous kidney cell population can be used to prepare a medicament for treating kidney disease. Kidney disease may be at any stage or degree of acute or chronic kidney failure. It may originate in the kidneys and may be secondary to another disease, such as heart failure, hypertension, diabetes, autoimmune disease or liver disease. Alternatively, the kidney disease may be a kidney disease caused by acute damage to the kidney, or as a result of an abnormality of the kidney and/or urinary tract. Kidney diseases may further include endocrine dysfunctions, such as anemia, e.g. erythropoietin deficiency, and mineral imbalances, e.g. vitamin D deficiency.

If the enriched heterogeneous kidney cell population is identified as having therapeutic potential, administration of the enriched heterogeneous kidney cell population or a pharmaceutical composition comprising the enriched heterogeneous kidney cell population can treat kidney disease. It can treat kidney disease by restoring kidney function, stabilizing kidney function, improving kidney function, reducing kidney fibrosis, reducing kidney inflammation, inducing the kidneys of a patient in need of such treatment. Treatment of kidney disease may restore mineral balance or alleviate anemia in patients in need of such treatment. Treatment of kidney disease may delay or prevent the need for dialysis in a patient in need of treatment of kidney disease, or may delay or prevent the need for performing kidney transplantation. If the treatment of kidney disease delays the patient's need for dialysis or the need for kidney transplantation, the delay may be at least 1 year, at least 1.5 years, at least 2 years, at least 2.5 years, at least 3 years, at least 3.5 years, at least 4 years, at least 4.5 years, at least 5 years, at least 5.5 years, at least 6 years, at least 6.5 years, at least 7 years, at least 7.5 years, at least 8 years, at least 8.5 years, at least 9 years, at least 9.5 years, or at least 10 years. Treatment of kidney disease can be determined by observing improvements in patient serum albumin, albumin to globulin ratio (a/G ratio), serum phosphorus, serum sodium, kidney size (measurable by ultrasound), serum calcium, phosphorus to calcium ratio, serum potassium, proteinuria, urinary creatinine, serum creatinine, blood nitrogen urea (BUN), cholesterol levels, triglyceride levels and Glomerular Filtration Rate (GFR), body weight, blood pressure (mean systemic, diastolic or systolic) and physical endurance performance.

If the enriched heterogeneous kidney cell population is identified as having therapeutic potential, it can be administered to the patient by any suitable route of administration known in the art. For example, the enriched heterogeneous kidney cell population or a pharmaceutical composition comprising the enriched heterogeneous kidney cell population can be administered systemically to a patient in need of kidney disease treatment. The enriched heterogeneous kidney cell population or the pharmaceutical composition comprising the enriched heterogeneous kidney cell population may be administered to the kidney or into the kidney of a patient in need of kidney disease treatment. If the enriched heterogeneous population of kidney cells is administered to the kidney or within the kidney of a patient in need of kidney disease treatment, it may be administered by a single or multiple injections. It may be administered via direct laparotomy, via direct laparoscopy, trans-abdominally, or transdermally. The enriched heterogeneous kidney cell population or the pharmaceutical composition comprising the enriched heterogeneous kidney cell population may be administered by percutaneous injection into the renal cortex of the kidney, or may be administered by percutaneous insertion of a guide cannula to pierce the kidney envelope and then injecting the enriched heterogeneous kidney cell population into the kidney.

The enriched heterogeneous kidney cell population or the pharmaceutical composition comprising the enriched heterogeneous kidney cell population is administered at a therapeutically effective dose by any suitable route. A therapeutically effective dose or amount for administration to a patient in need of kidney disease treatment may comprise about 1-9x10 kidney weight per gram estimated patient ⁶ And (3) enriched heterogeneous kidney cell population cells. A therapeutically effective amount of the pharmaceutical composition may be about 1x10 enriched heterogeneous population of renal cells per gram of estimated patient kidney weight ⁶ Individual cells, 1x10 ⁶ Individual cells, about 2x10 ⁶ Individual cells, 2x10 ⁶ Individual cells, about 3x10 ⁶ Individual cells, 3x10 ⁶ Individual cells, about 4x10 ⁶ Individual cells, 4x10 ⁶ Individual cells, 4x10 ⁶ Individual cells,About 5x10 ⁶ Individual cells, or 5x10 ⁶ Dose of individual cells.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein. Such equivalents are intended to be encompassed by the following claims.

All publications, patents, and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated herein by reference.

Examples

Example 1: preparation of enriched heterogeneous kidney cell populations

A solution. In a method of preparing an enriched heterogeneous population of kidney cells, the enriched heterogeneous population of kidney cells is prepared from kidney tissue or biopsy samples using the reagents provided in table 1.

Table 1: culture medium and solution

Dulbecco's Phosphate Buffered Saline (DPBS) was used for all cell washes.

Cells were isolated from kidney samples. In this method, kidney tissue obtained via a kidney biopsy is used as source material for preparing non-enriched kidney cells. In general, the kidney tissue used in the preparation may consist of one or more of cortical tissue, cortical medullary connective tissue, or medullary tissue. Cortical medullary connective tissue is preferred. Multiple biopsy cores (at least 2) from CKD kidneys may be required to avoid scar tissue. Renal tissue is obtained from a patient by a clinician. If necessary, the tissue may be transported in a tissue transport medium.

The tissue is washed with a tissue wash solution to reduce the biological load of the ingress prior to cell extraction of the treated tissue.

Kidney tissue was minced and dissociated in the digestive solution. The resulting cell suspension was neutralized, washed and resuspended in Dulbecco's Modified Eagle Medium (D-MEM) +10% Fetal Bovine Serum (FBS) (Invitrogen, carlsbad Calif.), washed and resuspended in Renal Cell Growth Medium (RCGM). Cultures were initiated in RCGM on tissue culture-treated polystyrene flasks or petri dishes. For example, one biopsy sample is plated in a T25 Nunc flask containing 8mL RCGM.

Amplification of non-enriched heterogeneous kidney cultures. Renal cell expansion depends on the amount of tissue received and the success or failure of isolating the renal cells from the incoming tissue. If desired, the isolated cells may be cryopreserved. Due to the inherent variability of cells isolated from individual patients, kidney cell growth kinetics may vary from sample to sample.

A well-defined cell expansion process has been developed and accommodates the range of cell recovery due to variability of the incoming tissue. TABLE 2 expansion of kidney cells involves the use of a closed culture vessel (e.g., T-flask, cell factory,) Serial passages were performed.

Table 2: yield of human biopsied kidney cells

Once cell growth was observed in the original T-flask (passage 0) and there was no visible sign of contamination, the medium was then changed every 2-4 days. Cells were evaluated by visual observation of the cultures under a microscope to verify renal cell morphology. Cultures typically exhibit a tight pavement or cobblestone appearance due to the cell aggregation. These morphological features may change during the amplification process and may not occur at every passage. Cell culture confluency was estimated using a pool of cell images at different confluency levels in culture vessels used throughout the cell expansion process.

When the culture vessels are at least 50% confluent, the kidney cells are passaged by trypsin digestion. Isolated cells were collected into a vessel containing kidney cell growth medium, counted and cell viability was calculated. At each passage of cells, the cells are grown at 500-4000 cells/cm ² Inoculated into a sufficient number of culture vessels to expand the cell count to that required for therapeutic formulation or evaluation. The culture vessel was placed in 5% CO ₂ The environment was in an incubator at 37 ℃. Cell morphology and confluency were monitored as described above, and tissue culture medium was changed every 2-4 days. Table 3 lists the viability of human kidney cells observed during cell isolation and expansion of kidney biopsies from human donors.

Table 3: cell viability of human kidney cells in culture

Passage of	Cell viability (average%)	Range (%)
			P0(n＝82)	89.2	68.7–99.1
P1(n＝82)	96.4	89.3–99.1
			P2(n＝82)	97.4	91.9–100
P3(n＝91)	94.7	78.1–100

The inherent variability of tissue from patient to patient may lead to differences in cell yield in culture. Thus, it is impractical to strictly define the time of passage of the cells or the number and type of culture vessels required for each passage to reach the target cell number. Typically kidney cells will undergo 2 or 3 passages; however, the duration of culture and cell yield may vary depending on the cell growth rate.

Cells were isolated using TrypLE (Invitrogen) for harvesting or passaging. Viability was assessed via trypan blue exclusion and using a hemocytometer or using automatedThe counting system (Nexcelom Bioscience, lawrence Mass.) or manual counting was performed using the AO/DAPI of NC-200 nucleic.

Cryopreservation of cultured cells. The expanded kidney cells can be cryopreserved to accommodate inherent variability in cell growth in individual patients and deliver therapeutic agents, such as enriched heterogeneous populations of kidney cells, on a predetermined clinical schedule, if desired. Cryopreserved cells may also provide a source of spare cells if a therapeutic/additional therapeutic dose is needed (e.g., an additional dose is needed if delayed due to patient illness, unpredictable process events, etc.). Conditions have been established for cryopreserving cells and recovering viable functional cells after thawing.

If the expanded kidney cells are cryopreserved, the cells are suspended in a cryopreservation solution to a final concentration of about 50X10 ⁶ Individual cells/mL and dispensed into vials. Will contain about 50x10 ⁶ A 1mL vial of individual cells/mL was placed in the freezer of a controlled rate freezer and frozen at pre-harvest Cheng Sulv. After freezing, the cells are transformedAnd transferring to a liquid nitrogen refrigerator for storage in the process.

Preparation of enriched heterogeneous kidney cell populations. The enriched heterogeneous kidney cell population can be prepared from the final culture vessel in which the cryopreserved cells are grown or directly from the expanded culture.

For cryopreserved cells, the cells are thawed and plated on tissue culture containers for a final expansion step. When the final culture vessel is about 50% -100% confluent, the cells are ready for treatment to enrich for heterogeneous kidney cell separation. The medium exchange and final washing of NKA dilutes any residual cryopreservation solution in the final product.

Once the final cell culture vessel reaches at least 50% confluence, the culture vessel is transferred to a hypoxic incubator at 37℃with 5% CO ₂ The environment was set up with 2% oxygen and incubated overnight. Cells may be kept in an oxygen-controlled incubator with an oxygen concentration set to 2% for less than 24 hours or overnight. Exposure to a physiologically more relevant hypoxic (2%) environment increases cell separation efficiency and enables better detection of hypoxia-induced markers such as VEGF.

After exposing the cells to hypoxic conditions for a sufficient period of time (overnight to 48 hours), the cells were isolated with TrypLE (Invitrogen). Viability was assessed via trypan blue exclusion and using a hemocytometer or using automatedThe counting system (Nexcelom Bioscience, lawrence Mass.) or manual counting was performed using the AO/DAPI of NC-200 nucleic. Cells were washed once with OptiMEM and resuspended to approximately 850X10 in DPBS ⁶ Individual cells/mL.

Centrifugation across the density boundary/interface is used to separate the harvested renal cell populations based on their buoyant density. The renal cell suspension was isolated by centrifugation over 7% iodixanol solution (OptiPrep; 60% (w/v)) in OptiMEM.

A 7% optiprep density interface solution was prepared and the refractive index (r.i. 1.3456 +/-0.0004) indicative of the desired density was measured prior to use. The harvested kidney cells layered on top of the solution. The density interface was centrifuged at 800x g for 20 minutes at room temperature (without braking) in a centrifuge tube or cell processor (e.g., COBE 2991). Cell fractions exhibiting a buoyancy density greater than about 1.045g/mL were collected as distinct pellet after centrifugation. Cells that maintained a buoyancy density of less than 1.045g/mL were excluded and discarded.

The enriched heterogeneous kidney cell population pellet was resuspended in DPBS. Residues of OptiPrep, FBS, medium and auxiliary materials remaining in the final product can be minimized by 4 DPBS washing steps.

Example 2: identification and measurement of the expression of nephrogenic markers enriched for heterogeneous renal cell populations indicates their therapeutic potential

Introduction to the following. Regeneration of complex tissues and organs (including kidneys) may take advantage of the mechanisms common in development and organogenesis. Expression of multiple markers, typically associated with the earliest signaling event during embryogenesis, would indicate that an enriched heterogeneous population of kidney cells, e.g., prepared via the method described in example 1, has dedifferentiated and acquired more kidney progenitor-like properties. The introduction of enriched heterogeneous populations of renal cells with these characteristics into the diseased renal parenchyma can trigger the occurrence of a critical signaling cascade that normally mediates nephrogenesis, but it can be interpreted (in the context of adult renal parenchyma) as regeneration.

The method. The enriched heterogeneous kidney cell population prepared, for example, as described in example 1, was tested for expression of nephrogenic markers by FACS analysis. Antibodies for detecting the expression of the nephrogenic marker of the cells are identified in table 4.

Table 4: antibodies useful for FACS analysis to detect developmental markers expressed by renal cell populations

Antigens	Antibodies to	Conjugate(s)	Catalog #	Suppliers (suppliers)
					Pax2	Rabbit monoclonal	Without any means for	EP3251/ab79389	Abcam
FoxD1	Rabbit, polyclonal, N-terminal	Without any means for	ab179940	Abcam
					Six2	Monoclonal Six1, H4	PE	sc377193-PE	Scbt
Osr1	Monoclonal IgGa, C8	PE	sc376545-PE	Scbt
					Bmp7	Rabbit polyclonal, full length	Without any means for	ab27569	Abcam
Bim	Rabbit monoclonal, HY36	AlexaFluor488	ab200667	Abcam
					Lim1	Rabbit polyclonal	Without any means for	NB110-12933	Novus bio
Sal1	Monoclonal IgG2a	Without any means for	ab41974	Abcam
					GDNF	Rabbit monoclonal	Without any means for	ab176564	Abcam
RET	Rabbit monoclonal	AlexaFluor488	ab237105	Abcam
					Wnt9b	Rabbit polyclonal	Without any means for	NBP1-44348	Novus bio
Bmp4	Rabbit monoclonal	AlexaFluor488	ab200794	Abcam
					Wt1	Rabbit monoclonal	Without any means for	ab89901	Abcam
Wnt4	Rabbit polyclonal	Without any means for	ab91226	Abcam
					Pax8	Monoclonal IgG1	Without any means for	ab53490	Abcam
Fgf8	Monoclonal, holoprotein	Without any means for	ab89550	Abcam
					Lhx1	Monoclonal IgG2a	PE	sc-515631PE	scbt
Notch1	Rabbit polyclonal	Without any means for	ab118824	Abcam
					Muc1	Rabbit monoclonal	Without any means for	ab109185	Abcam

Cell preparation for FACS. For FACS analysis, cells of the enriched heterogeneous kidney cell population were harvested by trypsinization and centrifuged at 400g for 10 min to form a cell pellet. The cell pellet was washed twice with Phosphate Buffered Saline (PBS) and resuspended in 4% paraformaldehyde solution containing saponin for cell permeabilization for fixation when detection of intracellular antigens is desired. After fixation, cells were pelleted and washed twice with 1x wash buffer (Becton Dickenson, proprietary composition) and resuspended in 100 μl to 200 μl wash buffer.

FACS staining and analysis. As shown in Table 4, FACS staining was performed by adding 2. Mu.g of primary antibody (directly labeled or unlabeled) and staining was performed at 4℃for 20 minutes. After incubating the cells with the antibodies, the cells were washed twice with wash buffer. If desired, the cells are further immunolabeled, for example if the primary antibody is unlabeled, with an appropriate secondary antibody bearing a detectable fluorophore (e.g., alexaFluor 488). To allow for proper channel gating during FACS analysis, appropriate isotype controls and secondary antibody controls were set up in parallel with the primary staining reactions.

All cell populations were again washed with wash buffer and resuspended in 200 μl PBS. Immunostaining enriched heterogeneous kidney cell populations were analyzed on a MACQUANT analyzer.

As a result. In a total of 18 clinically enriched heterogeneous renal cell population samples, specific expression levels of 5 nephrogenic markers were observed. See tables 5 and 6.

Table 5: expression of nephrogenic markers in enriched heterogeneous populations of kidney cells with therapeutic potential

Sample of	Six2	OSR1	LHx1	RET	FGF8
						NKA-013	0	43.77	13.64	2.49	0
NKA-031	0	78.66	21.48	10.15	0
						NKA-056	4.36	65.19	3.84	14.25	0.49
NKA-048	0.12	71.53	11.66	9.34	0.25
						NKA-028	0.18	72.77	13.92	18.8	0.63
NKA-039	0	84.49	5.65	6.49	1
						NKA-030	0.18	96.28	25.14	19.1	1.39
NKA-023	0.58	94.37	31.78	10.86	0.54
						NKA-032	0.99	96.72	38.33	2.07	0.23
NKA-070	4.08	97.19	42.58	1.33	1.08
						NKA-087	8.11	98.29	90.79	1.98	0.31
NKA-089	1.7	91.95	31.46	1.29	1.15
						NKA-042	0.24	52.82	0.87	1.42	0.01
NKA-034	0.98	57.23	2.53	1.26	2.65
						NKA-092	3.11	74.95	3.97	0.58	0.11
NKA-063	0.32	67.2	8.68	1.45	0.16
						NKA-097	0.06	69.29	6.43	1.09	0.08
NKA-075	1.12	81.8	6.63	11.37	0.52

Table 6: summary of the range of cell percentages expressing nephrogenic markers in enriched heterogeneous populations of kidney cells with therapeutic potential

Marker(s)	Low (cell%)	High (cell%)
			SIX2	0.04	6
OSR1	36.3	84.43
			LHX1	8.5	57.1
RET	49.1	89.4
			FGF8	0.48	58.55

It was also found that about 4.34 to 98.72 percent of cells in the enriched heterogeneous kidney cell population with therapeutic potential expressed kidney disease proteins.

Further analysis was performed on the clinically enriched heterogeneous kidney cell population samples, again determining the expression levels of the 5 nephrogenic markers. In addition to the 5 nephrogenic markers, specific levels of kidney disease proteins and podoprotein (which may be important for glomerular development during nephrogenesis) and RACK-1 were also determined. See fig. 1B.

Regeneration of the kidneys may require the use of the mechanism elements common in development and organogenesis. Expression of the marker normally associated with the earliest signaling event in embryogenesis will indicate that an enriched heterogeneous population of kidney cells, e.g., prepared as described in example 1, has dedifferentiated and acquired more kidney progenitor-like properties. Thus, the introduction of such an enriched heterogeneous population of kidney cells into a diseased kidney may trigger the occurrence of a critical signaling cascade that normally mediates nephrogenesis, but (in the substantial context of adults) may be interpreted as regeneration. Expression of the nephrogenic markers SIX2, OSR1, LHX1, RET and FGF8 may explain the mechanism of how the enriched heterogeneous kidney cell population can provide therapeutic benefit, for example via regeneration of the diseased kidney. Enriched heterogeneous populations of kidney cells, e.g., prepared as described in example 1, have been shown to have therapeutic effects in patients in need of kidney disease treatment, such as delayed dialysis, reduced albumin to creatinine ratio, and increased egffr. Each of the nephrogenic markers and its role in nephrogenesis is briefly described below.

SIX2 is a member of the vertebrate gene family, which encodes a protein homologous to the drosophila "sine oculis" homeobox protein. The SIX2 protein is a transcription factor and plays an important role in the development of a plurality of organs such as kidneys, skull, stomach and the like. During kidney development, SIX2 maintains the hat mesenchymal multipotent nephron progenitor cells in an undifferentiated state by antagonizing the induction signals emitted by ureteric buds and promotes the proliferation of the updated progenitor cells in cooperation with WNT 9B. SIX2 may act through its interaction with TCF7L2 and OSR1 to prevent transcription of differentiation genes (e.g., WNT 4) in the hat mesenchyme in a typical WNT signaling independent manner. In addition, it functions independently of OSR1, activating the expression of many of the cap mesenchymal genes (including itself, GDNF and OSR 1).

OSR1 is the earliest marker of the mesoderm (which will form gonads and kidneys). This expression is not essential for the formation of the mesoderm, but is critical for differentiation into renal and gonadal structures. OSR1 acts upstream of and causes expression of transcription factors LHX1, PAX2 and WT1 involved in early urogenital development. In normal kidney development, activation of the PAX2-EYA1-HOX11 complex and subsequent activation of SIX2 and GDNF expression allows for the branching of ureteral buds and maintenance of nephrons forming the hat mesenchyme. SIX2 maintains the self-renewing state of the hat matrix and GDNF via the GDNF-RET signaling pathway, necessary to attract and branch the growing ureteral bud. In the developing kidney, cells expressing OSR1 will become mesangial cells, pericytes, ureteric smooth muscle and kidney capsule. The cell type into which the OSR1 expressing cells will differentiate is determined by the time of loss of expression-cells that will become part of the vasculature or ureteric epithelium will lose OSR1 expression early (E8.5) while those that become nephrons will lose expression later (E11.5). All three phases of kidney formation were affected in mice lacking OSR1 expression and were similar to mice with reduced expression of WT1 and PAX 2-Wollfian abnormalities, fewer midrenal tubules, and formation of postrenal and gonadal deletions in the kidneys. At day 10.5 of the embryo, embryos lacking OSR1 expression were unable to develop ureteral buds that migrated into the uncompacted postrenal interstitium. The lack of ureteral bud induction signals combined with the reduction of downstream PAX2 expression results in renal cell apoptosis and hypoplasia.

LHX1 is a transcription factor. In vertebrate embryos, the kidneys are derived from the mesoderm. LIM homologous box transcription factor LHX1 is expressed in early middle mesoderm and is one of the earliest expressed genes in renal interstitium. The loss of LHX1 in xenopus embryos results in near complete loss of kidney area.

RET, among other things, is essential for normal kidney development and spermatogenesis (spermatogenesis). RET proteins cross the cell membrane, so that one end of the protein remains inside the cell and the other end protrudes from the outer surface of the cell. The localization of this protein enables it to interact with specific factors outside the cell and to receive signals that help the cell respond to its environment. When molecules (growth factors) that stimulate growth and development attach to RET proteins, a complex series of chemical reactions within the cell are triggered. These responses indicate that the cells undergo certain changes, such as division or maturation, to assume a particular function.

The FGF8 gene encodes fibroblast growth factor 8 (FGF 8). Such proteins are part of a family of proteins known as fibroblast growth factors, which are involved in many processes including cell division, regulation of cell growth and maturation, and prenatal development. FGF8 attaches (binds) to another protein called fibroblast growth factor receptor 1 (FGFR 1) on the cell surface, triggering a series of chemical reactions within the cell.

A group of nephrogenic markers expressed by enriched heterogeneous populations of kidney cells with therapeutic potential have been identified. Expression of these markers provides evidence that cells of the enriched heterogeneous kidney cell population have the ability to influence the signaling cascade that mediates nephrogenesis or kidney regeneration, thereby providing a therapeutic effect. Notably, there are some differences in the expression of the markers, which are expected due to the autologous source of the cells.

Example 3: transcriptional regulation is identified that is of mechanistic interest for the therapeutic biological activity of enriched heterogeneous kidney cell populations.

Introduction: to further understand the underlying mechanisms of repair, restoration and/or regeneration activity of the enriched heterogeneous kidney cell population, whole genome transcriptome analysis was performed on the kidney cell subpopulations included therein. Four component enriched kidney cell subsets (B2, B3, B4 and B5) prepared by density gradient separation of PreG (pre-gradient fractionation) material are contained in the enriched heterogeneous kidney cell population. In this example, transcriptome spectra of rodent PreG material and four component enriched kidney cell subpopulations (B2, B3, B4 and B5) comprising rodent enriched heterogeneous kidney cell populations were analyzed, compared and controlled, respectively. By analyzing each of the four component enriched kidney cell subsets (B2, B3, B4 and B5) separately and comparing their transcriptome profile with that of the PreG material, the contribution of each component to the repair, restoration and regeneration bioactivity observed for enriched heterogeneous kidney cell populations can be understood.

Materials and methods/preparation of renal cell populations: kidneys from 3 independent donors (5 week old, male, syngeneic Lewis laboratory rats)The viscera were used as starting material for 3 independent biological replicates of all renal cell populations and subpopulations characterized in this assay. Preparation of selected bioactive primary kidney cells from whole rat kidneys has been described in detail (a.t., guthrie, k.i., keley, r.methods Mol Biol 1001,53,2013; keley, r., et al, american Journal of Physiology)&The Renal physiolog 299,1026,2010; keley, r., et al cell transformation 22,1023,2013). Briefly, whole kidneys were harvested from 5 week old male Lewis rats (Hilltop Labs, scottsdale, PA, USA) and kidney tissue was digested in a buffer containing 4.0 units/mL of dispase (Stem Cell Technologies, inc., vancouver, BC, canada) and 300 units/mL collagenase IV (Worthington Biochemical, lakewood, NJ, USA). Through 15% iodixanolAxis Shield, norton, mass., USA) to remove red blood cells and debris. Primary kidney cells were seeded onto tissue culture treated polystyrene plates (NUNC, rochester, NY, USA) and cultured in a 1:1 mixture containing 5% Fetal Bovine Serum (FBS), 2.5 μg EGF, 25mg Bovine Pituitary Extract (BPE), 1X ITS (insulin/transferrin/sodium selenite medium supplement) and antibiotic/antifungal agents (all from Invitrogen, carlsbad, CA, USA) in 50:50 medium (i.e. high glucose Dulbecco's Modified Eagle Medium (DMEM): keratinocyte Serum Free Medium (KSFM)). Primary kidney cell cultures were transferred from atmospheric oxygen conditions (21%) to a more physiologically relevant hypoxic (2%) environment for 24 hours prior to cell separation after culture to increase cell separation efficiency. Prepared as 75x10 in 2mL of uncompensated KSFM (uKSFM) ⁶ Isolation of primary kidney cell cultures of individual cells were isolated by density gradient centrifugation (16%, 13%, 11% and 7%) of four-step iodixanol (OptiPrep; 60% w/v in uKSFM) exclusively for rodents in 15mL conical polypropylene tubes and centrifuged at 800xg for 20 min (no brake) at room temperature. Following centrifugation, cell subfractions were extracted from the gradients via pipettes and collected as 4 distinct bands (B1-B4) and pellet (B5). All strips were washed 3 times with sterile Phosphate Buffered Saline (PBS) before use. Gradient pretreatment from each rodentThe product ("PreG") and whole kidney tissue samples ("Macro") were used for comparison purposes. The culture conditions for each rodent cell population are summarized in table 7 below.

Table 7: culture conditions and gradient loadings ¹

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¹ Gradient loading refers to the number of PreG cells loaded onto an iodixanol gradient for separation into a component enriched population of kidney cells contained in an enriched heterogeneous population of kidney cells (e.g., SRC).

Materials and methods/transcript analysis: RNA for whole genome transcript analysis was prepared using the Qiagen RNA isolation kit according to the manufacturer's instructions. 2. Mu.g RNA from each sample was normalized to Table 8 below for whole genome transcript analysis on a Affymetrix GeneChip rat genome 230.0 array (Wei Ke forest university health science microarray core facility (Wake Forest University Health Sciences Microarray Core Facility), winston-Salem, NC).

Table 8: RNA concentration and normalization

RNA was normalized by resuspension of a total of 2. Mu.g RNA in a total volume of 20. Mu.l. In Table 2, the column "vol, 2. Mu.g" lists the volumes of RNA preparations representing a total of 2. Mu.g RNA. The column "Norm 20. Mu.l" lists the volume of additional buffer to be added to make up the final volume of 20. Mu.l.

Materials and methods/data analysis: affymetrix GeneChip data were normalized using RMA (Rafael et al 2003.Nucleic Acids Research 31:e15). The gene expression profile of each component enriched kidney cell subpopulation (B1, B2, B3, B4, B5) was compared to the gene expression profile in the PreG material using paired t-test. Detection of P by paired t-test<0.05, followed by DAVID (http:// david. Abcc. Ncifcrf. Gov) analysis was performed to examine the Gene Ontology (GO) class and the pathways of the Kyoto gene and genome encyclopedia (KEGG) that differ significantly between each fraction and the PreG. For GO analysis, GO-BP-FAT is used, which is a GO bioprocess category provided by DAVID to minimize redundancy and increase GO entry specificity. Bonferroni adjusts P values applied to GO and KEGG pathway analysis to make multiple test corrections. KEGG pathway patterns were generated using Pathview (Weijun Luo and Cory brouwer. Bioenginics, 29 (14): 1830-1831, 2013) in Bioconductor. The GO type gene network is Ingenuity Pathway Analysis using QIAGEN QIAGEN Redwood City, www.qiagen.com/ingness).

Results/subfractions B1: GO analysis of the B1 subpopulation showed significant down-regulation of GO categories associated with cell cycle (p=9.40E-04), cell division (p=9.81E-05) and cell cycle phase (p=0.016) regulation relative to PreG material (table 9). KEGG pathway analysis also produced similar results (table 9). Down-regulated cyclical pathway genes are distributed throughout various phases of the cell cycle, including cell growth (G1, G2), DNA replication (S), and mitosis (M). For example, key cell cycle regulatory proteins that are down-regulated in B1 relative to PreG include CDK2, CDK7, CYCE, CDC45, ORC, CHK1, CHK2, MAD2, CDC20, and APC. In addition, GO/KEGG categories associated with transcriptional regulation, including transcriptional regulation (p=0.001), RNA metabolic regulation (p=0.044), and spliceosome (p=0.002) were all significantly down-regulated (table 9).

Table 9: GO category and KEGG pathways significantly up-or down-regulated in B1 compared to PreG.

These transcriptome data indicate significantly lower proliferation and regeneration potential of the B1 subpopulation, consistent with the observed no functional impact of B1 cells on kidney pathophysiology in CKD rodent models (Bruce, a.t., guthrie, k.i., keley, r.methods Mol Biol 1001,53,2013; keley, r., et al, american Journal of Physiology & Renal physioloy 299,1026,2010; keley, r., et al, cell transformation 22,1023,2013).

The GO/KEGG class significantly up-regulated in subgroup B1 relative to PreG material includes cytokine-mediated signaling pathways involved in immune response (p=0.023) and lysosomes involved in digestion of foreign substances and cellular waste (p=4.44E-04). Cell adhesion (p=0.045) was also up-regulated (table 9). These data are again consistent with the observation that B1 cells in the rodent model of CKD have no functional impact on renal pathophysiology. B1-enriched kidney cell subpopulations are not a component of the enriched heterogeneous kidney cell population.

Results/subfractions B2: the transcriptome profile of B2 was found to be similar to PreG at the pathway level. No GO/KEGG class was identified as significantly up-regulated. Only one pathway, ECM-receptor interaction (rno 04512), was found to be significantly down-regulated relative to PreG material (p=0.039). Consistent with ECM-receptor interactions down-regulated, many ECM proteins, including collagen, laminin, filaggrin, tenascin, vitronectin, and Thrombospondin (THBS), are significantly down-regulated in B2; it is well known that excessive deposition of extracellular matrix (ECM) components can lead to tissue fibrosis (Liu Y. Kidney International 69,213,2006;Kisseleva and Brenner,2008.Proc Am Thorac Soc 5:338-42; el-Nahas,2003.Kidney Int 64:1553-63). Furthermore, renal fibrosis is a hallmark of disease progression from chronic kidney disease (CDK) to end-stage renal disease.

In contrast to the negative-regulatory transcriptome profile observed for ECM-related genes, hyaluronic acid-mediated movement receptor (RHAMM; also known as CD168 or hyaluronic acid-mediated movement receptor (HMMR)) expression was specifically up-regulated in the B2 subpopulation relative to PreG material (fold change = 1.114, p = 0.030). In many in vitro and in vivo model systems, RHAMM expression is directly related to regenerative biological activity. RHAMM has also been shown to promote each of angiogenesis, 3D salivary gland sphere regeneration, and Xenopus) tadpole tail and cell movement, for example. Thus, an increase in RHAMM expression levels may be beneficial for tissue regeneration (Pradhan-Bhatt et al, 2014.Laryngoscope 124:456-461;Contreras et al, 2009.Development 136:2987-96; tolg et al, 2006.J Cell Biol 175:1017-28;Savani RC,et al.2001.J.Biol.Chem.276 (39): 36770-8;Hall CL,et al.1994. J.cell biol.126 (2): 575-88;Hamilton SR,et al.2007.J.Biol.Chem.282 (22): 16667-80). In addition, hyaluronic Acid (HA) (RHAMM is its receptor) is an ECM component that shows the following: is present in the entire interstitial space of the developing kidney (Bakala, h., et al, 1988.J Morphol 196:1-14); stimulating ureteral buds to differentiate into collecting tubes; and a transition of the postrenal mesenchyme from mesenchyme to epithelium in a molecular weight independent manner. RHAMM is significantly upregulated in the B2 subgroup, and these data present interesting possibilities for renal tissue engineering and cytomedicine. For example, scaffolds designed to release HA of defined molecular weight at a certain concentration may be implanted into the kidney in the hope of modulating regeneration outcome.

Results/subfractions B3: KEGG pathway analysis found significant upregulation (p=0.008) of the class associated with complement and coagulation cascade in subgroup B3 relative to PreG material (table 10). Upregulated genes include complement component genes such as C2, C3 and C4, and coagulation-related genes such as Fibrinogen (FG) and factor XIII (F13). The complement and coagulation system is considered to be the "first line of defense" against injury and intruders (Choi G, et al Swiss Med Wkly.2006; 136:139-144). Thus, upregulation of this pathway indicates an enhanced immune response. Consistent with the KEGG analysis, the GO analysis also found a significant increase in humoral immune response (p=0.025) (table 10). It is well known that multiple components of the cellular and humoral immune system contribute to repair, restoration or regeneration of multiple organs and tissues, including the extremities, skeletal muscles, heart and nervous system (Aurora and Olson,2014.Cell Stem Cell 15:14-25).

Table 10: GO category and KEGG pathway significantly up-or down-regulated on B3 compared to PreG

Significant downregulation of GO class in subgroup B3 was associated with actin polymerization and regulation of actin filament length (table 10). Dynamic reorganization of actin cytoskeleton is the primary driving force for cell migration. Inhibition of actin polymerization may adversely affect cell migration and therapeutic efficacy of the B3 subpopulation.

Results/subfractions B4: the B4 subpopulation was observed to have functional consequences in CKD rodent models. Consistent with the observed in vivo therapeutic bioactivity, a variety of regeneration-related GO categories, including vasculature development (p=7.76E-07), vascular development (p=1.36E-06), vascular morphogenesis (p=1.76E-06), angiogenesis (p=1.82E-06), and response to injury (p=1.35E-05) were found to be significantly upregulated in subgroup B4 compared to PreG material (table 11). GO class angiogenesis is critical for repair, restoration or regeneration of kidneys, heart and a number of other organs and tissues (Takiya and Borojevic,2011.Kidney Int Suppl 1:99-102;Kramann and Humphreys,2014.Semin Nephrol 34:374-83;Park and Gerecht,2014.Development 141:2760-9; kaully et al, 2009.Tissue Eng Part B15:159-69). Key receptors (including CXCR4, TEK, FGFR1 and KDR) found to be upregulated in angiogenesis and other regeneration-related signaling pathways. Among them, the SDF1/CXCR4 axis is critical for kidney repair, restoration or regeneration in response to ex-situ cell-based therapies (Togel and Westenfelder,2011.Kidney Int Suppl 1:87-89, shamama et al, 2011.Stem Cells Dev20:933-46; sagrinati, C., et al trends Mol Med 14,277,2008). In the GO class response to injury, many up-regulated genes, such as Notch1, notch3, timp3, vwf, adam15, gas6, igfbp1 and Tm4sf4, also belong to the GO class of wound healing and tissue recovery, repair or regeneration. Significant upregulation of these genes relative to PreG is also consistent with regenerative bioactivity of the B4 subgroup.

Table 11: significantly up-regulated GO class in B4 compared to PreG

Furthermore, it was found that the GO class associated with cell adhesion was significantly up-regulated in B4 compared to PreG material (table 11). Cell adhesion is critical for the incorporation and functional integration of transplanted cells into the host environment. Induction of cell adhesion genes may be beneficial for homing of transplanted cells.

No significant downregulation of the pathway analyzed in B4 was found.

Results/subfractions B5: subgroup B5 was found to be the most unique fraction relative to PreG material at the gene expression level. Forty GO/KEGG categories differ significantly from PreG (Table 12 and Table 13).

Table 12: significantly up-regulated GO class and KEGG pathway in B5 compared to PreG

Table 13: significantly down-regulated GO class and KEGG pathway in B5 compared to PreG

Upregulated GO/KEGG classes include cell adhesion, angiogenesis, branching morphogenesis of the tube, cellular morphogenesis, regulation of epithelial cell proliferation, vascular development, vascular morphogenesis, vasculature development, cellular component morphogenesis, cellular morphogenesis involved in differentiation, tubular morphogenesis, cell process tissue and morphogenesis, cell fraction morphogenesis, morphogenesis of branching structures, developmental maturation, and response to nutrition, among others. Most of these significantly upregulated GO/KEGG classes are clearly involved in catalytic repair, restoration, or regeneration outcomes.

As observed in B4, the key pro-angiogenic receptors CXCR4, TEK and KDR are significantly up-regulated in B5. Furthermore, significant upregulation of pro-angiogenic ligands PGF, ANGPT2, ANGPT4, VEGFA and PDGFA relative to PreG material in the B5 subgroup was observed. The regenerative biological activity of these ligands has been well established.

Several significant differences of particular interest regulate pathways, including the canonical Wnt/b-catenin signaling pathway. Typical Wnt/B-catenin signaling pathways were significantly down-regulated in subgroup B5 relative to PreG material (table 13). Wnt signaling is closely related to nephrogenesis and organogenesis, but may also be pro-fibrotic and contribute to the maintenance of disease states where appropriate (Kawakami et al, 2013.J Pathol 229:221-31; shkreli et al, 2011.Nat Med 18:111-9;Cisternas et al, 2014.Curr Mol Med 14:510-22). In the context of SRC, the relevant Wnt signaling bioactivity of B5 may be its pro-fibrotic effect; thus, any reduction in Wnt signaling may be the desired outcome after implantation of fibrotic diseased kidneys (Cuevas et al 2015.Biomed Res Int article ID 726012).

Also interesting is the up-regulation of the expression of GO class "branching morphogenesis of tubes" in subgroup B5 relative to PreG material. This is interesting because mammalian nephrogenesis is driven by an iterative process of branching morphogenesis between ureteric buds (derivatives of the renal tubular) and the surrounding postrenal interstitium. Ureteral bud epithelium continues branch morphogenesis in response to signaling from the proximal postrenal interstitium, which in turn leads to the induction of new postrenal interstitium aggregates and persistent nephrogenic events at the tips of ureteral buds. This iterative process continues along the radial axis of the developing kidney, with the youngest nephron induced to the periphery (reviewed by Basu and Ludlow,2012.Birth Defects Research Part C96:30-38). To this end, implantation of an enriched heterogeneous population of kidney cells (e.g., SRC) in adult rodent kidneys is associated with de novo induction of new nephron-like structures (Basu, j., et al cell transformation 20,1771,2011).

Finally, interestingly, the modulation of activation of GO-like epithelial cell proliferation by TGF- β1 signaling pathway is consistent with the potential role of an enriched heterogeneous kidney cell population (e.g., SRC) in promoting host tubular epithelial cell proliferation. Indeed, for this purpose, it has been observed that the transplanted enriched heterogeneous kidney cell population promotes tubular cell proliferation in a 5/6Nx model. Treatment of enriched heterogeneous populations of kidney cells (e.g., SRC) specifically increases the number of ki67+ proliferating cells in tubular epithelial cells. Such compartment-specific proliferation may be a direct indicator of therapeutic outcome: epithelial proliferation leads to replenishment of renal function, while interstitial proliferation leads to fibrosis.

A significant down-regulation of multiple GO/KEGG categories associated with protein metabolism in the B5 subpopulation was observed compared to the PreG material, which may simply indicate a decrease in the overall metabolic rate of the B5 subpopulation relative to the PreG material.

Summary. Enriched heterogeneous kidney cell populations, such as SRC, are prepared by iodixanol gradient centrifugation of the PreG material. Iodixanol gradient centrifugation of the PreG material results in separation of the PreG material into five subpopulations (B1-B5). Transcriptome analysis of the B1-B5 subpopulations identified key transcriptome networks and accompanying signaling pathways that may be the basis for an enriched heterogeneous kidney cell population (e.g., SRC) as demonstrated by its repair, restoration and regeneration bioactivity in the treatment of chronic kidney disease. Significant differences in the B2-B5 subpopulations contained in the final enriched heterogeneous kidney cell population product relative to the PreG material include: downregulation of GO/KEGG class associated with ECM-receptor interactions in subgroup B2; up-regulation of GO/KEGG class associated with immune response and down-regulation of GO/KEGG class associated with actin polymerization regulation in subgroup B3; up-regulation of GO/KEGG class in B4 associated with repair, recovery, regeneration and cell adhesion; and forty differentially regulated GO/KEGG classes in B5, including multiple classes that are clearly associated with repair, restoration, or regeneration activity. Subpopulation B1, which is not included in the enriched heterogeneous kidney cell population, showed significant downregulation in the GO/KEGG class associated with cell cycle and transcriptional control and significant upregulation in the GO/KEGG class associated with inflammation, relative to the PreG material.

In summary, transcriptome data suggests that enriched heterogeneous populations of kidney cells (e.g., SRC) for therapeutic use catalyze kidney repair, restoration, or regeneration outcomes in part by activating multiple transcriptome networks associated with promoting regeneration while inhibiting alternative transcriptional pathways that may promote kidney disease and pathophysiological progression.

Example 4: identifying developmental pathways that are mechanically significant for regenerative bioactivity of enriched heterogeneous human renal cell populations for the treatment of chronic kidney disease.

Introduction: further based on rodent data in example 3, transcriptome and epigenomic analyses were performed on human enriched heterogeneous kidney cell populations prepared for clinical trials. Renal autologous cell therapy (REACT) is a novel regenerative medicine candidate for the treatment of chronic kidney disease, diabetic nephropathy, and congenital anomalies of the kidneys and urinary tract (Stavas 2021.Protocol and Baseline Data on Renal Autologous Cell Therapy Injection in Adults with Chronic Kidney Disease Secondary to Congenital Anomalies of the Kidney and Urinary Tract.Blood Purif 50 (4-5): 678-683; stenvenkel P,j, bertram T, detwiler R, gerber D, brismar TB, et al imaging of autologous selected renal cells in diabetic chronic kidney disease stages 3and 4-clinical experience of a "First in Human" student Kidney Int Rep.2016 (3): 105-13). REACT is formulated from an enriched heterogeneous kidney cell population (referred to as SRC or SRC population in this example), prepared or manufactured from an initial cell population derived from a subject's own kidney biopsy (referred to as BRC0 or BRC0 population in this example). Transcriptome and epigenomic profiles of human SRC and BRC0 populations were compared to identify and further explore potential mechanisms of repair and restoration activity of human SRC for use in treating kidney disease.

Materials and methods/preparation of SRC for use in the treatment of chronic kidney disease: methods for preparing selected kidney cells from kidney tissue biopsies have been described in detail previously (Bruce, a.t., guthrie, k.i., keley, r.ex vivo culture and separation of functional renal cells methods Mol Biol 1001,53,2013;Halberstadt et al, 2013,Methods Mol.Biol).

Materials and methods/cDNA library construction: to construct the library, RNA was extracted from each cell population (n=6). After DNase I treatment and Quality Control (QC), library construction was performed using 200ng of high quality total RNA. mRNA was isolated using magnetic beads with Oligo (dT). mRNA was randomly fragmented by adding a fragmentation buffer, and cDNA was then synthesized using mRNA templates and random hexamer primers after addition of dNTPs, RNase H and DNA polymerase I. The short fragments were purified and resolved with EB buffer for end repair and single nucleotide a (adenine) addition. Thereafter, the short fragments are ligated to the sequencing aptamer. Double stranded cDNA library was completed by size selection and PCR enrichment. In the QC step, sample libraries were quantified and identified using an Agilent 2100 bioanalyzer and an ABI StepOnePlus real-time PCR system. Finally, after combining according to effective concentration and expected data volume, qualified RNA-seq libraries were sequenced using the Illumina Novaseq6000 of CD Genomics (Shirley, N.Y.).

Materials and methods/bioinformatics analysis: the FastQC tool (http:// www.bioinformatics.babraham.ac.uk/subjects/fastq) was used to make basic statistics on the quality of the original reading. Trimmomatic (version 0.36) deleted sequencing aptamers and low quality data (Juhling, F., et al, metilene: fast and sensitive calling of differentially methylated regions from bisulfite sequencing data. Genome Res,2016.26 (2): p.256-62). Clean reads after filtration were mapped to the reference genome by HISAT2 (Kanehisa, M., et al KEGG for linking genomes to life and the environmental.nucleic Acids Res,2008.36 (Database isue): p.D480-4) and position and gene signature were obtained. The cuffquat and Cuffnorm components of Cufflinks software are used to quantify transcript and gene expression levels using mapping read location information on genes. The number of genes at different expression levels was analyzed, and the gene expression level of each individual gene. DESeq was used to analyze DEG of samples with biological replicates, while EBSeq was used to analyze samples without replicates. During the analysis, the samples were first grouped so that every two groups could later be compared pairwise as control-treatment groups. In this process, fold change was set to be 2 or more and FDR <0.01 was used as a screening criterion. The gene ontology (GO-Gene Ontology Consortium, 2000) enrichment analysis is a set of internationally standardized classification systems for gene function descriptions that attempt to identify GO terminology that is significantly related to differentially expressed protein-encoding genes. GO molecules fall into three major categories: 1) Cell composition: for describing subcellular structure, location, and recognition of macromolecular complexes (e.g., nucleoli, telomeres) and initial complexes; 2) Molecular function: for describing genes, gene products, individual functions such as carbohydrate binding or ATP hydrolase activity; 3) Biological process: are used to describe the products encoded by genes involved in biological processes such as mitosis or purine metabolism. The KEGG pathway was used to identify pathways that were significantly enriched in a particular gene compared to the entire genomic background. The formula for this analysis is:

Where "N" is the number of all genes with pathway notes; "N" is the number of candidate genes within N; and "m" is the number of genes annotated with a particular pathway. The pathways with FDR.ltoreq.0.05 are defined as significantly enriched pathways. Pathway enrichment analysis was performed using KOBAS (2.0).

Materials and methods/simplified representative bisulfite sequencing (RRBS): using Nucleon ^TM BACC genomic DNA extraction kit (GE Healthcare, life Sciences) genomic DNA was isolated from cell samples (BRC 0, SRC, n=4) and double stranded DNA content was quantified using a Qubit high sensitivity assay (Life Technologies). Mu.g of genomic DNA in each cell sample was digested with MspI (NEB) and then end-prepared and aptamer ligated using the Premium RRBS kit (reagent). Size selection was performed using AMPure XP beads (Beckman Coulter, inc.) to obtain DNA fractions of MspI digestion products enriched for the most CpG rich regions in the 150-350bp range. Subsequently, bisulphite treatment was performed using the ZYMO EZ DNA methylation-Gold kit. The transformed DNA was then amplified by twelve PCR cycles using 25. Mu. l KAPA HiFi HotStart Uracil +Readymix (2X) and an 8-bp index primer at a final concentration of 1. Mu.M, and purified using AMPure XP beads. The constructed RRBS library was sent to CD Genomics (Shirley, NY) for sequencing and bioinformatic analysis. In short The library was then quantitated using a Qubit fluorometer and the Quant-iT dsDNA HS assay kit (Invitrogen) and sequenced on the Illumina Hiseq platform using a double-ended 150bp strategy.

Materials and methods/RRBS data analysis: the FastQC tool (www.bioinformatics.babraham.ac.uk/subjects/fastq) was used to make basic statistics on the quality of the original readings. Then, low quality data (Juhling, F., et al, metilene: fast and sensitive calling of differentially methylated regions from bisulfite sequencing data. Genome Res,2016.26 (2): p.256-62) of the sequencing aptamer and sequencing data were deleted by Trimmomatic (version 0.36). The bisulphite sequence was mapped to the reference genome using BSMAP software, with parameters of' -n 0-g 0-v 0.08-m 50-x 1000 (Kanehisa, M., et al KEGG for linking genomes to life and the environmental.nucleic Acids Res,2008.36 (Database issue): p.D480-4). Statistical information of the alignment was collected, leaving only unique mapping readings for the following analysis. Only methylated cytosines with a sequence depth coverage of at least 5 are used. Methylation occurs if the aligned base is C; in contrast, if the aligned base is T, methylation does not occur. The methylation level of an individual cytosine is calculated as the ratio of the determined sequencing depth of methylated CpG cytosines to the total sequencing depth of individual CpG cytosines, i.e. ML = mC/(mc+ umC), where ML is the methylation level, mC and umC represent the number of reads supporting methylated C and the number of reads supporting unmethylated C, respectively. DMR (differential methylation region) was identified by binary segmentation algorithm (version 0.2-7) in combination with two-dimensional statistical test using metilene software (parameter: -M300-M5-d 0.1-t 1-f 1-v 0.7) (Bruce et al, 2013.Ex vivo culture and separation of functional renal cells.Methods Mol Biol 1001:53-640.Gene Ontology (referred to as GO, http:// www.geneontology.org /), enrichment analysis using DMR-related genes to reveal biological processes of interest. Pathways with Q value < 0.05 were considered significantly enriched for DMR-related genes. Functional enrichment analysis was performed on genes that were observed to overlap with DMR for the genome and upstream and downstream regions (upstream 2k, genome, downstream 2 k) based on DMR annotation results and KEGG database (Sha et al, 2021.Genome-wide transcriptional profiling of Selected Renal Cells (SRC): identification of functional pathways for regenerative bioactivity in treatment of chronic kidney query contribution).

Results/differentiation of differentially expressed transcripts in SRC and BRC0 population cells

A total of n=6 individual donors were used to prepare total RNA and cDNA for library construction and RNA-eq analysis as described in materials and methods. A total of 221 DEG were identified to distinguish between SRC and BRC0 populations. These 221 DEG consist of 119 genes specifically up-regulated in SRC relative to BRC0 and 102 genes specifically down-regulated in SRC relative to BRC 0. The list of the first 20 genes up-and down-regulated in SRC relative to BRC0 is shown in tables 14 and 15, respectively. These transcripts had a False Discovery Rate (FDR) of 10 ^-6 Or smaller, reflecting a very high statistical significance.

Table 14: the first 20 genes up-regulated in SRC relative to BRC0

Table 15: the first 20 genes down-regulated in SRC relative to BRC0

Examination of the genes listed in tables 14 and 15 shows that specifically distinguishing front up-regulated DEG of SRC and BRC0 includes genes that mediate aspects of cell cycle control (CDKN 2A, IL, tgfβ2), extracellular matrix recombination (FN 1, crisp 2, COL1A1, LOX) and signaling pathways involved in development (RUNX 2, LFNG, BDNF). Front down-regulated DEG, which specifically distinguishes SRC and BRC0, includes genes involved in tight junction assembly and organization (CLDN 3), glucose metabolism (UPP 1, KLF 15), protein glycosylation (GYLTL 1B, MAN C1, GALNT 9), and water and ion transport (AQP 1, SLC47A1, WNK2, CASR) and signaling pathways involved in development (RAI 2, PLVAP, SHISA3, PARM1, CASR). Other noteworthy upregulation DEG (not shown in table 14, but still very significant) included BDNF, FGF11, FOXE1, WNT5A, WNT10A, TGF β1, and IGFBP3, all of which were involved in development and morphogenesis-related intercellular signaling aspects.

Among these genes, CDKN2A expression may be only an expression of prolonged passage times of cell populations in vitro, as this gene is a well established marker of cell senescence (Famulski & haloran, 2005;Molecular events in kidney ageing,Curr Opin Nephrol Hyperten 14:243-248).

In the case of IL11, although it is closely related to many aspects of fibrosis and inflammatory diseases of the kidney and other organs (Cook and Schafer,2020;Hiding in Plain Sight:Interleukin-11Emerges as a Master Regulator of Fibrosis,Tissue Integrity,and Stromal Inflammation,Ann Rev Med.71:263), it has a potential role in particularly relevant regenerative biological activities. IL11 has been shown to be necessary for xenopus organ regeneration by inducing and maintaining a population of undifferentiated progenitor cells across multiple cell lineages during tail regeneration (Tsujioka et al, 2017; interleukin-11induces and maintains progenitors of different cell lineages during Xenopus tadpole tail regeneration.Nature Commun.8:495).

The TGF-beta 2 gene and other members of the TGF-beta superfamily are known to be important mediators of kidney development, directly or indirectly regulating the number of nephrons (Sims-Lucas et al, 2008,Augmented and accelerated nephrogenesis in TGF-beta 2heterozygous mutant mice,Pedr.Res 63:607-612). Furthermore, TGF- β2 acts synergistically with FGF2, and has been shown to induce multitube formation in isolated rodent postrenal mesenchymal explants (Plisov et al, 2001.TGF beta 2,LIF and FGF2 cooperate to induce nephrogenesis,Development 128:1045).

The branch morphogen CRISPLD2 (also known as LDL 1) has been shown to have a specific localization pattern for the developing Ureteral Branching Tip (UBT) (rutliege et al, 2017.Cellular heterogeneity in the ureteric progenitor niche and distinct profiles of branching morphogenesis in organ development,Development 144:3177). Observations of CRISPLD2/LDL1 expression indicate that CRISPLD2 secreted by postrenal mesenchymal cells enhances the branching morphogenesis of the renal aggregate system, similar to its putative effects on early stages of pulmonary bud branching (Quinlan et al 2007.LGL1,anovel branching morphogen in developing kidney,is induced by retinoic acid.Am J Physiol Renal Physiol;293 (4): F987-93).

LOX and LOX-like protein families are known to play a variety of roles in development and organogenesis through their biological activity in extracellular matrix assembly and recombination. LOX and LOX-like proteins are involved in the development of a variety of tissues and organs, including brain, spinal cord, lung, heart, aorta, teeth, bone, cartilage, muscle and tendon, skin and uterus (Wei et al 2020;Role of the lysyl oxidase family in organ development (reviewed), exp. Ther. Med. 20:163-172).

LFNG is one of three Notch ligands whose expression is involved in the establishment and division of proximal/distal polarity of kidney vesicles. The Notch pathway establishes proximal polarity, which is established by comma-and S-shaped stages and is essential for proximal tubule and podocyte development (O-Brian and McMahon,2014.Induction and patterning of the metanephric nephron.Semin Cell Dev Biol.36:31-38). Expression of LFNG and closely related glycosyltransferase free radical edges (radial fringes) was observed in the dorsal anterior zone of the proximal prokidney at the tail bud stage of xenopus development. LFNG expression was absent in distal and proximal parts of the developing nephron in E14.5 mouse embryos, but strongly detected in future proximal tubules (similar to expression of Notch ligand DLL 1). By E17.5, LFNG expression has been demonstrated to be limited to only a portion of the nephron in peripheral development of the kidney, suggesting that LFNG may be modulated by NOTCH signaling in future proximal tubule cells (Leimester et al 2003.Expression of Notch pathway genes in the embryonic mouse metanephros suggests a role in proximal tubule development.Gene Expression Patterns 3:595-598).

Analysis of expression of BDNF in human fetal kidneys indicated that BDNF is located in the apical region of the developing primitive glomerular structure and mesenchymally derived intratubular epithelial cells. Although BDNF was detected in the differentiated distal tubules, expression of BDNF was not observed in the uninduced mesenchyme, suggesting that BDNF is not involved in early induction events, but rather in later stages of nephron tissue (Huber et al 1996.Neurotrophins and neurotrophin receptors in human fetal kidney.Developmental biology 179:369-381). Interestingly, BDNF has also been shown to mediate podocyte repair in vitro and in vivo by micro-RNA dependent increase in cytoskeletal actin polymerization (Li et al 2015.BDNF repairs podocyte damage by microRNA-mediated increase of actin polymerization. J Pathol 235:731-744). The morphine-mediated knockdown of BDNF in zebra fish larvae resulted in abnormal kidney glomerulogenesis, reduced podocyte numbers, and erroneous expression of podocyte marker kidney disease proteins and podoproteins (Endlich et al, 2018.BDNF:mRNA expression in urine cells of patients with chronic kidney disease and its role in kidney function.J Cell Mol Med.22:5265-5277).

WNT5A has been shown to be critical for kidney development; congenital kidney and urinary tract abnormalities (CAKUT) in patients presenting with Robinow syndrome are associated with WNT5A gene mutations. Ureteric tree development, abnormalities in tubular epithelial cell tissue and basal membrane integrity (pietilla et al 2016.Wnt5a Deficiency Leads to Anomalies in Ureteric Tree Development,Tubular Epithelial Cell Organization and Basement Membrane Integrity Pointing to a Role in Kidney Collecting Duct Patterning.Plos One 11:e0147171), and repeated ureters and kidneys due to ectopic Ureteric Bud (UB) induction, were also observed in WNT5A deficient rodents. During initial UB formation, WNT5A mutant embryos exhibited deregulation of postrenal mesenchyme (MM), resulting in a space-time aberrant interaction between MM and wu's tube, resulting in inappropriate GDNF signaling with wu's tube. Furthermore, cell proliferation in mutant MM has been shown to be significantly reduced relative to controls, suggesting that WNT5A/ROR2 signaling plays a critical role in the morphogenesis of MM to ensure proper epithelial tubule formation of developing ureteral buds (Nishita et al, 2014.Role of Wnt5a-ROR2 Signaling in Morphogenesis of the Metanephric Mesenchyme during Ureteric Budding; mol Cell Biol 16:3096-3105). WNT5A signaling is also involved in podocyte development and recovery of glomerular podocytes from injury by recombining actin cytoskeleton via a planar cell polarity pathway (bayayiva et al, 2011.Planar cell polarity pathway regulates actin rearrangement,cell shape,motility,and nephrin distribution in podocytes.AJPRP F549-60).

IGFBP3 has been registered in mature and differentiated cells of the human fetal kidney collecting system and ureter, potentially modulating IGF bioactivity during renogenesis (Matsel et al 1994.Expression of insulin-like growth factor and binding protein genes during nephrogenis. Kidney Int 46:1031-42).

GO analysis of the results/DEG gene set showed enrichment of the development-related classifier: an analysis of the protein (Protein ANalysis THrough Evolutionary Relationships, panher) over-representation by evolutionary relationship was performed to identify GO categories that were particularly enriched in DEG gene sets relative to the entire human genome. The results of the panher analysis are approximately identical to those shown in tables 14 and 15, with DEG datasets showing enrichment of GO identifiers associated with development, including cellular communication, cellular differentiation, intercellular adhesion, developmental processes, neurological development, and phylogenetic development. Critical pathways of the entire DEG set emphasized in the REACTOME pathway analysis include developmental biology, cell cycle, intercellular communication, extracellular matrix tissue, neuronal systems, signal transduction, gene expression (transcription) pathways.

All DEG in SRC were also analyzed for GO-BP (biological process), GO-CC (cellular component) and GO-MF (molecular function) relative to BRC0, compared to the whole human genome. Specific enrichment of DEG relative to the entire human genome was observed for the following development-related GO-BP categories: cellular processes, single biological processes, biological regulation, metabolic processes, stimulus responses, multicellular biological processes, developmental processes, signaling, localization, cellular component tissue or biogenesis, multicellular processes, immune system processes, reproduction, reproductive processes, exercise, bioadhesion, behavior, presynaptic processes involving chemical synaptic transmission, cell killing and cell aggregation. Consistent with previous reports, indicating that exosome-mediated intercellular communication is an important mechanism of action for SRC (Bruce et al, 2013.Ex vivo culture and separation of functional renal cells.Methods Mol Biol 1001:53-64), the enrichment of the following GO-CC classes in the SRC/BRC0 DEG set was noted: extracellular regions, extracellular region portions, supramolecular complexes, synapses, other organisms, and other organism portions. Finally, enrichment of DEG relative to the whole human genome was observed for the following GO-MF categories: structural molecule activity, molecular function modulator, transporter activity, nucleic acid binding transcription factor activity, and molecular transducin activity. More detailed information on the genetic composition and ancestor/offspring of each of these GO classifiers can be found in amigo.

KEGG analysis of the results/DEG gene set showed enrichment of the classifier associated with development: KEGG notes were also made on the major identified metabolic and signaling pathways associated with the SRC/BRC0 DEG gene set. The DEG gene set-specific enriched KEGG cell process classifier is widely associated with epithelial cell link formation, tissue and maintenance, cell cycle control and stem cell pluripotency regulation: adhesive attachment, p53 signaling pathway, tight junctions, signaling pathway that modulates stem cell pluripotency, modulation of cytoskeletal effects, endocytosis, cell cycle, and focal adhesion. In addition, a number of key signal transduction pathways associated with development, cell cycle control and regeneration are also enriched in key KEGG categories: AMPK, mTOR, WNT, HIF-1, JAK-STAT, RAP-1, TNF, TGF beta, MAPK, FOXO, HIPPO and PI3K-AKT signaling pathways, as well as cytokine-cytokine receptor interactions, ECM receptor interactions.

The scatter plot shown in FIG. 2 is a graphical representation of the results of the KEGG enrichment analysis. In this figure, the KEGG enrichment is measured by the enrichment factor, q-value and the number of genes enriched in a particular pathway. Enrichment factor refers to the ratio of the number of differentially expressed genes located in the pathway to the total number of annotated genes located in the pathway. The greater the enrichment factor, the greater the degree of enrichment. The Q value is the corrected p value after multiple hypothesis testing. The q value is in the range of 0,1, and the closer to 0, the more remarkable the enrichment degree is. Key KEGG pathways involved in kidney regeneration include TGF- β signaling pathways, HIPPO signaling pathways, FOXO signaling pathways, cell cycle, ECM-receptor interactions, pyrimidine metabolism.

Particular attention to the up-regulated DEG set reveals the importance of the following KEGG classifier: adhesive attachment, endocytosis, cell cycle, regulation of actin cytoskeleton, p53 signaling pathway, signaling pathway regulating stem cell pluripotency, and focal adhesion. Other important pathways in the up-regulated DEG set include: the Rap1 signaling pathway, HIF1 signaling pathway, JAK-STAT signaling pathway, TGF- β signaling pathway, MAPK signaling pathway, TNF signaling pathway, and HIPPO signaling pathway (fig. 3). topGO analysis of up-regulated DEG gene sets is shown in figure 3. Key GO-BP identifiers particularly relevant to regeneration include: negative regulation of epidermal growth factor receptor signaling pathway, G protein coupled receptor signaling pathway, notch signaling pathway and epithelial cell proliferation in uterine embryo development.

A specific examination of the down-regulated DEG set showed enrichment of the following KEGG classifier: focal adhesion, apoptosis, lysosomes, cell cycle, tight junctions and endocytosis, and signaling pathways: HIPPO, hedgehog and AMPK. topGO analysis of the down-regulated DEG set showed enrichment of the BP classifier: EGFR signaling pathway, notch signaling pathway, GPCR signaling pathway, gut morphogenesis, cardiac circulation, epithelial cell proliferation.

Results/epigenomic analysis of SRC and BRC0 populations: a comparison of the Differential Methylation Region (DMR) between SRC and BRC0 is shown in the violin box plot of fig. 4. It shows that the SRC genome has proportionally more hypermethylation and less hypomethylation than the BRC0 genome (n=4). According to the result of DMR annotation, functional enrichment analysis is performed on genes overlapping with DMR in the genome and the upstream and downstream regions (upstream-2 k, genome, downstream-2 k). In contrast to BRC0, the most highly enriched GO-BP classifier in DMR-related genes in SRC includes cell adhesion, signal transduction, and down/up regulation of RNA polymerase transcription. In contrast to BRC0, the highly enriched KEGG pathway in the SRC population includes: modulation of actin cytoskeleton, proteoglycans in cancer, focal adhesions, endocytosis, cAMP signaling pathway, RAS signaling pathway, RAP1 signaling pathway, PI3-AKT signaling pathway, MAPK signaling pathway, and HIPPO signaling. In a broad sense, these classifiers can diagnose genes involved in extracellular matrix tissue and recombination, as well as signaling pathways associated with organism development and cell proliferation. These classifiers are also consistent with the classifiers previously identified in the DEG set analysis.

Also, according to the distribution of DMR on the genome, functional enrichment analysis was performed with the presence of overlapping genes with the gene promoter region (1.5K upstream of the transcription start site and 0.5K downstream of the transcription start site). According to this analysis, GO classifiers associated with DMR-related genes include actin cytoskeletal regulation, endocytosis, cAMP signaling pathway, cell adhesion molecule CAM, RAP1 signaling pathway. Also notable is that the greatest fold change in SRC relative to BRC0 is leukocyte transendothelial migration, a critical inflammatory stage associated with wound healing and tissue regeneration. In addition, the KEGG classifier associated with the DMR-related genes includes: modulation of actin cytoskeleton, cAMP signaling pathway, signaling pathway modulating stem cell pluripotency, RAS signaling pathway, RAP1 signaling pathway, PI3K-AKT signaling pathway, endocytosis, cell adhesion CAM, leukocyte transendothelial pathway. These classifiers are also approximately identical to the classifiers previously identified in the DEG set.

Results/summary: in general, human-derived SRCs provide DEG sets enriched in development-related GO and KEGG classifiers. The DEG gene set-specific enriched KEGG cell process classifier is widely associated with epithelial cell junction formation, tissue and maintenance, cell cycle control and stem cell pluripotency regulation. Similar enrichment patterns were observed in the DMR gene set. Based on these and other expression data presented herein, SRC may mediate in part in vivo regeneration of the kidney and improve clinical outcome in renal patients by activating a mechanical pathway parallel to embryogenesis and ultimately triggering formation of new kidney-like tissue at the site of injury or disease.

Example 5: administration of enriched heterogeneous renal cell populations to subjects with medium to late type 2 diabetes-associated CKD-assessment of potential relationship of nephrogenic marker expression levels to clinical outcome

Introduction: recognizing that an enriched heterogeneous population of kidney cells, such as SRC as mentioned in this example, can mediate regeneration of diseased kidneys by affecting the signaling cascade that mediates neonephrogenesis, SRC is administered to clinical test subjects with medium to late type 2 diabetes-related CKD, the expression levels of nephrogenesis markers are assessed and compared accordingly to the clinical response of the patient.

Materials and methods/preparation of SRC for use in applications: all patients in the clinical trial received standard percutaneous kidney biopsies to isolate kidney cells from which their SRC was prepared for administration. SRC was prepared from kidney biopsies as described in examples 1 and 4.

Materials and methods/phenotypic marker analysis of SRC: the SRC in suspension was centrifuged at 300Xg for 5 min at room temperature. The resulting SRC precipitate was washed once with Dulbecco's Phosphate Buffered Saline (DPBS) and then fixed in BD fixation/permeabilization solution (BD Biosciences, cat. No. 554714) for 20 minutes at 4 ℃. The fixed cells were centrifuged at 500Xg for 5 min at room temperature. The cell pellet was resuspended in BD Perm/Wash buffer. The resuspended cells were aliquoted (20. Mu.L containing 150,000 cells) and then incubated with 1. Mu.g of primary antibody for 1 hour at 4 ℃. At the end of incubation, cells were pelleted, washed with BD Perm/Wash buffer, reprecipitated, and resuspended in 150. Mu.L DPBS. For antibodies not directly conjugated to fluorophores, 1 μg of secondary antibody was incubated with cells for an additional 30 minutes, then washed and resuspended in DPBS. The cells were then analyzed by flow cytometry.

FACS analysis of materials and methods/SRC: to determine if SRC contains markers for the hat, nephrogenic and ureteric buds, cells were screened for membrane proteins that can identify these specific components. The following immunoreagents were used for screening: goat anti-rabbit IgG H&L, alexafluor488 (Abcam Inc. Cat#ab 150077); NPHS2 rabbit polyclonal antibody (Abcam Inc. Cat#ab 50339); kidney disease protein rabbit monoclonal antibodyY17R) (Abcam inc. Cat#ab 136894); RET Alexafluor488 rabbit monoclonal antibody (EPR 2871) (Abcam Inc. ab 237105); rabbit IgG, polyclonal, isotype control (Abcam inc. Abc37415); six2 antibody (H-4) PE (Santa Cruz, cat#sc-377193 PE); OSR1 antibody (Santa Cruz, cat#sc376545 PE); lhx1 monoclonal IgG2a (Santa Cruz, cat#sc515631 PE); RET rabbit monoclonal antibody (Abcam inc. Cat#ab 237105); RB IGG (H)&L) goat polyclonal secondary antibody-Alexa Fluor488 (Abcam Inc. Cat#ab 150077); alexa647 mouse IgG2a, kappa isotype control (BD, cat# 557715); fgf8 monoclonal, full length (Abcam, cat#ab 89550); rabbit IgG isotype control [ PE](Novus Biologicals, cat#NBP2-36463 PE) and PE mouse IgG1, kappa isotype control (BD, cat# 555749).

Materials and methods/FACS phenotypic marker analysis: phenotypic marker analysis was performed by precipitating at least 50,000 SRCs in a 1.5mL microcentrifuge tube and fixing the cells in 100. Mu.L BD fixation/permeabilization solution for 20 minutes at 4 ℃. The microcentrifuge tube was then placed in a microcentrifuge at 500Xg for 3 minutes to pellet the immobilized cells, and then washed once with 500. Mu.L BD Perm/Wash buffer. The washed pellet was resuspended in 20. Mu.L BD Perm/Wash buffer and 1. Mu.g primary antibody was added and incubated for 1 hour at 4 ℃. Cells were washed once with 500. Mu.L BD Perm/Wash buffer, then resuspended in 20. Mu.L BD Perm/Wash buffer, 1. Mu.g of secondary antibody was added and incubated for 30 min at 4 ℃. The last Wash was performed using 500. Mu.L BD Perm/Wash buffer. The pellet was resuspended in 150 μldpbs and the cells analyzed by flow cytometry.

Results/marker expression analysis of SRC: table 16 provides the results of marker expression analysis of SRC administered to subjects in clinical trials.

Table 16: sorting data for FACS analysis

Consistent with earlier assays such as presented in example 2, SRC expression from clinical trial subjects was used to express markers of the nephrogenic marker panel, as well as other markers including kidney disease proteins, podoproteins and RACK-1. SRC in clinical trial subjects also includes the percentage of cells expressing the marker, as previously described. Correlation of the various markers found evidence of their co-expression (see FIGS. 5A-H).

Further analysis of SRC marker expression indicated that subjects identified as medium/high responders (evfr improved) exhibited higher expression of the various biomarkers tending to be significant for Six2 and for LHx1 relative to those identified as low responders (evfr slope improved, but evfr did not improve) (table 16). These analyses provide further evidence that SRC may mediate new nephrogenesis, as cells of the SRC population have higher levels of nephrogenesis marker expression, they may have enhanced ability to mediate regeneration and recovery activity, thereby providing therapeutic effects to the diseased kidney.

Claims

1. A method of identifying an enriched heterogeneous population of kidney cells as having therapeutic potential, the method comprising:

determining whether cells of the enriched heterogeneous kidney cell population express at least one nephrogenic marker; and

identifying the enriched heterogeneous kidney cell population as having therapeutic potential if cells of the enriched heterogeneous kidney cell population are assayed for expression of the at least one nephrogenic marker;

wherein the at least one nephrogenic marker comprises one or more of SIX2, OSR1, LHX1, RET, and FGF 8.

2. The method of claim 1, wherein said step of determining comprises determining the percentage of cells in said enriched heterogeneous population of kidney cells that express said at least one nephrogenic marker.

3. The method of claim 1, wherein the at least one nephrogenic marker comprises SIX2.

4. The method of claim 2, wherein the at least one nephrogenic marker comprises SIX2 and wherein the enriched heterogeneous kidney cell population is identified as having therapeutic potential if greater than 0% but no more than about 6.0% of the cells in the enriched heterogeneous kidney cell population express SIX2.

5. The method of claim 1, wherein the at least one nephrogenic marker comprises OSR1.

6. The method of claim 2, wherein the at least one nephrogenic marker comprises OSR1 and wherein the enriched heterogeneous population of kidney cells is identified as having therapeutic potential if about 36% to about 85% of the cells in the enriched heterogeneous population of kidney cells express OSR 1.

7. The method of claim 1, wherein said at least one nephrogenic marker comprises LHX1.

8. The method of claim 2 wherein said at least one nephrogenic marker comprises LHX1 and wherein said enriched heterogeneous population of kidney cells is identified as having therapeutic potential if it is determined that from about 8% to about 58% of the cells in said enriched heterogeneous population of kidney cells express LHX1.

9. The method of claim 1, wherein the at least one nephrogenic marker comprises RET.

10. The method of claim 2, wherein the at least one nephrogenic marker comprises RET and wherein the enriched heterogeneous population of kidney cells is identified as having therapeutic potential if about 49% to about 90% of the cells in the enriched heterogeneous population of kidney cells express RET.

11. The method of claim 1, wherein the at least one kidney genesis marker comprises FGF8.

12. The method of claim 2, wherein the at least one nephrogenic marker comprises FGF8 and wherein the enriched heterogeneous population of kidney cells is identified as having therapeutic potential if about 0.48% to about 59% of the cells in the enriched heterogeneous population of kidney cells express FGF8.

13. The method of claim 1, wherein the at least one nephrogenic marker comprises:

(a) SIX2 and OSR1; or (b)

(b) SIX2 and LHX1; or (b)

(c) SIX2 and RET; or (b)

(d) SIX2 and FGF8; or (b)

(e) OSR1 and LHX1; or (b)

(f) OSR1 and RET; or (b)

(g) OSR1 and FGF8; or (b)

(h) LHX1 and RET; or (b)

(i) LHX1 and FGF8; or (b)

(j) RET and FGF8.

14. The method of claim 2, wherein the at least one nephrogenic marker comprises:

(a) SIX2 and OSR1, and

wherein the enriched heterogeneous kidney cell population is identified as having therapeutic potential if greater than 0% and up to about 6% of the cells in the enriched heterogeneous kidney cell population are determined to express SIX2 and at least about 36% of the cells in the enriched heterogeneous kidney cell population are determined to express OSR1; or (b)

(b) SIX2 and LHX1, and

wherein the enriched heterogeneous kidney cell population is identified as having therapeutic potential if greater than 0% and up to about 6% of the cells in the enriched heterogeneous kidney cell population are determined to express SIX2 and greater than about 8% of the cells in the enriched heterogeneous kidney cell population are determined to express LHX1; or (b)

(c) SIX2 and RET, and

wherein the enriched heterogeneous kidney cell population is identified as having therapeutic potential if greater than 0% and up to about 6% of the cells in the enriched heterogeneous kidney cell population are determined to express SIX2 and greater than about 49% of the cells in the enriched heterogeneous kidney cell population are determined to express RET; or (b)

(d) SIX2 and FGF8, and

wherein the enriched heterogeneous kidney cell population is identified as having therapeutic potential if greater than 0% and up to about 6% of the cells in the enriched heterogeneous kidney cell population are determined to express SIX2 and greater than about 0% and up to about 59% of the cells in the enriched heterogeneous kidney cell population are determined to express FGF 8; or (b)

(e) OSR1 and LHX1, and

wherein the enriched heterogeneous kidney cell population is identified as having therapeutic potential if at least about 36% of the cells in the enriched heterogeneous kidney cell population are determined to express OSR1 and greater than about 8% of the cells in the enriched heterogeneous kidney cell population are determined to express LHX 1; or (b)

(f) OSR1 and RET, and

wherein the enriched heterogeneous kidney cell population is identified as having therapeutic potential if at least about 36% of the cells in the enriched heterogeneous kidney cell population are determined to express OSR1 and greater than about 49% of the cells in the enriched heterogeneous kidney cell population are determined to express RET; or (b)

(g) OSR1 and FGF8, and

wherein the enriched heterogeneous kidney cell population is identified as having therapeutic potential if at least about 36% of the cells in the enriched heterogeneous kidney cell population are determined to express OSR1 and greater than 0% and up to about 59% of the cells in the enriched heterogeneous kidney cell population are determined to express FGF 8; or (b)

(h) LHX1 and RET, and

wherein the enriched heterogeneous kidney cell population is identified as having therapeutic potential if greater than about 8% of the cells in the enriched heterogeneous kidney cell population are determined to express LHX1 and greater than about 49% of the cells in the enriched heterogeneous kidney cell population are determined to express RET; or (b)

(i) LHX1 and FGF8, and

wherein the enriched heterogeneous kidney cell population is identified as having therapeutic potential if greater than about 8% of the cells in the enriched heterogeneous kidney cell population are determined to express LHX1 and greater than 0% and up to about 59% of the cells in the enriched heterogeneous kidney cell population are determined to express FGF 8; or (b)

(j) RET and FGF8, and

wherein the enriched heterogeneous kidney cell population is identified as having therapeutic potential if greater than about 49% of the cells in the enriched heterogeneous kidney cell population are determined to express RET and greater than 0% and up to about 59% of the cells in the enriched heterogeneous kidney cell population are determined to express FGF 8.

15. The method of claim 2, wherein the at least one nephrogenic marker comprises:

(a) SIX2 and OSR1, and

wherein the enriched heterogeneous kidney cell population is identified as having therapeutic potential if greater than 0% and up to about 6% of the cells in the enriched heterogeneous kidney cell population are determined to express SIX2 and about 36% to about 85% of the cells in the enriched heterogeneous kidney cell population are determined to express OSR 1; or (b)

(b) SIX2 and LHX1, and

wherein the enriched heterogeneous kidney cell population is identified as having therapeutic potential if greater than 0% and up to about 6% of the cells in the enriched heterogeneous kidney cell population are determined to express SIX2 and from about 8% to about 58% of the cells in the enriched heterogeneous kidney cell population are determined to express LHX 1; or (b)

(c) SIX2 and RET, and

wherein the enriched heterogeneous kidney cell population is identified as having therapeutic potential if greater than 0% and up to about 6% of the cells in the enriched heterogeneous kidney cell population are determined to express SIX2 and from about 49% to about 90% of the cells in the enriched heterogeneous kidney cell population are determined to express RET; or (b)

(d) SIX2 and FGF8, and

wherein the enriched heterogeneous kidney cell population is identified as having therapeutic potential if greater than 0% and up to about 6% of the cells in the enriched heterogeneous kidney cell population are determined to express SIX2 and from about 0.48% to about 59% of the cells in the enriched heterogeneous kidney cell population are determined to express FGF 8; or (b)

(e) OSR1 and LHX1, and

wherein the enriched heterogeneous kidney cell population is identified as having therapeutic potential if about 36% to about 85% of the cells in the enriched heterogeneous kidney cell population are determined to express OSR1 and about 8% to about 58% of the cells in the enriched heterogeneous kidney cell population are determined to express LHX 1; or (b)

(f) OSR1 and RET, and

wherein the enriched heterogeneous kidney cell population is identified as having therapeutic potential if about 36% to about 85% of the cells in the enriched heterogeneous kidney cell population are determined to express OSR1 and about 49% to about 90% of the cells in the enriched heterogeneous kidney cell population are determined to express RET; or (b)

(g) OSR1 and FGF8, and

wherein the enriched heterogeneous kidney cell population is identified as having therapeutic potential if about 36% to about 85% of the cells in the enriched heterogeneous kidney cell population are determined to express OSR1 and about 0.48% to about 59% of the cells in the enriched heterogeneous kidney cell population are determined to express FGF 8; or (b)

(h) LHX1 and RET, and

wherein the enriched heterogeneous kidney cell population is identified as having therapeutic potential if about 8% to about 58% of the cells in the enriched heterogeneous kidney cell population are determined to express LHX1 and about 49% to about 90% of the cells in the enriched heterogeneous kidney cell population express RET; or (b)

(i) LHX1 and FGF8, and

wherein the enriched heterogeneous kidney cell population is identified as having therapeutic potential if from about 8% to about 58% of the cells in the enriched heterogeneous kidney cell population are determined to express LHX1 and from about 0.48% to about 59% of the cells in the enriched heterogeneous kidney cell population are determined to express FGF 8; or (b)

(j) RET and FGF8, and

wherein the enriched heterogeneous kidney cell population is identified as having therapeutic potential if about 49% to about 90% of the cells in the enriched heterogeneous kidney cell population are determined to express RET and about 0.48% to about 59% of the cells in the enriched heterogeneous kidney cell population are determined to express FGF8.

16. The method of claim 1, wherein the at least one nephrogenic marker comprises:

(a) SIX2, OSR1 and LHX1; or (b)

(b) SIX2, OSR1, and RET; or (b)

(c) SIX2, OSR1 and FGF8; or (b)

(d) SIX2, LHX1 and RET; or (b)

(e) SIX2, LHX1 and FGF8; or (b)

(f) SIX2, RET and FGF8; or (b)

(g) OSR1, LHX1 and RET; or (b)

(h) OSR1, LHX1 and FGF8; or (b)

(i) OSR1, RET and FGF8; or (b)

(j) LHX1, RET and FGF8.

17. The method of claim 2, wherein the at least one nephrogenic marker comprises:

(a) SIX2, OSR1 and LHX1, and

wherein the enriched heterogeneous kidney cell population is identified as having therapeutic potential if greater than 0% and up to about 6% of the cells in the enriched heterogeneous kidney cell population express SIX2, at least about 36% of the cells in the enriched heterogeneous kidney cell population express OSR1, and greater than about 8% of the cells in the enriched heterogeneous kidney cell population express LHX1; or (b)

(b) SIX2, OSR1 and RET, and

wherein the enriched heterogeneous kidney cell population is identified as having therapeutic potential if greater than 0% and up to about 6% of the cells in the enriched heterogeneous kidney cell population express SIX2, at least about 36% of the cells in the enriched heterogeneous kidney cell population express OSR1 and greater than about 49% of the cells in the enriched heterogeneous kidney cell population express RET; or (b)

(c) SIX2, OSR1 and FGF8, and

wherein the enriched heterogeneous kidney cell population is identified as having therapeutic potential if greater than 0% and up to about 6% of the cells in the enriched heterogeneous kidney cell population express SIX2, at least about 36% of the cells in the enriched heterogeneous kidney cell population express OSR1, and greater than 0% and up to about 59% of the cells in the enriched heterogeneous kidney cell population express FGF 8; or (b)

(d) SIX2, LHX1 and RET, and

wherein the enriched heterogeneous kidney cell population is identified as having therapeutic potential if greater than 0% and up to about 6% of the cells in the enriched heterogeneous kidney cell population express SIX2, greater than about 8% of the cells in the enriched heterogeneous kidney cell population express LHX1, and greater than about 49% of the cells in the enriched heterogeneous kidney cell population express RET; or (b)

(e) SIX2, LHX1 and FGF8, and

wherein the enriched heterogeneous kidney cell population is identified as having therapeutic potential if greater than 0% and up to about 6.0% of the cells in the enriched heterogeneous kidney cell population are determined to express SIX2, greater than about 8% of the cells in the enriched heterogeneous kidney cell population are determined to express LHX1, and greater than 0% and up to about 59% of the cells in the enriched heterogeneous kidney cell population are determined to express FGF 8; or (b)

(f) SIX2, RET and FGF8, and

wherein the enriched heterogeneous kidney cell population is identified as having therapeutic potential if greater than 0% and up to about 6% of the cells in the enriched heterogeneous kidney cell population express SIX2, greater than about 49% of the cells in the enriched heterogeneous kidney cell population express RET, and greater than 0% and up to about 59% of the cells in the enriched heterogeneous kidney cell population express FGF 8; or (b)

(g) OSR1, LHX1 and RET, and

wherein the enriched heterogeneous kidney cell population is identified as having therapeutic potential if at least about 36% of the cells in the enriched heterogeneous kidney cell population express OSR1, greater than about 8% of the cells in the enriched heterogeneous kidney cell population express LHX1, and greater than about 49% of the cells in the enriched heterogeneous kidney cell population express RET; or (b)

(h) OSR1, LHX1 and FGF8, and

wherein the enriched heterogeneous kidney cell population is identified as having therapeutic potential if at least about 36% of the cells in the enriched heterogeneous kidney cell population are determined to express OSR1, greater than about 8% of the cells in the enriched heterogeneous kidney cell population express LHX1, and greater than 0% and up to about 59% of the cells in the enriched heterogeneous kidney cell population express FGF 8; or (b)

(i) OSR1, RET and FGF8, and

wherein the enriched heterogeneous kidney cell population is identified as having therapeutic potential if at least about 36% of the cells in the enriched heterogeneous kidney cell population express OSR1, greater than about 49% of the cells in the enriched heterogeneous kidney cell population express RET, and greater than 0% and up to about 59% of the cells in the enriched heterogeneous kidney cell population express FGF 8; or (b)

(j) LHX1, RET and FGF8, and

wherein the enriched heterogeneous kidney cell population is identified as having therapeutic potential if greater than about 8% of the cells in the enriched heterogeneous kidney cell population express LHX1, greater than about 49% of the cells in the enriched heterogeneous kidney cell population express RET, and greater than 0% and up to about 59% of the cells in the enriched heterogeneous kidney cell population express FGF 8.

18. The method of claim 2, wherein the at least one nephrogenic marker comprises:

(a) SIX2, OSR1 and LHX1, and

wherein the enriched heterogeneous kidney cell population is identified as having therapeutic potential if greater than 0% and up to about 6% of the cells in the enriched heterogeneous kidney cell population are determined to express SIX2, about 36% to about 85% of the cells in the enriched heterogeneous kidney cell population are determined to express OSR1, and about 8% to about 58% of the cells in the enriched heterogeneous kidney cell population are determined to express LHX 1; or (b)

(b) SIX2, OSR1 and RET, and

wherein the enriched heterogeneous kidney cell population is identified as having therapeutic potential if greater than 0% and up to about 6% of the cells in the enriched heterogeneous kidney cell population express SIX2, from about 36% to about 85% of the cells in the enriched heterogeneous kidney cell population express OSR1 and from about 49% to about 90% of the cells in the enriched heterogeneous kidney cell population express RET; or (b)

(c) SIX2, OSR1 and FGF8, and

wherein the enriched heterogeneous kidney cell population is identified as having therapeutic potential if greater than 0% and up to about 6% of the cells in the enriched heterogeneous kidney cell population express SIX2, from about 36% to about 85% of the cells in the enriched heterogeneous kidney cell population express OSR1, and from about 0.48% to about 59% of the cells in the enriched heterogeneous kidney cell population express FGF 8; or (b)

(d) SIX2, LHX1 and RET, and

wherein the enriched heterogeneous kidney cell population is identified as having therapeutic potential if greater than 0% and up to about 6% of the cells in the enriched heterogeneous kidney cell population express SIX2, from about 8% to about 58% of the cells in the enriched heterogeneous kidney cell population express LHX1, and from about 49% to about 90% of the cells in the enriched heterogeneous kidney cell population express RET; or (b)

(e) SIX2, LHX1 and FGF8, and

wherein the enriched heterogeneous kidney cell population is identified as having therapeutic potential if greater than 0% and up to about 6% of the cells in the enriched heterogeneous kidney cell population express SIX2, from about 8% to about 58% of the cells in the enriched heterogeneous kidney cell population express LHX1, and from about 0.48% to about 59% of the cells in the enriched heterogeneous kidney cell population express FGF 8; or (b)

(f) SIX2, RET and FGF8, and

wherein the enriched heterogeneous kidney cell population is identified as having therapeutic potential if greater than 0% up to about 6% of the cells in the enriched heterogeneous kidney cell population express SIX2, from about 49% to about 90% of the cells in the enriched heterogeneous kidney cell population express RET, and from about 0.48% to about 59% of the cells in the enriched heterogeneous kidney cell population express FGF 8; or (b)

(g) OSR1, LHX1 and RET, and

wherein the enriched heterogeneous kidney cell population is identified as having therapeutic potential if about 36% to about 85% of the cells in the enriched heterogeneous kidney cell population are determined to express OSR1, about 8% to about 58% of the cells in the enriched heterogeneous kidney cell population are determined to express LHX1, and about 49% to about 90% of the cells in the enriched heterogeneous kidney cell population are determined to express RET; or (b)

(h) OSR1, LHX1 and FGF8, and

wherein the enriched heterogeneous kidney cell population is identified as having therapeutic potential if about 36% to about 85% of the cells in the enriched heterogeneous kidney cell population are determined to express OSR1, about 8% to about 58% of the cells in the enriched heterogeneous kidney cell population are determined to express LHX1, and about 0.48% to about 59% of the cells in the enriched heterogeneous kidney cell population are determined to express FGF 8; or (b)

(i) OSR1, RET and FGF8, and

wherein the enriched heterogeneous kidney cell population is identified as having therapeutic potential if about 36% to about 85% of the cells in the enriched heterogeneous kidney cell population express OSR1, about 49% to about 90% of the cells in the enriched heterogeneous kidney cell population express RET, and about 0.48% to about 59% of the cells in the enriched heterogeneous kidney cell population express FGF 8; or (b)

(j) LHX1, RET and FGF8, and

wherein the enriched heterogeneous kidney cell population is identified as having therapeutic potential if it is determined that from about 8% to about 58% of the cells in the enriched heterogeneous kidney cell population express LHX1, from about 49% to about 90% of the cells in the enriched heterogeneous kidney cell population express RET, and from about 0.48% to about 59% of the cells in the enriched heterogeneous kidney cell population express FGF8.

19. The method of claim 1, wherein the at least one nephrogenic marker comprises:

(a) SIX2, OSR1, LHX1, and RET;

(b) SIX2, OSR1, LHX1 and FGF8;

(c) SIX2, LHX1, RET and FGF8;

(d) SIX2, OSR1, RET and FGF8;

(e) OSR1, LHX1, RET and FGF8.

20. The method of claim 2, wherein the at least one nephrogenic marker comprises:

(a) SIX2, OSR1, LHX1 and RET, and

wherein the enriched heterogeneous kidney cell population is identified as having therapeutic potential if greater than 0% and up to about 6% of the cells in the enriched heterogeneous kidney cell population express SIX2, at least about 36% of the cells in the enriched heterogeneous kidney cell population express OSR1, greater than about 8% of the cells in the enriched heterogeneous kidney cell population express LHX1, and greater than about 49% of the cells in the enriched heterogeneous kidney cell population express RET;

(b) SIX2, OSR1, LHX1 and FGF8, and

wherein if greater than 0% and up to about 6% of the cells in the enriched heterogeneous kidney cell population are determined to express SIX2, at least about 36% of the cells in the enriched heterogeneous kidney cell population express OSR1, greater than about 8% of the cells in the enriched heterogeneous kidney cell population express LHX1 and greater than 0% and up to about 59% of the cells in the enriched heterogeneous kidney cell population express FGF8, identifying the enriched heterogeneous kidney cell population as having therapeutic potential;

(c) SIX2, LHX1, RET and FGF8, and

wherein if greater than 0% and up to about 6% of the cells in the enriched heterogeneous kidney cell population are determined to express SIX2, greater than about 8% of the cells in the enriched heterogeneous kidney cell population express LHX1, greater than about 49% of the cells in the enriched heterogeneous kidney cell population express RET and greater than 0% and up to about 59% of the cells in the enriched heterogeneous kidney cell population express FGF8, identifying the enriched heterogeneous kidney cell population as having therapeutic potential;

(d) SIX2, OSR1, RET and FGF8, and

wherein if greater than 0% and up to about 6% of the cells in the enriched heterogeneous kidney cell population are determined to express SIX2, at least about 36% of the cells in the enriched heterogeneous kidney cell population express OSR1, greater than about 49% of the cells in the enriched heterogeneous kidney cell population express RET and greater than 0% and up to about 59% of the cells in the enriched heterogeneous kidney cell population express FGF8, identifying the enriched heterogeneous kidney cell population as having therapeutic potential;

(e) OSR1, LHX1, RET and FGF8, and

wherein the enriched heterogeneous kidney cell population is identified as having therapeutic potential if at least about 36% of the cells in the enriched heterogeneous kidney cell population express OSR1, greater than about 8% of the cells in the enriched heterogeneous kidney cell population express LHX1, greater than about 49% of the cells in the enriched heterogeneous kidney cell population express RET, and greater than 0% and up to about 59% of the cells in the enriched heterogeneous kidney cell population express FGF 8.

21. The method of claim 2, wherein the at least one nephrogenic marker comprises:

(a) SIX2, OSR1, LHX1 and RET, and

wherein the enriched heterogeneous kidney cell population is identified as having therapeutic potential if greater than 0% and up to about 6% of the cells in the enriched heterogeneous kidney cell population express SIX2, from about 36% to about 85% of the cells in the enriched heterogeneous kidney cell population express OSR1, from about 8% to about 58% of the cells in the enriched heterogeneous kidney cell population are determined to express LHX1, and from about 49% to about 90% of the cells in the enriched heterogeneous kidney cell population express RET;

(b) SIX2, OSR1, LHX1 and FGF8, and

wherein the enriched heterogeneous kidney cell population is identified as having therapeutic potential if greater than 0% and up to about 6% of the cells in the enriched heterogeneous kidney cell population express SIX2, from about 36% to about 85% of the cells in the enriched heterogeneous kidney cell population express OSR1, from about 8% to about 58% of the cells in the enriched heterogeneous kidney cell population are determined to express LHX1, and from about 0.48% to about 59% of the cells in the enriched heterogeneous kidney cell population express FGF 8;

(c) SIX2, LHX1, RET and FGF8, and

wherein the enriched heterogeneous kidney cell population is identified as having therapeutic potential if greater than 0% and up to about 6% of the cells in the enriched heterogeneous kidney cell population express SIX2, from about 8% to about 58% of the cells in the enriched heterogeneous kidney cell population express LHX1, from about 49% to about 90% of the cells in the enriched heterogeneous kidney cell population express RET, and from about 0.48% to about 59% of the cells in the enriched heterogeneous kidney cell population express FGF 8;

(d) SIX2, OSR1, RET and FGF8, and

wherein the enriched heterogeneous kidney cell population is identified as having therapeutic potential if greater than 0% and up to about 6% of the cells in the enriched heterogeneous kidney cell population express SIX2, from about 36% to about 85% of the cells in the enriched heterogeneous kidney cell population express OSR1, from about 49% to about 90% of the cells in the enriched heterogeneous kidney cell population express RET, and from about 0.48% to about 59% of the cells in the enriched heterogeneous kidney cell population express FGF 8;

(e) OSR1, LHX1, RET and FGF8, and

wherein the enriched heterogeneous kidney cell population is identified as having therapeutic potential if about 36% to about 85% of the cells in the enriched heterogeneous kidney cell population express OSR1, about 8% to about 58% of the cells in the enriched heterogeneous kidney cell population express LHX1, about 49% to about 90% of the cells in the enriched heterogeneous kidney cell population express RET, and about 0.48% to about 59% of the cells in the enriched heterogeneous kidney cell population express FGF 8.

22. The method of claim 1, wherein the at least one nephrogenic marker comprises SIX2, OSR1, LHX1, RET, and FGF8.

23. The method of claim 2, wherein the at least one kidney genesis marker comprises SIX2, OSR1, LHX1, RET, and FGF8, and

wherein the enriched heterogeneous kidney cell population is identified as having therapeutic potential if greater than 0% and up to about 6% of the cells in the enriched heterogeneous kidney cell population express SIX2, at least about 36% of the cells in the enriched heterogeneous kidney cell population express OSR1, greater than about 8% of the cells in the enriched heterogeneous kidney cell population express LHX1, greater than about 49% of the cells in the enriched heterogeneous kidney cell population express RET, and greater than 0% and up to about 59% of the cells in the enriched heterogeneous kidney cell population express FGF8.

24. The method of claim 2, wherein the at least one kidney genesis marker comprises SIX2, OSR1, LHX1, RET, and FGF8, and

wherein the enriched heterogeneous kidney cell population is identified as having therapeutic potential if greater than 0% and up to about 6% of the cells in the enriched heterogeneous kidney cell population express SIX2, from about 36% to about 85% of the cells in the enriched heterogeneous kidney cell population express OSR1, from about 8% to about 58% of the cells in the enriched heterogeneous kidney cell population are determined to express LHX1, from about 49% to about 90% of the cells in the enriched heterogeneous kidney cell population express RET, and from about 0.48% to about 59% of the cells in the enriched heterogeneous kidney cell population express FGF8.

25. The method of any one of claims 1-24, further comprising:

determining whether cells of the enriched heterogeneous kidney cell population express one or more of a kidney disease protein, a podoprotein, or RACK-1; and

if the cells of the enriched heterogeneous kidney cell population are determined to express one or more of a kidney disease protein, a podoprotein, or RACK-1, the enriched heterogeneous kidney cell population is identified as having therapeutic potential.

26. The method of claim 25, wherein the one or more comprises a kidney disease protein.

27. The method of claim 25, wherein the one or more comprises podoprotein.

28. The method of claim 26, wherein the one or more further comprises podoprotein.

29. The method of claim 25, wherein said one or more comprises RACK-1.

30. The method of claim 26, wherein said one or more further comprises RACK-1.

31. The method of claim 27, wherein said one or more further comprises RACK-1.

32. The method of claim 28, wherein said one or more further comprises RACK-1.

33. The method of claim 26, wherein the enriched heterogeneous kidney cell population is identified as having therapeutic potential if about 4% to about 99% of the cells in the heterogeneous kidney cell population are assayed for the expression of kidney disease proteins.

34. The method of claim 27, wherein the enriched heterogeneous kidney cell population is identified as having therapeutic potential if at least about 90% of the cells in the heterogeneous kidney cell population are determined to express podin.

35. The method of claim 28, wherein the enriched heterogeneous kidney cell population is identified as having therapeutic potential if it is determined that about 4% to about 99% of the cells in the heterogeneous kidney cell population express kidney disease protein and at least about 90% of the cells in the heterogeneous kidney cell population express podoprotein.

36. The method of claim 29, wherein said enriched heterogeneous kidney cell population is identified as having therapeutic potential if it is determined that at least about 85% of the cells in said heterogeneous kidney cell population express RACK-1.

37. The method of claim 30, wherein said enriched heterogeneous kidney cell population is identified as having therapeutic potential if it is determined that from about 4% to about 99% of the cells in said heterogeneous kidney cell population express kidney disease protein and at least about 85% of the cells in said heterogeneous kidney cell population express RACK-1.

38. The method of claim 31, wherein said enriched heterogeneous kidney cell population is identified as having therapeutic potential if it is determined that at least about 90% of the cells in said heterogeneous kidney cell population express podin and at least about 85% of the cells in said heterogeneous kidney cell population express RACK-1.

39. The method of claim 32, wherein said enriched heterogeneous kidney cell population is identified as having therapeutic potential if it is determined that from about 4% to about 99% of the cells in said heterogeneous kidney cell population express kidney disease protein, at least about 90% of the cells in said heterogeneous kidney cell population express podoprotein and at least about 85% of the cells in said heterogeneous kidney cell population express RACK-1.

40. The method of any one of claims 1-39, further comprising:

determining whether cells of the enriched heterogeneous kidney cell population express one or more of BMP4, BMP7, GDNF, HOX11, EYA1, SAL1, and SIX 4; and

if the cells of the enriched heterogeneous kidney cell population are assayed for the expression of one or more of BMP4, BMP7, GDNF, HOX11, EYA1, SAL1, and SIX4, the enriched heterogeneous kidney cell population is identified as having therapeutic potential.

41. The method of any one of claims 1-40, further comprising:

determining whether cells of the enriched heterogeneous kidney cell population express one or more of PAX2, CITED1, FGFR1, FGF7, FGF10, HOX10, POD1, and MUC 1; and

the enriched heterogeneous kidney cell population is identified as having therapeutic potential if the cells of the enriched heterogeneous kidney cell population are assayed for the expression of one or more of PAX2, CITED1, FGFR1, FGF7, FGF10, HOX10, POD1, and MUC 1.

42. The method of claim 41, wherein the one or more comprises RAX2.

43. The method of any one of claims 1-42, further comprising:

determining whether cells of the enriched heterogeneous kidney cell population express one or more of BMP7, SMAD, LIF, FOXD1, HOXB7, ALK3, DKK1, and SPRY 1; and

if the cells of the enriched heterogeneous kidney cell population are assayed for the expression of one or more of BMP7, SMAD, LIF, FOXD1, HOXB7, ALK3, DKK1, and SPRY1, then the enriched heterogeneous kidney cell population is identified as having therapeutic potential.

44. The method of any one of claims 1-43, further comprising:

determining whether cells of the enriched heterogeneous kidney cell population express one or more of the hyaluronic acid-mediated motility Receptor (RHAMM), C2, C3, C4, fibrinogen, clotting factor XIII, TEK, KDR, notch1, notch3, timp3, vwf, adam15, gas6, igfbp1, or Tm4sf4 genes; and

an enriched heterogeneous kidney cell population is identified as having therapeutic potential if the cells of the enriched heterogeneous kidney cell population are assayed for the expression of one or more of the RHAMM, C2, C3, C4, fibrinogen, clotting factor XIII, TEK, KDR, notch1, notch3, timp3, vwf, adam15, gas6, igfbp1, or Tm4sf4 genes.

45. The method of claim 44, wherein the one or more genes comprise RHAMM.

46. An enriched heterogeneous population of kidney cells identified according to the method of any one of claims 1-45.

47. A pharmaceutical composition comprising the enriched heterogeneous population of kidney cells of claim 46.

48. A method of treating kidney disease in a patient in need thereof, the method comprising:

a therapeutically effective amount of the pharmaceutical composition of claim 47.

49. The use of the pharmaceutical composition of claim 47 in the manufacture of a medicament for the treatment of kidney disease.

50. A method of identifying an enriched heterogeneous population of kidney cells as having therapeutic potential, the method comprising:

determining the expression level of one or more of the RHAMM, C2, C3, C4, fibrinogen, clotting factor XIII, TEK, KDR, notch1, notch3, timp3, vwf, adam15, gas6, igfbp1 and Tm4sf4 genes of cells of the enriched heterogeneous kidney cell population; and

an enriched heterogeneous kidney cell population is identified as having therapeutic potential if the expression level of the one or more genes in the cells of the enriched heterogeneous kidney cell population is increased relative to the expression level of the one or more genes in the cells of a control kidney cell population.

51. The method of claim 50, wherein said one or more genes comprise RHAMM.

52. The method of claim 50 or 51, wherein the enriched heterogeneous population of kidney cells is prepared via a method comprising a density gradient separation step, and the control population of kidney cells comprises cells that were subjected to the density gradient separation step.

53. The method of claim 52, wherein the enriched heterogeneous population of kidney cells produced via the method comprises cells having a buoyancy density greater than about 1.04 g/mL.

54. An enriched heterogeneous population of kidney cells identified according to the method of any one of claims 50-53.

55. A pharmaceutical composition comprising the enriched heterogeneous population of kidney cells of claim 54.

56. The pharmaceutical composition of claim 55, further comprising hyaluronic acid.

57. A method of treating kidney disease in a patient in need thereof, the method comprising:

administering a therapeutically effective amount of the pharmaceutical composition of claim 55 or 56.

58. Use of a pharmaceutical composition according to claim 55 or 56 in the manufacture of a medicament for the treatment of kidney disease.

59. A method of identifying an enriched heterogeneous population of kidney cells as having therapeutic potential, the method comprising:

determining whether cells of the enriched heterogeneous kidney cell population express SIX2, OSR1, RET and podin; and

If the cells of the enriched heterogeneous kidney cell population are determined to express SIX2, OSR1, RET, and podin, then the enriched heterogeneous kidney cell population is identified as having therapeutic potential.

60. The method of claim 59, wherein the enriched heterogeneous kidney cell population is identified as having therapeutic potential if greater than 0% and up to about 10% of the cells in the enriched heterogeneous kidney cell population are determined to express SIX 2.

61. The method of claim 60, wherein the enriched heterogeneous kidney cell population is identified as having therapeutic potential if greater than 0% and up to about 6% of the cells in the enriched heterogeneous kidney cell population are determined to express SIX 2.

62. The method of any one of claims 59-61, wherein the enriched heterogeneous kidney cell population is identified as having therapeutic potential if at least about 36% of the cells in the enriched heterogeneous kidney cell population are determined to express OSR 1.

63. The method of any one of claims 59-62, wherein the enriched heterogeneous kidney cell population is identified as having therapeutic potential if about 36% to about 85% of the cells in the enriched heterogeneous kidney cell population are determined to express OSR 1.

64. The method of any one of claims 59-63, wherein said enriched heterogeneous kidney cell population is identified as having therapeutic potential if greater than 0% and up to about 90% of the cells in said enriched heterogeneous kidney cell population are assayed for RET expression.

65. The method of any one of claims 59-64, wherein the enriched heterogeneous population of kidney cells is identified as having therapeutic potential if greater than 85% of the cells in the population are determined to express podin.

66. The method of any one of claims 59-65, further comprising:

determining whether cells of the enriched heterogeneous kidney cell population express one or more of LHX1, FGF8, RACK-1, or a kidney disease protein; and

the enriched heterogeneous kidney cell population is identified as having therapeutic potential if the cells of the enriched heterogeneous kidney cell population are assayed for the expression of one or more of LHX1, FGF8, RACK-1, or a kidney disease protein.

67. The method of claim 66 wherein said one or more comprises LHX1.

68. The method of claim 67, wherein said enriched heterogeneous kidney cell population is identified as having therapeutic potential if from about 8% to about 58% of the cells in said enriched heterogeneous kidney cell population are determined to express LHX1.

69. The method of any one of claims 66-68, wherein the one or more comprises FGF8.

70. The method of claim 69, wherein the enriched heterogeneous kidney cell population is identified as having therapeutic potential if about 0.48% to about 59% of the cells in the enriched heterogeneous kidney cell population are determined to express FGF8.

71. The method of any one of claims 66-70, wherein said one or more comprises RACK-1.

72. The method of claim 71, wherein said enriched heterogeneous kidney cell population is identified as having therapeutic potential if at least about 85% of the cells in said heterogeneous kidney cell population are determined to express RACK-1.

73. The method of any one of claims 66-72, wherein the one or more comprises a kidney disease protein.

74. The method of claim 73, wherein the enriched heterogeneous kidney cell population is identified as having therapeutic potential if about 4% to about 99% of the cells in the heterogeneous kidney cell population are determined to express kidney disease protein.

75. A method of identifying an enriched heterogeneous population of kidney cells as having therapeutic potential, the method comprising:

determining whether cells of the enriched heterogeneous kidney cell population express a kidney disease protein, podoprotein, and LHX1; and

if the cells of the enriched heterogeneous kidney cell population are determined to express kidney disease protein, podin and LHX1, the cell population is identified as having therapeutic potential.

76. The method of claim 75, wherein the enriched heterogeneous kidney cell population is identified as having therapeutic potential if greater than 70% of the cells in the enriched heterogeneous kidney cell population are determined to express kidney disease protein.

77. The method of claim 76, wherein the enriched heterogeneous kidney cell population is identified as having therapeutic potential if greater than 75% of the cells in the enriched heterogeneous kidney cell population are determined to express a kidney disease protein.

78. The method of any one of claims 75-77, wherein the enriched heterogeneous kidney cell population is identified as having therapeutic potential if greater than 80% of the cells in the enriched heterogeneous kidney cell population are determined to express podin.

79. The method of claim 78, wherein the enriched heterogeneous kidney cell population is identified as having therapeutic potential if greater than 90% of the cells in the enriched heterogeneous kidney cell population are determined to express podin.

80. The method of any one of claims 75-79, wherein said enriched heterogeneous kidney cell population is identified as having therapeutic potential if greater than 10% of the cells in said enriched heterogeneous kidney cell population are determined to express LHX 1.

81. The method of claim 80, wherein said enriched heterogeneous kidney cell population is identified as having therapeutic potential if greater than 20% of the cells in said enriched heterogeneous kidney cell population are determined to express LHX 1.

82. The method of any one of claims 75-81, further comprising:

Determining whether cells of the enriched heterogeneous kidney cell population express one or more of SIX2, OSR1, RET, FGF8, or RACK-1; and

the enriched heterogeneous kidney cell population is identified as having therapeutic potential if the cells of the enriched heterogeneous kidney cell population are assayed for the expression of one or more of SIX2, OSR1, RET, FGF8, or RACK-1.

83. The method of claim 82, wherein the one or more comprises SIX2.

84. The method of claim 83, wherein the enriched heterogeneous kidney cell population is identified as having therapeutic potential if greater than 0% and up to about 15% of the cells in the enriched heterogeneous kidney cell population are determined to express SIX2.

85. The method of any one of claims 82-84, wherein the one or more comprises OSR1.

86. The method of claim 85, wherein the enriched heterogeneous kidney cell population is identified as having therapeutic potential if about 36% to about 84% of the cells in the enriched heterogeneous kidney cell population are determined to express OSR1.

87. The method of any one of claims 82-86, wherein the one or more comprises RET.

88. The method of claim 87, wherein the enriched heterogeneous kidney cell population is identified as having therapeutic potential if greater than 0% and up to about 90% of the cells in the enriched heterogeneous kidney cell population are determined to express RET.

89. The method of any one of claims 82-88, wherein the one or more comprises FGF8.

90. The method of claim 89, wherein said enriched heterogeneous kidney cell population is identified as having therapeutic potential if about 0.48% to about 59% of the cells in said enriched heterogeneous kidney cell population are assayed for FGF8 expression.

91. The method of any one of claims 82-90, wherein said one or more comprises RACK-1.

92. The method of claim 91, wherein said enriched heterogeneous kidney cell population is identified as having therapeutic potential if at least about 85% of the cells in said heterogeneous kidney cell population are determined to express RACK-1.

93. A pharmaceutical composition comprising the enriched heterogeneous population of kidney cells identified as having therapeutic potential according to the method of any one of claims 75-92.

94. A method of treating kidney disease in a patient in need thereof, the method comprising:

administering a therapeutically effective amount of the pharmaceutical composition of claim 93.

95. Use of the pharmaceutical composition of claim 93 in the manufacture of a medicament for treating kidney disease.