WO2005075636A1

WO2005075636A1 - Molecular markers associated with metanephric development and renal progenitors

Info

Publication number: WO2005075636A1
Application number: PCT/AU2005/000162
Authority: WO
Inventors: Melissa Little; Grant Challen
Original assignee: The University Of Queensland; Monash University
Priority date: 2004-02-09
Filing date: 2005-02-09
Publication date: 2005-08-18

Abstract

Methods of identification, isolation and/or purification of metanephric mesenchyme cells and, more particularly, renal progenitor cells, are provided. These methods utilize a gene expression profile comprising genes that are differentially expressed in committed but uninduced metanephric mesenchyme compared to surrounding intermediate mesoderm tissue. Of these, eight genes were found to encode transmembrane proteins which are particularly advantageous for isolating and purifying renal progenitor cells. A cell surface phenotype of renal progenitor cells was determined to be CD24a+cadherin 11+c-kit +/lowSca-1+/low CD34-. Furthermore, metanephric mesenchyme cells and, more particularly, renal progenitor cells, isolated according to a gene expression profile may be used for in vivo and/or in vitro regeneration of renal tissue.

Description

TITLE MOLECULAR MARKERS ASSOCIATED WITH METANEPHRIC DEVELOPMENT AND RENAL PROGENITORS FIELD OF THE INVENTION THIS INVENTION relates to identifying, isolating and/or purifying metanephric mesenchyme cells and, in particular, renal progenitor cells, although without limitation thereto. More particularly, the invention relates to the use of specific molecular markers that correlate with particular stages of metanephric mesenchyme development to isolate and/or purify renal progenitor cells. This invention also relates to use of isolated and/or purified renal progenitor cells for kidney tissue repair and regeneration. BACKGROUND OF THE INVENTION Currently, there is a general belief that all organs contain stem cells, but that the technology to recognise them either by location or characteristic morphology and surface molecule expression (Al-Awqati & Oliver, 2002, Kidney

Int. 61 387-395) has not been developed. Although cell division is infrequent in the adult kidney, this organ possesses the capacity for regeneration, as witnessed by the cellular proliferation during recovery from conditions such as acute tubular necrosis (Shankland et al, 2000, Am. J. Physiol. 278 F515-F529). However, it is unknown whether this represents the persistence of renal progenitors or their origin. The developed kidney arises via reciprocal interactions between two tissues, the ureteric bud (UB) and the metanephric mesenchyme (MM). Each of these tissues is initially derived from the intermediate mesoderm (IM). The central dogma of kidney development implies that the UB forms the ureter and collecting duct system of the mature kidney while the MM gives rise to the remaining portions of the nephrons, from Bowmans's capsule to distal tubule. The undifferentiated MM can therefore be regarded as the renal progenitor population, a homogenous mass of cells with multipotent differentiation capacity, because it has the ability to differentiate into many more differentiated cell types than do the UB precursors (Herzlinger et al., 1992, Development 114 565-572). This suggests that of the two primordial tissues that interact to produce a mature kidney, the MM is more likely to be the source of a renal stem cell population rather than the UB. However, both entities are initially derived from IM tissue and the UB may be regarded as providing more specific progenitors for the collecting system of the kidney only. Several reports have generated evidence that supports the notion of the undifferentiated MM acting as or containing a mesodermal stem cell population. Lineage-tracing studies have suggested that the uncommitted MM not only has the potential to develop into all the epithelial regions of the nephron, but also can be incorporated into collecting duct epithelia (Qiao et al, 1995, Development 121 3207-3214). Further evidence for the developmental potential of this tissue was recognised when embryonic porcine metanephroi transplanted into immunodeficient mice developed non-renal derivatives such as cartilage and bone in addition to mature glomeruli and tubuli (Dekel et al, 2003, Nat. Med. 9 53-60). Several other studies of cell lines isolated from early, uninduced metanephric mesenchyme have indicated that the MM displays multipotentiality (Herzlinger et al, 1991, J. Am. Soc. Nephrol. 2 438; Oliver et al, 2002, Am., J. Physiol. 283 F799-F809; Herzlinger et al, 1992, supra). In vivo experiments have yet to confirm these findings. While many of the interactions between the UB and MM are now well characterised, the processes by which the MM differentiates from surrounding intermediate mesoderm (IM) and becomes committed to a renal fate remain poorly understood. The creation of the MM depends on the temporary embryonic kidneys, the pronephroi and mesonephroi, developing normally first (Davies & Fisher, 2002, Exp. Neph. 10 102-113). Gene-targeting studies have suggested that expression of the transcription factors Liml (Fujii et al, 1994, Dev. Dyn. 199

211-230), Pax2 (Torres et al, 1995, Development 121 4057-4065), Eyal (Xu et al, 1999, Nat. Gen. 23 113-117) and WT1 (Kreidberg et al, 1993, Cell 74 679- 691) are some of the earliest signs of commitment of the MM to a renal fate, even though the precise role of these genes in kidney development is not fully understood and their expression is not restricted to the MM but extend also to the adjacent mesonephros and gonad as well as to more distant sites, including eye and neural tissue. Microarrays have been used to generate temporal profiles of gene expression over the course of metanephric development (Stuart et al., 2001, Proc. Natl. Acad. Sci. USA 98 5649-5654; Schwab et al, 2003, Kidney Int. 64 1588-1604) and to analyse the expression profiles of discrete renal subcompartments (Stuart et al, Kidney Int. 64 1997-2008). Nevertheless, gene expression by the MM at the earlier renal progenitor stage is unknown. SUMMARY OF THE INVENTION The present invention broadly relates to defining a gene expression profile that facilitates identification, isolation and/or purification of committed but undifferentiated metanephric mesenchyme cells, including renal progenitor cells. In a first aspect, the invention provides a method of identifying a gene expression profile associated with metanephric mesenchyme development, said method including the step of identifying one or more genes that are differentially expressed by one or more metanephric mesenchyme cells at a particular stage of embryonic development compared to one or more intermediate mesoderm cells. In a second aspect, the invention provides a method of identifying a metanephric mesenchyme cell, said method including the step of determining a gene expression profile of said metanephric mesenchyme cell, wherein said gene expression profile comprises one or more genetic markers that are differentially expressed by one or more metanephric mesenchyme cells compared to one or more intermediate mesoderm cells. In a third aspect, the invention provides a method of isolating or purifying one or more metanephric mesenchyme cells including the step of identifying a gene expression profile that comprises one or more genetic markers that are differentially expressed by said one or more metanephric mesenchyme cells compared to one or more intermediate mesoderm cells. There has to be something said in this aspect and in the fifth aspect about the markers needing to be at the cell surface for the purposes of isolation. In one embodiment, gene expression is determined according to nucleic acid expression, such as mRNA expression. In another embodiment, gene expression is determined according to protein expression. In preferred embodiments, expressed genes that may be used in gene expression profiles associated with a particular stage of development of metanephric mesenchyme, which genes are differentially expressed with respect to DVI, are set forth in Tables 2 and 3. In a particularly preferred form, the gene expression profile comprises one or more genetic markers set forth in Table 4. In embodiments relating to isolation and purification of metanephric mesenchyme cells, advantageous cell surface markers include Neuropilin-1; CD 164 antigen; CD83 antigen; Stromal cell derived factor receptor 1; CD24a antigen; Serine protease inhibitor, Kunitz type 2; Tumor-associated calcium signal transducer 1; Receptor-like tyrosine kinase; Fibroblast growth factor receptor 2 ; Amyloid beta (A4) precursor protein; Bone morphogenetic protein receptor, type 1A; DNA segment, Chr 8, Wayne State University 49, expressed; Signal sequence receptor, alpha; Junction adhesion molecule 3; PTK7 protein tyrosine kinase 7; Cadherin 11; Syndecan binding protein; Integral membrane protein 2C; Tripartite motif-containing 59; NAD(P) dependent steroid dehydrogenase-like; DNA segment, Chr 3, University of California at Los Angeles 1; Solute carrier family 3 (activators of dibasic and neutral amino acid transport), member 2; RIKEN cDNA 1110018G07 gene; Purine rich element binding protein B; Solute carrier family 6 (neurotransmitter transporter, taurine), member 6; Gap junction membrane channel protein alpha 1 ; Tumor differentially expressed 1; CD81 antigen; Solute carrier family 35, member 5; Solute carrier family 39 (zinc transporter), member 7; Gene rich cluster, C3f gene; Claudin 6; Solute carrier family 20, member 1; Solute carrier family 16 (monocarboxylic acid transporters), member 1; ADP-ribosylation factor-like 6 interacting protein 2;

Autocrine motility factor receptor; Claudin 7; Calcitonin receptor-like; Tumor differentially expressed 2; Synaptophysin-like protein; Claudin 11; G protein- coupled receptor 89; ELOVL family member 6 (Elovl6), elongation of long chain fatty acids (yeast); Purinergic receptor (family A group 5); Non imprinted in Prader-Willi/Angelman syndrome 2 homolog (human); RIKEN cDNA

4930579A11 gene; Zinc finger, DHHC domain containing 6; Solute carrier family 37 (glycerol-3 -phosphate transporter), member 3; Sarcoma amplified sequence; RIKEN cDNA 1700022N24 gene; and Mid- 1 -related chloride channel

1, although without limitation thereto, either singly or in combination. In a fourth aspect, the invention provides a method of identifying a gene expression profile of a renal progenitor cell, said method including the step of identifying one or more genes that are differentially expressed by said renal progenitor cell compared to an intermediate mesenchyme cell. In a fifth aspect, the invention provides a method of identifying a renal progenitor cell, said method including the step of determining a gene expression profile of said renal progenitor cell, wherein the gene expression profile comprises one or more genetic markers differentially expressed compared to an intermediate mesenchyme cell. In a sixth aspect, the invention provides a method of isolating or purifying a renal progenitor cell, said method including the step of identifying a gene expression profile of said renal progenitor cell, wherein the gene expression profile comprises one or more genetic markers differentially expressed compared to an intermediate mesenchyme cell. Typically, said renal progenitor cell is isolated from differentiating embryonic or adult stem cells in culture or from any adult tissue, including the kidney. In preferred embodiments, expressed genes that may be used in gene expression profiles associated with renal progenitor cells are set forth in Tables 2 and 3. In a particularly preferred form, the gene expression profile comprises one or more genetic markers set forth in Table 4. In embodiments relating to isolation and purification of renal progenitor cells, advantageous cell surface markers include Neuropilin-1; CD 164 antigen;

CD83 antigen; Stromal cell derived factor receptor 1; CD24a antigen; Serine protease inhibitor, Kunitz type 2; Tumor-associated calcium signal transducer 1 ;

Receptor-like tyrosine kinase; Fibroblast growth factor receptor 2 ; Amyloid beta (A4) precursor protein; Bone morpho genetic protein receptor, type 1A; DNA segment, Chr 8, Wayne State University 49, expressed; Signal sequence receptor, alpha; Junction adhesion molecule 3; PTK7 protein tyrosine kinase 7; Cadherin 11; Syndecan binding protein; Integral membrane protein 2C; Tripartite motif- containing 59; NAD(P) dependent steroid dehydrogenase-like; DNA segment, Chr 3, University of California at Los Angeles 1; Solute carrier family 3 (activators of dibasic and neutral amino acid transport), member 2; RIKEN cDNA 1110018G07 gene; Purine rich element binding protein B; Solute carrier family 6

(neurotransmitter transporter, taurine), member 6; Gap junction membrane channel protein alpha 1; Tumor differentially expressed 1; CD81 antigen; Solute carrier family 35, member 5; Solute carrier family 39 (zinc transporter), member 7; Gene rich cluster, C3f gene; Claudin 6; Solute carrier family 20, member 1; Solute carrier family 16 (monocarboxylic acid transporters), member 1; ADP- ribosylation factor-like 6 interacting protein 2; Autocrine motility factor receptor; Claudin 7; Calcitonin receptor-like; Tumor differentially expressed 2; Synaptophysin-like protein; Claudin 11 ; G protein-coupled receptor 89; ELOVL family member 6 (Elovl6), elongation of long chain fatty acids (yeast); Purinergic receptor (family A group 5); Non imprinted in Prader-Willi/Angelman syndrome

2 homolog (human); RIKEN cDNA 4930579A11 gene; Zinc finger, DHHC domain containing 6; Solute carrier family 37 (glycerol-3 -phosphate transporter), member 3; Sarcoma amplified sequence; RIKEN cDNA 1700022N24 gene; and Mid- 1 -related chloride channel 1, although without limitation thereto, either singly or in combination. The cell surface markers profile may further comprise one or more cell surface markers together with one or more other stem cell markers as described in Table 6. Preferably, the one or more other stem cell markers are selected from the group consisting of CD34, c-kit and Sea 1, by virtue of their lack of, or low level of, expression. In a particularly preferred, non-limiting embodiment, a gene expression profile of a renal progenitor cell is defined as CD24a⁺cadherin 1 l⁺c-kit ^+/lowSca- j+Λow _CD34-_ In a seventh aspect, the invention provides use of metanephric mesenchyme cells, or more particularly renal progenitor cells, isolated or purified according to the invention, for in vitro and/or in vivo generation of renal tissue. hi an eighth aspect, the invention provides a nucleic acid array comprising a plurality of isolated nucleic acid molecules described herein, for use according to a method of any preceding aspect. In a ninth aspect, the invention provides a protein array comprising a plurality of isolated protein molecules described herein, for use according to a method of any preceding aspect. Preferably, according to the aforementioned aspects, the gene expression profile comprises a plurality of genetic markers, each of the genetic markers displaying at least 1.8 fold higher levels of expression in metanephric mesenchyme cells relative to intermediate mesenchyme cells. Suitably, according to the aforementioned aspects, said metanephric mesenchyme cells, renal progenitor cells and/or renal stem cells are of mammalian origin. Preferably, said metanephric mesenchyme cells, renal progenitor cells and/or renal stem cells are of human origin. Suitably, according to the aforementioned aspects, said genetic markers are of mammalian origin. Preferably, said genetic markers are of human origin. Throughout this specification, unless otherwise indicated, "comprise", "comprises" and "comprising" are used inclusively rather than exclusively, so that a stated integer or group of integers may include one or more other non-stated integers or groups of integers. BRIEF DESCRIPTION OF THE DRAWINGS In order that the present invention may be more readily understood and placed into practical effect, preferred embodiments of the invention will be described, by way of example only, with reference to the accompanying tables and figures, in which:

TABLE 1 : Classes of predicted membrane organization. TABLE 2: List of genes showing highest differential expression between intermediate mesoderm and metanephric mesenchyme at E10.5 genes from NIA

Microarrays according to a B-stat of >0 and a fold change >1.8. TABLE 3: List of genes showing highest differential expression between intermediate mesoderm and metanephric mesenchyme at El 0.5 genes from Compugen microarrays according to a B-stat of >0 and a fold change >1.8. TABLE 4: Preferred markers of El 0.5 metanephric mesenchyme / renal progenitors based on data from Compugen and NIA chips taking into account fold change (>1.8), B-stat (>0) and verification by in situ hybridisation. TABLE 5: Human gene equivalents of the mouse genes described in Table 4. Human genetic markers are listed in corresponding order to the mouse genetic markers in Table 4. Human homologs of the mouse genetic markers were obtained using Homologene (available from the NCBI website: http://www.ncbi.nlm.nih.gov/) or BLAST to compare two potential homologous full length cDNA sequences. In the case of murine CD24a, the human ortholog was identified at ENSML (http://www.ensembl.org/Homo_sapiens/geneview? gene=ENSG00000185275) which lists human CD24 as the ortholog of murine CD24a antigen.

TABLE 6: Expression of stem cell markers by renal progenitors in NIA microarray analysis.

FIG. 1. Analysis of microarray data. The three array comparison were normalised to each other to give a comparable range of log ratios. (A and B) Boxplot representations of the individual hybridisations before (A) and after (B) print-tip lowess normalisation. (C to E) Genespring scatterplots of each hybridisation. For replicate hybridisations (C and D), El 0.5 metanephric mesenchyme aRNA was labelled with Cy3 while El 0.5 intermediate mesoderm aRNA was labelled with Cy5. The sample labelling was reversed in a dye swap experiment to account for any dye bias (E). The outer lines represent 1.80-fold differences in expression between samples. Spots located outside the lines were identified as outliers and used to generate lists of genes differentially expressed between samples. (F) Genes were ranked in decreasing order of the duplicate- correlated B-value, with the highest B-values indicating those with the most significant changes in expression level.

FIG. 2. RNA in situ hybridisation of genes differentially displayed between IM and MM at El 0.5 in the mouse. The expression pattern of each gene was surveyed across El 0.5 embryos (A, E, I, M, Q, U, Y, c, g, k, o - Bars = 500 μm, IM = intermediate mesoderm, HL = hind limb bud; B, F, J, N, R, V, Z, d, h, 1, p - Bars = 100 μm, ND = nephric duct, MM = metanephric mesenchyme, arrows represent budding site of primitive ureteric bud), El 2.5 urogenital tracts (C, G, K, O, S, W, a, e, i, m, q - Bars = 500 μm, Ms = mesonephros, G = gonad,

Mt = metanephros) and metanephric explants (D, H, L, P, T, X, b, f, j, n, r - Bars = 300 μm). (A to D) Isletl, (E to H) Gata3, (I to L) Ewing sarcoma homolog, (M to P) p53, (Q to T) 14-3-3-theta, (U to X) Retinoic acid receptor alpha, (c to f) HI 9, (g to j) Stearoyl-coenzyme A desaturase 2, (k to n) Enolase and (o to r) RIKEN cDNA 1600029D21 gene.

FIG. 3. RNA in situ hybridisations of cell surface proteins representing renal progenitor cell markers. The expression pattern of each gene was surveyed across El 0.5 embryos (A, G, M, S, Y - Bars = 500 μm; B, H, N, T, Z - Bars = 100 μm, ND = nephric duct, MM = metanephric mesenchyme, arrows represent budding site of primitive ureteric bud), El 2.5 urogenital tracts (C, I, O, U, a -

Bars = 500 μm), metanephric explants (D, J, P, V, b - Bars = 300 μm; E, K, Q, W, c - Bars = 50 μm) and El 5.5 kidney sections (F, L, R, X, d; Bars = 200 μm). (A to F) Sρint-2, (G to L) Claudin-6, (M to R) CD24a antigen, (S to X) Cadherin-11 and (Y to d) Hypothetical protein BG072310. FIG. 4. Expression patterns of known stem cell markers during early kidney development. The expression pattern of each gene was analysed by RNA in situ hybridisation across El 0.5 embryos (A, E, I, M, Q, U, Y - Bars = 500 μm; B, F, J, N, R, V, Z - Bars = 100 μm, ND = nephric duct, MM = metanephric mesenchyme, arrows represent budding sit of primitive ureteric bud), El 2.5 urogenital tracts (C, G, K, O, S, W, a - Bars = 500 μm) and metanephric explants

(D, H, L, P, T, X, b - Bars = 300 μm). (A to D) Oct-4, (E to H) Nanog, (I to L) CD34, (M to P) C-kit, (Q to T) Sca-1, (U to X) Podocalyxin-like and (Y to b) Nestin. FIG. 5. Wholemount RNA in situ hybridisation of genes enriched identified as enriched in El 0.5 MM from Compugen microarray analysis. FIG. 6. In situ hybridisation analysis of CD24a and cadherin-11 throughout mouse kidney development.

FIG. 7. FACS analysis of embryonic and adult mouse kidneys for CD24a and cadherin-11 expressing cells demonstrating the presence of cell positive for both markers that decrease in population size over the course of kidney development. The presence of adult CD24a⁺cadherinl 1⁺ cells suggests the possibility of an adult renal progenitor or stem cell.

FIG. 8. CD24a expression in embryonic and adult mouse kidney side population cells (filled histogram) compared to isotype control (open histogram). FIG. 9. Nucleotide sequences for each of the human genetic markers set forth in Table 5. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention is predicated, at least in part, by the present inventors' identification of differential gene expression by metanephric mesenchyme cells developing along a metanephric lineage or pathway.

Specifically, differential gene expression was observed for El 0.5 metanephric mesenchyme (MM) and intermediate mesoderm (IM) tissue. While both of these tissues are formed from adjacent mesoderm, only the committed but uninduced MM has the capacity to form kidney tissue and therefore comprises a renal progenitor population. The present invention provides 554 non-redundant genes that are differentially expressed in committed but uninduced metanephric mesenchyme (MM) compared to the surrounding intermediate mesoderm tissue at El 0.5. Of these, 51 non-redundant genes were found to encode transmembrane proteins expressed at the cell surface which could therefore be used both to identify and isolate renal progenitors. Gene expression profiles may therefore be used to identify, isolate and/or purify renal progenitor cells during renal differentiation or upon induction or production of renal progenitors from stem cells. As used herein, the "Embryonic day or E" stages of the mouse embryo development can be compared to the defined Theiler Stages, wherein El 0.5 corresponds to Theiler Stage 17. This corresponds to E32 in the developing human embryo. rhttp://www.ana.ed.ac.uk/anatomy/database/hυmat/MouseComp.html). It will be appreciated that the invention has initially been elucidated using a murine model of human kidney development. Accordingly, the invention described herein may advantageously be applied to human kidney development.

It will therefore be appreciated that the murine gene expression profiles described herein and the developmental stages that they are associated with in mice, will also apply to human renal development. In this regard, the murine genetic markers identified herein as associated with metanephric mesenchyme and, in particular, renal progenitor cells, have human orthologs that may be readily utilized according to the invention in relation to human metanephric mesenchyme and, in particular, renal progenitor cells. For the purposes of this invention, by "isolated" is meant material that has been removed from its natural state or otherwise been subjected to human manipulation. Isolated material may be substantially or essentially free from components that normally accompany it in its natural state, or may be manipulated so as to be in an artificial state together with components that normally accompany it in its natural state. By "purified" and "purification" ', particularly in the context of cell purification from an initial cell population, is meant isolation of cells whereby the frequency or proportion of said cells in the isolated cell population is greater than in the initial cell population. The term "gene" is used herein to describe a discrete, structural unit of a genome that may comprise one or more of introns, exons, open reading frames and regulatory sequences such as promoters and polyadenylation sequences. As used herein a "gene expression profile" comprises one or more nucleic acid or protein products of gene expression (i.e genetic markers) that characterize a particular cell type and/or stage of development. In one particular embodiment, a "gene expression profile" comprises one or more nucleic acid or protein products of gene expression that are differentially expressed by one or more metanephric mesenchyme cells compared to one or more intermediate mesenchyme cells. In another particular embodiment, a "gene expression profile" comprises one or more nucleic acid or protein products of gene expression that are differentially expressed by one or more metanephric mesenchyme cells at a particular stage of development compared to metanephric mesenchyme cells at one or more other stages of development. An example is a gene expression profile of a renal progenitor cell or renal stem cell. Thus, it will be appreciated that gene expression profiles do not necessarily relate to the presence or absence of gene expression or to quantitatively measuring absolute levels of gene expression, but typically relate to relative or differential levels of gene expression. Typically, although not exclusively, a gene expression profile comprises a plurality of different nucleic acid or protein products of gene expression. As set forth in Tables 2 and 3, a plurality of genetic markers have been identified, at least some of which may be used to establish a gene expression profile of MM cells compared to IM cells. The genetic markers set forth in Tables 2 and 3 display at least 1.8 fold higher levels of expression in MM cells relative to IM cells. However, it will be appreciated that genetic markers set forth in Tables 2 and 3 may display at least 2, 3, 4, or 5-fold higher levels of expression in MM cells relative to IM cells. As will be understood by persons of skill in the art, statistical analyses and in situ hybridization studies have been performed to establish the degree of reliability and/or reproducibility of the genetic markers identified in Tables 2 and

3. Accordingly, Table 4 provides a list of murine genetic markers that have shown the highest reliability and/or reproducibility in terms of their association with MM cells compared to IM cells. Table 5 provides a corresponding list of human genetic markers corresponding to the murine markers set forth in Table 4. Human orthologs were identified using Homologene (available from the NCBI website: http://www.ncbi.nlm.nih.gov/) or BLAST to compare two potential homolog full length cDNA sequences. In the case of murine CD24a, ENSML(http://www.ensembl.org/Homo_sapiens/geneview?gene=ENSG0000018 5275) was consulted, which identified human CD24 as the human ortholog of murine CD24a. Preferably, the gene expression profile comprises one or more genetic markers selected from the group consisting of: Zinc finger protein 335; Ewing sarcoma homolog; t-complex protein 1; enolase 1, alpha non-neuron; tyrosine 3- monooxygenase/tryptophan 5-monooxygenase activation protein, theta polypeptide (CDK5 regulatory subunit associated protein 2); Cytoplasmic FMR1 interacting protein 1; Sine oculis-related homeobox 2 homolog (Drosophila); Minichromosome maintenance deficient 7 (S. cerevisiae); Karyopherin (importin) alpha 2; Heat shock protein 8; Ras-GTPase-activating protein SH3-domain binding protein; Homeo box A10; Crystallin, mu; RIKEN cDNA 2610312E17 gene; Opioid growth factor receptor; Retinoic acid receptor alpha (RARα); Glial cell line derived neurotrophic factor; Mesoderm development candiate 2; RIKEN cDNA 1300010F03 gene; RIKEN cDNA 2810037C14 gene; Neuropilin-1; CD 164 antigen; CD83 antigen; Stromal cell derived factor receptor 1; CD24a antigen; Serine protease inhibitor, Kunitz type 2; Tumor-associated calcium signal transducer 1; Receptor-like tyrosine kinase; Fibroblast growth factor receptor 2; Amyloid beta (A4) precursor protein; Bone morphogenetic protein receptor, type 1A; DNA segment, Chr 8, Wayne State University 49, expressed; Signal sequence receptor, alpha; Junction adhesion molecule 3; PTK7 protein tyrosine kinase 7; Cadherin 11; Syndecan binding protein; Integral membrane protein 2C; Tripartite motif-containing 59; NAD(P) dependent steroid dehydrogenase-like; Vesicle-associated membrane protein 3; DNA segment, Chr 3, University of California at Los Angeles 1; Solute carrier family 3 (activators of dibasic and neutral amino acid transport), member 2; RIKEN cDNA 1110018G07 gene; Purine rich element binding protein B; Solute carrier family 6

(neurotransmitter transporter, taurine), member 6; Gap junction membrane channel protein alpha 1; Tumor differentially expressed 1; CD81 antigen; Solute carrier family 35, member F5; Solute carrier family 39 (zinc transporter), member 7; Gene rich cluster, C3f gene; Claudin 6; Solute carrier family 20, member 1; Solute carrier family 16 (monocarboxylic acid transporters), member 1; ADP- ribosylation factor-like 6 interacting protein 2; Autocrine motility factor receptor;

Claudin 7; Calcitonin receptor-like; Tumor differentially expressed 2; Synaptophysin-like protein; Claudin 11; G protein-coupled receptor 89; ELOVL family member 6, elongation of long chain fatty acids (yeast); Purinergic receptor (family A group 5); Non imprinted in Prader-Willi/Angelman syndrome 2 homolog (human); RIKEN cDNA 4930579A11 gene; RIKEN cDNA 2610311119 gene; Zinc finger, DHHC domain containing 6; Solute carrier family 37 (glycerol- 3-phosphate transporter), member 3; Sarcoma amplified sequence; RIKEN cDNA 1700022N24 gene; Mid- 1 -related chloride channel 1; Stearoyl-Coenzyme A desaturase 2; RIKEN cDNA 1110034A24 gene; Homeo box D13; retinol dehydrogenase 10 (all-trans) (RdhlO); Sal-like 4 (Drosophila); Homeo box Al l, opposite strand transcript; and Roundabout homolog 2 (Drosophila). As set forth in FIG. 9, examples of human nucleotide sequences corresponding to each of the genetic markers in Table 5. For the purposes of cell isolation and/or purification, the invention contemplates particular use of genetic markers in the form of one or more proteins expressed at the cell surface of a metanephric mesenchyme cell, inclusive or renal progenitor cells and renal stem cells. With this in mind, Table 1 sets forth a classification of proteins that provides a code that is used in Tables 2-4. According to this code, preferred cell surface proteins are selected from the group consisting of:Neuropilin-l; CD 164 antigen; CD83 antigen; Stromal cell derived factor receptor 1; CD24a antigen; Serine protease inhibitor, Kunitz type 2; Tumor-associated calcium signal transducer 1; Receptor-like tyrosine kinase; Fibroblast growth factor receptor 2 ; Amyloid beta (A4) precursor protein; Bone morphogenetic protein receptor, type 1A; DNA segment, Chr 8, Wayne State

University 49, expressed; Signal sequence receptor, alpha; Junction adhesion molecule 3; PTK7 protein tyrosine kinase 7; Cadherin 11; Syndecan binding protein; Integral membrane protein 2C; Tripartite motif-containing 59; NAD(P) dependent steroid dehydrogenase-like; DNA segment, Chr 3, University of California at Los Angeles 1 ; Solute carrier family 3 (activators of dibasic and neutral amino acid transport), member 2; RIKEN cDNA 1110018G07 gene; Purine rich element binding protein B; Solute carrier family 6 (neurotransmitter transporter, taurine), member 6; Gap junction membrane channel protein alpha 1; Tumor differentially expressed 1; CD81 antigen; Solute carrier family 35, member 5; Solute carrier family 39 (zinc transporter), member 7; Gene rich cluster, C3f gene; Claudin 6; Solute carrier family 20, member 1; Solute carrier family 16 (monocarboxylic acid transporters), member 1; ADP-ribosylation factor-like 6 interacting protein 2; Autocrine motility factor receptor; Claudin 7; Calcitonin receptor-like; Tumor differentially expressed 2; Synaptophysin-like protein; Claudin 11; G protein-coupled receptor 89; ELOVL family member 6 (Elovl6), elongation of long chain fatty acids (yeast); Purinergic receptor (family A group 5); Non imprinted in Prader-Willi/Angelman syndrome 2 homolog

(human); RIKEN cDNA 4930579A11 gene; Zinc finger, DHHC domain containing 6; Solute carrier family 37 (glycerol-3 -phosphate transporter), member 3; Sarcoma amplified sequence; RIKEN cDNA 1700022N24 gene; and Mid-1 - related chloride channel 1. Although not wishing to be bound by any particular theory, the present invention postulates that genetic markers differentially expressed by metanephric mesenchyme cells in earlier stages of development compared to later stages may be genetic markers associated with renal progenitors and thereby facilitate their identification and isolation with respect to other metanephric mesenchyme cells. While the invention as described herein makes reference to genetic markers set forth variously in Tables 2-5 and FIG. 9, it will also be appreciated that the invention provides a principle capable of general application to the identification of other genetic markers that may be indicative of, or otherwise associated with metanephric mesenchyme development and, more particularly, renal progenitors inclusive of renal stem cells. A particular feature of the invention is the identification and use of gene expression profiles for the identification, isolation and/or purification of renal progenitor cells and/or renal stem cells. It is postulated that renal progenitor cells may comprise renal stem cells. As used herein, a "renal progenitor cell" is a metanephric mesenchyme cell which is a developmental antecedent of one or more mature renal cell types. This definition does not exclude the possibility that renal progenitor cells in later stages of the developing kidney may have the same phenotype as those defined in the metanephric mesenchyme. As used herein, a "stem cell " is a progenitor cell capable of self-renewal and differentiation into one or more mature cell types. As used herein, a "renal stem cell " is a progenitor cell capable of self- renewal and differentiation into one or more mature renal cell types. It will be appreciated that stem cells may be embryonic stem cells or adult stem cells. In a particularly advantageous form, the invention provides a gene expression profile of a renal progenitor cell in the form of a cell surface marker profile. This profile may comprise cell surface markers as hereinbefore identified together with one or more other stem cell markers as described in Table 6. Preferably, the one or more other stem cell markers are selected from the group consisting of CD34, c-kit and Sea 1, by virtue of their lack of, or low level of, expression. In a particular, non-limiting embodiment the cell surface marker profile of a renal progenitor cell is set forth as CD24a⁺cadherin l l⁺c-kit ^+/lowSca-l^{+ low} CD34^". Nucleic acid based determination of gene expression In particular embodiments of the present invention, gene expression profiles of metanephric mesenchyme, inclusive of renal progenitor cells and stem cells, may be determined by methods that employ nucleic acid detection. Typically, such methods use gene-specific primers and/or probes for nucleic acid detection and include but are not limited to, nucleic acid arrays (in microarrays), nucleic acid sequence amplification and blotting techniques. For the purposes of determining one or more gene expression profiles of temporal stages of metanephric mesenchyme development, the invention contemplates particular embodiments of such methods which may be used alone or in combination. Generally, these methods of the invention measure nucleic acid expression levels of intermediate mesenchyme and metanephric mesenchyme cells or tissues. The term "nucleic acid^'' as used herein designates single-or double-stranded mRNA, RNA, cRNA and DNA, said DNA inclusive of cDNA and genomic DNA. A "probe" may be a single or double-stranded oligonucleotide or polynucleotide. A "primer" is usually a single-stranded oligonucleotide, preferably having 15-50 contiguous nucleotides, which is capable of annealing to a complementary nucleic acid "template" and being extended in a template-dependent fashion by the action of a DNA polymerase such as Taq polymerase, RNA-dependent DNA polymerase or Sequenase™. It will also be appreciated that probes and/or primers may be labelled for the purpose of detecting amplification products and/or complementary sequences by hybridization and other uses as is well known in the art. The terms "hybridize and hybridization" are used herein to denote the pairing of at least partly complementary nucleotide sequences to produce a DNA- DNA, RNA-RNA or DNA-RNA hybrid. Hybrid sequences comprising complementary nucleotide sequences occur through base-pairing between complementary purine and pyrimidine bases, or between modified purines (for example, inosine, methylinosine and methyladenosine) and modified pyrimidines

(for example thiouridine and methylcytosine). In particular, non-limiting embodiments, the invention contemplates use of one or more molecular makers set forth in any one of Tables 2, 3, 4 and/or 5, in nucleic acid form, for identification, isolation and/or purification of MM cells and/or renal progenitor cells inclusive of renal stem cells. More particularly, examples of human nucleic acid sequences are provided in Table 5 which may be utilized according to the invention. It will be appreciated that fragments of the aforementioned nucleic acid markers may be utilized, such as primers for nucleic acid sequence amplification and/or as probes for nucleic acid hybridization, although without limitation thereto. Nucleic acid "fragments" may preferably comprise at least 20 contiguous nucleotides and up to 50, 100, 200, 300, 500 or more contiguous nucleotides, as required. According to the invention, a nucleic acid amplification technique may include polymerase chain reaction (PCR) and ligase chain reaction (LCR) as for example described in Chapter 15 of Ausubel et al. supra; strand displacement amplification (SDA) as for example described in U.S. Patent No 5,422,252; rolling circle replication (RCR) as for example described in Liu et al, 1996, J.

Am. Chem. Soc. 118 1587 and International application WO 92/01813, and

Lizardi et al, (International Application WO 97/19193); nucleic acid sequence- based amplification (NASBA) as for example described by Sooknanan et al,

199 , Biotechniques 17 1077; ligase chain reaction (LCR) as for example described in International Application WO89/09385; Q-β replicase amplification as for example described by Tyagi et al, 1996, Proc. Natl. Acad. Sci. USA 93

5395 and helicase-dependent amplification as for example described in International Publication WO 2004/02025. As used herein, an "amplification product" refers to a nucleic acid product generated by any nucleic acid amplification technique. In another particular form, quantitative PCR using primers corresponding to one or more genes expressed by MM or IM cells of particular stages of development may be used to quantify relative expression levels of one or many nucleic acids to thereby determine the relative gene expression for each MM or

IM tissue. In a particular, non-limiting embodiment described in more detail hereinafter, expressed RNA is linearly amplified using a messageAMP to provide "aRNA" kit supplied by Ambion. Preferably, according to this embodiment, the aRNA is reverse transcribed using random hexamers (Promega) into cDNA incorporating either Cy5- or Cy3- labelled dUTPs (Amersham). Nucleic acid arrays provide a particularly advantageous method of initially identifying or establishing a gene expression profile of a particular stage of metanephric mesenchyme development and also for subsequent detection of a gene expression profile when determining the stage of development of metanephric mesenchyme cells. Nucleic acid arrays typically use libraries of genomic DNA or cDNA. In one particular form, the invention provides a molecular library in the form of a nucleic acid array that comprises a substrate to which is immobilized, bound or otherwise coupled a plurality of nucleic acids that correspond to the expressed genes that are characteristic of a particular stage of metanephric mesenchyme development, or respective fragments thereof. Each immobilized, bound or otherwise coupled nucleic acid has an "address" on the array that signifies the location and identity of said nucleic acid. Generally, nucleic acid array technology has become well known in the art and examples of methods applicable to array technology are provided in Chapter 22 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel et al. (John Wiley & Sons NY USA 1995-2001). The array can have a density of at least 10, 50, 100, 200, 500, 1,000, 2,000, or 10,000 or more addresses/cm², and ranges there between. The substrate may be a two-dimensional substrate such as a glass slide, a wafer (e.g., silica or plastic), a mass spectroscopy plate, or a three-dimensional substrate such as a gel pad. Addresses in addition to the 15K mouse clone set of nucleic acids of the invention may also be disposed on the array. In certain embodiments, at least one address of the plurality includes a nucleic acid capture probe that hybridizes specifically to a member of a nucleic acid library, e.g., the sense or anti-sense strand. In one preferred embodiment, a subset of addresses of the plurality of addresses has a nucleic acid capture probe for a nucleic acid library member. Each address of the subset can include a capture probe that hybridizes to a different region of a library member. An array format may comprise glass slides having an immobilized, ordered grid of a plurality of cDNA fragments. In particular embodiments, said array has 5,000 to 19,000 or up to 40,000 cDNA fragments. Preferably, each said cDNA fragment corresponds to a particular gene or expressed sequence tag (EST) gene fragment. An array can be generated by various methods, e.g., by photolithographic methods (see, e.g., U.S. Patent Nos. 5,143,854; 5,510,270; and 5,527,681), mechanical methods (e.g., directed-flow methods as described in U.S. Patent No. 5,384,261), pin-based methods (e.g., as described in U.S. Pat. No. 5,288,514), and bead-based techniques (e.g., as described in PCT US/93/04145). In a preferred embodiment described in more detail hereinafter, nucleic acid arrays were performed using chips arrayed with the National Institute of Health - National Institute of Aging 15K mouse cDNA clone set (http://lgsun.grc.nia.nih.gov/cDNA/15k.html) in addition to a selection of custom clones submitted from the Institute for Molecular Bioscience (University of Queensland, Brisbane) and contained 15,989 elements in total (SRC NIA v3.0 chips). The National Institute of Ageing (NIA) array is a 15K mouse clone set containing 15,247 expressed sequence tags (ESTs) derived from pre-and periimplantation embryo E12.5 female gonad/mesonephros and newborn ovary cDNA libraries. The Compugen long oligonucleotide set used to create the additional Compugen lists are commercially available (http ://www.labonweb. com/chips/libraries .html) . Other human sets may be obtained from Agilent (for long oligonucleotides), Affymetrix (for short oligonucleotides synthesized on a substrate) or cDNA microarrays as produced by the Ontario Cancer Institute (http://www.oci.utoronto.ca/services/microarray/). In a preferred embodiment, gene expression is measured by isolating mRNA from samples MM and IM tissue and comparing expression with that of another sample (e.g. MM tissue of a different development stage or surrounding IM tissue). Other known conventional identification methods are contemplated. Typically, complementary nucleotide sequences are identified by blotting techniques that include a step whereby nucleotides are immobilized on a matrix (preferably a synthetic membrane such as nitrocellulose), a hybridization step, and a detection step (eg. northern hybridization). Dot blotting and slot blotting can be used to identify complementary RNA/RNA, DNA/RNA or DNA/DNA polynucleotide sequences. Such techniques are well known by those skilled in the art, and have been described in Ausubel et al, supra, at pages 2.9.1 through 2.9.20. Methods for detecting labelled nucleic acids hybridized to an immobilized nucleic acid are well known to practitioners in the art. Such methods include autoradiography, chemiluminescent, fluorescent and colorimetric detection. In particular embodiments, genes identified in Tables 2 and 3 may be used to determine whether a subpopulation of metanephric mesenchyme cells or stem cells from a variety of sources, including embryonic and adult stem cells from human tissues, is or has been, induced to become a renal progenitor based upon expression of one or more of these genes. This can be assessed in a variety of ways, including but not limited RT- PCR, Northern analysis and/or in situ hybridisation, such as hereinbefore described.

Protein based determination of gene expression and cell purification Gene expression profiles of metanephric mesenchyme, inclusive of renal progenitor cells and stem cells may be determined by methods that employ protein detection. Protein-based methods are also particularly useful for cell isolation and purification according to cell surface protein expression. By "protein" is meant an amino acid polymer. The amino acids may be natural or non-natural amino acids, D- or L- amino acids as are well understood in the art. As used herein, "protein" includes full-length proteins and fragments thereof including but not limited to, peptides, peptide-nucleic acid conjugates and epitopes capable of being recognized, bound or otherwise detected by an antibody. In one embodiment, identification of a gene expression profile may be performed using protein libraries or arrays. For example, a plurality of proteins may be used in a protein library displayed in any of a number of ways, e.g., in phage display or cell display systems, in protein arrays or by two-dimensional gel electrophoresis, or more specifically, differential two-dimensional gel electrophoresis (2D-DIGE). These particular embodiments may generally be referred to as "proteomic" or "protein profiling" methods, such as described in Chapters 3.9.1 and 22 of CURRENT

PROTOCOLS IN PROTEIN SCIENCE Eds. Coligan et al, John Wiley & Sons NY USA (1996-2002). Other particular examples include the SELDI protein chip technology of Ciphergen (www. Ciphergen.com/doclib/docFiles/262). In embodiments relating to protein arrays, preferably each of a plurality of expressed proteins of the invention is located at an identifiable address on the array. Preferably, the protein array comprises a substrate to which is immobilized, impregnated, bound or otherwise coupled a plurality of proteins described herein, or respective fragments thereof. Each immobilized, impregnated bound or otherwise coupled protein is at an "address" on the array that signifies the location and identity of each said protein. The substrate may be a chemically-derivatized aluminium chip, a synthetic membrane such as PVDF or nitrocellulose, a glass slide or microtiter plates. Detection of substrate-bound proteins may be performed using known methods such as mass spectrometry, ELISA, immunohistochemistry, fluorescence microscopy or colorimetric detection. Determination of protein expression may also conveniently be performed using antibodies or antibody fragments (such as Fab and Fab₂ fragments) directed to one or more proteins of a particular gene expression profile. These antibody-based methods may have particular efficacy in isolation and purification of renal progenitor cells. Typically, the antibody or antibody fragment further comprises a label. The label may be selected from a group including a chromogen, a catalyst, an enzyme, a fluorophore, a chemiluminescent molecule, a lanthanide ion such as Europium (Eu³⁴), a radioisotope (e.g. ¹²⁵I) and a direct visual label. In the case of a direct visual label, use may be made of a colloidal metallic or non-metallic particle, a dye particle, an enzyme or a substrate, an organic polymer, a latex particle, a liposome, or other vesicle containing a signal producing substance and the like. A large number of enzymes useful as labels is disclosed in United States Patent Specifications U.S. 4,366,241, U.S. 4,843,000, and U.S. 4,849,338, all of which are herein incorporated by reference. Enzyme labels useful in the present invention include alkaline phosphatase, horseradish peroxidase, luciferase, β- galactosidase, glucose oxidase, lysozyme, malate dehydrogenase and the like. The enzyme label may be used alone or in combination with a second enzyme in solution. By way of example, the fluorophore may be fluorescein isothiocyanate (FITC), Oregon green, tetramethylrhodamine isothiocyanate (TRITL), allophycocyanin (APC), R-Phycoerythrin (RPE), Cy3 and/or Cy5 although without limitation thereto. In a preferred embodiment, particularly with regard to the isolation and purification of metanephric mesenchyme and/or renal progenitor cells per se from metanephric mesenchyme, one or more antibodies are used in conjunction with a cell isolation technique such as any technique that selects cells (i.e positive selection) or depletes cells (i.e negative selection) according to cell surface protein expression. A non-exhaustive list includes panning, complement-mediated lysis, fluorescence-activated cell sorting (FACS) and magnetic activated cell sorting (MACS). For FACS enrichment, fluorescently-labelled antibodies are bound to the cells of interest. These cells are then passed through the excitation laser in a single cell stream and measured for size, granularity and fluorescent activity. Specific parameters are set and cells that fall within those parameters (e.g. fluorescence, forward light scatter, side scatter) are collected by a cell sorter. For MACS enrichment, monoclonal antibodies coupled to small magnetic particles are bound to the cells of interest. Using a magnet, the bound cells may be enriched from contaminating cells. Alternatively, contaminating cells may be removed with bound beads. Protein based detection of gene expression profiles according to the invention may also utilize immunoassays, for example ELISA, immunohistochemistry or immunoblotting to detect relative expression levels of one or more proteins to determine the stage of metanephric mesenchyme development. With regard to appropriate cell surface protein for use according to the invention, Tables 2 and 3 identify genetic markers that are, or are likely to be, expressed at the cell surface. Referring to Table 4 and Table 5, a preferred group of cell surface markers include Neuropilin-1 (Nrpl); CD164 antigen; CD83 antigen (CD83); Stromal cell derived factor receptor 1 ; CD24a antigen; Serine protease inhibitor, Kunitz type 2 (Spint-2); Tumor-associated calcium signal transducer 1; Receptor-like tyrosine kinase; Fibroblast growth factor receptor 2; Amyloid beta (A4) precursor protein; Bone morphogenetic protein receptor, type 1A; DNA segment, Chr 8, Wayne

State University 49, expressed; Signal sequence receptor, alpha; Junction adhesion molecule 3; PTK7 protein tyrosine kinase 7; Cadherin 11; Syndecan binding protein; Integral membrane protein 2C; Tripartite motif-containing 59; NAD(P) dependent steroid dehydrogenase-like; DNA segment, Chr 3, University of California at Los Angeles 1 ; Solute carrier family 3 (activators of dibasic and neutral amino acid transport), member 2; RIKEN cDNA 1110018G07 gene; Purine rich element binding protein B (purb); Solute carrier family 6 (neurotransmitter transporter, taurine), member 6; Gap junction membrane channel protein alpha 1; Tumor differentially expressed 1; CD81 antigen; Solute carrier family 35, member F5; Solute carrier family 39 (zinc transporter), member

7; Gene rich cluster, C3f gene; Claudin 6; Solute carrier family 20, member 1; Solute carrier family 16 (monocarboxylic acid transporters), member 1; ADP- ribosylation factor-like 6 interacting protein 2; Autocrine motility factor receptor; Claudin 7; Calcitonin receptor-like; Tumor differentially expressed 2; Synaptophysin-like protein; Claudin 11; G protein-coupled receptor 89; ELOVL family member 6, elongation of long chain fatty acids (yeast); Purinergic receptor (family A group 5); Non imprinted in Prader-Willi/Angelman syndrome 2 homolog (human); RIKEN cDNA 4930579A11 gene; Zinc finger, DHHC domain containing 6; Solute carrier family 37 (glycerol-3 -phosphate transporter), member 3; Sarcoma amplified sequence; RIKEN cDNA 1700022N24 gene; and Mid-1- related chloride channel 1, although without limitation thereto, either singly or in combination. In this regard, a gene expression profile particularly associated with renal progenitors and potentially renal stem cells is defined as CD24a⁺cadherin 1 l⁺c-kit ^+/lowSca-l^+/low CD34^". It will be appreciated with reference to Table 6 that CD34, Sca-1 and c-kit are additional cell surface markers that may be used with one or more of the genetic markers described herein that facilitate identification, isolation and/or purification of renal progenitors, inclusive of renal stem cells by virtue of their lack of, or low level of, expression. Antibodies to the aforementioned cell surface markers are readily available commercially, either alone or conjugated to fluorochromes as hereinbefore described. It is also well within the scope of a person skilled in the art to produce antibodies by immunization of a production species (such as rabbits, mice, rats etc) to produce monoclonal or polyclonal antibodies according to standard methods in the art.

Uses of isolated and/or purified renal progenitor cells In light of the foregoing, the skilled addressee will appreciate that the renal progenitor cells and renal stem cells purified according to the invention may find therapeutic use as adjunct therapy in the renal transplant procedures. The skilled person may further appreciate that any source of embryonic or adult stem cell (eg. human embryonic stem cell, neural stem cell, haematopoietic stem cell, mesodermal stem cell) could be induced towards a mesodermal lineage using co-culture with growth factors or conditioned medias. There are a number of mesodermal fates they may adopt, including muscle, bone, cartilage or haematopoietic cells. Hence the expression of the cell markers may find utility in demonstrating which stem cells had adopted or were adopting a renal fate. The skilled person may also appreciate the combination of cell surface markers which together define the renal progenitors allows antibodies to these cell surface markers to be used to enrich, purify and isolate specific subpopulations of renal progenitors. This aspect of the invention provides utility in isolating renal progenitor cells from mixed populations in which some subpopulations adopt a renal fate and others do not. It will be appreciated that the above markers may also be used to identify and then isolate adult renal stem cell or progenitor populations from an adult kidney. It will be appreciated to the skilled person that the isolated progenitor cells may be used in adjunct therapy. Specifically, the progenitor cells may be introduced into the renal parenchyma of the kidney or the renal capsule to elicit repair. They may further be introduced via the generalised or renal vasculature, which may involve injection into the renal artery. Any introduction of renal progenitor cells may be accompanied by an adjunct insult or stress to the kidney, such as mild ischaemia or radiation or other stresses designed to stimulate the receptivity of the kidney. Introduction of renal progenitor cells may include growth factors, cytokine or other agents selected to stimulate the integration and onward differentiation of renal progenitors into the receiving kidney or reduce the rejection of the progenitor cells by the receiving kidney. They may further be used in combination with biomatrices and growth factors to generate a replacement kidney organ de novo. The process of metanephric transplantation, where embryonic kidney is grown in the peritoneal cavity of a recipient animal may utilize methodology as previously described (Dekel et al, 2003, Nat. Med. 9 53-60; Hammerman, 2003, Nephron Exp. Nephrol. 2003, 93 e58; Hammeraian, 2003, Kidney Int. 63 1195-1204; Hammerman, 2000, Pediatr. Nephrol. 14 513-

517). In this regard, reference is also made to International Publication WO2004/090112 which describes examples of methodologies that may be utilized for treating acute renal failure, kidney transplant dysfunction and chronic renal failure by administration of kidney precursor cells to a patient. The present invention provides a particularly advantageous method and set of molecular markers whereby renal progenitors may be isolated and/or purified for use in vivo to treat diseases or conditions such as acute renal failure, kidney transplant dysfunction and/or chronic renal failure. In order that the invention may be readily understood and put into practical effect, particular embodiments will now be described by way of the following non-limiting examples. EXAMPLES The two main goals of this invention were (1) to identify the earliest markers of commitment to renal differentiation and (2) to determine the cell surface molecule phenotype of renal progenitor cells. The microarray expression profile of the uninduced MM from El 0.5 mice was examined by comparison to adjacent rostral IM, comprising the portion of the nephrogenic cord containing the mesonephros and which will give rise to the genital ridge. In this way, markers identifying the MM from UB and surrounding IM at the point of MM commitment were determined. We refer to the committed, undifferentiated MM as the renal progenitor timepoint. EXAMPLE 1 Identification of molecular markers of uninduced mesodermal mesenchyme Materials and Methods Tissue Collection and RNA Isolation Naturally mated outbred female CD1 mice were culled by cervical dislocation (Animal ethics committee approval number IMB/479/02/NIH) at embryonic day (E) 10.5. MM and rostral IM tissue (nephrogenic cord including mesonephros and genital ridge) was dissected from El 0.5 embryos and snap- frozen on dry ice. Embryos were defined as El 0.5 by the presence of between 8 and 10 tail somites. Pooled tissue was stored at -80 °C. Dissections were performed in cold phosphate-buffered saline (PBS). Total RNA was prepared using Trizoll (GibcoBRL) extraction in combination with RNeasy mini kits

(Qiagen).

Expression profiling using NIA microarrays Total RNA was linearly amplified using the messageAMP aRNA kit (Ambion). Briefly, 1000 nanograms of total RNA was reverse transcribed into cDNA using a T7 promoter-dT primer and amplified through an in vitro transcription reaction (12 hours) using T7 RNA polymerase to produce antisense RNA (aRNA). The aRNA was reverse transcribed using random hexamers (Promega) into cDNA incorporating either Cy5- or Cy3-labelled dUTPs (Amersham). Labelled targets were hybridised to microarray chips for 16 hours at 45°C. Arrays were produced by the SRC Microarray Facility, University of Queensland (ARC Centre for Functional and Applied Genomics). Experiments were performed using chips arrayed with the National Institute of Health - National Institute of Aging (NIA) 15K mouse cDNA clone set

(http://lgsun.grc.nia.nih.gov/cDNA/15k.html) in addition to a selection of custom clones submitted from the Institute for Molecular Bioscience (University of Queensland, Brisbane) and contained 15,989 elements in total (SRC NIA v3.0 chips). Every element was spotted in duplicate on each chip. The NIA 15K mouse clone set contained 15,247 expressed sequence tags (ESTs) derived from pre- and peri-implantation embryo, E12.5 female gonad / mesonephros and newborn ovary cDNA libraries (Tanaka et al, 2000, Proc. Natl. Acad. Sci. USA 97 9127-9132) thus making it an ideal gene set for this experiment. 14,428 of these ESTs sequences map to Unigene / TIGR / Ensembl and represent 11834 distinct transcripts after removal of duplicates. Up to 50% of clones were derived from novel genes. Hybridised slides were scanned with a GMS 418 array scanner (Genetic MicroSystems) and images were analysed with Imagene 5.5 (Biodiscovery). The microarray data was analysed with R statistical software using the LIMMA package (http://bioinf.wehi.edu.au/limma/) with scripts developed by Ola Spjuth of the Linnaeus Centre for Bioinformatics

(http://www.lcb.uu.se aseplugins.php). Mean foreground signals were taken for each spot and normalised within each array using print-tip lowess without background correction to give a mean value of zero for the log ratios of the two channels within each print block. The final normalised values were use for B- statistics calculations. B-statistics analysis included an allowance for the correlation between adjacent duplicate spots printed on the same array. A threshold B-score >0 was used to define differential expression. Normalised data were imported into Genespring 6.1 (Silicon Genetics) to generate lists of differentially expressed genes. All microarray data, protocols and lists of differentially expressed genes are available for download from the following website: http://kidney.scgap.org/base/index.phtml.

Expression profiling using Compugen Microarray Analysis Compugen (CGEN) chips contained the Compugen 22K mouse set of 65- mer oligonucleotides (http://www.labonweb.com/chips/libraries.html) and contained 22,329 elements in total. Total RNA was linearly amplified using the Amino Allyl messageAMP aRNA kit (Ambion). Briefly, 1000 nanograms of total RNA was reverse transcribed into cDNA using a T7 promoter-dT primer and amplified through an in vitro transcription reaction (12 hours) using T7 RNA polymerase to produce antisense RNA (aRNA). 5-(3-aminoallyl)-UTP was incorporated into the aRNA during in vitro transcription. A dye-coupling reaction was used to conjugate the amino allyl modified aRNA to mono-reactive NHS esters of either Cy3 or Cy5 moeities (Amersham). Labelled targets were hybridised to microarray chips for 16 hours at 42°C. Each array experiment was repeated in duplicate and included a dye reversal experiment to account for any dye bias. Hybridised slides were scanned with a GMS 418 array scanner (Genetic MicroSystems) and images were analysed with Imagene 5.5 (Biodiscovery). The microarray data was analysed with R statistical software using the LIMMA package (http://bioinf.wehi.edu.au/limma/) with scripts developed by Ola Spjuth of the Linnaeus Centre for Bioinformatics

(http://www.lcb.uu.se/baseplugins.php). Mean foreground signals were taken for each spot and normalised within each array using print-tip lowess without background correction to give a mean value of zero for the log ratios of the two channels within each print block. The final normalised values were used for B- statistics calculations as summarised in Table 3. B-statistics analysis included an allowance for the correlation between adjacent duplicate spots printed on the same array. A threshold B-score >0 was used to define differential expression. Normalised data were imported into Genespring 6.1 (Silicon Genetics) to generate lists of differentially expressed genes. Table 5 shows a subset of Table 3 whereby 35 non-redundant elements with a B-score >0 were shown to have an average increase in differential expression >1.80-fold greater in the uninduced MM compared to the surrounding IM. Bioinformatics and membrane organization predictions Representative sequences for differentially expressed ESTs or oligonucleotides were extracted from the National Centre for Biotechnology Information (NCBI) database (http://www.ncbi.nlm.nih.gov/). Using BLAST (Altshul et al, 1990, J. Mol. Biol., 215 403-410) each NIA EST sequence was mapped to an identical full-length RIKEN representative transcript / protein sequence (RIKEN RTPS 6.3 set). The RIKEN RTPS 6.3 set was recently annotated using a number of bioinformatic approaches (Kanapin et al, 2003, Gen. Res. 13 1335-1344) including the prediction of the membrane organisation of individual full-length proteins into one of six categories based on the presence or absence of endoplasmic reticulum-retention signal peptides and helical transmembrane (Table 1).

Metanephric Culture and Tissue Preparation For RNA in situ hybridisation, embryos were collected from outbred CD1 mice as above at days 10.5, 12.5 and 15.5 of gestation. E10.5 embryos were cut transversely below the forelimbs and longitudinally down the midline to expose the ND, UB and MM. At E12.5, complete urogenital (UG) tracts were collected.

For section RNA in situ analyses, metanephroi from El 5.5 embryos were dissected as above and fixed in 4% paraformaldehyde (PFA) in PBS for 2 hours at 4°C. Tissue was processed and embedded in paraffin. Sections were cut at 7 μm. For explant culture, metanephroi were isolated from El 2.0 embryos and grown as explants for two days at 5% CO2, 37°C on 3.0 μm polycarbonate transwell filters

(Costar) in minimum essential medium (MEM) supplemented with 10% fetal calf serum (FCS) and 20mM glutamine. Tissue for whole mount in situ hybridisation was fixed overnight in 4% PFA / PBS at 4°C. RNA in situ hybridisation Expression patterns were analysed by RNA in situ hybridisation using digoxigenin-labelled sense and antisense riboprobes. Probes were synthesized as described previously (Holmes et al, 1998, Mech. Dev. 79 57-72) using pSPORTl constructs containing the NIA EST of interest (SRC Microarray facility). Probes were not fragmented by hydrolysis and were purified using Sephadex columns (Roche) following digestion of the vector with DNasel (Promega) for 15 minutes at 37°C. Whole mount in situ hybridizations were performed as described by

(Christiansen et al, Mech. Dev. 51 341-350) with minor modifications. All probes were hybridised at 65°C and post-antibody washes reduced to 30 minutes. Tissue was mounted in Mount-Quick aqueous (Daido Sangyo) and photographs were taken using an Olympus AX70 compound microscope with Kodak Elite Ektachrome 160T colour reversal film. Section in situ hybridisations were hybridised at 65°C and post-hybridisation processing was performed using DIG wash and block buffer set (Roche). Sections were mounted in Mount-Quick (Daido Sangyo) and photographs were taken as above. Results Summary of Gene Expression Profile Analysis Until recently, it has been difficult to perform meaningful expression profile studies in embryological systems due to the limiting amount of nucleic acid available from small amounts of tissue. This problem was circumvented in this study by using mRNA linear amplification to produce amplified RNA (aRNA). aRNA produced by in vitro transcription has been shown to have a correlation coefficient >0.95 to total RNA and aRNA produced using different amounts of template total RNA (Luo et al, 1999, Nat. Med. 5 117-122; Baugh et al, Nucl. Acid Res. 25 e29). To minimise nonlinearity and ensure consistency, all samples were amplified under the exact same conditions at the same time. All array experiments were performed in duplicate and included a dye reversal experiment to account for any dye bias and the three arrays were normalised to each other to give a comparable range of log ratios. The scatter and volcano plots of the NIA expression profiling are provided as an example of data quality in Figure 1. The raw data for both NIA and Compugen expression profiling can be accessed via the webmaster at kidney.scgap.org. The skilled person will know that this data can be analysed in R tools, Genespring or other expression profiling software and a list of genes differentially expressed in the

E10.5 MM versus the adjacent IM determined based upon a variety of stringency criteria. Thee criteria might include fold change or statistical score (B-stat) or a combination of the two. Our preferred method of analysis is to take into account both B-stat and fold change. The data listed in Tables 2 and 3 represent a subset of the genes enriched in MM using either the NIA or Compugen gene sets. This includes all genes in which the fold change was greater than 1.8 fold and the B- stat was greater than 0. Both B-stat and fold change are indicated. A total of 26 genes from NIA and 528 genes from Compugen are detailed in Tables 2 and 3. Hence, this set comprises 554 markers. The sequence of each differentially expressed EST or Compugen oligo was mapped to a full-length protein sequence from the RIKEN RTPS 6.3 set and the membrane organisation predictions were adopted for each gene of interest. Of particular interest were candidate genes from class C (type I membrane proteins), class D (type II membrane proteins) and class E (multi-span membrane proteins) for their potential utility in antibody-based fluorescence-activated cell sorting

(FACS) for the purification of renal progenitors. Molecules Determining the Renal Progenitor Phenotype The temporospatial expression patterns of genes in Tables 2-4, most preferably Table 4, were determined using RNA in situ hybridisation (Figures 2- 6). Wholemount RNA in situ analysis (WISH) at E10.5 and E12.5 indicates that a proportion of genes identified using expression profiling were validated as MM- specific using WISH. In some cases, such as Islet 1 and Gata3, this was not shown to be the case. Isletl was shown to be highly expressed in the urogential sinus, suggesting that the differential expression between MM and IM was due to slight contamination of tissue caudal to the MM. In Figure 2, it can be seen that several genes were clearly verified as being expressed within the MM itself, particularly ewing sarcoma homolog (ewsh), tyrosine 3-monooxygenase / tryptophan 5-monooxygenase activation protein, theta polypeptide (14-3-3 theta), retinoic acid receptor alpha (RARα) and stearoyl-coenzyme A desaturase 2 (Scd2). hi explant cultures, 14-3-3 theta and Scd2 showed similar expression patterns being broadly expressed by epithelial segments, RARα was expressed in the renal interstitium, particularly the interstitium at the core of the explant, and ewsh was downregulated and expressed throughout the kidney. These markers may act as good RT-PCR probes to determine the renal potential of differentiating embryonic stem (ES) cell cultures. In Figure 5, a similar WISH verification screen was performed on a subset of genes from the Compugen expreesion profiling. Here it can be particularly seen that Crym, Hoxal ls, CD83, Ogfr, Smoc2, Itm2c, albumin 1, Anapc7, somatostatin and IGF2bpl show differential expression in the El 0.5 MM to varying extents. Of particular note was crystallin-mu (Crym) which showed a very specific expression profile. This gene was very highly and specifically expressed in the

MM at El 0.5 and was localised to cap mesenchyme in metanephric explants. The specific spatiotemporal expression pattern of this gene makes it attractive candidate for the production of transgenic mice to isolate specific cell populations during kidney development that may retain inherent differentiation plasticity. Also of interest was the secreted molecule Smco2 which was highly specific to the UB and MM at El 0.5 and was upregulated in ureteric bud tips in metanephric explants. Other highly verified genes included Ogfr, CD81, CD83, Nrpl, Hoxal Is and Gdnf, although not all are illustrated in the examples. Cell Surface Markers of Renal Progenitor Cells Within the data from both expression profiling platforms, a subset of predicted transmembrane proteins were identified which showed a significant increase in expression in the El 0.5 MM compared to the surrounding IM (Table 4). Although predicted as transmembrane, Scd2 is known to be an intracellular transmembrane protein localised to the endoplasmic reticulum and was not considered as a potential cell surface marker of renal progenitors. The in situ expression patterns of some of these genes were examined in El 0.5 embryo wholemounts to observe expression at the renal progenitor timepoint, El 2.5 urogenital tracts to determine specificity to the renal population and metanephric explants and El 5.5 kidney sections to resolve what renal cell types expressed these markers as development progressed (Figure 3 and 5 (Ogfr and CD83)). This also allowed the identification of genes that were outliers because of expression in the ND. These included claudin-6 and spint-2. Expression of these two genes was very specific to the ND and primordial UB in the El 0.5 embryo and to ND / UB derivatives in the El 2.5 UG tract (spint-2 also showed strong expression in the gonads at E12.5) and metanephric explants. Expression was also observed in the mesonephroi at E 12.5. Although present in other areas of the embryo, expression of CD24a antigen was strikingly specific to the uninduced MM population in the mesoderm of the E10.5 embryo (Figure 3). CD24a was expressed only in the MM and not throughout the nephrogenic cord. CD24a was specifically expressed in the kidneys of the E12.5 UG tract although weak expression was also observed in the

ND at this timepoint, made difficult to see due to the strong expression in the kidneys. In the explant, CD24a was expressed in epithelial cells of both UB and MM lineages, although not in the lower limbs of the S-shaped bodies that give rise to the glomeruli. Cadherin-11 showed expression in the mesoderm at El 0.5, but particularly the renal progenitor population. Expression of cadherin-11 became more widespread at E12.5 but was strongly expressed throughout the renal interstitium of the explant, particularly the cells surrounding the UB tips. The full-length transcript representing the EST BG072301 encodes for a protein for which little information is currently known. There was apparently ubiquitous expression of this molecule across the embryo at E10.5 and E12.5, although the levels of expression were elevated in the El 0.5 MM. In explants and sections, expression was observed in the interstitium but particularly the mesenchymal cells bordering epithelial structures. Using Known Stem Cell Markers to Identify Renal Progenitors As the renal progenitor population may represent potential stem cells, the expression of seven known markers of multipotential and pluripotential stem cell populations in the uninduced MM were analysed (Figure 4). While all of these stem cell markers were represented in the NIA gene set (Table 7), the signal strength was low resulting in B-scores <0 for all but Oct-4, podocalyxin-like (podxl) and nestin. All three of these genes were more strongly expressed in the IM than the MM. The transcription factors Oct-4 and nanog are markers of the pluripotential state. At El 0.5, expression of Oct-4 was restricted to primordial germ cells migrating through the urogenital ridge while nanog expression was observed in a non-specific fashion throughout the embryo, although seemingly higher in the El 0.5 MM. Neither of these genes showed any expression in metanephric explants. These stem cell markers would be unlikely to be of use according to the present invention The somatic stem cell markers CD34, podxl and nestin did not appear to mark the renal progenitor population. CD34 was expressed throughout the forming vasculature of the embryo and metanephric explant, but no expression was detected in El 0.5 MM. The recently identified haemangioblast marker podxl was expressed strongly in the aorta-gonad-mesonephros region at El 0.5, but not in the uninduced MM. In explants, expression of podxl was restricted to presumptive podocytes. The neural progenitor marker nestin was strongly expressed in the ectoderm of the El 0.5 embryo but not in the MM, although expression of nestin was observed in the S-shaped bodies of metanephric explants. A lack of expression of CD34 would be of value to determine a lack of heamatopoietic origin. Of the stem cell markers surveyed in this report, the receptor tyrosine kinase c-kit (CD 117) and the surface glycoprotein stem cell antigen- 1 (sca-1) showed the most potential to act as markers of renal progenitors. At El 0.5, both sca-1 and c-kit were expressed throughout the nephrogenic cord with sca-1 in particular showing an increase in expression in the El 0.5 MM. In metanephric explants, sca-1 was expressed by the primitive nephron tubules of the while c-kit expression was observed throughout the primary renal interstitium. Hence, renal progenitors would show a phenotype that was Oct-4^", nanog^lo/" , nestin^", Sca⁺, c- kit^Iow, CD34^", EXAMPLE 2 Molecular Markers of Renal Cells Methods and Materials

Fluorescent-Activated Cell Sorting 6-8 week old outbred CD1 mice were culled by cervical dislocation (Animal ethics committee approval number IMB/479/02/NIH) for harvest of kidneys and bone marrow. Adults kidneys were harvested into cold HANKS balanced salt solution (Sigma) and trimmed of all excess connective tissue. Femurs and tibias were flushed with cold PBS + 2% FCS using a 27 gauge needle to collect bone marrow. For embryonic kidney tissue collection, naturally mated outbred CD1 females were sacrificed as above at E15.5 and E10.5. Kidneys were dissected from embryos in cold L15 media (Gibco). Tissue was minced into a coarse slurry with scissors and digested in 10 mg/mL collagenase B (Roche), 1.2 U/mL dispase II (Roche), 0.01% DNase type I (Sigma) in HANKS for 20 minutes at 37°C with agitation. The concentration of collagenase was reduced to 1 mg/mL for dissociation of embryonic tissue.

Digested tissue was dissociated further using 23 gauge needles before being passed through a 40 μM cell strainer (BD Falcon). Red blood cells were lysed using Gey's solution and cells were resuspended in pre- warmed DMEM - phenol red (Gibco), 10 mM HEPES, 2% FCS at a concentration of 1 x 10⁶/mL. For hoechst staining, 5 μg/mL hoechst 33342 (Sigma) was added to each sample and incubated at 37°C for 90 minutes under protection from light. A control tube for each sample containing 50 μM verapamil (Sigma) was included in each preparation to set the side population gate. After hoechst staining, cells were washed in PBS + 2% FCS and subsequently stained with fluorescein isothiocyanate (FITC) and phycoerythrin (PE) conjugated antibodies for 20 minutes on ice. The following directly conjugated anti-mouse monoclonal antibodies were used - CD45-FITC (clone 30-F11), CD31-PE (clone MEC13.3), sca-l-PE (clone E13-161.7), c-kit-FITC (clone 2B8) and CD24a-PE (clone Ml/69)(Pharmingen). Non-conjugated CD34 (Zymed), CD24a (Pharmingen) and cadherin-11 (Santa Cruz) primary antibodies were also used which were then subsequently stained with anti-mouse-FITC, anti-rat-FITC and anti-goat-PE secondary antibodies (Sigma) respectively. Finally, 2 μg/mL 7- aminoactinomycin D (7-AAD, Sigma) was added to each sample to identify viable cells. Cells were analysed and sorted on a FACS Vantage SE (Becton Dickinson) with both 488 nm argon (200 mW power) and 365 nm ultra-violet (50 mW power) lasers. FITC and PE were excited with the 488 nm laser and emission signals were detected using 530/30 and 575/25 band pass filters respectively. Hoechst and 7-AAD were excited with the 365 nm laser and emission detected using a 670/40 filter for 7-AAD and 424/44 (blue) and 660/20 (red) filters for hoechst. Compensation was adjusted using samples stained with one fluorochrome only and the side population gate was set using verapamil control samples. Data were acquired using CellQuest software (Becton Dickinson) and analysed with winMDI 2.8. Results One of the major goals of this screen was to identify cell surface markers that may be used to isolate potential renal stem cells based on their similarity to El 0.5 MM. From the NIA microarray analysis, two cell surface molecules displayed expression patterns that make them attractive candidates for this purpose - CD24a and cadherin-11. From the Compugen array analysis, a further 45 cell surface markers of interest were identified which are indicated in Table 3 as non-intracellular C,D and E class genes, with the exception of Spint-2 and claudin 6. Additionally, analysis using NIA chips identified Spint-2 and claudin 6 as good markers for progenitors of the collecting tubules of the kidney, based on their expression in the ureteric bud of the El 0.5 metanephros. Focussing on CD24a and cadherin-11, we have further sought to validate their utility for the identification and isolation of renal progenitors and potentially also the identification and isolation or adult renal stem cells. Referring to Figure. 6, both of these genes were strongly and specifically expressed in the El 0.5 MM although in later metanephric development CD24a is located in all epithelial structures while cadherin-11 is expressed in the primary interstitium. However, both overlap clearly in the cap mesenchyme from E10.5 to around E15.5. Cap mesenchyme is derived from metanephric mesenhyme and surrounds the tips of the advancing ureteric buds, giving rise to all elements of the developing nephrons. Section in situ analysis was conducted to resolve what cell types expressed these genes throughout subsequent stages of kidney development. Cells co-expressing these molecules in the adult kidney may possess a phenotype more similar to that of the renal progenitor population and retain a degree of inherent differentiation capacity. In El 3.5 and El 5.5 kidney sections, CD24a and cadherin-11 expression remains in the structures observed in metanephric explants, namely epithelial nephron segments and the interstitium respectively. In the adult kidney, CD24a is expressed principally in distal convoluted tubules while cadherin-11 expression is also seen in distal tubules and loop of Henle segments. No expression of cadherin-11 is seen in any interstitial cell population in the adult. Immunophenotyping was done by FACS analysis to determine the proportion of renal cells, if any, which retained expression of both CD24a and cadherin-11 during development (FIG. 7). Three timepoints were analysed, the El 0.5 MM, El 5.5 metanephroi and adult kidneys. As anticipated, the proportion of CD24a⁺cad-ll⁺ cells in the kidney decreased throughout development, dropping from 16.22% of the total cell population at E10.5 to 8.13% at E15.5 to 4.39% in the adult. Specific markers of renal progenitor cells that continue to be expressed in a stem cell population should decrease in abundance as the kidney develops and differentiates and the progenitor pool becomes depleted. Of interest with regard to CD24a marking a potential stem cell population in the kidney, this molecule was shown to be strongly expressed by kidney side population cells. Side population cells are a specific subpopulation of cells isolated by FACS on the basis of their ability to rapidly efflux the vital dye Hoechst 33342 that have been shown to be highly enriched for stem cells from a number of organs (Goodell MA.Multipotential stem cells and 'side population' cells. Cytotherapy. 2002;4(6):507-8). The SP represent approximately 0.1-0.2% of the total cell population from El 5.5 and adult kidneys and approximately 91% and 67% of these embryonic and adult kidney SP cells respectively express CD24a when overlayed on isotype matched controls (FIG. 8). The observation that CD24a has been identified as a potential renal stem cell marker by two independent experiments (microarray analysis at the renal progenitor timepoint, FACS analysis of kidney side population cells) enhances the likelihood that this molecule may mark a renal stem cell population. Discussion Of the genes identified as enriched in the uninduced MM from microarray analysis, CD24a antigen and cadherin-11 appear to be the best candidates for renal progenitor cell surface markers. CD24a was strongly and specifically expressed in all uninduced MM cells at E10.5 while cadherin-11 is also strongly expressed by this population. Although these molecules both appear to mark the renal progenitor population, their expression patterns diverged greatly as kidney development progressed. In metanephric explants, CD24a expression was observed in all epithelial structures of the developing kidney except for the lower limbs of the S-shaped bodies while cadherin-11 was expressed by mesenchymal cells of the renal interstitium, most strongly by those surrounding the UB tips, but not in epithelial cells. The fact that CD24a marks cell types of both MM and UB derivatives suggests that it identifies renal progenitors committed to differentiating into epithelial segments of the nephron while cadherin- 11 may identify MM cells destined to form the renal interstitium. If individual cells of the uninduced MM co-expressed CD24a and cadherin-11, they may represent a renal stem cell population that has the ability to form all differentiated cell types found in the mature organ. There is some indirect evidence to suggest that CD24a may mark a renal stem cell population. The human ortholog, CD24, is strongly expressed in Wilms' tumours (Droz et al, 190, Hum. Pathol. 21 536-544) and renal cell carcinomas

(Droz et al, 1990, Am. J. Pathol. 137 895-905). Expression of human CD24 by renal tumour cells may indicate these cells are reverting to a more primitive or embryonic state, a condition analogous to the undifferentiated MM at E10.5. CD24a / CD24 may represent a marker of renal progenitor cells conserved between murine and human systems but its expression is not restricted to the uninduced MM and it will be necessary to use other markers in combination with CD24a to specifically purify renal progenitors. CD24a⁺ was successfully used to isolate cells from murine embryonic and adult kidneys by FACS, demonstrating the utility of the cell surface markers in this invention. Several other markers within the high priority list provided, including CD83, CD81 and CD 164, have associations with haematopoietic progenitors. CD83 is a marker of dendritic cells (Lechmann et al, 2002, Trends Immunol.

2002, 23 273-5). CD164 and CD81 are enriched in a population of bone marrow derived cells with multi-lineage potential (MIAMI cells) (DTppolito et al, 2004, J. Cell Sci. 1172971-81). While claudin-6 and spint-2 showed tremendous specificity of expression in the ND and UB, the UB has a much smaller differentiation spectrum than the

MM and is not likely to be the source of a stem cell population. However, the existence of a single nephrogenic progenitor is not clear because it is uncertain whether all epithelial cell types in the adult kidney can be derived from a single precursor cell or whether each cell type has its own precursors (Al Awqati & Oliver, 2002, supra). Therefore, cellular therapy of kidney diseases may require isolation of two distinct progenitor populations, one from the MM and one from the UB, in which case these markers would prove useful. The distinct expression of common stem cell markers in the uninduced MM at El 0.5 was not detected by microarray analysis or in situ hybridisation. The pluripotency markers Oct-4 and nanog were not observed in the uninduced

MM or metanpehric explants which is as expected from a progenitor population restricted to mesodermal differentiation. Of the somatic stem cell markers surveyed, c-kit and sca-1 were expressed in the El 0.5 MM but also throughout the nephrogenic cord, the tissue that gives rise to all three mammalian excretory entities. These cell surface proteins have traditionally been used to identify various lineages of bone-marrow derived stem cells (Ma et al, 2002, Br. J. Haematol. 116 401-408; Meirelles et al, 2003, Br. J. Haematol. 123 702- 711);,but more recently have also been used to isolate stem cells from other organs such as muscle (Howell et al, 2003, Ann. NY Acad. Sci. 996 158-173) testis (Kubota et al, 2003, Proc. Natl. Acad. Sci. USA 100 6487-6492) and mammary gland (Welm et al, 2002, 245 42-56). In metanephric explants, sca-1 was expressed by the primitive tubules of the nephrons. Recent studies with BrdU and label-retaining cells in the kidneys have demonstrated slow-cycling cells able to respond to kidney damage by proliferation located in these tubular structures in mature kidneys (Maeshima et al, 2003, J. Am. Scoc. Nephrol. 14 3138-3146), demonstrating the possible persistence of progenitor-like cells in this niche. C-kit expression was observed throughout the primary renal interstitium of the explant which is also of interest as certain interstitial cells of the adult kidney have been suggested to possess the ability to undergo a mesenchymal-epithelial transdifferentiation to replace injured tubular cells (Nadasdy et al, 199 A, Hum Pathol. 25 22-28). Gene knockout models have implied crucial roles for molecules such as WT-1 (Kreidberg et al, 1993, Cell, supra), Liml (Fujii et al, 1994, Dev. Dyn. supra), Eyal (Stuart et al, 2003, supra) and Pax2 (Torres et al, 1995, supra) in early metanephric development. Expression of these genes are thought to be some of the earliest signs of commitment of the metanephric mesenchyme to a renal fate. Many of these genes were not represented in the NIA clone set and some genes that were present did not produce a signal strength that would allow a reliable determination of differential expression to be made, resulting in a B-score <0. However, two genes involved in early metanephric development, the transcription factor WT-1 and the secreted morphogen wnt-4, showed higher expression in the IM compared to the MM in microarrays (fold change just below 1.80-fold threshold), findings confirmed by in situ hybridisation (results not shown). This highlights the fact that a comparison between IM and MM at El 0.5 essentially represents a spatial comparison between mesonephros / presumptive genital ridge and MM. Hence, some genes that play important roles in metanephric development will initially be expressed to a higher degree in the mesonephros / genital ridge than the MM. In summary, for the first time we have catalogued the expression profile of the MM at the point of commitment to a metanephric lineage. Using both the NIA cDNA and Compugen long oligonucleotide clone sets, this analysis has identified markers (Tables 2 and 3) as molecules highly expressed by renal progenitors which could be used as a set of markers for renal progenitor identification and isolation. A high priority subset of these markers, shown in Table 4 have been verified to display enriched expression in the region of the El 0.5 murine MM. This high priority list includesδ cell surface markers including those genes in Table 4 with a predicted membanre organization of Class C, D or E (excluding those known to reside on within an intracellular membrane). These 49 cell surface markers, comprising (all C,D and E genes in Table 4 other than Spint- 2, claudin 6 and the 2 or 3 marked as intracellular), could be used to isolate, enrich or purify renal progenitors at this timepoint or from stem cells induced to adopt a renal progenitor phenotype in vivo or in culture. Spint2 and claudin 6 mark the primitive ureteric bud rather than the MM and would therefore be useful in the isolation of prospective progenitors of the renal collecting ducts. By distinguishing some of the earliest genes expressed by the uninduced MM, this invention has not only identified novel molecules involved in metanephric development, but provided tools for the RT-PCR-based identification of ES cells adopting a renal fate. While the existence of an adult renal stem cell has not been established, recent discoveries in stem cell biology suggest that they might exist, although no markers for renal stem or progenitor cells had been discovered, prohibiting the identification and isolation of such cells. By defining a combination of cell surface proteins that specifically mark the renal progenitor population, this invention will facilitate purification of cells with this phenotype from mixed populations, such as kidneys at various stages of development or differentiating ES cell cultures, using antibody-based FACS. An isolation protocol based upon these renal progenitor / metanephric mesenchyme enriched cell surface proteins, together with the information on known stem cell markers that lie on the cell surface, renal progenitors would preferably show a phenotype that was CD24a⁺, cadherin l l⁺c-kit ^+/lowSca-l^+/low CD34^" It will be appreciated by the skilled person that the present invention is not limited to the embodiments described in detail herein, and that a variety of other embodiments may be contemplated which are nevertheless consistent with the broad spirit and scope of the invention. Table 1

a Membrane organization class b Presence of a signal peptide c Presence of a transmembrane domain

Table 2

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Claims

1. A method of identifying a gene expression profile associated with metanephric mesenchyme development, said method including the step of identifying one or more genes that are differentially expressed at least 1.8 fold by one or more metanephric mesenchyme cells at a particular stage of embryonic development compared to one or more intermediate mesoderm cells.

2. The method of Claim 1, wherein gene expression is determined according to nucleic acid expression.

3. The method of Claim 2, wherein nucleic acid expression is mRNA expression.

4. The method of Claim 1, wherein nucleic acid expression is determined using a nucleic acid array.

5. The method of Claim 1, wherein gene expression is determined according to protein expression.

6. The method of Claim 5, wherein protein expression is measured using a protein array.

7. The method of Claim 1, wherein the one or more metanephric mesenchyme cells are human cells.

8. The method of Claim 1, wherein the gene expression profile comprises one or more genetic markers selected from the group consisting of: Zinc finger protein 335; Ewing sarcoma homolog; t-complex protein 1; enolase 1, alpha non- neuron; tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, theta polypeptide (CDK5 regulatory subunit associated protein 2); Cytoplasmic FMR1 interacting protein 1; Sine oculis-related homeobox 2 homolog (Drosophila); Minichromosome maintenance deficient 7 (S. cerevisiae);

Karyopherin (importin) alpha 2; Heat shock protein 8; Ras-GTPase-activating protein SH3-domain binding protein; Homeo box AlO; Crystallin, mu; RIKEN cDNA 2610312E17 gene; Opioid growth factor receptor; Retinoic acid receptor alpha (RARα); Glial cell line derived neurotrophic factor; Mesoderm development candiate 2; RIKEN cDNA 1300010F03 gene; RIKEN cDNA

2810037C14 gene; Neuropilin-1; CD164 antigen; CD83 antigen; Stromal cell derived factor receptor 1; CD24a antigen; Serine protease inhibitor, Kunitz type 2; Tumor-associated calcium signal transducer 1; Receptor-like tyrosine kinase; Fibroblast growth factor receptor 2; Amyloid beta (A4) precursor protein; Bone morphogenetic protein receptor, type 1A; DNA segment, Chr 8, Wayne State University 49, expressed; Signal sequence receptor, alpha; Junction adhesion molecule 3; PTK7 protein tyrosine kinase 7; Cadherin 11; Syndecan binding protein; Integral membrane protein 2C; Tripartite motif-containing 59; NAD(P) dependent steroid dehydrogenase-like; Vesicle-associated membrane protein 3; DNA segment, Chr 3, University of California at Los Angeles 1; Solute carrier family 3 (activators of dibasic and neutral amino acid transport), member 2;

RIKEN cDNA 1110018G07 gene; Purine rich element binding protein B; Solute carrier family 6 (neurotransmitter transporter, taurine), member 6; Gap junction membrane channel protein alpha 1; Tumor differentially expressed 1; CD81 antigen; Solute carrier family 35, member F5; Solute carrier family 39 (zinc transporter), member 7; Gene rich cluster, C3f gene; Claudin 6; Solute carrier family 20, member 1; Solute carrier family 16 (monocarboxylic acid transporters), member 1; ADP-ribosylation factor-like 6 interacting protein 2; Autocrine motility factor receptor; Claudin 7; Calcitonin receptor-like; Tumor differentially expressed 2; Synaptophysin-like protein; Claudin 11; G protein- coupled receptor 89; ELOVL family member 6, elongation of long chain fatty acids (yeast); Purinergic receptor (family A group 5); Non imprinted in Prader- Willi/Angelman syndrome 2 homolog (human); RIKEN cDNA 4930579A11 gene; RIKEN cDNA 2610311119 gene; Zinc fmger, DHHC domain containing 6; Solute carrier family 37 (glycerol-3 -phosphate transporter), member 3; Sarcoma amplified sequence; RIKEN cDNA 1700022N24 gene; Mid-1-related chloride channel 1; Stearoyl-Coenzyme A desaturase 2; RIKEN cDNA 1110034A24 gene; Homeo box D13; retinol dehydrogenase 10 (all-trans) (RdhlO); Sal-like 4 (Drosophila); Homeo box Al l, opposite strand transcript; and Roundabout homolog 2 (Drosophila).

9. The method of Claim 8, wherein the gene expression profile comprises one or more cell surface markers selected from the group consisting of: Neuropilin-1; CD 164 antigen; CD83 antigen; Stromal cell derived factor receptor 1; CD24a antigen; Serine protease inhibitor, Kunitz type 2; Tumor-associated calcium signal transducer 1; Receptor-like tyrosine kinase; Fibroblast growth factor receptor 2; Amyloid beta (A4) precursor protein; Bone morphogenetic protein receptor, type 1A; DNA segment, Chr 8, Wayne State University 49, expressed; Signal sequence receptor, alpha; Junction adhesion molecule 3; PTK7 protein tyrosine kinase 7; Cadherin 11; Syndecan binding protein; Integral membrane protein 2C; Tripartite motif-containing 59; NAD(P) dependent steroid dehydrogenase-like; DNA segment, Chr 3, University of California at Los Angeles 1 ; Solute carrier family 3 (activators of dibasic and neutral amino acid transport), member 2; RIKEN cDNA 1110018G07 gene; Purine rich element binding protein B; Solute carrier family 6 (neurotransmitter transporter, taurine), member 6; Gap junction membrane channel protein alpha 1; Tumor differentially expressed 1; CD81 antigen; Solute carrier family 35, member 5; Solute carrier family 39 (zinc transporter), member 7; Gene rich cluster, C3f gene; Claudin 6; Solute carrier family 20, member 1; Solute carrier family 16 (monocarboxylic acid transporters), member 1; ADP-ribosylation factor-like 6 interacting protein 2; Autocrine motility factor receptor; Claudin 7; Calcitonin receptor-like; Tumor differentially expressed 2; Synaptophysin-like protein; Claudin 11; G protein- coupled receptor 89; ELOVL family member 6 (Elovl6), elongation of long chain fatty acids (yeast); Purinergic receptor (family A group 5); Non imprinted in

Prader-Willi/Angelman syndrome 2 homolog (human); RIKEN cDNA 4930579A11 gene; Zinc finger, DHHC domain containing 6; Solute carrier family 37 (glycerol-3-phosphate transporter), member 3; Sarcoma amplified sequence; RIKEN cDNA 1700022N24 gene; and Mid- 1 -related chloride channel 1.

10. A method of identifying a metanephric mesenchyme cell, said method including the step of determining a gene expression profile of said metanephric mesenchyme cell, wherein said gene expression profile comprises one or more genetic markers that are differentially expressed at least 1.8 fold by one or more metanephric mesenchyme cells compared to one or more intermediate mesoderm cells.

11. The method of Claim 10, wherein the gene expression profile is determined according to nucleic acid expression.

12. The method of Claim 11, wherein nucleic acid expression is mRNA expression.

13. The method of Claim 10, wherein the gene expression profile comprises one or more genetic markers selected from the group consisting of: Zinc finger protein 335; Ewing sarcoma homolog; t-complex protein 1; enolase 1, alpha non- neuron; tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, theta polypeptide (CDK5 regulatory subunit associated protein 2); Cytoplasmic FMR1 interacting protein 1; Sine oculis-related homeobox 2 homolog (Drosophila); Minichromosome maintenance deficient 7 (S. cerevisiae); Karyopherin (importin) alpha 2; Heat shock protein 8; Ras-GTPase-activating protein SH3-domain binding protein; Homeo box AlO; Crystallin, mu; RIKEN cDNA 2610312E17 gene; Opioid growth factor receptor; Retinoic acid receptor alpha (RARα); Glial cell line derived neurotrophic factor; Mesoderm development candiate 2; RIKEN cDNA 1300010F03 gene; RIKEN cDNA 2810037C14 gene; Neuropilin-1; CD164 antigen; CD83 antigen; Stromal cell derived factor receptor 1 ; CD24a antigen; Serine protease inhibitor, Kunitz type 2; Tumor-associated calcium signal transducer 1; Receptor-like tyrosine kinase; Fibroblast growth factor receptor 2; Amyloid beta (A4) precursor protein; Bone morphogenetic protein receptor, type 1A; DNA segment, Chr 8, Wayne State University 49, expressed; Signal sequence receptor, alpha; Junction adhesion molecule 3; PTK7 protein tyrosine kinase 7; Cadherin 11; Syndecan binding protein; Integral membrane protein 2C; Tripartite motif-containing 59; NAD(P) dependent steroid dehydrogenase-like; Nesicle-associated membrane protein 3;

DΝA segment, Chr 3, University of California at Los Angeles 1; Solute carrier family 3 (activators of dibasic and neutral amino acid transport), member 2; RIKEΝ cDΝA 1110018G07 gene; Purine rich element binding protein B; Solute carrier family 6 (neurotransmitter transporter, taurine), member 6; Gap junction membrane channel protein alpha 1; Tumor differentially expressed 1; CD 81 antigen; Solute carrier family 35, member F5; Solute carrier family 39 (zinc transporter), member 7; Gene rich cluster, C3f gene; Claudin 6; Solute carrier family 20, member 1; Solute carrier family 16 (monocarboxylic acid transporters), member 1; ADP-ribosylation factor-like 6 interacting protein 2; Autocrine motility factor receptor; Claudin 7; Calcitonin receptor-like; Tumor differentially expressed 2; Synaptophysin-like protein; Claudin 11; G protein- coupled receptor 89; ELOVL family member 6, elongation of long chain fatty acids (yeast); Purinergic receptor (family A group 5); Non imprinted in Prader- Willi/Angelman syndrome 2 homolog (human); RIKEN cDNA 4930579A11 gene; RIKEN cDNA 2610311119 gene; Zinc finger, DHHC domain containing 6; Solute carrier family 37 (glycerol-3-phosphate transporter), member 3; Sarcoma amplified sequence; RIKEN cDNA 1700022N24 gene; Mid- 1 -related chloride channel 1; Stearoyl-Coenzyme A desaturase 2; RIKEN cDNA 1110034A24 gene; Homeo box D13; retinol dehydrogenase 10 (all-trans) (RdhlO); Sal-like 4 (Drosophila); Homeo box Al l, opposite strand transcript; and Roundabout homolog 2 (Drosophila).

14. The method of Claim 10, wherein gene expression is determined according to protein expression.

15. The method of Claim 14, wherein protein expression is determined according to one or more cell surface markers.

16. The method of Claim 15, wherein the cell surface markers are selected from the group consisting of: Neuropilin-1; CD 164 antigen; CD83 antigen; Stromal cell derived factor receptor 1 ; CD24a antigen; Serine protease inhibitor, Kunitz type 2; Tumor-associated calcium signal transducer 1; Receptor-like tyrosine kinase; Fibroblast growth factor receptor 2; Amyloid beta (A4) precursor protein; Bone morphogenetic protein receptor, type 1A; DNA segment, Chr 8,

Wayne State University 49, expressed; Signal sequence receptor, alpha; Junction adhesion molecule 3; PTK7 protein tyrosine kinase 7; Cadherin 11; Syndecan binding protein; Integral membrane protein 2C; Tripartite motif-containing 59; NAD(P) dependent steroid dehydrogenase-like; DNA segment, Chr 3, University of California at Los Angeles 1; Solute carrier family 3 (activators of dibasic and neutral amino acid transport), member 2; RIKEN cDNA 1110018G07 gene; Purine rich element binding protein B; Solute carrier family 6 (neurotransmitter transporter, taurine), member 6; Gap junction membrane channel protein alpha 1; Tumor differentially expressed 1; CD81 antigen; Solute carrier family 35, member 5; Solute carrier family 39 (zinc transporter), member 7; Gene rich cluster, C3f gene; Claudin 6; Solute carrier family 20, member 1; Solute carrier family 16 (monocarboxylic acid transporters), member 1; ADP-ribosylation factor-like 6 interacting protein 2; Autocrine motility factor receptor; Claudin 7; Calcitonin receptor-like; Tumor differentially expressed 2; Synaptophysin-like protein; Claudin 11; G protein-coupled receptor 89; ELONL family member 6 (Elovl6), elongation of long chain fatty acids (yeast); Purinergic receptor (family A group 5); Νon imprinted in Prader-Willi/Angelman syndrome 2 homolog

(human); RIKEΝ cDΝA 4930579A11 gene; Zinc finger, DHHC domain containing 6; Solute carrier family 37 (glycerol-3 -phosphate transporter), member 3; Sarcoma amplified sequence; RIKEΝ cDΝA 1700022Ν24 gene; and Mid-1- related chloride channel 1.

17. The method of Claim 10, wherein the metanephric mesenchyme cell is a human cell.

18. The method of Claim 10, wherein the gene expression profile is identified according to Claim 1.

19. A method of isolating or purifying one or more metanephric mesenchyme cells including the steps of: (i) identifying one or more metanephric mesenchyme cells according to a gene expression profile that comprises one or more genetic markers that are differentially expressed at least 1.8 fold by said one or more metanephric mesenchyme cells compared to one or more intermediate mesoderm cells; and (ii) isolating or purifying said one or more cells.

20. The method of Claim 19, wherein gene expression is determined according to protein expression.

21. The method of Claim 20, wherein protein expression is determined according to one or more cell surface markers.

22. The method of Claim 21, wherein the cell surface markers are selected from the group consisting of: Neuropilin-1; CD 164 antigen; CD83 antigen; Stromal cell derived factor receptor 1; CD24a antigen; Serine protease inhibitor, Kunitz type 2; Tumor-associated calcium signal transducer 1; Receptor-like tyrosine kinase; Fibroblast growth factor receptor 2; Amyloid beta (A4) precursor protein; Bone morphogenetic protein receptor, type 1A; DNA segment, Chr 8, Wayne State University 49, expressed; Signal sequence receptor, alpha; Junction adhesion molecule 3; PTK7 protein tyrosine kinase 7; Cadherin 11; Syndecan binding protein; Integral membrane protein 2C; Tripartite motif-containing 59; NAD(P) dependent steroid dehydrogenase-like; DNA segment, Chr 3, University of California at Los Angeles 1; Solute carrier family 3 (activators of dibasic and neutral amino acid transport), member 2; RIKEN cDNA 1110018G07 gene; Purine rich element binding protein B; Solute carrier family 6 (neurotransmitter transporter, taurine), member 6; Gap junction membrane channel protein alpha 1; Tumor differentially expressed 1; CD81 antigen; Solute carrier family 35, member 5; Solute carrier family 39 (zinc transporter), member 7; Gene rich cluster, C3f gene; Claudin 6; Solute carrier family 20, member 1; Solute carrier family 16 (monocarboxylic acid transporters), member 1; ADP-ribosylation factor-like 6 interacting protein 2; Autocrine motility factor receptor; Claudin 7; Calcitonin receptor-like; Tumor differentially expressed 2; Synaptophysin-like protein; Claudin 11; G protein-coupled receptor 89; ELOVL family member 6 (Elovl6), elongation of long chain fatty acids (yeast); Purinergic receptor (family A group 5); Non imprinted in Prader-Willi/Angelman syndrome 2 homolog

(human); RIKEN cDNA 4930579A11 gene; Zinc finger, DHHC domain containing 6; Solute carrier family 37 (gly cerol-3 -phosphate transporter), member 3; Sarcoma amplified sequence; RIKEN cDNA 1700022N24 gene; and Mid-1- related chloride channel 1.

23. The method of Claim 19, wherein the metanephric mesenchyme cells are human cells.

24. A method of identifying a gene expression profile of a renal progenitor cell, said method including the step of identifying one or more genes that are differentially expressed at least 1.8 fold by said renal progenitor cell compared to an intermediate mesenchyme cell.

25. The method of Claim 24, wherein the gene expression profile is determined according to nucleic acid expression.

26. The method of Claim 25, wherein nucleic acid expression is mRNA expression.

27. The method of Claim 26, wherein nucleic acid expression is measured by a nucleic acid array.

28. The method of Claim 24, wherein gene expression is determined according to protein expression.

29. The method of Claim 28, wherein protein expression is measured using a protein array.

30. The method of Claim 24, wherein the gene expression profile comprises one or more genetic markers selected from the group consisting of: Zinc finger protein 335; Ewing sarcoma homolog; t-complex protein 1; enolase 1, alpha non- neuron; tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, theta polypeptide (CDK5 regulatory subunit associated protein 2); Cytoplasmic FMR1 interacting protein 1; Sine oculis-related homeobox 2 homolog (Drosophila); Minichromosome maintenance deficient 7 (S. cerevisiae);

Karyopherin (importin) alpha 2; Heat shock protein 8; Ras-GTPase-activating protein SH3-domain binding protein; Homeo box AlO; Crystallin, mu; RIKEN cDNA 2610312E17 gene; Opioid growth factor receptor; Retinoic acid receptor alpha (RARα); Glial cell line derived neurotrophic factor; Mesoderm development candiate 2; RLKEN cDNA 1300010F03 gene; RIKEN cDNA

2810037C14 gene; Neuroρilin-1; CD164 antigen; CD83 antigen; Stromal cell derived factor receptor 1; CD24a antigen; Serine protease inhibitor, Kunitz type 2; Tumor-associated calcium signal transducer 1; Receptor-like tyrosine kinase; Fibroblast growth factor receptor 2; Amyloid beta (A4) precursor protein; Bone morphogenetic protein receptor, type 1A; DNA segment, Chr 8, Wayne State

University 49, expressed; Signal sequence receptor, alpha; Junction adhesion molecule 3; PTK7 protein tyrosine kinase 7; Cadherin 11; Syndecan binding protein; Integral membrane protein 2C; Tripartite motif-containing 59; NAD(P) dependent steroid dehydrogenase-like; Vesicle-associated membrane protein 3; DNA segment, Chr 3, University of California at Los Angeles 1; Solute carrier family 3 (activators of dibasic and neutral amino acid transport), member 2; RIKEN cDNA 1110018G07 gene; Purine rich element binding protein B; Solute carrier family 6 (neurotransmitter transporter, taurine), member 6; Gap junction membrane channel protein alpha 1; Tumor differentially expressed 1; CD81 antigen; Solute carrier family 35, member F5; Solute carrier family 39 (zinc transporter), member 7; Gene rich cluster, C3f gene; Claudin 6; Solute carrier family 20, member 1; Solute carrier family 16 (monocarboxylic acid transporters), member 1; ADP-ribosylation factor-like 6 interacting protein 2; Autocrine motility factor receptor; Claudin 7; Calcitonin receptor-like; Tumor differentially expressed 2; Synaptophysin-like protein; Claudin 11; G protein- coupled receptor 89; ELONL family member 6, elongation of long chain fatty acids (yeast); Purinergic receptor (family A group 5); on imprinted in Prader- Willi/Angelman syndrome 2 homolog (human); RIKEΝ cDΝA 4930579A11 gene; RIKEΝ cDΝA 2610311119 gene; Zinc finger, DHHC domain containing 6; Solute carrier family 37 (glycerol-3-phosphate transporter), member 3; Sarcoma amplified sequence; RIKEΝ cDΝA 1700022Ν24 gene; Mid-1-related chloride channel 1; Stearoyl-Coenzyme A desaturase 2; RIKEN cDNA 1110034A24 gene; Homeo box D13; retinol dehydrogenase 10 (all-trans) (RdhlO); Sal-like 4 (Drosophila); Homeo box Al l, opposite strand transcript; and Roundabout homolog 2 (Drosophila).

31. The method of Claim 30, wherein protein expression is determined according to one or more cell surface markers.

32. The method of Claim 31, wherein the cell surface markers are selected from the group consisting of: Neuropilin-1; CD 164 antigen; CD83 antigen; Stromal cell derived factor receptor 1 ; CD24a antigen; Serine protease inhibitor, Kunitz type 2; Tumor-associated calcium signal transducer 1; Receptor-like tyrosine kinase; Fibroblast growth factor receptor 2; Amyloid beta (A4) precursor protein; Bone morphogenetic protein receptor, type 1A; DNA segment, Chr 8, Wayne State University 49, expressed; Signal sequence receptor, alpha; Junction adhesion molecule 3; PTK7 protein tyrosine kinase 7; Cadherin 11; Syndecan binding protein; Integral membrane protein 2C; Tripartite motif-containing 59;

NAD(P) dependent steroid dehydrogenase-like; DNA segment, Chr 3, University of California at Los Angeles 1 ; Solute carrier family 3 (activators of dibasic and neutral amino acid transport), member 2; RIKEN cDNA 1110018G07 gene; Purine rich element binding protein B; Solute carrier family 6 (neurotransmitter transporter, taurine), member 6; Gap junction membrane channel protein alpha 1 ; Tumor differentially expressed 1; CD81 antigen; Solute carrier family 35, member 5; Solute carrier family 39 (zinc transporter), member 7; Gene rich cluster, C3f gene; Claudin 6; Solute carrier family 20, member 1; Solute carrier family 16 (monocarboxylic acid transporters), member 1; ADP-ribosylation factor-like 6 interacting protein 2; Autocrine motility factor receptor; Claudin 7; Calcitonin receptor-like; Tumor differentially expressed 2; Synaptophysin-like protein; Claudin 11; G protein-coupled receptor 89; ELONL family member 6

(Elovl6), elongation of long chain fatty acids (yeast); Purinergic receptor (family A group 5); Νon imprinted in Prader-Willi/Angelman syndrome 2 homolog (human); RIKEΝ cDΝA 4930579A11 gene; Zinc finger, DHHC domain containing 6; Solute carrier family 37 (glycerol-3 -phosphate transporter), member 3; Sarcoma amplified sequence; RIKEΝ cDΝA 1700022Ν24 gene; and Mid-1- related chloride channel 1.

33. The method of Claim 32, wherein the cell surface markers are selected from the group consisting of: CD24a; Cadherin 11; and CD83.

34. The method of Claim 33, wherein the gene expression profile is defined as CD24a⁺cadherin 1 lVkit ^+/lowSca-l^{+ low} CD34^".

35. The method of Claim 24, wherein the renal progenitor cell is a human cell.

36. The method of Claim 24, wherein the renal progenitor cell is a stem cell.

37. A method of identifying a renal progenitor cell, said method including the step of determining a gene expression profile of said renal progenitor cell, wherein the gene expression profile comprises one or more genetic markers differentially expressed at least 1.8 fold compared to an intermediate mesenchyme cell.

38. The method of Claim 37, wherein the gene expression profile is determined according to nucleic acid expression.

39. The method of Claim 38, wherein nucleic acid expression is mRNA expression.

40. The method of Claim 37, wherein the gene expression profile comprises one or more genetic markers selected from the group consisting of: Zinc finger protein 335; Ewing sarcoma homolog; t-complex protein 1; enolase 1, alpha non- neuron; tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, theta polypeptide (CDK5 regulatory subunit associated protein 2);

Cytoplasmic FMR1 interacting protein 1; Sine oculis-related homeobox 2 homolog (Drosophila); Minichromosome maintenance deficient 7 (S. cerevisiae); Karyopherin (importin) alpha 2; Heat shock protein 8; Ras-GTPase-activating protein SH3-domain binding protein; Homeo box AlO; Crystallin, mu; RIKEN cDNA 2610312E17 gene; Opioid growth factor receptor; Retinoic acid receptor alpha (RARα); Glial cell line derived neurotrophic factor; Mesoderm development candiate 2; RIKEN cDNA 1300010F03 gene; RIKEN cDNA 2810037C14 gene; Neuroρilin-1; CD164 antigen; CD83 antigen; Stromal cell derived factor receptor 1 ; CD24a antigen; Serine protease inhibitor, Kunitz type 2; Tumor-associated calcium signal transducer 1; Receptor-like tyrosine kinase;

Fibroblast growth factor receptor 2; Amyloid beta (A4) precursor protein; Bone morphogenetic protein receptor, type 1A; DNA segment, Chr 8, Wayne State University 49, expressed; Signal sequence receptor, alpha; Junction adhesion molecule 3; PTK7 protein tyrosine kinase 7; Cadherin 11; Syndecan binding protein; Integral membrane protein 2C; Tripartite motif-containing 59; NAD(P) dependent steroid dehydrogenase-like; Nesicle-associated membrane protein 3; DΝA segment, Chr 3, University of California at Los Angeles 1; Solute carrier family 3 (activators of dibasic and neutral amino acid transport), member 2; RIKEΝ cDΝA 1110018G07 gene; Purine rich element binding protein B; Solute carrier family 6 (neurotransmitter transporter, taurine), member 6; Gap junction membrane channel protein alpha 1; Tumor differentially expressed 1; CD 81 antigen; Solute carrier family 35, member F5; Solute carrier family 39 (zinc transporter), member 7; Gene rich cluster, C3f gene; Claudin 6; Solute carrier family 20, member 1; Solute carrier family 16 (monocarboxylic acid transporters), member 1; ADP-ribosylation factor-like 6 interacting protein 2;

Autocrine motility factor receptor; Claudin 7; Calcitonin receptor-like; Tumor differentially expressed 2; Synaptophysin-like protein; Claudin 11; G protein- coupled receptor 89; ELONL family member 6, elongation of long chain fatty acids (yeast); Purinergic receptor (family A group 5); Νon imprinted in Prader- Willi/Angelman syndrome 2 homolog (human); RIKEΝ cDΝA 4930579A11 gene; RIKEΝ cDΝA 2610311119 gene; Zinc finger, DHHC domain containing 6;

Solute carrier family 37 (glycerol-3-phosphate transporter), member 3; Sarcoma amplified sequence; RIKEΝ cDΝA 1700022Ν24 gene; Mid- 1 -related chloride channel 1; Stearoyl-Coenzyme A desaturase 2; RIKEN cDNA 1110034A24 gene; Homeo box D13; retinol dehydrogenase 10 (all-trans) (RdhlO); Sal-like 4 (Drosophila); Homeo box Al l, opposite strand transcript; and Roundabout homolog 2 (Drosophila).

41. The method of Claim 38, wherein gene expression is determined according to protein expression.

42. The method of Claim 40, wherein protein expression is determined according to one or more cell surface markers.

43. The method of Claim 42, wherein the wherein the cell surface markers are selected from the group consisting of: Neuropilin-1; CD 164 antigen; CD83 antigen; Stromal cell derived factor receptor 1; CD24a antigen; Serine protease inhibitor, Kunitz type 2; Tumor-associated calcium signal transducer 1; Receptor- like tyrosine kinase; Fibroblast growth factor receptor 2; Amyloid beta (A4) precursor protein; Bone morphogenetic protein receptor, type 1 A; DNA segment, Chr 8, Wayne State University 49, expressed; Signal sequence receptor, alpha; Junction adhesion molecule 3; PTK7 protein tyrosine kinase 7; Cadherin 11; Syndecan binding protein; Integral membrane protein 2C; Tripartite motif- containing 59; NAD(P) dependent steroid dehydrogenase-like; DNA segment,

Chr 3, University of California at Los Angeles 1; Solute carrier family 3 (activators of dibasic and neutral amino acid transport), member 2; RIKEN cDNA 1110018G07 gene; Purine rich element binding protein B; Solute carrier family 6 (neurotransmitter transporter, taurine), member 6; Gap junction membrane channel protein alpha 1; Tumor differentially expressed 1; CD81 antigen; Solute carrier family 35, member 5; Solute carrier family 39 (zinc transporter), member 7; Gene rich cluster, C3f gene; Claudin 6; Solute carrier family 20, member 1; Solute carrier family 16 (monocarboxylic acid transporters), member 1; ADP- ribosylation factor-like 6 interacting protein 2; Autocrine motility factor receptor; Claudin 7; Calcitonin receptor-like; Tumor differentially expressed 2; Synaptophysin-like protein; Claudin 11; G protein-coupled receptor 89; ELONL family member 6 (Elovl6), elongation of long chain fatty acids (yeast); Purinergic receptor (family A group 5); Νon imprinted in Prader-Willi/Angelman syndrome 2 homolog (human); RIKEΝ cDΝA 4930579A11 gene; Zinc finger, DHHC domain containing 6; Solute carrier family 37 (glycerol-3 -phosphate transporter), member 3; Sarcoma amplified sequence; RIKEΝ cDΝA 1700022Ν24 gene; and Mid- 1 -related chloride channel 1.

44. The method of Claim 42, wherein the cell surface markers are selected from the group consisting of: CD24a; Cadherin 11; and CD83.

45. The method of Claim 44, wherein the gene expression profile is defined as CD24a⁺cadherin llVkit ^+/1°^wSca-l^{+ Iow} CD34^".

46. The method of Claim 37, wherein the renal progenitor cell is a human cell.

47. The method of Claim 37, wherein the gene expression profile is identified according to Claim 24.

48. A method of isolating or purifying a renal progenitor cell, said method including the steps of: (i) identifying said renal progenitor cell according to a gene expression profile, wherein the gene expression profile comprises one or more genetic markers differentially expressed at least 1.8 fold compared to an intermediate mesenchyme cell; and (ii) isolating or purifying said renal progenitor cell.

49. The method of Claim 48, wherein gene expression is determined according to protein expression.

50. The method of Claim 49, wherein protein expression is determined according to one or more cell surface markers.

51. The method of Claim 49, wherein the cell surface markers are selected from the group consisting of: Neuropilin-1; CD 164 antigen; CD83 antigen;

Stromal cell derived factor receptor 1; CD24a antigen; Serine protease inhibitor, Kunitz type 2; Tumor-associated calcium signal transducer 1; Receptor-like tyrosine kinase; Fibroblast growth factor receptor 2 ; Amyloid beta (A4) precursor protein; Bone morphogenetic protein receptor, type 1 A; DNA segment, Chr 8, Wayne State University 49, expressed; Signal sequence receptor, alpha; Junction adhesion molecule 3; PTK7 protein tyrosine kinase 7; Cadherin 11; Syndecan binding protein; Integral membrane protein 2C; Tripartite motif- containing 59; NAD(P) dependent steroid dehydrogenase-like; DNA segment, Chr 3, University of California at Los Angeles 1; Solute carrier family 3 (activators of dibasic and neutral amino acid transport), member 2; RIKEN cDNA 1110018G07 gene; Purine rich element binding protein B; Solute carrier family 6 (neurotransmitter transporter, taurine), member 6; Gap junction membrane channel protein alpha 1; Tumor differentially expressed 1; CD81 antigen; Solute carrier family 35, member 5; Solute carrier family 39 (zinc transporter), member 7; Gene rich cluster, C3f gene; Claudin 6; Solute carrier family 20, member 1; Solute carrier family 16 (monocarboxylic acid transporters), member 1; ADP- ribosylation factor-like 6 interacting protein 2; Autocrine motility factor receptor;

Claudin 7; Calcitonin receptor-like; Tumor differentially expressed 2; Synaptophysin-like protein; Claudin 11 ; G protein-coupled receptor 89; ELONL family member 6 (Elovl6), elongation of long chain fatty acids (yeast); Purinergic receptor (family A group 5); Νon imprinted in Prader-Willi/Angelman syndrome 2 homolog (human); RIKEΝ cDΝA 4930579A11 gene; Zinc f ger, DHHC domain containing 6; Solute carrier family 37 (glycerol-3 -phosphate transporter), member 3; Sarcoma amplified sequence; RIKEΝ cDΝA 1700022Ν24 gene; and Mid- 1 -related chloride channel 1.

52. The method of Claim 49, wherein the cell surface markers are selected from the group consisting of: CD24a; Cadherin 11; and CD83.

53. The method of Claim 52, wherein the gene expression profile is defined as CD24a⁺cadherin ll⁺c-kit ^{+ low}Sca-l^{+ low} CD34\

54. The method of Claim 48, wherein the renal progenitor cells are human cells.

55. The method of Claim 48, wherein the renal progenitor cells comprise a renal stem cell.

56. Use of metanephric mesenchyme cells isolated or purified according to Claim 19, for in vitro and/or in vivo generation of renal tissue.

57. Use of one or more renal progenitor cells isolated or purified according to Claim 46, for in vitro and/or in vivo generation of renal tissue.

58. Use according to Claim 57, wherein the renal progenitors are introduced i into the renal parenchyma of a human kidney or renal capsule to thereby elicit repair in vivo.

59. Use according to Claim 57, wherein the renal progenitors are used in combination with a biomatrix and/or one or more growth factors to generate a replacement kidney organ in vitro.