CN118222633A

CN118222633A - Method and product for inducing fibroblast to transform and differentiate into tubular epithelial cells

Info

Publication number: CN118222633A
Application number: CN202410452596.2A
Authority: CN
Inventors: 王林辉; 钟涛; 胡雪丽; 孙建建
Original assignee: East China Normal University; First Affiliated Hospital of Naval Military Medical University of PLA
Current assignee: East China Normal University; First Affiliated Hospital of Naval Military Medical University of PLA
Priority date: 2024-04-15
Filing date: 2024-04-15
Publication date: 2024-06-21

Abstract

The present disclosure provides a plasmid, cell, kit and method that can induce the transdifferentiation of fibroblasts into renal epithelial cells, which can greatly improve the efficiency of transdifferentiation of fibroblasts into tubular epithelial cells.

Description

Method and product for inducing fibroblast to transform and differentiate into tubular epithelial cells

Technical Field

The present disclosure relates to the field of cell reprogramming technology, and in particular to a plasmid, cell, kit and method for more conveniently, efficiently and stably inducing the transformation and differentiation of fibroblasts into tubular epithelial cells.

Background

Chronic kidney disease is a major cause of morbidity and mortality worldwide. Current interventions for end-stage renal disease include dialysis and kidney transplantation, but both therapies have limited efficacy or availability, and thus cell therapy may be a potential approach to future treatment of renal disease.

Tubular epithelial cell reprogramming is a potential therapeutic strategy for repairing damaged kidneys and improving fibrotic kidney function, and can convert fibroblasts activated by damaged kidneys into functional tubular epithelial cells, so as to promote recovery of damaged kidney functions. It has been shown that overexpression of Hnf1β, emx2, pax8 and Hnf4a enables the transdifferentiation of fibroblasts into tubular epithelial cells. For example, in prior art CN115851600a, mouse embryonic fibroblasts (Mouse Embryonic Fibroblast, MEF) were reprogrammed to tubular cells by constructing a bicistronic 4 factor vector that overexpresses hnf1β, emx2, pax8, and hnf4α, although the reprogramming efficiency of this method was 5% -15.3% after 2 weeks of culture, reprogramming Cheng Xiaolv (15% -48%) after 5 weeks or more of culture was still poor. Prior art CN115851600a also relates to reprogramming MEFs into tubular cells by constructing bicistronic 3-factor vectors (e.g., H1EP, H1H4P, and H4 EP) that overexpress 3 factors in hnf1β, emx2, pax8, and hnf4α, although the three-factor combination that eliminates Hnf4a can activate the fate of kidney epithelial cells, the recombination efficiency of the bicistronic combination is not materially improved. Also, although the short-term reprogramming efficiency after 2 weeks of culture is high, there is still a great room for improvement in reprogramming efficiency after 5 weeks or more of culture.

As noted above, the inefficiency of reprogramming fibroblasts into tubular epithelial cells limits potential therapeutic applications and clinical transformations. To the best of the inventors' knowledge, the effect of different Hnf1 beta/Emx 2/Pax8 expression levels on the efficiency of induction of reprogramming of tubular epithelial cells (iREC) has not been studied nor is its reprogramming efficiency and transdifferentiated cell quality clear. Minimizing reprogramming factors has been a very interesting problem in the reprogramming field, and constructing them on the same expression plasmid, achieves more efficient, simpler and more stable induction of the transdifferentiation of fibroblasts into kidney epithelial cells, and helps to promote the direct conversion of kidney fibroblasts into kidney epithelial cells and their therapeutic conversion in injured kidneys.

Accordingly, there is a need to provide a more convenient, efficient and stable method of inducing the transformation and differentiation of fibroblasts into tubular epithelial cells, as well as corresponding plasmids and kits.

Disclosure of Invention

The inventors have unexpectedly found that: by inducing fibroblasts with specific 3 reprogramming factors hnf1β (H1), emx (E) and Pax8 (P) in a specific order combined polycistronic vector, the efficiency of transdifferentiation of fibroblasts into kidney epithelial cells can be significantly improved.

The inventors have found for the first time that the relative expression levels of the reprogramming factors hnf1β, emx2 and Pax8 have an effect on the efficiency of transdifferentiation of fibroblasts into kidney epithelial cells, based on which viral expression plasmids were designed and used in methods and kits for inducing transdifferentiation of fibroblasts into kidney epithelial cells.

In a first aspect, the present disclosure provides a plasmid for inducing transdifferentiation of a fibroblast cell into a kidney epithelial cell comprising the transcription factors Emx, pax8, hnf1β and 2A polypeptide cleavage site P2A, T2A, linked in the following order to the transcription factors Emx, pax8, hnf1β and 2A polypeptide cleavage site P2A, T a:

Emx2-P2A-Pax8-T2A-Hnf1 beta-PuroR (also abbreviated herein as EPH 1),

Emx2-P2A-Hnf1 beta-T2A-Pax 8-PuroR (also abbreviated herein as EH 1P) or

Hnf1 beta-P2A-Emx-T2A-Pax 8-PuroR (also abbreviated herein as H1 EP);

Preferably, transcription factors Emx2, pax8, hnf1 β and 2A polypeptide cleavage site P2A, T a are linked in the following order: emx2-P2A-Pax8-T2A-Hnf1 beta-PuroR or Emx-P2A-Hnf 1 beta-T2A-Pax 8-PuroR.

Preferably, transcription factors Emx2, pax8, hnf1 β and 2A polypeptide cleavage site P2A, T a are linked in the following order: emx2-P2A-Pax8-T2A-Hnf1 beta-PuroR.

Preferably, the fibroblasts are derived from mammalian embryos, skin, bone and/or muscle, e.g. from mouse embryos, e.g. from 12.5 to 14 day old mouse embryos.

In an exemplary embodiment, the fibroblasts are derived from Cdh16-Cre; mT/mG mice.

In an exemplary embodiment, the fibroblast is Cdh 16-Cre-bearing; mouse Embryonic Fibroblasts (MEFs) with mT/mgs alleles.

Preferably, the plasmid is selected from the group consisting of a retrovirus expression plasmid, a lentivirus expression plasmid, an adenovirus expression plasmid and an adeno-associated virus expression plasmid, preferably a retrovirus expression plasmid.

In a second aspect, the present disclosure provides a cell comprising a plasmid provided by the present disclosure.

In a third aspect, the present disclosure provides a kit for inducing transdifferentiation of fibroblasts into kidney epithelial cells comprising a plasmid provided by the present disclosure.

Preferably, the kit may further comprise a sequence selected from the group consisting of SEQ ID NOs: 1 to SEQ ID NO: 18.

In a fourth aspect, the present disclosure provides a method of inducing transdifferentiation of fibroblasts into kidney epithelial cells comprising the steps of:

s1: preparing a fibroblast;

S2: construction of a viral expression plasmid comprising transcription factors Emx2, pax8, hnf1 β and 2A polypeptide cleavage site P2A, T a, said transcription factors Emx, pax8, hnf1 β and 2A polypeptide cleavage site P2A, T a being linked in the following order:

Emx2-P2A-Pax8-T2A-Hnf1β-PuroR、

emx2-P2A-Hnf1 beta-T2A-Pax 8-PuroR or

Hnf1β-P2A-Emx2-T2A-Pax8-PuroR；

Wherein PuroR represents a puromycin resistance gene;

s3: transfecting the plasmid in a packaging cell to obtain a virus;

s4: and (3) transfecting the fibroblasts by using the virus obtained in the step S3, and inducing the fibroblasts to transdifferentiate into kidney epithelial cells.

In an exemplary embodiment, cdh16-Cre is obtained by hybridizing mice that control Cre recombinase with the kidney epithelial cell specific promoter cadherein-16 (Cdh 16-Cre) with mice that carry tdtomao and GFP dual fluorescent reporter genes that fluoresce cell membranes; mT/mG mice, cre-mediated recombinant expression of membrane GFP (mGFP) in tubular epithelial cells, while other non-Cre-expressing cells, including mouse embryonic fibroblasts, still express red fluorescence, thereby allowing monitoring of tubular epithelial cell transdifferentiation.

Preferably, the virus is selected from the group consisting of retroviruses, lentiviruses, adenoviruses and adeno-associated viruses, such as retroviruses.

Preferably, step S1 comprises: tissue from a mammal is digested with pancreatin (including trypsin), the digestion is stopped by adding medium, and the resulting fibroblasts are cultured.

Preferably, the concentration of pancreatin (including trypsin) is 0.05% to 0.25% (w/v). In exemplary embodiments, the concentration of pancreatin (e.g., trypsin) is 0.05%, 0.1%, 0.15, 0.2, or 0.25% (w/v).

Preferably, the medium is DMEM, RPMI1640, eagles and/or L-15 medium supplemented with 5% to 20% (v/v) FBS.

In an exemplary embodiment, embryonic fibroblasts can be prepared as follows: carefully removing the envelope outside the embryo, removing the head, tail and viscera of the embryo, cutting the tissue, adding 1mL of 0.05% trypsin (w/v), transferring the tissue into a 15mL centrifuge tube, digesting at 37 ℃, uniformly mixing every 5min, adding into a DMEM medium containing 10% (v/v) FBS after about 15min, and stopping digestion; the primary embryo fibroblast cells which are completely digested are transferred into a culture flask for culture.

Preferably, step S2 includes: the fragment of interest was amplified by PCR, recovered and purified, and each element was ligated into the backbone plasmid by homologous recombination.

Preferably, the backbone plasmid is pMXs plasmid.

Preferably, the primers used in the PCR amplification are selected from the group consisting of SEQ ID NOs: 1 to SEQ ID NO:18.

In an exemplary embodiment, step S2 includes: amplifying the target fragment by PCR, recovering the target fragment, purifying, recombining, extracting the plasmid, sequencing and detecting the expression of the plasmid.

In an exemplary embodiment, step S2 includes: amplifying the fragment of interest by PCR, recovering and purifying the fragment of interest, ligating the elements into the backbone plasmid by homologous recombination, transforming (i.e., introducing the exogenous nucleic acid molecule into competent cells), plating, colony identification, extracting the plasmid, sequencing and detecting plasmid expression.

In an exemplary embodiment, PCR amplification is performed using a PCR instrument using a high-fidelity DNA polymerase (e.g., KOD Plus neo enzyme, KOD FX enzyme).

Preferably, in step S3, packaging cells are transfected with the plasmid constructed in step S2, puromycin and blasticidin S are added prior to transfection, and after a period of time (e.g.48 hours) the supernatant is collected and the viral particles in the supernatant are concentrated.

In an exemplary embodiment, the virus (e.g., retrovirus) is packaged using packaging cells (e.g., PLAT-E cells or similar cell lines) that express genes (e.g., reverse transcriptase and envelope proteins) required for the virus (e.g., retrovirus). In order to screen and retain plasmids in these packaging cells (e.g., PLAT-E cells, etc.), for packaging of viruses (e.g., retroviruses), they can be screened using media containing antibiotics (e.g., puromycin and blasticidin S).

In an exemplary embodiment, puromycin (G418) and blasticidin S (hygromycin B) are used as selective antibiotics, which are capable of selectively killing cells without the corresponding resistance gene. Packaging cells (e.g., PLAT-E cells) typically contain a plasmid carrying puromycin and blasticidin S resistance genes. Thus, by adding these antibiotics to the medium, it is ensured that only packaging cells (e.g., PLAT-E cells) harboring the viral expression plasmid survive the culture conditions. During packaging of the virus, these antibiotics ensure that only packaging cells (e.g., PLAT-E cells) expressing the desired genes of the virus (e.g., retrovirus) survive, thereby ensuring the effectiveness and success of the packaging process for the retrovirus.

Preferably, in step S3, the packaging cells are cultured prior to transfection using a medium selected from the group consisting of DMEM, RPMI1640, eagles and/or L-15 medium supplemented with 5% to 20% (v/v) FBS.

Preferably, in step S3, the packaging cell is selected from Plat-E cells, 293T cells and/or PA317 cells.

Preferably, in step S3, puromycin is used in an amount of 0.5 to 2.5 μg/mL (e.g. 0.5, 0.75, 1.0, 1.25, 1.5, 1.75, 2.0, 2.25, 2.5 μg/mL); and/or blasticidin S in an amount of 5 to 20 μg/mL (e.g., 5, 7.5, 10, 12.5, 15, 17.5, 20 μg/mL).

Preferably, in step S3, the virus particles are concentrated using polyethylene glycol (e.g., PEG 8000).

In an exemplary embodiment, step S3 involves obtaining a retrovirus from packaging a retrovirus packaging cell Plat-E, wherein the Plat-E cells are cultured using DMEM medium containing 10% (v/v) FBS, and 1 μg/mL puromycin is added to the medium while maintaining the culture; optionally passaging Plat-E and then carrying out plasmid transfection; fresh medium is changed 6-8 hours after transfection, and the supernatant is collected 48 hours after transfection; viral particles were released in the culture supernatant and virus concentration was performed using PEG 8000.

Preferably, step S4 includes: fibroblasts were inoculated at a density of 1X 10 ³ to 1X 10 ⁵/cm ² (e.g., 1X 10 ⁴/cm ²), the fibroblasts were cultured, the fibroblasts were infected with the virus obtained in step S3 in an amount of 2X 10 ⁶ to 1X 10 ⁷ IU/mL, and puromycin at a concentration of 0.5 to 2.5. Mu.g/mL (e.g., 0.5, 0.75, 1.0, 1.25, 1.5, 1.75, 2.0, 2.25, 2.5. Mu.g/mL, e.g., 1.0 to 2.0. Mu.g/mL) was added 60-72 hours after virus infection for screening.

Preferably, in step S4, the amount of puromycin screened for the first 3 days is 1.5 to 2.5 μg/mL (e.g. 1.5, 1.75, 2.0, 2.25, 2.5 μg/mL, e.g. 2.0 μg/mL), followed by 0.5 to 1.5 μg/mL (e.g. 0.5, 0.75, 1.0, 1.25, 1.5 μg/mL, e.g. 1.0 μg/mL). Illustratively, the screening duration is 2 to 3 weeks.

Preferably, in step S4, a concentration of 4.0 to 8.0 μg/mL polybrene (e.g. 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5 or 8.0 μg/mL) is added at the time of viral infection to facilitate viral transport into the cell.

In an exemplary embodiment, step S4 further comprises analyzing the efficiency of renal tubular epithelial cell reprogramming by flow cytometry. For example, cdh16 expression is activated in successfully transdifferentiated kidney epithelial cells, cdh16 promoter activates Cre expression, cleaves loxp site, and thus changes from tdTomato expression to mGFP expression, and mouse embryo fibroblasts are tracked for transdifferentiation into kidney tubular epithelial cells by observing GFP expression.

In an exemplary embodiment, 4×10 ⁴ fibroblasts are plated in a 12-well plate and reprogramming factor viruses are added after the cells adhere. The addition of 4. Mu.g/mL of polybrene at the time of viral infection promotes viral transport into the cell, with a three-factor retrovirus dose of about 15. Mu.L. After 48 hours of virus infection, fresh medium was changed; virus infection was performed for 60-72 hours and screening was performed using puromycin. The concentration of puromycin used in the first 3 days of screening can be 2 mug/mL, and the subsequent reduction of puromycin used concentration by 1 time can be carried out for maintenance culture.

Preferably, in step S4, after infection of the fibroblasts with the virus obtained in step S3, western blot detection is performed on the cell lysates, with the expression level ratio of Hnf1β, emx2 to Pax8 being (2.0 to 4.5): 1 (2.0 to 4.5). In some embodiments, the ratio of expression levels of hnf1β, emx2 to Pax8 may be (2.2 to 4.5): 1 (2.2 to 4.5), (2.5 to 4.5): 1 (2.5 to 4.5), (3.0 to 4.5): 1 (3.0 to 4.5), (3.0 to 4.0): 1 (3.0 to 4.0), (3.1 to 4.0): 1 (3.0 to 3.8), (3.1 to 3.8): 1 (3.0 to 3.6), or (3.1 to 3.7): 1 (3.0 to 3.5). In some embodiments, the ratio of expression levels of hnf1β, emx2 to Pax8 may be (3.1 to 3.6): 1 (3.0 to 3.4), (3.1 to 3.5): 1 (3.0 to 3.3), (3.1 to 3.4): 1 (3.0 to 3.3) or (3.2 to 3.4): 1 (3.1 to 3.3). In some embodiments, the ratio of expression levels of hnf1β, emx2 to Pax8 may be about 3.2:1:3.2, 3.2:1:3.3, 3.3:1:3.2, 3.3:1:3.3, about 3.4:1:3.2, or 3.4:1:3.3.

Preferably, in step S4, after the fibroblasts are infected with the virus obtained in step S3, western blot detection is performed on the cell lysate, wherein the expression level of hnf1β relative to Hsp90 is 5.0 times or more, the expression level of Emx2 relative to Hsp90 is 1.2 times or more, and the expression level of Pax8 relative to Hsp90 is 4.3 times or more.

In some embodiments, the expression level of hnf1β relative to Hsp90 is 5.1-fold or more, 5.2-fold or more, 5.3-fold or more, 5.4-fold or more, 5.5-fold or more, 5.6-fold or more, 5.7-fold or more, 5.8-fold or more, 5.9-fold or more, 6.0-fold or more, or 6.1-fold or more. In some embodiments, the expression level of hnf1β relative to Hsp90 is 10-fold or less, 9-fold or less, 8-fold or less, 7-fold or less, 6.9-fold or less, 6.8-fold or less, 6.7-fold or less, 6.6-fold or less, 6.5-fold or less, 6.4-fold or less, 6.3-fold or 6.2-fold or less.

In some embodiments, emx2 is expressed at a level of 1.3-fold or more, 1.4-fold or more, 1.5-fold or more, 1.6-fold or more, 1.7-fold or more, or 1.8-fold or more relative to Hsp 90. In some embodiments, emx2 is expressed at a level of 3-fold or less, 2.5-fold or less, 2.4-fold or less, 2.3-fold or less, 2.2-fold or less, 2.1-fold or less, 2-fold or less, or 1.9-fold or less relative to Hsp 90.

In some embodiments, the expression level of Pax8 relative to Hsp90 is 4.4-fold or more, 4.5-fold or more, 4.6-fold or more, 4.7-fold or more, 4.8-fold or more, 4.9-fold or more, 5-fold or more, 5.1-fold or more, 5.2-fold or more, 5.3-fold or more, 5.4-fold or more, 5.5-fold or more, 5.6-fold or more, 5.7-fold or more, 5.8-fold or more, 5.9-fold or more, or 6.0-fold or more. In some embodiments, the expression level of Pax8 relative to Hsp90 is 10-fold or less, 9-fold or less, 8-fold or less, 7-fold or less, 6.9-fold or less, 6.8-fold or less, 6.7-fold or less, 6.6-fold or less, 6.5-fold or less, 6.4-fold or less, 6.3-fold or less, 6.2-fold or 6.1-fold or less.

The present inventors have found through extensive studies that by controlling the expression ratio of the above-described specific reprogramming factors of the present invention to be in the above-described range, the transdifferentiation efficiency of fibroblasts into kidney epithelial cells can be further improved.

In exemplary embodiments, the present disclosure provides methods for inducing in vitro the transformation and differentiation of fibroblasts into tubular epithelial cells for non-therapeutic purposes.

In the present application, the inventors regulate the relative expression levels of 3 reprogramming factors hnf1β (H1), emx (E) and Pax8 (P) in polycistronic vectors by specifically selecting 2 different cleavage sites (P2A and T2A) and designing the positions of P2A and T2A in the expression vectors, thereby changing the efficiency of inducing the transdifferentiation of fibroblasts into kidney epithelial cells. It should be noted that vectors employing polycistronic, 2 cleavage sites (P2A and T2A), and 3 factor combinations do not necessarily lead to improved results in transdifferentiation efficiency, but may even lead to completely opposite results (transdifferentiation efficiency does not increase or decrease). For example, the transdifferentiation efficiency of the comparative examples vectors H1PE, PH1E and PEH1 decreased to nearly 0% after 2 weeks (as detailed below), indicating that they were not able to induce transdifferentiation efficiently.

The plasmids, cells, kits and methods provided herein for inducing the transformation and differentiation of fibroblasts into tubular epithelial cells unexpectedly achieve better transdifferentiation efficiency. Although the specific mechanism of interaction between the factors hnf1β (H1), emx (E) and Pax8 (P) is not clear, the applicant observed that there was a difference in the relative expression levels of the factors hnf1β (H1), emx2 (E) and Pax8 (P) in the 3 example vectors and the 3 comparative example vectors, which may be a direct cause of the large difference in their transdifferentiation efficiency.

Any feature or any combination of features described for the plasmid of the first aspect of the disclosure is also applicable to the cell according to the second aspect, the kit according to the third aspect and the method according to the fourth aspect, and vice versa, unless otherwise indicated herein or clearly contradicted by context.

Drawings

Fig. 1: according to an exemplary embodiment of the present disclosure, a schematic representation of 6 polycistronic vectors (i.e., EH1P, EPH1, H1EP, H1PE, PH1E, and PEH 1) with different splicing sequences, wherein reprogramming factors hnf1β, emx2, and Pax8 (separated by P2A and T2A sequences) are cloned into retroviral expression plasmid pMXs;

Fig. 2: according to exemplary embodiments of the present disclosure, an experimental strategy for iREC reprogramming using a three-factor retrovirus is performed. Preparing a retrovirus containing 3RF or a control retrovirus alone (control is MEF transfected with empty virus), and infecting the cells from Cdh16-Cre; mT/mgs report embryonic fibroblasts (MEFs) of mice; cdh16 expression in successfully transdifferentiated tubular cells is activated, cre cleaves the Loxp site, and the cells are converted from the original expression of red fluorescence tdTomato to the expression of green fluorescence GFP;

fig. 3: according to exemplary embodiments of the present disclosure, three factors induce immunofluorescence analysis of fibroblast transdifferentiation into kidney epithelial cells. Fluorescence image results analysis of EPH1, EH1P and H1EP (examples) induced iREC at 2 weeks of transfection;

Fig. 4: according to an exemplary embodiment of the present disclosure, the flow chart shows the efficiency of H1PE, PH1E and PEH1 (comparative) to induce re-programming of tubular epithelial cells after 2 weeks;

Fig. 5: according to exemplary embodiments of the present disclosure, EPH1, EH1P, and H1EP induce the efficiency of MEF to differentiate into tubular epithelial cells (expressed as gfp+ cells%) at weeks 2, 3, and 5;

Fig. 6: according to exemplary embodiments of the present disclosure, (a) expression of hnf1β, emx2 and Pax8 in 6 three factor vectors (EPH 1, EH1P, H1EP, H1PE, PH1E and PEH 1) was detected by Western blot; (B) The relative expression levels of hnf1β, emx2 and Pax8 (relative to Hsp 90) were counted by ImageJ software;

Fig. 7: according to exemplary embodiments of the present disclosure, GO enrichment analysis of EPH 1-induced tubular epithelial cells compared to up-regulated gene (a) and down-regulated gene (B) of MEF;

Fig. 8: the heat map shows the MEF, primary tubular epithelial cells (pREC), EPH1, EH1P induced expression of genes associated with tubular epithelial ECM;

Fig. 9: according to exemplary embodiments of the present disclosure, immunostaining detects the expression of ZO-1, E-cadherin, epcam, ATP A1, AQP1, and Vimentin in EPH 1-induced tubular epithelial cells;

Fig. 10: according to exemplary embodiments of the present disclosure, immunostaining detects Epcam, E-cadherein, and P2A expression in EPH 1-induced tubular epithelial cells;

Fig. 11: according to an exemplary embodiment of the present disclosure, immunostaining detects the expression of β -catenin, ZO-1, epcam, and Vimentin in 3D culture iREC, wherein nuclei are stained with DAPI, and arrows show basolateral membrane localization of β -catenin and apical membrane localization of ZO-1;

Fig. 12: according to exemplary embodiments of the present disclosure, the induced tubular epithelial cells have a certain tubular epithelial cell function. Schematic of a mouse kidney reconstitution experiment: the kidney of the mice is subjected to decellularization treatment, induced tubular epithelial cells are injected, and the tubular structure can be formed automatically after continuous culture. (B) Partially reconstructed mouse kidneys, fixed dehydrated and embedded slices are co-dyed with GFP through an epithelial cell marker gene Epcam, and the capability of inducing tubular epithelial cells to autonomously form a tubular structure is detected;

Fig. 13: emx2 (SEQ ID NO: 19);

fig. 14: cDNA sequence of Hnf1 beta (SEQ ID NO: 20);

fig. 15: pax8 cDNA sequence (SEQ ID NO: 21);

Fig. 16: emx2 the 2-P2A-Pax8-T2A-Hnf1 beta sequence (SEQ ID NO: 22);

fig. 17: emx2 the 2-P2A-Hnf1 beta-T2A-Pax 8 sequence (SEQ ID NO: 23);

Fig. 18: hnf1 beta-P2A-Emx-T2A-Pax 8 sequence (SEQ ID NO: 24);

Fig. 19: hnf1 beta-P2A-Pax 8-T2A-Emx sequence (SEQ ID NO: 25);

fig. 20: pax8-P2A-Hnf1 beta-T2A-Emx sequence (SEQ ID NO: 26); and

Fig. 21: pax 8-P2A-Emx-T2A-Hnf 1 beta sequence (SEQ ID NO: 27).

Detailed Description

The specific embodiments presented herein are intended to describe the present disclosure by way of example and are not intended to limit the disclosure in any way, including but not limited to the specific embodiments described herein.

Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure have the meanings commonly understood by one of ordinary skill in the art. Furthermore, unless the context requires otherwise, singular terms include the plural and plural terms shall include the singular. In general, the terms described herein are those commonly used as known to those skilled in the art.

As used herein, "comprising," "including," "having," and the like are intended to be open ended, meaning that there are additional, non-listed elements, components, or steps in addition to the listed elements, components, or steps, which do not materially affect the basic novel characteristics of the methods or products of this disclosure. Unless expressly stated otherwise, the expressions "comprising," "including," "containing," "having" and "having" also encompass the fact that "consisting of … …" are intended to be inclusive, i.e., that they may consist of only the recited elements, components, or steps.

As used herein, the term "and/or" as used in the phrase "a and/or B" is intended to include: both A and B, A or B, (separate) A and (separate) B. Likewise, the term "and/or" as used in the phrase "A, B and/or C" is intended to cover the following embodiments: A. b and C; A. b or C; a or C; a or B; b or C; a and C; a and B; b and C; (separate) A; (separate) B; and (separate) C.

As used herein, "above" and "below" include the present numbers. For example, "more than one" means "one or more", wherein "multiple" means "two or more".

As used herein, when referring to a numerical value or range of numerical values, unless otherwise expressly specified or contradicted by context: ranges of values provided herein are understood to encompass the endpoints of the range, and one tenth of the unit of the lower limit, each intermediate value, and each intermediate value; ranges excluding one or both of the endpoints are also included within the disclosure; any combination of the upper and lower endpoints of the ranges or sub-ranges of values provided herein is possible.

As used herein Emx2 is referred to collectively as EMPTY SPIRACLES Homeobox 2 (empty helix Homeobox 2), and herein refers to the relevant genes and their in vivo and in vitro expression products, as the case may be, including but not limited to cDNA (whose sequence is shown in SEQ ID NO: 19), mRNA and protein.

As used herein, hnf 1. Beta. Is referred to collectively as Hepatocyte nuclear factor 1. Beta. (hepatocyte nuclear factor 1. Beta.) and refers herein to the relevant genes and their in vivo and in vitro expression products, as the case may be, including but not limited to cDNA (whose sequence is shown in SEQ ID NO: 20), mRNA and protein.

As used herein, pax8 is designated as Paired Box (cassette 8), and related genes and their in vivo and in vitro expression products are represented herein as the case may be, including but not limited to cDNA (whose sequence is shown in SEQ ID NO: 21), mRNA and protein.

As used herein, P2A, T a represents genes associated with self-cleaving P2A, T a peptide and their in vivo appearance products, including but not limited to cDNA, mRNA, and peptide.

As used herein PuroR denotes herein puromycin or puromycin resistance genes as the case may be.

Unless otherwise indicated, terms involved in the present disclosure have meanings commonly understood or commonly used in the art.

Example 1: induction of transdifferentiation of mouse embryonic fibroblasts into kidney epithelial cells

In the present example, specific embodiments of the present disclosure are described in detail using mouse embryonic fibroblasts and retroviral expression plasmids as examples, but it should be noted that the present disclosure is not limited to these embodiments.

1. Experimental method

A. preparation and culture of primary embryonic fibroblasts

Pregnant mouse embryos from 12.5 to 14 days pregnant are used to extract mouse embryo fibroblasts. Carefully remove extra-embryonic membranes, remove head, tail and viscera, shear tissue, add 1mL of 0.05% (w/v) trypsin, transfer tissue to a 15mL centrifuge tube, digest in a 37℃water bath, mix every 5min, add to DMEM medium (Gibco, cat# 11966025) containing 10% (v/v) FBS (Gibco, cat# 10270106) after about 15min, and terminate digestion. The fully digested primary embryonic fibroblasts were plated into culture flasks for subsequent experiments.

B. Reverse transcription packaging plasmid construction

In this example, 6 polycistronic retroviral elements separated by 2A polypeptide cleavage sites (P2A and T2A) were constructed in a retroviral backbone plasmid pMXs, and cDNA fragments of 3 transcription factors were obtained by PCR and each element was ligated into a pMXs backbone plasmid by homologous recombination.

Primer design was performed using the homologous recombination method, and the amplification primers used in this example are as follows. The gene fragment of interest containing Hnf1 beta, pax8 and Emx was amplified using a PCR apparatus (ProFlex ^TM PCR 4484073,Thermo Fisher Scientific), KOD Plus Neo enzyme (TOYOBO LIFE SCIENCE, cat# KOD-401) and KOD FX Neo enzyme (TOYOBO LIFE SCIENCE, cat# KFX-201), PCR product recovery was performed using a Promega DNA recovery kit (Promega, cat# A9281), cDNA fragments of Hnf1 beta, emx2 and Pax8 genes were ligated to a retrovirus pMXs vector by a homologous recombination method, recombination, transformation (introduction of exogenous nucleic acid molecules into competent cells), plating, colony PCR identification, plasmid extraction, sequencing and plasmid expression detection, and the like. The retroviral plasmids of the present disclosure were obtained: EPH1, EH1P, H.sup.1EP, H1PE, PH1E and PEH1 (FIG. 1).

Hnf 1. Beta. Forward primer (SEQ ID NO: 1):

TAGTTAATTAAGGATCTACCGCCACCATGGTGTCCAAGCTCACG

hnf1 beta-P2A reverse primer (SEQ ID NO: 2):

CGGTCCAGGATTCTCTTCGACATCTCCGGCTTGTTTCAGCAGAGAGAAGTTTGTTGCCCAGGCTTGCAGTGG

Emx2 forward primer (SEQ ID NO: 3):

TAGTTAATTAAGGATCTACCGCCACCATGTTTCAGCCGGCGCCCAAG

emx2-P2A reverse primer (SEQ ID NO: 4):

CGGTCCAGGATTCTCTTCGACATCTCCGGCTTGTTTCAGCAGAGAGAAGTTTGTTGCATCGTCTGAGGTCAC

pax8 forward primer (SEQ ID NO: 5):

TAGTTAATTAAGGATCTACCGCCACCATGCCTCACAACTCGATCAG

pax8-P2A reverse primer (SEQ ID NO: 6):

CGGTCCAGGATTCTCTTCGACATCTCCGGCTTGTTTCAGCAGAGAGAAGTTTGTTGCCAGATGGTCAAAGGC

P2A-Hnf 1. Beta. Forward primer (SEQ ID NO: 7):

GCAACAAACTTCTCTCTGCTGAAACAAGCCGGAGATGTCGAAGAGAATCCTGGACCGATGGTGTCCAAGCTC

hnf1 beta-T2A reverse primer (SEQ ID NO: 8):

GGGACCGGGATTCTCCTCCACATCCCCGCAAGTAAGCAGTGATCCGCGTCCCTCCCAGGCTTGCAGTGG

P2A-Pax8 forward primer (SEQ ID NO: 9):

GCAACAAACTTCTCTCTGCTGAAACAAGCCGGAGATGTCGAAGAGAATCCTGGACCGATGCCTCACAACTCG

pax8-T2A reverse primer (SEQ ID NO: 10):

GGGACCGGGATTCTCCTCCACATCCCCGCAAGTAAGCAGTGATCCGCGTCCCTCCAGATGGTCAAAGGC

P2A-Emx2 forward primer (SEQ ID NO: 11):

GCAACAAACTTCTCTCTGCTGAAACAAGCCGGAGATGTCGAAGAGAATCCTGGACCGATGTTTCAGCCGGCG

emx2-T2A reverse primer (SEQ ID NO: 12):

GGGACCGGGATTCTCCTCCACATCCCCGCAAGTAAGCAGTGATCCGCGTCCCTCATCGTCTGAGGTCAC

T2A-Pax8 forward primer (SEQ ID NO: 13):

GAGGGACGCGGATCACTGCTTACTTGCGGGGATGTGGAGGAGAATCCCGGTCCCATGCCTCACAACTCG

Pax8 reverse primer (SEQ ID NO: 14):

TGTGCTGGCGGCCGCTCGAGCTACAGATGGTCAAAGGCTG

T2A-Hnf 1. Beta. Forward primer (SEQ ID NO: 15):

GAGGGACGCGGATCACTGCTTACTTGCGGGGATGTGGAGGAGAATCCCGGTCCCATGGTGTCCAAGCTC

Hnf 1. Beta. Reverse primer (SEQ ID NO: 16):

TGTGCTGGCGGCCGCTCGAGTCACCAGGCTTGCAGTGGACACTG

T2A-Emx2 forward primer (SEQ ID NO: 17):

GAGGGACGCGGATCACTGCTTACTTGCGGGGATGTGGAGGAGAATCCCGGTCCCATGTTTCAGCCGGCG

emx2 reverse primer (SEQ ID NO: 18):

TGTGCTGGCGGCCGCTCGAGTTAATCGTCTGAGGTCAC

C. retroviral packaging and preparation

The packaging cells Plat-E (CELL BIOLABS, cat. No. RV-101) were resuscitated and cultured using DMEM medium containing 10% (v/v) FBS, 1. Mu.g/mL puromycin and 10. Mu.g/mL blasticidin S. Plat-E was passaged and cells plated into 10cm dishes. Plasmid transfection was performed the next day, fresh medium was changed 6-8h after transfection, medium supernatant (virus particles released in medium supernatant) was collected 48h after transfection, and PEG8000 was added for virus concentration.

D. inducing the conversion and differentiation of MEF into tubular epithelial cells

Two transgenic mice, cdh16-Cre and mTmG, were used in the experiments to follow the process of MEF conversion into tubular epithelial cells. In Cdh16-Cre, mTmG mice, cre is expressed in kidney epithelial cells, and in Cre-expressing cells, tdTomato is knocked out, instead expressing green fluorescent protein GFP. Mating the two mice, then Cdh16-Cre from E12.5 to E14 days; embryo Fibroblasts (MEFs) were isolated from mTmG mouse embryos and used in reprogramming experiments. After cell attachment, the reprogramming factor virus (i.e., the virus particles concentrated as above) is added. To facilitate viral transport into the cells, 4. Mu.g/mL Polybrene (Polybrene) was added to the medium. After 48 hours of virus infection of MEF, fresh medium was changed. Screening was performed 60-72 hours after viral infection using puromycin. The puromycin was used at a concentration of 2 μg/mL for the first 3 days of screening, after which the concentration was reduced by half and cultivation continued for 1-2 weeks. In Cdh16 activated cells Cre expression was activated and the cells were transformed from tdTomato expression to GFP expression. The expression of MEF cells GFP was continuously observed, and the conversion efficiency of MEF into tubular epithelial cells was tracked and quantified by fluorescence microscopy (OLYMPUS, BX 53) and flow cytometry (LSRFortessa, BD Biosciences), and an exemplary experimental procedure is shown in fig. 2.

E. Flow cytometer analysis and sorting of cells

Cells were first digested with 0.05% (w/v) trypsin and resuspended in appropriate amounts of medium or FACS buffer. After filtering the cells using a 40 μm cell screen, the cells were checked on the machine. The experiments used specific transgenic mice and Cre strain mice, wherein GFP-specific markers of the target cell population were detected without staining.

F. cell removal treatment and cell re-implantation of mouse kidney

The kidneys of mice were rinsed with heparinized PBS, then kidney perfusion was performed with 1% (w/v) SDS, and cells in the kidneys were digested until decellularization. Next, 1% (v/v) Triton X-100 was used to perfuse the kidneys for about 24 hours. The kidneys were then rinsed with PBS containing antibiotics (penicillin-streptomycin, gibco, cat# 15140122) and bacteriostat Primocin (InvivoGen, cat# ant-pm-05) to remove residual detergent. Thereafter, the kidney samples were sent to irradiation company (Shanghai Nuclear Ming radiation technology Co., ltd.) for sterilization. Finally, iREC cells were injected into the organ scaffolds and cultured in DMEM medium for 14 days.

G.3D cell culture

First, cells were digested with 0.05% (w/v) trypsin and filtered, then resuspended in DMEM and mixed well with growth factor-containing matrigel and inoculated in 8-well slides. Matrigel was allowed to polymerize for 15min in an incubator at 37℃and then kidney epithelial cell growth medium (Lonza, cat. No. CC-3190) was added. Immunofluorescent staining was performed with 4% pfa fixed cells when sphere formation was evident. Adult mice were obtained from adult (age. Gtoreq.6 weeks) C57/BL6J mice.

Western blot assay

After 2 weeks of infection of fibroblasts with viral particles, the cells were in the process of transdifferentiation. Cells during transdifferentiation were lysed using RIPA lysis buffer (SB-BR 040) from Saint Biotech, shanghai with CELL SIGNALING Technology protease inhibitor Cocktail kit (5871). Lysis buffer was added to the cell culture dish and the cells were gently scraped off with a cell scraper. The lysate was then transferred to a microcentrifuge tube and centrifuged at 12,000rpm for 10 minutes at 4℃to pellet the cell debris. Then adding 1/4 volume of instant protein loading buffer (LT 101) of Shanghai elegance enzyme biological medicine technology Co., ltd into the sample, mixing, and heating the mixture with boiling water bath for 5-10 min; After the completion of heating, the supernatant was subjected to high-speed centrifugation at 12,000rpm for 5 minutes, and protein extraction was performed. The concentration of extracted protein was determined using the BCA protein quantification kit of Thermo FISHER SCIENTIFIC (a 55864). Bovine Serum Albumin (BSA) from Thermo FISHER SCIENTIFIC was purchased and used as a standard (23209). mu.L of BSA standard was diluted to 100. Mu.L with PBS (samples were typically diluted with PBS) to a final concentration of 0.5mg/mL. Standards were added to protein standard wells of 96-well plates at 0,2, 4, 6, 8, 12, 16, 20 μl, with PBS added to make up to 20 μl. The standard substances are added into protein standard substance holes of a 96-well plate according to 0, 1, 2, 4, 8, 12, 16 and 20 mu L, and PBS is added to complement 20 mu L. BCA reagent was added to microwell plates containing standards and samples and absorbance at 562nm was measured using a microwell plate reader. The protein concentration of the samples was calculated from the resulting BSA standard curve. SDS-PAGE gels were prepared with separation gels at 7.5% or 10% and concentrated gels at 4%. Acrylamide/bisacrylamide stock solution, SDS, ammonium Persulfate (APS), and tetramethyl ethylenediamine (TEMED) were mixed with Tris-glycine running buffer and then poured into a gel card. The gel was allowed to polymerize for about 30 minutes. The preparation was carried out using Tris/glycine/SDS electrophoresis buffer (PS 105) of Shanghai elegance enzyme biomedical technologies Co.Ltd. By dissolving an appropriate amount of buffer in deionized water. The pH of the buffer was adjusted to the desired value using HCl or NaOH and the final volume was adjusted accordingly. The prepared buffer solution is used for gel preparation and electrophoresis operation. The treated protein sample mixture (typically the protein-containing extraction supernatant) is mixed with a gel loading buffer and then loaded into a gel tank. Before the start of electrophoresis, it is ensured that the electrophoresis tank is filled with electrophoresis buffer, and then electrophoresis separation is performed using an appropriate current and time. Isolated proteins were transferred onto PVDF membranes (Millipore, IPVH, 00010) using a transfer buffer containing Tris-HCl and glycine (e.g.PS 109 from Shanghai enzyme Biomedicine technologies Co.). The transfer in ice water can prevent degradation or migration of proteins. The membrane is usually transferred by using a constant current of 260mA, and the membrane transfer time is adjusted according to the size and the characteristics of the target protein. After transfer, the PVDF membrane is blocked with a protein solution such as skim milk to prevent non-specific binding. The membrane was then rinsed with PBS and a solution of primary antibodies (target protein antibodies hnf1β, pax8 and Emx, and reference antibody Hsp 90) was prepared, typically diluted with 3% bsa. The incubation conditions for the primary antibody may be selected to be either overnight at 4℃or for 2-3 hours at 37 ℃. After incubation of the primary antibodies, unbound primary antibodies were removed by washing the membrane with PBST solution. Appropriately diluted secondary antibodies (secondary antibodies are typically anti-primary antibodies) are incubated, typically at room temperature for 1-2 hours. After incubation, unbound secondary antibodies were removed by washing the membrane with PBST solution. After addition of the chemiluminescent reaction substrate, an image of the film was taken using a chemiluminescent imaging system (glider ChemiScope 3300,3300). Imaging results were quantitatively analyzed using ImageJ software (Fiji version) to assess the expression level of the protein of interest and other relevant parameters. For quantitative analysis of Western blot protein results, imageJ software (Fiji version) was used. The following are the detailed steps: opening an image: first, a Western blot membrane image taken in a chemiluminescent imaging system is opened. In ImageJ, click on the "files" menu, then select "open", find and select image files. A scale is arranged in the image to convert the pixel values into actual distances. Clicking on the "straight line selection tool" in the toolbar (the toolbar on the left) then drags a line segment in the image, the length of which corresponds to the distance that should be known. The "set scale" is selected in the "analyze" menu and the actual distance value and pixel length are entered. A rectangular selection tool or an elliptical selection tool is used to select a region of interest in the image, such as a band region or a region of a protein dot. The "measure" is selected in the "analyze" menu, which will measure the pixel density of the selected region. This step is repeated and measurements are made on all regions of interest. The measured pixel density value is compared with a standard sample or negative control to calculate the relative protein expression amount. The measurement may be optionally normalized to a negative control or total protein load. The same measurement and analysis procedure is performed for all areas to be analyzed. All data are collated into a data table and data analysis is performed using appropriate statistical methods, such as mean calculation, standard deviation calculation, and statistical significance testing. Finally, charts or graphs are generated using ImageJ's drawing tool or export data into other software to display accurate relative protein expression data, thereby yielding expression levels of hnf1β, emx2 and Pax8 proteins. Antibodies for Western blot in this study were as follows ：Anti-Hnf1β(Abcam,ab128912,1:1000),Anti-Emx2(Sigma,HPA065294,1:1000),Anti-Pax8(Abcam,ab239363,1:1000),Anti-Hsp90(Abcam,ab32568,1:1000),HRP- anti-rabbit IgG (Cwbio, CW0103, 1:5000) and HRP-anti-mouse IgG (Cwbio, CW0102S, 1:5000). Wherein the Hnf1 beta is about 62KD, emx2 is 28KD, pax8 is 49KD and Hsp90 is 90KD.

RNA extraction

1ML Trizol was added to 1X 10 ⁷ cells, transferred to a 1.5mL centrifuge tube, lysed on ice for 30 minutes, centrifuged at 12000rpm at 4℃for 20 minutes, and after centrifugation, the supernatant was observed for the presence of precipitation, transferred to a new centrifuge tube, 1/5 volume of chloroform was added, mixed upside down, left to stand for 5 minutes, and centrifuged at 12000rpm at 4℃for 15 minutes. The aqueous phase was removed, transferred to a new centrifuge tube, and 1/5 volume of chloroform was added again and the process repeated once. Centrifugation was performed at 12000rpm for 5 minutes at 4℃and the upper aqueous phase was transferred to a new centrifuge tube, an equal volume of isopropanol was added, the RNA was precipitated at-20℃after inversion and mixing, and centrifugation was performed at 12000rpm for 20 minutes. After centrifugation, a white precipitate was observed. The supernatant was discarded, the pellet was washed with 75% ethanol and then centrifuged at 12000rpm for 5 minutes at 4℃and repeated once. Air-separating for 1 minute, and then uncovering and airing. DEPC water was added to dissolve RNA. After determining the RNA concentration, RNA was frozen at-80 ℃.

J. Cell immunostaining

First, 2.5X10 ⁴ induced tubular epithelial cells and 3X 10 ⁴ MEF cells were transferred to a culture plate, and fixed after waiting for the cells to grow to an appropriate density. Next, immunostaining of E-cadherein, ZO-1, AQP1, ATP1A1 and Epcam was performed, with the cell density of the Epcam staining being slightly higher. Fixation with 4% pfa or pre-chilled methanol for 15 min. After fixation, cells were washed with PBS. Cells are usually treated with 0.1% triton x-100 for 15min, but if fixed with methanol, permeabilization is usually not performed. AQP1 staining required 5 minutes treatment with 1% sds after fixation. The cells were then washed multiple times with PBS to prevent SDS from affecting the binding of the antibody to the protein of interest. Blocking solution was PBS containing 5% BSA and 5% goat serum, and blocked at 37℃for 1 hour. The antibody was diluted in PBS containing 1% BSA and 5% goat serum, and 1% DMSO was added to the AQP1 primary antibody dilution. An anti-dilution was added in accordance with the antibody instructions using the proportions, typically incubated overnight at 4 ℃. Wash 3 times with PBS. Secondary antibodies were diluted in PBS containing 1% bsa, 5% goat serum, and the secondary antibodies were typically used at 1:1000. After incubation for 1 hour at 37 ℃, the secondary antibody is incubated, and the subsequent operation can be judged by preliminarily observing the dyeing condition through a fluorescence microscope. PBST washed cells 3 times before sealing. DAPI is contained in the tablet, DAPI staining is not needed in advance, and if the tablet does not contain DAPI, DAPI staining is carried out on the tablet. Firstly, preparing a glass slide, cleaning with 75% alcohol, then using, dripping a proper amount of anti-quenching agent, transferring the cell climbing sheet to the glass slide by using tweezers, sealing the glass slide by using nail polish, observing the dyeing effect by using a microscope after the nail polish is solidified, and taking a picture.

PBS buffer composition: naCl (sodium chloride), KCl (potassium chloride), na ₂HPO₄ (disodium hydrogen phosphate), KH ₂PO₄ (monopotassium phosphate); pH of 7.2-7.4

The information of one antibody is as follows:

Antibody name	Manufacturer (S)	Goods number	Use of the same
				Hnf1β	Abcam	ab128912	Western blot analysis
Emx2	Sigma-Aldrich	HPA065294	Western blot analysis
				Pax8	Abcam	ab239363	Western blot analysis
ZO-1	Abcam	ab221547	Cell immunostaining assay
				Epcam	Abcam	ab223582	Cell immunostaining assay
E-cadherin	Invitrogen	13-1900	Cell immunostaining assay
				ATP1A1	Abcam	ab7671	Cell immunostaining assay
AQP1	Santa Cruz	sc-515770	Cell immunostaining assay
				Vimentin	Abcam	ab8978	Cell immunostaining assay
β-catenin	Abcam	ab6302	Cell immunostaining assay
				Hsp90	Abcam	ab32568	Western blot analysis

The secondary antibody information is as follows:

Second antibody name	Manufacturer (S)	Goods number	Use of the same
				HRP-anti-rabbit IgG	Cwbio	CW0103S	Western blot analysis
HRP-anti-mouse IgG	Cwbio	CW0102S	Western blot analysis
				Alexa Fluor 594 goat anti-rabbit IgG(H+L)	Invitrogen	A-11012	Cell immunostaining assay
Rabbit Anti-Rat IgG(H&L)-Alexa Fluor 594	Abmart	M213629	Cell immunostaining assay
				Alexa Fluor 647 goat anti-mouse IgG(H+L)	Invitrogen	A-21235	Cell immunostaining assay

2. Results and discussion

The inventors regulate the relative expression levels of 3 reprogramming factors hnf1β (H1), emx (E) and Pax8 (P) in polycistronic vectors by specifically selecting 2 different cleavage sites (P2A and T2A) and designing the positions of P2A and T2A in the expression vector, thereby changing the efficiency of inducing the transdifferentiation of fibroblasts into kidney epithelial cells.

To investigate the effect of the relative expression levels between reprogramming factors hnf1β (H1), emx (E) and Pax8 (P) on transdifferentiation efficiency, the present application provided 6 vectors in example 1: EPH1, EH1P and H1EP (examples), and H1PE, PH1E and PEH1 (comparative examples).

It should be noted that vectors employing polycistronic, 2 cleavage sites (P2A and T2A), and 3 factor combinations do not necessarily lead to improved results in transdifferentiation efficiency, but may even lead to completely opposite results (transdifferentiation efficiency does not increase or decrease). For example, the transdifferentiation efficiency of vectors H1PE, PH1E and PEH1 (comparative) decreased to nearly 0% after 2 weeks (FIG. 4), indicating that they were not able to induce transdifferentiation efficiently.

1. The invention obviously promotes the transformation of cell fate and improves the induction efficiency of the tubular epithelial cells

After MEF transfection with virus and further incubation for 2 weeks, transformation efficiency was examined by flow cytometry. The results show that the reprogramming efficiencies of the example vectors EPH1, EH1P and H1EP (examples) of the present disclosure at week 2 are 19.2%, 10.1% and 9.11%, respectively (fig. 3 and 5), significantly higher than the factor 3 alone mediated iREC reprogramming efficiency, and also overall higher than the bicistronic 4 factor 4 vectors H1EH4P, H1H4EP and H1EH4P of prior art CN115851600a (15.3%, 10% and 5%, respectively) at week 2 post-culture reprogramming Cheng Xiaolv. After 3 weeks of culture, the efficiencies of the vectors EPH1, EH1P and H1EP (examples) of the present disclosure to induce tubular epithelial cells were 28%, 20% and 10%, respectively, which are not significantly different from the double cistron 4 factor 4 vectors H1EH4P, H H4EP and H1EH4P of prior art CN115851600a as to reprogramming Cheng Xiaolv (29.9%, 16% and 10%, respectively) after 3 weeks of culture.

Notably, the advantages of the vectors of the present disclosure are highlighted after 5 weeks or more of incubation. As shown in FIG. 5, after 5 weeks of culture, EPH1, EH1P and H1EP (examples) induced re-programming efficiencies of tubular epithelial cells increased to 62%, 53% and 33%, respectively, significantly higher than that of the bicistronic 4 factor 4 vectors H1EH4P, H H4EP and H1EH4P of prior art CN115851600A (48%, 31% and 15%, respectively) were reprogrammed Cheng Xiaolv after 5 weeks of culture.

Furthermore, it is also notable that prior art CN115851600a, although also mentioning several bicistronic 3 factor vectors H1EP, H1H4P and H4EP (prior art), is unaware of the effect of the relative expression levels between different reprogramming factors on reprogramming efficiency. Unlike the present disclosure, prior art CN115851600a employs a bicistronic, single-cut site P2A design without regulation of the relative expression levels of several reprogramming factors. The reprogramming efficiencies of the bicistronic 3 factor vectors H1EP, H1H4P and H4EP (prior art) after 2 weeks of culture were higher, but the reprogramming Cheng Xiaolv (28%, 12%, 2% respectively) after 3 weeks of culture had begun to be entirely lower than the present disclosure, and the reprogramming Cheng Xiaolv (36%, 20%, 0% respectively) after 5 weeks of culture for the 3 prior art vectors were all significantly lower than the vectors of the present disclosure (62%, 53% and 33% respectively).

It follows that the polycistronic 3 factor vectors EPH1, EH1P and H1EP (examples) provided by the present disclosure achieve overall improved reprogramming efficiency compared to the prior art, and that such improvements of the present disclosure will be more apparent after 5 weeks or more of culture.

2. The expression levels of the polynucleotide homology cassettes Hnf1 beta, emx2 and Pax8 protein in fibroblasts are different

As shown in fig. 3 to 5, the 6 vectors EPH1, EH1P and H1EP (examples) and H1PE, PH1E and PEH1 (comparative examples) provided by the present application have differences in transdifferentiation efficiency.

There was a significant difference in transdifferentiation efficiency between the example vector and the comparative example vector. The transdifferentiation efficiencies of vectors EPH1, EH1P and H1EP (examples) after 2 weeks were 19.2%, 10.1% and 9.11%, respectively (FIG. 3), whereas the transdifferentiation efficiencies of vectors H1PE, PH1E and PEH1 (comparative examples) after 2 weeks were reduced to nearly 0% (FIG. 4), indicating that they were not able to induce transdifferentiation efficiently. Although the specific mechanism of interaction between the factors hnf1β (H1), emx (E) and Pax8 (P) is not clear, the applicant observed that the relative expression levels of the factors hnf1β (H1), emx2 (E) and Pax8 (P) in the 3 comparative vectors did not fall within the specific ranges claimed by the present application, which the inventors speculated to be a direct cause of the reduced transdifferentiation efficiency to nearly 0%.

There was also a difference in the efficiency of the three EPH1, EH1P and H1EP (examples) polynucleotide cassettes to induce the transdifferentiation of MEF into tubular epithelial cells, which the inventors speculated to be due to the different expression levels of the three transcription factors Hnf1β, emx2 and Pax 8.

The inventors analyzed the expression levels of transcription factors in MEFs transduced with these 6 different polynucleotide vectors by Western blot, see table 1 below and fig. 6.

Table 1: expression level of transcription factor (relative to Hsp 90)

Carrier body	Hnf1β	Emx2	Pax8	Hnf1β/Emx2	Pax8/Emx2
						EPH1	6.11	1.85	6.04	3.31	3.27
EH1P	5.53	1.74	5.32	3.19	3.07
						H1EP	5.60	1.51	5.28	3.70	3.49
H1PE	4.96	0.89	4.25	5.57	4.78
						PH1E	4.39	0.88	2.85	5.01	3.24
PEH1	2.51	1.19	1.86	2.11	1.57

The inventors have unexpectedly found that: when the relative expression amounts of the 3 reprogramming factors hnf1β (H1), emx (E), and Pax8 (P) are within a specific range, the efficiency of inducing the transdifferentiation of fibroblasts into kidney epithelial cells is significantly improved. Specifically, the present inventors unexpectedly found that: the efficiency of the transdifferentiation of fibroblasts into kidney epithelial cells may be significantly improved when the expression level of hnf1β relative to Hsp90 is 5.0 fold or more, emx2 relative to Hsp90 is 1.2 fold or more, and Pax8 relative to Hsp90 is 4.3 fold or more, and/or the ratio of hnf1β, emx2 to Pax8 expression levels is (2.0 to 4.5): 1 (2.0 to 4.5), e.g., (3.0 to 4.0): 1 (3.0 to 4.0).

The higher levels of Emx protein expression in EPH1, EH1P and H1EP (examples) and lower levels of Emx protein expression in H1PE, PH1E and PEH1 (comparative examples) indicate that Emx2 plays an important role in the reprogramming of tubular epithelial cells. The expression levels of Emx2 were similar in the three combinations of EPH1, EH1P and H1EP (examples), but the expression levels of Hnf1β or Pax8 protein were highest in the EPH1 combination. These results indicate that high abundance of hnf1β and Pax8 protein expression levels can promote the transition of cells to tubular epithelial cell fate. The combination EPH1, EH1P and H1EP each unexpectedly achieved improved transdifferentiation efficiency compared to the prior art, wherein the expression ratios of Hnf1β (H1), emx (E) and Pax8 (P) in EPH1 and EH1P were better and the expression ratios of Hnf1β (H1), emx2 (E) and Pax8 (P) in EPH1 were optimal.

3. The tubular epithelial cells induced by the invention have epithelial cell characteristics

The RNA-seq analysis was further performed on iREC transformed with EPH1, EH1P to investigate the epithelial characteristics. The RNA was extracted and sequenced from iREC induced by EPH1, EH1P and the individual 3-factor group. The control group was Mouse Embryonic Fibroblasts (MEFs) transfected with empty virus and primary kidney epithelial cells (pREC). Transcript profiles of EPH1, EH1P, the individual 3-factor group, MEF and pREC were generated and analyzed. The results show that there is a difference in the characteristics of the induced epithelial cells under different factor combinations. EPH 1-induced upregulation genes of tubular epithelial cells are enriched in the "ion transport", "transport regulation", "cell communication regulation" and "transmembrane transport" categories (FIG. 7A); in contrast, downregulated genes were enriched in "motility," "cell migration," "cell motility," and "extracellular matrix tissue" (fig. 7B), indicating that EPH1, EH 1P-induced iREC acquired a kidney epithelial-like phenotype while inhibiting fiber-related genes. Reprogramming with EPH1 significantly reduced expression of ECM-related genes (fig. 8), indicating establishment of molecular features of kidney epithelial cells. Finally, the results of immunostaining assays for the epithelial marker genes ZO-1, E-cadherein and Epcam showed that they were expressed in the iREC induced by EPH1 and distributed across the cell membrane (FIG. 9). In addition, epo 1-induced iREC expressed the tubular epithelial marker genes ATP1A1 and Aquaporin (AQP 1) (fig. 9), and no mesenchymal marker gene Vimentin was expressed in iREC (fig. 9). Immunostaining detects Epcam, E-cadherin and P2A expression in induced tubular epithelial cells, indicating that the EPH 1-induced iREC of the application has cell autonomy (fig. 10). Thus, iREC transduced by EPH1, EH1P and H1EP of the present application acquire the characteristics of tubular epithelial cells and lose the mesenchymal phenotype.

4. The induced tubular epithelial cells have certain functions of tubular epithelial cells

In order to study whether iREC can independently form a three-dimensional structure, induced tubular epithelial cells are subjected to 3D culture, the cells are resuspended in matrigel, and cultured in a specific culture chamber, wherein the matrigel comprises laminin, collagen IV, entactin and heparin sulfate proteoglycan as main components, and an extracellular matrix growth environment is provided for the cells. EPH1 (and EH1P, H EP, not shown) induced iREC continued to form spheres with a central lumen when cultured in Matrigel (FIG. 11). The spheroids formed by the induced tubular epithelial cells had polarity, β -catenin was expressed on the basolateral membrane, epcam was expressed on the cell surface, ZO-1 was expressed on the apical membrane, and the interstitial marker Vimentin was not expressed in the spheroids formed by the induced tubular epithelial cells (fig. 11). Next, it was investigated whether iREC could self-organize into tubular structures by means of extracellular matrix alone as a guiding framework in the absence of other cells (fig. 12A). The harvested mouse kidneys were decellularized by detergent perfusion leaving only the extracellular matrix framework and the decellularized kidney scaffold was used as a guide structure. iREC was infused into the scaffold, iREC was observed to grow and organize into a partially crimped tubular structure and presented Epcam positive, epcam distributed in the tubular between adjacent iREC continuous basement membranes (fig. 12B). Therefore, the research result of the application shows that iREC tubular epithelial cells induced by the present disclosure have certain tubular epithelial cell functions.

5. Summary

The present disclosure provides an advanced system and method that can greatly increase the efficiency of transdifferentiation of fibroblasts into tubular epithelial cells. The inventor carries Cdh16-Cre; mouse Embryo Fibroblasts (MEFs) and retroviral expression plasmids of mT/mgs alleles are exemplified, and the technical effects (in particular, reprogramming efficiency) of the viral expression plasmids constructed in the present disclosure, methods of inducing the transdifferentiation of fibroblasts into kidney epithelial cells using the viral expression plasmids, are validated by monitoring single cells for fate switching of renal tubular epithelial cell reprogramming. The results show that the use of polycistronic 3 factor combinations EPH1, EH1P and H1EP of the present disclosure significantly improved the efficiency of tubular epithelial reprogramming, with the direct reprogramming efficiencies of EPH1 and EH1P being better, whereas the direct reprogramming efficiency of EPH1 was highest (eventually reaching 62% transdifferentiation efficiency after 5 weeks of culture). The transdifferentiation efficiency of these 3 vectors is greatly superior to the previously reported 4-factor bicistronic and 3-factor bicistronic combinations, and the advantages of the disclosed system are particularly pronounced, especially after 5 weeks or more of culture. Thus, the present disclosure provides a very valuable platform for kidney disease modeling and regenerative medicine. In particular, the methods, viral expression plasmids and kits provided by the present disclosure have the following technical benefits:

(1) The direct induction of tubular epithelial cells (iREC) from fibroblasts provides a great opportunity for renal cell replacement therapy. The problems of low reprogramming efficiency, uncontrollable expression of reprogramming factors, multi-site integration of viral genome and the like generated by the current method have become main obstacles of kidney disease modeling and regeneration methods. Since the first report that repeated infection with four lentiviruses encoding hnf1β, emx2, pax8 and hnf4α can convert mouse embryonic fibroblasts to iREC, few studies have been associated with increasing the efficiency and quality of iREC production, which has greatly lagged the progress in the area of cardiac, liver and neuronal reprogramming. The strategy of using a bicistronic retroviral vector and a dual selection antibiotic to induce kidney reprogramming has been previously reported, and the optimized four-factor combination significantly improves the efficiency of kidney epithelial cell transdifferentiation; the three-factor combination with the Hnf4a removed can activate the fate of the kidney epithelial cells, and the transdifferentiation efficiency is higher than that of the four factors reported before. However, the construction of the previously reported bicistronic combinations (whether factor 4 or factor 3) adds complexity to the combination and there is no substantial improvement in recombination efficiency. The invention point of the present disclosure is that it is found for the first time that polycistronic systems expressing three factors (different from the previously reported bicistronic systems) can induce the fibroblast to be transformed into the tubular epithelial cell, and pay attention to the influence of different hnf1β/Emx2/Pax8 differential expression levels and different ratios on iREC reprogramming efficiency, thereby realizing simple and efficient direct reprogramming of the fibroblast into the tubular epithelial cell and promoting the clinical transformation application thereof. Accordingly, the present disclosure provides an advanced system that can greatly increase the efficiency of fibroblast transdifferentiation into iREC. The method connects 3 different transcription factors through 2 different cleavage sites P2A and T2A, and utilizes the difference of the cleavage efficiency of P2A and T2A in the mRNA translation process, thereby realizing the regulation and control of the relative expression level of the reprogramming factors. We found that the combination of EPH1, EH1P and H1EP all significantly improved the efficiency of tubular epithelial cell reprogramming, with the highest efficiency of EPH1 transdifferentiation (up to 62%), suggesting that differential expression levels and different ratios of reprogramming factors do affect tubular epithelial cell production.

(2) The system provided by the present disclosure (particularly EPH 1) induced iREC successfully exhibits superior characteristics of tubular epithelial cells, such as expression of characteristic tubular epithelial cell markers, formation of 3D cavity structures, assembly in decellularized kidney scaffolds to form tubular structures.

(3) The research of the present disclosure will have a wide impact on the fields of cell reprogramming, tissue regeneration and kidney disease replacement therapy, providing a valuable iREC induction platform for kidney disease modeling and regeneration methods.

The present disclosure describes preferred embodiments, including the best mode known to the inventors for carrying out the present disclosure. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading this disclosure. These variations may be suitably employed by those skilled in the art, and the present disclosure is intended to cover such variations as may be practiced in a manner different than that specifically described herein. Accordingly, this disclosure includes all modifications and equivalents of the subject matter recited in the claims appended hereto. Moreover, unless otherwise indicated herein or clearly contradicted by context, this disclosure is intended to cover any combination of all possible variations of the elements described herein.

Claims

1. A plasmid comprising transcription factors Emx2, pax8, hnf1 β, and 2A polypeptide cleavage site P2A, T a, the transcription factors Emx, pax8, hnf1 β, and 2A polypeptide cleavage site P2A, T a being linked in the following order:

Emx2-P2A-Pax8-T2A-Hnf1β-PuroR、

emx2-P2A-Hnf1 beta-T2A-Pax 8-PuroR or

Hnf1β-P2A-Emx2-T2A-Pax8-PuroR；

Wherein PuroR denotes a puromycin resistance gene.

2. The plasmid of claim 1, wherein the transcription factors Emx2, pax8, hnf1 β, and 2A polypeptide cleavage site P2A, T a are linked in the following order:

Emx2-P2A-Pax8-T2A-Hnf1 beta-PuroR or

Emx2-P2A-Hnf1β-T2A-Pax8-PuroR；

Preferably, the transcription factors Emx2, pax8, hnf1 β and 2A polypeptide cleavage site P2A, T a are linked in the following order:

Emx2-P2A-Pax8-T2A-Hnf1β-PuroR。

3. Plasmid according to claim 1 or 2, wherein the fibroblasts are derived from mammalian embryos, skin, bone and/or muscle, preferably from mouse embryos, more preferably from 12.5 to 14 day old mouse embryos; and/or the plasmid is selected from the group consisting of a retrovirus expression plasmid, a lentivirus expression plasmid, an adenovirus expression plasmid and an adeno-associated virus expression plasmid, preferably a retrovirus expression plasmid.

4. A cell comprising the plasmid of any one of claims 1-3.

5. A kit comprising the plasmid of any one of claims 1-3; preferably, the kit further comprises a sequence selected from the group consisting of SEQ ID NOs: 1 to SEQ ID NO: 18.

6. A method of inducing transdifferentiation of fibroblasts into kidney epithelial cells comprising the steps of:

s1: preparing a fibroblast;

Emx2-P2A-Pax8-T2A-Hnf1β-PuroR、

emx2-P2A-Hnf1 beta-T2A-Pax 8-PuroR or

Hnf1β-P2A-Emx2-T2A-Pax8-PuroR；

Emx2-P2A-Pax8-T2A-Hnf1 beta-PuroR or

Emx2-P2A-Hnf1β-T2A-Pax8-PuroR；

More preferably, the transcription factors Emx2, pax8, hnf1 β, and 2A polypeptide cleavage site P2A, T a are linked in the following order:

Emx2-P2A-Pax8-T2A-Hnf1β-PuroR；

wherein PuroR represents a puromycin resistance gene;

s3: transfecting the plasmid in a packaging cell to obtain a virus;

7. The method of claim 6, wherein step S1 comprises: digesting a tissue obtained from a mammal by trypsin, adding a culture medium to terminate digestion, and culturing fibroblasts obtained by digestion;

Preferably, the concentration of trypsin is 0.05% to 0.25% (w/v), and/or the medium is selected from DMEM, RPMI1640, eagles and/or L-15 medium supplemented with 5% to 20% (v/v) FBS.

8. The method according to claim 6 or 7, wherein step S2 comprises: amplifying the target fragment by PCR, recovering and purifying the target fragment, and connecting each element into a backbone plasmid by homologous recombination; preferably, the backbone plasmid is pMXs plasmid; preferably, the primers used in the PCR amplification are selected from the group consisting of SEQ ID NOs: 1 to SEQ ID NO:18.

9. The method according to claim 6 or 7, wherein in step S3, packaging cells are transfected with the plasmid constructed in step S2, puromycin and blasticidin S are added before transfection, and after a period of time, the supernatant is collected and virus particles in the supernatant are concentrated;

Preferably, step S3 satisfies one or more of the following conditions: prior to transfection, the packaging cells were cultured using a medium of DMEM, RPMI1640, eagles and/or L-15 supplemented with 5% to 20% (v/v) FBS; the packaging cell is selected from Plat-E cells, 293T cells and/or PA317 cells; puromycin is used in an amount of 0.5 to 2.5 μg/mL; the dosage of the blasticidin S is 5 to 20 mug/mL; the virus particles were concentrated using polyethylene glycol.

10. The method according to claim 6 or 7, wherein step S4 comprises: inoculating fibroblasts at a density of 1×10 ³ to 1×10 ⁵/cm ², culturing the fibroblasts, infecting the fibroblasts with the virus obtained in step S3 in an amount of 2×10 ⁶ to 1×10 ⁷ IU/mL, and adding puromycin at a concentration of 0.5 to 2.5 μg/mL after 60-72 hours of virus infection for screening;

preferably, the amount of puromycin for the first 3 days of screening is 1.5 to 2.5 μg/mL, followed by 0.5 to 1.5 μg/mL of puromycin; the concentration of polybrene was added at the time of virus infection at 4 to 8. Mu.g/mL.

11. The method according to claim 6 or 7, wherein in step S4, after infection of the fibroblasts with the virus obtained in step S3, western blot detection is performed on the cell lysates with the expression level ratio of Hnf1β, emx2 to Pax8 being (2.0 to 4.5): 1 (2.0 to 4.5); preferably, the ratio of expression levels of Hnf1β, emx2 to Pax8 is (3.0 to 4.0): 1 (3.0 to 4.0); and/or the number of the groups of groups,

The expression level of hnf1β relative to Hsp90 is 5.0 fold or more, the expression level of Emx2 relative to Hsp90 is 1.2 fold or more, and the expression level of Pax8 relative to Hsp90 is 4.3 fold or more; preferably, the expression level of hnf1β relative to Hsp90 is 5.5-fold or more, emx2 relative to Hsp90 is 1.5-fold or more, and Pax8 relative to Hsp90 is 5.0-fold or more.