Selected renal cell populations, characteristics and uses thereof

Abstract

Methods of identifying an enriched heterogeneous renal cell population having therapeutic potential, enriched heterogeneous renal cell populations having therapeutic potential and uses for same.

Description

Attorney Docket No.55801WO 0728.000558WO01 SELECTED RENAL CELL POPULATIONS, CHARACTERISTICS AND USES THEREOF RELATED APPLICATIONS

[0001] This application claims the benefit of US Provisional Patent Application No, 63 / 705,886 filed on October 10, 2024, US Provisional Patent Application No, 63 / 705,946 filed on October 10, 2024, US Provisional Patent Application No, 63 / 711,042 filed on October 23, 2024, US Provisional Patent Application No, 63 / 711,048 filed on October 23, 2024, and US Provisional Patent Application No, 63 / 733,355 filed on December 12, 2024, which applications are hereby incorporated herein by reference in their entireties to the extent that they do not conflict with the disclosure presented herein. BACKGROUND

[0002] Chronic kidney disease (CKD) is reaching, or has reached, pandemic proportions. Prevalence data from the U.S. to Europe show that approximately 10% of the general population has stage 1-3 CKD. CKD has increased >33% between 1996 and 2006 in the US alone. Meanwhile, management of CKD remains a challenge for nephrologists. Most standard-of-care treatments for CKD are small molecules targeting biochemical pathways in the kidney to affect single or related co-morbidities. However, these treatments do not affect the underlying CKD kidney’s glomerular and tubulointerstitial dysfunction. Ultimately, in patients that progress to end stage renal disease (ESRD), renal replacement therapy (RRT; dialysis or transplantation) is required for survival. There remains an unmet need for a therapeutic modality that directly addresses the diseased kidney’s tissue biology. Such a therapeutic modality can potentially arrest or even reverse CKD progression and mitigate or avert the need for RRT.

[0003] Selected renal cells (SRC), a heterogeneous renal cell population enriched for renal epithelial cells, are being advanced as autologous cell-based therapy for treatment of CKD. Derived from the donor kidney, SRC is primarily composed of kidney epithelial cells. Results from a Phase II clinical trial in a diabetic kidney disease cohort suggested that SRC administration is well tolerated and may stabilize or improve renal function.Attorney Docket No.55801WO 0728.000558WO01

[0004] There is a need in the art to identify enriched heterogenous renal cell populations, such as SRCs, that have potential therapeutic activity. BRIEF SUMMARY

[0005] The present disclosure relates generally to a method of identifying an enriched heterogeneous renal cell population as having a therapeutic potential. In particular, some embodiments of the disclosed methods include (a) profiling expression levels of a panel of genes associated with (i) renal cell identity, (ii) tissue healing response (renewal and repair), (iii) fibrosis, (iv) inflammation, and / or (v) senescence-associated secretory phenotype (SASP); (b) determining the presence of a gene signature biomarker in the enriched heterogeneous renal cell population based at least in part upon the profiled expression levels; and (c) identifying the enriched heterogeneous renal cell population as having therapeutic potential if the gene signature biomarker is determined to be present in the enriched heterogeneous renal cell population.

[0006] In one aspect, the present disclosure relates to a method of identifying an enriched heterogeneous renal cell population as having a therapeutic potential, comprising: (a) profiling expression levels of a panel of genes associated with (i) renal cell identity, and / or (ii) tissue healing response (renewal and repair) in the enriched heterogeneous renal cell population; (b) determining the presence of a gene signature biomarker in the enriched heterogeneous renal cell population based at least in part upon the profiled expression levels; and (c) identifying the enriched heterogeneous renal cell population as having therapeutic potential if the gene signature biomarker is determined to be present in the enriched heterogeneous renal cell population.

[0007] In one aspect, the present disclosure relates to a method of preparing a formulation comprising an enriched heterogeneous renal cell population, comprising: (a) identifying the enriched heterogeneous renal cell population as suitable for formulation, wherein identifying comprises: (i) profiling expression levels of a panel of genes associated with (i) renal cell identity, and / or (ii) tissue healing response (renewal and repair) in the enriched heterogeneous renal cell population; (ii) determining the presence of a gene signature biomarker in the enriched heterogeneous renal cell population based at least in part upon the profiled expression levels; and (iii) identifying the enriched heterogeneous renal cell population as suitable for formulation if the gene signature biomarker is determined to be present in the enriched heterogeneous renal cell population, and (b) combining the enriched heterogeneous renal cell population that has beenAttorney Docket No.55801WO 0728.000558WO01 identified as being suitable for formulation with a pharmaceutically acceptable carrier or excipient to prepare the formulation.

[0008] In one aspect, the present disclosure relates to a method of treating a subject in need of treatment for chronic kidney disease (CKD) comprising: (a) identifying an enriched heterogeneous renal cell population as suitable for treating the subject according to steps comprising: (i) profiling expression levels of a panel of genes associated with (i) renal cell identity, and / or (ii) tissue healing response (renewal and repair) in the enriched heterogeneous renal cell population; (ii) determining the presence of a gene signature biomarker in the enriched heterogeneous renal cell population based at least in part upon the profiled expression levels; and (iii) identifying the enriched heterogeneous renal cell population as suitable for treating the subject if the gene signature biomarker is determined to be present in the enriched heterogeneous renal cell population, and (b) administering the enriched heterogeneous renal cell population that has been identified as suitable for treating the subject to the subject to treat the CKD.

[0009] In another aspect, the present disclosure relates to a method of identifying an enriched heterogeneous renal cell population as having a therapeutic potential, comprising: (a) profiling expression levels of a panel of genes associated with (i) fibrosis, (ii) inflammation, and / or (iii) senescence-associated secretory phenotype (SASP); in the enriched heterogeneous renal cell population; (b) determining the presence of a gene signature biomarker in the enriched heterogeneous renal cell population based at least in part upon the profiled expression levels; and (c) identifying the enriched heterogeneous renal cell population as having therapeutic potential if the gene signature biomarker is determined to be present in the enriched heterogeneous renal cell population.

[0010] In another aspect, the present disclosure relates to a method of identifying an enriched heterogeneous renal cell population as having a therapeutic potential, the method comprising: (a) profiling expression levels of a panel of genes expressed by cells of the enriched heterogeneous renal cell population; (b) identifying one or more cell types in the enriched heterogeneous renal cell population, wherein the one or more cell types are identified based at least in part on presence of a cell type / state gene signature biomarker comprising one or more differentially expressed genes; and (c) identifying the enriched heterogeneous renal cell population as having therapeutic potential if one of the one or more cell types comprises an indicator gene signature biomarker.Attorney Docket No.55801WO 0728.000558WO01 BRIEF DESCRIPTION OF FIGURES

[0011] FIG.1A - 1G: Show percentage of cells expressing renal cell type identity markers (CD10 (FIG.1A), CD13 (FIG.1B), CD224 (FIG.1C), CD227 (FIG.1D), CD326 (FIG.1E), GATA3 (FIG.1F) and podoplanin (PDPN; FIG.1G)) at timepoint 1 (T1), timepoint 2 (T2), timepoint 3 (T3) and selected renal cell (SRC) stages of rilparencel manufacturing.

[0012] FIG.2A - 2D: Co-expression of renal cell type identity markers on cells from samples taken at varying stages, from renal biopsy to final product, of rilparencel manufacturing. FIG. 2A provides flow cytometry data showing expression of CD13 (x-axis) and CD10 (y-axis) on cells at biopsy, timepoint 0 (T0), T2, and SRC stages of rilparencel manufacturing. FIG.2B provides flow cytometry data showing expression of CD13 (x-axis) and CD227 (y-axis) on cells at biopsy, T0, T2, and SRC stages of rilparencel manufacturing. FIG.2C provides flow cytometry data showing expression of CD13 (x-axis) and CD224 (y-axis) on cells at T0, T2, and SRC stages of rilparencel manufacturing. FIG.2D provides a plot showing that of the four quadrants in FIG.2B for biopsy cells, CD10 is largely present on CD13+CD227- cells.

[0013] FIG.3: Shows flow cytometry data for expression of proximal tubular markers CD13 (x-axis) and CD224 (y-axis) on a sample of cells taken at the T0 stage of rilparencel manufacturing.

[0014] FIG.4: Provides t-distribute stochastic neighbor embedding (tSNE) analysis of flow cytometry data generated using the antibody clones described in Table 1 to identify cell clusters at the T0 stage of rilparencel manufacturing.

[0015] FIG.5A - 5B: Provide tSNE analyses of concatenated timepoints. The concatenation of cells from T0, T2, and SRC samples showed clustering through markers CD13, CD224, CD227, and PDPN. FIG.5A shows clustering of cells delineated by both concatenation and individual contributions of each manufacturing stage. Shading of cell populations shows them becoming more CD13+CD224+ during manufacturing process, with relative loss of other cell types. FIG. 5B shows that there are two primary clusters in SRCs, and that they are separated by CD227 expression.Attorney Docket No.55801WO 0728.000558WO01

[0016] FIG.6A - 6B: Provides flow cytometry data showing expression of proximal tubular marker CD13 (x-axis) and glomerular marker PDPN (y-axis) by cells of each of seven different rilparencel samples (FIG.6A) and flow cytometry data showing expression of PDPN and CD106 by rilparencel cells (FIG.6B).

[0017] FIG.7A - 7G: Show flow cytometry data demonstrating that some cells having a proximal tubule (CD13+CD10+) phenotype at T1, T2, T3, and SRC stages of manufacturing also express distal tubule cell markers such as CD227 or GATA3. FIG.7A shows the gating strategy for phenotyping live single CD13+CD10+ cells in FIG.7B - 7G. FIG.7B - 7E show that some CD13+CD10+ cells at each of rilparencel manufacturing stages T1 (FIG.7B), T2 (FIG.7C), T3 (FIG.7D), and SRC (FIG.7E) are also positive for CD227; each histogram provided in FIG.7B - 7E shows CD227 expression for each of seven separate samples taken through manufacturing to SRCs. FIG.7F provides percent CD13+CD10+ cells that are also positive for CD227 expression at each of the T1, T2, T3, and SRC stages of rilparencel manufacturing. FIG.7G provides percent CD13+CD10+ cells that are also positive for GATA3 expression at each of the T1, T2, T3, and SRC stages of rilparencel manufacturing.

[0018] FIG.8A - 8G: Show percentage of cells from each of the T1, T2, T3, and SRC stages of rilparencel manufacturing that stain positive for renewal and repair markers CD24 (FIG.8A), CD44 (FIG.8B), CD90 (FIG.8C), CD106 (FIG.8D), CD133 (FIG.8E), CD146 (FIG.8F), and Vimentin (FIG.8G).

[0019] FIG.9A - 9C: Provide flow cytometry data showing co-expression of renewal and repair markers by cells at the T0, T2, and SRC stages of rilparencel manufacturing. FIG.9A provides flow cytometry data for expression of markers CD24 (x-axis) and CD133 (y-axis) on cells at the T0, T2, and SRC stages of rilparencel manufacturing. FIG.9B shows, for each quadrant in the flow cytometry data immediately above it in FIG.9A, CD90 expression on those cells. FIG.9C shows, for each quadrant in the flow cytometry data immediately above it in FIG. 9A, CD146 expression on those cells.

[0020] FIG.10: Provides a heatmap showing differences in levels of secreted proinflammatory cytokines by cells at an early timepoint (T2) in, relative to the SRC (rilparencel) stage of, rilparencel manufacturing.Attorney Docket No.55801WO 0728.000558WO01

[0021] FIG.11: Provides a heatmap showing differences in levels of secreted fibrosis and kidney toxicity markers by cells at an early timepoint (T2) in, relative to the SRC (rilparencel) stage of, rilparencel manufacturing.

[0022] FIG.12: Provides a principal component analysis (PCA) plot of secretome analysis, demonstrating that the early timepoint (T2) and final SRC (rilparencel) product samples from five CKD patients form distinct clusters.

[0023] FIG.13: Provides a heatmap showing differences in levels of secreted canonical senescence markers by cells at an early timepoint (T2) in, relative to the SRC (rilparencel) stage of, rilparencel manufacturing.

[0024] FIG.14A - 14B: Show different representations of senescence associated secretory phenotype (SASP) from the SASP atlas.

[0025] FIG.15: Show percentage of cells from each of the T1, T2, T3, and SRC stages of rilparencel manufacturing that stain positive for renewal and repair markers CD90 and CD106.

[0026] FIG.16: Show percentage of cells from each of the T1, T2, T3, and SRC stages of rilparencel manufacturing that stain positive for renewal and repair markers CD90 and CD133.

[0027] FIG.17A - 17B: Shows percentage of cells at each of the T0, T1, T2, T3, and SRC stages of rilparencel manufacturing that stain positive for proximal tubule markers CD13 and CD10 (FIG.17A) and proximal tubule markers CD13 and CD224 (FIG.17B).

[0028] FIG.18A - 18E: Provides evidence of a reduced inflammatory response in the majority of a subpopulation of rilparencel cells, (CD90+CD106+, self-renewing cells). The rilparencel cells used to test whether CD90+CD106+ cells are associated with reduced inflammation were generated from a kidney obtained from National Disease Research Interchange (NDRI). Rilparencel cells generated from the NDRI kidney were sorted into CD90- / CD106-, CD90+ / CD106-, CD90- / CD106+, and CD90+ / CD106+ populations. Each of the four sorted cell populations, and a sample of the cells before being sorted (pre-sorted cells), were plated. Level of inflammation-associated molecules IL-6 (FIG.18A), IL-8 (FIG.18B), CCL2 (FIG.18C), MIP-1b (FIG.18D), and TNFa (FIG.18E) secreted by cells were quantitated. Overall, CD90+CD106+ cells secreted at least two-fold less of the inflammation-associated molecules. In each of FIG.18A - FIG.18E quantity secreted molecules by cells for which the data areAttorney Docket No.55801WO 0728.000558WO01 shown, displayed as clustered dots from left to right are: pre-sorted cells, CD90-CD106- cells, CD90+ / CD106- cells, CD90- / CD106+ cells, and CD90+ / CD106+ cells (with quantity secreted molecule by CD90+ / CD106+ cells being in circles).

[0029] FIG.19: Provides evidence that rilparencel (SRC) cells secrete molecules with potential for tissue remodeling. Secretome of T2 and rilparencel cells manufactured from kidney biopsies of patients having diabetes and chronic kidney disease in a phase 2 clinical trial (NCT05018416; n=12) were compared for differences in quantity of secreted factors (SRC / T2). Additionally, secretome of rilparencel cells manufactured from the patient samples (n=12) and rilparencel manufactured from a non-diseased kidney (n=2; SRC / Control) were compared. Secretome evaluation was for 80 molecules, including 29 associated with pro- and anti-inflammatory processes (e.g., IL-6, IL-8, CCL2, MIP-1b, TGFb1-3, TNFa, sTNF-R1, and CD137), 18 associated with kidney toxicity (e.g., A1M, b2M, calbindin, clusterin, cystatin C, GSTA1, KIM- 1, NGAL, osteoactivin, OPN, TIMP-1, and VEGF), 8 involved in tissue remodeling (e.g., MMP- 1, MMP-7, and MMP-9), and 10 linked to growth factors (e.g., LIF and uPAR). Molecules of the secretomes were detected 24 hours after cell plating. Overall, with the exception of TGFb1, secretome of rilparencel manufactured from patient kidneys was comparable to secretome of rilparencel manufactured from non-diseased kidneys. Notably, comparing secretome of the T2 and rilparencel cells of the clinical trial patients revealed evidence of cells showing increased anti-inflammatory and remodeling type responses over the course of manufacturing (e.g., greater fold-change for MMP-1, NGAL, sTNF-R1, and clusterin).

[0030] FIG.20A - 20B: Identification and distribution of cell types in a cortical kidney biopsy sample obtained from a National Disease Research Interchange (NDRI) kidney. FIG.20A shows flow cytometry data for expression of CD13 (x-axis) and CD326 (y-axis) by cells in the cortical kidney biopsy sample. Cells gated as CD13+CD326- were identified as proximal tubular cells and cells gated as CD13-CD326+ were identified as distal tubule cells. The proximal tubule cells made up a higher percentage of cells (at 31.4%) in the sample than did distal tubular cells (at 0.21%). Around 35% of the cells in this biopsy sample were non-renal, e.g., lymphocyte, cells. FIG.20B shows that proximal tubule markers (CD10 and CD224), and distal tubule markers (CD227 and CD171) were expressed by distinct, e.g., either proximal or distal tubule, cell populations in the biopsy sample.Attorney Docket No.55801WO 0728.000558WO01

[0031] FIG.21: Provides flow cytometry data showing renal progenitor-like marker expression, CD24 (x-axis) and CD133 (y-axis), by cortical kidney biopsy cells and cells from samples taken at the T0 and SRC (final) stages of rilparencel manufacturing. At biopsy, a small population of CD24+CD133+ cells was present. At the T0 stage of manufacturing, the CD24+CD133+ population greatly increased, becoming the prevalent cell population. At the final, SRC, stage of manufacturing, the CD24+CD133+ cells present at the T0 stage of manufacturing appeared to have dispersed into a largely CD24+ population. The starting cortical kidney biopsy samples and T0 and SRC cell samples subject to flow cytometry to generate this figure were prepared from biopsies obtained from NDRI kidneys.

[0032] FIG.22A - 22C: Development of renal cell plasticity over the course of rilparencel manufacture. FIG.22A shows flow cytometry data presenting the evolution of cell proliferation (CD133, y-axis) and de- / re-differentiation (CD90, x-axis) marker expression by cells over the course, from the T0 to SRC (or final) stage, of rilparencel manufacturing. The T0, T1, T2, T3, and SRC cell samples used to generate the flow cytometry data in FIG.22A were from a single manufacturing run that began with a kidney biopsy sample obtained from an NDRI kidney. FIG. 22B and 22C graphically display how cells from samples at early, e.g., T0 and T1, stages of manufacturing go from a more CD133+CD90- phenotype to a more CD133-CD90+ phenotype at later, e.g., T3 and SRC, stages of manufacturing. The T1, T2, T3, and SRC cell samples used to generate the graphical data in FIG.22B and 22C were from manufacturing runs that began with clinical trial patient cortical kidney biopsies (REGEN-007 / NCTG05018416 patients (n=7)) that produced investigational rilparencel product.

[0033] FIG.23A - 23G: Over the course of rilparencel manufacturing, cells acquire a proximal and distal nephron marker co-expression phenotype. FIG.23A provides flow cytometry data showing that, at the early T0 stage of manufacturing, groups of cells were found to express proximal tubule epithelial marker CD13 (x-axis)), or proximal nephron marker CD224 (y-axis), or both CD13 and CD224. It also shows that, at the SRC (final) rilparencel stage of manufacturing, cells largely came to express both the CD13 and CD224 proximal tubule markers. FIG.23B and 23C graphically show the percent increase in cells taken from samples at the T0, T1, T2, T3, and SRC stages of rilparencel manufacturing that expressed both CD13 and CD10 (FIG.23B) or both CD13 and CD224 (FIG.23C). FIG.23D provides flow cytometry data showing how cells at the early, T0, stage of manufacturing tended to express proximal tubuleAttorney Docket No.55801WO 0728.000558WO01 epithelial marker CD13 (x-axis), or distal nephron marker CD171 (y-axis), with some cells co- expressing both CD13 and CD171, while cells at the SRC (final) stage of rilparencel manufacturing came to largely express both CD13 and CD171. FIG.23E graphically depicts the percent cells that expressed CD171 at the T0 and SRC (final) rilparencel manufacturing time points. FIG.23F provides a graph showing the percent CD13+CD10+ cells, e.g., cells having a proximal tubule epithelial phenotype, that additionally expressed either distal nephron marker CD227 or distal nephron marker CD171, FIG.23G graphically displays the increase in expression of distal nephron marker CD171 (left panel) and distal marker CD227 (right panel) by CD13 expressing cells. The T0, T1, T2, T3, and SRC cell samples used to generate the data in FIG.23A and 23G were from a rilparencel manufacturing run that produced SRC from an NDRI kidney biopsy.

[0034] FIG.24A - 24B: Acquisition of a proximal and distal nephron marker co-expression phenotype by cells over the course of rilparencel manufacturing. FIG.24A graphically shows the percent cells in samples taken at the T0, T2, and SRC stages of manufacturing that expressed proximal nephron marker CD13 (LAP), proximal nephron marker GGT1 (CD224), or distal nephron marker GATA3. FIG.24B graphically shows the percentage cells in samples taken at the T0, T2, and SRC stages of manufacturing that were CD13 (LAP)+GATA3+, CD224 (GGT1)+GATA3+, CD13 (LAP)-GATA3+, and CD224 (GGT1)-GATA3+. The T0 cell samples used to generate the data in FIG.24A and 24B were taken from a rilparencel manufacturing run that produced SRC from an NDRI kidney biopsy. The T2 and SRC cell samples used to generate the data in FIG.24A and 24B were from manufacturing runs that began with clinical trial patient cortical kidney biopsies (REGEN-007 / NCTG05018416 patients (n=7)) that produced investigational rilparencel product.

[0035] FIG.25A - 25B: Provide flow cytometry data showing that from the T1 to SRC (final) stages of rilparencel manufacturing, cells that expressed proximal tubule markers CD13 (FIG. 25A) and CD224 (FIG.25B) less frequently co-expressed CD133, and did so as they become more strongly proximal in nature. The, T1, T2, T3, and rilparencel cell samples used to generate the data in FIG.25A and 25B were from a manufacturing run that produced SRC from an NDRI kidney biopsy.

[0036] FIG.26A - 26B: Provide flow cytometry data showing that from the T1 to SRC (final) stages of rilparencel manufacturing, cells that expressed distal nephron markers CD227Attorney Docket No.55801WO 0728.000558WO01 (FIG.26A) and CD171 (FIG.26B) less frequently co-expressed CD133. The, T1, T2, T3, and rilparencel cell samples used to generate the data in FIG.26A and 26B were from a manufacturing run that produced SRC from an NDRI kidney biopsy.

[0037] FIG.27A - 27B: Provide flow cytometry data showing that from the T1 to SRC (final) stages of rilparencel manufacturing CD90+ cells transitioned to become more proximal tubule in nature through their greater expression of proximal markers CD13 (FIG.27A) and CD224 (FIG.27B). The, T1, T2, T3, and rilparencel cell samples used to generate the data in FIG.27A and 27B were from a manufacturing run that produced rilparencel from an NDRI kidney biopsy.

[0038] FIG.28A - 28B: Provide flow cytometry data showing that from the T1 to SRC (final) stages of rilparencel manufacturing CD90+ cells more readily acquired, or co-expressed, distal nephron marker CD227 (FIG.28A) than CD171 (FIG.28B). The, T1, T2, T3, and rilparencel cell samples used to generate the data in FIG.28A and 28B were from a manufacturing run that produced rilparencel from an NDRI kidney biopsy.

[0039] FIG.29: Provides a table summarizing the percent cells in T0, T1, T2, T3, or SRC cell samples that expressed each renal cell identity marker presented in Table 16. The SRC samples used to generate the data in FIG.29 are based on the number of as-noted retains of investigational rilparencel product that had been manufactured for patients enrolled in a phase 2 clinical trial (“(Clinical)”; REGEN-002 / NCT02836574 and / or REGEN-007 / NCTG05018416) and, separately, prepared from biopsies of kidneys obtained from NDRI (“(PK)”). SD = standard of deviation.

[0040] FIG.30: Provides a table summarizing the percent cells in T0, T1, T2, T3, or SRC cell samples that express each renal cell progenitor and self-renewal marker presented in Table 17. The SRC samples used to generate the data in FIG.30 are based on the number of as-noted retains of investigational rilparencel product that had been manufactured for patients enrolled in a phase 2 clinical trial (“(Clinical)”; REGEN-002 / NCT02836574 and / or REGEN- 007 / NCTG05018416) and, separately, prepared from biopsies of kidneys obtained from NDRI (“(PK)”). SD = standard of deviation.

[0041] FIG.31: Provides a heatmap showing differences in levels of secreted pro-inflammatory cytokines by cells at the T0, T1, T2, T3, and final (SRC) stages of rilparencel manufacturing. The T1, T2, T3, and SRC cell samples used to generate the data in FIG.31 were fromAttorney Docket No.55801WO 0728.000558WO01 manufacturing runs that began with clinical trial patient cortical kidney biopsies (REGEN- 007 / NCTG05018416 patients (n=6)) and produced investigational rilparencel product.

[0042] FIG.32A-32B: Shows evolution of renal epithelial cell self-renewal marker expression by cells as they progress from the T0 to SRC stage of rilparencel manufacturing. FIG.32A provides a graphical representation of percentage of cells in samples taken from the T0, T1, T2, T3, and SRC stages of manufacturing determined to be CD90-CD133+ or CD90+CD133-. FIG. 32B provides a graphical representation of percentage of cells in samples taken from the T0, T1, T2, T3, and SRC stages of manufacturing determined to be CD106-CD133+ or CD106+CD133-. The T0 cell samples used to generate the data in FIG.32A and 32B were prepared from biopsies of NDRI kidneys (n=6 for FIG.32A and n=4 for FIG.32B). The T1, T2, T3, and SRC cell samples used to generate the data in FIG.32A were prepared from manufacturing runs that began with clinical trial patient (REGEN-002 / NCT02836574 and REGEN-007 / NCTG05018416) cortical kidney biopsies (n=12) and biopsies of NDRI kidneys (n=6). The T1, T2, T3, and SRC cell samples used to generate the data in FIG.32B were prepared from manufacturing runs that began with both clinical trial patient (REGEN-002 / NCT02836574 and REGEN- 007 / NCTG05018416) cortical kidney biopsies (n=12) and biopsies of NDRI kidneys (n=4).

[0043] FIG.33A-33D: Graphically displays relative level of intracellular proinflammatory cytokine staining in unsorted rilparencel (SRC) cells relative to each of CD90+CD106+, CD90+CD106-, CD90-CD106+, and CD90-CD106- sorted rilparencel (SRC) cells. IL-6 (FIG.33A); IL-8 (FIG. 33B); MCP-1 (FIG. 33C); and TNF- (FIG. 33D). The SRC sample used togenerate the data in FIG.33A -33D was prepared from a manufacturing run that began with a biopsy of an NDRI kidney. Samples were run with ≥2 technical replicates.

[0044] FIG. 34A-34D: Provides characterization of intracellular TNF- expression by cellsharvested at multiple steps during rilparencel manufacturing. FIG.34A shows a representativestaining of cells for intracellular TNF- + / - treatment with a protein transport inhibitor (PTI)cocktail at the T0, T1, and final SRC stages of rilparencel manufacturing. FIG.34B provides a plot presenting the percent PTI-treated T0, T1, and rilparencel (SRC) cells that expressed TNF- .FIG. 34C and 34D provide percent of PTI-treated TNF- + or TNF- - cells at the T0 (FIG.34C), and T1 (FIG.34D) stages of manufacturing that expressed proximal nephron (CD13 and CD224), distal nephron (CD171) or self-repair (CD90, CD106, CD133) markers. The T0, T1, and SRC samples used to generate the data in FIG.34A and the T0 cell sample used to generateAttorney Docket No.55801WO 0728.000558WO01 the data in FIG.34B-34D were prepared from a manufacturing run that began with the biopsy of an NDRI kidney. The T1 and SRC cell samples used to generate the data in FIG.34B and the T1 cell sample used to generate the data in FIG.34C-34D were prepared from manufacturing runs that began with clinical trial patient (REGEN-007 / NCTG05018416) cortical kidney biopsies (n=4) that produced investigational rilparencel product and a biopsy of an NDRI kidney (n=1).

[0045] FIG.35A - 35G: Uniform manifold approximation and projection (UMAP) showing distribution of cell types and cell states in each of five (FIG.35A - 35E) rilparencel drug product samples. For comparison, UMAPs generated from a single cell atlas of chronic kidney disease and healthy patients (Kidney Precision Medicine Project (KPMP); FIG.35F) and a single cell atlas of developing kidney (GenitoUrinary Development Molecular Anatomy Project (GUDMAP); FIG.35G) are provided.

[0046] FIG.36: Provides the proportion of five KPMP-like cell types within each of five rilparencel drug product samples.

[0047] FIG.37: Provides a plot showing, for each of the five KPMP-like cell types in each rilparencel drug product sample: (i) percent cells, and level of expression by cells, within that cell type expressing the top 5 differentially expressed genes (DEGs) for that cell type; and (ii) percent cells, and level of expression by cells, within that cell type expressing canonical KPMP gene markers for that cell type. Top 5 DEGs assigned by average Log2 fold change. * denotes canonical KPMP gene markers for the cell types.

[0048] FIG.38: Provides a graphical representation of estimated glomerular filtration rate (eGFR) slopes, from time of first rilparencel injection to 12 months post first rilparencel injection, for each of five chronic kidney disease patients enrolled in a phase 2 clinical trial (NCT02836574).

[0049] FIG.39: Shows results of an analysis conducted to identify biologic processes associated with rilparencel samples from patients who had +eGFR slopes versus -eGFR slopes from time of first rilparencel injection to 12 months post first rilparencel injection. The number of genes identified from each pathway are shown on the x-axis and the adjusted p value is shown as the dot shade. The gene ratio (circle size) is the number inputs DEGs associated with the Biological Pathway / the total number of input DEGs (e.g., higher gene ratios indicates that theAttorney Docket No.55801WO 0728.000558WO01 input DEGs make up a larger proportion of the total DEGs found between the two clinical outcomes groups).

[0050] FIG.40A - 40D: Provide plots showing DEGs, and associated pathways, within ascending thin limb / adaptive thick ascending limb (ATL / aTAL; FIG.40A and 40C) or adaptive proximal tubule (aPT; FIG.40B and 40D) cell types of rilparencel samples from patients’ who had +eGFR slopes versus -eGFR slopes from time of first rilparencel injection to 12 months post first rilparencel injection. DEFINITIONS

[0051] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0052] Although various features of the disclosure can be described in the context of a single embodiment, the features can also be provided separately or in any suitable combination. Conversely, although the present disclosure can be described herein in the context of separate embodiments for clarity, the present disclosure can also be implemented in a single embodiment. It is to be understood that the present disclosure is not limited to the particular embodiments described herein and as such can vary. Those of skill in the art will recognize that there are variations and modifications of the present disclosure, which are encompassed within its scope.

[0053] All terms are intended to be understood as they would be understood by a person skilled in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains.

[0054] It is intended that every maximum numerical limitation given throughout the present disclosure includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.Attorney Docket No.55801WO 0728.000558WO01

[0055] As used in the specification and claims, “about” with reference to a particular number or value means within 10% of the particular number or value. It should be understood that if the percentage of cells expressing a certain marker is provided as being a percentage of “about” a particular number e.g., about 5%, the percentage of cells need not be exactly the particular number, e.g., exactly 5%. Rather, it should be understood that if the percentage of cells expressing a certain marker is provided as being “about” a particular number, e.g., about 5%, then the percentage of cells expressing the certain marker can be within up to 10% of that particular number, e.g., between 4.5% and 5.5%. It should also be understood that if cells express a certain marker by secreting it at an amount of “about” a particular number, e.g., about 5 ng, the amount need not be exactly that particular number, e.g., exactly 5 ng. Rather, it should be understood that the secreted amount of the marker can be within 10% of the particular number, e.g., between 4.5 and 5.5 ng.

[0056] It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, can also be provided in combination in a single embodiment. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, can also be provided separately or in any suitable sub- combination. All combinations of the embodiments pertaining to the disclosure are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present disclosure and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

[0057] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. It should also be noted that the term “or” is generally employed in its sense including “and / or” unless the content clearly dictates otherwise. The terms “and / or” and “any combination thereof” and their grammatical equivalents as used herein can be used interchangeably. These terms can convey that any combination is specifically contemplated. Solely for illustrative purposes, the following phrases “A, B, and / or C” or “A, B, C, or any combination thereof” can mean “A individually; B individually; C individually; A and B; B and C; A and C; and A, B, and C.” The term “or” can be used conjunctively or disjunctively unless the context specifically refers to a disjunctive use. TheAttorney Docket No.55801WO 0728.000558WO01 use of the term “or” implies that the disjunctive sense is contemplated. That is, while “A, B, or C” may mean “A, B, and / or C” or “A, B, C, or any combination thereof,” “A, B, or C” will also include “A or B or C, but not (A and B), (A and C), (B and C), and (A, B, and C).”

[0058] As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the present disclosure, and vice versa. Furthermore, compositions of the present disclosure can be used to achieve methods of the present disclosure.

[0059] An article, composition, method, or the like that comprises one or more elements encompasses “consists” of the one or more elements and / or “consists essentially” of the one or more elements. As used in this specification and claim(s), “consisting of” (and any form of consisting of, such as “consists of” and “consist of”) means including and limited to. As used in this specification and claim(s), an article, composition, method, or the like “consisting essentially of” (and any form of consisting essentially of, such as “consists essentially of” and “consist essentially of”) means the article, composition, method, or the like includes the specified enumerated elements; such as components, compounds, materials, steps, or the like, and may include additional elements that do not materially affect the basic and novel characteristics of the article, composition, method, or the like. For example, subsumed within a panel of genes comprising GGT1, CK18, GATA3, PDPN, VEGF, and KIM-1 is a panel of genes consisting essentially of GGT1, CK18, GATA3, PDPN, VEGF, and KIM-1 and a panel of genes consisting of GGT1, CK18, GATA3, PDPN, VEGF, and KIM-1.

[0060] Reference in the specification to “some embodiments,” “an embodiment,” “one embodiment,” “one or more embodiments,” “embodiments” or “other embodiments” means that a particular feature, structure, or characteristic described in connection with the embodiments is included in at least one or more embodiments, but not necessarily all embodiments, of the present disclosure. To the extent that the disclosure describes aspects, components, or elements associated with a particular embodiment in more detail or breadth, it is contemplated that theAttorney Docket No.55801WO 0728.000558WO01 aspects, components, or elements associated with such embodiment should be understood to encompass the additional detail and breadth described in the disclosure.

[0061] Ranges provided herein are understood to be shorthand for all the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, as well as all intervening decimal values between the aforementioned integers such as, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9. With respect to sub-ranges, “nested sub-ranges” that extend from either end point of the range are specifically contemplated. For example, a nested sub-range of an exemplary range of 1 to 50 may comprise 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or 50 to 40, 50 to 30, 50 to 20, and 50 to 10 in the other direction.

[0062] Numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0063] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements.

[0064] In several places throughout the application, guidance is provided through examples, which examples, including the particular aspects thereof, can be used in various combinations and be the subject of claims. In each instance, the recited elements serve only as a representative group and should not be interpreted as an exclusive list. It is to be understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention as set forth herein, and it is contemplated that the variousAttorney Docket No.55801WO 0728.000558WO01 aspects set forth in the examples and the disclosure may be combined and set forth in patentably distinct claims.

[0065] For any method disclosed herein that includes discrete steps, the steps may be conducted in any feasible order, and, as appropriate, any combination of two or more steps may be conducted simultaneously. DETAILED DESCRIPTION

[0066] The present disclosure relates generally to methods of identifying an enriched heterogeneous renal cell population, such as a selected renal cell population (SRC), as having a therapeutic potential, a composition including an enriched heterogeneous renal cell population identified as having a therapeutic potential, and methods and uses of an enriched heterogeneous renal cell population identified as having a therapeutic potential.

[0067] In the methods of identifying an enriched heterogeneous renal cell population as having a therapeutic potential, the therapeutic potential of the enriched heterogeneous renal cell population may be in the treatment of a kidney disease, a tubular transport deficiency, or a glomerular filtration deficiency.

[0068] The therapeutic potential of the enriched heterogeneous renal cell population identified by the methods may be a restoration of kidney function, stabilization of kidney function, improvement in kidney function, reduction of renal fibrosis, reduction in renal inflammation, induction of tubulogenesis in a kidney, induction of nephrogenesis in a kidney, or induction of glomerulogenesis in a kidney of a patient in need of such treatment. The therapeutic potential of the heterogeneous renal cell population identified by the methods may be a restoration of mineral balance, electrolyte balance, fluid homeostasis, reabsorption of essential nutrients, cystatin C metabolism, or an alleviation of anemia in a patient in need of such treatment. The therapeutic potential of the enriched heterogeneous renal cell population identified by the methods may be the potential to delay or prevent the need for dialysis, or delay or prevent the need for a kidney transplant in a patient in need of a treatment for a kidney disease.

[0069] As described in greater detail below, the identification of an enriched heterogeneous renal cell population having a therapeutic potential in accordance with some embodiments of the disclosure may be achieved by using a combination of approaches and techniques. For example,Attorney Docket No.55801WO 0728.000558WO01 the experimental data presented herein illustrate that the characterization of an enriched heterogeneous renal cell population using immunophenotyping by flow cytometry in combination with immunoassays can provide expanded understanding of the biological functions of the cell components that make up the enriched heterogeneous renal cell population. Furthermore, better understanding of renal epithelial cell plasticity during preparation of the enriched heterogeneous renal cell population can inform on potential favorable implications to clinical outcome. The methods provided herein may involve profiling expression level of a panel of genes, or of differentially expressed genes (DEGs), of cells of the enriched heterogeneous renal cell populations.

[0070] In one aspect, some embodiments of the disclosure provide a method of identifying an enriched heterogeneous renal cell population as having a therapeutic potential, including: (a) profiling expression levels of a panel of genes associated with (i) renal cell identity, (ii) tissue healing response (renewal and repair), (iii) fibrosis, (iv) inflammation, and / or (v) senescence- associated secretory phenotype (SASP); (b) determining the presence of a gene signature biomarker in the enriched heterogeneous renal cell population based at least in part upon the profiled expression levels; (c) identifying the enriched heterogeneous renal cell population as having therapeutic potential if the gene signature biomarker is determined to be present in the enriched heterogeneous renal cell population.

[0071] Non-limiting exemplary embodiments of the methods according to the present disclosure include one or more of the following features. In some embodiments, the gene signature biomarker may include one or more genes selected from the group consisting of CD227 (EMA), CD326 (EpCAM), CD13 (ANPEP), CD224 (GGT), PDPN, CD10 (MME), GATA3, CD171, OCT2 (SLC22A2), NBCe1 (SLC4A4), NDCBE1 (SLC4A8), NKCC2 (SLC12A1), NHE3 (SLC9A3), NCC (SLC12A3), and SLC26A4 (Pendrin). In some embodiments, the gene signature biomarker may include one or more genes selected from the group consisting of CD90 (Thy-1), CD24 (heat stable antigen (HSA)), Vimentin, CD106 (VCAM-1), CD146 (MCAM), CD44 (H-CAM), and CD133 (Prom1). In some embodiments, the gene signature biomarker may include one or more genes selected from the group consisting of IP-10, MCP-1, MIP-1a, MIP-1b, tumor necrosis factor alpha (referred to herein as TNFa or TNFα), IL-6, IL-8, IL-18, Calbindin, Clusterin, GSTA1, KIM-1, Osteoactivin, RBP4, Renin, VEGF-A, a-1 microglobulin, b-2 microglobulin, Cystatin, Osteopontin, TFF3, and TIMP-1. In some embodiments, the geneAttorney Docket No.55801WO 0728.000558WO01 signature may include one or more genes selected from the group consisting of CD62E, CD62P,GM-CSF, IFN , IFNγ, IP-10, TNF , IL-1 , IL-1 , IL-6, IL-8, IL-18, IL-12p70, IL-17A, MCP-1, MIP1 , MIP-1b, IL-4, IL-10, and IL-13. In some embodiments, the gene signature biomarkermay include one or more of genes selected from the group consisting of PTER, ANPEP (CD13), THBS2, Clusterin, CXCL8 (IL-8), Cystatin C, TIMP-1, and MMP9. In some embodiments, the gene signature may include one or more genes selected from the group consisting of Bmi-1, Oct- 4, Pax-2, HoxA11, Six-2, Sox-9, and Foxm1.

[0072] In some embodiments, the gene signature biomarker may include one or more gene combinations. Non-limiting examples of gene combinations suitable for the methods discloses herein include:

[0073] CD10+ and GATA3+ (also referred to herein as C10+GATA3+);

[0074] CD10+ and CD13+;

[0075] CD10+ and CD224+;

[0076] CD10+ and CD227+;

[0077] CD10+ and CD171+;

[0078] CD13+ and CD10+;

[0079] CD13+ and CD224+;

[0080] CD13+ and CD227+;

[0081] CD13+ and GATA3+;

[0082] CD13+ and CD171+;

[0083] CD13+, CD10+, and CD227+;

[0084] CD13+, CD10+, and GATA3+;

[0085] CD24+, CD133+, and CD106-;

[0086] CD24+, CD133+, and CD106+;

[0087] CD90+ and CD106-;

[0088] CD90+ and CD106+Attorney Docket No.55801WO 0728.000558WO01

[0089] CD90+ and CD106-

[0090] CD90+ and CD133-;

[0091] CD133+ and CD24-;

[0092] CD133+ and CD24+;

[0093] CD133- and CD24+;

[0094] CD133+, CD24+, and CD90+;

[0095] CD133+, CD24+, and CD146+;

[0096] PDPN+ and CD106+.

[0097] In some embodiments, the gene signature biomarker may include: (a) elevated expression of CD10, CD13, CD224, and GATA3; and (b) essentially unchanged expression of CD227, CD326, and PDPN, relative to cells from a kidney biopsy.

[0098] In some embodiments, the method may further include determining the relative percentages of cells that express the one or more genes and / or gene combinations. In some such embodiments, the determining the relative percentages may be a determining that: at least a certain percentage of cells express one of the one or more genes, and / or at most a certain percentage of cells express one of the one or more genes, and / or that cells express one of the one or more genes within a range of percentage values. Renal cell identity

[0099] In some embodiments, the gene signature biomarker may include one or more genes associated with renal cell identity. Non-limiting examples of genes associated with renal cell identity include CD227 (EMA), CD326 (EpCAM), CD13 (ANPEP), CD224 (GGT), PDPN, CD10 (MME), GATA3, CD171, OCT2 (SLC22A2), NBCe1 (SLC4A4), NDCBE1 (SLC4A8), NKCC2 (SLC12A1), NHE3 (SLC9A3), NCC (SLC12A3), and SLC26A4 (Pendrin). In some embodiments, the gene signature biomarker may include GATA3+. In some embodiments, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95% ofAttorney Docket No.55801WO 0728.000558WO01 cells in the enriched heterogeneous renal cell population express GATA3. In some embodiments, at least about 80% of the cells in the enriched heterogeneous renal cell population express GATA3.

[0100] In some embodiments, the gene signature biomarker may include CD227+. In some embodiments, about 30% to about 95% of cells in the enriched heterogeneous renal cell population express CD227. In some embodiments about 30% to about 70%, about 35% to about 75%, about 40% to about 80%, about 45% to about 85, about 50% to about 90%, about 55% to about 95%, about 40% to about 90%, about 35% to about 95%, or about 45% to about 85% of cells in the enriched heterogeneous renal cell population express CD227.

[0101] In some embodiments, the gene signature biomarker may include PDPN+ (podoplanin). In some embodiments, greater than about 0% and at most about 30% of cells in the enriched heterogeneous renal cell population express PDPN. In some embodiments, greater than about 0% and at most about 28% of cells in the enriched heterogeneous renal cell population express PDPN. In some embodiments, greater than about 0% and at most about 25% of cells in the enriched heterogeneous renal cell population express PDPN. In some embodiments, greater than about 0% and at most about 22% of cells in the enriched heterogeneous renal cell population express PDPN. In some embodiments, greater than about 0% and at most about 20% of cells in the enriched heterogeneous renal cell population express PDPN. In some embodiments, greater than about 0% and at most about 19% of cells in the enriched heterogeneous renal cell population express PDPN. In some embodiments, greater than about 0% and at most about 18% of cells in the enriched heterogeneous renal cell population express PDPN. In some embodiments, greater than about 0% and at most about 17% of cells in the enriched heterogeneous renal cell population express PDPN. In some embodiments, greater than about 0% and at most about 16% of cells in the enriched heterogeneous renal cell population express PDPN. In some embodiments, greater than about 0% and at most about 30%, greater than about 0% and at most about 25%, greater than about 0% and at most about 20%, greater than about 0% and at most about 15% of cells in the enriched heterogeneous renal cell population express PDPN. In some embodiments, greater than about 0% and at most about 10%, greater than about 0% and at most about 11%, greater than about 0% and at most about 12%, greater than about 0% and at most about 13%, greater than about 0% and at most about 14%, or greater than about 0%Attorney Docket No.55801WO 0728.000558WO01 and at most about 15% of cells in the enriched heterogeneous renal cell population express PDPN.

[0102] In some embodiments, greater than about 1% and at most about 10%, greater than about 1% and at most about 11%, greater than about 1% and at most about 12%, greater than about 1% and at most about 13%, greater than about 1% and at most about 14%, greater than about 1% and at most about 15%, greater than about 1% and at most about 16%, greater than about 1% and at most about 17%, greater than about 1% and at most about 18, greater than about 1% and at most about 19%, greater than about 1% and at most about 20%, greater than about 1% and at most about 22%, greater than about 1% and at most about 25%, greater than about 1% and at most about 28%, or greater than about 1% and at most about 30% of cells in the enriched heterogeneous renal cell population express PDPN. In some embodiments, greater than about 2% and at most about 10%, greater than about 2% and at most about 11%, greater than about 2% and at most about 12%, greater than about 2% and at most about 13%, greater than about 2% and at most about 14%, greater than about 2% and at most about 15%, greater than about 2% and at most about 16%, greater than about 2% and at most about 17%, greater than about 2% and at most about 18%, greater than about 2% and at most about 19%, greater than about 2% and at most about 20%, greater than about 2% and at most about 22%, greater than about 2% and at most about 25%, greater than about 2% and at most about 28%, or greater than about 2% and at most about 30% of cells in the enriched heterogeneous renal cell population express PDPN.

[0103] In some embodiments, the gene signature biomarker may include CD10+. In some embodiments, about 60% to about 98% of cells in the enriched heterogeneous renal cell population express CD10. In some embodiments, about 60% to about 80%, about 65% to about 85%, about 70% to about 90%, about 75% to about 95%, about 75% to about 96%, about 75% to about 97%, at least about 80% to about 95%, or about 85% to about 98% of cells in the enriched heterogeneous renal cell population express CD10.

[0104] In some embodiments, the gene signature biomarker may include CD13+CD227+. In some embodiments, about 30% to about 95% of cells in the enriched heterogeneous renal cell population express both CD13 and CD227. In some embodiments, about 30% to about 70%, about 35% to about 75%, about 40% to about 80%, about 45% to about 85, about 50% to about 90%, about 55% to about 95%, about 35% to about 90%, about 35% to about 95%, or about 45%Attorney Docket No.55801WO 0728.000558WO01 to about 85% of cells in the enriched heterogeneous renal cell population express both CD13 and CD227.

[0105] In some embodiments, the gene signature biomarker may include CD13+GATA3+. In some embodiments, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of cells in the enriched heterogeneous renal cell population express both CD13 and GATA3. In some embodiments, about 85% to about 95%, about 90% to about 100%, about 90% to about 95%, about 85% to about 95%, or about 95% to about 100% of cells in the enriched heterogeneous renal cell population express CD13+GATA3+.

[0106] In some embodiments, the gene signature biomarker may include CD10+GATA3+. In some embodiments, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, or at least about 90% of cells in the enriched heterogeneous renal cell population express both CD10 and GATA3. In some embodiments, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, or at least about 90% of cells in the enriched heterogeneous renal cell population express CD10+GATA3+.

[0107] In some embodiments, the gene signature biomarker may include CD10+CD13+. In some embodiments, about 60% to about 98% of cells in the enriched heterogeneous renal cell population express CD10. In some embodiments, about 60% to about 80%, about 65% to about 85%, about 70% to about 90%, about 75% to about 95%, about 75% to about 96%, about 75% to about 97%, about 80% to about 95%, or about 85% to about 98% of cells in the enriched heterogeneous renal cell population express both CD10 and CD13.

[0108] In some embodiments, the gene signature biomarker may include CD13+CD224+. In some embodiments, at least about 80%, at least about 81%, at least about 82%, at least aboutAttorney Docket No.55801WO 0728.000558WO01 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of cells in the enriched heterogeneous renal cell population express both CD13 and CD224.

[0109] In some embodiments, the gene signature biomarker may include CD10+CD227+.

[0110] In some embodiments, the gene signature biomarker may include CD13+CD10+CD227+. In some embodiments, about 20% to about 85% of cells in the enriched heterogeneous renal cell population express all three CD13, CD10, and CD227. In some embodiments, about 20% to about 80%, about 25% to about 75%, about 30% to about 75%, about 35% to about 80%, about 30% to about 70%, about 35% to about 70%, about 25% to about 80%, about 35% to about 80%, or about 20% to about 75% of cells in the enriched heterogeneous renal cell population express all three CD13, CD10, and CD227.

[0111] In some embodiments, the gene signature biomarker may include CD13+CD10+GATA3+. In some embodiments, at least about 60%, for example, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of cells in the enriched heterogeneous renal cell population express all three CD13, CD10, and GATA3.

[0112] In some embodiments, the gene signature biomarker may include CD13+CD10+CD171+. In some embodiments, about 30% to about 75% of cells in the enriched heterogeneous renal cell population express all three CD13, CD10, and CD171. In some embodiments, about 32% to about 75%, about 30% to about 72%, about 30% to about 70%, about 30% to about 72%, about 32% to about 70%, about 35% to about 70%, about 37% to about 70%, about 37% to about 72%, about 40% to about 70%, about 37% to about 70%, about 35% to about 75%, about 35% to about 72%, or about 40% to about 70% of cells in the enriched heterogeneous renal cell population express all three CD13, CD10, and CD171,

[0113] In some embodiments, it is further contemplated that determining that cells of an enriched heterogeneous renal cell population co-express, or that a certain percentage of cells of the enriched heterogeneous renal cell population co-express, certain renal cell identity markersAttorney Docket No.55801WO 0728.000558WO01 may be predictive of the enriched heterogeneous renal cell population’s degree of clinical benefit to a chronic kidney disease patient. For example, co-expression of one or more of proximal tubule markers CD13, CD10, CD224 with one or more of distal nephron markers CD171, CD227, and GATA3 may predict that the enriched heterogeneous renal cell population will have a higher degree of clinical benefit to a chronic kidney disease patient. Tissue healing response

[0114] In some embodiments, the gene signature biomarker may include one or more genes associated with tissue healing response (e.g., renewal and repair markers). Non-limiting examples of genes associated with tissue healing response (e.g., renal and repair) include CD90 (Thy-1), CD24 (HSA), Vimentin, CD106 (VCAM-1), CD146 (MCAM), CD44 (H-CAM), and CD133 (PROM-1). In some embodiments, the gene signature biomarker may include CD90+. In some embodiments, about 30% to about 95% of cells in the enriched heterogeneous renal cell population express CD90. In some embodiments, about 30% to about 70, about 35% to about 75%, about 40% to about 80%, about 45% to about 85, about 50% to about 80%, about 50% to about 90%, about 55% to about 95%, about 40% to about 90%, about 35% to about 95%, or about 45% to about 85% of cells in the enriched heterogeneous renal cell population express CD90.

[0115] In some embodiments, the gene signature biomarker may include CD90+CD106-. In some embodiments, about 10% to about 65% of cells in the enriched heterogeneous renal cell population express CD90 but lack CD106. In some embodiments, about 10% to about 50%, about 10% to about 55%, about 10% to about 60%, about 10% to about 65%, about 15% to about 50%, about 15% to about 55%, about 15% to about 60%, about 15% to about 65%, about 20% to about 50%, about 20% to about 55%, about 20% to about 60%, or about 20% to about 65% of cells in the enriched heterogeneous renal cell population express CD90 but lack CD106.

[0116] In some embodiments, the gene signature biomarker may include CD90+CD106+. In some embodiments, about 10% to about 50% of cells in the enriched heterogeneous renal cell population co-express CD90 and CD106. In some embodiments, about 10% to about 45%, about 10% to about 40%, about 15% to about 50%, about 15% to about 45%, or about 15% to about 40%, or about 20% to about 50% of cells in the enriched heterogeneous renal cell population co- express CD90 and CD106. In some embodiments, if cells of the enriched heterogeneous renalAttorney Docket No.55801WO 0728.000558WO01 cell population co-express CD90 and CD106 at any of these percentages, the enriched heterogeneous renal cell population may be predicted to be of clinical benefit, or of a greater degree of clinical benefit, to a chronic kidney disease patient in need of administration thereof.

[0117] In some embodiments, the gene signature biomarker may include CD90+CD133-. In some embodiments, about 25% to about 75% of cells in the enriched heterogeneous renal cell population express CD90 but lack CD133. In some embodiments, about 25% to about 55%, about 25% to about 60%, about 25% to about 65%, about 25% to about 70%, about 25% to about 75%, about 30% to about 55%, about 30% to about 60%, about 30% to about 65%, about 30% to about 70%, about 30% to about 75%, about 35% to about 55%, about 35% to about 60%, about 35% to about 65%, about 35% to about 70%, or about 35% to about 75% of cells in the enriched heterogeneous renal cell population express CD90 but lack CD133.

[0118] In some embodiments, the gene signature biomarker may include CD24+CD133+CD106+. In some embodiments, about 3% to about 45% of cells in the enriched heterogeneous renal cell population express all three CD24, CD133, and CD106. In some embodiments, about 0% to about 50%, about 0% to about 40%, about 0% to about 35%, about 0% to about 30%, about 0% to about 25%, about 1% to about 50%, about 1% to about 40%, about 1% to about 35%, about 1% to about 30%, about 1% to about 25%, about 2% to about 45%, about 2% to about 40%, about 2% to about 35%, about 2% to about 30%, about 2% to about 25%, about 2% to about 20%, about 3% to about 25%, about 3% to about 30%, about 3% to about 35%, about 3% to about 40%, about 3% to about 45%, about 4% to about 25%, about 4% to about 30%, about 4% to about 35%, about 4% to about 40%, about 4% to about 45%, about 5% to about 25%, about 5% to about 30%, about 5% to about 35%, about 5% to about 40%, about 5% to about 45%, about 6% to about 25%, about 6% to about 30%, about 6% to about 35%, about 6% to about 40%, or about 6% to about 45% of cells in the enriched heterogeneous renal cell population express all three CD24, CD133, and CD106.

[0119] In some embodiments, the gene signature biomarker may include CD24+CD133+CD106-. In some embodiments, about 3% to about 45% of cells in the enriched heterogeneous renal cell population express CD24 and CD133 but lack CD106. In some embodiments, about 0% to about 50%, about 0% to about 40%, about 1% to about 45%, about 1% to about 35%, about 2% to about 35%, about 2% to about 25%, about 3% to about 25%,Attorney Docket No.55801WO 0728.000558WO01 about 3% to about 30%, about 3% to about 35%, about 3% to about 40%, about 3% to about 45%, about 4% to about 25%, about 4% to about 30%, about 4% to about 35%, about 4% to about 40%, about 4% to about 45%, about 5% to about 25%, about 5% to about 30%, about 5% to about 35%, about 5% to about 40%, about 5% to about 45%, about 6% to about 25%, about 6% to about 30%, about 6% to about 35%, about 6% to about 40%, or about 6% to about 45% of cells in the enriched heterogeneous renal cell population express CD24 and CD133 but lack CD106.

[0120] In some embodiments, the gene signature biomarker may include a ratio of CD24+CD133+CD106+ cells to CD24+CD133+CD106- cells being about 0.5 to about 1:5. In some embodiments, the ratio of CD24+CD133+CD106+ cells to CD24+CD133+CD106- cells ranges from about 0.5 to about 1, from about 0.5 to about 1:25, from about 0.75 to about 1, from about 0.75 to about 1:25, from about 0.75 to about 1:5, from about 1 to about 1.25, or from about 1 to about 1.5. In some embodiments, the ratio of CD24+CD133+CD106+ cells to CD24+CD133+CD106- cells is about 0.5, about 0.17, about 1.0, about 1.25, or about 1.5.

[0121] In some embodiments, the gene signature biomarker may include (i) at least one gene associated with renal cell identity and (ii) at least one gene associated with tissue healing response (e.g., renewal and repair markers). In some embodiments, the at least one gene associated with renal cell identity includes PDPN and the at least one gene associated with tissue healing response includes CD106. Fibrosis, inflammation, and senescence

[0122] In some embodiments, the gene signature biomarker may include one or more genes associated with fibrosis, inflammation or senescence. Non-limiting examples of genes associated with fibrosis, inflammation or senescence include IL-6, IL-8, IL-18, IL-1b, IP-10, TNFa, MCP-1, MIP-1a, and MIP-1b. In some embodiments, the gene signature biomarker may include one or more proteins selected from the group consisting of IL-6, IL-8, IL-18, IL-1b, IP- 10, TNFa, MCP-1, MIP-1a, and MIP-1b. In some embodiments, the gene signature biomarker may include a single protein selected from the group consisting of IL-6, IL-8, IL-18, IL-1b, IP- 10, TNFa, MCP-1, MIP-1a, and MIP-1b. In some embodiments, the gene signature biomarker may include a combination of any one or more, e.g., combinations of two, three, four, five, six,Attorney Docket No.55801WO 0728.000558WO01 seven, eight, or nine proteins selected from the group consisting of IL-6, IL-8, IL-18, IL-1b, IP- 10, TNFa, MCP-1, MIP-1a, and MIP-1b.

[0123] In some embodiments, the gene signature biomarker may include a secreted level of protein expressed from any one or more of the fibrosis, inflammation, or senescence genes. In some such embodiments, level of proteins secreted by cells may be determined to be below a certain threshold level. In some embodiments, the threshold level may be a level secreted in about 24 hours. In some embodiments, the determining levels of secreted proteins by cells may be a determining that the cells of the population secrete one of the one or more proteins at lower levels than is secreted by a reference population. Non-limiting examples of suitable reference populations include (i) cells from a biopsy or tissue sample of a patient from which the enriched heterogeneous renal cell population is isolated; or (ii) cells from (i) that have been plated; or (iii) cells from (ii) that have been passaged once or twice.

[0124] In some embodiments, the gene signature biomarker may include, in addition to one or more genes associated with renal cell identity as described earlier herein, one or more genes associated with fibrosis, inflammation or senescence as described herein. In some embodiments, the gene signature biomarker may include, in addition to one or more genes associated with tissue healing response (e.g., renewal and repair) as described earlier herein, one or more genes associated with fibrosis, inflammation or senescence as described herein. In some embodiments, the gene signature biomarker may include, in addition to one or more genes associated with renal cell identity and with tissue healing response (e.g., renewal and repair) as described herein, one or more genes associated with fibrosis, inflammation or senescence as described herein

[0125] In any embodiment described herein, the gene signature biomarker may additionally include one or more further inflammation genes. In instances where the gene signature biomarker additionally includes one or more further inflammation genes, the further inflammation genes may encode one or more proteins selected from the group consisting of CD62E, CD62P, GM-CSF, IFN , IFNγ, IL-1 , IL-12p-70, IL-17a, IL-4, IL-10, and IL-13. In some embodiments, theinflammation genes may encode a protein selected from the group consisting of IL-6, IL-8, IL-18, IL-1 , IP-10, TNF , MCP-1, MIP-1 , and MIP-1 . In some embodiments, the inflammationgene may encode one or more, e.g., combinations of two, three, four, five, six, seven, eight, orAttorney Docket No.55801WO 0728.000558WO01 nine proteins selected from the group consisting of IL-6, IL-8, IL-18, IL-1b, IP-10, TNFa, MCP- 1, MIP-1a, and MIP-1b. In some embodiments, the gene signature biomarker may include a secreted level of protein expressed from any one or more of the further inflammation genes. In some such embodiments, level of proteins secreted by cells may be determined to be below a certain threshold level. In some embodiments, the threshold level may be a level secreted in about 24 hours. In some embodiments, the determining levels of secreted proteins by cells may be a determining that the cells of the population secrete proteins at lower levels than is secreted by a reference population. Non-limiting examples of suitable reference populations include (i) cells from a biopsy or tissue sample of a patient from which the enriched heterogeneous renal cell population is isolated; or (ii) cells from (i) that have been plated; or (iii) cells from (ii) that have been passaged once or twice. Additional genes and markers

[0126] In some embodiments, the methods disclosed herein further include measuring expression levels of at least one additional gene or marker.

[0127] In instances where the expression level of at least one additional marker may further be determined, the at least one additional marker may be at least one (at least one, at least two, or three) of cytokeratin (CK)18, vascular endothelial growth factor (VEGF) or kidney injury molecule (KIM)-1. The at least one additional marker may be or may be CK18, or may be VEGF, or may be KIM-1, or may be CK18 and VEGF, or may be CK18 and KIM-1, or may be VEGF and KIM-1, or may be GGT-1, CK18 and VEGF, or may be CK18, VEGF and KIM-1.

[0128] Determining that the cells of the enriched heterogeneous renal cell population, e.g., SRCs, further express the at least one additional marker may identify the enriched heterogeneous renal cell population as having a therapeutic potential.

[0129] In some instances, the determining cells of the enriched heterogeneous renal cell population further express the at least one additional marker may be a determining that a certain percentage of cells of the enriched heterogeneous renal cell population express the at least one additional marker or may be a determining that cells of the enriched heterogeneous renal cell population secrete the at least one additional marker. Determining that a certain percentage of cells of the enriched heterogeneous renal cell population express the at least one additional marker, and / or that cells of the enriched heterogeneous renal cell population secrete the at leastAttorney Docket No.55801WO 0728.000558WO01 one additional marker, may identify the enriched heterogeneous renal cell population as having a therapeutic potential.

[0130] In instances in which the at least one additional marker is CK18, a certain percentage of cells of the enriched heterogeneous renal cell population may further be determined to express CK18 to identify the enriched heterogeneous renal cell population as having a therapeutic potential. In these instances, the percent cells that express CK18 that may identify the enriched heterogeneous renal cell population as having a therapeutic potential may be greater than about 80%, greater than about 82%, greater than about 84%, greater than about 86%, greater than about 88%, greater than about 90%, greater than about 92%, or greater than about 94%.

[0131] In instances in which the at least one additional marker is VEGF and / or KIM-1, cells of the enriched heterogeneous renal cell population may further be determined to secrete VEGF and / or KIM-1 to identify the enriched heterogeneous renal cell population as having a therapeutic potential.

[0132] In some embodiments in which cells of the enriched heterogeneous renal cell population are determined to express at least one additional marker, the gene signature biomarker may be GATA3 and CD224, and the at least one biomarker may be CK18. In such embodiments, the enriched heterogenous renal cell population may be identified as having therapeutic potential if at least about 70% of cells of an enriched heterogeneous renal cell population are CD224+, at least about 70% of cells of the enriched heterogeneous renal cell population are CK18+, and at least about 70% of cells of the enriched heterogeneous renal cell population are GATA3+. Alternatively, in such embodiments, the enriched heterogeneous renal cell population may be identified as having therapeutic potential if at least about 80% of cells of the population are CD224+, at least about 80% of cells of the population are GATA3+, and at least about 80% of cells of the population are CK18+.

[0133] In some embodiments in which cells of the enriched heterogeneous renal cell population are determined to express at least one additional marker, the gene signature biomarker may be GATA3 and PDPN, and the at least one biomarker may be CK18. In such embodiments, the enriched heterogenous renal cell population may be identified as having therapeutic potential if at least about 70% of cells of an enriched heterogeneous renal cell population are GATA3+, at most about 30% of cells of the enriched heterogeneous renal cell population are PDPN+, and atAttorney Docket No.55801WO 0728.000558WO01 least 70% of cells of the enriched heterogeneous renal cell population are CK18+. Alternatively, in such embodiments, the enriched heterogeneous renal cell population may be identified as having therapeutic potential if at least about 80% of cells of the population are GATA3+, at most about 30% of cells of the population are PDPN+, and at least about 80% of cells of the population are CK18+. Alternatively, in such embodiments, the enriched heterogeneous renal cell population may be identified as having therapeutic potential if at least about 80% of cells of the population are GATA3+, at most about 18% of cells of the population are PDPN+, and at least about 80% of cells of the population are CK18+.

[0134] In some embodiments in which cells of the enriched heterogeneous renal cell population are determined to express at least one additional marker, the gene signature biomarker may be CD224 and PDPN, and the at least one biomarker may be CK18. In such embodiments, the enriched heterogenous renal cell population may be identified as having therapeutic potential if at least about 70% of cells of an enriched heterogeneous renal cell population are CD224+, at most 30% of cells of the enriched heterogeneous renal cell population are PDPN+, and at least 70% of cells of the enriched heterogeneous renal cell population are CK18+. Alternatively, in such embodiments, the enriched heterogeneous renal cell population may be identified as having therapeutic potential if at least about 80% of cells of the population are CD224+, at most about 30% of cells of the population are PDPN+, and at least about 80% of cells of the population are CK18+. Alternatively, in such embodiments, the enriched heterogeneous renal cell population may be identified as having therapeutic potential if at least about 80% of cells of the population are CD224+, at most about 18% of cells of the population are PDPN+, and at least about 80% of cells of the population are CK18+.

[0135] In some embodiments in which cells of the enriched heterogeneous renal cell population are determined to express at least one additional marker, the gene signature biomarker may be CD224, GATA3, and PDPN, and the at least one biomarker may be CK18. In such embodiments, the enriched heterogenous renal cell population may be identified as having therapeutic potential if at least about 70% of the cells of an enriched heterogeneous renal cell population are CD224+, at least 70% of the cells of the enriched heterogeneous renal cell population are GATA3+, at most about 30% of cells of the enriched heterogeneous renal cell population are PDPN+, and at least 70% of the cells of the enriched heterogeneous renal cell population are CK18+. Alternatively, in such embodiments, the enriched heterogeneous renalAttorney Docket No.55801WO 0728.000558WO01 cell population may be identified as having therapeutic potential if at least about 80% of cells of the population are CD224+, at least 80% of the cells of the enriched heterogeneous renal cell population are GATA3+, at most about 18% of cells of the enriched heterogeneous renal cell population are PDPN+, and at least 80% of the cells of the enriched heterogeneous renal cell population are CK18+.

[0136] In some embodiments in which cells of the enriched heterogeneous renal cell population are determined to express at least one additional marker, the gene signature biomarker may be GATA3 and CD224, and the at least one biomarker may be CK18 and VEGF. In such embodiments, the enriched heterogenous renal cell population may be identified as having therapeutic potential if at least about 70% of cells of an enriched heterogeneous renal cell population are CD224+, at least about 70% of cells of the enriched heterogeneous renal cell population are CK18+, at least about 70% of cells of the enriched heterogeneous renal cell population are GATA3+, and cells of the enriched heterogeneous renal cell population are determined to express VEGF. Alternatively, in such embodiments, the enriched heterogeneous renal cell population may be identified as having therapeutic potential if at least about 80% of cells of the population are CD224+, at least about 80% of cells of the population are GATA3+, at least about 80% of cells of the population are CK18+, and cells of the enriched heterogeneous renal cell population are determined to express VEGF.

[0137] In some embodiments in which cells of the enriched heterogeneous renal cell population are determined to express at least one additional marker, the gene signature biomarker may be GATA3 and PDPN, and the at least one biomarker may be CK18 and VEGF. In such embodiments, the enriched heterogenous renal cell population may be identified as having therapeutic potential if at least about 70% of cells of an enriched heterogeneous renal cell population are GATA3+, at most 30% of cells of the enriched heterogeneous renal cell population are PDPN+, at least 70% of cells of the enriched heterogeneous renal cell population are CK18+, and cells of the enriched heterogeneous renal cell population are determined to express VEGF. Alternatively, in such embodiments, the enriched heterogeneous renal cell population may be identified as having therapeutic potential if at least about 80% of cells of the population are GATA3+, at most about 30% of cells of the population are PDPN+, at least about 80% of cells of the population are CK18+, and cells of the enriched heterogeneous renal cell population are determined to express VEGF. Alternatively, in such embodiments, the enrichedAttorney Docket No.55801WO 0728.000558WO01 heterogeneous renal cell population may be identified as having therapeutic potential if at least about 80% of cells of the population are GATA3+, at most about 18% of cells of the population are PDPN+, at least about 80% of cells of the population are CK18+, and cells of the enriched heterogeneous renal cell population are determined to express VEGF.

[0138] In some embodiments in which cells of the enriched heterogeneous renal cell population are determined to express at least one additional marker, the gene signature biomarker may be CD224 and PDPN, and the at least one biomarker may be CK18 and VEGF. In such embodiments, the enriched heterogenous renal cell population may be identified as having therapeutic potential if at least about 70% of cells of an enriched heterogeneous renal cell population are CD224+, at most 30% of cells of the enriched heterogeneous renal cell population are PDPN+, at least 70% of cells of the enriched heterogeneous renal cell population are CK18+, and cells of the enriched heterogeneous renal cell population are determined to express VEGF. Alternatively, in such embodiments, the enriched heterogeneous renal cell population may be identified as having therapeutic potential if at least about 80% of cells of the population are CD224+, at most about 30% of cells of the population are PDPN+, at least about 80% of cells of the population are CK18+, and cells of the enriched heterogeneous renal cell population are determined to express VEGF. Alternatively, in such embodiments, the enriched heterogeneous renal cell population may be identified as having therapeutic potential if at least about 80% of cells of the population are CD224+, at most about 18% of cells of the population are PDPN+, at least about 80% of cells of the population are CK18+, and cells of the enriched heterogeneous renal cell population are determined to express VEGF.

[0139] In some embodiments in which cells of the enriched heterogeneous renal cell population are determined to express at least one additional marker, the gene signature biomarker may be CD224, GATA3, and PDPN, and the at least one biomarker may be CK18 and VEGF. In such embodiments, the enriched heterogenous renal cell population may be identified as having therapeutic potential if at least about 70% of the cells of the enriched heterogeneous renal cell population are CD224+, at least 70% of the cells of the enriched heterogeneous renal cell population are GATA3+, at most about 30% of cells of the enriched heterogeneous renal cell population are PDPN+, at least 70% of the cells of the enriched heterogeneous renal cell population are CK18+, and cells of the enriched heterogeneous renal cell population are determined to express VEGF. Alternatively, in such embodiments, the enriched heterogeneousAttorney Docket No.55801WO 0728.000558WO01 renal cell population may be identified as having therapeutic potential if at least about 80% of cells of the population are CD224+, at least 80% of the cells of the enriched heterogeneous renal cell population are GATA3+, at most about 18% of cells of the enriched heterogeneous renal cell population are PDPN+, at least 80% of the cells of the enriched heterogeneous renal cell population are CK18+, and cells of the enriched heterogeneous renal cell population are determined to express VEGF.

[0140] In some embodiments in which cells of the enriched heterogeneous renal cell population are determined to express at least one additional marker, the gene signature biomarker may be GATA3 and CD224, and the at least one biomarker may be CK18 and KIM-1. In such embodiments, the enriched heterogenous renal cell population may be identified as having therapeutic potential if at least about 70% of cells of an enriched heterogeneous renal cell population are CD224+, at least about 70% of cells of the enriched heterogeneous renal cell population are CK18+, at least about 70% of cells of the enriched heterogeneous renal cell population are GATA3+, and cells of the enriched heterogeneous renal cell population are determined to express KIM-1. Alternatively, in such embodiments, the enriched heterogeneous renal cell population may be identified as having therapeutic potential if at least about 80% of cells of the population are CD224+, at least about 80% of cells of the population are GATA3+, at least about 80% of cells of the population are CK18+, and cells of the enriched heterogeneous renal cell population are determined to express KIM-1.

[0141] In some embodiments in which cells of the enriched heterogeneous renal cell population are determined to express at least one additional marker, the gene signature biomarker may be GATA3 and PDPN, and the at least one biomarker may be CK18 and KIM-1. In such embodiments, the enriched heterogenous renal cell population may be identified as having therapeutic potential if at least about 70% of cells of an enriched heterogeneous renal cell population are GATA3+, at most 30% of cells of the enriched heterogeneous renal cell population are PDPN+, at least 70% of cells of the enriched heterogeneous renal cell population are CK18+, and cells of the enriched heterogeneous renal cell population are determined to express KIM-1. Alternatively, in such embodiments, the enriched heterogeneous renal cell population may be identified as having therapeutic potential if at least about 80% of cells of the population are GATA3+, at most about 30% of cells of the population are PDPN+, at least about 80% of cells of the population are CK18+, and cells of the enriched heterogeneous renal cellAttorney Docket No.55801WO 0728.000558WO01 population are determined to express KIM-1. Alternatively, in such embodiments, the enriched heterogeneous renal cell population may be identified as having therapeutic potential if at least about 80% of cells of the population are GATA3+, at most about 18% of cells of the population are PDPN+, at least about 80% of cells of the population are CK18+, and cells of the enriched heterogeneous renal cell population are determined to express KIM-1.

[0142] In some embodiments in which cells of the enriched heterogeneous renal cell population are determined to express at least one additional marker, the gene signature biomarker may be CD224 and PDPN, and the at least one biomarker may be CK18 and KIM-1. In such embodiments, the enriched heterogenous renal cell population may be identified as having therapeutic potential if at least about 70% of cells of an enriched heterogeneous renal cell population are CD224+, at most 30% of cells of the enriched heterogeneous renal cell population are PDPN+, at least 70% of cells of the enriched heterogeneous renal cell population are CK18+, and cells of the enriched heterogeneous renal cell population are determined to express KIM-1. Alternatively, in such embodiments, the enriched heterogeneous renal cell population may be identified as having therapeutic potential if at least about 80% of cells of the population are CD224+, at most about 30% of cells of the population are PDPN+, at least about 80% of cells of the population are CK18+, and cells of the enriched heterogeneous renal cell population are determined to express KIM-1. Alternatively, in such embodiments, the enriched heterogeneous renal cell population may be identified as having therapeutic potential if at least about 80% of cells of the population are CD224+, at most about 18% of cells of the population are PDPN+, at least about 80% of cells of the population are CK18+, and cells of the enriched heterogeneous renal cell population are determined to express KIM-1.

[0143] In some embodiments in which cells of the enriched heterogeneous renal cell population are determined to express at least one additional marker, the gene signature biomarker may be CD224, GATA3 and PDPN and the at least one biomarker may be CK18 and KIM-1. In such embodiments, the enriched heterogenous renal cell population may be identified as having therapeutic potential if at least about 70% of the cells of an enriched heterogeneous renal cell population are CD224+, at least 70% of the cells of the enriched heterogeneous renal cell population are GATA3+, at most about 30% of cells of the enriched heterogeneous renal cell population are PDPN+, at least 70% of the cells of the enriched heterogeneous renal cell population are CK18+, and cells of the enriched heterogeneous renal cell population areAttorney Docket No.55801WO 0728.000558WO01 determined to express KIM-1. Alternatively, in such embodiments, the enriched heterogeneous renal cell population may be identified as having therapeutic potential if at least about 80% of cells of the population are CD224+, at least 80% of the cells of the enriched heterogeneous renal cell population are GATA3+, at most about 18% of cells of the enriched heterogeneous renal cell population are PDPN+, at least 80% of the cells of the enriched heterogeneous renal cell population are CK18+, and cells of the enriched heterogeneous renal cell population are determined to express KIM-1.

[0144] In some embodiments in which cells of the enriched heterogeneous renal cell population are determined to express at least one additional marker, the gene signature biomarker may be GATA3 and CD224, and the at least one biomarker may be CK18, VEGF, and KIM-1. In such embodiments, the enriched heterogenous renal cell population may be identified as having therapeutic potential if at least about 70% of cells of an enriched heterogeneous renal cell population are CD224+, at least about 70% of cells of the enriched heterogeneous renal cell population are CK18+, at least about 70% of cells of the enriched heterogeneous renal cell population are GATA3+, and cells of the enriched heterogeneous renal cell population are determined to express VEGF and KIM-1. Alternatively, in such embodiments, the enriched heterogeneous renal cell population may be identified as having therapeutic potential if at least about 80% of cells of the population are CD224+, at least about 80% of cells of the population are GATA3+, at least about 80% of cells of the population are CK18+, and cells of the enriched heterogeneous renal cell population are determined to express VEGF and KIM-1.

[0145] In some embodiments in which cells of the enriched heterogeneous renal cell population are determined to express at least one additional marker, the gene signature biomarker may be GATA3 and PDPN, and the at least one biomarker may be CK18, VEGF, and KIM-1. In such embodiments, the enriched heterogenous renal cell population may be identified as having therapeutic potential if at least about 70% of cells of an enriched heterogeneous renal cell population are GATA3+, at most 30% of cells of the enriched heterogeneous renal cell population are PDPN+, at least 70% of cells of the enriched heterogeneous renal cell population are CK18+, and cells of the enriched heterogeneous renal cell population are determined to express VEGF and KIM-1. Alternatively, in such embodiments, the enriched heterogeneous renal cell population may be identified as having therapeutic potential if at least about 80% of cells of the population are GATA3+, at most about 18% of cells of the population are PDPN+, atAttorney Docket No.55801WO 0728.000558WO01 least about 80% of cells of the population are CK18+, and cells of the enriched heterogeneous renal cell population are determined to express VEGF and KIM-1.

[0146] In some embodiments in which cells of the enriched heterogeneous renal cell population are determined to express at least one additional marker, the gene signature biomarker may be CD224 and PDPN, and the at least one biomarker may be CK18, VEGF, and KIM-1. In such embodiments, the enriched heterogenous renal cell population may be identified as having therapeutic potential if at least about 70% of cells of an enriched heterogeneous renal cell population are CD224+, at most 30% of cells of the enriched heterogeneous renal cell population are PDPN+, at least 70% of cells of the enriched heterogeneous renal cell population are CK18+, and cells of the enriched heterogeneous renal cell population are determined to express VEGF and KIM-1. Alternatively, in such embodiments, the enriched heterogeneous renal cell population may be identified as having therapeutic potential if at least about 80% of cells of the population are CD224+, at most about 18% of cells of the population are PDPN+, at least about 80% of cells of the population are CK18+, and cells of the enriched heterogeneous renal cell population are determined to express VEGF and KIM-1.

[0147] In some embodiments in which cells of the enriched heterogeneous renal cell population are determined to express at least one additional marker, the gene signature biomarker may be CD224, GATA3, and PDPN and the at least one biomarker may be CK18, VEGF, and KIM-1. In such embodiments, the enriched heterogenous renal cell population may be identified as having therapeutic potential if at least about 70% of the cells of an enriched heterogeneous renal cell population are CD224+, at least 70% of the cells of the enriched heterogeneous renal cell population are GATA3+, at most about 30% of cells of the enriched heterogeneous renal cell population are PDPN+, at least 70% of the cells of the enriched heterogeneous renal cell population are CK18+, and cells of the enriched heterogeneous renal cell population are determined to express VEGF and KIM-1. Alternatively, in such embodiments, the enriched heterogeneous renal cell population may be identified as having therapeutic potential if at least about 80% of cells of the population are CD224+, at least 80% of the cells of the enriched heterogeneous renal cell population are GATA3+, at most about 18% of cells of the enriched heterogeneous renal cell population are PDPN+, at least 80% of the cells of the enriched heterogeneous renal cell population are CK18+, and cells of the enriched heterogeneous renal cell population are determined to express VEGF and KIM-1.Attorney Docket No.55801WO 0728.000558WO01

[0148] In any of the disclosed methods of identifying an enriched heterogeneous renal cell population as having a therapeutic potential, the determining whether cells of the enriched heterogeneous renal cell population express any marker(s), may be by detection of the marker(s) in a nucleic acid, e.g., mRNA or miRNA, or polypeptide form and may be by any suitable assay / technique. For example, if expression is determined in polypeptide form, it may be determined by assays such as Western blot, fluorescence activated cell sorting (FACS), flow cytometry, or enzyme linked immunosorbent assay (ELISA). If expression is determined in nucleic acid form, it may be determined by an assay such as, or that uses, Southern blot, polymerase chain reaction (PCR), or reverse transcriptase quantitative PCR (RT-qPCR), serial analysis of gene expression (SAGE), Mass ARRAY, scRNAseq, or fluorescence in situ hybridization (FISH). Expression may be determined via a bulk or a single cell assay or may be determined after collection of conditioned media from a sample of cells from an enriched heterogeneous renal cell population. The assay may be one that employs a labeled detection reagent. The labeled detection reagent may include (a) a portion that complexes, directly or indirectly, with marker(s) and (b) a detection moiety. Non-limiting detection moieties include radioactive isotopes, e.g.,35S,14C,125I,3H and131I, colloidal gold particles, fluorescent labels, e.g., Texas Red, rhodamine, fluorescein,phycocryterin, phycocyanin, SPECTRUM ORANGE, SPECTRUM GREEN1 and enzyme substrates, e.g., firefly luciferase, bacterial luciferase, luciferin, horseradish peroxidase, alkaline phosphatase, or beta galactosidase.

[0149] The enriched heterogeneous renal cell population, which may be identified as having a therapeutic potential in any of the methods, may be enriched for one or more renal cell types such as renal epithelial cells, renal tubular cells, renal tubular epithelial cells, or renal proximal tubular cells. The enrichment of the enriched heterogeneous renal cell population for these one or more of renal cell types may be a reference to the enriched heterogeneous renal cell population having a greater percentage of the one or more renal cell types than does a kidney tissue of a patient, a kidney biopsy of a patient, or an in vitro culture of cells established from a kidney tissue or kidney biopsy of a patient, (which, collectively, may be referred to as a “starting renal cell population”). A starting renal cell population, if an in vitro culture of cells established from a kidney tissue of a patient or a kidney biopsy of a patient, may be a renal cell preparation comprising dissociated cells of a kidney tissue or kidney biopsy (e.g., cells dissociated from theAttorney Docket No.55801WO 0728.000558WO01 kidney tissue or kidney biopsy via mincing and / or enzyme digestion), that may or may not have been treated to remove red blood cells and debris. The enriched heterogeneous renal cell population, in addition to being enriched for renal epithelial cells, renal tubular cells, renal tubular epithelial cells, and / or renal proximal tubular cells, may also include other renal cell types such as glomerular cells, podocytes, collecting duct cells and / or vascular cells. In another aspect, the present disclosure relates to methods including a step of providing a renal cell population, such as a starting renal cell population. A renal cell population may be provided as part of a method of preparing an enriched heterogeneous renal cell population.

[0150] The starting renal cell population may be non-fetal renal cells, such as adult renal cells. A starting renal cell population may be, for example, from a biopsy of non-fetal subject, such as an adult human subject. A starting renal cell population, if an in vitro culture of cells established from a patient or a kidney sample of a patient, may be an in vitro culture of cells established from a non-fetal kidney sample of a subject, such as an adult human subject. The enriched heterogenous renal cell populations of the present disclosure does not include fetal cells, such as fetal renal cells.

[0151] In some embodiments, the starting renal cell population is not genetically manipulated, is not genetically modified at any point during preparation of an enriched heterogenous renal cell population and the enriched heterogeneous renal cell population does not include cells including a genetic modification. In some embodiments, the starting renal cell population is not genetically manipulated, is not genetically modified at any point during preparation of an enriched heterogenous renal cell population and the enriched heterogeneous renal cell population does not include cells including a genetic modification, other than a genetic modification that knocks out a major histocompatibility complex (MHC) class 1, MHC class 2 or beta-2 microglobulin (B2M) gene. For example, a starting renal cell population is not genetically manipulated by transient or stable introduction of exogeneous genetic material, and an enriched heterogeneous renal cell population does not include cells including exogenous genetic material. Exogenous genetic material may include plasmid DNA, siRNA, shRNA, miRNA, and editing reagents such as CRISPR / Cas9. In some embodiments, neither the starting renal cell population nor the enriched heterogeneous renal cell population are or have been genetically manipulated by deletion, mutation, or replacement of endogenous genetic material, other than to knock out or knock down expression of an MHC class I, MHC class II or B2M gene. In instances, none of the genome ofAttorney Docket No.55801WO 0728.000558WO01 starting renal cell population, the enriched heterogenous renal cell population, and / or any population of renal cells prepared to arrive at the enriched heterogeneous renal cell population from the starting renal cell population have been manipulated such that the enriched heterogeneous renal cell population de-differentiates or deliberately expresses markers that would otherwise not be expressed. In some embodiments, the cells of an enriched heterogeneous renal cell population have not been genetically modified, such as by deletion in or replacement of any one or more nucleotides in a native genomic sequence.

[0152] In some embodiments, none of the starting renal cell population, the enriched heterogeneous renal cell population and / or any cell population prepared in the process of arriving at the enriched heterogeneous renal cell population from the starting renal cell population has been contacted with a demethylating agent.

[0153] In some embodiments, none of the starting renal cell population, the enriched heterogeneous renal cell population and / or any cell population prepared in the process of arriving at the enriched heterogeneous renal cell population from the starting renal cell population has been treated with nephrogenic growth factors. Examples of nephrogenic growth factors include retinoic acid, activin-A, and bone morphogenic protein 7 (BMP7).

[0154] In some embodiments, none of the starting renal cell population, the enriched heterogeneous renal cell population and / or any cell population prepared in the process of arriving at the enriched heterogeneous renal cell population from the starting renal cell population has been treated with a histone acetylation regulator. Non-limiting examples of histone acetylation regulators include HDAC inhibitors, such as trichostatin A (TSA), valproic acid (VPA, also referred to as a chromatin modifying agent), and / or 5-aza-2’-deoxycytidine. In some embodiments, none of the starting renal cell population, the enriched heterogeneous renal cell population and / or any cell population prepared in the process of arriving at the enriched heterogeneous renal cell population from the starting renal cell population has treated with a Wnt agonist.

[0155] The enriched heterogeneous renal cell population may be enriched for the one or more renal cell types as a result of having been prepared from a starting renal cell population, (e.g., a kidney tissue of a patient, a kidney biopsy of a patient, or an in vitro culture of cells established from a kidney tissue or kidney biopsy of a patient), via a method that includes a separation step.Attorney Docket No.55801WO 0728.000558WO01 The separation step may be one that separates cells of the starting renal cell population that have been passaged no more than one, two, or three times, on the basis of their buoyant density. If the separation step is one that separates cells on the basis of their buoyant density, the separation step may utilize a single or multi-step continuous or discontinuous density gradient using a density gradient media such as glycerol, glucose OptiPrep, Percoll, or Ficoll-Paque. The use of such a density gradient media in this manner may result in cells of the starting renal cell population (or starting renal cell population having been passaged at most one, two or three times) separating into one or more distinguishable fractions from which cells of the enriched heterogeneous renal cell population may be distinctly identified and isolated. The distinguishable fraction(s) may be one / those in which the buoyant density of cells in the fraction(s) is greater than about 1.045 g / mL, or greater than 1.045 g / mL, or greater than or equal to 1.045 g / mL. The distinguishable fraction(s) may be one / those in which the buoyant density of cells in the fraction(s) is greater than about 1.04 g / mL, or greater than 1.04 g / mL, or greater than or equal to 1.04 g / mL, or greater than about 1.0419 g / mL, or greater than 1.0419 g / mL, or greater than or equal to 1.0419 g / mL. The distinguishable fraction(s) may be one / those in which the buoyant density is between about 1.045 g / mL and about 1.091 g / mL, or between about 1.045 g / mL and about 1.052 g / mL. Alternatively, the separation step may be one that separates cells of the starting renal cell population (or cells of the starting renal cell population that have been passaged no more than one, two or three times), on the basis of whether they express particular markers on their surface. If the separation step separates cells on the basis of their expression of particular cell surface markers, the separation step may be one that utilizes flow cytometry. The flow cytometry may sort out cells from the starting renal cell population (or starting renal cell population having been passaged at most one, two or three times) if they express particular surface markers, such as nephrin or a cytokeratin (CK), e.g., CK18, characteristic of, e.g., renal epithelial cells, renal tubular cells, renal tubular epithelial cells, or renal proximal tubular cells, to thereby form the isolated enriched heterogeneous renal cell population.

[0156] The enriched heterogeneous renal cell population, having been prepared from a starting renal cell population (or starting renal cell population having been passaged at most one, two or three times) may be cultured under hypoxic conditions prior to the separation step. If the cells are cultured under hypoxic conditions prior to the separation step, the cells may be cultured under conditions in which the oxygen levels are less than about 20%, or less than about 15%, orAttorney Docket No.55801WO 0728.000558WO01 less than about 10%, or less than about 9%, or less than about 8%, or less than about 7%, or less than about 6%, or less than about 5% oxygen, or less than about 4% oxygen, or less than about 3% oxygen or less than about 2% oxygen. If the cells are cultured under hypoxic conditions, the cells may be cultured under the hypoxic conditions for at least 6 hours, at least 8 hours, at least 10 hours, at least 12 hours, at least 14 hours, at least 16 hours, at least 20 hours, at least 24 hours, at least 30 hours, at least 36 hours, at least 42 hours, at least 48 hours, about 6 hours to about 48 hours, about 6 hours to about 36 hours, about 6 hours to about 24 hours, about 12 hours to about 48 hours, about 12 hours to about 36 hours, or about 12 hours to about 24 hours.

[0157] In general, the preparation of an enriched heterogeneous renal cell population may be from any starting cell population, for example, an in vitro culture of cells established from a kidney tissue of a patient or a kidney biopsy of a patient. If the enriched heterogeneous renal cell population is prepared from the in vitro culture of cells established from the kidney tissue or kidney biopsy of the patient, the cells of the in vitro culture may be expanded by passaging at most one, or at most two or at most three times. Alternatively, if desired, cells of the in vitro culture of cells established from the kidney tissue or kidney biopsy may be cryopreserved and then expanded by passaging at most one, or at most two or at most three times. Once the cells have been expanded, the expanded cells may be cryopreserved. The expanded cells, whether or not cryopreserved, may then be subject to a separation step, or may then be subject to hypoxic conditions, or may then be subject to hypoxic culture conditions followed by a separation step. The enriched heterogeneous renal cell population may be isolatable after having been subject to the hypoxic conditions or after having been subject to the hypoxic conditions and the separation step. Once the enriched heterogeneous renal cell population has been isolated, it may be frozen and / or analyzed prior to use as a therapeutic.

[0158] In some instances, the enriched heterogeneous renal cell population may be enriched for the one or more renal cell types as a result of having been prepared from a starting renal cell population, (e.g., a kidney tissue of a patient, a kidney biopsy of a patient, or an in vitro culture of cells established from a kidney tissue or kidney biopsy of a patient), via a method in which cells of the starting renal cell population have been passaged no more than one, two, or three times. In such instances, the starting renal cell population (or starting renal cell population having been passaged at most one, two or three times) may be cultured under hypoxic conditions. If the cells are cultured under hypoxic conditions, the cells may have been culturedAttorney Docket No.55801WO 0728.000558WO01 under conditions in which the oxygen levels are less than about 20%, or less than about 15%, or less than about 10%, or less than about 9%, or less than about 8%, or less than about 7%, or less than about 6%, or less than about 5% oxygen, or less than about 4% oxygen, or less than about 3% oxygen or less than about 2% oxygen. If the cells are cultured under hypoxic conditions, the cells may be cultured under the hypoxic conditions for at least 6 hours, at least 8 hours, at least 10 hours, at least 12 hours, at least 14 hours, at least 16 hours, at least 20 hours, at least 24 hours, at least 30 hours, at least 36 hours, at least 42 hours, at least 48 hours, about 6 hours to about 48 hours, about 6 hours to about 36 hours, about 6 hours to about 24 hours, about 12 hours to about 48 hours, about 12 hours to about 36 hours, or about 12 hours to about 24 hours. In such instances, the cells having been passaged no more than one, two, or three times and then exposed to the hypoxic conditions may result in the enriched heterogeneous renal cell population. It will be further understood that once the enriched heterogeneous renal cell population has been isolated, it may be frozen and / or analyzed prior to use as a therapeutic. Cells at any step of preparing the enriched heterogeneous renal cell population from the starting renal cell population, (e.g., the starting renal cell population or the starting renal cell population having been passaged once or twice or three times) may be cryopreserved.

[0159] It should be understood that if the percentage of cells expressing a certain marker is provided as being a percentage of “about” a particular number, e.g., about 5%, the percentage of cells need not be exactly the particular number, e.g., exactly 5%. Rather, it should be understood that if the percentage of cells expressing a certain marker is provided as being “about” a particular number, e.g., about 5%, then the percentage of cells expressing the certain marker may be within up to 10% of that particular number, e.g., between 4.5% and 5.5%. It should also be understood that if cells express a certain marker by secreting it at an amount of “about” a particular number, e.g., about 5 ng, the amount need not be exactly that particular number, e.g., exactly 5 ng. Rather, it should be understood that the secreted amount of the marker may be within 10% of the particular number, e.g., between 4.5 and 5.5 ng. Pharmaceutical compositions

[0160] In some embodiments, the present disclosure relates to pharmaceutical compositions, or methods of preparing pharmaceutical compositions, including an enriched heterogeneous renal cell population in a pharmaceutically acceptable carrier. The pharmaceutical composition can beAttorney Docket No.55801WO 0728.000558WO01 formulated for administration to a subject in need of treatment for kidney disease, such as chronic kidney disease (CKD) or related conditions. The enriched heterogeneous renal cell population can be suspended in a suitable liquid carrier, such as saline, buffered saline, or cell culture media. The composition can include additional components, such as stabilizers, cryoprotectants, or other.

[0161] If the enriched heterogeneous renal cell population is identified as having a therapeutic potential in accordance with any of the methods disclosed herein, it may be included in a pharmaceutical composition, or administered in a method of treating kidney disease in a patient in need thereof, and / or used in the manufacture of a medicament to treat kidney disease. If the enriched heterogeneous renal cell population is identified as having a therapeutic potential and is included in a pharmaceutical composition, it may be formulated as a hydrogel composition or as a liquid composition. The pharmaceutical composition may, or may not, include hyaluronic acid.

[0162] If the pharmaceutical composition is formulated as a hydrogel composition, cells of the enriched heterogeneous renal cell composition may be combined with a temperature-sensitive cell-stabilizing biomaterial. The temperature-sensitive cell-stabilizing biomaterial may be a biomaterial in a gel state at certain temperatures and a liquid state at others. For example, if the biomaterial is temperature-sensitive, the biomaterial may be in a gel state at about 8 °C or below, a substantially liquid state at about ambient temperature or above, and a solid-to-liquid transitional state between about 8 °C and about ambient temperature; or a gel state at about 4 °C or below, a liquid state at about 37 °C or above, and a solid-to-liquid transitional state between about 8 °C and about 18 °C; or a gel state at about 2 °C or below, a liquid state at about 37 °C or above, and a solid-to-liquid transitional state between about 8 °C and about 18 °C; or a gel state at about 2 °C or below and a liquid state at about 37 °C or above; or a gel state at about 4 °C or below and a liquid state at about 34 °C or above; or a gel state at about 6 °C or below and a liquid state at about 32 °C or above. The temperature-sensitive cell-stabilizing the biomaterial may include, or be made up of, one or more naturally sourced or recombinant proteins or peptides. The naturally sourced or recombinant proteins or peptides may be extracellular matrix proteins of recombinant origin, or extracellular matrix sourced from kidney or another tissue or organ, or gelatin. If the temperature-sensitive cell-stabilizing biomaterial is, or includes, gelatin, the gelatin may be derived from a Type I, alpha I collagen such as porcine Type I, alpha IAttorney Docket No.55801WO 0728.000558WO01 collagen or recombinant human Type I, alpha I collagen. If the temperature-sensitive cell- stabilizing biomaterial is, or includes, gelatin, the gelatin may present in the therapeutic composition at about 0.5% to about 1% weight per volume (w / v), or about 0.8% to about 0.9% (w / v), or about 0.75% (w / v) or about 0.88% (w / v). Cells of the enriched heterogeneous renal cell population may be dispersed throughout the biomaterial, or substantially uniformly distributed throughout the biomaterial. Cells of the enriched heterogeneous renal cell population may be formulated in the biomaterial, e.g., gelatin, such that the number of cells per mL biomaterial is about 20 × 106cells per mL, about 40 × 106cells per mL, about 60 × 106cells per mL, about 100 × 106cells per mL, about 120 × 106cells per mL, about 140 × 106cells per mL, about 160 × 106cells per mL, about 180 × 106cells per mL, or about 200 × 106cells per mL.

[0163] If the pharmaceutical composition is formulated as a liquid composition, the enriched heterogeneous renal cell population may be combined with any suitable liquid, e.g. appropriate cell storage or culture medium, a saline, or combinations thereof, for immediate use or for cryopreservation up until the timing of its use. If the therapeutic composition is a liquid composition, the cells of the enriched heterogeneous renal cell population may be suspended in a pharmaceutically acceptable carrier or excipient, such as saline, buffered saline, dextrose, water, polyethyleneglycol, and / or any combinations thereof. Cells of the enriched heterogeneous renal cell population may be combined with the suitable liquid, e.g., cell storage or culture medium, such that the number of cells per mL liquid is about 20 × 106cells per mL, about 40 × 106cells per mL, about 60 × 106cells per mL, about 100 × 106cells per mL, about 120 × 106cells per mL, about 140 × 106cells per mL, about 160 × 106cells per mL, about 180 × 106cells per mL, or about 200 × 106cells per mL. Methods of preparing a formulation

[0164] In another aspect, the present disclosure relates to a method of preparing a formulation. A method of preparing a formulation may include combining an enriched heterogeneous renal cell population and a pharmaceutically acceptable carrier or excipient. The method of preparing a formulation may include combining an enriched heterogeneous renal cell population, identified as being suitable for formulation, with a pharmaceutically acceptable carrier or excipient. Methods of identifying an enriched heterogeneous renal cell population as suitable for formulation may include the steps, and / or elements, as described for any of the methods ofAttorney Docket No.55801WO 0728.000558WO01 identifying an enriched heterogeneous renal cell population as having a therapeutic potential herein. Pharmaceutically acceptable carriers and excipients are known in the art. Examples of suitable pharmaceutically acceptable carriers and excipients are also described elsewhere herein, such as in the section entitled “Pharmaceutical Compositions”. Methods of administering a formulation

[0165] In another aspect, the present disclosure relates to a method of administering a formulation. The formulation may include an enriched heterogeneous renal cell population.

[0166] A method of administering a formulation including an enriched heterogeneous renal cell population may be an in vitro method or an in vivo method. Examples of in vitro methods include cell culture, organoid culture, or ex vivo organ culture methods. Examples of in vivo methods include treating a subject, such as a human subject.

[0167] If the enriched heterogeneous renal cell population is identified as having a therapeutic potential, the enriched heterogeneous renal cell population, or a pharmaceutical composition comprising the enriched heterogeneous renal cell population, may be administered to a patient in a method of treating kidney disease or may be for use in a method of treating kidney disease. If the enriched heterogeneous renal cell population is identified as having a therapeutic potential, the enriched heterogeneous renal cell population, or a pharmaceutical composition comprising the enriched heterogeneous renal cell population, may be used in the manufacture of a medicament to treat kidney disease. The kidney disease may be at any stage or degree of acute or chronic renal failure. The kidney disease may originate in the kidney or it may be secondary to another condition, e.g., heart failure, hypertension, diabetes, autoimmune disease or liver disease. Alternatively, the kidney disease may be a kidney disease arising from an acute injury to the kidney, or the result of an anomaly of the kidney and / or urinary tract. The kidney disease may further include endocrine dysfunctions such as anemia, e.g., erythropoietin-deficiency, and mineral imbalance, e.g., Vitamin D deficiency.

[0168] If the enriched heterogeneous renal cell population is identified as having a therapeutic potential, then administering the enriched heterogeneous renal cell population, or a pharmaceutical composition comprising the enriched heterogeneous renal cell population, may treat the kidney disease. It may treat the kidney disease by restoring kidney function, stabilizing kidney function, improving kidney function, reducing renal fibrosis or reducing renalAttorney Docket No.55801WO 0728.000558WO01 inflammation in a kidney of a patient in need of such treatment. To determine whether a treatment stabilizes kidney function, the kidney function of a subject may be compared before treatment, during treatment, and / or after treatment. Kidney function may be measured using any suitable metric, such as measuring the estimated glomerular filtration rate (eGFR) or GFR. Methods of measuring eGFR are known to the art. Typically, a treatment that stabilizes kidney function slows or halts the rate of eGFR decline in a subject. To determine whether the rate of eGFR decline is reduced, the eGFR of a treated subject may be compared to a baseline eGFR of the subject. A baseline eGFR may be established by measuring the subject’s eGFR prior to treatment. In some embodiments, a treatment that stabilizes kidney function in a subject reduces the rate of decline in eGFR of at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 100% relative to a baseline eGFR of the subject. In some embodiments, stabilizing kidney function comprises reducing annual rate of decline in eGFR to no more than 3.0 mL / min / 1.73m2, no more than 2.75 mL / min / 1.73m2, no more than 2.5 mL / min / 1.73m2, no more than 2.25 mL / min / 1.73m2, no more than 2.0 mL / min / 1.73m2, no more than 1.75 mL / min / 1.73m2, no more than 1.5 mL / min / 1.73m2, no more than 1.25 mL / min / 1.73m2, no more than 1.0 mL / min / 1.73m2, no more than 0.75 mL / min / 1.73m2, no more than 0.5 mL / min / 1.73m2, or no more than 0.25 mL / min / 1.73m2. The treating the kidney disease may restore mineral balance, electrolyte balance, fluid homeostasis, reabsorption of essential nutrients, or Cystatin C metabolism, or alleviate anemia in a patient in need of such treatment. The treating the kidney disease may delay or prevent the need for dialysis, or it may delay or prevent the need for a kidney transplant in a patient in need of a treatment for kidney disease. If the treating the kidney disease delays the need for dialysis or the need for a kidney transplant in the patient, the delay may be by at least 1 year, at least 1.5 years, at least 2 years, at least 2.5 years, at least 3 years, at least 3.5 years, at least 4 years, at least 4.5 years, at least 5 years, at least 5.5 years, at least 6 years, at least 6.5 year, at least 7 years, at least 7.5 years, at least 8 years, at least 8.5 years, at least 9 years, at least 9.5 years, or at least 10 years. The treating the kidney disease may be determined by observation in an improvement in the patient’s serum albumin, albumin to globulin ratio (A / G ratio), serum phosphorous, serum sodium, kidney size (measurable by ultrasound), serum calcium, phosphorous:calcium ratio, serum potassium, proteinuria, urine creatinine, serum creatinine, blood nitrogen urea (BUN), cholesterol levels, triglyceride levels, and glomerular filtration rate (GFR), weight, bloodAttorney Docket No.55801WO 0728.000558WO01 pressure (mean systemic blood pressure, diastolic blood pressure, or systolic blood pressure), and physical endurance performance.

[0169] If the enriched heterogeneous renal cell population is identified as having a therapeutic potential, it may be administered to a patient by any suitable administration route known in the art. For instance, the enriched heterogeneous renal cell population, or a pharmaceutical composition comprising the enriched heterogeneous renal cell population, may be systemically administered to a patient in need of treatment for kidney disease. The enriched heterogeneous renal cell population, or a pharmaceutical composition comprising the enriched heterogeneous renal cell population, may be administered at or into the kidney(s) of a patient in need of treatment for kidney disease. If the enriched heterogeneous renal cell population is administered at or into the kidney(s) of the patient in need of treatment for kidney disease, it may be administered over a single or over multiple injection(s). It may be administered via direct laparotomy, via direct laparoscopy, transabdominally, or percutaneously. The enriched heterogeneous renal cell population, or pharmaceutical composition comprising the enriched heterogeneous renal cell population, may be administered by percutaneous injection into the renal cortex of a kidney, or may be administered by inserting a guiding cannula percutaneously to puncture the kidney capsule and then injecting the enriched heterogeneous renal cell population into the kidney. The enriched heterogeneous renal cell population, or pharmaceutical composition comprising the enriched heterogeneous renal cell population, may be administered by injection into the renal cortex of one or both kidneys of the patient. The administration of the enriched heterogeneous renal cell population, or pharmaceutical composition comprising the enriched heterogeneous renal cell population, may be by two injections wherein the first injection is into the renal cortex of one kidney and a second injection is into the renal cortex of the other kidney of the patient.

[0170] The enriched heterogeneous renal cell population, or pharmaceutical composition comprising the enriched heterogeneous renal cell population, may be administered by any suitable route, at a therapeutically effective dose. A therapeutically effective dose, or amount, for administration to the patient in need of treatment for kidney disease may include about 1-9 × 106enriched heterogeneous renal cell population cells per gram estimated kidney weight of the patient. A therapeutically effective amount of the pharmaceutical composition may be a dose of about 1.0 × 106, about 2.0 × 106, about 3.0 × 106, about 4.0 × 106, about 5.0 × 106, about 6.0 ×Attorney Docket No.55801WO 0728.000558WO01 106, about 7.0 × 106, about 8.0 × 106, about 9.0 × 106, about 2.0 - 7.0 × 106, between about 4.0 - 7.0 × 106, or between about 5.0 × 106- 7.0 × 106cells of an enriched heterogeneous renal cell population per gram estimated kidney weight of the patient.

[0171] The administration of the therapeutic composition that includes the heterogeneous renal cell population to a patient in a method of treating kidney disease, may be by first and second injections. In some embodiments, the first injection is into a first kidney, and the second injection is into a second kidney. In some embodiments, both the first injection and second injection are into the same kidney. The first and second injections may be administered to a first and second kidney on the same day. In some embodiments, a first injection is administered to a first kidney on a first day, and a second injection is administered to a second kidney on a second day. The first and second injections may be administered between approximately 3 and 12 months apart. The first and second injections may be administered approximately 3 months apart, approximately 4 months apart, approximately 5 months apart, approximately 6 months apart, approximately 7 months apart, approximately 8 months apart, approximately 9 months apart, approximately 10 months, approximately 11 months apart or approximately 12 months apart. The first and second injections may be administered between approximately 3 and 6 months apart, between approximately 6 and 9 months apart, between approximately 9 and 12 months apart, between approximately 3 and 9 months apart, between approximately 6 and 9 months apart or between approximately 6 and 12 months apart. In some embodiments, a method includes administering a third injection, a fourth injection, a fifth injection, and / or a sixth injection. Methods of treating a subject

[0172] In another aspect, the present disclosure relates to a method of treating a subject. The subject may have kidney disease, such as chronic kidney disease (CKD). A method of treating a subject may include administering to the subject an enriched heterogeneous renal cell population or formulation including an enriched heterogeneous renal cell population as described herein. A method of treating a subject may include administering to the subject an enriched heterogeneous renal cell population identified as suitable for treating the subject, or administering to the subject a formulation that includes an enriched heterogeneous renal cell population that had been identified as suitable for treating the subject. Methods of identifying an enriched heterogeneous renal cell population as suitable for treating a subject may include the steps, and / or elements, asAttorney Docket No.55801WO 0728.000558WO01 described for any of the methods of identifying an enriched heterogeneous renal cell population as having a therapeutic potential herein. The enriched heterogeneous renal cell population or formulation including an enriched heterogeneous renal cell population may be administered to the subject in a therapeutically effective dose. In some embodiments, the administering the enriched heterogenous renal cell population or formulation including the enriched heterogeneous renal cell population, as set forth herein, treats the CKD. It may treat the kidney function as disclosed herein in the section entitled “Methods of administering a formulation.” Alternative methods of identifying an enriched heterogeneous renal cell population as having therapeutic potential.

[0173] An alternative method of identifying an enriched heterogeneous renal cell population as having therapeutic potential may profile expression levels of a panel of genes by cells of the enriched heterogeneous renal cell population. One or more cell types within the enriched heterogeneous renal cell population may be identified. The one or more cell types may be identified, at least in part, by presence of a cell type / state gene signature biomarker. The cell type / state gene signature biomarker may include one or more differentially expressed genes. The enriched heterogeneous renal cell population may be identified as having therapeutic potential if one of the one or more cell types includes an indicator gene signature biomarker.

[0174] In any of the instances of the alternative method of identifying an enriched heterogeneous renal cell population as having therapeutic potential, the panel of genes (which may or may not include genes associated with renal cell identity and / or tissue response) may include at least 1, at least 10, at least 100, at least 1000, at least 10,000, or at least 100,000 genes.

[0175] In any of the instances of the alternative method of identifying an enriched heterogeneous renal cell population as having a therapeutic potential, the profiling of the expression level of the panel of genes may be by a sequencing method or a transcriptomic technique. The sequencing method may be single-cell RNA sequencing (scRNA-seq).

[0176] In any of the instances of the alternative method of identifying an enriched heterogeneous renal cell population as having therapeutic potential, the one or more cell types of the enriched heterogeneous renal cell population that may be identified by the cell type / state gene signature biomarker may be or include: (i) proximal tubule (PT) cell type, (ii) Loop of Henle cell type (e.g., ascending thin limb / adaptive thin ascending limb-2 (ATL / aTAL-2) cell types); (iii) parietalAttorney Docket No.55801WO 0728.000558WO01 epithelial cell (PEC) type, (iv) distal convoluted tubule (DCT) cell type, and / or (v) inner medullary collect duct (IMCD) cell type. In some such instances, the one or more cell types may include a PT cell type and / or ATL / aTAL cell type.

[0177] In any of the instances of the alternative method of identifying an enriched heterogeneous renal cell population as having therapeutic potential, the one or more differentially expressed genes that may be included in the cell type / state gene signature biomarker, and that may identify the one or more cell types in the enriched heterogeneous renal cell population, may be genes whose expression levels, e.g., level and / or percentage cells, are sufficiently and / or quantifiably different between cells of one cell type relative to cells of other cell type(s) in the enriched renal cell population. The differentially expressed genes, e.g., genes that show sufficient and / or quantifiable differences in expression levels between cells of one cell type relative to cells of other cell types in the enriched heterogeneous renal cell population, may be genes whose expression is upregulated or downregulated in cells of the one cell type relative to cells of the other cell type(s) in the enriched heterogeneous renal cell population. Additionally, or alternatively, they may be genes that are expressed by higher or lower percentages of cells in the one cell type relative to cells of other cell type(s) in the enriched heterogeneous renal cell population. In some instances, the differentially expressed genes may be the top-most differentially expressed genes of one cell type relative to the other cell type(s) in the enriched heterogeneous renal cell population.

[0178] In any of the instances of the alternative method of identifying an enriched heterogeneous renal cell population as having therapeutic potential, the one or more differentially expressed genes that may be included in the cell type / state gene signature biomarker, and that may identify the one or more cell types in the enriched heterogeneous renal cell population, may include any one or more of: Complement Factor H (CFH), ST8 Alpha-N-Acetyl-Neuraminide Alpha-2,8- Sialyltransferase 2 (ST8SIA2), Aquaporin 1 (AQP1), MIR548A1 Host Gene (MIR548A1HG), Solute Carrier Family 14 Member 1 (SLC14A1), Vascular Cell Adhesion Molecule 1 (VCAM1), Thrombospondin 2 (THBS2), AC010789.1, AL079338.1, Dedicator of Cytokinesis 8 (DOCK8), Solute Carrier Family 7 Member 7 (SLC7A7), Acyl-CoA Synthetase Medium Chain Family Member 2A (ACSM2A), Alanyl Aminopeptidase (ANPEP), Spondin 2 (SPON2), Single-Minded Family BHLH Transcription Factor 2 (SIM2), Insulin-Like Growth Factor Binding Protein 2 (IGFBP2), Transcription Factor AP-2 Beta (TFAP2B), Iroquois Homeobox 2 (IRX2),Attorney Docket No.55801WO 0728.000558WO01 Chromosome 5 Open Reading Frame 38 (C5orf38), Secretin Receptor (SCTR), Transmembrane Protein 37 (TMEM37), Secreted Frizzled Related Protein 1 (SFRP1), Secretory Leukocyte Peptidase Inhibitor (SLPI), Secreted Phosphoprotein 1 (SPP1), GATA Binding Protein 3 (GATA3), AC019197.1, Transmembrane Protein 52B (TMEM52B), GATA3 Antisense RNA 1 (GATA3-AS1), Dual Specificity Phosphatase 2 (DUSP2), Transient Receptor Potential Cation Channel Subfamily M Member 6 (TRPM6), Transient Receptor Potential Cation Channel Subfamily M Member 7 (TRPM7), Solute Carrier Family 8 Member A1 (SLC8A1), Kallikrein 1 (KLK1), Calbindin 1 (CALB1), Kallikrein 8 (KLK8), Kallikrein 7 (KLK7), Vestigial Like Family Member 1 (VGLL1), Mucin 16 (MUC16), S100 Calcium Binding Protein A9 (S100A9), Prostaglandin-Endoperoxide Synthase 1 (PTGS1), and Lipocalin 2 (LCN2).

[0179] In any of the instances of the alternative method of identifying an enriched heterogeneous renal cell population as having a therapeutic potential, the one or more cell type identified by the cell type / state gene signature biomarker may be PECs and the cell type / state gene signature biomarker that identifies the cell type as PECs may include one or more CFH, ST8SIA2, AQP1, MIR548A1HG, SLC14A1, VCAM1, and THBS2 as differentially expressed genes. In instances in which the one or more cell type identified by the cell type / state gene signature biomarker is PT, the cell type / state gene signature biomarker that may identify the cell type as PTs may include one or more of AC010789.1, AL079338.1, DOCK8, SLC7A7, ACSM2A, and ANPEP as differentially expressed genes. In instances in which the one or more cell type identified by the cell type / state gene signature biomarker is ATL / aTAL-2, the cell type / state gene signature biomarker that identifies the cell type as ATL / aTAL-2 may include one or more of SPON2, SIM2, IGFBP2, TFAP2B, IRX2, C5orf38, SCTR, TMEM37, SFRP1, SLPI, and SPP1 as differentially expressed genes. In instances in which the one or more cell type identified by the cell type / state gene signature biomarker is DCT / CNT, the cell type / state gene signature biomarker that identifies the cell type as DCT / CNT may include one or more of GATA3, AC019197.1, TMEM52B, GATA3-AS1, DUSP2, TRPM6, TRPM7, SLC8A1, KLK1, CALB1, and KLK8 as differentially expressed genes. In instances in which the one or more cell type identified by the cell type / state gene signature biomarker is IMCD, the cell type / state gene signature biomarker that may identify the cell type as IMCD may include one or more of KLK7, VGLL1, MUC16, S100A9, PTGS1, and LCN2 as differentially expressed genes.Attorney Docket No.55801WO 0728.000558WO01

[0180] In any of the instances of the alternative method of identifying an enriched heterogeneous renal cell population as having a therapeutic potential, the one or more cell types may further be identified by comparison to a reference database. The reference database may be the Kidney Precision Medicine Project (KPMP) atlas or the GenitoUrinary Development Molecular Anatomy Project (GUDMAP) atlas. The comparison to the reference database may be by unsupervised clustering and projection methods. Examples of unsupervised clustering techniques include, but are not limited to, k-means clustering, hierarchical clustering, density- based spatial clustering (DBSCAN), uniform manifold approximation and projection (UMAP), t- distributed stochastic neighbor embedding (t-SNE), and graph-based clustering (Louvain algorithm). In some instances, the application of the unsupervised clustering techniques can confirm identification of the one or more cell types based on their cell type / state gene signature biomarker.

[0181] In any of the instances of the alternative method of identifying an enriched heterogeneous renal cell population as having a therapeutic potential, an indicator gene biomarker signature of the identified one of the one or more cell types may identify the enriched heterogenous renal cell population as having the therapeutic potential. In such instances, if the one or more cell types identified by the cell type / state gene signature biomarker is ATL / aTAL-2, the indicator gene biomarker signature that may identify the enriched heterogenous renal cell population as having the therapeutic potential may include one or more of genes Lymphocyte Cytosolic Protein 1 (LCP1), Wnt Family Member 7B (WNT7B), Retinoic Acid Receptor Responder 2 (RARRES2), - SRY-Box Transcription Factor 11 (SOX11), Nerve Growth Factor (NGF), Apolipoprotein E (APOE), Contactin Associated Protein-Like 2 (CNTNAP2), G Protein Regulated Inducer of Neurite Outgrowth 3 (GPRIN3), SLIT-ROBO Rho GTPase-Activating Protein 3 (SRGAP3), Cytochrome P450 Family 1 Subfamily B Member 1 (CYP1B1), Thymosin Beta 4 Y-Linked (TMSB4Y), Somatostatin (SST), Erb-B2 Receptor Tyrosine Kinase 4 (ERBB4), and Cadherin 1 (CDH1). In such instances, the indicator biomarker signature may include elevated expression, e.g., level of expression or percentage of cells in the ATL / aTAL2 cell type of the population that are positive for expression, of one or more of genes SYK, RARRES2 or EFNB2. In other such instances, the indicator biomarker signature may include elevated expression, e.g., level of expression or percentage of cells in the ATL / aTAL2 cell type, positive for expression of one or more of RARRES, GPRIN3, SRGAP3, CYP1B1, TMSB4Y, SST, CDH1, and ERBB4.Attorney Docket No.55801WO 0728.000558WO01

[0182] Alternatively, in any of the instances of the alternative method of identifying an enriched heterogeneous renal cell population as having a therapeutic potential, where the one or more cell types identified by the cell type / state gene signature biomarker is PT, the indicator gene biomarker signature that may identify the enriched heterogenous renal cell population as having the therapeutic potential may include one or more of genes Major Histocompatibility Complex, Class II, DR Beta 1 (HLA-DRB1), Apolipoprotein E (APOE), Spleen Tyrosine Kinase (SYK), Retinoic Acid Receptor Responder 2 (RARRES2), Ephrin B2 (EFNB2), and Matrix Metallopeptidase 2 (MMP2). In such instances, the indicator biomarker signature may include elevated expression, e.g., level of expression or percentage of cells in the aPT cell type of the population positive for expression, of one or more of WNT7B, RARRES2, NGF, CYP1B1, or ERBB4. In a further such instance, the indicator biomarker signature may include elevated expression, e.g., level of expression or percentage of cells in the aPT cell type of the population positive for expression, of RARRES.

[0183] In any of the methods of any of the instances of the alternative method of identifying an enriched heterogeneous renal cell population as having a therapeutic potential, the methods may further include a step of determining expression levels of at least one additional gene or marker, e.g., CK18, KIM-1, and / or VEGF, and / or gene signature biomarker, e.g., CD224 aka GGT1, as discussed earlier herein above. See, in particular, section entitled “Additional genes and markers”. Such methods may include a yet further step of identifying the enriched heterogeneous renal cell population as having a therapeutic potential if cells of the population are determined to express CK18, KIM-1, and / or VEGF, and / or gene signature biomarker, e.g., CD224, also known asGGT1 as discussed earlier herein.

[0184] Furthermore, the enriched heterogeneous renal cell population, that may be subject to the alternative method of identifying an enriched heterogeneous renal cell population as having therapeutic potential, may be prepared from any starting renal cell population according to any method provided herein. The enriched heterogeneous renal cell population, having been prepared from such a starting renal cell population, and according to such methods, may be enriched for certain renal cells, e.g., renal epithelial cells or tubule cells, as discussed earlier herein above. The enriched heterogeneous renal cell population, if identified by any of the alternative methods as having a therapeutic potential, may be included in any pharmaceutical composition as provided in the disclosure and may be included in the pharmaceuticalAttorney Docket No.55801WO 0728.000558WO01 composition according to any of the methods of preparing a pharmaceutical composition provided in the present disclosure.

[0185] Additionally, the enriched heterogeneous renal cell population, if identified as having therapeutic potential by any of the alternative methods, may be administered to a subject according to any of the methods of treatment, e.g., treatment for CKD, provided herein. In some instances, the enriched heterogeneous renal cell population identified by any of the alternative methods as having a therapeutic potential, may be used in a method of treatment, e.g., treatment for CKD, and may be administered to a subject in a pharmaceutical composition by any route as provided herein.

[0186] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein. Such equivalents are intended to be encompassed by the appended claims.

[0187] All publications, patents and patent applications mentioned in this specification are herein incorporated by reference into the specification to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference in their entirety. EXAMPLES EXAMPLE 1 – Phenotypic characterization of a renal cell therapy product through its manufacture

[0188] Introduction.

[0189] Rilparencel, which has also been referred to a selected renal cell (SRC) population or enriched heterogeneous renal cell population, is an autologous renal cell therapy in phase 3 clinical trials for treatment of type 2 diabetes and chronic kidney disease (CKD). To better characterize rilparencel, contribute to an understanding of its mechanism of action, and assess any unique features that may explain its potential clinical activity, an investigation into rilparencel’s phenotypic characteristics was conducted.

[0190] Methods / scRNA-seq to map gene expression:

[0191] SRC aliquots were stained with acridine orange and propidium iodide, and cells were assessed for viability, concentration, and singleness using the LUNA-FX7 Dual Fluorescence Cell Counter (Logos Biosystems). Cells were then processed using the 10x Genomics ChromiumAttorney Docket No.55801WO 0728.000558WO01 Controller and the Chromium Single Cell 3′ GEM, Library & Gel Bead Kit v3.1 Dual Index Kit (PN-1000268) following the manufacturer’s user guide (https: / / tinyurl . com / 4855859x). Briefly, approximately 5,000 cells per sample were loaded onto the Chromium Chip G with a target recovery of 3,000 cells per sample for library preparation. Single cells, reverse transcription reagents and gel beads coated with barcoded oligos were encapsulated together in an oil droplet to produce gel beads in emulsion (GEMs). Reverse transcription was performed using a C1000 thermal cycler (Bio-Rad) to generate complementary DNA (cDNA) libraries tagged with a cell barcode and unique molecular index (UMI). GEMs were then broken and cDNA was purified using Dynabeads MyOne SILANE beads (Invitrogen) prior to 12 cDNA amplification cycles. Amplified cDNA libraries were purified with SPRIselect magnetic beads (Beckman Coulter) and were quantified using an Agilent Bioanalyzer High Sensitivity DNA chip (Agilent Technologies). Fragmentation, end repair, A-tailing and double-sided size selection using SPRIselect beads were then performed. Illumina-compatible adapters were ligated onto the size-selected cDNA fragments. Adapter-ligated cDNA was then purified using SPRIselect beads. Uniquely identifiable indexes were added to this cDNA during 12 amplification cycles. The completed sequencing libraries were then purified using SPRIselect beads, visualized using the Bioanalyzer High Sensitivity DNA chip, and pooled in an equimolar ratio. Pooled libraries were sequenced on a NextSeq 2000 machine (Illumina) at the UNC High Throughput Sequencing Facility. Libraries were denatured and diluted following standard Illumina protocol, spiked with 1% PhiX sequencing control (Illumina), and sequenced on one P3 flow cell in paired-end format (Read 1: 28 cycles, i7 index: 10 cycles, i5 Index: 10 cycles, Read 2: 90 cycles) to a total depth of 1.2 billion read pairs passing quality filters. Demultiplexing and preliminary analysis were performed using Cell Ranger 7.1.0 with default settings, and analysis was performed using Seurat.

[0192] Methods / Flow Cytometry Phenotypic Analysis:

[0193] Kidney biopsy cell samples and T0 cell samples for flow cytometry analysis were prepared from kidneys obtained from National Disease Research Interchange (NDRI). T1, T2, T3, and rilparencel drug product (SRC) cell samples for flow cytometry analysis were prepared from biopsies of kidneys obtained from NDRI (n=1) or biopsies of kidneys from chronic kidney disease patients enrolled in a phase 2 clinical trial (n=6, NCT02836574). To prepare rilparencel, cells were isolated from the kidney biopsies by enzymatic digestion and then plated (T0). TheAttorney Docket No.55801WO 0728.000558WO01 T0 cells were subjected to an expansion process having at least first (T1), second (T2) and third (T3) steps. Following further manufacturing steps, cells of rilparencel drug product (SRCs) were selected.

[0194] Cell samples for surface phenotyping were placed into cell culture media, washed once with Becton Dickenson (BD) flow stain buffer (BD # 554656), and counted using a NucleoCounter NC-202. Cells (1×106) were plated for surface phenotyping with cocktails of titrated conjugated monoclonal antibodies (BioLegend, BD, and ThermoFisher) containing eBioScience SuperBright Staining Buffer (Thermo # SB-4401-42). See Tables 1 and 2 for conjugated monoclonal antibodies in the cocktails. Live cells were surface stained in 100 μL volume for 30 minutes at 4 °C in the dark. Cells were then washed with flow buffer and incubated with Live Dead NIR for 15 minutes at 4 °C in the dark (Thermo # L34975).

[0195] Following surface staining cells were fixed and permeabilized as per manufacturer instructions using eBioScience FoxP3 Fix / Perm kit (Thermo # 00-5523-00). Cells were blocked with 2% mouse serum then incubated with conjugated antibodies (BioLegend) targeting cytoplasmic and nuclear antigens for 45 minutes at room temperature in the dark. Cells were washed with permeabilization buffer, resuspended in flow buffer, and acquired on a BD FACSLYRIC Flow Cytometer. Single-color compensation controls were made using both cells and UltraComp beads (Thermo # 01-2222-41). Compensation was calculated in FLOWJO. Single cells in the live gate were analyzed and analysis was performed using FLOWJO and GraphPad Prism. Table 1: Multiplex Flow Cytometry Panel – Renal Cell Identity Protein Clone Fluorophore Cell type CD227 EMA 16A BV421 Di t l h L fAttorney Docket No.55801WO 0728.000558WO01 Table 2: Multiplex Flow Cytometry – Renewal and Repair Protein Clone Fluorophore CD90 (Thy-1) 5E10 BV421Marker Expression by scRNAseq:

[0197] Two immunophenotyping panels were developed to assess phenotype characteristics of rilparencel drug product (SRC) as well as precursors to rilparencel during the manufacturing process. One panel focused on renal cell identity in the nephron (proximal tubules, distal tubules, collecting duct, and podocytes). See Table 1. The other panel focused on markers associated with a tissue healing response. See Table 2. SRCs had been found to express, by scRNAseq, each marker selected for inclusion in the immunophenotyping panels. Table 3. Table 3: Percent SRCs Expressing, by scRNAseq, Markers Used in Flow Cytometry Analysis Gene Average RangeAttorney Docket No.55801WO 0728.000558WO01 CD146 (MCAM) 93.67 91.34-96.94 CD44 (H-CAM) 99.93 99.83-99.99s at Manufacturing Stages from Renal Biopsy to Product:

[0199] Expression of renal cell type identity markers by cells at varying stages of rilparencel manufacturing were analyzed by flow cytometry. Percent cells at the T1, T2, T3, and SRC stages of manufacturing that stained positive for expression of each renal cell identity marker in Table 1 was determined. See Table 4 and FIG.1A-1G. Table 4: Percent Cells Positive for Expression of Renal Cell Identity Markers at Different Stages of rilparencel Manufacturing Marker T1 T2 T3 SRC Average Range Average Range Average Range Average Range

[0200] As seen from Table 4, the percentage cells expressing certain renal cell identity markers CD10 (proximal tubule), CD13 (proximal tubule), CD224 (proximal tubule), and GATA3 (collecting duct) progressively increase over the course of the manufacturing process. CD227 (distal nephron) and CD326 (distal tubule) are both also relatively abundant, while PDPN (glomerulus) is and remains relatively low.

[0201] A further analysis looked at co-expression of several of renal cell identity markers on cells at biopsy, T0, T2, and SRC stages of the manufacturing process. It was found that cells at earlier stages in the manufacturing process, e.g., biopsy and T0 cells, had a similar pattern of CD10 (proximal tubule) and CD13 (proximal tubule) expression / co-expression, but that at laterAttorney Docket No.55801WO 0728.000558WO01 stages, e.g., T2 and SRC, the CD10 and CD13 expression pattern changed to become largely CD13+CD10+. See FIG.2A and Table 5. When expression of CD227 (distal tubule) and CD13 were analyzed, it was found that at the T0 early stage of manufacturing, CD227 was highest on CD13- cells, but that at the late, SRC, stage of manufacturing, CD227 had decreased and became co-expressed with CD13. See FIG.2B and Table 5. Further, over time in manufacturing, cells increased co-expression of proximal tubule markers CD13 and CD227. See FIG.2C and Table 5. Overall, from these analyses, proximal tubule marker expression by cells was found to increase over the course of the rilparencel manufacturing process. Kidney biopsy cells, analyzed in FIG.2B for CD13 and CD227 expression, were additionally evaluated for expression of CD10. CD10 was largely found on the CD13+CD227- cells. See FIG.2D. Table 5: Percent Co-Expression / Expression of Cell Identity Phenotype Markers by Cells at Different Stages of Rilparencel Manufacturing Process Marker(s) Biopsy T0 T2 SRC A R A R

[0202] To better understand cells at an early, e.g., T0, stage of manufacturing, dimensionality reduction analysis was performed. As shown in FIG.3, when analyzed for CD224 and CD13 expression, T0 cells separated into four relatively distinct cell populations. tSNE analysis of flow cytometry data obtained from use of the panel of clones provided in Table 1 revealed that atAttorney Docket No.55801WO 0728.000558WO01 T0, cell clustering is largely driven by expression of CD224 and CD13 (four cell population clusters) and PDPN (fifth cluster). See FIG.4.

[0203] Dimensionality reduction analysis was also performed for cells from T0, T2, and SRC stages of the manufacturing process. Concatenation of T0, T2, and SRC samples showed clustering through CD13, CD224, CD227, and PDPN. Clustering of cells was delineated by both concatenation and individual contributions of each sample timepoints. As the manufacturing progressed, cells populations became more CD13+CD224+. FIG.5A. Also observed in FIG. 2C. The two primary clusters in SRCs were separated based on expression of CD227. See FIG. 5B.

[0204] PDPN (glomerular) marker expression, while low throughout the stages of the manufacturing process, was found to be a driver in cell clustering in dimensionality reduction analysis. Flow cytometry data showing expression of PDPN, and its lack of co-expression with CD13, by cells of seven different SRC samples is provided at FIG.6A. Co-expression of PDPN and CD106 is shown in FIG.6B.

[0205] Finally, as earlier mentioned in reference to FIG.2A and Table 5, SRCs are largely CD10+CD13+, e.g., largely express markers characteristic of the renal proximal tubule cells. To further explore phenotypic characteristics of CD13+CD10+ cells through manufacturing, CD13+CD10+ T1, T2, T3, and SRC cells were analyzed for whether they co-express CD227 (distal tubule) or GATA3 (collecting duct) renal cell identity markers. As shown in FIG.7B-7G, CD13+CD10+ cells at each stage of the manufacturing process co-express, to varying degrees, CD227 (FIG.7B-7F) and GATA3 (FIG.7G), with CD227 generally becoming more downregulated, and GATA3 becoming more highly expressed, by cells as they progress through manufacturing. See also Table 6. Table 6: Percent Co-Expression of Cell Identity Phenotype Markers by CD13+CD10+ Cells at Different Stages of Rilparencel Manufacturing Process Marker(s) T1 T2 T3 SRCAttorney Docket No.55801WO 0728.000558WO01

[0206] Results / Phenotype Characterization of Cells, at Manufacturing Stages from T0, or T1 to Product, by Renewal and Repair Markers

[0207] Expression of renewal and repair markers on cells from samples taken at varying stages of rilparencel manufacturing were also analyzed. Percent cells positive by flow cytometry for expression of each renewal and repair marker in Table 2 was determined for cells at the T1, T2, T3 and SRC stages of manufacturing. See Table 7 and FIG.8A-8G. Table 7: Percent Cells Positive for Expression of Renewal and Repair Markers at Different Stages of rilparencel Manufacturing Process Marker T1 T2 T3 SRC A R A R A R A R 7 4 5 8 6

[0208] Co-expression analysis of various of the renewal and repair markers revealed that cells undergo dynamic phenotypic changes during the SRC manufacturing process. Cells early in manufacturing (T0) were CD24 positive and highly CD133 positive. CD133 downregulated during manufacturing, and CD133’s downregulation was accompanied by both increased CD90 expression and significantly increased CD146 expression. See FIG.9A-9C and Table 8.Attorney Docket No.55801WO 0728.000558WO01 Table 8: Percent Expression / Co-Expression of Renewal and Repair Markers CD133 and CD24 at Different Stages of rilparencel Manufacturing Process Marker(s) T0 T2 SRC Avg Range Avg Range

[0209] CD90 and CD106 have been reported to be involved in kidney repair. From the T1 to SRC stage of manufacturing, cells became less CD90-CD106- and more CD90+CD106-. See FIG.15 and Table 9. Table 9: Percent Expression / Co-Expression of Renewal and Repair Markers CD90 and CD106 at Different Stages of rilparencel Manufacturing Process Markers T1 T2 T3 SRC Av Ran e Av Ran e Av Ran e Av Ran e

[0210] CD90 and CD133 were also examined for expression / co-expression. A notable change in expression of these markers over manufacturing was observed. The percentage CD90-CD133+ cells became reduced while the percentage CD90+CD133- cells increased. See FIG.16 and Table 10. Table 10: Percent Expression / Co-Expression of Renewal and Repair Markers CD90 and CD133 at Different Stages of rilparencel Manufacturing Process Markers T1 T2 T3 SRCAttorney Docket No.55801WO 0728.000558WO01 CD90-CD133+ 54.46 42.6- 70.3 29.39 17.8- 39.8 13.65 6.48- 19 14.82 8.96-- 18.9 CD90+CD133+ 22.4 9.4- 35 18.05 7.42- 25.8 12.13 3.32- 17.9 21.22 8.83- 31.8 5 5CD106. In general, the CD24+CD133+CD106+ phenotype appeared to remain relatively constant through manufacturing, while the CD24+CD133+CD106- phenotype was reduced. See Table 11. Table 11: Percent Expression / Co-Expression of Renewal and Repair Marker CD106 by CD24+CD133+ Cells at Different Stages of rilparencel Manufacturing Process Markers T1 T2 T3 SRC Avg Range Avg Range Avg Range Avg Range 9

[0212] Discussion.

[0213] A multiparameter flow cytometry analysis revealed dynamic expression profiling changes during manufacture of SRCs, e.g., rilparencel, from biopsy tissue. Among the changes are that as cells progressed through the manufacturing process, they became increasing positive for proximal tubule kidney markers such as CD13 and CD10. Cells also mostly came to express CD224. Distal tubule marker, CD227, was highly expressed at early stages of manufacturing but was largely downregulated at later stages of manufacturing. CD90 expression became upregulated, as did CD106 and CD146, but less so. Further, CD133 was highly expressed early in manufacturing, but became significantly downregulated. Finally, biopsy data showed that CD10 was largely expressed on CD13+CD227- cells, but SRCs were characterized by co- expression of CD10, CD13, CD224, and significant amounts of CD227, raising the possibility that during manufacturing phenotypic adaptation (e.g., de- / re-differentiation) occurs.

[0214] It was also noted that several markers were highly (CD24, CD44, GATA3, and vimentin) or lowly (PDPN) expressed on cells throughout manufacturing.Attorney Docket No.55801WO 0728.000558WO01 EXAMPLE 2 –Reduction of inflammatory and fibrotic secretomic signatures of renal cell therapy product through the manufacturing process

[0215] Introduction.

[0216] Inflammation, fibrosis, and senescence are three contributing factors leading to loss of kidney function in acute kidney injury and CKD. A study was performed to investigate whether cells at the final product (SRC) stage of rilparencel manufacturing secreted lower levels of markers associated with inflammation and fibrosis than did cells at an early (T2) stage of rilparencel manufacture.

[0217] Methods.

[0218] Rilparencel was prepared from biopsies of a healthy kidney obtained from NDRI (n=1) or biopsies of kidneys from chronic kidney disease patients enrolled in a phase 2 clinical trial (n=5, NCT02836574). To prepare rilparencel, cells were isolated from the kidney biopsies by enzymatic digestion and then plated (T0). The T0 cells were subjected to an expansion process having at least first (T1), second (T2), and third (T3) steps. Following further manufacturing steps, cells of rilparencel drug product (SRCs) were selected.

[0219] To analyze proteins secreted by cells at an early, e.g., T2, versus late, e.g., SRC, stage of manufacturing, cells were plated and then cultured for 24 hours. After the 24 hours, conditioned media was harvested and screened using a LUMINEX INTELLIFLEX multiplex instrument using multiplex assay panels from ThermoFisher PROCARTAPLEX. Heatmaps were generated using GraphPad Prism 10.2.0.

[0220] Results.

[0221] Analysis of conditioned media harvested from cells of each of the five CKD patients at the early (e.g., T2) and final (e.g., SRC) rilparencel manufacturing stages revealed that proteins associated with inflammation were secreted at lower levels by SRCs, e.g., rilparencel cells, than cells at the earlier, e.g., T2, stage of manufacturing. See FIG.10 and Table 12.Attorney Docket No.55801WO 0728.000558WO01 Table 12: Quantity of Proteins Associated with Inflammation Secreted by Cells at T2 and SRC Stages of rilparencel Manufacture Protein secreted (pg per 10,000 cells at 24 hr) Protein Patient 1 Patient 2 Patient 3 Patient 4 Patient 5 Healthy 2 3 07 2 5

[0222] Analysis of conditioned media harvested from cells of each of the five CKD patients at the early (e.g., T2) and final (e.g, SRC) rilparencel manufacturing stages also revealed that proteins associated with fibrosis / kidney toxicity were, mostly, secreted at lower levels by SRCs, e.g., rilparencel cells, than cells at the earlier, e.g., T2, stage of manufacturing. See FIG.11 andAttorney Docket No.55801WO 0728.000558WO01 Table 13. Of note, levels of secreted kidney toxicity associated proteins appeared to be more variable than those associated with inflammation. Table 13: Quantity of Proteins Associated with Fibrosis / Kidney Toxicity Secreted by Cells at T2 and SRC Stages of rilparencel Manufacture Protein secreted (pg per million cells at 24 hr) Protein Patient 1 Patient 2 Patient 3 Patient 4 Patient 5 HealthySRC T2 SRC T2 SRC7 5 5 4 5Attorney Docket No.55801WO 0728.000558WO01

[0223] Using the data from the secretomic analysis, PCA was performed. A PCA plot from the analysis showed distinct clustering between the five patients’ cells at the early (T2), and the five patients’ cells at the late (SRC or rilparencel), stages of manufacturing. See FIG.12.

[0224] Overall, in this cohort of five CKD patients, it appeared that levels of secreted inflammation and fibrosis associated markers was reduced in rilparencel cells relative to cells from an earlier, e.g., T2, stage of rilparencel manufacturing. EXAMPLE 3 – Reduced canonical senescence marker expression by a renal cell therapy product relative to a precursor to the renal cell therapy product in manufacturing

[0225] Senescence, a characteristic of CKD, is a process of permanent cell growth arrest. The relationship between CKD and senescence is cyclical. A study was performed to investigate whether cells at the final product (SRC) stage of rilparencel manufacturing secreted lower levels of senescence markers than did cells at an early (T2) stage of rilparencel manufacture, and / or had a senescence-associated secretory phenotype (SASP).

[0226] Methods.

[0227] Rilparencel was prepared from biopsies of a healthy kidney obtained from NDRI (n=1) or biopsies of kidneys from chronic kidney disease patients enrolled in a phase 2 clinical trial (n=5, NCT02836574) as described in Example 2. Analysis of proteins secreted by cells at an early, e.g., T2, versus late, e.g., SRC, stage of manufacturing, was also performed as described in Example 2. scRNAseq and DEG analysis were performed as described in Example 1.

[0228] The SASP of rilparencel was assessed using the publicly available SASP atlas database (https: / / saspatlas . herokuapp . com / ). The SASP atlas database is a proteomic database of soluble proteins and exosomal cargo SASP factors originating from senescence inducers, e.g., X- ray irradiation (genotoxic stress-induced), RAS overexpression (oncogene induced), and atazanavir treatment (treatment induced) and cell types, e.g., human primary fibroblasts and epithelial cells. Ninety rilparencel DEGs and eighty secreted proteins that had been measured for rilparencel were input into the renal cortical epithelial cell SASP atlas to identify whether rilparencel expressed senescence biomarkers.

[0229] Results.Attorney Docket No.55801WO 0728.000558WO01

[0230] Analysis of conditioned media harvested from cells of each of the five CKD patients at the early (e.g., T2) and final (e.g, SRC) rilparencel manufacturing stages revealed that canonical senescence markers were secreted at lower levels by SRCs, e.g., rilparencel cells, than cells at the earlier, e.g., T2, stage of manufacturing. See FIG.13 and Table 14. Table 14: Quantity of Canonical Senescence Proteins Secreted by Cells at T2 and SRC Stages of rilparencel Manufacture Protein secreted (pg per 10,000 cells at 24 hr) Protein Patient 1 Patient 2 Patient 3 Patient 4 Patient 5 Healthy C 07 2 5

[0231] The SASP of rilparencel was assessed using the SASP atlas database. Ninety rilparencel DEGs and 80 rilparencel secreted proteins were input in the SASP atlas database to identify those that may be senescence biomarkers. Of the input biomarkers, only 10 were identified via the SASP atlas, suggesting that rilparencel is, at most, weakly associated with senescence. See Table 15 for quantity of senescence associated proteins, as identified by the SASP atlas, secreted by rilparencel. Table 15: Quantity of rilparencel Secreted SASP-Associated Proteins Protein Protein secreted (pg per 10,000 SRCs at 24 hr)Attorney Docket No.55801WO 0728.000558WO01 IL-8 88.53 136.16 89.69 105.05 79.87 TIMP-1 2365.71 5102.15 1506.47 3084.35 3231.96rilparencel, through its manufacture process using multiparameter flow cytometry

[0232] Introduction.

[0233] A further characterization of rilparencel, or SRC, through its manufacturing process was performed to better determine the renal cell types and states within rilparencel, and to further elucidate its mechanism of action.

[0234] Methods:

[0235] Kidney biopsy cell samples and T0 cell samples for analysis were prepared from kidneys obtained from National Disease Research Interchange (NDRI; n=5). T1, T2, T3, and rilparencel (SRC) cell samples were prepared from biopsies of kidneys obtained from NDRI kidneys and from kidneys of patients with type 2 diabetes (T2D) and chronic kidney disease (CKD) enrolled in two Phase 2 investigative clinicals trials (n=5 from REGEN-002 / NCT02836574 and n=12 from REGEN-007 / NCTG05018416).

[0236] To prepare rilparencel (SRC), cells were isolated from kidney biopsies by enzymatic digestion and then plated (T0). The T0 cells were subjected to an expansion process having at least first (T1), second (T2), and third (T3) steps. Following further manufacturing steps, cells of rilparencel (SRCs) were selected.

[0237] Samples (e.g., biopsy, T0, T1, T2, T3, and SRC) were evaluated by multi-color flow cytometry using antibody reagents specific to canonical renal cell markers associated with renal cell identity or tissue healing. See Tables 16 (renal cell identity) and 17 (tissue healing) for antibody cocktails used to characterize cell samples through the rilparencel manufacturing process. Table 16: Flow Assay Cocktail for Renal Cell Identity Protein Clone Fluor Cell TypeAttorney Docket No.55801WO 0728.000558WO01 CD227 (EMA) 16A BV421 Distal nephron CD326 (EpCAM) 9C4 BV510 Distal tubulea e : ow ssay oc a or ena e rogen ors an e -Renewal Protein Clone Fluor [0g y p Table 16 or the antibody cocktail provided Table 17 to characterize the samples, assays were performed using a combination of select antibodies from both Table 16 and 17.

[0239] To evaluate the cell samples, cells from the samples were placed into cell culture media, washed once with Becton Dickenson (BD) flow stain buffer (BD # 554656), and counted using a NucleoCounter NC-202. Cells (1x106) were plated for surface phenotyping with cocktails of titrated conjugated monoclonal antibodies containing eBioScience SuperBright Staining Buffer (Thermo # SB-4401-42). Live cells were surface stained in 100 μL volume for 30 minutes at 4 °C in the dark. Cells were then washed with flow buffer and incubated with Live Dead NIR for 15 minutes at 4 °C in the dark (Thermo # L34975).

[0240] Following surface staining, cells were fixed and permeabilized as per manufacturer instructions using eBioScience FoxP3 Fix / Perm kit (Thermo # 00-5523-00). Cells were blocked with 2% mouse serum then incubated with conjugated antibodies (BioLegend) targeting cytoplasmic and nuclear antigens for 45 minutes at room temperature in the dark. Cells were washed with permeabilization buffer, resuspended in flow buffer, and acquired on a BD FACSLyric Flow Cytometer. Single-color compensation controls were made using both cells and UltraComp beads (Thermo # 01-2222-41). Compensation was calculated in FLOWJO. Single cells in the live gate were analyzed and analysis was performed using FLOWJO and GraphPad Prism.Attorney Docket No.55801WO 0728.000558WO01

[0241] Results from the analysis are expressed as the % cell positivity for each marker tested in a sample. Mean ± standard deviation (SD) is used to describe data for a sample set and was calculated using Excel. Variability is reported as the coefficient of variation (%CV) based on the SD expressed as a percentage of the mean.

[0242] Results / Phenotype Characterization of rilparencel (SRC) Cells – Individual Markers.

[0243] Expression of the eight renal cell identity markers in Table 16 and six renal progenitor and self-renewal markers in Table 17 were evaluated for each of seventeen clinical rilparencel cell samples (n=5 from REGEN-002 / NCT02836574 and n=12 from REGEN- 007 / NCTG05018416). The mean percentage cells positive, and associated variability (reported as % CV), were calculated for each marker across the 17 samples. See Table 18 for the mean percentage cells positive, and associated variability, for each renal cell identity marker. See Table 19 for the mean percentage cells positive, and associated variability, for each renal progenitor and self-renewal marker. Table 18: Renal cell identity marker expression by rilparencel cells Marker Nephron Segment Expression in rilparencel (Mean (% Cell Distribution and % CV))Table 19: Renal cell progenitor and self-renewal marker expression by rilparencel cells Marker Purpose Expression inAttorney Docket No.55801WO 0728.000558WO01 CD24 (HSA) Stem cell marker associated with scattered 98.6 (2.2%) tubular progenitor cells or h i i i l i h li l ll [was no e a e mean percen age o ce s a were pos ve or o prox ma and distal nephron markers was elevated. As shown in Table 18, greater than 85% of rilparencel cells were positive for each of the three tested proximal nephron (CD13, CD10, and CD224) markers and greater than 70% of rilparencel cells were positive for each of the four tested distal nephron (CD227, CD326, GATA3, and PDPN) markers. Such co-expression of proximal and distal nephron markers suggested renal epithelial cell plasticity.

[0245] To further investigate renal epithelial cell plasticity in the rilparencel cell samples, expression of renal cell progenitor / self-renewal markers was examined. Interestingly, rilparencel cells exhibited a counterbalance between the progenitor and self-renewal markers. See Table 19, which shows that: (i) about a third of rilparencel cells expressed progenitor cell marker CD133, and (ii) around 55% of rilparencel cells expressed self-renewal / repair marker CD90. Also, rilparencel cells were observed to highly co-express stem cell markers CD24, CD44, and CD146, e.g., markers which drive self-renewal and dedifferentiation.

[0246] Low variability was observed for the percentage cells expressing certain renal cell identity markers across the 17 rilparencel clinical samples. For example, a range of 1.2% CV to 5.1% CV was calculated for proximal markers CD13 (LAP), CD10 (MMe), CD224 (GGT1), and collecting duct system marker GATA3 (1.2% CV). For renal distal nephron markers CD227, CD326, and CD171 a variability of around 20%, was observed. Finally, higher variability wasAttorney Docket No.55801WO 0728.000558WO01 calculated for glomerular podocyte marker PDPN. The higher variability for PDPN was likely due to a wide range in the data for a very small cell population size (<6% cell-positivity) overall in the rilparencel cell samples. See Table 18.

[0247] Similar levels of variability in percentage cells expressing progenitor / self-renewal markers across the 17 rilparencel clinical samples was observed. Of the six progenitor / self- renewal markers, <2.5% variability was calculated for the three stem cells markers (CD24, CD44, and CD146) and a higher, e.g., 19-39% variability, was calculated for the three self- renewal markers (CD133, CD106, and CD90). See Table 19.

[0248] Results / Phenotype Characterization of Biopsy Cell Samples in Comparison to rilparencel Cell Samples.

[0249] To gain insight into the cell dynamics associated with the suggested renal epithelial cell plasticity of rilparencel cell samples, a longitudinal study was conducted. The longitudinal study explored expression of the markers in Tables 16 and 17 by samples of cells from renal cortical biopsies, and samples of cells from the T0, T1, T2, and T3 stages of rilparencel’s manufacturing process. Due to restrictions to accessing biopsy samples from clinical trial participants for a longitudinal study such as this, e.g., clinical biopsy samples are a limited cell resource and are needed as starting material for rilparencel manufacture, data for biopsy samples and samples from the T0 stage of manufacturing were established from donor kidneys procured from the NDRI. The phenotype of biopsy cell samples differed greatly from that of the clinical rilparencel cell samples. As seen in FIG.20A - 20B, unlike rilparencel cell samples, which displayed extensive co-expression of proximal and distal nephron markers, biopsy cell samples exhibited distinct proximal and distal cell clusters representative of their origin in native cortical tissue. Also, proximal tubule epithelial cells, in comparison to cells of the distal nephron compartment, were the predominant cell type in the biopsy cell samples.

[0250] The resident epithelial cells of the biopsy cell samples were tested for expression of the renal cell progenitor and self-renewal markers in Table 17. As shown in the first panel of FIG. 21, about 10% of the cells in the biopsy cell samples were CD24+CD133+. After a single culture passage (which generated T0 cells), the percentage of CD24+CD133+ cells increased dramatically. However, by the end of manufacturing (SRC or rilparencel cells), the percentage CD24+CD133+ cells decreased with a concomitant increase in CD24+CD133- cells.Attorney Docket No.55801WO 0728.000558WO01

[0251] Additional support for renal epithelial cell plasticity gained by cells as a result of the manufacturing process was observed by examining expression of self-renewal marker CD133 and de- / re-differentiation marker CD90 by cells of biopsy, T0, T1, T2, T3, and rilparencel cell samples. As shown in FIG.22A, at the earliest, T0, stage of manufacturing, CD133+CD90- cells were the predominant cell type. However, as the manufacturing process progressed, at the T3 stage of manufacturing and at the final rilparencel manufacturing stage, the dynamic had shifted and CD133-CD90+ cells became the predominant cell type. FIG.22B-22C provide graphical representations of the shift of cells from a CD133+CD90- to a CD133-CD90+ phenotype over the course of the rilparencel manufacturing process.

[0252] Overall, the data obtained from tracking the phenotype of renal epithelial cells from biopsy to rilparencel confirmed that there are fundamental differences between cell samples from kidney biopsy tissue and rilparencel. Among these differences are that in biopsy cell samples the predominant cell type was the proximal tubular epithelial cell, while in rilparencel cell samples the cells came to express both proximal and distal nephron markers. Also, in the biopsy cell samples epithelial cells were infrequently associated with tissue repair, e.g., there were a low number of CD133-CD90+ cells, while in rilparencel cell samples, CD133-CD90+ cells came to be at a higher frequency.

[0253] Results / Over the Course of rilparencel (SRC) Manufacturing, Cells Come to Co-Express Both Proximal and Distal Tubule Markers.

[0254] As proximal tubular epithelial cells were the predominant cell type in kidney biopsies, and as cells in rilparencel samples largely came to co-express proximal and distal nephron markers, distal marker acquisition by proximal tubular epithelial cells at different stages of rilparencel manufacturing was evaluated. As shown in FIG.23A-23G, over the course of manufacturing, cells came to co-express sets of proximal nephron markers, sets of distal nephron markers, and combinations of proximal and distal nephron markers. For instance, FIG.23A-23C show that proximal tubule epithelial cells became co-expressers of three proximal nephron markers tested in Table 16, e,g.,CD10 and CD13, and CD224 and CD13. Not only did the cells become co-expressers of these proximal nephron markers, they also acquired expression of distal markers CD227 and CD171. See, in particular, FIG.23D-23G, which shows co-expression of proximal nephron marker CD13 or proximal markers CD13 and CD10 with distal nephron marker CD171 or CD227. In addition, it was determined that over the course of manufacturing,Attorney Docket No.55801WO 0728.000558WO01 the percentage of cells co-expressing collecting duct marker GATA3 and either of proximal nephron markers CD224 or CD13 increased to near 100%, while the percentage of cells that were CD13-GATA3+ or CD224-GATA3+ decreased to less than 10%. See FIG.24B and Table 20. It appeared that cells with a proximal nephron phenotype had acquired characteristics of distal nephron cells. Table 20: Expression of Proximal Nephron and Collecting Duct Markers by Cells in Samples Over the Course of rilparencel Manufacturing Marker(s) T0 T2 (SD) SRC (SD) CD13 (LAP) 41.4 94.9 (5.41) 98.7 (1.51) uring clinical manu ac ur ng o r parence ce s ( ) or - / pa en s (n=6).

[0256] Results / Co-Expression of Proximal Tubule and Tissue Repair Makers by Cells Over the Course of rilparencel Manufacture.

[0257] It was also examined whether rilparencel cells expressing proximal nephron markers had acquired, or were co-expressing, markers for tissue repair or response to injury. To this end, cells of five clinical rilparencel samples (REGEN-002 / NCT02836574) were analyzed for percent cells having the following phenotypes: CD24+CD133+, CD24+CD133-, CD224+CD133+, CD224+CD133-, CD224+CD90+, and CD326+CD13+. The results of the analysis revealed that rilparencel cells expressed markers consistent with cells having the characteristic of epithelial plasticity. See Table 21. Table 21: Expression of Repair Markers with or without Co-Expression of Proximal Nephron Markers by rilparencel Cells Mk E i% Cell PositiveC ll Ph t C i t t ith:Attorney Docket No.55801WO 0728.000558WO01 CD224+CD133+ <20% (8.7 to 19.7%)Intermediate (progenitor &differentiated)cell) marker expression by cells along the manufacturing process was explored. Flow cytometry analysis examined the percent cells, at each of the T1,T2, T3, T3a, and rilparencel stages of manufacturing, that co-expressed a (i) proximal (CD13 or CD224) or distal (CD227 or CD171) nephron markers and (ii) progenitor (CD133) / de-re-differentiation (CD90) marker. With respect to cells expressing either CD13 or CD224 proximal nephron markers, it was found that co- expression with CD133 was highest at the early, T1, stage of manufacturing and overall decreased beginning with the T2 stage of manufacturing. While CD133 expression was decreasing beginning with the T2 stage of manufacturing, both CD13 and CD224 expression were increasing. See FIG.25A and 25B. Meanwhile, cells expressing the CD13 and CD224 proximal nephron markers gained CD90 expression and become predominantly CD90+CD13+ and CD90+CD224+ over the course of manufacturing. See FIG.27A - 27B. Cells expressing distal nephron marker CD227 exhibited similar trends in CD133 co-expression (FIG.26A) and CD90 co-expression (FIG.28A) as did the CD13 and CD224 cells. Cell expressing distal nephron marker CD171 showed different CD133 and CD90 co-expression patterns than did the cells expressing proximal nephron markers CD13 and CD224 and distal nephron marker CD227. Cells expressing CD171 were more likely to become negative for both CD133 (FIG.26B) and CD90 (FIG.28B). Overall, the results suggested that the dynamics of CD90 and CD133 expression by cells as they progressed through the manufacturing process were largely driven by those having a proximal epithelial cell phenotype.

[0259] Results / Cumulative Phenotype Characterization of Cells at each Stage of rilparencel Manufacture

[0260] Based on the cumulative data obtained from the flow cytometry panels, a phenotypic profile of cell populations at each of the T0, T1, T2, T3, and rilparencel (SRC) stages of manufacturing was assembled. A phenotype profile of T0, T1, T2, T3, and rilparencel (SRC) cells based on expression of the renal cell identity markers presented in Table 16 can be found inAttorney Docket No.55801WO 0728.000558WO01 FIG.29. A phenotype profile of T0, T1, T2, T3, and rilparencel (SRC) cells based on expression of the renal cell progenitor and self-renewal markers presented in Table 17 can be found in FIG. 30. Of note, the phenotypic profiles for cell populations at each manufacturing stage was similar, regardless of whether the source biopsy material used for manufacturing was obtained from a T2D and CKD patient biopsy an NDRI kidney biopsy.

[0261] Of the renal cell identity markers examined in Table 16, there was a strong trend in increased proximal nephron marker expression over the course of manufacturing and a mixed change in distal nephron marker expression over the course of manufacturing. For the distal nephron markers, the percent cells expressing CD227 and CD171 decreased, but the percent cells expression GATA3 increased over the course of manufacturing.

[0262] Of the renal cell progenitor and self-renewal markers examined in Table 17, dynamic trends were observed with the percent cells expressing CD133, or CD133 and CD24 decreasing over the course of manufacturing, the percent cells expressing CD90 or expressing CD106 increasing over the course of manufacturing, and the percent cells expressing CD146 remaining mostly steady over the course of manufacturing.

[0263] By having performed a detailed phenotypic characterization of a series of canonical renal epithelial cell markers representative of the entire nephron, it was possible to demonstrate that manufacture of rilparencel causes large scale changes in the tubular epithelial cell composition inherent to the native cortical biopsy tissue source material. The changes included an evolution of the cells to largely express both proximal and distal nephron markers, and markers of the collecting duct, in tandem with a dynamic evolution in markers linked to self-renewal and tissue repair. EXAMPLE 5: Inflammatory Profile Characterization of Specific Rilparencel Cell Types

[0264] Introduction.

[0265] Based on phenotypic characteristics of rilparencel (SRC) cells, including that rilparencel (SRC) cells secrete lower levels of proteins associated with inflammation than do cells at early stages of rilparencel manufacturing, cells with different phenotypes were profiled for pro- inflammatory cytokine secretion at multiple rilparencel manufacturing stages.

[0266] Methods.Attorney Docket No.55801WO 0728.000558WO01

[0267] T0 cell samples for analysis were prepared from donor kidneys obtained from National Disease Research Interchange (NDRI). T1, T2, T3, and rilparencel (SRC) cell samples were prepared from biopsies of kidneys obtained from NDRI kidneys and / or from kidneys of patients with type 2 diabetes (T2D) and chronic kidney disease (CKD) enrolled in two Phase 2 investigative clinicals trials (REGEN-002 / NCT02836574 and / or REGEN-007 / NCTG05018416).

[0268] Methods / SRC preparation. To prepare rilparencel (SRC), cells were isolated from kidney biopsies by enzymatic digestion and then plated (T0). The T0 cells were subjected to an expansion process having at least first (T1), second (T2), and third (T3) steps. Following further manufacturing steps, cells of rilparencel (SRCs) were selected.

[0269] Methods / Secretome Analysis. To analyze proteins secreted by T0, T1, T2, T3, and rilparencel (SRC) cell samples, cells were plated and then cultured for 24 hours. After 24 hours, conditioned media was harvested. Harvested media was screened using a LUMINEX INTELLIFLEX multiplex instrument using multiplex assay panels from ThermoFisher PROCARTAPLEX. Heatmaps were generated using GraphPad Prism 10.2.0.

[0270] Methods / Flow Cytometry. Flow Cytometry was performed on T1, T2, T3, and rilparencel (SRC) viably frozen cell samples prepared from biopsies of kidneys of patients with T2D and CKD (n=5 from REGEN-002 / NCT02836574 and n=12 from REGEN- 007 / NCTG05018416) and from NDRI kidneys (n=8). T0 cells samples were prepared from biopsies of NDRI kidneys.

[0271] Briefly, viably frozen cells from the samples were thawed in a 37 °C water bath, then were placed into cell culture media, washed once with Becton Dickinson (BD) flow stain buffer (BD # 554656), and counted using a NucleoCounter NC-200. Cells (1×106) were plated for surface phenotyping with cocktails of titrated conjugated monoclonal antibodies containing EBIOSCIENCE SuperBright Staining Buffer (Thermo # SB-4401-42). Live cells were surface stained in 100 μL volume for 20 minutes at 4 °C in the dark. Cells were then washed with flow buffer and incubated with Live Dead NIR (Thermo # L34975) for 10 minutes at 4 °C in the dark.

[0272] Following surface staining, cells were fixed and permeabilized as per manufacturer instructions using EBIOSCIENCE FoxP3 Fix / Perm kit (Thermo # 00-5523-00). Cells were blocked with 2% mouse serum then incubated with conjugated antibodies (BioLegend) targeting cytoplasmic and nuclear antigens for 30 minutes at room temperature in the dark. Cells were washed with permeabilization buffer, resuspended in flow buffer, and analyzed via a BDAttorney Docket No.55801WO 0728.000558WO01 FACSLYRIC Flow Cytometer or CYTEK Aurora Spectral Flow Cytometer. Single-color compensation controls were made using both cells and UltraComp beads (Thermo # 01-2222- 41). Data analysis was performed using FLOWJO v10 and GraphPad Prism v10.3.1.

[0273] Intracellular cytokine analysis was performed using T1 and rilparencel (SRC) cell samples prepared from biopsies of kidneys of patients with T2D and CKD enrolled in Phase 2 clinical trial REGEN-007 / NCTG05018416 (n=4) and from an NDRI kidney (n=1). A T0 cell sample was prepared from the biopsy of an NDRI kidney (n=1). To perform the intracellular cytokine analysis, cells from the samples were cultured overnight at 37°C in media. A cocktail of Monensin and Brefeldin-A (Thermo #00-4980-03) was then added to the cultures for 4 hours to inhibit the cells’ protein transport. Cells were then counted and plated at 1x106per well in a 96-well U-Bottom plate. Surface and intracellular staining was performed as previously described: surface staining, viability staining, fixing / permeabilization, followed by intracellular staining. Cells were analyzed via a CYTEK Aurora spectral flow cytometer. Single-color compensation controls were made using both cells and UltraComp beads (Thermo # 01-2222- 41). Data analysis was performed using Spectroflo v3.3.0, FLOWJO v10 and GraphPad Prism v10.3.1.

[0274] Methods / Cell Enrichment Based on CD90 and / or CD106 Expression. Rilparencel (SRC) cell populations were sorted and enriched based on their CD90 and CD106 expression status (e.g., CD90+CD106+, CD90+CD106-, CD90-CD106+, CD90-CD106-) using two magnetic cell selection kits (StemCell Technologies #100-0031 and #17684) following manufacturer’s instructions. Cells were sorted sequentially. First, the cells were incubated with anti-CD90-APC followed by incubation with releasable magnetic beads. Next, cells were placed in an EasySepTMmagnet (StemCell Technologies) to separate CD90+ and CD90- populations. Beads were then released from cells. The separated CD90+ and CD90- cell populations were then each incubated with anti-CD106-PE followed by incubation with magnetic beads. Next, cells were placed in magnet to separate the four cell fractions (CD90+CD106+, CD90+CD106-, CD90-CD106+, CD90-CD106-).

[0275] Results / rilparencel (SRC) Cells Secrete Pro-Inflammatory Cytokines Less than Cells at Earlier Stages of rilparencel’s Manufacturing Process. Analysis of the culture media collected from cells at the T1, T2, T3, and rilparencel (SRC) stages of manufacturing revealed that cell samples from early, e.g., T1, stage of manufacturing secreted higher levels of pro-inflammatoryAttorney Docket No.55801WO 0728.000558WO01 cytokines and chemokines than did cells from samples at later stages of rilparencel manufacturing. See FIG.31. See also Example 2.

[0276] Results / Evolution of Renal Epithelial Cell Self-Renewal Marker Expression Over the Course of rilparencel Manufacturing. When examining pro-inflammatory cytokine secretion, cell phenotypes of interest include those where cells express renal epithelial cell self-renewal, e.g., CD90, CD106, and CD133, markers. Data showing percentage of cells expressing each of self-renewal markers CD90, CD106, and CD133 in T1, T2, T3, and SRC cell samples have been presented in Tables 1 and 19, and in FIG.8A-8G, and 30. As shown in these tables and figures, the percentage of cells that expressed each of CD90 and CD106 increased over the T0 to SRC stages of manufacturing, while the percentage of cells expressing CD133 decreased over the T0 to SRC stages of manufacturing. Co-expression of CD106 and CD133 and co-expression of CD90 and CD133 by cells at each stage of the rilparencel manufacturing was next determined. It was found that, with respect to CD90 and CD133 co-expression, the percent CD90-CD133+ cells decreased over the course of manufacturing, while the percentage of CD90+CD133- cells increased over the course of manufacturing. See FIG.32A. Similarly, with respect to CD106 and CD133 co-expression, CD106-CD133+ populations decreased over the course of manufacturing, while CD106+CD133- populations increased over the course of manufacturing. See FIG.32B. Co-expression of CD90 and CD106 and co-expression of CD90 and CD133 at the T1, T2, T3, and SRC stages of manufacturing had already been investigated and are presented in FIG.15 and 16, respectively.

[0277] Results / rilparencel Cells Expressing Certain Self-Renewal Markers Secrete Lower Levels of Pro-Inflammatory Cytokines. The increased percentage of cells expressing self-renewal markers CD90 and CD106 over the course of rilparencel manufacturing led to an investigation into cytokine secretion by CD90- and CD106-expressing cells in rilparencel (SRC) samples. Cells from SRC, the final stage of rilparencel manufacturing were sorted and enriched according to their CD90 and CD106 expression status (e.g., CD90+CD106+, CD90+CD106-, CD90- CD106+, CD90-CD106-). After overnight culture, supernatants of the sorted SRC subpopulations were analysed using the LUMINEX INTELLIFLEX 20-Plex inflammation panel. CD106+ cells in the SRC cell samples, whether CD90+ or CD90-, secreted the lowest levels of proinflammatory cytokines. CD106+CD90+ expressing cells, for the most part, secreted lowerAttorney Docket No.55801WO 0728.000558WO01 levels of proinflammatory cytokines than did CD106+CD90- cells. See FIG.33A-33D. See also FIG.30.

[0278] Results / TNF-α Expression by Cells at Early and Late Stages of rilparencelManufacturing. High levels of TNF- are associated with increased inflammation and fibrosis inCKD. TNF- is produced by multiple cell types in the kidney including immune cells, proximaltubule epithelial cells, podocytes, mesangial cells, and cells of the thick ascending limb andcollecting duct. To assess T0, T1, and final (SRC) samples for TNF- expression, cells fromeach of the T0, T1, and SRC stages of manufacturing were cultured in the presence or absence ofa PTI cocktail, stained for intracellular TNF- and analyzed by a CYTEK Aurora. As shown inFIG.34A and 34B, a higher percentage of cells from the early, T0 and T1, stages ofmanufacturing secreted TNF- (approximately 10% of T0 and 5% of T1 cells) compared to cellsat the end (SRC) stage of manufacturing (approximately 2% of SRC cells).

[0279] T0 and T1 samples were further analysed to determine the phenotype of cells in theirsamples that expressed TNF- . To conduct this analysis, proximal (CD13 or CD224), distal(CD171), or self-repair (CD90, CD106 or CD133) markers were gated on the PTI-treated cellsthat were TNF- + or that were TNF- -. It was found that the TNF- + and TNF- - cells havedistinct phenotypes, with CD90 and CD106 expressing cells secreting less TNF- than cells ofother phenotypes. See FIG.34C and 34D. EXAMPLE 6 – Identification of biological pathways that may be associated with rilparencel’s effect on kidney function

[0280] Introduction.

[0281] Rilparencel, which has also been referred to as a selected renal cell (SRC) population or as an example of an enriched heterogeneous renal cell population, is an autologous renal cell therapy in phase 3 clinical trials for treatment of type 2 diabetes and chronic kidney disease (CKD). To better characterize rilparencel, contribute to an understanding of its mechanism of action, and assess any unique features that can explain its potential clinical activity, a study was conducted to investigate differentially expressed genes (DEGs) and regulated pathways in rilparencel cells that can potentially explain its ability, or degree of ability, to effect kidney function.

[0282] Methods - rilparencel Cell Samples:Attorney Docket No.55801WO 0728.000558WO01

[0283] Banked human rilparencel samples from five patients with type 2 diabetes and CKD enrolled in a regulatory approved phase 2 investigative trial (eGFR of 20 to 50 mL / min / 1.73m2; NCT02836574) were used in the analyses. Rilparencel had been prepared from patient kidney biopsy samples that had been enzymatically digested, passaged, and subject to a selection process.

[0284] Methods - Single Cell Transcriptomic Studies.

[0285] Single cell RNA-seq (scRNAseq) was conducted by the Advanced Analytics Core at University of North Carolina at Chapel Hill (UNC, Chapel Hill, NC). Briefly, aliquots from each of five banked rilparencel samples were stained with acridine orange and propidium iodide, and cells were assessed for viability, concentration, and singleness using the LUNA-FX7 Dual Fluorescence Cell Counter (Logos Biosystems). Cells were then processed using the 10x Genomics Chromium Controller and the Chromium Single Cell 3′ GEM, Library & Gel Bead Kit v3.1 Dual Index Kit (PN-1000268) following the manufacturer’s user guide (https: / / tinyurl . com / 4855859x). Briefly, approximately 5,000 cells per sample were loaded onto the Chromium Chip G with a target recovery of 3,000 cells per sample for library preparation. Single cells, reverse transcription reagents and gel beads coated with barcoded oligos were encapsulated together in an oil droplet to produce gel beads in emulsion (GEMs). Reverse transcription was performed using a C1000 thermal cycler (Bio-Rad) to generate complementary DNA (cDNA) libraries tagged with a cell barcode and unique molecular index (UMI). GEMs were then broken and cDNA was purified using Dynabeads MyOne SILANE beads (Invitrogen) prior to 12 cDNA amplification cycles. Amplified cDNA libraries were purified with SPRIselect magnetic beads (Beckman Coulter) and were quantified using an Agilent Bioanalyzer High Sensitivity DNA chip (Agilent Technologies). Fragmentation, end repair, A-tailing and double-sided size selection using SPRIselect beads were then performed. Illumina-compatible adapters were ligated onto the size-selected cDNA fragments. Adapter-ligated cDNA was then purified using SPRIselect beads. Uniquely identifiable indexes were added to this cDNA during 12 amplification cycles. The completed sequencing libraries were then purified using SPRIselect beads, visualized using the Bioanalyzer High Sensitivity DNA chip, and pooled in an equimolar ratio. Pooled libraries were sequenced on a NextSeq 2000 machine (Illumina) at the UNC High Throughput Sequencing Facility. Libraries were denatured and diluted following standard Illumina protocol, spiked with 1% PhiX sequencing control (Illumina), and sequenced on one P3 flow cell in paired-end formatAttorney Docket No.55801WO 0728.000558WO01 (Read 1: 28 cycles, i7 index: 10 cycles, i5 Index: 10 cycles, Read 2: 90 cycles) to a total depth of 1.2 billion read pairs passing quality filters. Demultiplexing and preliminary analysis were performed using Cell Ranger 7.1.0 with default settings. For each sample, quality control was performed to remove damaged cells and doublets. Counts were log-normalized and scaled.

[0286] Methods – Identification of Kidney Cell Types and States.

[0287] Identification of kidney cell types and states mapped in FIG.35A-35G, was determined using unsupervised clustering and prediction methods to the single cell atlases of CKD and healthy patients (KPMP) & developing kidneys (GUDMAP).

[0288] In FIG.36, cell types were assigned using a combination of both automated cell type calling as well as by comparing gene expression to established kidney cell type markers derived from the KPMP (Lake et al., DOI: 10.1038 / s41586-023-05769-3). For automated cell type calling, cell types were identified by the highest probability cell type match to the KPMP single cell RNA-seq reference (V1.5) using both the R packages Seurat and SingleR. Co-expression of multiple KPMP markers was common among rilparencel cell types and indicated by an annotation of the most similar cell types (e.g., ATL / aTAL2, DCT / CNT, IMCD / CNT / DCT).

[0289] Methods – rilparencel cell type DEGs and pathway analysis.

[0290] For each rilparencel cell-type, cell-level and pseudo-bulk DEGs (|Log2FC|>1.5, adj. P<0.05) were identified by Wilcoxon Rank Sum test or DESeq2, respectively, based on each patient’s response to rilparencel (eGFR slope computed across 1-year post-treatment calculated using a generalized linear model (GLM)). Biological pathways were identified using both Enrichr and clusterProfileR analysis tools.

[0291] Results / Distribution of rilparencel Cells into Cell Types.

[0292] To contribute to a better understanding of rilparencel and its cell composition, rilparencel’s kidney cell types and states were determined using unsupervised clustering and projection methods to the single cell KPMP and GUDMAP atlases. FIG.35A - 35E show separate UMAPs for each of five patient rilparencel samples. FIG.35F and FIG.35G provide UMAPs for the KPMP and GUDMAP single cell atlases, respectively. Of note, distribution of cell types across the five patient rilparencel samples, as shown in FIG.35A - 35E, were consistent.Attorney Docket No.55801WO 0728.000558WO01

[0293] KPMP-anchored molecularly profiled neighborhoods found rilparencel to be constituted with cell types including: glomerular parietal epithelial cells (PECs), adaptive proximal tubules (aPTs), Loop of Henle (ascending thin limb (ATL) and adaptive thick ascending limb-2 (aTAL- 2), distal convoluting tubules (DCT), and connecting tubule (CNT) & inner medullary collecting ducts (IMCD). Proportions of cells in each of these types, for each of the five rilparencel patient samples, is shown in FIG.36 and Table 22 below. Table 22: Proportion of KPMP-Like Cell Types Within rilparencel Patient # aPT ATL / aTAL2 DCT / CNT IMCD / CNT / DCT PEC 1 45.21 45.28 5.28 2.34 1.89

[0294] Rilparencel cells were assigned to one of these five KPMP-like cell types based on the top five differentially expressed genes (DEGs), by Log2FC, in that cell type relative to other cells or cell types in the rilparencel sample. These cell type assignments were supported by differential expression of canonical KPMP genes, for and by cells of the cell type, in the rilparencel sample. In some cases, the KPMP markers for a cell type were identified as being within the top 5 DEGs for that cell type. See FIG.37. See also Table 23 for percentage cells, within each of the five cell types, that express the top 5 DEGs and KPMP canonical markers across all five cell types. Percentage numbers in bold and italics indicate the top DEGs and the KPMP canonical markers for the particular cell type specified in the column header. Table 23: Percentage Cells that Express, within each Rilparencel Cell Type, the Top 5 DEGs (by Log2Fold Change) and KPMP Canonical Markers Across all Five Cell Types GenePercentage Cells within Indicated Cell Type Expressing GeneAttorney Docket No.55801WO 0728.000558WO01 SLC14A1 3.04 1.85 1.46 4.24 49.18 VCAM1 19.94 9.26 31.05 83.83 95.08parence sampes were groupe y pa en e sope oucome - e sope. The two groups, e.g., +eGFR / -eGFR slope outcome groups, were compared for differences in gene expression and biological pathways. Patient eGFR slope outcomes for the five patients areAttorney Docket No.55801WO 0728.000558WO01 provided in FIG.38. Clinical cohort baseline characteristics for the five patients is provided in Table 24. Table 24: Clinical Cohort Baseline Characteristics Patient Sex Age eGFR UACR Calcium Serum Albumin Hemoglobin # (mL / min / 1.73m2) (mg / g) (mg / dL) Bicarbonate (g / dL) A1C (%)

[0296] Grouping the samples by +eGFR slope (n=3) and -eGFR slope (n=2) outcomes identified DEGs between the two that were associated with biological processes and pathways that included cell differentiation and migration, extracellular matrix (ECM) organization, proteolysis, and kidney development. Biological pathways between the samples were identified using clusterProfileR from a list of filtered DEG genes (DEG filtered for |Log2FC| > 1.5, adj. p- value < 0.05, n=267 ). See FIG.39.

[0297] Further, DEGs within only the ATL / aTAL-2 cell type or only the aPT cell type between the +eGFR slope and -eGFR slope samples were identified and subject to pathway analysis. DEGs were then analyzed for enrichment of genes associated with known biological pathways using the Biological_Processes_2023 GO database and the R package EnrichR. A subset of biological pathways containing the greatest number of DEGs are provided in FIG.40A - 40D as the average gene expression of these DEGs between the two clinical outcome groups for each of the ATL / aTAL-2 and aPT cell types. DEGs associated with processes such as regulation of cell migration, regulation of inflammatory response, and regulation of a cell differentiation were identified.. See also Table 25 for percentage cells expressing, and level of expression of, each identified DEG within the ATL / aTAL2 cell type in rilparencel samples of the +eGFR slope versus -eGFR slope patients. See Table 26 for percentage cells expressing, and level ofAttorney Docket No.55801WO 0728.000558WO01 expression of, each identified DEG within aPT cells in rilparencel samples of the +eGFR slope versus -eGFR slope patients. Table 25: Percent and Level Expression of Identified DEGs within ATL / aTAL2 Cells between +eGFR slope versus -eGFR slope patients Gene Percent expression within Level expression*within ATL / aTAL2 cells ATL / aTAL2 cells fTable 26: Percent and Level Expression of Identified DEGs within aPT Cells between +eGFR slope versus -eGFR slope patients GenePercent expression within aPT cellsLevel expression* within aPT cellsfAttorney Docket No.55801WO 0728.000558WO01 OTHER EMBODIMENTS

[0298] From the foregoing description, it will be apparent that variations and modifications may be made to the disclosure described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the claims.

[0299] The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or sub-combination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment, any portion of the embodiment, or in combination with any other embodiments or any portion thereof.

[0300] While specific examples and numerous embodiments have been provided to illustrate aspects and combinations of aspects of the foregoing, it should be appreciated and understood that any aspect, or combination thereof, of an exemplary or disclosed embodiment may be excluded therefrom to constitute another embodiment without limitation and that it is contemplated that any such embodiment can constitute a separate and independent claim. Similarly, it should be appreciated and understood that any aspect or combination of aspects of one or more embodiments may also be included or combined with any aspect or combination of aspects of one or more embodiments and that it is contemplated herein that all such combinations thereof fall within the scope of this disclosure and can be presented as separate and independent claims without limitation. Accordingly, it should be appreciated that any feature presented in one claim may be included in another claim; any feature presented in one claim may be removed from the claim to constitute a claim without that feature; and any feature presented in one claim may be combined with any feature in another claim, each of which is contemplated herein. The following enumerated clauses are further illustrative examples of aspects and combination of aspects of the foregoing embodiments and examples:

[0301] Following is an example of enumerated clauses:

[0302] Clause A1 is a method of identifying an enriched heterogeneous renal cell population as having a therapeutic potential, comprising:Attorney Docket No.55801WO 0728.000558WO01 (a) profiling expression levels of a panel of genes associated with (i) renal cell identity, and / or (ii) tissue healing response (renewal and repair) in the enriched heterogeneous renal cell population; (b) determining the presence of a gene signature biomarker in the enriched heterogeneous renal cell population based at least in part upon the profiled expression levels; and (c) identifying the enriched heterogeneous renal cell population as having therapeutic potential if the gene signature biomarker is determined to be present in the enriched heterogeneous renal cell population.

[0303] Clause A2 is a method of preparing a formulation comprising an enriched heterogeneous renal cell population, comprising: (a) identifying the enriched heterogeneous renal cell population as suitable for formulation, wherein identifying comprises: (i) profiling expression levels of a panel of genes associated with (i) renal cell identity, and / or (ii) tissue healing response (renewal and repair) in the enriched heterogeneous renal cell population; (ii) determining the presence of a gene signature biomarker in the enriched heterogeneous renal cell population based at least in part upon the profiled expression levels; and (iii) identifying the enriched heterogeneous renal cell population as suitable for formulation if the gene signature biomarker is determined to be present in the enriched heterogeneous renal cell population, and (b) combining the enriched heterogeneous renal cell population that has been identified as being suitable for formulation with a pharmaceutically acceptable carrier or excipient to prepare the formulation.

[0304] Clause A3 is a method of treating a subject in need of treatment for chronic kidney disease (CKD) comprising: (a) identifying an enriched heterogeneous renal cell population as suitable for treating the subject according to steps comprising: (i) profiling expression levels of a panel of genes associated with (i) renal cell identity, and / or (ii) tissue healing response (renewal and repair) in the enriched heterogeneous renal cell population;Attorney Docket No.55801WO 0728.000558WO01 (ii) determining the presence of a gene signature biomarker in the enriched heterogeneous renal cell population based at least in part upon the profiled expression levels; and (iii) identifying the enriched heterogeneous renal cell population as suitable for treating the subject if the gene signature biomarker is determined to be present in the enriched heterogeneous renal cell population, and (b) administering the enriched heterogeneous renal cell population that has been identified as suitable for treating the subject to the subject to treat the CKD.

[0305] Clause A4 is the method of clause A3, further comprising formulating a pharmaceutical composition comprising the enriched heterogeneous renal cell population that has been identified as suitable for treating before (b) administering, and wherein the pharmaceutical composition is administered to the subject.

[0306] Clause A5 is the method of clause A4, wherein formulating the pharmaceutical composition comprises combining the enriched heterogeneous renal cell population with a pharmaceutically acceptable carrier or excipient.

[0307] Clause A6 is the method of any of clauses A3 to A5, wherein treating the CKD comprises stabilizing kidney function.

[0308] Clause A7 is the method of clause A6, wherein stabilizing kidney function comprises reducing rate of decline in eGFR by at least 20% relative to a baseline eGFR of the subject, wherein the baseline eGFR of the subject is determined prior to a first administering of the enriched heterogeneous renal cell population.

[0309] Clause A8 is the method of clause A6, wherein stabilizing kidney function comprises reducing annual rate of decline in eGFR to no more than 2.0 mL / min / 1.73m2.

[0310] Clause A9 is the method of any preceding clause, wherein the panel of genes comprises genes associated with renal cell identity.

[0311] Clause A10 is the method of any preceding clause, wherein the panel of genes comprises genes associated with tissue healing response (renewal and repair).

[0312] Clause A11 is the method of any preceding clause, wherein the panel of genes comprises one or more of CD10, GATA3, CD227, CD13, podoplanin (PDPN), CD24, CD171, CD90, CD133, CD106.Attorney Docket No.55801WO 0728.000558WO01

[0313] Clause A12 is the method of any preceding clause, wherein the gene signature biomarker comprises GATA3 expression by at least about 80% of cells in the enriched heterogeneous renal cell population.

[0314] Clause A13 is the method of any preceding clause, wherein the gene signature biomarker comprises PDPN expression by at most about 30% of cells of the enriched heterogeneous renal cell population.

[0315] Clause A14 is the method of any preceding clause, wherein the panel of genes comprises at least two genes.

[0316] Clause A15 is the method of clause A14, wherein the at least two genes comprise CD10 and GATA3, CD10 and CD227, CD13 and CD227, CD13 and GATA3, or CD90 and CD106.

[0317] Clause A16 is the method of clause A15, wherein the gene signature biomarker comprises CD13 and CD227, wherein at least about 30% of cells of the enriched heterogeneous renal cell population are CD13+CD227+.

[0318] Clause A17 is the method of clause A16, wherein about 30% to about 95% of cells in the enriched heterogeneous renal cell population are CD13+CD227+

[0319] Clause A18 is the method of clause A15, wherein the gene signature biomarker comprises CD13 and GATA3, wherein at least about 80% of cells in the enriched heterogeneous renal cell population are CD13+GATA3+.

[0320] Clause A19 is the method of clause A15, wherein the gene signature biomarker comprises CD10 and GATA3, wherein at least about 70% of cells in the enriched heterogeneous renal cell population are CD10+GATA3+.

[0321] Clause A20 is the method of clause A15, wherein the gene signature biomarker comprises CD90 and CD106, wherein at least about 20% to about 50% of cells of the enriched heterogeneous renal cell population are CD90+CD106+.

[0322] Clause A21 is the method of any preceding clause, wherein the panel of genes comprises at least three genes.

[0323] Clause A22 is the method of clause A21, wherein the at least three genes comprise CD13, CD10, and CD227; CD13, CD10, and GATA3; CD24, CD133, and CD106; CD13, CD10 and CD171; or CD24, CD133, and CD106.Attorney Docket No.55801WO 0728.000558WO01

[0324] Clause A23 is the method of clause A22, wherein the gene signature biomarker comprises CD13, CD10, and CD227, wherein at least about 20% of cells of the enriched heterogeneous renal cell population are CD13+CD10+CD227+.

[0325] Clause A24 is the method of clause A23, wherein about 20% to about 85% of cells of the enriched heterogeneous renal cell population are CD13+CD10+CD227+.

[0326] Clause A25 is the method of any clause A22, wherein the gene signature biomarker comprises CD13, CD10, and GATA3, wherein at least about 60% of cells of the enriched heterogeneous renal cell population are CD13+CD10+GATA3+.

[0327] Clause A26 is the method of clause A22, wherein the gene signature biomarker comprises CD24, CD133, and CD106, wherein about 3% to about 30% of cells of the enriched heterogeneous renal cell population are CD24+CD133+CD106+.

[0328] Clause A27 is the method of clause A26, wherein about 9% to about 30% of cells of the enriched heterogenous renal cell population are CD24+CD133+CD106+.

[0329] Clause A28 is the method of clause A22, wherein the gene signature biomarker comprises CD24, CD133, and CD106, wherein about 3% to about 50% of cells of the enriched heterogenous renal cell population are CD24+CD133+CD106-.

[0330] Clause A29 is the method of clause A28, wherein about 3% to about 45% of cells of the enriched heterogeneous renal cell population are CD24+CD133+CD106-.

[0331] Clause A30 is the method of clause A22, wherein the gene signature biomarker comprises CD13, CD10 and CD171, wherein about 40% to about 70% of cells of the enriched heterogeneous renal cell population are CD13+CD10+CD171+.

[0332] Clause A31 is the method of any preceding clause, further comprising determining whether cells of the enriched heterogeneous renal cell population express one or more additional markers.

[0333] Clause A32 is the method of clause A31, wherein the one or more additional markers comprise cytokeratin (CK)18, vascular endothelial growth factor (VEGF) and kidney injury molecule (KIM)-1.

[0334] Clause A33 is the method of clause A32, wherein the gene signature biomarker comprises GATA3 and CD224, and wherein the one or more additional markers comprise and CK18.Attorney Docket No.55801WO 0728.000558WO01

[0335] Clause A34 is the method of clause A33, wherein the enriched heterogeneous renal cell population is identified as having therapeutic potential, being suitable for formulation, or suitable for treating a subject if: at least about 70% of cells of the population are CD224+, at least about 70% of cells of the population are GATA3+, and at least about 70% of cells of the population are CK18+.

[0336] Clause A35 is the method of clause A34, wherein the enriched heterogeneous renal cell population is identified as having therapeutic potential, being suitable for formulation, or suitable for treating the subject if: at least about 80% of cells of the population are CD224+, at least about 80% of cells of the population are GATA3+, and at least about 80% of cells of the population are CK18+.

[0337] Clause A36 is the method of clause A32, wherein the gene signature biomarker comprises PDPN, GATA3, and CD224 and the additional markers comprise CK18.

[0338] Clause A37 is the method of clause A36, wherein the enriched heterogeneous renal cell population is identified as having therapeutic potential, being suitable for formulation, or suitable for treating a subject if: at least about 70% of cells of the population are CD224+, at least about 70% of cells of the population are GATA3+, at most about 30% of cells of the population are PDPN+, and at least about 70% of cells of the population are CK18+.

[0339] Clause A38 is the method of clause A36, wherein the enriched heterogeneous renal cell population is identified as having therapeutic potential, being suitable for formulation, or suitable for treating a subject if: at least about 80% of cells of the population are CD224+, at least about 80% of cells of the population are GATA3+, at most about 18% of cells of the population are PDPN+, and at least about 80% of cells of the population are CK18+.

[0340] Clause A39 is the method of clause A32, wherein the gene signature biomarker comprises GATA3 and PDPN, and wherein the one or more additional markers comprise CK18.

[0341] Clause A40 is the method of clause A39, wherein the enriched heterogeneous renal cell population is identified as having therapeutic potential, being suitable for formulation, or suitable for treating a subject if: at least about 70% of cells of the population are GATA3+, at least about 70% of cells of the population are CK18+, and at most about 30% of cells of the population are PDPN+.

[0342] Clause A41 is the method of clause A39, wherein the enriched heterogeneous renal cell population is identified as having therapeutic potential, being suitable for formulation, or suitableAttorney Docket No.55801WO 0728.000558WO01 for treating a subject if: at least about 80% of cells of the population are GATA3+, at least about 80% of cells of the population are CK18+, and at most about 18% of cells of the population are PDPN+.

[0343] Clause A42 is the method of clause A32, wherein the gene signature biomarker comprises CD224, and PDPN, and wherein the one or more additional markers comprise CK18.

[0344] Clause A43 is the method of clause A42, wherein the enriched heterogeneous renal cell population is identified as having therapeutic potential, being suitable for formulation, or suitable for treating a subject if: at least about 80% of cells of the population are CD224+, at most about 30% of cells of the population are PDPN+, and at least about 80% of cells of the population are CK18+.

[0345] Clause A44 is the method of any of clauses A31 to A43, further comprising determining whether cells of the enriched heterogeneous renal cell population secrete the one or more additional markers, and identifying the enriched heterogeneous renal cell population as having therapeutic potential, being suitable for formulation, or suitable for treating a subject if cells of the population secrete the one or more additional markers.

[0346] Clause A45 is the method of clause 44, wherein the one or more additional markers comprise VEGF and / or KIM-1.

[0347] Clause A46 is the method of any preceding clause, wherein the enriched heterogeneous renal cell population is prepared from cells isolated from a non-fetal kidney.

[0348] Clause A47 is the method of any preceding clause, wherein the enriched heterogeneous renal cell population is prepared from primary kidney cells.

[0349] Clause A48 is the method of clause A47, wherein the primary kidney cells are cells of an adult kidney.

[0350] Clause A49 is the method of any preceding clause, wherein cells of the enriched heterogeneous renal cell population have not been genetically modified and wherein the population lacks cells comprising a genetic modification relative to native kidney cells of an adult subject.

[0351] Clause A50 is the method of clause A49, wherein the genetic modification comprises introduction of exogenous genetic material.Attorney Docket No.55801WO 0728.000558WO01

[0352] Clause A51 is the method of clause A49, wherein the genetic modification comprises a deletion in, or replacement of, any one or more nucleotides in a native genomic sequence of the cells of the enriched heterogeneous renal cell population.

[0353] Clause A52 is the method of any preceding clause, wherein cells of the enriched heterogeneous renal cell population have not been contacted with a demethylating agent.

[0354] Clause A53 is the method of any preceding clause, (a) wherein cells of the enriched heterogeneous renal cell population have not been treated with a WNT agonist; or (b) wherein cells of the enriched heterogeneous renal cell population have not been treated with one or more nephrogenic growth factors; or (c) wherein cells of the enriched heterogeneous renal cell population have not been treated with a histone acetylation regulator; or any combination of two or more of (a), (b), and (c).

[0355] Clause B1 is a method of identifying an enriched heterogeneous renal cell population as having a therapeutic potential, comprising: (a) profiling expression levels of a panel of genes associated with (i) fibrosis, (ii) inflammation, and / or (iii) senescence-associated secretory phenotype (SASP); in the enriched heterogeneous renal cell population; (b) determining the presence of a gene signature biomarker in the enriched heterogeneous renal cell population based at least in part upon the profiled expression levels; and (c) identifying the enriched heterogeneous renal cell population as having therapeutic potential if the gene signature biomarker is determined to be present in the enriched heterogeneous renal cell population.

[0356] Clause B2 is the method of clause B1, wherein the panel comprises genes associated with inflammation.

[0357] Clause B3 is the method of clause B2, wherein the gene signature biomarker comprises tumor necrosis factor .

[0358] Clause C1 is a method of identifying an enriched heterogeneous renal cell population as having a therapeutic potential, the method comprising:Attorney Docket No.55801WO 0728.000558WO01 (a) profiling expression levels of a panel of genes expressed by cells of the enriched heterogeneous renal cell population; (b) identifying one or more cell types in the enriched heterogeneous renal cell population, wherein the one or more cell types are identified based at least in part on presence of a first, e.g., cell type / state, gene signature biomarker comprising one or more differentially expressed genes; and (c) identifying the enriched heterogeneous renal cell population as having therapeutic potential if one of the one or more cell types comprises a second, e.g., indicator, gene signature biomarker.

[0359] Clause C2 is the method of clause C1, wherein the first (e.g., cell type / state) and / or second (e.g., indicator) gene signature biomarkers comprise the top most differentially expressed genes.

[0360] Clause C3 is the method of clause C1 or C2, wherein the one or more cell types comprises: (i) a proximal tubule cell type or (ii) ascending thin limb / adaptive thick ascending limb-2 cell type.

[0361] Clause C4 is the method of any one of clauses C1-C3, wherein the profiling comprises a sequencing method.

[0362] Clause C5 is the method of clause C4, the sequencing method is single-cell RNA sequencing (scRNA-seq).

[0363] Clause C6 is the method of any of clauses C1-C5, wherein the one or more cell types are further identified by comparison to a reference database.

[0364] Clause C7 is the method of clause C6, wherein the reference database is the Kidney Precision Medicine Project atlas or the GenitoUrinary Development Molecular Anatomy Project atlas.

[0365] Clause C8 is the method of clause C7, wherein the comparison comprises unsupervised clustering and projection methods.

[0366] It will be appreciated from reviewing the present disclosure that it is contemplated that the one or more aspects or features presented in one of or a group of related clauses may also be included in other clauses or in combination with the one or more aspects or features in other clauses.Attorney Docket No.55801WO 0728.000558WO01

[0367] While the disclosure has been particularly shown and described with reference to specific embodiments (some of which are preferred embodiments), it should be understood by those having skill in the art that various changes in form and detail can be made therein without departing from the spirit and scope of the present disclosure as disclosed herein. There is no intention, therefore, of limitations to the exact abstract and disclosure herein presented.

[0368] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein. Such equivalents are intended to be encompassed by the appended claims.

[0369] All publications, patents and patent applications mentioned in this specification are herein incorporated by reference into the specification to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference in their entirety.

Claims

Attorney Docket No.55801WO 0728.000558WO01 CLAIMS 1. A method of identifying an enriched heterogeneous renal cell population as having a therapeutic potential, comprising: (a) profiling expression levels of a panel of genes associated with (i) renal cell identity, and / or (ii) tissue healing response (renewal and repair) in the enriched heterogeneous renal cell population; (b) determining the presence of a gene signature biomarker in the enriched heterogeneous renal cell population based at least in part upon the profiled expression levels; and (c) identifying the enriched heterogeneous renal cell population as having therapeutic potential if the gene signature biomarker is determined to be present in the enriched heterogeneous renal cell population.

2. A method of preparing a formulation comprising an enriched heterogeneous renal cell population, comprising: (a) identifying the enriched heterogeneous renal cell population as suitable for formulation, wherein identifying comprises: (i) profiling expression levels of a panel of genes associated with (i) renal cell identity, and / or (ii) tissue healing response (renewal and repair) in the enriched heterogeneous renal cell population; (ii) determining the presence of a gene signature biomarker in the enriched heterogeneous renal cell population based at least in part upon the profiled expression levels; and (iii) identifying the enriched heterogeneous renal cell population as suitable for formulation if the gene signature biomarker is determined to be present in the enriched heterogeneous renal cell population, and (b) combining the enriched heterogeneous renal cell population that has been identified as being suitable for formulation with a pharmaceutically acceptable carrier or excipient to prepare the formulation.Attorney Docket No.55801WO 0728.000558WO01 3. A method of treating a subject in need of treatment for chronic kidney disease (CKD) comprising: (a) identifying an enriched heterogeneous renal cell population as suitable for treating the subject according to steps comprising: (i) profiling expression levels of a panel of genes associated with (i) renal cell identity, and / or (ii) tissue healing response (renewal and repair) in the enriched heterogeneous renal cell population; (ii) determining the presence of a gene signature biomarker in the enriched heterogeneous renal cell population based at least in part upon the profiled expression levels; and (iii) identifying the enriched heterogeneous renal cell population as suitable for treating the subject if the gene signature biomarker is determined to be present in the enriched heterogeneous renal cell population, and (b) administering the enriched heterogeneous renal cell population that has been identified as suitable for treating the subject to the subject to treat the CKD.

4. The method of claim 3, further comprising formulating a pharmaceutical composition comprising the enriched heterogeneous renal cell population that has been identified as suitable for treating before (b) administering, and wherein the pharmaceutical composition is administered to the subject.

5. The method of claim 4, wherein formulating the pharmaceutical composition comprises combining the enriched heterogeneous renal cell population with a pharmaceutically acceptable carrier or excipient.

6. The method of any of claims 3 to 5, wherein treating the CKD comprises stabilizing kidney function.

7. The method of claim 6, wherein stabilizing kidney function comprises reducing rate of decline in eGFR by at least 20% relative to a baseline eGFR of the subject, wherein the baselineAttorney Docket No.55801WO 0728.000558WO01 eGFR of the subject is determined prior to a first administering of the enriched heterogeneous renal cell population.

8. The method of claim 6, wherein stabilizing kidney function comprises reducing annual rate of decline in eGFR to no more than 2.0 mL / min / 1.73m2.

9. The method of any preceding claim, wherein the panel of genes comprises genes associated with renal cell identity.

10. The method of any preceding claim, wherein the panel of genes comprises genes associated with tissue healing response (renewal and repair).

11. The method of any preceding claim, wherein the panel of genes comprises one or more of CD10, GATA3, CD227, CD13, podoplanin (PDPN), CD24, CD171, CD90, CD133, and CD106.

12. The method of any preceding claim, wherein the gene signature biomarker comprises GATA3 expression by at least about 80% of cells in the enriched heterogeneous renal cell population.

13. The method of any preceding claim, wherein the gene signature biomarker comprises PDPN expression by at most about 30% of cells of the enriched heterogeneous renal cell population.

14. The method of any preceding claim, wherein the panel of genes comprises at least two genes.

15. The method of claim 14, wherein the at least two genes comprise CD10 and GATA3, CD10 and CD227, CD13 and CD227, CD13 and GATA3, or CD90 and CD106.

16. The method of claim 15, wherein the gene signature biomarker comprises CD13 and CD227, wherein at least about 30% of cells of the enriched heterogeneous renal cell population are CD13+CD227+.Attorney Docket No.55801WO 0728.000558WO01 17. The method of claim 16, wherein about 30% to about 95% of cells in the enriched heterogeneous renal cell population are CD13+CD227+.

18. The method of claim 15, wherein the gene signature biomarker comprises CD13 and GATA3, wherein at least about 80% of cells in the enriched heterogeneous renal cell population are CD13+GATA3+.

19. The method of claim 15, wherein the gene signature biomarker comprises CD10 and GATA3, wherein at least about 70% of cells in the enriched heterogeneous renal cell population are CD10+GATA3+.

20. The method of claim 15, wherein the gene signature biomarker comprises CD90 and CD106, wherein at least about 20% to about 50% of cells of the enriched heterogeneous renal cell population are CD90+CD106+.

21. The method of any preceding claim, wherein the panel of genes comprises at least three genes.

22. The method of claim 21, wherein the at least three genes comprise CD13, CD10, and CD227; CD13, CD10, and GATA3; CD24, CD133, and CD106; CD13, CD10 and CD171; or CD24, CD133, and CD106.

23. The method of claim 22, wherein the gene signature biomarker comprises CD13, CD10, and CD227, wherein at least about 20% of cells of the enriched heterogeneous renal cell population are CD13+CD10+CD227+.

24. The method of claim 23, wherein about 20% to about 85% of cells of the enriched heterogeneous renal cell population are CD13+CD10+CD227+.Attorney Docket No.55801WO 0728.000558WO01 25. The method of any claim 22, wherein the gene signature biomarker comprises CD13, CD10, and GATA3, wherein at least about 60% of cells of the enriched heterogeneous renal cell population are CD13+CD10+GATA3+.

26. The method of claim 22, wherein the gene signature biomarker comprises CD24, CD133, and CD106, wherein about 3% to about 30% of cells of the enriched heterogeneous renal cell population are CD24+CD133+CD106+.

27. The method of claim 26, wherein about 9% to about 30% of cells of the enriched heterogenous renal cell population are CD24+CD133+CD106+.

28. The method of claim 22, wherein the gene signature biomarker comprises CD24, CD133, and CD106, wherein about 3% to about 50% of cells of the enriched heterogenous renal cell population are CD24+CD133+CD106-.

29. The method of claim 28, wherein about 3% to about 45% of cells of the enriched heterogeneous renal cell population are CD24+CD133+CD106-.

30. The method of claim 22, wherein the gene signature biomarker comprises CD13, CD10, and CD171, wherein about 40% to about 70% of cells of the enriched heterogeneous renal cell population are CD13+CD10+CD171+.

31. The method of any preceding claim, further comprising determining whether cells of the enriched heterogeneous renal cell population express one or more additional markers.

32. The method of claim 31, wherein the one or more additional markers comprise cytokeratin (CK)18, vascular endothelial growth factor (VEGF) and kidney injury molecule (KIM)-1.

33. The method of claim 32, wherein the gene signature biomarker comprises GATA3 and CD224, and wherein the one or more additional markers comprise and CK18.Attorney Docket No.55801WO 0728.000558WO01 34. The method of claim 33, wherein the enriched heterogeneous renal cell population is identified as having therapeutic potential, being suitable for formulation, or suitable for treating a subject if: at least about 70% of cells of the population are CD224+, at least about 70% of cells of the population are GATA3+, and at least about 70% of cells of the population are CK18+.

35. The method of claim 34, wherein the enriched heterogeneous renal cell population is identified as having therapeutic potential, being suitable for formulation, or suitable for treating the subject if: at least about 80% of cells of the population are CD224+, at least about 80% of cells of the population are GATA3+, and at least about 80% of cells of the population are CK18+.

36. The method of claim 32, wherein the gene signature biomarker comprises PDPN, GATA3, and CD224 and the additional markers comprise CK18.

37. The method of claim 36, wherein the enriched heterogeneous renal cell population is identified as having therapeutic potential, being suitable for formulation, or suitable for treating a subject if: at least about 70% of cells of the population are CD224+, at least about 70% of cells of the population are GATA3+, at most about 30% of cells of the population are PDPN+, and at least about 70% of cells of the population are CK18+.

38. The method of claim 36, wherein the enriched heterogeneous renal cell population is identified as having therapeutic potential, being suitable for formulation, or suitable for treating the subject if: at least about 80% of cells of the population are CD224+, at least about 80% of cells of the population are GATA3+, at most about 18% of cells of the population are PDPN+, and at least about 80% of cells of the population are CK18+.

39. The method of claim 32, wherein the gene signature biomarker comprises GATA3 and PDPN, and wherein the one or more additional markers comprise CK18.

40. The method of claim 39, wherein the enriched heterogeneous renal cell population is identified as having therapeutic potential, being suitable for formulation, or suitable for treating aAttorney Docket No.55801WO 0728.000558WO01 subject if: at least about 70% of cells of the population are GATA3+, at least about 70% of cells of the population are CK18+, and at most about 30% of cells of the population are PDPN+.

41. The method of claim 39, wherein the enriched heterogeneous renal cell population is identified as having therapeutic potential, being suitable for formulation, or suitable for treating a subject if: at least about 80% of cells of the population are GATA3+, at least about 80% of cells of the population are CK18+, and at most about 18% of cells of the population are PDPN+.

42. The method of claim 32, wherein the gene signature biomarker comprises CD224, and PDPN, and wherein the one or more additional markers comprise CK18.

43. The method of claim 42, wherein the enriched heterogeneous renal cell population is identified as having therapeutic potential, being suitable for formulation, or suitable for treating a subject if: at least about 80% of cells of the population are CD224+, at most about 30% of cells of the population are PDPN+, and at least about 80% of cells of the population are CK18+.

44. The method of any of claims 31 to 43, further comprising determining whether cells of the enriched heterogeneous renal cell population secrete the one or more additional markers, and identifying the enriched heterogeneous renal cell population as having therapeutic potential, being suitable for formulation, or suitable for treating a subject if cells of the population secrete the one or more additional markers.

45. The method of claim 44, wherein the one or more additional markers comprise VEGF and / or KIM-1.

46. The method of any preceding claim, wherein the enriched heterogeneous renal cell population is prepared from cells isolated from a non-fetal kidney.

47. The method of any preceding claim, wherein the enriched heterogeneous renal cell population is prepared from primary kidney cells.Attorney Docket No.55801WO 0728.000558WO01 48. The method of claim 47, wherein the primary kidney cells are cells of an adult kidney.

49. The method of any preceding claim, wherein cells of the enriched heterogeneous renal cell population have not been genetically modified and wherein the population lacks cells comprising a genetic modification relative to native kidney cells of an adult subject.

50. The method of claim 49, wherein the genetic modification comprises introduction of exogenous genetic material.

51. The method of claim 49, wherein the genetic modification comprises a deletion in, or replacement of, any one or more nucleotides in a native genomic sequence of the cells of the enriched heterogeneous renal cell population.

52. The method of any preceding claim, wherein cells of the enriched heterogeneous renal cell population have not been contacted with a demethylating agent.

53. The method of any preceding claim, (a) wherein cells of the enriched heterogeneous renal cell population have not been treated with a WNT agonist; or (b) wherein cells of the enriched heterogeneous renal cell population have not been treated with one or more nephrogenic growth factors; or (c) wherein cells of the enriched heterogeneous renal cell population have not been treated with a histone acetylation regulator; or any combination of two or more of (a), (b), and (c).

54. A method of identifying an enriched heterogeneous renal cell population as having a therapeutic potential, comprising: (a) profiling expression levels of a panel of genes associated with (i) fibrosis, (ii) inflammation, and / or (iii) senescence-associated secretory phenotype (SASP); in the enriched heterogeneous renal cell population;Attorney Docket No.55801WO 0728.000558WO01 (b) determining the presence of a gene signature biomarker in the enriched heterogeneous renal cell population based at least in part upon the profiled expression levels; and (c) identifying the enriched heterogeneous renal cell population as having therapeutic potential if the gene signature biomarker is determined to be present in the enriched heterogeneous renal cell population.

55. The method of claim 54, wherein the panel comprises genes associated with inflammation.

56. The method of claim 55, wherein the gene signature biomarker comprises tumor necrosis factor .

57. A method of identifying an enriched heterogeneous renal cell population as having a therapeutic potential, the method comprising: (a) profiling expression levels of a panel of genes expressed by cells of the enriched heterogeneous renal cell population; (b) identifying one or more cell types in the enriched heterogeneous renal cell population, wherein the one or more cell types are identified based at least in part on presence of a cell type / state gene signature biomarker comprising one or more differentially expressed genes; and (c) identifying the enriched heterogeneous renal cell population as having therapeutic potential if one of the one or more cell types comprises an indicator gene signature biomarker.

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