Selected renal cell populations, their characteristics and uses

Abstract

Method for identifying an enriched heterogeneous renal cell population having a therapeutic ability, an enriched heterogeneous renal cell population having a therapeutic ability, and use thereof.

Description

Background Art

[0001] Chronic kidney disease (CKD) is spreading or is already widespread (Non-Patent Document 1, Non-Patent Document 2, Non-Patent Document 3). According to morbidity data from the United States to Europe, it has been shown that approximately 10% of the general population suffers from stage 1 to stage 3 CKD (Non-Patent Document 4, Non-Patent Document 5, Non-Patent Document 6). CKD increased by more than 33% in the United States alone between 1996 and 2006 (Non-Patent Document 7). On the other hand, the management of CKD still remains a challenge for nephrologists. The most standard treatment for CKD is small molecules that target biochemical pathways in the kidney that act on single or related comorbidities. However, these treatments do not act on the glomerular and tubulointerstitial dysfunction of the underlying CKD kidney. Ultimately, in patients who have progressed to end-stage renal disease (ESRD), renal replacement therapy (RRT, dialysis, or transplantation) is required to survive. There are still unmet needs for treatments that directly address the tissue biology of the affected kidney. Such treatments could potentially prevent the progression of CKD or even reverse it, reducing or avoiding the need for RRT.

[0002] Selected renal cells (SRC), a heterogeneous renal cell population enriched in renal epithelial cells, have shown promise as an autologous cell-based therapy for the treatment of CKD (Non-Patent Document 8, Non-Patent Document 9). SRC is derived from donor kidneys and is mainly a mixture of proximal tubular epithelial cells (Non-Patent Document 10). The results of a phase II clinical trial in a cohort of patients with diabetic kidney disease suggested that SRC administration is safe and is associated with an improvement in renal function (Non-Patent Document 9).

[0003] In the art, there is a need to identify heterogeneous renal cell populations such as SRC that can improve renal function in CKD patients and reduce or avoid the need for RRT.

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Non-Patent Document 10

Summary of the Invention

[0005] The present disclosure describes a method for identifying an enriched heterogeneous renal cell population as having therapeutic potential. In the method, it is determined whether the cells of the enriched heterogeneous renal cell population express (i) NEPH1 and (ii) one or more markers, where the one or more markers can be any one or more of nephrin (NPHS1), podocin (NPHS2), homeobox protein sine oculis homeobox homolog 2 (SIX2), odd-skipped related 1 (OSR1), lim homeobox protein 1 (LHX1), and fibroblast growth factor 8 (FGF8), receptor for activated C kinase 1 (RACK1), or rearranged during transfection (RET). If the cells of the enriched heterogeneous renal cell population express (i) NEPH1 and (ii) one or more markers, the enriched heterogeneous renal cell population is identified as having therapeutic potential.

Brief Description of the Drawings

[0006]

Fig. 1A-1B

Fig. 2A-2H

Fig. 3

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Fig. 5A-5B

Fig. 6

Modes for Carrying Out the Invention

[0007] This specification describes a method for identifying an enriched heterogeneous renal cell population as having a therapeutic ability, a composition comprising the enriched heterogeneous renal cell population identified as having a therapeutic ability, and methods and uses of the enriched heterogeneous renal cell population having a therapeutic ability.

[0008] In the method for identifying an enriched heterogeneous renal cell population as having a therapeutic ability, the therapeutic ability of the enriched heterogeneous renal cell population can be in the treatment of kidney diseases, tubular transport abnormalities, or glomerular filtration abnormalities.

[0009] The therapeutic ability of the enriched heterogeneous renal cell population identified by these methods can be the restoration of kidney function, the stabilization of kidney function, the improvement of kidney function, the reduction of renal fibrosis, the reduction of kidney inflammation, the induction of tubulogenesis in the kidney, the induction of nephrogenesis in the kidney, or the induction of glomerulogenesis in the kidney in a patient in need of such treatment. When the enriched heterogeneous renal cell population is identified as having a therapeutic ability, the enriched heterogeneous renal cell population can restore the mineral balance or reduce anemia in a patient in need of such treatment. When the enriched heterogeneous renal cell population is identified as having a therapeutic ability, the enriched heterogeneous renal cell population can also delay or prevent the need for dialysis, or delay or prevent the need for kidney transplantation in a patient in need of treatment for kidney disease.

[0010] In a method of identifying an enriched population of heterogeneous renal cells as having a therapeutic ability, it can be determined whether the cells of the enriched population of heterogeneous renal cells express (i) NEPH1 (also known variously as KIRREL1, KIRREL, kin of IRRE like (Drosophila), Kin of IRRE-like protein 1, kirre-like nephrin family adhesion molecule 1, NPHS23) and (ii) one or more markers. The one or more markers can be any one or more of, or can include, nephrin, podocin, homeobox protein sine oculis homeobox homolog 2 (SIX2), odd-skipped related 1 (OSR1), lim homeobox protein 1 (LHX1), fibroblast growth factor 8 (FGF8), receptor for activated C kinase 1 (RACK1), or rearranged during transfection (RET). The one or more markers can be any one, any two, any three, any four, any five, any six, any seven, or all eight of, or can include, nephrin, podocin, SIX2, OSR1, LHX1, RET, FGF8, and RACK1.

[0011] In a method of identifying an enriched population of heterogeneous kidney cells as having a therapeutic ability, it can be determined whether the cells of the enriched population of heterogeneous kidney cells express (i) NEPH1 and (ii) at least two of the markers nephrin, podocin, SIX2, OSR1, LHX1, RET, FGF8, and RACK1. When the expression of at least two markers is determined, the at least two markers are (a) SIX2 and OSR1, (b) SIX2 and LHX1, (c) SIX2 and RET, (d) SIX2 and FGF8, (e) OSR1 and LHX1, (f) OSR1 and RET, (g) OSR1 and FGF8, (h) LHX1 and RET, (i) LHX1 and FGF8, (j) RET and FGF8, (k) nephrin and SIX2, (l) nephrin and OSR1, (m) nephrin and LHX1, (n) nephrin and RET, (o) nephrin and FGF8, (p) podocin and nephrin, (q) podocin and SIX2, (r) podocin and OSR1, (s) podocin and LHX1, (t) podocin and RET, (u) podocin and FGF8, (v) RACK1 and nephrin, (w) RACK1 and podocin, (x) RACK1 and SIX2, (y) RACK1 and OSR1, (z) RACK1 and LHX1, (aa) RACK1 and RET, or (bb) RACK1 and FGF8, or can include these.

[0012] In a method of identifying an enriched heterogeneous renal cell population as having a therapeutic ability, it can be determined whether the cells of the enriched heterogeneous renal cell population express (i) NEPH1 and (ii) at least three of the markers of nephrin, podocin, SIX2, OSR1, LHX1, RET, FGF8, and RACK1. When the expression of at least three markers is determined, the at least three markers are (a) SIX2, OSR1, and LHX1, (b) SIX2, OSR1, and RET, (c) SIX2, OSR1, and FGF8, (d) SIX2, LHX1, and RET, (e) SIX2, LHX1, and FGF8, (f) SIX2, RET, and FGF8, (g) OSR1, LHX1, and RET, (h) OSR1, LHX1, and FGF8, (i) OSR1, RET, and FGF8, (j) LHX1, RET, and FGF8, (k) nephrin, SIX2, and OSR, (l) nephrin, SIX2, and LHX1, (m) nephrin, SIX2, and RET, (n) nephrin, SIX2, and FGF8, (o) nephrin, OSR, and LHX1, (p) nephrin, OSR, and RET, (q) nephrin, OSR, and FGF8, (r) nephrin, LHX1, and RET, (s) nephrin, LHX1, and FGF8, (t) nephrin, RET, and FGF8, (u) podocin, SIX2, and OSR, (v) podocin, SIX2, and LHX1, (w) podocin, SIX2, and RET, (x) podocin, SIX2, and FGF8, (y) podocin, OSR, and LHX1, (z) podocin, OSR, and RET, (aa) podocin, OSR, and FGF8, (bb) podocin, LHX1, and RET, (cc) podocin, LHX1, and FGF8, (dd) podocin, RET, and FGF8, (ee) RACK1, SIX2, and OSR, (ff) RACK1, SIX2, and LHX1, (gg) RACK1, SIX2, and RET, (hh) RACK1, SIX2, and FGF8, (ii) RACK1, OSR, and LHX1, (jj) RACK1, OSR, and RET, (kk) RACK1, OSR, and FGF8, (ll) RACK1, LHX1, and RET, (mm) RACK1, LHX1, and FGF8, (nn) RACK1, RET, and FGF8, (oo) nephrin, podocin, and RACK1.(pp) Nephrin, podocin, and SIX2, (qq) Nephrin, podocin, and OSR, (rr) Nephrin, podocin, and RET, (ss) Nephrin, podocin, and LHX1, (tt) Nephrin, podocin, and FGF8, (uu) Nephrin, RACK1, and SIX2, (vv) Nephrin, RACK1, and OSR, (ww) Nephrin, RACK1, and RET, (xx) Nephrin, RACK1, and LHX1, (yy) Nephrin, RACK1, and FGF8, (zz) Podocin, RACK1, and SIX2, (aaa) Podocin, RACK1, and OSR, (bbb) Podocin, RACK1, and RET, (ccc) Podocin, RACK1, and LHX1, or (ddd) Podocin, RACK1, and FGF8, or may contain these.,

[0013] In a method of identifying an enriched heterogeneous renal cell population as having a therapeutic ability, it can be determined whether the cells of the enriched heterogeneous renal cell population express (i) NEPH1 and (ii) at least four of the markers nephrin, podocin, SIX2, OSR1, LHX1, RET, FGF8, and RACK1. When the expression of at least four markers is determined, the at least four markers are (a) SIX2, OSR1, LHX1, and RET, (b) SIX2, OSR1, LHX1, and FGF8, (c) SIX2, LHX1, RET, and FGF8, (d) SIX2, OSR1, RET, and FGF8, (e) OSR1, LHX1, RET, and FGF8, (f) nephrin, SIX2, OSR1, and LHX1, (g) nephrin, SIX2, OSR1, and RET, (h) nephrin, SIX2, OSR1, and FGF8, (i) nephrin, SIX2, LHX1, and RET, (j) nephrin, SIX2, LHX1, and FGF8, (k) nephrin, SIX2, RET, and FGF8, (l) nephrin, OSR1, LHX1, and FGF8, (m) nephrin, OSR1, LHX1, and RET, (n) nephrin, OSR1, RET, and FGF8, (o) nephrin, LHX1, RET, and FGF8, (p) nephrin, SIX2, OSR1, and LHX1, (q) podocin, SIX2, OSR1, and RET, (r) podocin, SIX2, OSR1, and FGF8, (s) podocin, SIX2, LHX1, and RET, (t) podocin, SIX2, LHX1, and FGF8, (u) podocin, SIX2, RET, and FGF8, (v) podocin, OSR1, LHX1, and FGF8, (w) podocin, OSR1, LHX1, and RET, (x) podocin, OSR1, RET, and FGF8, (y) podocin, LHX1, RET, and FGF8, (z) RACK1, SIX2, OSR1, and FGF8, (aa) RACK1, SIX2, LHX1, and RET, (bb) RACK1, SIX2, LHX1, and FGF8, (cc) RACK1, SIX2, RET, and FGF8, (dd) RACK1, OSR1, LHX1, and FGF8, (ee) RACK1, OSR1, LHX1, and RET, (ff) RACK1, OSR1, RET, and FGF8, (gg) RACK1, LHX1, RET, and FGF8,(hh) Nephrin, podocin, SIX2, and OSR1, (ii) Nephrin, podocin, SIX2, and LHX1, (jj) Nephrin, podocin, SIX2, and RET, (kk) Nephrin, podocin, SIX2, and FGF8, (ll) Nephrin, podocin, OSR1, and LHX1, (mm) Nephrin, podocin, OSR1, and RET, (nn) Nephrin, podocin, OSR1, and FGF8, (oo) Nephrin, podocin, LHX1, and RET, (pp) Nephrin, podocin, LHX1, and FGF8, (qq) Nephrin, podocin, RET, and FGF8, (rr) RACK1, podocin, SIX2, and OSR1, (ss) RACK1, podocin, SIX2, and LHX1, (tt) RACK1, podocin, SIX2, and RET, (uu) RACK1, podocin, SIX2, and FGF8, (vv) RACK1, podocin, OSR1, and LHX1, (ww) RACK1, podocin, OSR1, and RET, (xx) RACK1, podocin, OSR1, and FGF8, (yy) RACK1, podocin, LHX1, and RET, (zz) RACK1, podocin, LHX1, and FGF8, (aaa) RACK1, podocin, RET, and FGF8, (bbb) RACK1, nephrin, SIX2, and OSR1, (ccc) RACK1, nephrin, SIX2, and LHX1, (ddd) RACK1, nephrin, SIX2, and RET, (eee) RACK1, nephrin, SIX2, and FGF8, (fff) RACK1, nephrin, OSR1, and LHX1, (ggg) RACK1, nephrin, OSR1, and RET, (hhh) RACK1, nephrin, OSR1, and FGF8, (iii) RACK1, nephrin, LHX1, and RET, (jjj) RACK1, nephrin, LHX1, and FGF8, (kkk) RACK1, nephrin, RET, and FGF8, (lll) RACK1, nephrin, podocin, and SIX2, (mmm) RACK1, nephrin, podocin, and RET, (nnn) RACK1, nephrin, podocin, and LHX1, (ooo) RACK, nephrin, podocin, and OSR1, or (ppp) RACK, nephrin, podocin, and FGF8, or may include these.,

[0014] In a method of identifying an enriched heterogeneous renal cell population as having a therapeutic ability, it can be determined whether the cells of the enriched heterogeneous renal cell population express (i) NEPH1 and (ii) at least 5 of the markers nephrin, podocin, SIX2, OSR1, LHX1, RET, FGF8, and RACK1. When the expression of at least 5 markers is determined, the at least 5 markers are (a) SIX2, OSR1, LHX1, RET, and FGF8, (b) nephrin, SIX2, OSR1, LHX1, and RET, (c) nephrin, SIX2, OSR1, LHX1, and FGF8, (d) nephrin, SIX2, LHX1, RET, and FGF8, (e) nephrin, SIX2, OSR1, RET, and FGF8, (f) nephrin, OSR1, LHX1, RET, and FGF8, (g) podocin, SIX2, OSR1, LHX1, and RET, (h) podocin, SIX2, OSR1, LHX1, and FGF8, (i) podocin, SIX2, LHX1, RET, and FGF8, (j) podocin, SIX2, OSR1, RET, and FGF8, (k) podocin, OSR1, LHX1, RET, and FGF8, (l) RACK1, SIX2, OSR1, LHX1, and RET, (m) RACK1, SIX2, OSR1, LHX1, and FGF8, (n) RACK1, SIX2, LHX1, RET, and FGF8, (o) RACK1, SIX2, OSR1, RET, and FGF8, (p) RACK1, OSR1, LHX1, RET, and FGF8, (q) nephrin, podocin, SIX2, OSR1, and LHX1, (r) nephrin, podocin, SIX2, OSR1, and RET, (s) nephrin, podocin, SIX2, OSR1, and FGF8, (t) nephrin, podocin, SIX2, LHX1, and RET, (u) nephrin, podocin, SIX2, LHX1, and FGF8, (v) nephrin, podocin, SIX2, RET, and FGF8, (w) nephrin, podocin, OSR1, LHX1, and RET, (x) nephrin, podocin, OSR1, LHX1, and FGF8, (y) nephrin, podocin, OSR1, RET, and FGF8, (z) nephrin, podocin, LHX1, RET, and FGF8, (aa) nephrin, RACK1, SIX2, OSR1, and LHX1, (bb) nephrin, RACK1, SIX2,OSR1, and RET, (cc) nephrin, RACK1, SIX2, OSR1, and FGF8, (dd) nephrin, RACK1, SIX2, LHX1, and RET, (ee) nephrin, RACK1, SIX2, LHX1, and FGF8, (ff) nephrin, RACK1, SIX2, RET, and FGF8, (gg) nephrin, RACK1, OSR1, LHX1, and RET, (hh) nephrin, RACK1, OSR1, LHX1, and FGF8, (ii) nephrin, RACK1, OSR1, RET, and FGF8, (jj) nephrin, RACK1, LHX1, RET, and FGF8, (kk) podocin, RACK1, SIX2, OSR1, and LHX1, (ll) podocin, RACK1, SIX2, OSR1, and RET, (mm) podocin, RACK1, SIX2, OSR1, and FGF8, (nn) podocin, RACK1, SIX2, LHX1, and RET, (oo) podocin, RACK1, SIX2, LHX1, and FGF8, (pp) podocin, RACK1, SIX2, RET, and FGF8, (qq) podocin, RACK1, OSR1, LHX1, and RET, (rr) podocin, RACK1, OSR1, LHX1, and FGF8, (ss) podocin, RACK1, OSR1, RET, and FGF8, (tt) podocin, RACK1, LHX1, RET, and FGF8, (uu) nephrin, podocin, RACK1, SIX2, and OSR1, (vv) nephrin, podocin, RACK1, SIX2, and LHX1, (ww) nephrin, podocin, RACK1, SIX2, and RET, (xx) nephrin, podocin, RACK1, SIX2, and FGF8, (yy) nephrin, podocin, RACK1, OSR1, and LHX1, (zz) nephrin, podocin, RACK1, OSR1, and RET, (aaa) nephrin, podocin, RACK1, OSR1, and FGF8, (bbb) nephrin, podocin, RACK1, LHX1, and RET, (ccc) nephrin, podocin, RACK1, LHX1, and FGF8, or (ddd) nephrin, podocin, RACK1, RET, and FGF8, or may include these.,

[0015] In a method of identifying an enriched heterogeneous renal cell population as having therapeutic potential, it can be determined whether the cells of the enriched heterogeneous renal cell population express (i) NEPH1 and (ii) at least 6 of the markers nephrin, podocin, SIX2, OSR1, LHX1, RET, FGF8, and RACK1. If the expression of at least 6 markers is determined, the at least 6 markers are (a) nephrin, SIX2, OSR1, LHX1, RET, and FGF8, (b) podocin, SIX2, OSR1, LHX1, RET, and FGF8, (c) RACK1, SIX2, OSR1, LHX1, RET, and FGF8, (d) nephrin, podocin, SIX2, OSR1, LHX1, and RET, (e) nephrin, podocin, SIX2, OSR1, LHX1, and FGF8, (f) nephrin, podocin, SIX2, LHX1, RET, and FGF8, (g) nephrin, podocin, SIX2, OSR1, RET, and FGF8, (h) nephrin, podocin, OSR1, LHX1, RET, and FGF8, (i) nephrin, RACK1, SIX2, OSR1, LHX1, and RET, (j) nephrin, RACK1, SIX2, OSR1, LHX1, and FGF8, (k) nephrin, RACK1, SIX2, LHX1, RET, and FGF8, (l) nephrin, RACK1, SIX2, OSR1, RET, and FGF8, (m) nephrin, RACK1, OSR1, LHX1, RET, and FGF8, (n) podocin, RACK1, SIX2, OSR1, LHX1, and RET, (o) podocin, RACK1, SIX2, OSR1, LHX1, and FGF8, (p) podocin, RACK1, SIX2, LHX1, RET, and FGF8, (q) podocin, RACK1, SIX2, OSR1, RET, and FGF8, (r) podocin, RACK1, OSR1, LHX1, RET, and FGF8, (s) nephrin, podocin, RACK1, SIX2, OSR1, and LHX1, (t) nephrin, podocin, RACK1, SIX2, OSR1, and RET, (u) nephrin, podocin, RACK1, SIX2, OSR1, and FGF8, (v) nephrin, podocin, RACK1, SIX2, LHX1, and RET, (w) nephrin, podocin, RACK1, SIX2, LHX1, and FGF8, (x) nephrin, podocin,RACK1, SIX2, RET, and FGF8, (y) nephrin, podocin, RACK1, OSR1, LHX1, and RET, (z) nephrin, podocin, RACK1, OSR1, LHX1, and FGF8, (aa) nephrin, podocin, RACK1, OSR1, RET, and FGF8, or (bb) nephrin, podocin, RACK1, LHX1, RET, and FGF8, or may include these.

[0016] In a method of identifying an enriched heterogeneous renal cell population as having therapeutic ability, it can be determined whether the cells of the enriched heterogeneous renal cell population express (i) NEPH1 and (ii) at least 7 of the markers of nephrin, podocin, SIX2, OSR1, LHX1, RET, FGF8, and RACK1. When the expression of at least 7 markers is determined, the at least 7 markers can be (a) podocin, RACK1, SIX2, OSR1, LHX1, RET, and FGF8, (b) nephrin, RACK1, SIX2, OSR1, LHX1, RET, and FGF8, (c) nephrin, podocin, SIX2, OSR1, LHX1, RET, and FGF8, (d) nephrin, podocin, RACK1, OSR1, LHX1, RET, and FGF8, (e) nephrin, podocin, RACK1, SIX2, LHX1, RET, and FGF8, (f) nephrin, podocin, RACK1, SIX2, OSR1, RET, and FGF8, (g) nephrin, podocin, RACK1, SIX2, OSR1, LHX1, and FGF8, or (h) nephrin, podocin, RACK1, SIX2, OSR1, LHX1, and RET, or may include these.

[0017] In a method of identifying an enriched heterogeneous renal cell population as having therapeutic ability, it can be determined whether the cells of the enriched heterogeneous renal cell population express (i) NEPH1 and (ii) the markers of nephrin, podocin, SIX2, OSR1, LHX1, RET, FGF8, and RACK1.

[0018] In a method for identifying an enriched heterogeneous renal cell population as having therapeutic ability, when it is determined that the cells of the enriched heterogeneous renal cell population express (i) NEPH1 and (ii) at least one (e.g., any one, or any two, or any three, or any four, or any five, or any six, or any seven, or all eight) markers, the enriched heterogeneous renal cell population can be identified as having therapeutic ability.

[0019] In a method for identifying an enriched heterogeneous renal cell population as having therapeutic ability, determining the expression of (i) NEPH1 and (ii) one or more (or at least two, or at least three, or at least four, or at least five, or at least six, or at least seven, or eight) markers may further include determining the percentage of cells of the enriched heterogeneous renal cell population that express (i) NEPH1 and / or (ii) one or more (or at least one, at least two, or at least three, or at least four, or at least five, or at least six, or at least seven, or eight) markers. If the percentage of cells of the enriched heterogeneous renal cell population that express (i) NEPH1 and / or (ii) one or more (or at least one, or at least two, or at least three, or at least four, or at least five, or at least six, or at least seven, or eight) markers is determined, the enriched heterogeneous renal cell population can be identified as having therapeutic ability if approximately a certain or specific percentage of the cells of the enriched heterogeneous renal cell population express (i) NEPH1 and / or (ii) one or more (or at least one, or at least two, or at least three, or at least four, or at least five, or at least six, or at least seven, or eight) markers.

[0020] In the above method, when the percentage of cells in the enriched heterogeneous renal cell population expressing NEPH1 is determined, if between 2.0% and about 8.0%, or between about 2.0% and about 7.5%, or between about 2.0% and about 7.0%, or between about 2.0% and about 6.5%, or between about 2.0% and about 6.0%, or between about 2.0% and about 5.5%, or between about 2.0% and about 5.0%, or between about 2.5% and about 8.0%, or between about 3.0% and about 8.0%, or between about 3.5% and about 8.0%, or between about 4.0% and about 8.0%, or between about 2.5% and about 7.5%, or between about 3.0% and about 7.0%, or between about 3.5% and about 6.5%, or between about 4.0% and about 6.0%, or between about 4.5% and about 5.5%, or between about 4.0% and about 6.5%, or between about 4.0% and about 7.0%, or between about 3.5% and about 6.5%, or between about 3.5% and about 6.0%, or between about 3% and about 6.0% of the cells in the enriched heterogeneous population express NEPH1, the enriched heterogeneous renal cell population can be identified as having a therapeutic ability.

[0021] In the above method, (ii) the percentage of cells of an enriched heterogeneous renal cell population expressing one or more (or at least one, or at least two, or at least three, or at least four, or at least five, or at least six, or at least seven, or eight) markers is determined, and if one or more (at least one, or at least two, or at least three, or at least four, or at least five, or at least six, or at least seven, or eight) markers are SIX2 or include SIX2, and at least about 0.10% of the cells of the enriched heterogeneous renal cell population express SIX2, then the enriched heterogeneous renal cell population can be identified as having a therapeutic ability. Alternatively, if one or more (at least one, or at least two, or at least three, or at least four, or at least five, or at least six, or at least seven, or eight) markers are SIX2 or include SIX2, and at least about 0.15%, or at least about 0.20%, or at least about 0.50%, or at least about 1.0%, or at least about 1.5%, or at least about 2.0%, or at least about 2.5%, or at least about 3.0%, or at least about 3.5%, or at least about 4.0%, or at least about 4.5%, or at least about 5.0%, or at least about 5.5% of the cells of the enriched heterogeneous renal cell population express SIX2, then the enriched heterogeneous renal cell population can be identified as having a therapeutic ability. If one or more (at least one, or at least two, or at least three, or at least four, or at least five, or at least six, or at least seven, or eight) markers are SIX2 or include SIX2, and more than 0% up to a maximum of about 35.0%, or more than 0% up to a maximum of about 30.0%, or more than 0% up to a maximum of about 25.0%, or more than 0% up to a maximum of about 20.0%, or more than 0% up to a maximum of about 15.0%, or more than 0% up to a maximum of about 12.5%, or more than 0% up to a maximum of about 10.0%, or more than 0% up to a maximum of about 7.5%, or more than 0% up to a maximum of about 5.0% of the enriched heterogeneous renal cell population expresses SIX2, then the enriched heterogeneous renal cell population can be identified as having a therapeutic ability.Furthermore, if one or more (at least 1, or at least 2, or at least 3, or at least 4, or at least 5, or at least 6, or at least 7, or 8) markers are SIX2 or include SIX2, and between about 0.10% and about 15.0%, or between about 0.10% and about 10.0%, or between about 0.10% and about 8.0%, or between about 0.10% and about 6.0%, or between about 0.10% and about 4.0%, or between about 0.10% and about 2.0%, or between about 0.40% and about 15.0%, or between about 0.40% and about 10.0%, or between about 0.40% and about 8.0%, or between about 0.40% and about 6.0%, or between about 0.40% and about 4.0%, or between about 0.40% and about 2.0%, or between about 0.60% and about 15.0%, or between about 0.60% and about 10.0%, or between about 0.60% and about 8.0%, or between about 0.60% and about 6.0%, or between about 0.60% and about 4.0%, or between about 0.60% and about 2.0% of the cells in the enriched heterogeneous kidney cell population express SIX2, then the enriched heterogeneous kidney cell population can be identified as having a therapeutic ability.

[0022] In the above method, the percentage of cells in an enriched heterogeneous renal cell population expressing one or more (at least 1, or at least 2, or at least 3, or at least 4, or at least 5, or at least 6, or at least 7, or 8) markers is determined, and if one or more (at least 1, or at least 2, or at least 3, or at least 4, or at least 5, or at least 6, or at least 7, or 8) markers are OSR1 or include OSR1, and at least about 35% of the cells in the enriched heterogeneous renal cell population express OSR1, then the enriched heterogeneous renal cell population can be identified as having a therapeutic ability. Alternatively, if one or more (at least 1, or at least 2, or at least 3, or at least 4, or at least 5, or at least 6, or at least 7, or 8) markers are OSR1 or include OSR1, and at least about 45%, or at least about 47.5%, or at least about 50%, or at least about 52.5%, or at least about 55%, or at least about 57.5%, or at least about 60%, or at least about 62.5%, or at least about 65%, or at least about 67.5%, or at least about 70%, or at least about 72.5%, or at least about 75% of the cells in the enriched heterogeneous renal cell population express OSR1, then the enriched heterogeneous renal cell population can be identified as having a therapeutic ability.If one or more (at least one, or at least two, or at least three, or at least four, or at least five, or at least six, or at least seven, or eight) markers are OSR1 or include OSR1, and between about 35% and about 99%, or between about 35% and about 98%, or between about 35% and about 96%, or between about 35% and about 94%, or between about 35% and about 92%, or between about 35% and about 90%, or between 40% and 99%, or between about 40% and about 98%, or between about 40% and about 96%, or between about 40% and about 94%, or between about 40% and about 92%, or between about 40% and about 90%, or between about 45% and about 99%, or between about 45% and about 98%, or between about 45% and about 96%, or between about 45% and about 94%, or between about 45% and about 92%, or between about 45% and about 90%, or between about 50% and about 99%, or between about 50% and about 98%, or between about 50% and about 96%, or between about 50% and about 94%, or between about 50% and about 92%, or between about 50% and about 90%, or between about 55% and about 99%, or between about 55% and about 98%, or between about 55% and about 96%, or between about 55% and about 94%, or between about 55% and about 92%, or between about 55% and about 90%, or between about 60% and about 99%, or between about 60% and about 98%, or between about 60% and about 96%, or between about 60% and about 94%, or between about 60% and about 92%, or between about 60% and about 90% of the cells of the enriched heterogeneous renal cell population express OSR1, then the enriched heterogeneous renal cell population can be identified as having therapeutic ability.

[0023] In the above method, the percentage of cells in an enriched heterogeneous renal cell population expressing one or more (at least 1, or at least 2, or at least 3, or at least 4, or at least 5, or at least 6, or at least 7, or 8) markers is determined, and if one or more (at least 1, or at least 2, or at least 3, or at least 4, or at least 5, or at least 6, or at least 7, or 8) markers are LHX1 or include LHX1, and at least about 0.80% of the cells in the enriched heterogeneous renal cell population express LHX1, then the enriched heterogeneous renal cell population can be identified as having therapeutic potential. Alternatively, if one or more (at least 1, or at least 2, or at least 3, or at least 4, or at least 5, or at least 6, or at least 7, or 8) markers are LHX1 or include LHX1, and at least about 1%, or at least about 3%, or at least about 5%, or at least about 7%, or at least about 9%, or at least about 11%, or at least about 13%, or at least about 15% of the cells in the enriched heterogeneous renal cell population express LHX1, then the enriched heterogeneous renal cell population can be identified as having therapeutic potential. If one or more (at least 1, or at least 2, or at least 3, or at least 4, or at least 5, or at least 6, or at least 7, or 8) markers are LHX1 or include LHX1, and more than 0% and up to a maximum of 99%, or more than 0% and up to a maximum of 90%, or more than 80% and up to approximately 75%, or more than 0% and up to approximately 70%, or more than 0% and up to approximately 65%, or more than 0% and up to approximately 60%, or more than 0% and up to approximately 55%, or more than 0% and up to approximately 50%, or more than 0% and up to approximately 45%, or more than 0% and up to approximately 40%, or more than 0% and up to approximately 35%, or more than 0% and up to approximately 30% of the cells in the heterogeneous renal cell population express LHX1, then the enriched heterogeneous renal cell population can be identified as having therapeutic potential.Furthermore, if one or more (at least 1, or at least 2, or at least 3, or at least 4, or at least 5, or at least 6, or at least 7, or 8) markers are LHX1 or include LHX1, and between about 0.80% and about 99%, or between about 0.80% and about 90%, or between about 0.80% and about 80%, or between about 0.80% and about 70%, or between about 0.80% and about 60%, or between about 0.80% and about 50%, or between about 0.80% and about 40%, or between about 5% and about 99%, or between about 5% and about 90%, or between about 5% and about 80%, or between about 5% and about 70%, or between about 5% and about 60%, or between about 5% and about 50%, or between about 5% and about 40%, or between about 10% and about 99%, or between about 10% and about 90%, or between about 10% and about 80%, or between about 10% and about 70%, or between about 10% and about 60%, or between about 10% and about 50%, or between about 10% and about 40% of the cells of the enriched heterogeneous kidney cell population express LHX1, then the enriched heterogeneous kidney cell population can be identified as having a therapeutic ability.

[0024] In the above method, the percentage of cells in an enriched heterogeneous renal cell population expressing one or more (at least 1, or at least 2, or at least 3, or at least 4, or at least 5, or at least 6, or at least 7, or 8) markers is determined, and if one or more (at least 1, or at least 2, or at least 3, or at least 4, or at least 5, or at least 6, or at least 7, or 8) markers are RET or include RET, and at least about 1% of the cells in the enriched heterogeneous renal cell population express RET, then the enriched heterogeneous renal cell population can be identified as having a therapeutic ability. Alternatively, if one or more (at least 1, or at least 2, or at least 3, or at least 4, or at least 5, or at least 6, or at least 7, or 8) markers are RET or include RET, and at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, or at least about 12% of the cells in the enriched heterogeneous renal cell population express RET, then the enriched heterogeneous renal cell population can be identified as having a therapeutic ability. If one or more (at least 1, or at least 2, or at least 3, or at least 4, or at least 5, or at least 6, or at least 7, or 8) markers are RET or include RET, and more than 1% up to a maximum of about 85%, or more than 1% up to a maximum of about 83%, or more than 1% up to a maximum of about 80%, or more than 1% up to a maximum of about 77%, or more than 1% up to a maximum of about 75%, or more than 1% up to a maximum of about 72%, or more than 1% up to a maximum of about 70%, or more than 1% up to a maximum of about 67%, or more than 1% up to a maximum of about 65%, or more than 1% up to a maximum of about 63%, or more than 1% up to a maximum of about 60% of the cells in the enriched heterogeneous renal cell population express RET, then the enriched heterogeneous renal cell population can be identified as having a therapeutic ability.Furthermore, if one or more (at least one, or at least two, or at least three, or at least four, or at least five, or at least six, or at least seven, or eight) markers are RET or contain RET, and between about 1% and about 80%, or between about 1% and about 77%, or between about 2% and about 80%, or between about 2% and about 77%, or between about 4% and about 80%, or between about 4% and about 78%, or between about 5% and about 70%, or between about 5% and about 65%, or between about 5% and about 60%, or between about 10% and about 80%, or between about 10% and about 75%, or between about 10% and about 70%, or between about 10% and about 65%, or between about 10% and about 60% of the cells in the enriched heterogeneous renal cell population express RET, then the enriched heterogeneous renal cell population can be identified as having a therapeutic ability.

[0025] In the above method, the percentage of cells in an enriched heterogeneous renal cell population expressing one or more (at least 1, or at least 2, or at least 3, or at least 4, or at least 5, or at least 6, or at least 7, or 8) markers is determined, and if one or more (at least 1, or at least 2, or at least 3, or at least 4, or at least 5, or at least 6, or at least 7, or 8) markers are FGF8 or include FGF8, and at least about 0.01% of the cells in the enriched heterogeneous renal cell population express FGF8, then the enriched heterogeneous renal cell population can be identified as having a therapeutic ability. Alternatively, if one or more (at least 1, or at least 2, or at least 3, or at least 4, or at least 5, or at least 6, or at least 7, or 8) markers are FGF8 or include FGF8, and at least about 0.01%, or at least about 0.03%, or at least about 0.05%, or at least about 0.07%, or at least about 0.09%, or at least about 0.10%, or at least about 0.20%, or at least about 0.30%, or at least about 0.40%, or at least about 0.50%, or at least about 0.60%, or at least about 0.70%, or at least about 0.80%, or about 0.90% of the cells in the enriched heterogeneous renal cell population express FGF8, then the enriched heterogeneous renal cell population can be identified as having a therapeutic ability. If one or more (at least 1, or at least 2, or at least 3, or at least 4, or at least 5, or at least 6, or at least 7, or 8) markers are FGF8 or include FGF8, and more than 0% and up to a maximum of about 60%, or more than 0% and up to a maximum of about 57%, or more than 0% and up to a maximum of about 55%, or more than 0% and up to a maximum of about 52%, or more than 0% and up to a maximum of about 50%, or more than 0% and up to a maximum of about 47%, or more than 0% and up to a maximum of about 45%, or more than 0% and up to a maximum of about 42%, or more than 0% and up to a maximum of about 40%, or more than 0% and up to a maximum of about 37%, or more than 0% and up to a maximum of about 35% of the cells in the enriched heterogeneous renal cell population express FGF8, then the enriched heterogeneous renal cell population can be identified as having a therapeutic ability.Furthermore, if one or more (at least one, or at least two, or at least three, or at least four, or at least five, or at least six, or at least seven, or eight) markers are FGF8 or contain FGF8, and between about 0.01% and about 60%, or between about 0.01% and about 58%, or between about 0.01% and about 50%, or between about 0.01% and about 45%, or between about 0.01% and about 40%, or between about 0.01% and about 35%, or between about 0.01% and about 30%, or between about 0.01% and about 25%, between 0.10% and about 60%, or between about 0.10% and about 58%, or between about 0.10% and about 50%, or between about 0.10% and about 45%, or between about 0.10% and about 40%, or between about 0.10% and about 35%, or between about 0.10% and about 30%, or between about 0.10% and about 25%, between 0.20% and about 60%, or between about 0.20% and about 58%, or between about 0.20% and about 50%, or between about 0.20% and about 45%, or between about 0.20% and about 40%, or between about 0.20% and about 35%, or between about 0.20% and about 30%, or between about 0.20% and about 25% of the cells in the enriched heterogeneous kidney cell population express FGF8, then the enriched heterogeneous kidney cell population can be identified as having therapeutic ability.

[0026] In the above method, the percentage of cells in an enriched heterogeneous renal cell population that express one or more (at least 1, or at least 2, or at least 3, or at least 4, or at least 5, or at least 6, or at least 7, or 8) markers is determined, and if one or more (at least 1, or at least 2, or at least 3, or at least 4, or at least 5, or at least 6, or at least 7, or 8) markers are RACK1 or include RACK1, and at least about 75% of the cells in the enriched heterogeneous renal cell population express RACK1, then the enriched heterogeneous renal cell population can be identified as having a therapeutic ability. Alternatively, if one or more (at least 1, or at least 2, or at least 3, or at least 4, or at least 5, or at least 6, or at least 7, or 8) markers are RACK1 or include RACK1, and 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%, at least about 99%, or about 100% of the cells in the enriched heterogeneous renal cell population express RACK1, then the enriched heterogeneous renal cell population can be identified as having a therapeutic ability.

[0027] In the above method, the percentage of cells in an enriched heterogeneous renal cell population expressing one or more (at least 1, or at least 2, or at least 3, or at least 4, or at least 5, or at least 6, or at least 7, or 8) markers is determined, and if one or more (at least 1, or at least 2, or at least 3, or at least 4, or at least 5, or at least 6, or at least 7, or 8) markers are nephrin or include nephrin, and at least about 65% of the cells in the enriched heterogeneous renal cell population express nephrin, then the enriched heterogeneous renal cell population can be identified as having a therapeutic ability. Alternatively, if one or more (at least 1, or at least 2, or at least 3, or at least 4, or at least 5, or at least 6, or at least 7, or 8) markers are nephrin or include nephrin, and at least about 68%, at least about 70%, at least about 72%, at least about 74%, at least about 76%, at least about 78%, at least about 80%, at least about 82%, at least about 84%, at least about 86%, at least about 88%, at least about 90%, at least about 92%, at least about 94%, at least about 96%, at least about 98%, or about 100% of the cells in the enriched heterogeneous renal cell population express nephrin, then the enriched heterogeneous renal cell population can be identified as having a therapeutic ability.If one or more (at least one, or at least two, or at least three, or at least four, or at least five, or at least six, or at least seven, or eight) markers are nephrin or contain nephrin, and between about 65% and about 99%, or between about 67% and about 99%, or between about 69% and about 99%, or between about 70% and about 99%, or between about 72% and about 99%, and between about 74% and about 99%, or between 35% and about 95%, or between about 40% and about 95%, or between about 45% and 95%, or between about 50% and about 95%, or between about 55% and about 95%, or between about 60% and about 95%, or between about 65% and about 95%, or between 70% and about 95%, or between about 75% and about 95%, or between about 80% and about 95%, or between about 85% and about 95% of the cells of the enriched heterogeneous renal cell population express nephrin, then the enriched heterogeneous renal cell population can be identified as having a therapeutic ability.

[0028] In the above method, if the percentage of cells in an enriched heterogeneous renal cell population expressing one or more (at least one, or at least two, or at least three, or at least four, or at least five, or at least six, or at least seven, or eight) markers is determined, and one or more (at least one, or at least two, or at least three, or at least four, or at least five, or at least six, or at least seven, or eight) of the markers are podocin or include podocin, and at least about 90% of the cells in the enriched heterogeneous renal cell population express podocin, then the enriched heterogeneous renal cell population can be identified as having a therapeutic ability. Alternatively, if one or more (at least one, or at least two, or at least three, or at least four, or at least five, or at least six, or at least seven, or eight) of the markers are podocin or include podocin, and 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%, at least about 98%, or about 100% of the cells in the enriched heterogeneous renal cell population express podocin, then the enriched heterogeneous renal cell population can be identified as having a therapeutic ability.

[0029] In the above method, when the percentage of cells in a heterogeneous renal cell population expressing (i) NEPH1 and (ii) one or more (at least 1, or at least 2, or at least 3, or at least 4, or at least 5, or at least 6, or at least 7, or 8) markers is determined, the heterogeneous renal cell population can be identified as having a therapeutic ability by determining the percentage of cells expressing any combination of (i) NEPH1 and (ii) one or any two, any three, any four, any five, any six, any seven, or all eight of the markers SIX2, OSR1, LHX1, RET, FGF8, RACK1, nephrin, and podocin. For example, (a) between about 2% and about 8.0%, between about 3.0% and about 7.0%, or between about 4.0% and about 6.0% of the cells in the heterogeneous renal cell population express NEPH1, and (b) any two, any three, any four, any five, any six, any seven, or all eight of the following are determined: (i) more than 0% to a maximum of about 35.0% of the cells in the heterogeneous renal cell population express SIX2; (ii) at least about 35% of the cells in the heterogeneous renal cell population express OSR1; (iii) at least about 1% of the cells in the heterogeneous renal cell population express LHX1; (iv) at least about 1% of the cells in the heterogeneous renal cell population express RET; (v) more than 0% to a maximum of about 60% of the cells in the heterogeneous renal cell population express FGF8; (vi) at least about 80% of the cells in the heterogeneous renal cell population express RACK1; (vii) at least about 65% of the cells in the enriched heterogeneous renal cell population express nephrin; and / or (viii) about 90% of the cells in the enriched heterogeneous renal cell population express podocin, the enriched heterogeneous renal cell population can be identified as having a therapeutic ability.In another example, (a) between about 2% and about 8.0%, between about 3.0% and about 7.0%, or between about 4.0% and about 6.0% of the cells of the heterogeneous renal cell population express NEPH1, and (b) (i) between about 0.1% and about 30.0% of the cells of the heterogeneous renal cell population express SIX2, (ii) between about 40% and about 99% of the cells of the heterogeneous renal cell population express OSR1, (iii) at least about 1% of the cells of the heterogeneous renal cell population express LHX1, (iv) between about 1% and about 80% of the cells of the heterogeneous renal cell population express RET, (v) between about 0.01% and about 60% of the cells of the heterogeneous renal cell population express FGF8, (vi) at least about 80% of the cells of the enriched heterogeneous renal cell population express RACK1, (vii) at least about 65% of the cells of the enriched heterogeneous renal cell population express nephrin, and / or (viii) about 90% of the cells of the enriched heterogeneous renal cell population express podocin. When any two, any three, any four, any five, any six, any seven, or all eight combinations are determined, the enriched heterogeneous renal cell population can be identified as having a therapeutic ability. Any two, any three, any four, any five, any six, any seven, or all eight combinations of markers whose expression can be determined at these percentages can be any of the following combinations: SIX2 and OSR1; SIX2 and LHX1; SIX2 and RET; SIX2 and FGF8; OSR1 and LHX1; OSR1 and RET; OSR1 and FGF8; LHX1 and RET; LHX1 and FGF8; RET and FGF8; nephrin and SIX2; nephrin and OSR1; nephrin and LHX1; nephrin and RET; nephrin and FGF8; podocin and nephrin; podocin and SIX2; podocin and OSR1; podocin and LHX1; podocin and RET; podocin and FGF8; RACK1 and nephrin; RACK1 and podocin; RACK1 and SIX2; RACK1 and OSR1; RACK1 and LHX1; RACK1 and RET; RACK1 and FGF8; SIX2, OSR1, and LHX1; SIX2, OSR1, and RET; SIX2, OSR1, and FGF8; SIX2, LHX1, and RET; SIX2, LHX1, and FGF8; SIX2, RET, and FGF8; OSR1, LHX1, and RET; OSR1, LHX1, and FGF8; OSR1, RET, and FGF8; LHX1, RET, and FGF8; Nephrin, SIX2, and OSR; Nephrin, SIX2, and LHX1; Nephrin, SIX2, and RET; Nephrin, SIX2, and FGF8; Nephrin, OSR, and LHX1; Nephrin, OSR, and RET; Nephrin, OSR, and FGF8; Nephrin, LHX1, and RET; Nephrin, LHX1, and FGF8; Nephrin, RET, and FGF8; Podocin, SIX2, and OSR; Podocin, SIX2, and LHX1; Podocin, SIX2, and RET; Podocin, SIX2, and FGF8; Podocin, OSR, and LHX1; Podocin, OSR, and RET; Podocin, OSR, and FGF8; Podocin, LHX1, and RET; Podocin, LHX1, and FGF8; Podocin, RET, and FGF8; RACK1, SIX2, and OSR; RACK1, SIX2, and LHX1; RACK1, SIX2, and RET; RACK1, SIX2, and FGF8; RACK1, OSR, and LHX1; RACK1, OSR, and RET; RACK1, OSR, and FGF8; RACK1, LHX1, and RET; RACK1, LHX1, and FGF8; RACK1, RET, and FGF8; Nephrin, Podocin, and RACK1; Nephrin, Podocin, and SIX2; Nephrin, Podocin, and OSR; Nephrin, Podocin, and RET; Nephrin, Podocin, and LHX1; Nephrin, Podocin, and FGF8; Nephrin, RACK1, and SIX2; Nephrin, RACK1, and OSR; Nephrin, RACK1, and RET; Nephrin, RACK1, and LHX1; Nephrin, RACK1, and FGF8; Podocin, RACK1, and SIX2; Podocin, RACK1, and OSR; Podocin, RACK1, and RET; Podocin, RACK1, and LHX1; Podocin, RACK1, and FGF8; SIX2, OSR1, LHX1, and RET; SIX2, OSR1, LHX1, and FGF8; SIX2, LHX1, RET, and FGF8; SIX2, OSR1, RET, and FGF8; OSR1, LHX1, RET, and FGF8; Nephrin, SIX2, OSR1, and LHX1; Nephrin, SIX2, OSR1, and RET; Nephrin, SIX2, OSR1, and FGF8; Nephrin, SIX2, LHX1, and RET; Nephrin, SIX2, LHX1, and FGF8; Nephrin, SIX2, RET, and FGF8; Nephrin, OSR1, LHX1, and FGF8; Nephrin, OSR1, LHX1, and RET; Nephrin, OSR1, RET, and FGF8; Nephrin, LHX1, RET, and FGF8; Nephrin, SIX2, OSR1, and LHX1; Podocin, SIX2, OSR1, and RET; Podocin, SIX2, OSR1, and FGF8; Podocin, SIX2, LHX1, and RET; Podocin, SIX2, LHX1, and FGF8; Podocin, SIX2, RET, and FGF8; Podocin, OSR1, LHX1, and FGF8; Podocin, OSR1, LHX1, and RET; Podocin, OSR1, RET, and FGF8; Podocin, LHX1, RET, and FGF8; RACK1, SIX2, OSR1, and FGF8; RACK1, SIX2, LHX1, and RET; RACK1, SIX2, LHX1, and FGF8; RACK1, SIX2, RET, and FGF8; RACK1, OSR1, LHX1, and FGF8; RACK1, OSR1, LHX1, and RET; RACK1, OSR1, RET, and FGF8; RACK1, LHX1, RET, and FGF8; Nephrin, Podocin, SIX2, and OSR1; Nephrin, Podocin, SIX2, and LHX1; Nephrin, Podocin, SIX2, and RET; Nephrin, Podocin, SIX2, and FGF8; Nephrin, Podocin, OSR1, and LHX1; Nephrin, Podocin, OSR1, and RET; Nephrin, Podocin, OSR1, and FGF8; Nephrin, Podocin, LHX1, and RET; Nephrin, Podocin, LHX1, and FGF8; Nephrin, Podocin, RET, and FGF8; RACK1, Podocin, SIX2, and OSR1; RACK1, Podocin, SIX2, and LHX1; RACK1, Podocin, SIX2, and RET;RACK1, podocin, SIX2, and FGF8; RACK1, podocin, OSR1, and LHX1; RACK1, podocin, OSR1, and RET; RACK1, podocin, OSR1, and FGF8; RACK1, podocin, LHX1, and RET; RACK1, podocin, LHX1, and FGF8; RACK1, podocin, RET, and FGF8; RACK1, nephrin, SIX2, and OSR1; RACK1, nephrin, SIX2, and LHX1; RACK1, nephrin, SIX2, and RET; RACK1, nephrin, SIX2, and FGF8; RACK1, nephrin, OSR1, and LHX1; RACK1, nephrin, OSR1, and RET; RACK1, nephrin, OSR1, and FGF8; RACK1, nephrin, LHX1, and RET; RACK1, nephrin, LHX1, and FGF8; RACK1, nephrin, RET, and FGF8; RACK1, nephrin, podocin, and SIX2; RACK1, nephrin, podocin, and RET; RACK1, nephrin, podocin, and LHX1; RACK, nephrin, podocin, and OSR1; RACK, nephrin, podocin, and FGF8; SIX2, OSR1, LHX1, RET, and FGF8; nephrin, SIX2, OSR1, LHX1, and RET; nephrin, SIX2, OSR1, LHX1, and FGF8; nephrin, SIX2, LHX1, RET, and FGF8; nephrin, SIX2, OSR1, RET, and FGF8; nephrin, OSR1, LHX1, RET, and FGF8; podocin, SIX2, OSR1, LHX1, and RET; podocin, SIX2, OSR1, LHX1, and FGF8; podocin, SIX2, LHX1, RET, and FGF8; podocin, SIX2, OSR1, RET, and FGF8; podocin, OSR1, LHX1, RET, and FGF8; RACK1, SIX2, OSR1, LHX1, and RET; RACK1, SIX2, OSR1, LHX1, and FGF8; RACK1, SIX2, LHX1, RET, and FGF8; RACK1, SIX2, OSR1, RET, and FGF8; RACK1, OSR1, LHX1, RET, and FGF8; nephrin, podocin, SIX2, OSR1, and LHX1; nephrin, podocin, SIX2, OSR1, and RET; nephrin, podocin, SIX2, OSR1, and FGF8; nephrin, podocin, SIX2, LHX1, and RET; nephrin, podocin, SIX2, LHX1, and FGF8; nephrin, podocin, SIX2, RET, and FGF8; nephrin, podocin, OSR1, LHX1, and RET; nephrin, podocin, OSR1, LHX1, and FGF8; nephrin, podocin, OSR1, RET, and FGF8; nephrin, podocin, LHX1, RET, and FGF8; nephrin, RACK1, SIX2, OSR1, and LHX1; nephrin, RACK1, SIX2, OSR1, and RET; nephrin, RACK1, SIX2, OSR1, and FGF8; nephrin, RACK1, SIX2, LHX1, and RET; nephrin, RACK1, SIX2, LHX1, and FGF8; nephrin, RACK1, SIX2, RET, and FGF8; nephrin, RACK1, OSR1, LHX1, and RET; nephrin, RACK1, OSR1, LHX1, and FGF8; nephrin, RACK1, OSR1, RET, and FGF8; nephrin, RACK1, LHX1, RET, and FGF8; podocin, RACK1, SIX2, OSR1, and LHX1;Podocin, RACK1, SIX2, OSR1, and RET; Podocin, RACK1, SIX2, OSR1, and FGF8; Podocin, RACK1, SIX2, LHX1, and RET; Podocin, RACK1, SIX2, LHX1, and FGF8; Podocin, RACK1, SIX2, RET, and FGF8; Podocin, RACK1, OSR1, LHX1, and RET; Podocin, RACK1, OSR1, LHX1, and FGF8; Podocin, RACK1, OSR1, RET, and FGF8; Podocin, RACK1, LHX1, RET, and FGF8; Nephrin, Podocin, RACK1, SIX2, and OSR1; Nephrin, Podocin, RACK1, SIX2, and LHX1; Nephrin, Podocin, RACK1, SIX2, and RET; Nephrin, Podocin, RACK1, SIX2, and FGF8; Nephrin, Podocin, RACK1, OSR1, and LHX1; Nephrin, Podocin, RACK1, OSR1, and RET; Nephrin, Podocin, RACK1, OSR1, and FGF8; Nephrin, Podocin, RACK1, LHX1, and RET; Nephrin, Podocin, RACK1, LHX1, and FGF8; Nephrin, Podocin, RACK1, RET, and FGF8; Nephrin, SIX2, OSR1, LHX1, RET, and FGF8; Podocin, SIX2, OSR1, LHX1, RET, and FGF8; RACK1, SIX2, OSR1, LHX1, RET, and FGF8; Nephrin, Podocin, SIX2, OSR1, LHX1, and RET; Nephrin, Podocin, SIX2, OSR1, LHX1, and FGF8; Nephrin, Podocin, SIX2, LHX1, RET, and FGF8; Nephrin, Podocin, SIX2, OSR1, RET, and FGF8; Nephrin, Podocin, OSR1, LHX1, RET, and FGF8; Nephrin, RACK1, SIX2, OSR1, LHX1, and RET; Nephrin, RACK1, SIX2, OSR1, LHX1, and FGF8; Nephrin, RACK1, SIX2, LHX1, RET, and FGF8; Nephrin, RACK1, SIX2, OSR1, RET, and FGF8; Nephrin, RACK1, OSR1, LHX1, RET, and FGF8; Podocin, RACK1, SIX2, OSR1, LHX1, and RET; Podocin, RACK1, SIX2, OSR1, LHX1, and FGF8; Podocin, RACK1, SIX2, LHX1, RET, and FGF8; Podocin, RACK1, SIX2, OSR1, RET, and FGF8; Podocin, RACK1, OSR1, LHX1, RET, and FGF8; Nephrin, Podocin, RACK1, SIX2, OSR1, and LHX1; Nephrin, Podocin, RACK1, SIX2, OSR1, and RET; Nephrin, Podocin, RACK1, SIX2, OSR1, and FGF8; Nephrin, Podocin, RACK1, SIX2, LHX1, and RET; Nephrin, Podocin, RACK1, SIX2, LHX1, and FGF8; Nephrin, Podocin, RACK1, SIX2, RET, and FGF8; Nephrin, Podocin, RACK1, OSR1, LHX1, and RET; Nephrin, Podocin, RACK1, OSR1, LHX1, and FGF8; Nephrin, Podocin, RACK1, OSR1, RET, and FGF8; Nephrin, Podocin, RACK1, LHX1, RET, and FGF8; Podocin, RACK1, SIX2, OSR1, LHX1, RET, and FGF8; Nephrin, RACK1, SIX2, OSR1, LHX1, RET, and FGF8; Nephrin, Podocin, SIX2, OSR1, LHX1, RET, and FGF8; Nephrin, Podocin, RACK1, OSR1, LHX1, RET, and FGF8; Nephrin, Podocin, RACK1, SIX2, LHX1, RET, and FGF8; Nephrin, Podocin, RACK1, SIX2, OSR1, RET, and FGF8; Nephrin, Podocin, RACK1, SIX2, OSR1, LHX1, and FGF8; Nephrin, Podocin, RACK1, SIX2, OSR1, LHX1, and RET; or SIX2, OSR1, LHX1, RET, FGF8, RACK1, Nephrin, and Podocin.

[0030] In a method of identifying an enriched heterogeneous renal cell population as having a therapeutic ability, it can be determined whether the cells of the enriched heterogeneous renal cell population express one or more additional markers, i.e., markers other than NEPH1, Nephrin, Podocin, RACK1, SIX2, OSR1, LHX1, RET, and / or FGF8. The one or more additional markers can be or include one or more of Leftie, Nodal, or CD24.

[0031] If the percentage of cells expressing a particular marker is indicated to be "about" a particular percentage value, e.g., about 5%, it should be understood that the percentage of cells does not have to be exactly the particular numerical value, e.g., exactly 5%. Rather, if the percentage of cells expressing a particular marker is indicated to be "about" a particular percentage value, e.g., about 5%, it should be understood that the percentage of cells expressing a particular marker can be within up to 10% of that particular numerical value, e.g., between 4.5% and 5.5%.

[0032] In any of the methods of identifying an enriched heterogeneous renal cell population as having a therapeutic ability, determining whether the cells of the enriched heterogeneous renal cell population express (i) NEPH1 and (ii) one or more markers (e.g., SIX2, OSR1, LHX1, FGF8, RACK1, RET, nephrin, and / or podocin), and optionally (iii) one or more additional markers, can be determining whether the cells of the enriched heterogeneous renal cell population express (i) NEPH1 and (ii) one or more markers, and optionally (iii) one or more additional markers, in the form of nucleic acids, such as mRNA or miRNA, or polypeptides. Expression of (i) NEPH1 and (ii) one or more markers (e.g., SIX2, OSR1, LHX1, FGF8, RACK1, RET, nephrin, and / or podocin), and optionally (iii) one or more additional markers, by the cells of the enriched heterogeneous renal cell population can be determined via any assay suitable for detecting the presence of (i) NEPH1 and (ii) one or more markers, and optionally (iii) one or more additional markers. For example, when expression is determined in the form of a polypeptide, this can be determined by assays such as Western blot, fluorescence-activated cell sorting (FACS), enzyme-linked immunosorbent assay (ELISA), etc. When expression is determined in the form of nucleic acids, this can be determined by assays such as Southern blot, polymerase chain reaction (PCR) or reverse transcriptase PCR, serial analysis of gene expression (SAGE), MassARRAY, or fluorescence in situ hybridization (FISH), or assays using them. The assay can use a labeled detection reagent to determine whether (i) NEPH1 and (ii) one or more markers, and optionally (iii) one or more additional markers are present, and / or the percentage of cells expressing (i) NEPH1 and (ii) one or more markers, and optionally (iii) one or more additional markers. The labeled detection reagent can comprise (a) a moiety that forms a complex directly or indirectly with NEPH1 and one or more markers and / or additional markers, and (b) a detection moiety. Non-limiting detection moieties include radioisotopes, such as 35 S,14 C, 125 I, 3 H, and 131 I, colloidal gold particles, fluorescent labels such as Texas Red, rhodamine, fluorescein, dansyl, Lissamine, phycocryterin, phycocyanin, SPECTRUM ORANGE, SPECTRUM GREEN1, and enzyme substrates such as firefly luciferase, bacterial luciferase, luciferin, horseradish peroxidase, alkaline phosphatase, or beta-galactosidase.

[0033] An enriched heterogeneous renal cell population that can be identified as having therapeutic potential in any of the above methods may be enriched in one or more renal cell types such as renal epithelial cells, renal tubular cells, renal tubular epithelial cells, or renal proximal tubular cells. Enrichment of these one or more renal cell types in the enriched heterogeneous renal cell population means that the enriched heterogeneous renal cell population has the one or more renal cell types at a higher percentage than the starting renal cell population, which can be collectively referred to as the "starting renal cell population" and includes a renal cell preparation that may or may not have undergone a process to remove red blood cells and debris and contains dissociated cells from the patient's kidney tissue, patient's kidney biopsy, or in vitro culture of cells established from the patient's kidney tissue or kidney biopsy. The starting renal cell population, when it is an in vitro culture of cells established from the patient's kidney tissue or patient's kidney biopsy, can be a renal cell preparation that may or may not have undergone a process to remove red blood cells and debris and contains dissociated cells (e.g., cells dissociated from the kidney tissue or kidney biopsy through mincing and / or enzymatic digestion) from the kidney tissue or kidney biopsy. In addition to being enriched in renal epithelial cells, renal tubular cells, renal tubular epithelial cells, and / or renal proximal tubular cells, the enriched heterogeneous renal cell population may also include other renal cell types such as glomerular cells, podocytes, collecting duct cells, and / or vascular cells.

[0034] Enriched heterogeneous renal cell populations may result from being prepared from a starting renal cell population (e.g., a patient's kidney tissue, a patient's kidney biopsy, or an in vitro culture of cells established from a patient's kidney tissue or kidney biopsy) via a method that includes a separation step, where one or more renal cell types may be enriched. The separation step may be one that separates the cells of the starting renal cell population that have not been passaged more than once, twice, or three times based on their buoyant density. When the separation step is one that separates the cells based on their buoyant density, the separation step may utilize a single-step or multi-step continuous density gradient or discontinuous density gradient using a density gradient medium such as glycerol, glucose, OptiPrep, Percoll, or Ficoll-Paque. As a result of using such a density gradient medium in this way, the cells of the starting renal cell population (or the starting renal cell population passaged at most once, twice, or three times) can be separated into one or more distinguishable fractions, and the cells of the enriched heterogeneous renal cell population can be clearly identified and isolated from that fraction. The distinguishable fraction(s) can be a fraction where the buoyant density of the cells in the fraction(s) is greater than about 1.045 g / mL, or greater than 1.045 g / mL, or 1.045 g / mL or more. The distinguishable fraction(s) can be a fraction where the buoyant density of the cells in the fraction(s) is greater than about 1.04 g / mL, or greater than 1.04 g / mL, or 1.04 g / mL or more, or greater than about 1.0419 g / mL, or greater than 1.0419 g / mL, or 1.0419 g / mL or more. The distinguishable fraction(s) can be a fraction where 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 can be one that separates the cells of the starting renal cell population (or the cells of the starting renal cell population that have not been passaged more than once, twice, or three times) based on whether they express a particular marker on their surface. When the separation step is one that separates the cells based on the expression of their particular cell surface markers, the separation step can be one that utilizes flow cytometry.Flow cytometry can select cells from a starting renal cell population (or a starting renal cell population passaged at most once, twice, or three times) when they express certain surface markers such as nephrin characteristic of, for example, renal epithelial cells, renal tubular cells, renal tubular epithelial cells, or renal proximal tubular cells, thereby forming an enriched heterogeneous renal cell population isolated thereby.

[0035] An enriched heterogeneous renal cell population prepared from a starting renal cell population (or a starting renal cell population passaged at most once, twice, or three times) can be cultured under hypoxic conditions prior to the separation step. When the cells are cultured under hypoxic conditions prior to the separation step, the cells can be cultured under conditions where the oxygen level is 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. When the cells are cultured under hypoxic conditions, the cells can be cultured for at least 6 hours, at least 8 hours, at least 10 hours, at least 12 hours, at least 14 hours, at least 16 hours, at least 20 hours, at least 24 hours, at least 30 hours, at least 36 hours, at least 42 hours, or at least 48 hours under hypoxic conditions.

[0036] Generally, an enriched heterogeneous renal cell population can be prepared from any starting cell population, such as a patient's kidney tissue or an in vitro culture of cells established from a patient's kidney biopsy. When an enriched heterogeneous renal cell population is prepared from a patient's kidney tissue or an in vitro culture of cells established from a kidney biopsy, the cells of the in vitro culture can be expanded by subculturing them at most once, or at most twice, or at most three times. Alternatively, if desired, the cells of the in vitro culture of cells established from kidney tissue or a kidney biopsy can be cryopreserved and then expanded by subculturing them at most once, or at most twice, or at most three times. Once the cells have been expanded, the expanded cells may be cryopreserved. Whether cryopreserved or not, the expanded cells can then be subjected to a separation step, or first subjected to hypoxic culture conditions followed by a separation step. The enriched heterogeneous renal cell population can be isolated by performing a separation step. Once the enriched heterogeneous renal cell population has been isolated, it may be used as a therapeutic agent after being frozen and / or analyzed.

[0037] If an enriched heterogeneous renal cell population is identified as having therapeutic potential according to any of the methods disclosed herein, it can be included in a pharmaceutical composition, or administered in a method of treating a kidney disease in a patient in need thereof, and / or used in the manufacture of a medicament for treating a kidney disease. If an enriched heterogeneous renal cell population is identified as having therapeutic potential and is included in a pharmaceutical composition, it can be formulated as a hydrogel composition or a liquid composition. The pharmaceutical composition may or may not contain hyaluronic acid.

[0038] When the pharmaceutical composition is formulated as a hydrogel composition, the cells of the enriched heterogeneous kidney cell composition can be combined with a temperature-sensitive cell-stabilizing biomaterial. The temperature-sensitive cell-stabilizing biomaterial can be a biomaterial that is in a gel state at a certain temperature and liquid at other temperatures. For example, if the biomaterial is temperature-sensitive, the biomaterial is in a gel state at about 8°C or below, substantially in a liquid state at about ambient temperature or above, and in a solid-liquid transition state between about 8°C and about ambient temperature, or in a gel state at about 4°C or below, in a liquid state at about 37°C or above, and in a solid-liquid transition state between about 8°C and about 18°C, or in a gel state at about 2°C or below, in a liquid state at about 37°C or above, and in a solid-liquid transition state between about 8°C and about 18°C, or in a gel state at about 2°C or below and in a liquid state at about 37°C or above, or in a gel state at about 4°C or below and in a liquid state at about 34°C or above, or in a gel state at about 6°C or below and in a liquid state at about 32°C or above. The temperature-sensitive cell-stabilizing biomaterial may contain or be composed of one or more proteins or peptides of natural origin or recombinant origin. The protein or peptide of natural origin or recombinant origin can be a cell-derived extracellular matrix protein, or an extracellular matrix originating from the kidney or another tissue or organ, or gelatin. When the temperature-sensitive cell-stabilizing biomaterial is gelatin or contains it, the gelatin can be derived from type I αI collagen such as porcine type I αI collagen or recombinant human type I αI collagen. When the temperature-sensitive cell-stabilizing biomaterial is gelatin or contains it, the gelatin can be present in the therapeutic composition at about 0.5 weight / volume (w / v)% to about 1 weight / volume (w / v)%, or about 0.8% (w / v) to about 0.9% (w / v), or about 0.75% (w / v) or about 0.88% (w / v). The cells of the enriched heterogeneous kidney cell population may be distributed throughout the biomaterial or substantially uniformly distributed throughout the biomaterial. The number of cells per milliliter of the enriched heterogeneous kidney cell population is about 20×10 6 cells per milliliter, about 40×10 6 cells per milliliter, about 60×10 6cells, about 100×10 per mL 6 cells, about 120×10 per mL 6 cells, about 140×10 per mL 6 cells, about 160×10 per mL 6 cells, about 180×10 per mL 6 cells, or about 200×10 per mL 6 cells, can be formulated in a biomaterial such as gelatin.

[0039] When the pharmaceutical composition is formulated as a liquid composition, the enriched heterogeneous kidney cell population can be combined with any suitable liquid, such as a suitable cell storage medium or cell culture medium, physiological saline, or a combination thereof, and used immediately or cryopreserved until the timing of its use. When the therapeutic composition is a liquid composition, the cells of the enriched heterogeneous kidney cell population can be suspended in a pharmaceutically acceptable carrier or additive such as physiological saline, buffered physiological saline, dextrose, water, polyethylene glycol, etc., and / or any combination thereof. The cells of the enriched heterogeneous kidney cell population are such that the number of cells per mL of the liquid is about 20×10 per mL 6 cells, about 40×10 per mL 6 cells, about 60×10 per mL 6 cells, about 100×10 per mL 6 cells, about 120×10 per mL 6 cells, about 140×10 per mL 6 cells, about 160×10 per mL 6 cells, about 180×10 per mL 6 cells, or about 200×10 per mL 6 cells, can be combined with a suitable liquid such as a cell storage medium or cell culture medium.

[0040] When an enriched heterogeneous renal cell population is identified as having therapeutic potential, the enriched heterogeneous renal cell population, or a pharmaceutical composition comprising the enriched heterogeneous renal cell population, can be administered to a patient in a method of treating a kidney disease, or can be used in a method of treating a kidney disease. When an enriched heterogeneous renal cell population is identified as having therapeutic potential, the enriched heterogeneous renal cell population, or a pharmaceutical composition comprising the enriched heterogeneous renal cell population, can be used in the manufacture of a medicament for treating a kidney disease. The kidney disease can be at any stage or degree of acute or chronic renal failure. The kidney disease can be kidney - caused, or can be secondary to another condition, such as heart failure, hypertension, diabetes, autoimmune disease, or liver disease. Alternatively, the kidney disease can be a kidney disease resulting from acute injury to the kidney, or can be a kidney disease resulting from an abnormality of the kidney and / or urinary tract. The kidney disease can further include endocrine dysfunctions such as anemia, for example, erythropoietin deficiency, and mineral imbalances, for example, vitamin D deficiency.

[0041] When an enriched heterogeneous renal cell population is identified as having therapeutic ability, a renal disease can be treated by administering the enriched heterogeneous renal cell population, or a pharmaceutical composition comprising the enriched heterogeneous renal cell population. A renal disease can be treated by restoring renal function, stabilizing renal function, improving renal function, reducing renal fibrosis, or reducing renal inflammation in the kidney of a patient in need of treatment for a renal disease. Treating a renal disease can also restore the mineral balance or alleviate anemia in a patient in need of such treatment. Treating a renal disease can also delay or prevent the need for dialysis, or delay or prevent the need for kidney transplantation in a patient in need of treatment for a renal disease. When the need for dialysis or kidney transplantation in a patient is delayed by treatment of a renal disease, the delay can be at least 1 year, at least 1.5 years, at least 2 years, at least 2.5 years, at least 3 years, at least 3.5 years, at least 4 years, at least 4.5 years, at least 5 years, at least 5.5 years, at least 6 years, at least 6.5 years, at least 7 years, at least 7.5 years, at least 8 years, at least 8.5 years, at least 9 years, at least 9.5 years, or at least 10 years. Treatment of a renal disease can be determined by observing improvement in the patient's serum albumin, albumin to globulin ratio (A / G ratio), serum phosphorus, serum sodium, kidney size (measurable by ultrasound), serum calcium, phosphorus:calcium ratio, serum potassium, proteinuria, urinary creatinine, serum creatinine, blood urea nitrogen (BUN), cholesterol level, triglyceride level, and glomerular filtration rate (GFR), body weight, blood pressure (mean systemic blood pressure, diastolic blood pressure, or systolic blood pressure), and physical endurance.

[0042] When an enriched heterogeneous renal cell population is identified as having therapeutic potential, the enriched heterogeneous renal cell population can be administered to a patient by any suitable route of administration known in the art. For example, the enriched heterogeneous renal cell population, or a pharmaceutical composition comprising the enriched heterogeneous renal cell population, can be administered systemically to a patient in need of treatment for a renal disease. The enriched heterogeneous renal cell population, or a pharmaceutical composition comprising the enriched heterogeneous renal cell population, can be administered to the kidney(s) or into the kidney(s) of a patient in need of treatment for a renal disease. When the enriched heterogeneous renal cell population is administered to the kidney(s) or into the kidney(s) of a patient in need of treatment for a renal disease, the enriched heterogeneous renal cell population can be administered via a single or multiple injections. The enriched heterogeneous renal cell population can be administered directly via an open laparotomy, via a direct laparoscopy, transabdominally, or percutaneously. The enriched heterogeneous renal cell population, or a pharmaceutical composition comprising the enriched heterogeneous renal cell population, can also be administered by percutaneous injection into the renal cortex of the kidney, or by inserting a guide cannula percutaneously and puncturing the renal capsule and then injecting the enriched heterogeneous renal cell population into the kidney. The enriched heterogeneous renal cell population, or a pharmaceutical composition comprising the enriched heterogeneous renal cell population, can be administered by injection into the renal cortex of one or both kidneys of the patient. Administration of the enriched heterogeneous renal cell population, or a pharmaceutical composition comprising the enriched heterogeneous renal cell population, can be by two injections, where the first injection is an injection into the renal cortex of one kidney of the patient and the second injection is an injection into the renal cortex of the other kidney of the patient.

[0043] The enriched heterogeneous renal cell population, or a pharmaceutical composition comprising the enriched heterogeneous renal cell population, is administered in a therapeutically effective dose by any suitable route. The therapeutically effective dose or amount administered to a patient in need of treatment for a renal disease can be, for example, from about 1×10 6 cells to 9×10 6 cells of the enriched heterogeneous renal cell population per gram of the patient's estimated kidney weight. The therapeutically effective amount of the pharmaceutical composition can be about 1.0×106 cells, about 2.0×10 6 cells, about 3.0×10 6 cells, about 4.0×10 6 cells, about 5.0×10 6 cells, about 6.0×10 6 cells, about 7.0×10 6 cells, about 8.0×10 6 cells, about 9.0×10 6 cells, about 2.0×10 6 cells to 7.0×10 6 cells, about 4.0×10 6 cells to 7.0×10 6 cells, or about 5.0×10 6 cells to 7.0×10 6 cells, and can be a dose of cells of an enriched heterogeneous renal cell population between.

[0044] Administering a therapeutic composition comprising a heterogeneous renal cell population to a patient in a method of treating a kidney disease may be by the first and second injections. The first and second injections can be administered at intervals of approximately 3 months to 12 months. The first and second injections can 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 apart, approximately 11 months apart, or approximately 12 months apart. The first and second injections can be administered at intervals of approximately 3 months to 6 months, approximately 6 months to 9 months, approximately 9 months to 12 months, approximately 3 months to 9 months, approximately 6 months to 9 months, or approximately 6 months to 12 months.

[0045] One of ordinary skill 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 accompanying claims.

[0046] All publications, patents, and patent applications cited in this specification are hereby incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application were specifically and individually indicated to be incorporated by reference as part of this specification.

Example

[0047] Example 1 - Selective renal cells (SRC) stabilize and improve renal function by partially co-expressing a unique set of markers involved in kidney development. Introduction. Selective renal cells (SRC) are an example of an enriched heterogeneous renal cell population enriched in renal epithelial cells. SRC has been advancing as an autologous cell-based therapy for the treatment of chronic kidney disease (CKD). A study was conducted that linked empirical SRC gene expression data with gene ontology to determine whether human SRC, as a stand-alone platform, has the potential to restore the function of diseased CKD kidneys and initiate de novo kidney development by at least partially co-expressing proteins involved in kidney development.

[0048] Methods / Human SRC Nodes: Proteomic analysis showed that SRC, a human enriched heterogeneous renal cell population, co-expresses eight markers: Six2, Osr1, Lhx1, Ret, Fgf8, Rack1, Nphs1, and Nphs2 (Non-Patent Document 9). These eight markers were selected as nodes for the analysis in this study. The co-expression data for each of the eight markers are shown in Table 1.

[0049]

Table 1

[0050] Method / Co-expression analysis.SRC: For example, eight nodes co-expressed by a heterologous kidney cell population enriched as an example were converted to their gene symbols (homo sapien), namely, six2, osr1, lhx1, ret, fgf8, rack1 / gnb2l1, nphs1, and nphs2, and queried in STRING (string-DB.org, last access June 6, 2022) and Genemania (https: / / genemania.org, last access June 6, 2022) for their co-expression across the entire human tissue.

[0051] Method / Tracking of renal progenitor cells: To map the expression of eight markers across all cells involved in kidney development, their gene symbols were queried using the Human Fetal Kidney Atlas (Human Fetal Kidney Atlas - Semraulab(leidenuniv.nl), last access June 6, 2022). A violin plot was generated to report the expression levels of the eight markers across up to 22 cell types.

[0052] Method / Analysis of the human SRC interactome and biological processes: The gene symbols for eight nodes co-expressed by human SRC were seeded into Genemania, and the human SRC interactome was constructed by querying gene interactions. Maps of biological processes were created using ShinyGO (bioinformatics.sdstate.edu / go / http: / / bioinformatics.sdstate.edu / go / , last access June 3, 2022, false discovery rate cutoff = 0.05, number of pathways shown = 40) and Humanbase (https: / / hb.flatironisntiute.org, last access June 4, 2022, gene network of biological processes, default value of 0.1 for minimum interaction confidence and maximum number of genes = 15).

[0053] Method for mapping kirrel1 expression by scRNA-seq: SRC was sent for scRNA-seq to map gene expression. Briefly, SRC aliquots were stained with acridine orange and propidium iodide, and the viability, concentration, and singleness of the cells were evaluated using a LUNA-FX7 Dual Fluorescence Cell Counter (Logos Biosystems). Cells were then processed using a 10x Genomics Chromium Controller and Chromium Single Cell 3' GEM, Library & Gel Bead Kit v3.1 Dual Index Kit (PN-1000268) according to the manufacturer's user guide (https: / / tinyurl.com / 4855859x). Briefly, approximately 5000 cells per sample were loaded onto a Chromium Chip G, and libraries were prepared targeting a recovery of 3000 cells per sample. Single cells, reverse transcription reagents, and gel beads coated with barcoded oligos were encapsulated together in oil droplets to generate gel beads in emulsion (GEM). Reverse transcription was performed using a C1000 thermal cycler (Bio-Rad) to generate a complementary DNA (cDNA) library tagged with cell barcodes and unique molecular identifiers (UMI). The GEMs were then disrupted, and the cDNA was purified using Dynabeads MyOne SILANE beads (Invitrogen), followed by 12 cycles of cDNA amplification. The amplified cDNA library was purified using SPRIselect magnetic beads (Beckman Coulter) and quantified using an Agilent Bioanalyzer High Sensitivity DNA chip (Agilent Technologies).Next, fragmentation, end repair, A-tailing, and size selection on both sides using SPRIselect beads were performed. Illumina-compatible adapters were ligated to the size-selected cDNA fragments. Next, the cDNA ligated to the adapters was purified using SPRIselect beads. A uniquely distinguishable index was added to this cDNA during 12 amplification cycles. Next, the completed sequencing library was purified using SPRIselect beads, visualized using a Bioanalyzer high-sensitivity DNA chip, and pooled at an equimolar ratio. The pooled library was sequenced on a NextSeq 2000 machine (Illumina) at the UNC High Throughput Sequencing Facility. The library was denatured and diluted according to the standard Illumina protocol, spiked with 1% PhiX sequencing control (Illumina), and sequenced in paired-end format (Read 1: 28 cycles, i7 index: 10 cycles, i5 index: 10 cycles, Read 2: 90 cycles) on one P3 flow cell until a total depth of 1.2 billion read pairs passing the quality filter was achieved. Demultiplexing and preliminary analysis were performed using Cell Ranger 7.1.0 with default settings, and the analysis was performed using the 10X Genomics Loupe Browser.

[0054] Results / Profiling of human SRC: Two knowledge bases, STRING and Genemania, were queried to determine whether the co-expression of six2, osr1, lhx1, ret, fgf8, rack1, nphs1, and nphs2 has been reported in human tissues. Since a network lacking rack1 and ret was returned when these eight genes were seeded into either database (Figures 1A and 1B), this suggests that there is no known human tissue in which all eight nodes are co-expressed.

[0055] For SRC, for example, eight markers co-expressed by an enriched heterogeneous renal cell population as an example were queried in the human fetal kidney atlas to map whether they are expressed by cells involved in early kidney development. The human fetal kidney atlas is a repository of single-cell transcriptomics related to 22 cell types involved in human kidney development. Querying this repository showed that human SRC markers are variably expressed by various renal progenitor cells, including pools of epithelial and mesenchymal progenitors. The genes six2 (Figure 2A), osr1 (Figure 2B), and lhx1 (Figure 2C) are mainly expressed by nephron progenitor cells, where it was also observed that the gene lhx1 is expressed in renal vesicles, comma-shaped bodies, S-shaped bodies, podocyte precursors, connecting tubules, and loops of Henle cells. Expression of ret (Figure 2D) occurred in connecting tubule cells and ureteric buds, whereas expression of fgf8 (Figure 2E) was observed in nephron progenitors, pretubular aggregates, and comma-shaped body cells. Expression of rack1 / guanine nucleotide-binding protein subunit beta 2-like 1 (gnb2l) (Figure 2F) was relatively uniform across the aforementioned cells, as well as a number of cell types, including podocytes, mesangial cells, and endothelial cells. Nphs1 (Figure 2G) and nphs2 (Figure 2H) were expressed in podocyte precursors and podocytes. These findings suggest that SRC, as a single platform, may represent a number of renal progenitors involved in the spatio-temporal patterning of the kidney.

[0056] Results / Human SRC interactome: The human SRC interactome was constructed by mapping eight markers co-expressed by SRC using gene ontology, such as interactions across nodes. In addition to the eight nodes, the human SRC interactome (Figure 3) may contain additional nodes, the main of which was Kirre-like nephrin family adhesion molecule 1 (kirrel1). NEPH1, the gene product of kirrel1, is a member of the nephrin-like protein family and interacts with podocin to maintain the integrity of the podocyte barrier. Mutations in kirrel1 are known to be associated with nephrotic syndrome (Solanki AK, et al., Kidney Int, 96(2019):883-889).

[0057] Analysis of gene ontology biological processes showed that elements within the human SRC interactome were involved in playing an essential role in all aspects of early kidney development, including ureteric bud induction, mesenchymal cell differentiation, metanephric glomerulus development, pronephric tubule aggregation, and comma and S-shaped body formation (Figure 4). The roles of elements within the human SRC interactome in these aspects obtained from the analysis of gene ontology biological processes were consistent with the results obtained from the above SRC profiling. That is, SRC serves as a single platform that accommodates elements typically expressed by kidney progenitor cells.

[0058] Finally, using HumanBase, an investigation was conducted into other renal processes that are likely to be associated with the human SRC interactome. HumanBase applies machine learning algorithms to learn biological relevance from a vast genomic data collection. Through its integrated analysis, novel data-driven associations are identified beyond the existing biological knowledge shown in the literature. By HumanBase analysis, it was identified that angiogenesis is very likely to be associated with the SRC interactome, as demonstrated by the robust network strength for this process (Figure 5A, network strength 0.45). Consistent with the findings of HumanBase, analysis of the human SRC secretome revealed an increase in VEGF-A levels (Non-Patent Document 9). Na + Transport emerged as another process that is very likely to be associated with the SRC network (Figure 5B, network strength 0.74). Sodium transport in distal tubule cells and connecting tubule cells occurs via the Na + -Cl - cotransporter (Hadchouel J and Jeunemaitre X, Cell Metab 4(2006):335-7).

[0059] Results / scRNAseq: To further confirm that cells expressing SRC also contain cells expressing kirrel1, SRC was sent for scRNAseq to examine the expression of kirrel1. The scRNAseq data confirmed the expression of kirrel1 in SRC. See Figure 6 and Table 2.

[0060]

Table 2

[0061] Based on SRC gene expression data, a mechanism by which SRC may mediate the regenerative and repair activities of diseased kidneys has been elucidated. Through the expression of genes such as six2, osr1, lhx1, ret, fgf8, rack1, nphs1, and nphs2, SRC has been identified as a cell that can affect the signaling cascade mediating kidney formation or kidney regeneration. SRC includes cells that express genes also expressed by renal progenitor cells, such as nephron progenitor cells. Furthermore, the identification of kirrel1 expression in SRC suggests that the biological activity of SRC may be through an impact on or involvement in glomerular barrier function. Overall, by combining empirical gene expression data with interactome analysis, SRC, a heterogeneous renal cell population that is enriched, is supported as an independent platform for treating CKD, and consistent with clinical activity, SRC is supported as a therapy with the potential to initiate de novo kidney generation along with the mechanical ability to restore endogenous repair and function maintenance in diseased CKD kidneys.

Claims

1. A method for identifying a population of enriched heterologous kidney cells that possesses therapeutic properties, The cells of the enriched heterologous renal cell population mentioned above (i) NEPH1, and (ii) One or more markers, To determine whether or not it is expressing, Here, the one or more markers include one or more of the following: lim homeobox protein 1 (LHX1), homeobox protein sineoculis homeobox homolog 2 (SIX2), odd skip-related protein 1 (OSR1), fibroblast growth factor 8 (FGF8), activated C kinase receptor 1 (RACK1), or transfection-induced rearrangement (RET); The cells of the enriched heterologous renal cell population mentioned above (i) NEPH1, and (ii) The one or more markers, If it is determined that the gene is expressed, the enriched heterologous renal cell population is identified as having therapeutic activity. Methods that include...

2. The method according to claim 1, wherein the one or more markers include LHX1.

3. The method according to claim 1, wherein the one or more markers include OSR1.

4. The method according to claim 1, wherein the one or more markers include SIX2.

5. The method according to claim 1, wherein the one or more markers include FGF8.

6. The method according to claim 1, wherein the one or more markers include RACK1.

7. The method according to claim 1, wherein the one or more markers include RET.

8. The one or more markers include at least two markers, where the at least two markers are (a) SIX2 and OSR1, (b) SIX2 and LHX1, (c) SIX2 and RET, (d) SIX2 and FGF8, (e) OSR1 and LHX1, (f) OSR1 and RET, (g) OSR1 and FGF8, (h) LHX1 and RET, (i) LHX1 and FGF8, (j) RET and FGF8, (k) RACK1 and SIX2, (l) RACK1 and OSR1, (m) RACK1 and LHX1, (n) RACK1 and RET, or (o) RACK1 and FGF8, The method according to claim 1, including the method described in claim 1.

9. The one or more markers include at least three markers, where the at least three markers are (a) SIX2, OSR1, and LHX1, (b) SIX2, OSR1, and RET, (c) SIX2, OSR1, and FGF8, (d) SIX2, LHX1, and RET, (e) SIX2, LHX1, and FGF8, (f) SIX2, RET, and FGF8, (g) OSR1, LHX1, and RET, (h) OSR1, LHX1, and FGF8, (i) OSR1, RET, and FGF8, (j) LHX1, RET, and FGF8, (k) RAC K1, SIX2, and OSR1, (l) RACK1, SIX2, and LHX1, (m) RACK1, SIX2, and RET, (n) RACK1, SIX2, and FGF8, (o) RACK1, OSR1, and LHX1, (p) RACK1, OSR1, and RET, (q) RACK1, OSR1, and FGF8, (r) RACK1, LHX1, and RET, (s) RACK1, LHX1, and FGF8, (t) RACK1, RET, and FGF8, The method according to claim 1, including the method described in claim 1.

10. The one or more markers include at least four markers, where the at least four markers are (a) SIX2, OSR1, LHX1, and RET, (b) SIX2, OSR1, LHX1, and FGF8, (c) SIX2, LHX1, RET, and FGF8, (d) SIX2, OSR1, RET, and FGF8, (e) OSR1, LHX1, RET, and FGF8, (f) RACK1, SIX2, OSR1, and FGF8, (g) RACK1, SIX2, L HX1 and RET, (h) RACK1, SIX2, LHX1 and FGF8, (i) RACK1, SIX2, RET and FGF8, (j) RACK1, OSR1, LHX1 and FGF8, (k) RACK1, OSR1, LHX1 and RET, (l) RACK1, OSR1, RET and FGF8, (m) RACK1, LHX1, RET and FGF8, The method according to claim 1, including the method described in claim 1.

11. The one or more markers include at least five markers, where the at least five markers are (a) SIX2, OSR1, LHX1, RET, and FGF8, (b) RACK1, SIX2, OSR1, LHX1, and RET, (c) RACK1, SIX2, OSR1, LHX1, and FGF8, (d) RACK1, SIX2, LHX1, RET, and FGF8, (e) RACK1, SIX2, OSR1, RET, and FGF8, (f) RACK1, OSR1, LHX1, RET, and FGF8, The method according to claim 1, including the method described in claim 1.

12. The one or more markers mentioned above are RACK1, SIX2, OSR1, LHX1, RET, and FGF8. The method according to claim 1, including the method described in claim 1.

13. The method according to any one of claims 1 to 12, wherein the determination comprises determining the percentage of cells in the enriched heterogeneous kidney cell population that express NEPH1.

14. The method according to claim 13, wherein the enriched heterologous renal cell population is identified as having therapeutic activity if it is determined that between approximately 2.0% and approximately 8.0% of the cells in the population express NEPH1.

15. The method according to claim 1, wherein the determination comprises determining the percentage of cells in the enriched heterogeneous population expressing the one or more markers.

16. The method according to claim 8, wherein the determination comprises determining the percentage of cells in the enriched heterogeneous population expressing the at least two markers.

17. The method according to claim 9, wherein the determination comprises determining the percentage of cells in the enriched heterogeneous population expressing the at least three markers.

18. The method according to claim 10, wherein the determination comprises determining the percentage of cells in the enriched heterogeneous population expressing the at least four markers.

19. The method according to claim 11, wherein the determination comprises determining the percentage of cells in the enriched heterogeneous population expressing the at least five markers.

20. The method according to claim 12, wherein the determination comprises determining the percentage of cells in the enriched heterogeneous population expressing RACK1, SIX2, OSR1, LHX1, RET, and FGF8.

21. The method according to any one of claims 15 to 20, further comprising determining the percentage of cells in the enriched heterogeneous kidney cell population that express NEPH1.

22. The method according to claim 21, wherein the enriched heterologous renal cell population is identified as having therapeutic activity if it is determined that between approximately 2.0% and approximately 8.0% of the cells in the population express NEPH1.

23. The method according to any one of claims 1 to 12, further comprising determining whether the cells of the enriched heterogeneous kidney cell population express Lefty, and if it is further determined that the cells of the enriched heterogeneous kidney cell population express Lefty, identifying the enriched heterogeneous kidney cell population as having therapeutic activity.

24. The method according to any one of claims 1 to 12, further comprising determining whether the cells of the enriched heterogeneous kidney cell population express nodal, and if it is further determined that the cells of the enriched heterogeneous kidney cell population express nodal, identifying the enriched heterogeneous kidney cell population as having therapeutic activity.

25. The method according to any one of claims 1 to 12, further comprising determining whether the cells of the enriched heterogeneous kidney cell population express CD24, and if it is further determined that the cells of the enriched heterogeneous kidney cell population express CD24, identifying the enriched heterogeneous kidney cell population as having therapeutic activity.

26. The method according to any one of claims 1 to 12, wherein the enriched heterologous kidney cell population is prepared by a method including a density gradient separation step.

27. The method according to claim 26, wherein the enriched heterologous kidney cell population prepared by the method described above comprises cells having a suspension density greater than approximately 1.04 g / mL.

28. The method according to claim 26, wherein the enriched heterologous kidney cell population is hypoxic-tolerant and is cultured under hypoxic conditions prior to the density gradient separation step.

29. A composition comprising the enriched heterogeneous renal cell population identified by the method of any one of claims 1 to 12.

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