# US 10,870,834 B2 · Isolated populations of adult renal cells and methods of isolating and using same

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- Number: US 10,870,834 B2
- Document ID: US10870834B2
- Status: Granted
- Kind: B2
- Application number: 16/045,784
- Filed: 2018-07-26
- Published: 2018-12-06
- Granted: 2020-12-22
- Earliest claimed: 2010-05-13
- Assignees: Tel HaShomer Medical Research Infrastructure and Services Ltd.; Ramot at Tel-Aviv University Ltd.
- Applicants: Tel Hashomer Medical Research Infrastructure and Services Ltd.; Ramot At Tel-Aviv University Ltd.
- Inventors: Benjamin Dekel; Orit Harari-Steinberg; Ella Buzhor
- Web page: https://patentgrep.com/patent/US10870834B2

## CPC

- C12N 5/0686: {Kidney cells}
- C12N 2500/90: Serum-free medium, which may still contain naturally-sourced components
- C12N 2501/11: Epidermal growth factor [EGF]
- C12N 2501/115: Basic fibroblast growth factor (bFGF, FGF-2)
- C12N 2501/33: Insulin
- C12N 2501/392: Sexual steroids
- C12N 2533/40: Polyhydroxyacids, e.g. polymers of glycolic or lactic acid (PGA, PLA, PLGA); Bioresorbable polymers
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

A method of generating a nephrospheroid is disclosed. The method comprises culturing human adult kidney cells in a culture medium under non-adherent conditions. Uses thereof and other renal cell populations are also disclosed.

## Claims

### Claim 1

A method for forming tubular structures in the kidney of a subject in need thereof, comprising:
expanding human kidney epithelial cells from a human adult kidney in a first culture medium under adherent conditions, wherein the first culture medium is a serum containing medium comprising at least one growth factor comprising IMDM supplemented with 10% FBS, 1% L-glutamine, 1% Pen-Strep, and growth factors: 50 ng/ml of bFGF, 50 ng/ml of EGF, and 5 ng/ml of SCF;
culturing the cells in a second culture medium under non-adherent conditions, thereby generating a plurality of nephrospheroid cells, wherein the second culture medium is a serum free medium comprising 500 ml DMEM:F12 in a 1:1 ratio, 1% pen-strep, 2 ml B27 supplement, 4 μg/ml heparin, 1% non-essential amino acids, 1% sodium pyruvate, 1% L-glutamine, 1 ml lipid mix, 5 ml of 100× N2 supplement, 10 ng/ml FGF, 20 ng/ml EGF, and 5 ml growth factor mix; wherein a 200 ml solution of growth factor mix comprises 100 ml DMEM:F12, 4 ml of 30% glucose, 200 mg transferin, 50 mg insulin in 20 ml of water, 19.33 mg putrescine in 20 ml ddw, 200 μl of 0.3 mM sodium selenite, and 20 μl of 2 mM progesterone;
isolating a population of nephrospheroid cells from the second culture medium; and
administering to the subject a therapeutically effective amount of the isolated nephrospheroid cells by injection to a damaged kidney of the subject.

### Claim 2 (depends on 1)

The method according to claim 1 further comprising removing kidney cells from a subject suffering from renal damage, prior to expanding the cells.

### Claim 3 (depends on 1)

The method according to claim 1 further comprising testing a nephrospheroid cell for the presence of a renal progenitor cell marker.

### Claim 4 (depends on 3)

The method according to claim 3 wherein the nephrospheroid cells express NCAM.

### Claim 5 (depends on 4)

The method according to claim 4 wherein the nephrospheroid cells have increased levels of sall1, pax2, six2 and WT1.

### Claim 6 (depends on 1)

The method according to claim 1 wherein the isolated nephrospheroid cells are administered with a pharmaceutically acceptable carrier.

### Claim 7 (depends on 1)

The method according to claim 1 wherein the subject suffers from renal failure.

### Claim 8 (depends on 1)

The method according to claim 1 wherein the subject suffers from acute or chronic kidney disease, diabetic nephropathy, renal disease associated with hypertension, hypertensive acute tubular injury, interstitial nephritis, Aplasia, dysplasia, obstructive uropathy, reflux nephropathy, Juvenile nephronophtisis, autosomal recessive polycystic kidney disease, Alport, Cystinosis, Primary Hyperoxaluria, Glomerulonephritides, Focal Segmental Glomerulosclerosis, systemic lupus erythematosus, Henoch-Schönlein purpura, or Hemolytic-uremic syndrome.

### Claim 9 (depends on 1)

The method according to claim 1 wherein at least 50% of the nephrospheroid cells are NCAM+.

### Claim 10 (depends on 9)

The method according to claim 9 wherein the nephrospheroid cells are EpCAM+.

### Claim 11 (depends on 1)

The method according to claim 1 wherein the cells from the human adult kidney are expanded for two passages or fewer.

### Claim 12 (depends on 1)

The method according to claim 1 wherein the tubular structures are proximal and distal tubules.