# US 12,180,509 B2 · Methods for reprograming non-pluripotent cells into pluripotent stem cells

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- Number: US 12,180,509 B2
- Document ID: US12180509B2
- Status: Granted
- Kind: B2
- Application number: 17/383,019
- Filed: 2021-07-22
- Published: 2022-01-27
- Granted: 2024-12-31
- Earliest claimed: 2021-07-22
- Assignees: BeiHao Stem Cell and Regenerative Medicine Research Institute Co., Ltd.; Hong Guan Ltd.
- Applicants: Beihao Stem Cell and Regenerative Medicine Research Institute Co., Ltd.; Hong Guan Ltd.
- Inventors: Hongkui Deng; Yang Zhao; Ting Zhao; Jingyang Guan; Xu Zhang; Yao Fu; Junqing Ye
- Web page: https://patentgrep.com/patent/US12180509B2

## CPC

- C12N 5/0696: {Artificially induced pluripotent stem cells, e.g. iPS}
- A61K 9/0019: {Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner (non-active ingredients are additionally classified in A61K47/00)}
- A61K 35/545: Embryonic stem cells; Pluripotent stem cells; Induced pluripotent stem cells; Uncharacterised stem cells
- C12N 5/0018: {Culture media for cell or tissue culture (media for specific animal cell type C12N5/06)}
- C12N 5/0606: {Pluripotent embryonic cells, e.g. embryonic stem cells [ES] (embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696)}
- C12N 5/0656: {Adult fibroblasts}
- C12N 5/0667: {Adipose-derived stem cells [ADSC]; Adipose stromal stem cells}
- C12N 2501/01: Modulators of cAMP or cGMP, e.g. non-hydrolysable analogs, phosphodiesterase inhibitors, cholera toxin
- C12N 2501/06: Anti-neoplasic drugs, anti-retroviral drugs, e.g. azacytidine, cyclophosphamide
- C12N 2501/115: Basic fibroblast growth factor (bFGF, FGF-2)
- C12N 2501/15: Transforming growth factor beta (TGF-β)
- C12N 2501/385: of the family of the retinoic acid recptor, e.g. RAR, RXR; Peroxisome proliferator-activated receptor [PPAR]
- C12N 2501/415: Wnt; Frizzeled
- C12N 2501/602: Sox-2
- C12N 2501/604: Klf-4
- C12N 2501/606: c-Myc
- C12N 2501/727: Kinases (EC 2.7.)
- C12N 2501/73: Hydrolases (EC 3.)
- C12N 2501/999: Small molecules not provided for elsewhere
- C12N 2506/1307: from adult fibroblasts
- C12N 2506/23: from cells of the gastro-intestinal tract
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)

## Abstract

Provided are chemical inducers of pluripotency (CIP) which include glycogen synthase kinase inhibitors, TGFβ receptor inhibitors, cyclic AMP agonists and S-adenosylhomocysteine hydrolase (SAH) inhibitors or histone acetylators. A method of inducing pluripotency in a partially or completely differentiated cell by using such chemical inducers of pluripotency is also provided. The method includes: (i) contacting a cell with the CIPs for a sufficient period of time to result in reprograming the cell into a pluripotent stem cell having ESC-like characteristics (CiPSC). Isolated chemically induced pluripotent stem cells (CiPSCs) and their progeny, produced by inducing differentiation of the CiPSCs, can be used in a number of applications, including but not limited to cell therapy and tissue engineering.

## Claims

### Claim 1

A method of inducing pluripotency in partially or completely differentiated cells, the method comprising:
(a) contacting the partially or completely differentiated cells with a first cocktail comprising VC6TFAE (XEN-Cocktail) for a sufficient period of time to obtain XEN-like cells;
(b) contacting the XEN-like cells for a sufficient period of time to reprogram the XEN-like cells into chemically induced pluripotent stem cells (CiPSCs) with a second cocktail comprising VC6TFZASD (XEN-CiPSC cocktail); and
(c) culturing the CiPSCs in 2i-medium,
wherein V is valproic acid,
C is [6-[[2-[[4-(2,4-Dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile] (CHIR99021),
6 is [2-(3-(6-Methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine] (616452),
T is tranylcypromine,
F is Forskolin,
A is (4-[(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl) carboxamido]benzoic acid) (AM-580),
E is 1-(3-((((2R,3S,4R,5R)-5-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl) (isopropyl)amino) propyl)-3-(4-(tert-butyl)phenyl) urea (EPZ004777),
Z is 3-deazaneplanocin A (DZNep),
S is (1-[3-[[[(2R,3S,4R,5R)-5-(4-Amino-5-bromo-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-3,4-dihydroxytetrahydrofuran-2-yl]methyl](isopropyl)amino]propyl]-3-[4-(2,2-dimethylethyl)phenyl]urea) (SGC 0946), and
D is 5-azacytidine.

### Claim 2 (depends on 1)

The method of claim 1, wherein the differentiated cells are selected from the group consisting of multipotent stem cells, cells of hematological origin, cells of embryonic origin, skin derived cells, fibroblasts, adipose cells, epithelial cells, endothelial cells, mesenchymal cells, parenchymal cells, neurological cells, and connective tissue cells.

### Claim 3 (depends on 1)

The method of claim 1, wherein the partially or completely differentiated cells are selected from the group consisting of fibroblasts, adipose-derived cells, neural derived cells and intestinal epithelial cells.

### Claim 4 (depends on 1)

The method of claim 1, wherein the partially or completely differentiated cells are not transfected to express markers selected from the group comprising Oct4, KLF4, SOX2, C-Myc and NANOG.

### Claim 5 (depends on 1)

The method of claim 1, wherein the partially or completely differentiated cells are cultured in reprograming medium comprising the XEN-CiPSC cocktail for a period between 26-30 days.

### Claim 6 (depends on 1)

The method of claim 1 wherein the XEN-Cocktail comprises VC6TFA the entire time before the use of 2i-medium.

### Claim 7 (depends on 1)

The method of claim 1 wherein the partially or completely differentiated cells in step (a) are replated at a density between 50,000 to 100,000 per well in a 6-well plate between day 6-10.

### Claim 8 (depends on 1)

The method of claim 1 wherein the chemically induced pluripotent stem cells are transferred to 2i-medium between day 26 and day 30.

### Claim 9 (depends on 8)

The method of claim 8 wherein the 2i-medium further comprises N2B27, wherein N2 is N2 supplement, and wherein B27 is B27 supplement.

### Claim 10 (depends on 8)

The method of claim 8 wherein the chemically induced pluripotent stem cells are cultured in 2i-medium for about 10-14 days.

### Claim 11 (depends on 1)

The method of claim 1 wherein the XEN-Cocktail, the XEN-CiPSC cocktail, or both the XEN-Cocktail and XEN-CiPSC cocktail further comprise small molecules that facilitate late reprograming selected from the group consisting of cAMP agonists, epigenetic modulators and small molecules that boost chemical reprograming efficiency.

### Claim 12 (depends on 1)

The method of claim 1 wherein the XEN-cocktail does not comprise SGC 0946.

### Claim 13 (depends on 1)

The method of claim 1, wherein the XEN-Cocktail, the XEN-CiPSC cocktail, or both the XEN-Cocktail and XEN-CiPSC cocktail comprise a small molecule which boosts reprograming efficiency, wherein the small molecule is [4-[(E)-2-(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoic acid] (TTNPB).

### Claim 14 (depends on 1)

The method of claim 1, comprising identifying the pluripotent cells based on possession of one or more ESC-like properties selected from the group consisting of morphology, doubling time, the ability of the cell to differentiate into tissues of the three embryonic germ layers, and expression of ESC markers.

### Claim 15 (depends on 14)

The method of claim 14, wherein the ESC markers are selected from the group consisting of alkaline phosphatase (AP); nanog; Rex1; Sox2; Dax1; Sall4; undifferentiated embryonic cell transcription factor (Utf1); stage specific embryonic antigen-4 (SSEA-4) and combinations thereof.

### Claim 16 (depends on 1)

The method of claim 1, further comprising isolating the pluripotent cells.

### Claim 17 (depends on 3)

The method of claim 3, wherein the partially or completely differentiated cells do not express Oct4.

### Claim 18 (depends on 1)

The method of claim 1 wherein the cells in step (a) are replated at a density between 50,000 to 100,000 per well in a 6-well plate at day 8.

### Claim 19 (depends on 1)

The method of claim 1 wherein the chemically induced pluripotent stem cells are transferred to 2i-medium on day 28.

### Claim 20 (depends on 8)

The method of claim 8 wherein the chemically induced pluripotent stem cells are cultured in 2i-medium for 12 days.