# US 10,370,638 B2 · Primitive gut endoderm cells and method for producing same

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- Number: US 10,370,638 B2
- Document ID: US10370638B2
- Status: Granted
- Kind: B2
- Application number: 15/534,205
- Filed: 2015-12-08
- Published: 2017-11-23
- Granted: 2019-08-06
- Earliest claimed: 2014-12-09
- Assignees: Public University Corporation Yokohama City University
- Applicants: Public University Corporation Yokohama City University
- Inventors: Takanori Takebe; Hideki Taniguchi; Ran ran Zhang
- Web page: https://patentgrep.com/patent/US10370638B2

## CPC

- A61K 35/38: Stomach; Intestine; Goblet cells; Oral mucosa; Saliva
- C12N 5/0607: {Non-embryonic pluripotent stem cells, e.g. MASC (induced pluripotent stem cells C12N5/0696)}
- A01N 1/10: Preservation of living parts
- A61K 35/407: Liver; Hepatocytes
- C12N 5/10: Cells modified by introduction of foreign genetic material
- C12N 5/068: {Stem cells; Progenitors}
- C12N 5/0697: {Artificial constructs associating cells of different lineages, e.g. tissue equivalents (blood vessels C12N5/0691)}
- C12N 2501/11: Epidermal growth factor [EGF]
- C12N 2501/115: Basic fibroblast growth factor (bFGF, FGF-2)
- C12N 2501/12: Hepatocyte growth factor [HGF]
- C12N 2501/155: Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
- C12N 2501/16: Activin; Inhibin; Mullerian inhibiting substance
- C12N 2501/165: Vascular endothelial growth factor [VEGF]
- C12N 2501/415: Wnt; Frizzeled
- C12N 2501/727: Kinases (EC 2.7.)
- C12N 2506/45: from artificially induced pluripotent stem cells
- C12N 2533/52: Fibronectin; Laminin
- C12N 2533/90: Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A01N: PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF (preservation of food or foodstuff A23); BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES (preparations for medical, dental or toiletry purposes which kill or prevent the growth or proliferation of unwanted organisms A61K); PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS

## Abstract

The present invention provides a technique that serves as a platform for inducing human organ cells at a low cost, stably and in a large quantity. A cell inducible after differentiating pluripotent stem cells and then passaging the resultant cells at least once or more times, which is negative for undifferentiated (pluripotent) cell markers NANOG, OCT4, MYC and LIN28A, negative for endoderm cell markers CXCR4, CER1, HHEX and GATA4, positive for intestinal endoderm cell markers CDX2 and HOXB9, negative for a mesenchymal cell marker brachyury (T), negative for a pancreatic cell marker PDX1, and capable of differentiating into at least a hepatocyte, a pancreatic cell and an intestinal cell. Also provided are methods of preparing and amplifying the above cells; a method of preparing organ cells using the above cells; and a method of constructing a working cell bank for preparing organ cells, comprising cryopreserving the above cells.

## Claims

### Claim 1

Cells prepared by differentiating human pluripotent stem cells and then passaging the resultant cells at least once or more times, which are negative for undifferentiated cell markers NANOG, OCT4, MYC and LIN28A, negative for endoderm cell markers CXCR4, CER1, HHEX and GATA4, positive for intestinal endoderm cell markers CDX2 and HOXB9, negative for a mesenchymal cell marker brachyury (T), negative for a pancreatic cell marker PDX1, and capable of differentiating into at least hepatocytes, pancreatic cells and intestinal cells.

### Claim 2 (depends on 1)

A method of preparing the resultant cells of claim 1, comprising culturing pluripotent stem cells without feeder cells in the presence of ROCK inhibitor, wherein said ROCK inhibitor is Y27632, at the first stage, in the presence of Activin A and Wnt3a at the second stage, in the presence of BMP4, bFGF, VEGF and Activin A at the third stage and in the presence of BMP4, bFGF, VEGF and Activin A at the fourth stage, to thereby effect differentiation and then passaging the resultant cells at least once or more times.

### Claim 3 (depends on 1)

A method of amplifying the resultant cells of claim 1, comprising culturing the cells in the presence of ROCK inhibitor at the first stage after passage or on the first day of passage and thereafter in the presence of SFD, FGF2, VEGF, EGF, A83-01 and Chir99021.

### Claim 4 (depends on 1)

A method of differentiating the resultant cells of claim 1, comprising:
directing differentiation and induction of the resultant cells into at least one of hepatocyte, pancreatic, or intestinal cells.

### Claim 5 (depends on 1)

A method of constructing a working cell bank for preparing at least one of hepatocytes, pancreatic cells, and intestinal cells comprising:
cryopreserving the resultant cells of claim 1 at 1 to 20 passages.