# US 10,370,639 B2 · Method for preparing pluripotent stem cells

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- Number: US 10,370,639 B2
- Document ID: US10370639B2
- Status: Granted
- Kind: B2
- Application number: 15/651,691
- Filed: 2017-07-17
- Published: 2018-01-18
- Granted: 2019-08-06
- Earliest claimed: 2013-09-04
- Assignees: Otsuka Pharmaceutical Factory, Inc.
- Applicants: Otsuka Pharmaceutical Factory, Inc.
- Inventors: Risa Okairi; Masuhiro Nishimura; Tamaki Wada
- Web page: https://patentgrep.com/patent/US10370639B2

## CPC

- C12N 5/0607: {Non-embryonic pluripotent stem cells, e.g. MASC (induced pluripotent stem cells C12N5/0696)}
- A61K 35/28: Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- C12N 5/0663: {Bone marrow mesenchymal stem cells (BM-MSC)}
- C12N 2506/1353: from bone marrow mesenchymal stem cells (BM-MSC)
- C12N 2506/1384: from adipose-derived stem cells [ADSC], from adipose stromal stem cells
- 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)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS

## Abstract

An object of the present invention is to provide a method capable of inexpensively and conveniently preparing cells having pluripotency and a very low risk of tumorigenic transformation. The cells can be prepared by suspension-culturing mammalian mesenchymal stem cells such as human mesenchymal stem cells from bone marrow (hMSC-BM) and human adipose tissue-derived mesenchymal stem cells (hAT-MSC) (also referred to as “human adipose-derived stem cells [hADSC]”), 7 types of human adherent mature cells (human hepatocyte cells [hHEP cells], human umbilical vein endothelial cells [HUVEC cells], human dermal lymphatic microvascular endothelial cells [HMVEC cells], human epidermal keratinocyte cells [NHEK cells], human bronchial epithelial cells [NHBE cells], human melanocyte cells [NHEM cells], and human smooth muscle cells [UASMC cells]), and 3 types of human adherent precursor cells (human dermal fibroblast cells [NHDF cells], human skeletal muscle myoblast cells [HSMM cells], and human osteoblast cells [NHOst cells]) to form cell masses (spheroids).

## Claims

### Claim 1

A method for preparing a pluripotent stem cell, comprising
spheroid-culturing mammalian mesenchymal stem cells to form a spheroid of pluripotent stem cells; and
confirming a pluripotency of the spheroid of the pluripotent stem cells using a detected expression of a pluripotency marker Oct3/4, Sox2, SSEA3, or TRA-1-60 as an index,
wherein the spheroid-culturing is performed in a physiological aqueous solution free from serum or a serum substitute.

### Claim 2 (depends on 1)

The method according to claim 1, wherein the mammalian mesenchymal stem cells are human mesenchymal stem cells from bone marrow or human adipose tissue-derived mesenchymal stem cells.

### Claim 3 (depends on 1)

The method according to claim 1, wherein the pluripotent stem cell expresses Nanog, Oct3/4, or Sox2.

### Claim 4 (depends on 2)

The method according to claim 2, wherein the pluripotent stem cell expresses Nanog, Oct3/4, or Sox2.