Induced extended pluripotent stem cells, method of making and using

Patent metadata

Field	Value
Publication number	US-11028369-B2
Application number	US-201615746398-A
Country	US
Kind code	B2
Filing date	Aug 12, 2016
Priority date	Aug 13, 2015
Publication date	Jun 8, 2021
Grant date	Jun 8, 2021

Abstract

Factors for extending the ability of isolated pluripotent stem cells to generate extraembryonic lineages in vivo, following in vitro culture, herein, chemical extenders of pluripotency (CEP). Methods of extending the ability of a pluripotent cell to generate embryonic and extraembryonic lineages. The cell to be reprogrammed is contacted with effective amounts of the CEPs for a sufficient period of time to reprogram the cell into a chemically induced extended pluripotent cell (ciEPSC). ciEPSC are identified as an extended pluripotent cell based on properties including: (i) morphologically and (ii) functionally for example, based on their ability contribute to both TE and ICM, in vivo. The ciEPSCs can be cultured or induced to differentiate into cells of a desired type, and used in a number of applications, including but not limited to cell therapy and tissue engineering.

First claim

We claim:

1. A cell culture medium for extending cell potency of an isolated pluripotent stem cells, the cell culture medium comprising
(i) 1-100 ng/ml of human leukemia inhibitory factor (LIF),
(ii) 0.5-5.0 μM of 6-[[2-[[4-(2,4-Dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidin-yl]amino]ethyl]amino]-3-pyridinecarbonitrile,
(iii) 1.0-5.0 μM of (S)-(+)-Dimethindene maleate (DiM), and
(iv) 0.5-5.0 μM of Minocycline hydrochloride (MiH), wherein said culture medium maintains the normal karyotype and extend the pluripotent state of the pluripotent stem cells for at least 5 passages.

2. The cell culture medium of claim 1 , further comprising 0.2 to 20 μM of a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor selected from the group consisting of [(+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide+++dihydrochloride)] (Y27632) and fusadil.

3. The cell culture medium of claim 1 , further comprising 4-[(3aR,4S,7R,7aS)-1,3,3a,4,7,7a-hexahydro-1,3-dioxo-4,7-methano-2H-isoindol-2-yl]-N-8-quinolinyl-benzamide) (endo-IWR1) or 3,5,7,8-Tetrahydro-2-[4-(trifluoromethyl)phenyl]-4H-thiopyrano [4,3-d]pyrimidin-4-one (XAV939).

4. A method for culturing pluripotent stem cells, the method comprising:
(a) seeding pluripotent stem cells as single cell in an ES cell culture medium containing 1.0 to 10 μM of [(+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide+++dihydrochloride)] for 12 to 24 hours; and
(b) culturing the pluripotent stem cells from step (a) in a cell culture medium comprising (i) 1-100 ng/ml of human leukemia inhibitory factor (LIF), (ii) 0.5-5.0 μM of 6-[[2-[[4-(2,4-Dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidin-yl]amino]ethyl]amino]-3-pyridinecarbonitrile, (iii) 1.0-5.0 μM of (S)-(+)-Dimethindene maleate (DiM), and (iv) 0.5-5.0 μM of Minocycline hydrochloride (MiH) to maintain pluripotency in said pluripotent stem cells, wherein said pluripotent stem cells maintain normal karyotype in the culture for at least 5 passages.

5. A method for culturing pluripotent stem cells, the method comprising:
culturing pluripotent stem cells in a cell culture medium comprising (i) 1-100 ng/ml of human leukemia inhibitory factor (LIF), (ii) 0.5-5.0 μM of 6-[[2-[[4-(2,4-Dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidin-yl]amino]ethyl]amino]-3-pyridinecarbonitrile, (iii) 1.0-5.0 μM of (S)-(+)-Dimethindene maleate (DiM), and (iv) 0.5-5.0 μM of Minocycline hydrochloride (MiH) to maintain pluripotency in said pluripotent stem cells, wherein said pluripotent stem cells maintain normal karyotype in the culture for at least 5 passages.

Assignees

• Beihao Stem Cell And Regenerative Medicine Res Institute Co Ltd
• Univ Beijing
• Hong Guan Ltd

Inventors

• Deng Hongkui
• Yang Yang
• Liu Bei
• Xu Jun

Classifications

• C07K16/38

  against protease inhibitors of peptide structure · CPC title

• C12N2501/603

  Oct-3/4 · CPC title

• C12N5/0606

  Pluripotent embryonic cells, e.g. embryonic stem cells [ES] (embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696) · CPC title

• A01K2217/00

  Genetically modified animals · CPC title

• C12N2503/02

  Drug screening · CPC title