Reprogramming methods and cell culture platforms

Patent metadata

Field	Value
Publication number	US-11268069-B2
Application number	US-201515123217-A
Country	US
Kind code	B2
Filing date	Mar 4, 2015
Priority date	Mar 4, 2014
Publication date	Mar 8, 2022
Grant date	Mar 8, 2022

Abstract

The invention provides compositions and methods for manufacturing pluripotent cells. In particular, the invention provides improved culture platforms for manufacturing pluripotent cells with ground state pluripotency. In various embodiments, the invention contemplates, in part, a composition comprising: (a) a Wnt pathway agonist; (b) a MEK inhibitor; and (c) a ROCK inhibitor. In certain embodiments, the composition further comprises bFGF or LIF.

First claim

The invention claimed is:

1. A method of culturing human or mouse induced pluripotent stem cells (iPSCs) using a culture medium, the method comprising:
reprogramming human or mouse non-pluripotent cells to human or mouse iPSCs, wherein the reprogramming comprises (i) introducing into the human or mouse non-pluripotent cells one or more reprogramming factors comprising Oct4, and (ii) culturing the human or mouse non-pluripotent cells in a reprogramming medium comprising a Wnt pathway agonist, a MEK inhibitor, a ROCK inhibitor, and a TGFβR inhibitor; and
passaging said human or mouse iPSCs in the culture medium, thereby maintaining pluripotency of the cultured cells, wherein (i) the culture medium comprises a Wnt pathway agonist, a MEK inhibitor, and a ROCK inhibitor; and (ii) the culture medium does not comprise a TGFβR inhibitor.

2. The method of claim 1 , wherein the human or mouse iPSCs comprise a homogenous population of human or mouse iPSCs.

3. The method of claim 2 , wherein (i) at least 95% of the population of the human or mouse iPSCs expresses SSEA4-FITC and TRA1-81 or TRA1-60; or (ii) at most 5% of the population of the human or mouse iPSCs expresses α-smooth muscle actin (SMA), TUJ1, or FoxA2.

4. The method of claim 1 , further comprising dissociating the human or mouse iPSCs into single dissociated human or mouse iPSCs before or during passaging.

5. The method of claim 1 , wherein the cultured human or mouse iPSCs in said culture medium exhibit (i) reduced spontaneous differentiation; (ii) a greater proportion of cells having a ground state of pluripotency; (iii) increased genomic stability; and/or (iv) increased viability during passaging, as compared to counterpart human or mouse iPSCs cultured in a medium without the mixture of a Wnt pathway agonist, a MEK inhibitor, and a ROCK inhibitor.

6. The method of claim 5 , wherein the cultured human or mouse iPSCs comprise decreased expression of one, two, three, four, five or more differentiation marker genes by at least about 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% compared to the expression of the differentiation marker genes in a human or mouse pluripotent cell cultured in a medium comprising a TGFβR inhibitor; wherein the one, two, three, four, five or more differentiation marker genes are (i) selected from the group consisting of: FOXA2, FGF5, SOX17, XIST, NODAL, COL3A1, OTX2, DUSP6, EOMES, NR2F2, NROB1, CXCR4, CYP2B6, GATA3, GATA4, ERBB4, GATA6, HOXC6, INHA, SMAD6, RORA, NIPBL, TNFSF11, CDH11, ZIC4, GAL, SOX3, PITX2, APOA2, CXCL5, CER1, FOXQ1, MLL5, DPP10, GSC, PCDH10, CTCFL, PCDH20, TSHZ1, MEGF10, MYC, DKK1, BMP2, LEFTY2, HES1, CDX2, GNAS, EGR1, COL3A1, TCF4, HEPH, KDR, TOX, FOXA1, LCK, PCDH7, CD1D FOXG1, LEFTY1, TUJ1, T gene (Brachyury) and ZIC1; or (ii) selected from the group consisting of: T gene, CXCR4, NODAL, GATA4, SOX17, FOXA2, OTX2, and TUJ1.

7. The method of claim 5 , wherein the ground state of pluripotency and/or genomic stability of the human or mouse iPSCs is maintained for (i) at least 50 passages, or (ii) at least 100 passages.

8. The method of claim 1 , further comprising, prior to the step of passaging the human or mouse iPSCs in said culture medium, isolating the human or mouse iPSCs that are cultured in the presence of feeder cells and/or a TGFβR inhibitor, wherein said culture medium is feeder-free.

9. The method of claim 4 , further comprising dissociating the human or mouse iPSCs, and passaging the human or mouse iPSCs as single cells.

10. The method of claim 1 , wherein the reprogramming comprises introducing into the human or mouse non-pluripotent cells at least one of the following:
(1) one or more vectors comprising at least one OCT4 encoding polynucleotide, at least one ECAT1 encoding polynucleotide, and at least one UTF1 encoding polynucleotide;
(2) one or more vectors comprising at least one OCT4 encoding polynucleotide, at least one ECAT1 encoding polynucleotide, at least one UTF1 encoding polynucleotide, and at least one NANOG encoding polynucleotide;
(3) one or more vectors comprising at least two OCT4 encoding polynucleotides, and at least one NANOG encoding polynucleotide; and/or
(4) one or more vectors comprising at least one OCT4 encoding polynucleotide, at least one DPPA2 encoding polynucleotide, and at least one ESRRB encoding polynucleotide.

11. The method of claim 10 , wherein the one or more vectors comprises a retrovirus vector, a lentivirus vector, a Sendai virus vector, an adenovirus vector, an episomal vector, mini-circle, a vector system with expression cassette, or mRNA.

12. The method of claim 1 , wherein the human or mouse iPSCs are single cell dissociated cells.

13. The method of claim 5 , wherein the cultured human or mouse iPSCs exhibit reduced spontaneous differentiation.

Assignees

• Fate Therapeutics Inc

Inventors

• Valamehr Bahram
• Flynn Peter
• Abujarour Ramzey
• Robinson Megan

Classifications

• C12N2501/603

  Oct-3/4 · CPC title

• C07K14/4702

  Regulators; Modulating activity · 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

• C12N15/63 Primary

  Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression · CPC title

• C12N2501/724

  Glycosyltransferases (EC 2.4.) · CPC title