Improved reprogramming methods and cell culture platforms

Abstract

The invention provides methods for manufacturing pluripotent cells. In particular, the invention provides improved methods for manufacturing pluripotent cells with ground state pluripotency. In various embodiments, the invention contemplates, in part, use of 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.

Description

Abstract This invention relates to methods for producing pluripotent cells. In particular, the invention provides an improved method for producing pluripotent cells having ground-state pluripotency. In various embodiments, the invention partially contemplates the use of compositions comprising: (a) a Wnt pathway agonist; (b) a MEK inhibitor; and (c) a ROCK inhibitor. In some embodiments, the compositions further comprise bFGF or LIF.

Claims

1. A method of culturing a population of human induced pluripotent stem cells (hiPSCs) for at least 10 passages, the method comprising: (a) dissociating the population of hiPSCs to produce a population of dissociated hiPSCs, wherein the dissociating comprises dissociation to single cells; (b) culturing the population of dissociated hiPSCs in a feeder-free culture medium to produce an expanded population of hiPSCs, wherein (i) the culturing comprises specifically inhibiting each of GSK3, MEK, and ROCK, and (ii) the culturing does not comprise specifically inhibiting TGFβR; and (c) repeating the dissociating and culturing steps at least 9 times to produce a population of passaged hiPSCs, wherein the passaged hiPSCs maintain ground state pluripotency.

2. The method of claim 1, wherein the step of dissociating comprises sorting the dissociated cells to enrich for hiPSCs with one or more markers of pluripotency.

3. The method of claim 2, wherein the one or more markers of pluripotency comprise SSEA and TRA1-81.

4. The method of any one of claims 1-3, wherein at least 70%, at least 80%, or at least 90% of cells in the population of passaged hiPSCs are positive for both SSEA and TRA1-81.

5. The method of any one of claims 1-4, wherein the dissociating comprises enzymatic dissociation.

6. The method of any one of claims 1-5, wherein the passaged hiPSCs maintain one or more of genomic stability, or a normal karyotype.

7. The method of any one of claims 1-6, wherein the at least 10 passages comprises at least 50 passages or at least 100 passages.

8. The method of any one of claims 1-7, wherein the population of hiPSCs comprises one or more exogenous polynucleotides encoding one or more of OCT4, SOX2, KLF4, NANOG, ECAT1, UTF1, ESRRB, and SV40LT.

9. The method of any one of claims 1-8, wherein specifically inhibiting each of GSK3, MEK, and ROCK comprises culturing the dissociated hiPSCs with: (a) thiazovivin or Y27632, (b) PD98059 or PD0325901, and (c) CHIR99021 or BIO.

10. The method of any one of claims 1-5, further comprising reprogramming non-pluripotent human cells to produce the population of hiPSCs.

11. The method of claim 10, wherein the reprogramming comprises introducing into the non-pluripotent human cells one or more copies of at least one reprogramming factor encoded by one or more exogenous polynucleotides, wherein the at least one reprogramming factor is selected from the group consisting of: OCT4, SOX2, KLF4, NANOG, ECAT1, UTF1, ESRRB, and SV40LT.

12. The method of claim 11, wherein the reprogramming further comprises culturing the non-pluripotent human cells in a reprogramming medium, wherein culturing in the reprogramming medium comprises specifically inhibiting each of GSK3, MEK, ROCK, and TGFβR.

13. The method of claim 11, wherein the one or more exogenous polynucleotides comprise a vector comprising: (a) at least two copies of OCT4 polynucleotides, one copy of ECAT1 polynucleotide, one copy of UTF1 polynucleotide, one copy of NANOG polynucleotide, and one copy of ESRRB polynucleotide; or (b) at least one copy of OCT4 polynucleotide, one copy of ECAT1 polynucleotide, and one copy of UTF1 polynucleotide.

14. The method of claim 11, wherein the one or more exogenous polynucleotides: (a) are introduced to cells by a retrovirus, Sendai virus, an adenovirus, an episome, mini-circle, vector system with expression cassette, or mRNA; (b) comprise polycistronic polynucleotides that are separated by at least one 2A peptide; or (c) comprise polycistronic polynucleotides each encodes an OCT4 polypeptide.

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