Resetting pluripotent stem cells

Patent metadata

Field	Value
Publication number	US-10894948-B2
Application number	US-201515505707-A
Country	US
Kind code	B2
Filing date	Aug 21, 2015
Priority date	Aug 22, 2014
Publication date	Jan 19, 2021
Grant date	Jan 19, 2021

Abstract

The invention provides methods and materials for resetting or sustaining a human stem cell in a “naïve” or “ground” state, based on the use of media including combinations of inhibitors. An example naïve culture medium comprises a PKC inhibitor, a MEK inhibitor. Also provided are methods of obtaining or propagating such cells, cells obtained using these methods, and novel culture media, which can be used in these methods.

First claim

The invention claimed is:

1. A method of resetting a human stem cell to a naive state, the method comprising:
(a) providing a human stem cell to be reset,
(b) inducing a naive state by
(i) optionally introducing one or more heterologous reprogramming factors into the cell for expression thereof,
(ii) culturing the cell in a resetting medium, wherein the resetting medium comprises a MEK inhibitor, a tankyrase inhibitor and a STAT3 activator,

(c) sustaining the cell in a naive culture medium, wherein the naive culture medium is different from the resetting medium and comprises a MEK inhibitor, a PKC inhibitor, a GSK3 inhibitor, and a STAT3 activator,
thereby resetting a human stem cell to a naive state.

2. The method according to claim 1 , wherein step (b)(i) is performed to reprogram the cell to a naive state and comprises expressing reprogramming factors in the cell, wherein the reprogramming factors comprise NANOG and KLF2.

3. The method according to claim 2 , wherein the reprogramming factors consist of NANOG and KLF2.

4. The method according to claim 1 , wherein the resetting medium further comprises the presence of a GSK3 inhibitor and/or an FGF inhibitor.

5. The method of claim 2 , comprising expressing reprogramming factors in the cell, wherein the reprogramming factors comprise NANOG and KLF2, and culturing the cell in the resetting medium, wherein the resetting medium comprises a MEK inhibitor, and optionally either (i) a GSK3 inhibitor and a STAT3 activator, or (ii) a FGF inhibitor and a STAT3 activator.

6. The method according to claim 5 , wherein expression of the reprogramming factors is transient.

7. The method according to claim 6 , comprising introducing into the cell a plasmid preparation which expresses the reprogramming factors in the cell.

8. The method according to claim 6 , wherein heterologous nucleic acid encoding the reprogramming factors is not maintained following reprogramming to the nave state.

9. The method according to claim 1 , wherein step (b) (i) is not performed and the resetting medium further comprises a HDAC inhibitor.

10. The method of claim 1 , wherein the resetting medium further comprises a HDAC inhibitor.

11. The method according to claim 1 , wherein the naive culture medium further comprises a ROCK inhibitor.

12. The method according to claim 1 , wherein
(i) the GSK3 inhibitor is CHIR99021;
(ii) the MEK inhibitor is PD0325901;
(iii) the PKC inhibitor is Gδ6983 or Ro-31-8425;
(iv) the STAT3 activator is LIF, which is optionally human LIF; or
(v) the tankyrase inhibitor is XAV939.

13. The method according to claim 1 , wherein
(i) the cells are cultured in an FGF supplemented serum replacement medium prior to inducing the naive state in step (b)(i),
(ii) the resetting medium or naive culture medium of step (b)(ii) or step (c), respectively is replaced daily, or
(iii) the resetting is performed in the presence of 5% oxygen.

14. The method according to claim 1 , wherein maintenance in a nave state is confirmed by one of more of the following phenotypes or genotypes:
a) the ability to continuously and clonally self-renew in culture and retain pluripotency;
b) a global transcriptome more similar to that of mouse embryonic stem cells cultured in defined media than to mouse post-implantation epiblast stem cells (EpiSCs) or conventional human pluripotent stem cells;
c) a global transcriptome more similar to pre-implantation epiblast than post-implantation epiblast;
d) expression of mRNA and protein of pre-implantation epiblast specific transcription factors, optionally 1, 2, 3, 4, 5, 6 or all of: KLF2, KLF4, TFCP2L1, TBX3, REX1, GBX2 and STELLA (DPPA3), and expression of mRNA and protein of general pluripotency factors, such as OCT4, SOX2 and SALL4, and optionally elevated mRNA and protein levels of NANOG;
e) reliance on critical transcription factors defined in mouse embryonic stem cells, particularly TFCP2L1 and KLF4;
f) nuclear localisation of TFE3;
g) low level expression or absence of expression of early lineage markers that are typically expressed in convention human pluripotent stem cells, such as AFP, EOMES and/or BRACHURY;
h) active mitochondrial respiration;
i) genome-wide hypomethylation;
j) lower levels of histone modifications associated with gene, such as reduced levels of H3K27me3 and H3K9me3;
k) capable of incorporation into a host embryo inner cell mass and a pre-implantation epiblast to form embryo chimaeras;
l) able to colonise a post-implantation epiblast and derivative tissues in chimaeras formed with the same, or closely related, species;
m) self-renewal in the presence of complete inhibition of Erk/MAP kinase signalling;
n) self-renewal in the presence of growth factor receptor tyrosine kinase signalling inhibition;
o) self-renewal in the presence of TGFbeta/activin signalling inhibition;
p) self-renewal in the presence of PKC inhibition or knockdown;
q) self-renewal in the presence of partial inhibition of (GSK3) glycogen synthase kinase-3 activity;
r) self-renewal in the presence of STAT3 agonists, such as LIF;
s) self-renewal from dissociated single cells with or without Rho associated kinase inhibition (ROCKi);
t) self-renewal in the absence of serum or serum substitutes;
u) self-renewal in the absence of feeder cells;
v) self-renewal in the absence of transgene expression or other genetic perturbation;
w) retention of diploid karyotype without rearrangement in long-term passaging, for example over more than 40 population doublings;
x) differentiation into conventional primed pluripotent phenotype in the presence of growth factor stimulation of Erk/MAP kinase signalling and activin;
y) able to differentiate in vitro into primordial germ cells as well as somatic germ layers;
z) able to establish continuous culture in vitro by transition from a pre-implantation epiblast;
aa) tightly packed domed appearance; and/or
bb) reduction in expression of DNMT3a and DNMT3b.

15. The method according to claim 14 , wherein expression of KLF2, KLF4, TFCP2L1, TBX3, REX1, GBX2 and STELLA is induced in the reprogrammed cells.

16. The method according to claim 5 , wherein the human stem cell to be reset in step (a) is selected from:
(i) an induced pluripotent stem cell;
(ii) a cell from an embryonic cell line; or
(iii) an embryonic stem cell obtained by biopsy without destruction of the respective embryo.

17. The method according to claim 1 , wherein the resetting medium comprises a PKC inhibitor.

18. The method of claim 4 , wherein the FGF inhibitor is PD173074.

Assignees

• Cambridge Entpr Ltd

Inventors

• Smith Austin
• Guo Ge
• Takashima Yasuhiro

Classifications

• C12N2501/415

  Wnt; Frizzeled · CPC title

• C12N2501/605

  Nanog · CPC title

• C12N2500/38

  Vitamins · CPC title

• C12N5/0696 Primary

  Artificially induced pluripotent stem cells, e.g. iPS · CPC title

• C12N2501/999

  Small molecules not provided for elsewhere · CPC title