Methods and compositions for generating or maintaining pluripotent cells

Patent metadata

Field	Value
Publication number	US-10428310-B2
Application number	US-201514884293-A
Country	US
Kind code	B2
Filing date	Oct 15, 2015
Priority date	Oct 15, 2014
Publication date	Oct 1, 2019
Grant date	Oct 1, 2019

Abstract

Methods and compositions are provided for generating or maintaining human iPS cells in culture. Methods include the use of a low osmolality medium to make human iPS cells, or use of a low osmolality medium to maintain human iPS cells. Methods for making targeted genetic modification to human iPS cells cultured in low osmolality medium are also included. Compositions include human iPS cells cultured and maintained using the low osmolality medium defined herein.

First claim

That which is claimed:

1. An in vitro culture comprising:
(a) a population of nave human induced pluripotent stem cells (hiPSCs) that display a morphology characterized by compact, dome-shaped colonies; and
(b) a low osmolality medium comprising a base medium and supplements, wherein the low osmolality medium comprises:
(i) a leukemia inhibitory factor (LIF) polypeptide;
(ii) a glycogen synthase kinase 3 (GSK3) inhibitor; and
(iii) a MEK inhibitor;
wherein the low osmolality medium has an osmolality of about 200 mOsm/kg to about 250 mOsm/kg.

2. The in vitro culture of claim 1 , wherein the hiPSCs:
(a) express one or more pluripotency markers;
(b) can differentiate into cells of any one of the endoderm, ectoderm, or mesoderm germ layers;
(c) have a doubling time of between about 16 hours and about 24 hours; or
(d) any combination of (a) to (c).

3. The in vitro culture of claim 1 , wherein the hiPSCs have a normal karyotype.

4. The in vitro culture of claim 2 , wherein the hiPSCs express the one or more pluripotency markers, and wherein the pluripotency markers comprise NANOG, alkaline phosphatase, or a combination thereof.

5. The in vitro culture of claim 1 , wherein the hiPSCs are derived from non-pluripotent cells transformed to express a pluripotent state.

6. The in vitro culture of claim 5 , wherein the transformed cells express reprogramming genes comprising Oct4, Sox2, Klf4, Myc, or any combination thereof.

7. The in vitro culture of claim 5 , wherein the transformed cells are first cultured in a high osmolality medium prior to culturing in the low osmolality medium, wherein the high osmolality medium comprises bFGF.

8. The in vitro culture of claim 7 , wherein the high osmolality medium has an osmolality of at least 290 mOsm/kg.

9. The in vitro culture of claim 7 , wherein:
(a) the transformed cells are first cultured in the high osmolality medium until they express characteristics of a nave state;
(b) the transformed cells are first cultured in the high osmolality medium for a period of about two months;
(c) the transformed cells are first cultured in the high osmolality medium until they display a morphology characterized by three-dimensional cell clumps; or
(d) a combination thereof.

10. The in vitro culture of claim 1 , wherein the base medium comprises NaCl at about 3 mg/mL, sodium bicarbonate at about 2.2 mg/mL, and has an osmolality of about 200 mOsm/kg.

11. The in vitro culture of claim 1 , wherein the base medium comprises glucose at about 4.5 mg/mL.

12. The in vitro culture of claim 1 , wherein the base medium has an osmolality of about 180 mOsm/kg to about 250 mOsm/kg.

13. The in vitro culture of claim 12 , wherein the low osmolality medium has an osmolality of about 233 mOsm/kg.

14. The in vitro culture of claim 1 , wherein:
(a) the supplements comprise:
(i) F-12 medium;
(ii) N2 supplement;
(iii) B-27 supplement;
(iv) L-glutamine;
(v) 2-mercaptoethanol; or
(vi) any combination of (i) to (v);

(b) the LIF polypeptide is a human LIF (hLIF) polypeptide;
(c) the GSK3 inhibitor is CHIR99021;
(d) the MEK inhibitor is PD0325901; or
(e) any combination of (a) to (d).

15. The in vitro culture of claim 1 , wherein the low osmolality medium comprises inhibitors consisting essentially of the glycogen synthase kinase 3 (GSK3) inhibitor and the MEK inhibitor.

16. The in vitro culture of claim 1 , wherein the low osmolality medium comprises the base medium at about 24.75% (v/v), F-12 medium at about 24.75% (v/v), N2 supplement at about 0.5% (v/v), B-27 supplement at about 1% (v/v), L-glutamine at about 2 mM, 2-mercaptoethanol at about 0.1 mM, hLIF at about 100 units/mL, CHIR99021 at about 3 μM, and PD0325901 at about 0.5 μM.

17. The in vitro culture of claim 1 , wherein the low osmolality medium does not comprise one or more of the following: bFGF supplement; TGF-β1 supplement; JNK inhibitor; p38 inhibitor; ROCK inhibitor; and PKC inhibitor.

18. The in vitro culture of claim 1 , wherein the hiPSCs are cultured on newborn human foreskin fibroblast (NuFF) feeder cells.

19. The in vitro culture of claim 1 , wherein the hiPSCs are in a single-cell suspension.

20. The in vitro culture of claim 19 , wherein the hiPSCs in the single-cell suspension:
(a) maintain expression of one or more pluripotency markers;
(b) maintain a naïve state; or
(c) a combination thereof.

21. The in vitro culture of claim 19 , wherein the hiPSCs in the single-cell suspension maintain a normal karyotype.

22. The in vitro culture of claim 12 , wherein the low osmolality medium further comprises F-12 medium.

23. The in vitro culture of claim 17 , wherein the low osmolality medium does not comprise bFGF supplement.

Assignees

• Regeneron Pharma

Inventors

• Kuno Junko
• Auerbach Wojtek
• Valenzuela David M

Classifications

• C12N5/0696 Primary

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

• C12N2501/235

  Leukemia inhibitory factor [LIF] · CPC title

• C12N2500/44

  Thiols, e.g. mercaptoethanol · CPC title

• C12N2500/60

  Buffer, e.g. pH regulation, osmotic pressure · CPC title

• C12N2509/00

  Methods for the dissociation of cells, e.g. specific use of enzymes · CPC title