US 12,365,875 B2
Methods for generating skeletal muscle progenitor cells
April D. Pyle, Los Angeles, CA (US); and Michael R. Hicks, Los Angeles, CA (US)
Assigned to The Regents of the University of California, Oakland, CA (US)
Appl. No. 16/474,895
Filed by The Regents of the University of California, Oakland, CA (US)
PCT Filed Dec. 15, 2017, PCT No. PCT/US2017/066794
§ 371(c)(1), (2) Date Jun. 28, 2019,
PCT Pub. No. WO2018/128779, PCT Pub. Date Jul. 12, 2018.
Claims priority of provisional application 62/443,499, filed on Jan. 6, 2017.
Prior Publication US 2021/0139854 A1, May 13, 2021
Int. Cl. C12N 5/077 (2010.01)
CPC C12N 5/0658 (2013.01) [C12N 2501/105 (2013.01); C12N 2501/33 (2013.01)] 17 Claims
 
1. A method for generating skeletal muscle progenitor cells (SMPCs), the method comprising:
a) culturing pluripotent stem cells in a chemically defined liquid culture medium for a period of time to generate a population of SMPCs that express paired box protein-7 (PAX7), wherein said culturing comprises:
i) culturing the pluripotent stem cells in a chemically defined liquid culture medium comprising a Rho-associated kinase inhibitor from day 1 to day 2 only;
ii) (1) culturing the cells from step i) in a chemically defined liquid culture medium comprising a glycogen synthase kinase-3 (GSK3) inhibitor from day 2 to day 3 only;
ii) (2) culturing the cells from step ii) (1) in a chemically defined liquid culture medium from day 3 to day 12 only;
iii) culturing the cells from step ii) (2) in a chemically defined liquid culture medium comprising fibroblast growth factor from day 12 to day 20 only;
iv) culturing the cells from step iii) in a chemically defined liquid culture medium from day 20 to day 35 only; and
v) culturing the cells from step iv) in a chemically defined liquid culture medium comprising insulin-like growth factor-1 from day 35 to day 50 only,
thereby generating a population of cells comprising at least 50% PAX7* SMPCs, which also express nerve growth factor receptor (NGFR), Erb-B2 receptor tyrosine kinase 3 (ERBB3) or both; and
b) enriching for SMPCs that are nerve growth factor receptor (NGFR)-positive and Erb-B2 receptor tyrosine kinase 3 (ERBB3)-positive.