US 12,365,872 B2
Methods for differentiating pluripotent stem cells in dynamic suspension culture
Rekha R. Nair, Mountain View, CA (US); Stephanie Kayser, Mountain View, CA (US); Abhirath S. Parikh, Los Angeles, CA (US); Uzma Shoukat-Mumtaz, Fremont, CA (US); Erik Michael Whiteley, Concord, CA (US); Nathan C. Manley, San Jose, CA (US); and Craig R. Halberstadt, Pleasanton, CA (US)
Assigned to LINEAGE CELL THERAPEUTICS, INC., Carlsbad, CA (US)
Appl. No. 17/277,548
Filed by Lineage Cell Therapeutics, Inc., Carlsbad, CA (US)
PCT Filed Sep. 19, 2019, PCT No. PCT/US2019/052015
§ 371(c)(1), (2) Date Mar. 18, 2021,
PCT Pub. No. WO2020/061371, PCT Pub. Date Feb. 6, 2020.
Claims priority of provisional application 62/733,621, filed on Sep. 19, 2018.
Prior Publication US 2022/0267723 A1, Aug. 25, 2022
Int. Cl. C12N 5/00 (2006.01); C12N 5/079 (2010.01)
CPC C12N 5/0622 (2013.01) [C12N 5/0068 (2013.01); C12N 2500/46 (2013.01); C12N 2501/11 (2013.01); C12N 2501/115 (2013.01); C12N 2501/135 (2013.01); C12N 2501/15 (2013.01); C12N 2501/155 (2013.01); C12N 2501/16 (2013.01); C12N 2501/385 (2013.01); C12N 2501/41 (2013.01); C12N 2506/02 (2013.01); C12N 2506/03 (2013.01); C12N 2506/45 (2013.01); C12N 2533/52 (2013.01)] 15 Claims
OG exemplary drawing
 
1. A method for obtaining a population of cells comprising glial progenitor cells from undifferentiated human pluripotent stem cells, the method comprising:
a) obtaining a suspension culture of non-embryoid body (non-EB) aggregates of undifferentiated human pluripotent stem cells, wherein the human pluripotent stem cells remain in an undifferentiated state;
b) culturing the non-EB aggregates from a) in dynamic suspension in the presence of at least one inhibitor of transforming growth factor beta (TGFβ)/Activin/Nodal signaling selected from an inhibitor of activin receptor-like kinase 5 (ALK5), SB431542, LY2157299, GW788388, A-77-01, A-83-01 or SB505124 and at least one inhibitor of bone morphogenetic protein (BMP) signaling selected from an activin receptor-like kinase 2 (ALK2), Dorsomorphin, DMH-1, K02288, ML3467, LDN193189 or Noggin protein for a first time period, thereby inducing differentiation to neuroectoderm lineage cells, wherein the first time period is three to four days;
c) culturing the neuroectoderm lineage cells from b) in dynamic suspension in the presence of retinoic acid and at least one agonist of Smoothened receptor for a second time period, wherein the second time period is three to four days; and
d) culturing the neuroectoderm lineage cells from c) in dynamic suspension in the presence of basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) for a further time period, until the cells have matured into glial progenitor cells, wherein steps a) through d) are performed over a period of about 21 days, wherein about refers to a variation of ±10%.