US 12,188,047 B2
Preparation of retinal pigment epithelium cells
Benjamin Eithan Reubinoff, Doar-Na HaEla (IL); Orna Singer, Jerusalem (IL); Osnat Bohana-Kashtan, Tel-Mond (IL); and Ofer Wiser, Jerusalem (IL)
Assigned to HADASIT MEDICAL RESEARCH SERVICES AND DEVELOPMENT LTD., Jerusalem (IL); and CELL CURE NEUROSCIENCES LTD., Jerusalem (IL)
Appl. No. 15/771,046
Filed by CELL CURE NEUROSCIENCES LTD., Jerusalem (IL); and HADASIT MEDICAL RESEARCH SERVICES AND DEVELOPMENT LTD., Jerusalem (IL)
PCT Filed Oct. 26, 2016, PCT No. PCT/IL2016/051155
§ 371(c)(1), (2) Date Apr. 25, 2018,
PCT Pub. No. WO2017/072763, PCT Pub. Date May 4, 2017.
Claims priority of provisional application 62/246,214, filed on Oct. 26, 2015.
Prior Publication US 2018/0312805 A1, Nov. 1, 2018
Int. Cl. C12N 5/079 (2010.01); C12N 5/00 (2006.01); C12N 5/0735 (2010.01); G01N 33/53 (2006.01)
CPC C12N 5/0621 (2013.01) [C12N 5/0018 (2013.01); C12N 5/0606 (2013.01); C12N 2501/15 (2013.01); C12N 2501/16 (2013.01); C12N 2501/999 (2013.01); C12N 2506/02 (2013.01); C12N 2506/45 (2013.01); C12N 2509/00 (2013.01); C12N 2533/52 (2013.01); C12N 2533/54 (2013.01); G01N 33/53 (2013.01)] 29 Claims
 
1. A method of generating retinal pigment epithelial (RPE) cells, the method comprising:
(a) differentiating a population of undifferentiated human pluripotent stem cells on an adherent surface comprising at least one molecule selected from the group consisting of laminin, fibronectin, vitronectin, collagen I and collagen IV and under feeder cell free conditions in a medium comprising between 1 mM and 100 mM nicotinamide as a differentiating agent to obtain differentiating cells, wherein between about 50%-100% of the cells of said population of undifferentiated human pluripotent stem cells are Oct4+TRA-1-60+; and
(b) further differentiating said differentiating cells on said adherent surface for 1 day to 2 weeks in a medium which comprises between 1 mM and 100 mM nicotinamide, and between 20 and 200 ng/ml Activin A;
(c) further differentiating the cells obtained from step (b) in a medium comprising nicotinamide and devoid of Activin A to obtain a culture of differentiated cells comprising pigmented and non-pigmented cells;
(d) enzymatically harvesting the culture of differentiated cells from said adherent surface to obtain a population of isolated differentiated cells; and
(e) expanding the population of isolated differentiated cells to obtain an expanded population of differentiated cells, wherein at least 70% of the expanded population of differentiated cells are CRALBP+PMEL17+, thereby generating RPE cells,
wherein at least one of step (a) or step (b) occurs in 1% to 10% oxygen.