	US 12,421,499 B2
	Method of differentiation of human pluripotent stem cells to monohormonal cells
Shuibing Chen, Pelham, NY (US); and Sadaf Amin, New York, NY (US)
Assigned to Cornell University, Ithaca, NY (US)
Appl. No. 17/054,633
Filed by Cornell University, Ithaca, NY (US)
PCT Filed May 10, 2019, PCT No. PCT/US2019/031805 § 371(c)(1), (2) Date Nov. 11, 2020, PCT Pub. No. WO2019/217875, PCT Pub. Date Nov. 14, 2019.
Claims priority of provisional application 62/670,451, filed on May 11, 2018.
Prior Publication US 2021/0071146 A1, Mar. 11, 2021
Int. Cl. C12N 5/071 (2010.01)

CPC C12N 5/0676 (2013.01) [C12N 2510/00 (2013.01)]

4 Claims

1. A method of generating an enriched population of monohormonal cells from pluripotent stem cells comprising the following steps exposing pluripotent stem cells sequentially to:

a) a basal medium supplemented with at least CHIR99021 and Activin A;

b) a basal medium supplemented with Activin A;

c) a basal medium supplemented with at least FGF7;

d) a basal medium supplemented with retinoic acid, LDN 193189, and SANT1;

e) a basal medium supplemented with components consisting of 2 μM retinoic acid, 200 nM LDN193189, 0.25 UM SANT1, 10 ng/ml EGF and 10 ng/ml FGF2; and

f) a basal differentiation medium,

wherein step a is carried out for 1 day, step b is carried out for 2 days, step c is carried out for 2 days, step d is carried out for 4 days, step e is carried out for 14 days and step f is carried out for 7 days; and

wherein at least 90% of the cells in the resulting population are monohormonal and express insulin, but not glucagon, somatostatin, or ghrelin and are Glis3+.