	US 12,404,493 B2
	Methods for the re-derivation of diverse pluripotent stem cell-derived brown fat cells
Michael D. West, Mill Valley, CA (US); and Hal Sternberg, Berkeley, CA (US)
Assigned to AgeX Therapeutics, Inc., Alameda, CA (US)
Filed by AgeX Therapeutics, Inc., Alameda, CA (US)
Filed on May 31, 2018, as Appl. No. 15/994,302.
Application 15/994,302 is a continuation of application No. PCT/US2016/065366, filed on Dec. 7, 2016.
Claims priority of provisional application 62/264,311, filed on Dec. 7, 2015.
Prior Publication US 2020/0157505 A1, May 21, 2020
Int. Cl. C12N 5/077 (2010.01); C12N 5/0735 (2010.01); C12Q 1/6881 (2018.01)

CPC C12N 5/0653 (2013.01) [C12N 5/0606 (2013.01); C12Q 1/6881 (2013.01); C12N 2501/385 (2013.01); C12N 2506/02 (2013.01); C12Q 2600/158 (2013.01)]

12 Claims

1. A method of producing brown adipocytes comprising:

a. differentiating a human pluripotent stem cell in a neural differentiation medium consisting of DMEM/F12, B27 supplement, and basic Fibroblast Growth Factor (bFGF), thereby forming an embryoid body;

b. producing a clonal embryonic progenitor cell line from the embryoid body, wherein said clonal embryonic progenitor cell line expresses DIO3, SLC1A3, SBSN, DLK1 and ZIC2, but does not express COX7A1, DLX5, HOXA5, IL13RA2, CRABP1, NEFM, PRG4, and RBP1;

c. differentiating the clonal embryonic progenitor cell line in the presence of a PPARgamma (PPARy) agonist, a thyroid hormone, and a TGFβ family member polypeptide, thereby producing a differentiated progeny from the clonal embryonic progenitor cell line;

d. screening the differentiated progeny from the clonal embryonic progenitor cell line for the expression of a brown adipocyte marker comprising UCP1; and

e. isolating UCP1 expressing brown adipocytes.