US 11,920,164 B2
Media for culturing naive human pluripotent stem cells
Yaqub Hanna, Tel-Aviv (IL); Noa Novershtern, Rehovot (IL); and Yoach Rais, Rehovot (IL)
Assigned to Yeda Research and Development Co. Ltd., Rehovot (IL)
Appl. No. 15/500,163
Filed by Yeda Research and Development Co. Ltd., Rehovot (IL)
PCT Filed Jul. 30, 2015, PCT No. PCT/IL2015/050785
§ 371(c)(1), (2) Date Jan. 30, 2017,
PCT Pub. No. WO2016/016894, PCT Pub. Date Feb. 4, 2016.
Claims priority of provisional application 62/167,469, filed on May 28, 2015.
Claims priority of provisional application 62/030,792, filed on Jul. 30, 2014.
Prior Publication US 2017/0275593 A1, Sep. 28, 2017
Int. Cl. C12N 5/00 (2006.01); C12N 5/0735 (2010.01); C12N 5/074 (2010.01)
CPC C12N 5/0696 (2013.01) [C12N 5/0037 (2013.01); C12N 5/0606 (2013.01); C12N 2500/38 (2013.01); C12N 2500/90 (2013.01); C12N 2501/01 (2013.01); C12N 2501/115 (2013.01); C12N 2501/125 (2013.01); C12N 2501/15 (2013.01); C12N 2501/155 (2013.01); C12N 2501/16 (2013.01); C12N 2501/235 (2013.01); C12N 2501/415 (2013.01); C12N 2501/50 (2013.01); C12N 2501/60 (2013.01); C12N 2501/71 (2013.01); C12N 2501/72 (2013.01); C12N 2501/727 (2013.01); C12N 2501/73 (2013.01); C12N 2501/999 (2013.01); C12N 2506/00 (2013.01)] 1 Claim
 
1. A cell culture comprising naïve human pluripotent stem cells (PSC) and a culture medium comprising about 1-300 ng/ml of LIF, about 0.05-5 μM of BIRB796, about 0.1-5 μM of CHIR99021, about 0.1-50 μM of PD032590, about 0.5-25 μM of SP600125, about 0.1-25 μM of Y27632, about 0.2-10 μM of CGP77675, about 0.1-20 ng/ml of TGFβ1 and about 0.5-10 μM of IWR1, and
wherein the culture medium is capable of maintaining the human naive PSC in a naive state for at least 5 passages, and wherein
(i) when said naive PSC is a female PSC, then said naive female PSC has two unmethylated alleles of the promoter of the X-inactive specific transcript (XIST) gene; and
(ii) when said naive PSC is a male PSC, then said naive male PSC has an unmethylated allele of said promoter of said XIST gene, and
said naive state is characterized by: ability to maintain pluripotency the absence of DNA methyltransferase 1 (DNMT1) expression as determined by OCT4 expression in an immunostaining assay; and
an expression level of transcription factor E3 (TFE3) in said naive PSC is characterized by a nucleus to cytoplasm expression ratio which is equal to or higher than 1 as determined by an immunostaining assay.