Media for culturing naive human pluripotent stem cells
A culture medium is disclosed which comprises STAT3 activator, an ERK1/2 inhibitor and an Axin stabilizer, and optionally also a PKC inhibitor. Cell cultures comprising same and uses thereof are also disclosed.
1 . A cell culture comprising human or rhesus monkey naive pluripotent stem cell (PSC) and a culture medium comprising a STAT3 activator at a concentration of 1-300 ng/ml, an ERK1/2 inhibitor at a concentration of 0.1-50 μM, a Tankyrase inhibitor at a concentration of 0.5-10 μM and at least one agent selected from the group consisting of a p38 inhibitor at a concentration of about 0.05-5 μM, an SRC inhibitor at a concentration of 0.2-10 μM, Activin A at a concentration of 1-40 ng/ml and a NOTCH inhibitor at a concentration of 0.05-50 μM,
wherein the culture is capable of maintaining the human or rhesus monkey naive PSC in a naïve 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 naïve 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.
2 . The cell culture of claim 1 , wherein said STAT3 activator is selected from the group consisting of leukemia inhibitory factor (LIF), interleukin 6 (IL6) and epidermal growth factor (EGF), said ERK1/2 inhibitor is selected from the group consisting of PD0325901, PD98059 and PD184352, said Tankyrase inhibitor is selected from the group consisting of IWR-1 and XAV939, said p38 inhibitor is r selected from the group consisting of SB203580, SB 202190, LY 2228820, BIRB796 and PD169316, said SRC inhibitor is CGP77675, and said NOTCH inhibitor is a gamma secretase inhibitor selected from the group consisting of DAPT, LY2886721 hydrochloride and DBZ.
3 . The cell culture of claim 1 , wherein said culture medium further comprises at least one agent selected from the group consisting of a GSK3β inhibitor, a PKC inhibitor, a ROCK inhibitor, a JNK inhibitor, a PKA agonist, a YAP/TAZ inhibitor, an SHH inhibitor, a transforming growth factor receptor (TGFR) inhibitor, a BMP inhibitor, an FGFR inhibitor, an ERK5 inhibitor, a BRAF inhibitor, a CRAF inhibitor, a PI3K activator, basic fibroblast growth factor (bFGF), a SMAD activator, insulin-like growth factor 1 (IGF1), insulin-like growth factor II (IGFII), a bone morphogenetic protein (BMP) signaling inhibitor, a Sonic Hedgehog pathway (SHH) inhibitor, an ERK5 inhibitor, Forskolin, BayK8644, an inhibitor of G9a and/or Glp, stem cell factor (SCF), YAP/TAZ inhibitor, L-Ascorbic Acid, an LSD1 inhibitor, a DOT1L inhibitor and a TGF inducer.
4 . The cell culture of claim 1 , wherein said culture medium further comprises a GSK3β inhibitor.
5 . The cell culture of claim 4 , wherein said GSK3b inhibitor is selected from the group consisting of CHIR99021, BIO and Kenpaullone.
6 . The cell culture of claim 1 , wherein said culture medium further comprises a PKC inhibitor.
7 . The cell culture of claim 6 , wherein said PKC inhibitor is Go6983.
8 . The cell culture of claim 1 , wherein said culture medium comprises said STAT3 activator, said ERK1/2 inhibitor, said Tankyrase inhibitor and said p38 inhibitor.
9 . The cell culture medium of claim 8 , wherein said culture medium further comprises a GSK3β inhibitor.
10 . The cell culture of claim 9 , wherein said culture medium further comprises a PKC inhibitor.
11 . The cell culture of claim 1 , wherein said culture medium being devoid of serum.