Chemical conversion of human conventional PSCs to TSCs following transient naive gene activation

Abstract

In human embryos, naive pluripotent cells of the inner cell mass (ICM) generate epiblast, primitive endoderm and trophectoderm (TE) lineages, whence trophoblast cells derive. In vitro, naive pluripotent stem cells (PSCs) retain this potential and efficiently generate trophoblast stem cells (TSCs), while conventional PSCs form TSCs at low efficiency. Transient histone deacetylase and MEK inhibition combined with LIF stimulation is used to chemically reset conventional to naive PSCs. Here, we report that chemical resetting induces the expression of both naive and TSC markers and of placental imprinted genes. A modified chemical resetting protocol allows for the fast and efficient conversion of conventional PSCs into TSCs, entailing shutdown of pluripotency genes and full activation of the trophoblast master regulators, without induction of amnion markers. Chemical resetting generates a plastic intermediate state, characterised by co‐expression of naive and TSC markers, after which cells steer towards one of the two fates in response to the signalling environment. The efficiency and rapidity of our system will be useful to study cell fate transitions and to generate models of placental disorders.

KLF7 is a general inducer of human pluripotency

“…After 7 days naive compact colonies emerged and were expanded. In EMPTY-iPSCs, resetting gave rise to a mixed population, as previously reported 17,33 , while overexpression of KLF7 resulted in a morphologically homogeneous population of naive colonies (Fig. 5b, left).…”

“…Induction of pluripotency in human somatic cells can be achieved via the expression of different combinations of the factors OCT4, SOX2, KLF4, MYC, NANOG, LIN28A (OSKM and OSNL), which leads to the generation of induced Pluripotent Stem Cells (iPSCs) 4,6 . We verified the absolute expression of such reprogramming factors in several human PSC and iPSC lines by interrogating previously published data 10,12,27–33 and observed that expression of the pluripotency factor KLF4 was barely detectable (Fig. 1a).…”

“…b: Barplot showing the absolute expression, measured by RNAseq, of Kruppel-like factors, pluripotency and differentiation genes in conventional hPSCs, cultured in 3 distinct culture conditions. Data were obtained from 10,12,27–33 . Mean +/- SD of at least 8 biological replicates is shown.…”

“…To further investigate if the mechanism of action of KLF7 in promoting pluripotency genes and blocking GATA3 is direct or indirect, we analysed the transcriptome of conventional PSCs over-expressing KLF7, as several trophoblast markers are already expressed in conventional PSCs 33 (e.g. GATA2/3, KRT7 and TFAP2A), thus, if KLF7 is a direct repressor of trophoblast markers, we should detect a reduction in their expression in conventional PSCs.…”

“…Chemical resetting generates an initial plastic state in which naive- and TSC- specific markers are co-expressed, allowing the acquisition of pluripotent or extraembryonic fates depending on the environmental signals to which cells are later exposed 33 . We showed that KLF7 overexpression during chemical resetting promotes acquisition of the naive fate, in spite of the extraembryonic lineage.…”