Pluripotency-Independent Induction of Human Trophoblast Stem Cells from Fibroblasts

Abstract

SUMMARYRecent studies demonstrated that human trophoblast stem-like cells (hTS-like cells) can be derived from naïve embryonic stem cells or be induced from somatic cells by the pluripotency factors, OSKM. This raises two main questions; (i) whether human induced TSCs (hiTSCs) can be generated independently to pluripotent state or factors and (ii) what are the mechanisms by which hTSC state is established during reprogramming. Here, we identify GATA3, OCT4, KLF4 and MYC (GOKM) as a pluripotency-independent combination of factors that can generate stable and functional hiTSCs, from both male and female fibroblasts. By using single and double knockout (KO) fibroblasts for major pluripotency genes (i.e. SOX2 or NANOG/PRDM14) we show that GOKM not only is capable of generating hiTSCs from the KO cells, but rather that the efficiency of the process is increased. Through H3K4me2 and chromatin accessibility profiling we demonstrate that GOKM target different loci and genes than OSKM, and that a significant fraction of them is related to placenta and trophoblast function. Moreover, we show that GOKM exert a greater pioneer activity compared to OSKM. While GOKM target many specific hTSC loci, OSKM mainly target hTSC loci that are shared with hESCs. Finally, we reveal a gene signature of trophoblast-related genes, consisting of 172 genes which are highly expressed in blastocyst-derived TSCs and GOKM-hiTSCs but absent or mildly expressed in OSKM-hiTSCs.Taken together, these results imply that not only is the pluripotent state, and SOX2 specifically, not required to produce functional hiTSCs, but that pluripotency-specific factors actually interfere with the acquisition of the hTSC state during reprogramming.

Analysis of regulatory network modules in hundreds of human stem cell lines reveals complex epigenetic and genetic factors contribute to pluripotency state differences between subpopulations

“…We directly calculated the enrichment of the 92 hiPSC TF groups in the 54 fetal cell-type specific ATAC-seq peaks (Figure 4D, Table S14). TEAD Family hiPSC TFBSs exhibited a strong enrichment in the three trophoblast cell types cells (Figure 4D), recapitulating their importance in the development of placental lineages (Figure 4C) 42,53 . Trophoblast giant cells and villous cytotrophoblasts were also enriched with GRHL2, which has been reported to be a master regulator of placental branching morphogenesis 54 .…”

“…We demonstrate that trophoblast-specific peaks are enriched with RNM 3 peaks and TEAD Family binding sites (Figure 4C-D), which is consistent with previous observations that TEAD4 is a pioneering factor for the placental lineage commitment 63 . Recently much effort has been put into developing efficient trophoblast differentiation protocols to study common pregnancy-related diseases, such as preeclampsia 41,43,44,53 . RNM 3 may serve as a resource to identify regulatory elements that are early determinants of trophoblast cell fate commitment.…”

Transcription factor-based transdifferentiation of human embryonic to trophoblast stem cells

“…In addition, the establishment of a trophoblast organoid culture system has allowed hTSCs to be cultured in 3D, somewhat resembling the architecture of villous CTB cells and STB of the placenta (Haider et al, 2018; Turco et al, 2018). Most recently, induced trophoblast stem cells (iTSCs) which resemble primary tissue-derived hTSCs have been captured in the process of transdifferentiation of fibroblasts to human embryonic stem cells (hESCs) in naïve cell culture conditions (Castel et al, 2020; Cinkornpumin et al, 2020; Dong et al, 2020; Liu et al, 2020; Naama et al, 2023). In addition, established naïve ES cells cultured cultured in conditions which inhibit ERK and Nodal signalling also yield iTSCs (Guo et al, 2021).…”