Establishment of human induced trophoblast stem-like cells from term villous cytotrophoblasts

Abstract

Human trophoblast stem cells (hTSC) can be isolated from first trimester placenta but not from term placenta. Here we demonstrate that villous cytotrophoblasts (vCTB) from term placenta can be reprogrammed into induced trophoblastic stem-like cells (iTSC) by introducing sets of transcription factors. The iTSCs express TSC markers such as GATA3, TEAD4 and ELF5, and are multipotent, validated by their differentiation into both extravillous trophoblasts (EVT) and syncytiotrophoblasts (STB) in vitro and in vivo . The iTSC can be passaged indefinitely in vitro without slowing of growth. The transcriptome profile of these cells closely resembles the profile of hTSC isolated from first trimester placentae but different from the term placental vCTB from which they originated. The ability to reprogram cells from term placenta into iTSC will allow study of early gestation events which impact placental function later in gestation, including preeclampsia and spontaneous preterm birth.

Derivation of functional trophoblast stem cells from primed human pluripotent stem cells

“…Here, we applied a new medium to CTB stem-like cells at the end of step 1 of our trophoblast differentiation protocol ( Figure 1 A). This medium was developed originally by the Kessler lab for the reprogramming of term CTB into TSC-like cells (iTSCs) ( Bai et al., 2021 ) and is a modification of the Okae medium (see experimental procedures ) used for derivation of TSCs from human embryos and early-gestation placentae ( Okae et al., 2018 ). After passage in this medium at least 5 times, we were able to derive cells morphologically resembling primary TSCs ( Figure 1 B) and uniformly expressing cell-surface TSC markers EGFR and ITGA6 ( Figure 1 C).…”

“…Here, we have applied a culture medium, recently developed for maintenance of induced TSCs (iTSCs), reprogrammed from term CTB ( Bai et al., 2021 ) to hPSC-derived CTB, and show that this transitions the cells into bona fide TSCs. These cells resemble placenta-derived TSCs and can be differentiated into functional syncytiotrophoblast (STBs) and extravillous trophoblast (EVTs), both in vitro and in vivo .…”

Stem Cell-Based Trophoblast Models to Unravel the Genetic Causes of Human Miscarriages

“…Remarkably, whereas mouse iTSCs can be derived by the overexpression of trophoblast-specific TFs EOMES, GATA3, TFAP2C, and MYC in mouse fibroblasts [ 139 , 140 ], human iTSCs were established by reprogramming somatic cells with induction with Yamanaka’s cocktail (OCT4, KLF4, SOX2, and MYC), coupled with hTSC culture conditions [ 137 , 138 ]. However, non-integrating viral expression of transcription factors TFAP2C, TEAD4, CDX2, ELF5, and ETS2, as shown, efficiently generated TSC-like cells from the term placenta villous cytotrophoblasts (vCTBs) [ 141 ]. The iTSCs express TSC markers such as GATA3, TEAD4 and ELF5, are capable of differentiation into both EVTs and STBs, and can be passaged indefinitely without a slowing of growth.…”

“…The iTSCs express TSC markers such as GATA3, TEAD4 and ELF5, are capable of differentiation into both EVTs and STBs, and can be passaged indefinitely without a slowing of growth. The transcriptome profile of these cells closely resembles the profile of hTSCs isolated from first-trimester placentae [ 141 ].…”

Transcription factor networks in trophoblast development

“…Similarly, transient, ectopic expression of Eomes , Tfap2c , Gata3 and optionally Ets2 or Myc in mouse fibroblast coupled to mouse TSC culture conditions (FGF4 and TGF-β) led to the establishment of self-renewing and multipotent induced TSCs (iTSCs) that contributed to the placenta in chimeras [ 27 , 28 ]. Moreover, a recent report showed reprogramming of human term CTB to iTSCs upon expression of 5 TFs ( TFAP2C , TEAD4 , CDX2 , ELF5 and ETS2 ) in hTSC culture conditions [ 29 ]. The role of TFs in translating the signaling inputs into the transcriptional outputs was demonstrated in the trophoblast context, as constitutive overexpression of Sox2 and Esrrb / Tfap2c was sufficient to confer FGF4-independent self-renewal and sustain multipotency in mTSCs [ 30 ].…”