Characterization of primary models of human trophoblast

Abstract

Although understanding of human placental development is still limited, two models, trophoblast organoids and trophoblast stem cells (TSC) provide new useful tools to study this. Both differentiate from villous cytotrophoblast (VCT) to either extravillous trophoblast (EVT) or syncytiotrophoblast (SCT). Here, we compare transcriptomes and miRNA profiles of these models to identify which trophoblast they resemble in vivo. Our findings indicate that TSC do not readily undergo SCT differentiation and closely resemble cells at the base of the cell columns from where EVT are derived. In contrast, organoids are similar to VCT and undergo spontaneous SCT differentiation. A defining feature of human trophoblast is that VCT and SCT are HLA null whilst EVT express HLA-C, -G, -E molecules. We find that trophoblast organoids retain these in vivo characteristics. In contrast, TSC do express classical HLA-A and HLA-B molecules and still maintain their expression after EVT differentiation with upregulation of HLA-G. Furthermore, HLA expression in TSC differs when grown in 3D rather than 2D suggesting mechanical cues are important. Our results will allow choice of the most suitable model to study trophoblast development, function and pathology.

Transcription factor networks in trophoblast development

“…These observations may suggest that TOs constitute a better model of chorionic villi as they recapitulate the co-existence of CTB and STB in a dynamic balance. Indeed, recent findings confirm that TOs represent villous CTB and STB, while hTSCs resemble cells at the base of the cell columns from where EVT derives [ 129 ]. Interestingly, EVT differentiation of both hTSC and TOs requires specific growth factor stimuli and seems less robust compared to STB differentiation.…”

WNT and NOTCH signaling in human trophoblast development and differentiation

“…As 2D TSCs, TB-ORGs expressed critical markers of trophoblast identity and vCTB proliferation such as the aforementioned key regulators GATA3, TEAD4, p63 and AP-2γ and displayed a hypo-methylated ELF5 promoter region. Noteworthy, growing TSCs in 3D could be favorable compared to cultivation in 2D, since the latter provoked artificial upregulation of HLA-A and HLA-B, two proteins that are absent from all human trophoblast subtypes in vivo [ 117 ].…”

“…The latter was downregulated by NOTCH1 ICD-dependent expression of interferon regulatory factor 6 (IRF6), a negative regulator of p63 stability [ 135 ]. NOTCH1 and ITGA2 proteins were recently detected in TB-ORGs prepared from primary CTBs suggesting that some organoids, containing EVT progenitors, could be maintained under self-renewing conditions [ 117 ]. Notably, TB-ORGs derived from TSCs showed elevated expression of NOTCH1 and NOTCH1 ICD suggesting that the cells share features with EVT progenitors [ 117 ].…”

“…NOTCH1 and ITGA2 proteins were recently detected in TB-ORGs prepared from primary CTBs suggesting that some organoids, containing EVT progenitors, could be maintained under self-renewing conditions [ 117 ]. Notably, TB-ORGs derived from TSCs showed elevated expression of NOTCH1 and NOTCH1 ICD suggesting that the cells share features with EVT progenitors [ 117 ]. Alternatively, upregulation of the receptor could improve survival of the TSCs in 3D.…”

Leveraging Optimized Transcriptomic and Personalized Stem Cell Technologies to Better Understand Syncytialization Defects in Preeclampsia

“…We will use this model to identify differences in lineage-specific features of normal and abnormal TB proliferation, differentiation, invasion, secretory capacity and cell cycling between control and EOPE lines. In addition, we are using these cells to develop a self-renewing TSC organoid culture system that mimics the villous placenta and allows for the study of cell subtype interactions in three dimensions ( Sheridan et al, 2021 ). In combination with the advances outlined in part one of this review, we also plan to apply snRNA-seq analyses to the iTSC-derived EOPE models, with a specific goal of improving our understanding of the STB functional phenotype.…”