Single Nucleus RNA Sequence (snRNAseq) Analysis of the Spectrum of Trophoblast Lineages Generated From Human Pluripotent Stem Cells in vitro

Abstract

One model to study the emergence of the human trophoblast (TB) has been the exposure of pluripotent stem cells to bone morphogenetic protein 4 (BMP4) in presence of inhibitors of ACTIVIN/TGFB; A83–01 and FGF2; PD173074 (BAP), which generates a mixture of cytotrophoblast, syncytiotrophoblast, and cells with similarities to extravillous trophoblast. Here, H1 human embryonic stem cells were BAP-exposed under two O2 conditions (20% and 5%, respectively). At day 8, single nuclei RNA sequencing was used for transcriptomics analysis, thereby allowing profiling of fragile syncytial structures as well as the more resilient mononucleated cells. Following cluster analysis, two major groupings, one comprised of five (2,4,6,7,8) and the second of three (1,3,5) clusters were evident, all of which displayed recognized TB markers. Of these, two (2 and 3) weakly resembled extravillous trophoblast, two (5 and 6) strongly carried the hallmark transcripts of syncytiotrophoblast, while the remaining five were likely different kinds of mononucleated cytotrophoblast. We suggest that the two populations of nuclei within syncytiotrophoblast may have arisen from fusion events involving two distinct species of precursor cells. The number of differentially expressed genes between O2 conditions varied among the clusters, and the number of genes upregulated in cells cultured under 5% O2 was highest in syncytiotrophoblast cluster 6. In summary, the BAP model reveals an unexpectedly complex picture of trophoblast lineage emergence that will need to be resolved further in time-course studies.

Early Human Trophoblast Development: From Morphology To Function

“…Advancements in single-cell transcriptomics and multi-omics technologies enable pro ling of thousands of human trophoblasts obtained either from rst trimester placenta specimens, 3D organoids or bone morphogenetic protein 4 (BMP4)-exposed pluripotent stem cells. These novel approaches will contribute to more precisely characterize new sub-clusters of previously poorly described trophoblast subpopulations [9,83,123,144,152]. Integration of spatial transcriptomics and advanced imaging techniques will further enhance the characterization of the spatiotemporal distribution of these trophoblast sub-clusters during human placental development and will shed light on altered compositions of these clusters in placenta-associated pregnancy pathologies.…”

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

“…The first involves extended embryo culture beyond the blastocyst stage and has been recently reviewed by Zhou et al ( Zhou et al, 2021 ). A second generates TB from human pluripotent stem cells (PSCs), and induced pluripotent stem cells (iPSCs) by exposing them to BMP4 and two small compounds, an activin A signaling inhibitor (A83-01) and a FGF2 signaling inhibitor (PD173074) and has been called the BAP model ( Yabe et al, 2016 ; Roberts et al, 2018 ; Khan et al, 2021 ). The third approach uses TB stem cells (TSCs) and induced TSCs (iTSCs) ( Oda et al, 2006 ; Roberts & Fisher, 2011 ; Dong et al, 2020 ).…”

“…Recognizing these limitations, we have recently employed snRNA-seq approaches to help to circumvent the cell size, cellular fragility and multinucleation challenges that STB presents to scRNA-seq approaches. Using snRNA-seq, we were able to generate a transcriptome profile for STB generated from human PSCs (H1) created by the BAP model ( Khan et al, 2021 ). A two-step approach is commonly used to obtain nuclei from cells for use in snRNA-seq, in order: 1) harvest a cellular fraction with an optimized percentage of viable cells and 2) lyse these harvested cells to obtain a high proportion of intact nuclei.…”

“…(C) Representative data from one of isolated nuclei samples, left panel is the image showing isolated nuclei, right panel showed concentration of the nuclei and percentage of the broken cells from which the nuclei were collected. Reproduced with permission from Khan et al ( Khan et al, 2021 ). BMP4, Bone morphogenic protein-4; A, A83-01 (Activin/Nodal inhibitor); P, PD173074; BAP, BMP4 + A83-01 + PD173074; AP, A83-01 + PD173074; hCG, human chorionic gonadotropin; D, day.…”

“…STB-1 and STB-2 referring cluster 5 and 6 respectively from cluster analysis of snRNA-seq ( Figure 3A ). Reproduced with permission from Khan et al ( Khan et al, 2021 ). TB, trophoblast; EVT, extra villous trophoblast cells; CTB, cytotrophoblast cells; STB, syncytiotrophoblast.…”