Morey Robert, Soncin Francesca, Kallol Sampada, Sah Nirvay, Manalo Zoe, Bui Tony, Slamecka Jaroslav, Cheung Virginia Chu, Pizzo Don, Requena Daniela F, Chang Ching-Wen, Farah Omar, Kittle Ryan, Meads Morgan, Horii Mariko, Fisch Kathleen, Parast Mana M
Department of Pathology, School of Medicine, University of California San Diego, La Jolla, CA, 92093, USA.
Sanford Consortium for Regenerative Medicine, La Jolla, CA, 92093, USA.
bioRxiv. 2024 Jul 16:2024.07.12.603155. doi: 10.1101/2024.07.12.603155.
Cytotrophoblast (CTB) of the early gestation human placenta are bipotent progenitor epithelial cells, which can differentiate into invasive extravillous trophoblast (EVT) and multinucleated syncytiotrophoblast (STB). Trophoblast stem cells (TSC), derived from early first trimester placentae, have also been shown to be bipotential. In this study, we set out to probe the transcriptional diversity of first trimester CTB and compare TSC to various subgroups of CTB. We performed single-cell RNA sequencing on six normal placentae, four from early (6-8 weeks) and two from late (12-14 weeks) first trimester, of which two of the early first trimester cases were separated into basal (maternal) and chorionic (fetal) fractions prior to sequencing. We also sequenced three TSC lines, derived from 6-8 week placentae, to evaluate similarities and differences between primary CTB and TSC. CTB clusters displayed notable distinctions based on gestational age, with early first trimester placentae showing enrichment for specific CTB subtypes, further influenced by origin from the basal or chorionic plate. Differential expression analysis of CTB from basal versus chorionic plate highlighted pathways associated with proliferation, unfolded protein response, and oxidative phosphorylation. We identified trophoblast states representing initial progenitor CTB, precursor STB, precursor and mature EVT, and multiple CTB subtypes. CTB progenitors were enriched in early first trimester placentae, with basal plate cells biased toward EVT, and chorionic plate cells toward STB, precursors. Clustering and trajectory inference analysis indicated that TSC were most like EVT precursor cells, with only a small percentage of TSC on the pre-STB differentiation trajectory. This was confirmed by flow cytometric analysis of 6 different TSC lines, which showed uniform expression of proximal column markers ITGA2 and ITGA5. Additionally, we found that ITGA5 CTB could be plated in 2D, forming only EVT upon spontaneous differentiation, but failed to form self-renewing organoids; conversely, ITGA5-CTB could not be plated in 2D, but readily formed organoids. Our findings suggest that distinct CTB states exist in different regions of the placenta as early as six weeks gestation and that current TSC lines most closely resemble ITGA5 CTB, biased toward the EVT lineage.
早孕期人胎盘的细胞滋养层细胞（CTB）是具有双向分化能力的祖上皮细胞，可分化为侵袭性绒毛外滋养层细胞（EVT）和多核合体滋养层细胞（STB）。来源于孕早期胎盘的滋养层干细胞（TSC）也显示出双向分化潜能。在本研究中，我们旨在探究孕早期CTB的转录多样性，并将TSC与CTB的各个亚组进行比较。我们对六个正常胎盘进行了单细胞RNA测序，其中四个来自孕早期（6 - 8周），两个来自孕晚期（12 - 14周），其中两个孕早期病例在测序前被分离为基底（母体）和绒毛膜（胎儿）部分。我们还对来源于6 - 8周胎盘的三个TSC系进行了测序，以评估原代CTB和TSC之间的异同。CTB簇根据孕周显示出显著差异，孕早期胎盘显示特定CTB亚型富集，这进一步受到基底或绒毛膜板来源的影响。对基底板与绒毛膜板CTB的差异表达分析突出了与增殖、未折叠蛋白反应和氧化磷酸化相关的途径。我们鉴定出了代表初始祖CTB、前体STB、前体和成熟EVT以及多种CTB亚型的滋养层细胞状态。CTB祖细胞在孕早期胎盘中富集，基底板细胞偏向于EVT，而绒毛膜板细胞偏向于STB前体。聚类和轨迹推断分析表明，TSC最类似于EVT前体细胞，只有一小部分TSC处于前体STB分化轨迹上。对6个不同TSC系的流式细胞术分析证实了这一点，该分析显示近端柱状标记物ITGA2和ITGA5表达一致。此外，我们发现ITGA5 CTB可以在二维培养，自发分化时仅形成EVT，但不能形成自我更新的类器官；相反，ITGA5 - CTB不能在二维培养，但很容易形成类器官。我们的研究结果表明，早在妊娠六周时，胎盘不同区域就存在不同的CTB状态，并且当前的TSC系与ITGA5 CTB最相似，偏向于EVT谱系。
