Ballasy Noura, Radford Bethany N, Mohammad Shuhiba, Dean Wendy, Hemberger Myriam
Department of Biochemistry and Molecular Biology, Cumming School of Medicine, University of Calgary, 3330 Hospital Drive NW, Calgary, AB, T2N 4N1, Canada.
Alberta Children's Hospital Research Institute, University of Calgary, 3330 Hospital Drive NW, Calgary, AB, T2N 4N1, Canada.
Stem Cell Res Ther. 2025 Jul 15;16(1):371. doi: 10.1186/s13287-025-04487-z.
The characteristic constituents of the mature placenta are made up of highly specialised trophoblast cell types. Trophoblast stem cells (TSCs) possess the developmental plasticity to differentiate into all these mature placental cell types. However, TSCs are typically a heterogenous population with individual cells exhibiting varying degrees of stem cell marker expression. Moreover, standard differentiation protocols of mouse TSCs are based on a release from the stem cell state and result in a mixed population of various trophoblast cell types. This mix of differentiating cells is a particular impediment for functional investigations into the roles of specific trophoblast subtypes of the mouse placental labyrinth, i.e. the portion of the placenta that establishes the feto-maternal exchange unit.
Murine TSCs were used to establish culture protocols that enhance the stem cell state of mouse TSCs and that drive differentiation into specific labyrinth trophoblast cell types. TSCs were treated with a panel of 35 epigenetic inhibitors and with 12 selected small molecule compounds either alone or in combination, and across a wide range of doses. TSC stemness and differentiation was assessed by RT-qPCR to determine the relative expression of trophoblast cell type-specific marker genes, and by immunofluorescence staining to verify enrichment of the cell type(s) of interest.
TSC markers CDX2 and SOX2 were robustly enriched upon treatment of mouse TSCs with the KDM1A inhibitor GSK-LSD1, indicative of an enhanced stem cell state. Treatment of differentiating TSCs with the LIMK2 inhibitor BMS-3 in conjunction with either the PPARG agonist rosiglitazone (Rosi) or with Tunicamycin and GSK-LSD1 promoted differentiation of labyrinth trophoblast cell types in general, at the expense of junctional zone trophoblast. Rosi in combination with the KDM1A1 inhibitor GSK-LSD1 or BMS-3 enriched for syncytiotrophoblast layer I cells and sinusoidal trophoblast giant cells, while high doses of Rosi resulted specifically in sinusoidal trophoblast giant cell differentiation. Rosi in combination with the protein synthesis inhibitor Tunicamycin enriched for syncytiotrophoblast layer I cells only. Activin A and the WNT agonist Chiron99021 resulted in predominant syncytiotrophoblast layer II differentiation.
Collectively, we establish a roadmap of treatment regimens that promote the differentiation of mouse TSCs into specific trophoblast cell types of the feto-maternal exchange surface. These insights will enable refined biochemical and molecular assessment strategies on defined trophoblast cell types that govern reproductive outcome.
成熟胎盘的特征性成分由高度特化的滋养层细胞类型组成。滋养层干细胞（TSCs）具有发育可塑性，可分化为所有这些成熟的胎盘细胞类型。然而，TSCs通常是异质群体，单个细胞表现出不同程度的干细胞标志物表达。此外，小鼠TSCs的标准分化方案基于从干细胞状态释放，导致各种滋养层细胞类型的混合群体。这种分化细胞的混合对于研究小鼠胎盘迷路中特定滋养层亚型的作用（即建立胎儿-母体交换单元的胎盘部分）的功能研究来说是一个特别的障碍。
使用小鼠TSCs建立培养方案，以增强小鼠TSCs的干细胞状态并驱动其分化为特定的迷路滋养层细胞类型。用一组35种表观遗传抑制剂和12种选定的小分子化合物单独或联合处理TSCs，并设置广泛的剂量范围。通过RT-qPCR评估TSC的干性和分化，以确定滋养层细胞类型特异性标志物基因的相对表达，并通过免疫荧光染色验证感兴趣细胞类型的富集情况。
用KDM1A抑制剂GSK-LSD1处理小鼠TSCs后，TSC标志物CDX2和SOX2显著富集，表明干细胞状态增强。用LIMK2抑制剂BMS-3与PPARG激动剂罗格列酮（Rosi）或与衣霉素和GSK-LSD1联合处理分化中的TSCs，总体上促进了迷路滋养层细胞类型的分化，但以连接区滋养层为代价。Rosi与KDM1A1抑制剂GSK-LSD1或BMS-3联合使用可富集合体滋养层I型细胞和窦状滋养层巨细胞，而高剂量的Rosi则特异性地导致窦状滋养层巨细胞分化。Rosi与蛋白质合成抑制剂衣霉素联合使用仅富集合体滋养层I型细胞。激活素A和WNT激动剂Chiron99021导致主要为合体滋养层II型分化。
总体而言，我们建立了一个治疗方案路线图，可促进小鼠TSCs分化为胎儿-母体交换表面的特定滋养层细胞类型。这些见解将有助于对控制生殖结果的特定滋养层细胞类型进行精细的生化和分子评估策略。
