Genbacev O, Larocque N, Ona K, Prakobphol A, Garrido-Gomez T, Kapidzic M, Bárcena A, Gormley M, Fisher S J
Center for Reproductive Sciences, University of California San Francisco, San Francisco, CA 94143, USA Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California San Francisco, San Francisco, CA 94143, USA.
Center for Reproductive Sciences, University of California San Francisco, San Francisco, CA 94143, USA Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California San Francisco, San Francisco, CA 94143, USA Department of Obstetrics and Gynecology, Fundacion IVI, Instituto Universitario IVI, School of Medicine, Universidad de Valencia, INCLIVA, Valencia, Spain.
Hum Reprod. 2016 Jun;31(6):1300-14. doi: 10.1093/humrep/dew077. Epub 2016 Apr 15.
What are the functional characteristics and transcriptional regulators of human trophoblast progenitor cells (TBPCs)?
TBPC lines established from the human smooth chorion by cell sorting for integrin α4 expressed markers of stemness and trophoblast (TB) stage-specific antigens, invaded Matrigel substrates and contributed to the cytotrophoblasts (CTBs) layer of smooth chorion explants with high-mobility group protein HMGI-C (HMGA2) and transcription factor GATA-4 (GATA4) controlling their progenitor state and TB identity.
Previously, we reported the derivation of TBPC lines by trypsinization of colonies that formed in cultures of chorionic mesenchyme cells that were treated with an activin nodal inhibitor. Microarray analyses showed that, among integrins, α4 was most highly expressed, and identified HMGA2 and GATA4 as potential transcriptional regulators.
STUDY DESIGN, SIZE, DURATION: The aim of this study was to streamline TBPC derivation across gestation. High-cell surface expression of integrin α4 enabled the use of a fluorescence-activated cell sorter (FACS) approach for TBPC isolation from the human smooth chorion (n = 6 lines). To confirm their TBPC identity, we profiled their expression of stemness and TB markers, and growth factor receptors. At a functional level, we assayed their invasive capacity (n = 3) and tropism for the CTB layer of the smooth chorion (n = 3). At a molecular level, we studied the roles of HMGA2 and GATA4.
PARTICIPANTS/MATERIALS, SETTINGS, METHODS: Cells were enzymatically disassociated from the human smooth chorion across gestation. FACS was used to isolate the integrin α4-positive population. In total, we established six TBPC lines, two per trimester. Their identity was determined by immunolocalization of a suite of antigens. Function was assessed via Matrigel invasion and co-culture with explants of the human smooth chorion. An siRNA approach was used to down-regulate HMGA2 and GATA4 expression and the results were confirmed by immunoblotting and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analyses. The endpoints analyzed included proliferation, as determined by 5-bromo-2'-deoxyuridine (BrDU) incorporation, and the expression of stage-specific antigens and hormones, as determined by qRT-PCR and immunostaining approaches.
As with the original cell lines, the progenitors expressed a combination of human embryonic stem cell and TB markers. Upon differentiation, they primarily formed CTBs, which were capable of Matrigel invasion. Co-culture of the cells with smooth chorion explants enabled their migration through the mesenchyme after which they intercalated within the chorionic CTB layer. Down-regulation of HMGA2 showed that this DNA-binding protein governed their self-renewal. Both HMGA2 and GATA4 had pleitropic effects on the cells' progenitor state and TB identity.
LIMITATIONS, REASONS FOR CAUTION: This study supported our hypothesis that TBPCs from the chorionic mesenchyme can contribute to the subpopulation of CTBs that reside in the smooth chorion. In the absence of in vivo data, which is difficult to obtain in humans, the results have the limitations common to all in vitro studies.
The accepted view is that progenitors reside among the villous CTB subpopulation. Here, we show that TBPCs also reside in the mesenchymal layer of the smooth chorion throughout gestation. We theorize that they can contribute to the CTB layer in this region. This phenomenon may be particularly important in pathological situations when CTBs of the smooth chorion might provide a functional reserve for CTBs of the placenta proper.
STUDY FUNDING/COMPETING INTERESTS: Research reported in this publication was supported by the Eunice Kennedy Shriver National Institute of Child Health and Human Development of the National Institutes of Health under award P50HD055764. O.G., N.L., K.O., A.P., T.G.-G., M.K., A.B., M.G. have nothing to disclose. S.J.F. received licensing fees and royalties from SeraCare Life Sciences for trisomic TBPC lines that were derived according to the methods described in this manuscript.
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人类滋养层祖细胞（TBPCs）的功能特征和转录调节因子是什么？
通过对整合素α4表达的干性和滋养层（TB）阶段特异性抗原标志物进行细胞分选，从人平滑绒毛膜建立的TBPC系，能侵袭基质胶底物，并通过高迁移率族蛋白HMGI-C（HMGA2）和转录因子GATA-4（GATA4）控制其祖细胞状态和TB特性，从而对平滑绒毛膜外植体的细胞滋养层（CTBs）层有贡献。
此前，我们报道了通过胰蛋白酶消化经激活素节点抑制剂处理的绒毛膜间充质细胞培养物中形成的集落来获得TBPC系。微阵列分析显示，在整合素中，α4表达最高，并鉴定出HMGA2和GATA4为潜在的转录调节因子。
研究设计、规模、持续时间：本研究的目的是简化整个孕期的TBPC衍生过程。整合素α4的高细胞表面表达使得能够使用荧光激活细胞分选仪（FACS）从人平滑绒毛膜中分离TBPC（n = 6个系）。为了确认它们的TBPC身份，我们分析了它们干性和TB标志物以及生长因子受体的表达。在功能水平上，我们检测了它们的侵袭能力（n = 3）和平滑绒毛膜CTB层的嗜性（n = 3）。在分子水平上，我们研究了HMGA2和GATA4的作用。
参与者/材料、环境、方法：在整个孕期从人平滑绒毛膜中酶解分离细胞。使用FACS分离整合素α4阳性群体。总共建立了6个TBPC系，每个孕期2个。通过一组抗原的免疫定位确定它们的身份。通过基质胶侵袭和与人平滑绒毛膜外植体共培养来评估功能。使用小干扰RNA（siRNA）方法下调HMGA2和GATA4表达，并通过免疫印迹和定量逆转录-聚合酶链反应（qRT-PCR）分析确认结果。分析的终点包括通过5-溴-2'-脱氧尿苷（BrDU）掺入确定的增殖，以及通过qRT-PCR和免疫染色方法确定的阶段特异性抗原和激素的表达。
与原始细胞系一样，祖细胞表达人类胚胎干细胞和TB标志物的组合。分化后，它们主要形成能够侵袭基质胶的CTB。将细胞与人平滑绒毛膜外植体共培养可使其迁移穿过间充质，然后插入绒毛膜CTB层。HMGA2的下调表明这种DNA结合蛋白控制着它们的自我更新。HMGA2和GATA4对细胞的祖细胞状态和TB特性都有多种影响。
局限性、谨慎的原因：本研究支持了我们的假设，即来自绒毛膜间充质的TBPC可对存在于平滑绒毛膜中的CTB亚群有贡献。由于难以在人类中获得体内数据，在缺乏此类数据的情况下，结果具有所有体外研究共有的局限性。
普遍观点认为祖细胞存在于绒毛CTB亚群中。在这里，我们表明在整个孕期TBPC也存在于平滑绒毛膜的间充质层中。我们推测它们可以对该区域的CTB层有贡献。当平滑绒毛膜的CTB可能为胎盘固有CTB提供功能储备时，这种现象在病理情况下可能尤为重要。
研究资金/竞争利益：本出版物中报道的研究得到了美国国立卫生研究院尤妮斯·肯尼迪·施莱佛国家儿童健康与人类发展研究所的资助，资助编号为P50HD055764。O.G.、N.L.、K.O.、A.P.、T.G.-G.、M.K.、A.B.、M.G.无利益冲突需要披露。S.J.F.因根据本手稿所述方法衍生的三体TBPC系从SeraCare Life Sciences获得了许可费和版税。
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