Instituto de Biopatología y Medicina Regenerativa, Centro de Investigación Biomédica, Universidad de Granada, Granada, Spain.
Departamento de Estadística e Investigación Operativa, Universidad de Granada, Granada, Spain.
Stem Cell Res Ther. 2019 Jun 14;10(1):177. doi: 10.1186/s13287-019-1284-z.
Human decidual stromal cells (DSCs) are involved in the maintenance and development of pregnancy, in which they play a key role in the induction of immunological maternal-fetal tolerance. Precursors of DSCs (preDSCs) are located around the vessels, and based on their antigen phenotype, previous studies suggested a relationship between preDSCs and mesenchymal stromal/stem cells (MSCs). This work aimed to further elucidate the MSC characteristics of preDSCs.
We established 15 human preDSC lines and 3 preDSC clones. Physiological differentiation (decidualization) of these cell lines and clones was carried out by in vitro culture with progesterone (P4) and cAMP. Decidualization was confirmed by the change in cellular morphology and prolactin (PRL) secretion, which was determined by enzyme immunoassay of the culture supernatants. We also studied MSC characteristics: (1) In mesenchymal differentiation, under appropriate culture conditions, these preDSC lines and clones differentiated into adipocytes, osteoblasts, and chondrocytes, and differentiation was confirmed by cytochemical assays and RT-PCR. (2) The expression of stem cell markers was determined by RT-PCR. (3) Cloning efficiency was evaluated by limited dilution. (4) Immunoregulatory activity in vivo was estimated in DBA/2-mated CBA/J female mice, a murine model of immune-based recurrent abortion. (5) Survival of preDSC in immunocompetent mice was analyzed by RT-PCR and flow cytometry.
Under the effect of P4 and cAMP, the preDSC lines and clones decidualized in vitro: the cells became rounder and secreted PRL, a marker of physiological decidualization. PreDSC lines and clones also exhibited MSC characteristics. They differentiated into adipocytes, osteoblasts, and chondrocytes, and preDSC lines expressed stem cell markers OCT-4, NANOG, and ABCG2; exhibited a cloning efficiency of 4 to 15%; significantly reduced the embryo resorption rate (P < 0.001) in the mouse model of abortion; and survived for prolonged periods in immunocompetent mice. The fact that 3 preDSC clones underwent both decidualization and mesenchymal differentiation shows that the same type of cell exhibited both DSC and MSC characteristics.
Together, our results confirm that preDSCs are decidual MSCs and suggest that these cells are involved in the mechanisms of maternal-fetal immune tolerance.
人类蜕膜基质细胞（DSC）参与妊娠的维持和发育，在诱导免疫性母胎耐受中发挥关键作用。DSC 的前体（preDSC）位于血管周围，根据其抗原表型，先前的研究表明 preDSC 与间充质基质/干细胞（MSC）之间存在关系。本研究旨在进一步阐明 preDSC 的 MSC 特征。
我们建立了 15 个人类 preDSC 系和 3 个 preDSC 克隆。通过体外培养添加孕激素（P4）和环磷酸腺苷（cAMP）来实现这些细胞系和克隆的生理分化（蜕膜化）。通过细胞形态的变化和培养上清液中催乳素（PRL）的分泌来确认蜕膜化，PRL 的分泌通过酶免疫测定来确定。我们还研究了 MSC 特征：（1）在间充质分化中，在适当的培养条件下，这些 preDSC 系和克隆分化为脂肪细胞、成骨细胞和成软骨细胞，并通过细胞化学染色和 RT-PCR 确认分化。（2）通过 RT-PCR 确定干细胞标志物的表达。（3）通过有限稀释评估克隆效率。（4）在 DBA/2 交配的 CBA/J 雌性小鼠中评估体内免疫调节活性，这是一种基于免疫的复发性流产的小鼠模型。（5）通过 RT-PCR 和流式细胞术分析免疫活性小鼠中 preDSC 的存活情况。
在 P4 和 cAMP 的作用下，preDSC 系和克隆在体外发生蜕膜化：细胞变得更圆，并分泌 PRL，这是生理蜕膜化的标志。preDSC 系和克隆还表现出 MSC 特征。它们分化为脂肪细胞、成骨细胞和成软骨细胞，preDSC 系表达干细胞标志物 OCT-4、NANOG 和 ABCG2；具有 4 至 15%的克隆效率；显著降低了流产小鼠模型中的胚胎吸收率（P < 0.001）；并在免疫活性小鼠中长时间存活。3 个 preDSC 克隆同时经历蜕膜化和间充质分化表明同类型细胞同时具有 DSC 和 MSC 特征。
综上所述，我们的结果证实了 preDSC 是蜕膜 MSC，并表明这些细胞参与了母胎免疫耐受的机制。
