Derivation and characterization of induced trophoblast stem cells

项目说明 滋养外胚层是一种胚外组织，提供胚胎所需的指导性信号。 在原肠发育过程中形成图案，并产生胎盘，胎盘是连接发育中的胎儿和 子宫壁允许营养吸收，废物消除，气体交换和温度调节通过 母亲的血液供应。在小鼠中，滋养层干细胞(MTSCs)来源于植入前胚泡或 从成纤维细胞通过直接谱系转换已经成为解剖 负责TE规范、区分和功能的机制。然而，试图建立TSC 具有早期人类的特征的TE一直没有成功。我们的建议旨在通过以下方式填补这一知识空白 通过谱系转换建立诱导型人TSCs(i-hTSC)。为了更好地理解 TSCs是如何在人类胚胎中形成的，我们分析了之前收集的单细胞RNA测序数据 人类囊胚期胚胎。我们鉴定了一组转录因子，这些转录因子在 TE，但不存在于胚胎上皮细胞(EPI)。我们还鉴定了几种信号配体，它们在 EPI和/或TE本身可能负责TSC的规范和/或维护。为了方便 血统转换实验我们已经产生的人类胚胎干细胞携带一种 荧光mCherry报告与ALL中高度活跃的内源性GATA2或GATA3基因连锁 TE的细胞。我们对这些细胞进行了分化，以获得ESC来源的人成纤维细胞样细胞。 多西环素(Dox)诱导的TE特异性转录对这些报告细胞的转导作用 这些因素产生了几个GATA2/3-mCherry阳性克隆。这些克隆可以在有 DOX，并表达了调控TSC自我更新的关键基因。基于这些数据，我们假设 人成纤维细胞和/或人胚胎干细胞可以通过与TE-1的鸡尾酒直接谱系转换来诱导hTSC。 在存在维持细胞因子和/或信号抑制物时的特定因素。这一假设将是 通过两个特定的目标进行测试。在目标1中，我们将开发直接谱系转换为i-hTSCs的协议 现有的hESCs和/或成纤维细胞：(1A)定义诱导 HESCs或成纤维细胞的hTSCs；和(1B)定义一组细胞因子和/或信号抑制物，使 独立于DOX的I-hTSC维护。在目标2中，我们将进行功能和分子表征 I-HTSC系列。我们的方法是创新的，因为它将开发新的生物试剂，使 早期I-hTSC的派生和维护。这项拟议的研究意义重大，因为它将使 更好地了解人类胚泡谱系的基本生物学。这样的知识具有 为妊娠并发症、不孕症和个性化药物提供新治疗方案的潜力 生殖生物学的研究方法。

PROJECT DESCRIPTION The trophectoderm (TE) is an extraembryonic tissue that supplies instructive signals required for embryo patterning during gastrulation and gives rise to the placenta, an organ that connects the developing fetus to the uterine wall to allow nutrient uptake, waste elimination, gas exchange and thermoregulation via the mother's blood supply. In mice, trophoblast stem cells (mTSCs) derived from the preimplantation blastocyst or from fibroblasts via direct lineage conversion have become an important tool with which to dissect the mechanisms responsible for TE specification, differentiation and function. However, attempts to establish TSCs with features of early human TE have been unsuccessful. Our proposal seeks to fill this gap in knowledge by establishing induced human TSCs (i-hTSCs) through the process of lineage conversion. To better understand how TSCs form in the human embryo we analyzed previously collected single cell RNA sequencing data from human blastocyst stage embryos. We identified a set of transcription factors that are highly expressed in the TE but not in the embryonic epiblast (EPI) cells. We also identified several signaling ligands expressed in the EPI and/or in the TE itself that may be responsible for specification and/or maintenance of TSCs. To facilitate lineage conversion experiments we have generated human embryonic stem cells (hESCs) that carry a fluorescent mCherry reporter linked to the endogenous GATA2 or GATA3 genes which are highly active in all cells of the TE. We have differentiated these cells to obtain ESC-derived human fibroblast-like cells. Transduction of these reporter cells with the cocktail of doxycycline (Dox)-inducible TE-specific transcription factors yielded several GATA2/3-mCherry positive clones. These clones could be passaged in the presence of Dox and expressed the key genes known to regulate TSC self-renewal. Based on these data, we hypothesize that hTSCs can be induced from fibroblasts and /or hESCs via direct lineage conversion with a cocktail of TE- specific factors in the presence of maintenance cytokines and/or signaling inhibitors. This hypothesis will be tested via two specific aims. In Aim 1, we will develop protocols for direct lineage conversion into i-hTSCs from existing hESCs and/or fibroblasts: (1A) Define the minimal set of conversion factors required for induction of hTSCs from hESCs or fibroblasts; and (1B) Define a set of cytokines and/or signaling inhibitors which allow Dox-independent maintenance of i-hTSCs. In Aim 2, we will perform functional and molecular characterization of i-hTSC lines. Our approach is innovative, because it will develop new biological reagents that would allow derivation and maintenance of early i-hTSCs. The proposed research is significant, because it would enable better understanding of fundamental biology of blastocyst lineages in humans. Such knowledge has the potential to inform new treatments of pregnancy complications, infertility and personalized medicine approaches in reproductive biology.