Derivation and characterization of induced trophoblast stem cells

诱导滋养层干细胞的衍生和表征

• 批准号: 9808504
• 负责人: Natalia B Ivanova
• 金额: $ 22.65万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-12 至 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9808504
• 关键词: Affect; Biological; Biological Assay; Biology; Cause of Death; Cell Compartmentation; Cell Differentiation process; Cells; Cessation of life; DIF factor; Data; Derivation procedure; Development; Developmental Biology; Dissection; Doxycycline; Embryo; Epiblast; Epigenetic Process; Ethics; Exhibits; Fetal Development; Fibroblasts; GATA3 gene; Gases; Gene Expression; Genes; Genetic Transcription; Human; In Vitro; Individual; Infertility; Instruction; Knowledge; Ligands; Link; Maintenance; Maps; Metabolic; Mission; Molecular; Molecular Analysis; Mothers; Moths; Mus; Mutation; Nutrient; Organ; Pathway interactions; Pattern; Physiologic Thermoregulation; Placenta; Placentation; Pregnancy Complications; Process; Proliferating; Property; Protocols documentation; Public Health; Reagent; Reporter; Reproductive Biology; Research; Research Personnel; Ribosomes; Signal Pathway; Signal Transduction; Source; Stem cells; Testing; Tissues; United States National Institutes of Health; Uterus; Vascular blood supply; base; blastocyst; burden of illness; cell type; cytokine; disability; experimental study; gastrulation; human data; human embryonic stem cell; in vivo; inhibitor/antagonist; innovation; insight; mouse model; personalized medicine; preimplantation; self-renewal; single-cell RNA sequencing; small molecule inhibitor; stem cell fate specification; stem-like cell; stillbirth; tool; transcription factor; transcriptome; trophoblast; uptake; wasting

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.

项目描述 滋养外胚层 (TE) 是一种胚胎外组织，提供胚胎所需的指导信号 原肠胚形成过程中的模式并产生胎盘，胎盘是连接发育中的胎儿的器官 子宫壁允许营养吸收、废物排除、气体交换和体温调节 母亲的血液供应。在小鼠中，滋养层干细胞 (mTSC) 源自植入前囊胚或 通过直接谱系转换从成纤维细胞中分离出来的细胞已成为剖析 负责 TE 规范、区分和功能的机制。然而，尝试建立 TSC 具有早期人类特征的 TE 尚未成功。我们的建议旨在通过以下方式填补这一知识空白： 通过谱系转换过程建立诱导性人类 TSC（i-hTSC）。为了更好地理解 我们分析了 TSC 在人类胚胎中的形成方式，之前收集了来自以下来源的单细胞 RNA 测序数据： 人类囊胚期胚胎。我们鉴定了一组在 TE 但不在胚胎外胚层 (EPI) 细胞中。我们还鉴定了在 EPI 和/或 TE 本身可能负责 TSC 的规范和/或维护。为了方便 谱系转换实验我们已经生成了携带 荧光 mCherry 报告基因与内源性 GATA2 或 GATA3 基因相关，这些基因在所有细胞中都高度活跃 TE 的单元格。我们对这些细胞进行分化以获得ESC衍生的人成纤维细胞样细胞。 用多西环素 (Dox) 诱导的 TE 特异性转录混合物转导这些报告细胞 因素产生了几个 GATA2/3-mCherry 阳性克隆。这些克隆可以在存在以下物质的情况下传代 Dox 并表达已知调节 TSC 自我更新的关键基因。根据这些数据，我们假设 hTSC 可以通过 TE-混合物的直接谱系转换从成纤维细胞和/或 hESC 中诱导出来 维持细胞因子和/或信号抑制剂存在下的特定因素。这个假设将是 通过两个特定目标进行测试。在目标 1 中，我们将开发将谱系直接转化为 i-hTSC 的协议 现有的 hESC 和/或成纤维细胞：(1A) 定义诱导 hESC 和/或成纤维细胞所需的最小转换因子集 来自 hESC 或成纤维细胞的 hTSC； (1B) 定义一组细胞因子和/或信号传导抑制剂，允许 i-hTSC 的不依赖 Dox 的维护。在目标 2 中，我们将进行功能和分子表征 i-hTSC 系。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 的衍生和维护。拟议的研究意义重大，因为它将使 更好地了解人类囊胚谱系的基础生物学。这样的知识有 为妊娠并发症、不孕不育和个性化医疗的新疗法提供信息的潜力 生殖生物学方法。