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: 10017694
• 负责人: Natalia B Ivanova
• 金额: $ 18.88万
• 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 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10017694
• 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; Estrogen receptor positive; 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; 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 cells; 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（trophectoderm，TE）是一种胚外组织，提供胚胎发育所需的指导性信号 在原肠胚形成期间形成图案，并产生胎盘，胎盘是连接发育中的胎儿和 子宫壁，以允许营养吸收，废物消除，气体交换和体温调节，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一个重要的工具， 负责TE规格、分化和功能的机制。然而，建立TSC的尝试 与早期人类TE特征的结合并不成功。我们的建议旨在填补这一知识空白， 通过谱系转化过程建立诱导的人TSCs（i-hTSCs）。更好地了解 我们分析了先前收集的单细胞RNA测序数据， 人类囊胚期胚胎。我们鉴定了一组转录因子，这些转录因子在细胞中高度表达。 TE，而不是在胚胎外胚层（EPI）细胞。我们还鉴定了几种信号配体， EPI和/或TE本身，其可能负责TSC的规范和/或维护。以促进 谱系转换实验我们已经产生了人类胚胎干细胞（hESC）， 荧光mCherry报告基因与内源性GATA 2或GATA 3基因连接，这些基因在所有细胞中都是高度活性的。 TE的细胞。我们已经分化这些细胞以获得ESC衍生的人成纤维细胞样细胞。 用多西环素（Dox）诱导的TE特异性转录的混合物转导这些报告细胞 因子产生几个GATA 2/3-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并表达了已知调节TSC自我更新的关键基因。基于这些数据，我们假设 hTSCs可以从成纤维细胞和/或hESC通过用TE-1的混合物的直接谱系转化来诱导。 在维持细胞因子和/或信号传导抑制剂的存在下的特异性因子。这一假设将是 通过两个具体目标进行测试。在目标1中，我们将开发用于从人造血干细胞直接谱系转化为i-hTSCs的方案。 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;和（1B）定义一组细胞因子和/或信号传导抑制剂， i-hTSC的Dox非依赖性维持。在目标2中，我们将进行功能和分子表征 i-hTSC细胞系。我们的方法是创新的，因为它将开发新的生物试剂，从而允许 早期i-hTSC的衍生和维持。这项研究意义重大，因为它将使 更好地了解人类胚泡谱系的基础生物学。这样的知识， 为妊娠并发症、不孕症和个性化医疗提供新的治疗方法的潜力 生殖生物学方法。