Modeling human trophoblast stem cells using iPS cells derived from molar placenta

使用源自臼齿胎盘的 iPS 细胞模拟人类滋养层干细胞

• 批准号: 8511232
• 负责人: Mana M Parast
• 金额: $ 19.38万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-07-15 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8511232
• 关键词: Activities of Daily Living; Animal Model; BMP4; Biological Assay; Blood Vessels; Blood flow; CDKN1C gene; CDX2 gene; Cell Culture System; Cell Culture Techniques; Cell Line; Cell physiology; Cell surface; Cells; Chorionic villi; Chronic; Commit; Complete Hydatidiform Moles; Conceptus; DNA; Data; Derivation procedure; Development; Diagnosis; Disease; Dose; Embryo; Epithelial; Erythroblasts; Evaluation; Fertilization; Fetal Growth Retardation; Fetal Tissues; Fibroblasts; First Pregnancy Trimester; Gases; Gene Expression; Gene Expression Profiling; Genes; Genome; Growth Factor; HLA G antigen; Hospitals; Human; Human Chorionic Gonadotropin; Hydatidiform Mole; Hypoxia; In Vitro; Lead; Life; Maintenance; Mediating; Mesoderm; Modeling; Molecular; Molecular Profiling; Morbidity - disease rate; Morphology; Mus; Nutrient; Organ; Ovum; Partial Hydatidiform Mole; Phenotype; Placenta; Placentation; Pluripotent Stem Cells; Population; Pre-Eclampsia; Pregnancy; Pregnancy Complications; Protocols documentation; Sampling; Sendai virus; Side; Stem cells; Subfamily lentivirinae; Surface; Syncytiotrophoblast; System; Teratoma; Time; Tissues; Triploidy; Tumor Suppressor Proteins; Villous; Villus; Woman; base; blastocyst; cell behavior; cytotrophoblast; embryo tissue; fetal; fetal blood; human embryonic stem cell; human embryonic stem cell line; imprint; in vivo; induced pluripotent stem cell; mortality; mouse model; paternal imprint; pluripotency; pregnancy disorder; progenitor; public health relevance; sperm cell; success; transcription factor; trophoblast

DESCRIPTION (provided by applicant): The placenta is a fetal-derived organ, whose proper development and function are pivotal in pregnancy success. Trophoblast cells comprise the epithelial compartment of the placenta, and mediate nutrient/gas exchange functions and establishment of maternal blood flow into the feto-placental unit. Abnormal differentiation of trophoblast during early embryonic life is thought to lead to chronic feto-placental hypoxia and result in pregnancy complications associated with both maternal and fetal morbidity and mortality, such as preeclampsia and fetal growth restriction. One major limitation in studying these diseases is the lack of a useful human trophoblast stem cell culture system, where differentiation and lineage specification can be examined at the molecular level. Current human trophoblast cell lines (i.e. HTR8, BeWo, JEG3) are limited in their differentiation potential and functional resemblance to trophoblast in vivo. Also, despite the ability to derive trophoblast stem cells from blastocysts, mouse models are of limited use, due to major differences in placental morphology, trophoblast subtypes, and lineage-specific functional abilities. Human trophoblast stem cells have yet to be derived. Recently, several groups have shown trophoblast differentiation following BMP4 treatment of human embryonic stem cells (hESCs); however, it has been difficult to identify trophoblast stem cells in this system, partly due to the mixture of other resulting lineages, in particular mesoderm, in early cultures following BMP4 addition. We have recently shown that induced pluripotent stem cells (hiPSCs), similar to hESCs, are capable of differentiating into trophoblast following BMP4 treatment. We therefore propose to take advantage of complete molar gestations, which, being exclusively of paternal origin, are unable to give rise to any embryonic tissue and result in a subtype of gestational trophoblastic disease characterized by abnormal placental tissue with hydropic chorionic villi and trophoblastic proliferation. We will isolate fibroblast from these molar placentas and reprogram them into iPS cells using lenti- or Sendai virus expressing Klf4, Sox2, Oct4, and c-Myc. We will then differentiate these iPSCs into trophoblast using BMP4 and characterize the resulting cells at early timepoints following treatment. We hypothesize that these molar iPSC-derived trophoblast will provide a more uniform cell culture system for defining the human "trophoblast stem" cell signature. Characterization of these cells will further provide us with surface markers and lineage-specific transcription factors by which to define this cell population and will help to establish culture conditions for their maintenance in vitro.

描述（由申请人提供）：胎盘是胎儿来源的器官，其正常发育和功能对于妊娠成功至关重要。滋养层细胞构成胎盘的上皮区室，介导营养/气体交换功能以及母体血流进入胎儿胎盘单位的建立。早期胚胎生命期间滋养层的异常分化被认为会导致慢性胎儿胎盘缺氧，并导致与母体和胎儿发病率和死亡率相关的妊娠并发症，例如先兆子痫和胎儿生长受限。研究这些疾病的一个主要限制是缺乏有用的人类滋养层干细胞培养系统，可以在分子水平上检查分化和谱系规范。目前的人类滋养层细胞系（即 HTR8、BeWo、JEG3）的分化潜力和与体内滋养层的功能相似性有限。此外，尽管能够衍生滋养层干 来自囊胚的细胞，由于胎盘形态、滋养层亚型和谱系特异性功能能力的巨大差异，小鼠模型的用途有限。人类滋养层干细胞尚未产生。最近，多个研究小组在 BMP4 处理人胚胎干细胞 (hESC) 后显示出滋养层分化；然而，很难识别该系统中的滋养层干细胞，部分原因是在添加 BMP4 后的早期培养物中混合了其他所得谱系，特别是中胚层。我们最近发现，与 hESC 类似，诱导多能干细胞 (hiPSC) 在 BMP4 处理后能够分化为滋养层。因此，我们建议利用完全磨牙妊娠，这种妊娠完全是父系起源，无法产生任何胚胎组织，并导致妊娠滋养细胞疾病的亚型，其特征是胎盘组织异常，伴有绒毛膜绒毛水肿和滋养细胞增殖。我们将从这些臼齿胎盘中分离出成纤维细胞，并使用表达 Klf4、Sox2、Oct4 和 c-Myc 的慢病毒或仙台病毒将其重新编程为 iPS 细胞。然后，我们将使用 BMP4 将这些 iPSC 分化为滋养层，并在治疗后的早期时间点表征所得细胞。我们假设这些磨牙 iPSC 衍生的滋养层将为定义人类“滋养层干”细胞特征提供更均匀的细胞培养系统。这些细胞的表征将进一步为我们提供表面标记和谱系特异性转录因子，通过它们来定义该细胞群，并将有助于建立其体外维持的培养条件。