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

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妊娠成功至关重要。滋养层细胞构成胎盘的上皮区室，并介导营养/气体交换功能和母体血流进入胎儿-胎盘单位的建立。胚胎早期滋养层细胞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，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一个有用的人滋养层干细胞培养系统，其中分化和谱系特化可以在分子水平上进行检查。目前的人滋养层细胞系（即HTR 8、BeWo、JEG 3）在其体内分化潜力和与滋养层的功能相似性方面是有限的。此外，尽管滋养层干细胞能够 由于胎盘形态、滋养层亚型和谱系特异性功能能力的主要差异，小鼠模型的使用有限。人类滋养层干细胞还有待开发。最近，几个小组已经显示了人胚胎干细胞（hESC）的BMP 4处理后的滋养层分化;然而，很难在这个系统中鉴定滋养层干细胞，部分原因是在BMP 4添加后的早期培养物中，其他所得谱系，特别是中胚层的混合物。我们最近已经表明，诱导多能干细胞（hiPSC），类似于hESC，能够分化成滋养层后BMP 4处理。因此，我们建议利用完全的磨牙妊娠，这是完全的父亲的起源，不能产生任何胚胎组织，并导致一个亚型的妊娠滋养细胞疾病的特征是异常的胎盘组织与绒毛膜绒毛和滋养细胞增生。我们将从这些磨牙胎盘中分离成纤维细胞，并使用表达Klf 4、Sox 2、Oct 4和c-Myc的慢病毒或仙台病毒将其重编程为iPS细胞。然后，我们将使用BMP 4将这些iPSC分化为滋养层细胞，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衍生的滋养层细胞将提供一个更统一的细胞培养系统，用于定义人类“滋养层干细胞”的细胞特征。这些细胞的表征将进一步为我们提供表面标志物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。

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.