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.
Human pregnancy disorders, such as preeclampsia and fetal growth restriction, which result from aberrations during early placental development, are difficult to study, partly due to lack of a useful human trophoblast cell culture model. This project proposes to take advantage of cels from complete molar gestations, which are composed exclusively of placental tissue, in order to establish a model for human trophoblast stem cells. Such cells would provide, for the first time, a representative in vitro system for study of human trophoblast differentiation and lineage specification.