Prematurity/preterm birth is the leading cause of perinatal morbidity and mortality both in the U.S. and in California. These babies are at increased risk for long-term disabilities, including cerebral palsy, gastrointestinal problems, and vision and hearing loss. Many premature babies also suffer from low birth weight, which not only increases complications in the perinatal period, but also leads to increased cardiovascular disease and diabetes in adulthood. The majority of these perinatal complications result from abnormal development and function of the placenta, a transient organ that forms the interface between mother and baby. Trophoblasts are the primary cells which carry out major placental functions such as establishing blood supply from the mother to the fetus. In this application, we proposed the placenta as a novel target for stem cell therapy and sought to generate human trophoblast stem (TS) cells, which give rise to all subtypes of trophoblasts in the placenta.
During the past year, we have completed our characterization of p63, a protein we had previously identified as a potential marker of “cytotrophoblast,” the proliferative trophoblast “stem” cell in the placenta. We have a manuscript in review describing its role in maintaining the cytotrophoblast cell state in the placenta, as well as its role in inducing the trophoblast lineage in human embryonic stem cells (hESCs). In addition, we have established a protocol for differentiating hESCs first into a pure culture of cytotrophoblast, then differentiating them further into the two distinct functional, hormone-secreting trophoblast subtypes. Finally, we have learned that hypoxia both accelerates differentiation of hESCs into trophoblast, and also favors differentiation into the invasive type of trophoblast. We are now attempting to establish “defined” culture conditions, which can reproducibly differentiate hESCs into each specific trophoblast subtype.
We have also completed analysis of gene expression changes in different gestation placental samples, isolated cytotrophoblast, and differentiated cytotrophoblast, and identified multiple additional genes with potential role for maintaining the proliferative cytotrophoblast stem cell niche. A few of these have previously been shown to play a role in maintaining trophoblast stem cells in the mouse, but most appear to be specific to the human placenta. We are currently focusing on the genes involved in signaling (either transcription factors which control expression of other genes in the nucleus of the cell, or kinases which control signals in the cytoplasm of the cell) in order to determine how proliferation and the stem cell state is regulated in the human trophoblast. Over the next year, we will be evaluating the localization of these gene products in the human placenta and evaluating their function in trophoblast differentiation.
Finally, we are proceeding to address how the cytotrophoblast stem cell differentiates further into functional trophoblast. We are in process of isolating the different cell types in the placenta, then plan to characterize their gene expression, as we did with the placental and cytotrophoblast samples above, in order to identify genes involved in regulating differentiation decisions.