Prematurity/preterm birth remains 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 last year of this grant, we had established a protocol for differentiating human embryonic stem cells (hESCs) first into a pure culture of TS cells, then differentiating them further into the two distinct functional, hormone-secreting trophoblast subtypes. During the past few months, we tried several conditions in order to maintain the hESC-derived TS cells in their stem cell state. We learned that while low oxygen keeps them from differentiating further, it does not maintain their ability to divide. For the latter, we will likely need a combination of low oxygen, appropriate matrix for the cells to “sit” on, and other chemicals in the culture medium. We recently wrote and submitted a grant to NIH to pursue this line of questioning.

The previous year, we had started to analyze gene expression changes in placental samples from different gestational ages, as well as isolated cytotrophoblast, and differentiated trophoblast. We identified multiple additional genes with potential role for maintaining the proliferative cytotrophoblast stem cell niche. We performed extensive analysis on these data during the past year and found that very few have previously been shown to play a role in maintaining trophoblast stem cells in the mouse. Most appear to be specific to the human placenta. We are currently working on a new manuscript detailing these findings. In addition, we evaluated the localization and function of a few of these genes, focusing mostly on transcription factors, because these factors tend to act as “master switches,” regulating cell behavior. We plan to expand on these findings in the near future; to that end, we have incorporated these findings in the above-mentioned grant to NIH, and are hoping to be able to follow up these findings using this new source of funding.

In summary, thanks to this grant, we have identified the human TS cell niche in the placenta, have developed a protocol for their derivation from hESCs, and begun to unravel the mechanism by which they contribute to the development and function of this important organ. In the near future, we plan to evaluate this stem cell niche in placentas of abnormal pregnancies, including those complicated by diseases leading to preterm birth. By understanding mechanisms of placental cell differentiation, we will be able to identify ways of targeting the placenta in disease and hopefully decrease the need for preterm delivery.