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 published a manuscript describing its role in maintaining the cytotrophoblast stem cell state in the placenta, as well as its role in inducing the trophoblast lineage in human embryonic stem cells (hESCs). As part of the same study and publication, we also showed that, when compared to many different types of human cells and tissues, hESC-derived cytotrophoblast most closely resemble trophoblast isolated from the placenta. This was an important milestone, as a recent publication had challenged the validity of the hESC-derived cells as bona fide trophoblast.
In the previous year, we had established a protocol for differentiating hESCs first into a pure culture of cytotrophoblast stem cells, then differentiating them further into the two distinct functional, hormone-secreting trophoblast subtypes. This past year, we have fine-tuned the culture conditions further, learning that both oxygen tension and the extracellular matrix (ECM)—the material the cells “sit on” in culture–may direct them further into each of the trophoblast subtypes. We are currently testing a combination of oxygen tension and ECM materials in order to optimize differentiation of the cytotrophoblast stem cells into either of the two main trophoblast subtypes: “villous” trophoblast which secretes the pregnancy hormone hCG, and “extravillous” trophoblast which invades the uterine tissue in order to gain access to maternal blood for fetal growth.
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 presented this work at an international conference on the placenta this past year and are currently working on a new manuscript detailing these findings. In addition, we have been evaluating the localization and function of a handful of these genes, focusing mostly on transcription factors, because these factors tend to act as “master switches,” regulating cell behavior. Overall, these data have given us extensive insight into how the human placental develops over time. In the near future, we plan to evaluate these same genes 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.