Human Embryonic Stem Cell Differentiation to Trophoblast: Basic Biology and Clinical Translation to Improve Human Fertility
Statement of Benefit to California:
SYNOPSIS: This proposal will address the processes of early human development using hES cells, specifically the early phases of implantation and the embryo-uterine interactions during this early implantation. Briefly, the work focuses on the differentiation of hES cells to trophectoderm (TE), the development of the TE, and the paracrine interactions between the TE and the maternal endometrium. The 3 specific aims will: 1) identify key genes and biochemical pathways that are expressed during the differentiation of hES cells to TE, and to compare these to TE isolated from human blastocysts, 2)identify secreted markers expressed during hES to TE differentiation, and investigate their effects on human endometrial epithelium, and 3) identify secreted markers expressed in TE and hES-derived TE for potential diagnostic use to evaluate embryo quality after IVF, and as potential therapeutic agents to enhance the receptivity of maternal endometrium to implantation. INNOVATION AND SIGNIFICANCE: These highly significant proposed studies highlight a neglected and innovative potential use of hES cells, specifically, for the study of early developmental aspects of human embryogenesis. Such studies are currently extremely difficult to conduct due to scarcity of human blastocysts and to the federal restrictions on embryo research. Throughout early development there are a number of reciprocal interactions between the embryo and the uterine tissues. Abnormalities in these interactions lead to defects in implantation which can result in pregnancy disorders that can lead to morbidities of the fetus and the mother. This is a critically important area of research, and it can be argued that it is uniquely accessible by utilizing hES cells together with the appropriate in vitro experimental systems. The proposed approach of comparing primary trophoblast from IVF human embryos to trophoblast derived from hESCs in vitro to elucidate key genes, secreted markers, and pathways involved in embryonic implantation is highly novel and innovative. In addtition, these studies can potentially lead to improved assisted reproductive technologies, in-vitro diagnoses and pre-implantation screening of embryos that may have a relatively rapid impact on clinical practice. STRENGTHS: Overall, this is a very strong and unique proposal. Major strengths of this proposal lie in its significance and innovation, as stated above. The environment at USCF is excellent for this work; the applicant is highly qualified and has assembled a team for this project that is highly experienced in IVF, embryology, and developmental biology, and is well qualified to carry out the proposed experiments. Additionally, Dr. Susan Fisher, an hESC expert, has agreed to support this effort as a collaborator. Another major strength of this proposal is the unique suitability of hES cells to address questions that cannot be feasibly addressed in any other way, specifically, the differentiation of human embryo cells into trophectoderm. As noted above, this phenomenon cannot be studied in other ways because of the federal funding restriction, the limited availability of human blastocysts, and the fact that aborted fetal material is already at the more mature trophblast stage. It has been shown that, unlike murine ES cells, hES differentiate readily into trophoblasts. This sets the stage for the hES cells to differentiate into early trophectoderm. In the first studies, hBMP4 will be used to explore the molecular events that occur during the hES to trophblast differentiation pathway. Here, optimal doses of hBMP4 will be determined, and microarrays will be used to monitor gene expression changes during time. These data will be compared to gene expression in normal human trophectoderm obtained from discarded blastocysts donated to research. There may be complications in obtaining appropriate numbers of blastocysts; however, the PI adequately discusses these and other potential problems. The PI also addresses issues of data analysis in an adequate manner. In the second Aim, bioinformatics approaches will be used to identify secreted proteins from the microarray datasets obtained in the first Aim. This should be straightforward, and available secreted proteins will be used to treat human endometrial cells in culture, followed by microarray analyses to monitor possible effects on gene expression. In the third Aim, a list of molecules expressed by the trophectoderm and correlate these with fcandidate ligands expressed by endometrial cells. The goal is to generate a list of candidates for diagnostic and potentially therapeutic use to evaluate and improve embryo implantation. The gene expression profiles of endometrial cells are already available. The PI is in an excellent position to obtain valuable, and potentially clinically applicible data in a relatively short time period. WEAKNESSES: While there are no significant weaknesses to this proposal, the few weaknesses include a lack of detail in the experimental approach - e.g. the experiments in the second and third Aims are rather sketchy; there is no mention of which time points and which passage numbers will be examined for the hESC lines grown in culture. There is no clear hypothesis, rather the work is more descriptive in nature and finally, the genes identified by microarray should be validated by qPCR. However, one reviewer noted that even if only the first Aim is substantially successful, the information will be invaluable. Aside from these minor weaknesses this is a very good proposal focusing on an important field of research. DISCUSSION: This is an area of science that has been completely neglected and is not fundable by NIH due to Dickey amendment. This application is a way to address that neglect.