In addition to the important potential applications for transplantation and treatment of chronic diseases, human embryonic stem cells (hESC) are also a valuable resource to study early human development relevant to fertility and healthy pregnancies. After fertilization, the human zygote undergoes cell divisions and ultimately becomes the blastocyst that has an inner cell mass and a trophectoderm shell, the precursor of the placenta and the tissue that attaches to the surface of the lining of the uterus, initiating the process of embryonic implantation. After attachment, the placental cells invade into the mother’s uterine lining to secure the pregnancy and to establish the placenta for fetal growth and development. Abnormalities in the implantation process can lead to infertility, small babies, and pre-eclampsia that have significant health consequences for women and children. It has been extremely difficult to study the early phases of human implantation because of lack of available tissues (human blastocysts), restrictions on using federal funds for experimentation on human embryos, and the advanced differentiated state (i.e., beyond the trophectoderm stage) of placental cells derived from early terminations of pregnancies. This grant focuses on differentiating hESC to trophectoderm, development of this specialized tissue, and interactions of it with the maternal uterus, as a model of events in the early stages of human implantation.
hESC and human embryos are essential to this project. We propose to study biological processes, biochemical pathways, and key genes expressed during the transition of hESC to trophectoderm and compare them with those of the outer shell isolated from human blastocysts. We shall also identify secreted products from these specialized cells and investigate their effects on human endometrial epithelial cells to get information about how the blastocyst communicates with the maternal uterine lining just as it is about to implant. Finally, we shall identify secreted biomarkers that can be used in future studies as a diagnostic to assess embryo quality in human IVF and as therapies to enhance endometrial receptivity to embryonic implantation in women with implantation-based infertility. This proposal has promise for important translation to improve practical issues in human infertility and pregnancy disorders associated with abnormal embryonic implantation.
We believe that our proposal has the potential to be of benefit to California and its citizens from three perspectives: 1. Basic knowledge to impart to our students. This proposal has the potential to elucidate genes, their products, biochemical pathways, and biological processes involved in the earliest stages of implantation – specifically in the shell of the blastocyst and its communication with the maternal uterine endometrium. This could add significantly to the education of our students in this important field of human development. 2. Clinical benefits from development of a diagnostic for the implantation potential of an embryo for individuals undergoing in vitro fertilization/embryo transfer, and also to improve the ability of the maternal uterus to accept and nourish an implanting embryo, important in women who have implantation-based infertility and/or a history of implantation-based pregnancy disorders, such as pre-eclampsia, intrauterine fetal growth restriction, and miscarriage.3. Commercial benefit. The development of a diagnostic test of the implantation potential of an embryo in clinical practice and of developing therapies to enhance implantation can be of major benefit to the economy of our State.
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.