We understand little about human development especially at the earliest stages. Yet human developmental biology is very important to stem cell biology and regenerative medicine for two reasons: 1) Understanding human developmental pathways especially of embryonic differentiation will inform our efforts to derive pluripotent stem cells and differentiate them to stable progenitors that are suitable for transplantation or pharmaceutical applications. Clearly, human development follows well-defined pathways that we are just beginning to elucidate. 2) Understanding human development will allow us to translate findings to the clinic to alleviate common problems of women's and children's health. Errors in the earliest stages of development are the most common cause of all birth defects in the human population and yet we know little of the fundamental ways in which errors occur. Our lack of knowledge is likely enhanced by the complete ban of federal funding for this research in spite of the fact that each year there is increased clinical use of procedures such as IVF.
Thus, here we seek to build a map of human development that combines imaging (microscopic) data, molecular data, genetic and epigenetic data to describe human pluripotent blastomeres (cells) and their potentials. We note that events in the first few cell divisions, even before human embryos turn on their own genes, have repercussions to later generations of cells and the overall health and welfare of the embryo and fetus (and likely adult).
Our goals are based on our research over several years in which we initiated construction of a map of pathways and programs that function during embryo development. Our studies provide methods and algorithms for early diagnosis of embryo potential in clinics and should be extended to the diagnosis of the general health of pluripotent stem cell populations. We expect that via translation of our basic studies to the clinic, we will improve outcomes of IVF in terms of birth of healthy offspring and decrease devastating and common adverse outcomes such as multiple births with attending complications to organ development, epigenetic errors that may result in miscarriage, and need to reduce fetal number to increase odds of survival of siblings and/or mother. Thus, this research may yield benefits to both maternal/fetal health and stem cell biology and regenerative medicine.
Stem cell biology and regenerative medicine holds great promise for the citizens of California in terms of establishing a superior basic science infrastructure, translating findings to the clinic for improved health and designing new diagnostics to prevent disease and allow screening of new pharmaceutical agents. There are many promising applications with pluripotent human embryonic stem cells (hESCs) and clinical trials are beginning in several biomedical applications. Yet, we lack an understanding of fundamentals of human biology, especially of the earliest stages. Indeed, we know remarkably little about the relationship of human embryonic cells (blastomeres in the preimplantation embryo), hESCs and induced pluripotent stem cells. This lack of knowledge is costly to the health of Californians today and in the future. First, we have numerous practices in the reproductive arena that carry a substantial burden in terms of diverse adverse outcomes that include prenatal birth and associated risks, increased risk of epigenetic/genetic errors in development, and a need to balance the safety of carrying a multiple-pregnancy with the health of the mother. It is clear that we can improve the health of women and children through knowledge of human embryo development, even though federal funding is not allowed in this arena. Moreover, we can provide increased knowledge of the legitimacy (relationship to nature) of stem cell lines that we derive and thus employ natural programs to improve pluripotent stem cell lines. Successful completion of this research will lead to clinical applications and positively impact a relatively-large segment of the California population.
This proposal is focused on determining the normal processes of early human development from the one cell to the blastocyst stage with the ultimate goal of improving outcomes of in vitro fertilization (IVF) techniques by better understanding viability indices for embryo selection. This knowledge will then be applied to improve the derivation and analysis of human pluripotent stem cell lines. Specifically, in Aim 1, the applicants will examine the molecular basis of genome activation in human embryos. In Aim 2, they will determine the relationship between certain imaging parameters and aneuploidy as a means of predicting the quality of embryos. The goal of Aim 3 is to use this information to identify embryos optimal for derivation of human embryonic stem cells (hESC), to improve derivation of human induced pluripotent stem cells (hiPSC), and to develop a set of imaging parameters that correlate with the quality and stability of hESC and hiPSC lines.
Significance and Innovation:
- This proposal is highly significant since Aim 1 directly tackles the understanding of the human embryo - a neglected field in embryology. As human embryos display features that are not necessarily found in embryos of model systems, a direct and basic approach is necessary to understand the molecular basis of our own origin.
- During IVF treatments, it is highly preferable to transfer single embryos in order to avoid the problems associated with multiple births. If the applicants can develop improved non-invasive methods for identifying the best embryos for IVF as proposed in Aim 2, this could change practice and improve outcomes of IVF treatments. However, other techniques to detect aneuploidy in human embryos exist. Thus, while interesting, this is not as compelling as the goal of Aim 1.
- The approach used in Aim 3 is significantly different from that used by others in the field and has the potential to improve the quality of pluripotent stem cell lines over those derived using current technologies.
- Improvements are likely to be incremental rather than transformative. However, such incremental improvements in quality could be very important, especially in terms of hiPSC derivation, since current research is showing considerable variation and genetic alterations among different lines. The rationale for using better embryos to derive new hESC lines is not as strong, as good quality human embryos do not represent a limiting factor for hESC derivation.
- The proposed research is innovative, as it effectively brings together simple time-lapse imaging, state of the art chromosome analysis and molecular techniques, which should have a positive impact on the stem cell field.
Feasibility and Experimental Design:
- The proposal, although well written, is comprised of only loosely related aims.
- The background data provided for this study are very strong and compelling; most of it is already published.
- Due to a lack of detail in Aim 1, it remains unclear whether the challenges associated with single cell analysis can be met and whether the scope of the proposed studies is manageable, as a potentially large number of single cells will have to be analyzed.
- Zygotes donated for research tend to be lower quality, raising questions about the generalizability of the findings.
- Aim 2 is straightforward and achievable as it is supported by strong preliminary data. A reviewer suggested extending the analysis beyond the blastomere stage to the blastocyst stage, since aneuploid embryos do progress to that stage in the human and at least some cleavage-stage aneuploid embryos self-correct their ploidy.
- In Aim 3, some experimental detail is lacking; for example, it is unclear how the quality of the newly derived hESC will be evaluated relative to other lines. On the other hand, details regarding improvements to hiPSC derivation are well described and supported.
Principal Investigator (PI) and Research Team:
- The applicant is a highly accomplished, extremely well funded stem cell researcher with extensive publications and relevant experience and is ideally suited to oversee the proposed research.
- The assembled team is impressive. A co-investigator with bioinformatics expertise has a strong track record.
- The research facilities and environment are excellent, and the PI has access to a critical, unique resource.
- A large proportion of the budget is for personnel; since the proposed research is expensive, not enough funds have been allocated to supplies.
Responsiveness to the RFA:
- This proposal is only partially responsive to the RFA, since the goal to improve outcomes in IVF, while laudable, has minimal relevance to stem cell research.
- The goal of using knowledge gained about human embryo development to help improve generation of pluripotent stem cells is responsive to the RFA.
- While there is some attempt to place the experiments in the context of understanding the mechanisms of genome activation during development, this is not a proposal that is highly focused on molecular mechanisms. It really is focused on developing a practical approach to improve the quality of IVF embryos and of pluripotent stem cell lines.