Correlated time-lapse imaging and single cell molecular analysis of human embryo development
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.
As noted in our application, human embryo development is poorly understood in spite of its central importance to our understanding of the natural programs and mechanisms of human nuclear reprogramming and cell fate decisions, subjects critical to stem cell biology and regenerative medicine. Thus we sought to address the hypothesis that knowledge of human embryo development can be translated to improved clinical applications in both reproductive and regenerative medicine. Further, we hypothesized that the native programs of the human oocyte to embryo transition can inform our understanding and practices in human pluripotent stem cell biology. We proposed three aims to address our hypothesis, as follows: Specific Aim 1) Examine developmental genetic programs to identify factors required to activate the human embryonic genome. Here, we proposed to uncover the relationships between imaging parameters, mRNA programs especially degradation and EGA, and epigenetic programs. We test the hypothesis that destruction of maternal RNAs occurs concurrently with epigenetic remodeling and is a prerequisite for EGA through modeling. Specific Aim 2) Determine the relationship between predictive imaging parameters and aneuploidy. Here we proposed to examine the relationship between embryonic cell behaviors and underlying chromosomal composition and then develop a fully-automated algorithm that allows non-invasive diagnosis of viability and aneuploidy in single cells. Specific Aim 3) Extend our findings to human pluripotent stem cells. We have made substantial progress on all three aims, in particular in examining epigenetic programs of human embryos, examining the relationship between aneuploidy and imaging behavior and deriving methods amenable to screening of new factors that may impact pluripotent line characteristics.
We proposed three aims to increase knowledge of basic aspects of preimplantation human embryo development, to provide the first examination of cell behavior of aneuploid blastomeres, and to utilize embryology to assist in production/diagnosis of “gold standard” pluripotent cell lines. Results of the first aim followed up on those from the last year, with studies to functionally-probe requirements for human embryonic genome activation. Results indicate that the patterns of modifications in human embryo development bear much similarity to those in mice but also demonstrate important differences. Timing and reproducibility of the patterns of methyl- and hydroxyl-methylation (modifications of DNA) suggested to us a key role in embryo development, with inheritance likely to be maternal in the first few days. Thus, we have reduced maternal expression of key players during the murine oocyte to embryo transition in order to probe the relationship to global genome activation and activation of specific programs. Results indicate a critical role in progression of embryo development, with arrest ensuing in those with reduced expression. In the second aim, we extended our studies from year 1 that had focused on imaging to the 4-cell stage, in order to encompass imaging of development to the blastocyst stage. We performed non-invasive time-lapse imaging of human embryos from the zygote to the blastocyst stage and chromosomal analysis via trophectoderm biopsy and array-comparative genomic hybridization (A-CGH). We demonstrated the range of aneuploidies that are compatible with blastocyst development and demonstrate that previously identified cell cycle parameters that are predictive of blastocyst formation are strongly predictive of blastocyst ploidy, as well. In addition, we identified several non-invasive imaging parameters beyond Day 2 that also correlated with blastocyst ploidy status. A subset of parameters is also highly predictive of blastocyst quality and thus, may assist in embryo selection. Taken together, our findings over the last year suggest that human embryo development is characterized by precise timing in developmental windows; however, aneuploid embryos have altered timing suggesting perturbation of key cell cycle processes. It is likely that assembly of a large database of human embryo data should allow description of the limits of these parameters in conjunction with normal human development. Finally, based on our studies, as reported in year one, we sought to use mRNA reprogramming in order to allow for maximum flexibility in reprogramming with novel factors. Only a few studies have addressed the molecular and functional properties of iPSCs that may predispose contribution to germ line in the mouse or human. As noted, we have derived integration-free iPSCs via use of modified mRNAs that encode the Yamanaka factors (OSKM) alone or in combination with the germ cell specific mRNA, VASA, which encodes an RNA-binding protein (OSKMV). Global gene expression profiling could not distinguish between OSKM and OSKMV iPSCs and only subtle differences were observed in expression of germ cell specific genes, epigenetic profiles and in vitro differentiation studies. We then extended our studies from the last funding period and transplanted undifferentiated OSKM and OSKMV iPSCs to mouse seminiferous tubules. We observed that xenografting of both undifferentiated OSKM and OSKMV cells resulted in production of human germ cells in mouse seminiferous tubules; the germ cell differentiation is beyond that which has been previously observed with extensive colocalization of markers, of cells to the basement membrane and other germ cell characteristics. Moreover, OSKMV and OSKM cells differed in their behavior especially in regards to development of cell masses (tumors) that might perturb testicular function. The studies highlight the divergent fates linked to iPSCs derived via exogenous expression of different factors. Overall, studies combine basic and translational approaches and are yielding novel results important to human development, diagnosis of aneuploidies and derivation of new pluripotent stem cell lines.
We proposed three aims to increase knowledge of basic aspects of preimplantation human embryo development, to provide the first examination of cell behavior of aneuploid blastomeres, and to utilize embryology to assist in production/diagnosis of “gold standard” pluripotent cell lines. Over the course of the grant, we have functionally-probed requirements for human embryonic genome activation (both genetic and epigenetic determinants), examined gene expression in single embryos and single blastomeres of embryos, examined the correlations between cell cycle parameters as determined by non-invasive imaging, and identified long intergenic noncoding RNAs (lincRNAs) that are expressed specifically in human embryos and enhance reprogramming. Overall the studies shed significant light on normal and aberrant human embryo development and have been translated to studies of pluripotent stem cell derivation and differentiation.