Understanding the status of the X chromosomes in human ESCs and preimplantation embryos
Human embryonic stem cells (hESCs) are able to divide indefinitely and under the proper conditions, can essentially become any cell in the human body. They are derived from the developing human embryo and carry great promise for regenerative medicine. However, these cells demonstrate an instability surrounding the state of the X chromosome. Male (XY) cells and female (XX) cells use a mechanism called X chromosome inactivation (XCI) to achieve equal expression of genes on the X chromosomes. XCI happens specifically in female cells and shuts down one of the two X chromosomes. The silencing of the X chromosome is a life-or-death decision that is made early in development, but when exactly remains unclear. Female cells from humans always possess one inactive and one active X chromosome. hESCs with the same X inactivation pattern as adult cells and those that have not undergone XCI have been derived, yet the cause for this heterogeneity is unknown. Given that lines can change their XCI state over time, and this volatility may have far-reaching impact, we propose to study why this happens. In addition, we will test whether the different X chromosome states affect the utility of hESCs.
The proposed project will benefit the state of California and its citizens as follows:
1. Human embryonic stem cells could revolutionize modern medicine if used in cell-based therapies. However, the translational use of hESCs will not be realized unless we can ensure reproducible derivation of high quality stem cells. Our proposal works towards identifying markers that can be used as benchmarks to assess the quality of female hESCs. Therefore, our work will have practical implications for stem cell therapy and the use of hESCs in disease studies and basic biology.
2. Our team is composed of two research groups with expertise in stem cell biology. In addition to creating highly skilled jobs, the proposed research activities will create an interdisciplinary education environment for training the next generation of California citizens at all levels, including high school, undergraduate, graduate students, as well as postdoctoral fellows.
3. All scientific findings produced from these studies will be publicly available to non-profit and academic organizations in California, and any intellectual property developed by this project will be developed under the guidelines of CIRM to benefit the State of California.
In placental mammals, dosage compensation occurs by silencing one X-chromosome in female cells, a process known as X-chromosome inactivation. Unlike female mouse embryonic stem cells (ESCs), which possess two active X chromosomes and undergo XCI upon induction of differentiation, female human ESCs exhibit various epigenetic states of the X chromosome, indicating a surprising epigenetic instability of these cells under normal culturing conditions. Since this epigenetic variation could have implications for the use of female human ESCs in regenerative medicine, disease studies, and basic research, in this proposal, we are aiming to determine how the epigenetic variability of the X chromosome arises during derivation and maintenance of human ESCs, the causes and consequences of deregulation of XCI in human ESCs, and to devise methods of stabilizing Xist expression in human ESCs. During the first funding period, we have extensively characterized the epigenetic state of the X chromosome in many established and newly derived human ESC lines as well as in human blastocysts. Together, our findings reveal new insights into the relationship between different X chromosome states in undifferentiated female human ESCs, clarify how they arise during ESC derivation, and define the implications of these X chromosome status for differentiated cells. The findings from our study have implications for the utilization and quality assessment of human ESCs.
The application of human embryonic stem cells (hESCs) requires reliable cell sources that do not change over time and initiate proper transcriptional and chromatin changes upon induction of differentiation. Therefore, it is important to understand how and when aberrancies such as the epigenetic instability of the X chromosome arise, and to define their consequences for differentiation processes and the differentiated progeny. To this end, our goal is to understand how the inactive X chromosome is regulated in human pre-implantation embryos, during derivation of hESCs from blastocysts, and during their maintenance. We have applied a large number of single cell, genome-wide, and population-wide approaches to understand this problem at a systematic and comprehensive level. Our findings define the relationship between different X-inactivation states in female hESCs and demonstrate the consequences of different X-inactivation states for hESC differentiation. Moreover, we have started to assess strategies that would prevent the instability of the inactive X chromosome and allow normal dosage compensation upon differentiation of hESCs.