Discovery of mechanisms that control epigenetic states in human reprogramming and pluripotent cells
The CIRM Basic Biology Award III was developed to support basic research that enables the realization of the full potential of human stem cells and reprogrammed cells for therapies and as tools for biomedical innovation. This is particularly important since many fundamental issues related to the regulation of stem cell fate and reprogramming, especially with regard to human cells, remain to be resolved. X chromosome inactivation (XCI) is one of those fundamental processes of human development related to stem cell biology and reprogramming, that we know surprisingly little about, and we therefore propose to study the regulation of XCI in human cells in this proposal using human induced pluripotent stem cells (iPSCs) as model system.
A normal female has two X chromosomes and no Y chromosome and males have one X and one Y chromosome. To be equal with males, females must shut off one of two X chromosomes during embryonic development by inducing XCI, such that only one X chromosome remains active in every cell of the female body. Females even become genetic mosaics by randomly inactivating either the X chromosome inherited from the father or the mother, which has important consequences for the clinical phenotype of X-linked diseases between the two sexes.
Studies on XCI in the mouse model system have revealed that female embryonic stem cells (ESCs) carry two active X’s and that XCI must be initiated when these cells are induced to differentiate. XCI is an epigenetic phenomenon that occurs without alterations in the primary sequence of DNA by formation of a repressive heterochromatin structure. Intriguingly, this heterochromatin structure can be erased when adult murine cells are reprogrammed to the ESC-like state of iPSCs.
Findings in the human system are less clear as typical female human ESCs and iPSC lines have an inactive X chromosome that can change its composition with extended culturing indicating potential epigenetic instability. It is now also thought that these cells don't represent the same developmental state as mouse ESCs and iPSCs. In agreement with this notion, human pluripotent cells with two active X chromosomes have recently been generated that appear to resemble the mouse ESC state. Thus, there are now at least two different human pluripotent states that also differ in their X chromosome status. We believe that our proposed studies of XCI regulation during differentiation and reprogramming in human cells and in these different human ESC states will not only unveil mechanisms underlying this fundamental silencing process and human development, but also be instrumental for the careful characterization of these different human pluripotent states. This is particularly important given that human ESCs and iPSCs carry a tremendous promise for therapeutic applications and for modeling of human development and diseases, and that the XCI status in these cells also will have specific implications for modeling of X-linked diseases.
The proposed project will benefit the state of California and its citizens as follows:
1. Better characterization of human induced pluripotent cells (iPSCs) for use in disease studies and cell-based treatments. Human iPSCs hold great potential for regenerative medicine to treat many devastating injuries and diseases such as Alzheimer’s disease, Parkinson’s disease, diabetes, cancer, rheumatoid arthritis, and spinal cord injuries. Our studies will help to define different human iPSC states and assess their epigenetic stability. This would be beneficial to the people of California as tens of millions of Americans suffer from diseases and injuries that could benefit from a detailed characterization and understanding of the biology of human iPSCs. Such advances would benefit the health as well as the economy of the state of California.
2. Advances in understanding the reprogramming process to the iPSC state. X chromosome inactivation is one of the most dramatic forms of developmentally regulated heterochromatin formation that is reset during the reprogramming process. Understanding how the inactive X chromosome reactivates should reveal epigenetic mechanisms that stabilize the differentiated state and therefore eventually enable the development of safer and more efficient reprogramming approaches. In addition, the X chromosome status may be used as an indicator of complete reprogramming to the naïve pluripotent state.
3. Studies of X-linked diseases using human iPSCs. As X chromosome inactivation can alter the consequences of X-linked mutations, a better understanding of the X chromosome state in human hiPSCs is essential for their use in the modeling of X-linked diseases in vitro.
4. Education and jobs for next-generation California scientists. 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.
Fundamental issues related to the regulation of stem cell fate and reprogramming, especially with regard to human cells, remain to be resolved. X chromosome inactivation is one of those important processes of human development related to stem cell biology that we know surprisingly little about. A normal female has two X chromosomes and no Y chromosome and males have one X and one Y chromosome. To be equal with males, females must shut off one of two X chromosomes during embryonic development by inducing the silencing of one of the two X chromosomes (X-inactivation), such that only one X chromosome remains active in every cell of the female body. X-inactivation is an epigenetic phenomenon that occurs without alterations in the primary sequence of DNA by formation of a repressive heterochromatin structure early in development. However, how the initiation of X-inactivation is regulated in human development and how the inactive X chromosome is then stably maintained throughout cell division remains unclear. In this work, we study these questions using human induced pluripotent stem cells (iPSCs) as model system. Since various epigenetic states of the X chromosome have been observed in human iPSCs, our work is also important for the characterization and classification of the epigenetic state of human iPSCs, which has implications for the use of these cells in the laboratory and clinic. During the past funding period, we have worked towards the establishment of the iPSC lines that will be used for the characterization of the X-inactivation process.
Many fundamental issues related to the regulation of stem cell function and fate, especially with regard to human cells, remain to be resolved. X chromosome inactivation (XCI or X-inactivation) is one of those fundamental processes of human development related to stem cell biology, that we know surprisingly little about. To be equal with males, females shut off one of two X chromosomes during development by inducing X-inactivation, such that only one X chromosome remains active in every cell of the human female body. XCI is an epigenetic phenomenon that occurs without alterations in the primary sequence of DNA but instead by formation of a repressive heterochromatin structure on an entire X chromosome. Studies on X-inactivation in the mouse model system have revealed that female embryonic stem cells (ESCs) carry two active X’s and that the X-inactivation process is initiated when these cells differentiate such that all differentiated cells have an inactive X chromosome. Intriguingly, the heterochromatin structure of the inactive X chromosome can be erased when adult cells of the mouse are reprogrammed to the mouse ESC-like pluripotent state also coined induced pluripotency. Notably, the X-inactivation process is much less studied in the human system. Furthermore, some of the existing data already suggest that the process is regulated differently in human than in mouse. Therefore, in this project, our goal is to begin to understand how XCI is initiated in the human system. Towards this end, we are working towards the establishment of a pluripotent stem cell model that allows us to follow the initiation of XCI in a cell culture model. We are proposing that the identification of such pre-XCI human pluripotent cells, which undergo X-inactivation upon differentiation, has implications for the use of human pluripotent cells in therapeutic applications, modeling of human development and diseases, as the XCI status affects all these applications. During the past funding period, we have tested various approaches for the generation of pre-XCI human pluripotent cells and are currently further refining these methods. Along this line, we established tools for better identifying the pre-XCI human pluripotent cells and studying the initiation of X-inactivation. In addition, we made considerable progress in defining the epigenetic instability of the inactive X in certain pluripotent human stem cell lines.
X-inactivation is a fundamental process of mammalian development, leading to the transcriptional silencing of one of the two X chromosomes in female cells. Studies of the X-inactivation process in the human system have been limited due to the lack of a cellular system that faithfully recapitulates the initiation of X-inactivation. In the mouse, ESCs faithfully recapitulate the initiation of X-inactivation and induce expression of the long noncoding RNA Xist, the major regulator of X-inactivation, on one of the two X chromosomes upon differentiation, leading to the silencing of the chromosome. Thus, mouse ESCs carry two active X chromosomes (Xa’s) and gain an XIST RNA-coated inactive X (the Xi) upon differentiation. Contrary, conventionally cultured human pluripotent stem cells (hPSCs) (both ESCs and iPSCs) do not display a stable XaXa state that allows silencing of the X chromosome via induction of XIST upon induction of differentiation. Instead, these cells display varying epigenetic states of the X chromosome, predominantly carrying an inactive X (Xi) with XIST expression, an Xi without XIST expression, or a partially reactivated Xi, the eroded Xe, and even change their X state over time in culture. Furthermore, none of the X-states found in human PSCs resembles that of the human blastocyst (XaXa with both chromosomes expressing XIST). Therefore, typical human PSCs are in a developmental state that endows an epigenetic instability on the X chromosome, and is past the early developmental window that allows for de novo initiation of X-inactivation. Given the importance of X-inactivation for development and its close ties to pluripotency, surprisingly little is known about how X-inactivation is initiated in the human system. To overcome this gap in knowledge, in this proposal, we established and characterized a pluripotent cell culture system that resembles the blastocyst-like pattern of the X chromosome. With this tool in hand, we are studying the mechanisms of X-inactivation during human development, and are addressing how XIST function in human cells differs from that in the mouse. In addition, we are testing whether these cells are epigenetically better suited for both basic scientific and translational goals than hPSCs in grown under conventional culture conditions.