Embryonic stem (ES) cells are remarkable cells in that they can replicate themselves indefinitely and have the potential to turn into all possible cell type of the body under appropriate environmental conditions. These characteristics make ES cells a unique tool to study development in the culture dish and put them at center stage for regenerative medicine. Two techniques, one called somatic cell nuclear transfer (SCNT) and the other in vitro reprogramming, have shown that adult cells from the mouse can be reverted to an ES like state. In SCNT, adult cell nuclei are transferred into oocytes and allowed to develop as early embryos from which ES cells can be derived, while in the in vitro method four genes are ectopically activated in the adult cell nucleus to induce an embryonic state in the culture dish. Key requirement for both processes is to erase the memory of the adult cell that specifies it as an adult cell and set up the ES cell program. How this happens remains unclear, and if it can be reproduced with human adult cells is an open question. Therefore, we will attempt to use the in vitro reprogramming method to generate human ES cells from adult cells and begin to understand the mechanism of the reprogramming process in both human and mouse cells. In addition to being integral to improving our understanding of how ES cells develop, if successful, this work will provide an important milestone for regenerative medicine. Many debilitating diseases and conditions are caused by damage to cells and tissue. In vitro reprogramming could provide a way to generate patient-specific stem cells that, in culture, could be turned into the type of cell or tissue needed to cure the patient’s disease or injury and transplanted back into the patient’s body. For example, Parkinson’s disease is caused by the loss or destruction of nerve cells. If reprogramming becomes possible, we could take a skin biopsy from a patient with Parkinson’s disease, induce the embryonic state in those skin cells to then be able to turn them into nerve cells and transplant them back into the same donor patient. Reprogramming could also be used to repair spinal cord injuries, allowing people who are paralyzed by accidents to walk again, or be helpful for patients with juvenile diabetes. One important advantage of patient-specific self-transplants is that they obviate the need for immunosuppression, which is often problematic for the patient. In addition, human cell reprogramming could be a new way to study how diseases progress at the cellular level as reprogramming could generate ES cells from patients with complex diseases that can be studied in detail for what makes them go awry during development. This knowledge could speed the search for new treatments and possibly cures for some of the most complex diseases that affect societies. We hope that the knowledge gained from our studies on reprogramming can, someday, support research that will help to put these idea to clinical use.
Statement of Benefit to California:
Donated organs and tissues are often used to replace those that are diseased or destroyed, but unfortunately, the number of people needing a transplant exceeds the number of organs available for transplantation. Embryonic stem (ES) cells can be propagated in the laboratory for an unlimited period of time and can turn into all the specialized cell types that make us a human being. Therefore, ES cells offer the possibility of a renewable source of replacement cells and tissues to treat diseases, conditions, and disabilities such as Parkinson’s and Alzheimer’s, spinal cord injury, stroke, burns, heart disease, diabetes, osteoarthritis and rheumatoid arthritis. Our research is aimed to generate ES cells from adult cells through a method called in vitro reprogramming and to understand the mechanism by which the ES cell program can be reinstated in the adult cells. This work will not only provide the foundation for a better understanding of how human ES cells develop, but, if successful, be an important milestone for regenerative medicine. The advantage of using ES cells derived from adult cells by in vitro reprogramming would be that the patient’s own cells could be reprogrammed to an ES cell state and therefore, when transplanted back into the patient, not be attacked and destroyed by the body’s immune system. This would be beneficial to the people of California as tens of millions of Americans suffer from diseases and injuries that could benefit from research of in vitro reprogramming. Such advances would benefit the health as well as the economy of the state of California.
Year 1The discovery of induced pluripotent stem (iPS) cells by Shinya Yamanaka in 2006 marks a major landmark in the fields of stem cell biology and regenerative medicine. iPS cells can be obtained by co‐expression of four transcription factors in differentiated cells. The reprogramming process takes 2‐3 weeks and is very inefficient with about 1 in a 1000 somatic cells giving rise to an iPS cell. In previous work, we and others had demonstrated that mouse iPS cells are highly similar to ES cells in their molecular and functional characteristics as they for example can support adult chimerism with germline
contribution. The goal of the New Faculty Award proposal is to understand the molecular mechanisms underlying transcription factor‐ induced reprogramming of differentiated cells and to define the iPS cell state.
During this funding period, our efforts have focused on all three Aims. Within Aim 1, we have addressed a range of technical strategies to improve the reprogramming process. In Aim 2, we have analyzed human and mouse iPS cells in comparison to ES cells and attempted a better definition of the iPS cell state. In Aims 3, we are currently attempting to define barriers of the reprogramming process and begin to understand the transcriptional network that leads to reprogrammed cells.
Year 2The discovery of induced pluripotent stem (iPS) cells, which are derived from differentiated cells by simply overexpression a few transcription factors, by Shinya Yamanaka in 2006 marks a major landmark in the fields of stem cell biology and regenerative medicine. To unfold the full potential of reprogramming for disease studies and regenerative medicine, we believe that it is important to understand the molecular mechanisms underlying transcription factor‐ induced reprogramming and to carefully characterize the iPS cell state. To this end, during the third year of funding, we have devised a novel screen to identify factors important for the reprogramming process and allow replacement of the original reprogramming factors. We also studied the role of candidate transcriptional and chromatin regulators in the reprogramming process, which led us to identify novel barriers of the reprogramming process and to a better understanding of how chromatin interferes with the reprogramming process. We have also made progress in understanding the function of the reprogramming factors. Regarding human iPS cell lines, we have derived iPS cells from patients carrying X-linked diseases, and are beginning to characterize them molecularly. Together, we hope that our work will contribute to a better understanding of the reprogramming process.
Year 3Cellular reprogramming and the generation of induced pluripotent stem cells (iPSCs) from differentiated cells has enabled the creation of patient-specific stem cells for use in disease modeling. Reprogramming to the induced pluripotent state can be achieved through the ectopic expression of Oct4, Sox2, Klf4 and cMyc. Insight into the role that the reprogramming factors, various signaling pathways and epigenetic mechanisms play during the different stages of reprogramming remains limited, partly due to the low efficiency with which somatic cells convert to pluripotency. During the past year we have made great progress in (i) defining the molecular requirement for the reprogramming factors; (ii) gaining a better understanding of how repressive chromatin states control the reprogramming process; (iii) determining the differential regulation of chromatin states during reprogramming; (iv) identifying novel reprogramming stages; (v) assessing the three-dimensional organization of the genome during reprogramming; and (vi) determining the influence of a specific signaling pathway and its downstream effectors on different stages of the reprogramming process. Together, our findings provide novel mechanistic insights into the reprogramming process, which will form the basis of approaches to approve the efficiency of the process.
Year 4When this grant was awarded in 2008, reprogramming to the induced pluripotent state was just achieved by Shinya Yamanaka through the ectopic expression of Oct4, Sox2, Klf4 and cMyc in mouse fibroblasts. The overall goal of this proposal was to understand the molecular mechanisms underlying in vitro reprogramming of somatic cells of the mouse to iPSCs and to apply this knowledge to the reprogramming of human somatic cells. During the last funding period, our work particularly aimed at mechanistic questions: (i) determining the molecular origin of the spatio-temporal demarcation of the DNA binding sites of the reprogramming factors, and how the reprogramming factors induce chromatin changes, employing systematic and comprehensive mapping approaches; (ii) defining how the reprogramming factors induce a specific transcriptional output on target genes; (iii) identifying the steps of the reprogramming process to mouse iPSCs, which revealed an unprecedented detail of the reprogramming process and established that transition through a multitude of hierarchical stages is a fundamental feature of the reprogramming process; (iv) determining the dynamics of DNA methylation in reprogramming; (v) gaining a better understanding of how repressive Polycomb proteins control the reprogramming process; (vi) assessing the three-dimensional organization of the genome during reprogramming; and (vii) using the human iPSC approach for disease studies. Together, our findings provide novel mechanistic insights into the reprogramming process.