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.
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.
SYNOPSIS: There are currently two possible approaches toward the generation of patient-specific embryonic stem cells. One involves nuclear transfer and cloning (SCNT), and the other involves reprogramming of an adult differentiated state. SCNT is very inefficient and suffers from very limited source of donated biological material. Reprogramming of somatic cells into embryonic stem cells (ESC) has recently been shown to be possible in mouse cells by the Yamanaka group as well as the Jaenisch group. Both groups have reported that overexpression of 4 transcription factors (Oct4, Sox2, c-Myc, and Klf4) can convert murine fibroblasts to ESC-like cells (induced pluripotent cells, iPS cells). The overall goal of the proposal is to understand the molecular mechanisms underlying somatic cell reprogramming to an embryonic state in mice, to improve reprogramming protocols, to apply this knowledge to the reprogramming of human somatic cells, and examine chromatin and transcription factor targets and changes during the reprogramming process. The PI has recently published a paper with a former colleague (K. Hochedlinger), comfirming the Yamanaka data and showing that selection by inspection for ESC-like morphology is sufficient to isolate iPS cells with an improved phenotype. In that study she performed the chromatin experiments showing that the chromatin of iPS cells is virtually indistinguishable from that of ESC.
STRENGTHS AND WEAKNESSES OF THE RESEARCH PLAN: Understanding the reprogramming process is highly important within the stem cell field. If reprogramming could be improved and applied to human cells, it might revolutionize approaches to the creation of disease- and individual-specific ES cells and will inevitably move the field closer to clinical applications. While this work is on the forefront of stem cell biology, and the PI is experienced and successful, leaving little doubt that interesting results will be obtained, the actual methods and approaches proposed in this application are not particularly innovative. There are assuredly many laboratories engaged in similar types of experiments attempting to adapt the Yamanaka approach to human cells.
The proposed experiments are feasible and the PI has the requisite background to address the aims. In Aim 1, several different approaches are proposed for introducing the transcription factors into cells (adenovirus, retrovirus, inducible versions, reprogrammable mice, and the introduction of additional factors). These are all defensible but the possibilities are laid out without much prioritization as to the order and weight to be applied to each. The second aim is by far the most significant one, as one can argue that regardless of the mechanism of reprogramming that the PI proposes to dissect in the mouse in Aim 1, ultimately it is the human reprogramming experiment that would be relevant. One reviewer therefore strongly suggested the prioritization of the second aim. There is no doubt in the mind of the PI and the rest of us that many human ESC groups are focused to address this issue, so far without any success, since the same strategy that was successful in the mouse does not seem to work in humans. The PI should expect tremendous scrutiny in this area from other investigators. If maximium effort is not dedicated to this aim the goal might not be achieved, and she will be scooped. In Aim 3, the PI goes back to the mouse to address the mechanism of reprogramming. There is some naivete in scientific assumptions within this aim that might or might not turn out to be valid. For example, the PI states with confidence that: “The central issue of reprogramming is clearly an epigenetic one: how is the donor genome reprogrammed to ensure correct activation of those genes needed for embryonic development and inactivation of those that are donor cell-specific . Therefore the question becomes: how do the four transcription factors induce global epigenetic remodeling of the genome?” Epigenetic influences represent only one aspect of reprogramming.
Most importantly, though, the reviewers emphasized that the proposed studies on reprogramming are highly significant in that they have the potential to move the stem cell field significantly closer toward clinical applications.
QUALIFICATIONS AND POTENTIAL OF THE PRINCIPAL INVESTIGATOR: Dr. Plath is a talented investigator with many important contributions to her field, as shown by her excellent list of publications. The PI has superb training in cell biology, chromatin biology, and ESC, and she has recently contributed her chromatin expertise to a paper on somatic reprogramming. She has also been highly successful in grant applications as she is a Kimmel Scholar and was awarded an NIH New Innovator Award. A strong body of her prior work relates to the mechanisms of X chromosome inactivation. There is no hesitation affirming that she and her team are perfectly qualified to perform all
the specific aims of this grant.The PI’s career plan is excellent, she has a clear view of how she would like to advance academically and in her research.
INSTITUTIONAL COMMITMENT TO PRINCIPAL INVESTIGATOR: UCLA is the best institution for these studies, encompassing all of the necessary resources and centers to successfully execute the proposed specific aims. The institutional commitment to the PI is outstanding, she was recruited with a strong package and has the backing of her department and the institution. The presence of a stem cell program at UCLA is an asset.
DISCUSSION: Overall, the panel expressed tremendous enthusiasm about this proposal, because of its potential impact on the stem cell field. The proposed research on reprogramming of adult human somatic cells toward an ESC-like state is of highest priority in the field, potentially paving the way for the derivation of patient-specific cell lines. Moreover, the candidate is very talented, has an excellent track record, and she is well-positioned with all the right tools in place to address reprogramming in human cells. In short, the reviewers expressed confidence that the proposed work would get done.
Clearly, this field of highest scientific priority is highly competitive, which prompted some discussion about wheher this candidate could be “first” to complete the work. But, with the right funding, this PI could be the right person to develop this methodology. Therefore, the panel agreed that the work on the human cells should be prioritized. In addition to the work on human cells, the PI proposes detailed studies on mouse cells. The science was considered very good. However, the question was raised if the mouse work would yield data relevant to the human case, since data are emerging in the field suggesting that there are many differences in reprogramming between mouse and human cells.