Pluripotent stem cells can give rise to any cell type of the body and hold enormous promise for regenerative medicine. Pluripotent stem cells, such as embryonic stem (ES) cells, are derived from very young human embryos. It is of great interest to derive pluripotent stem cells from adult cells. In this way, one could potentially model in vitro genetic diseases that afflict patients. In addition, cells derived from patient-specific stem cells would not be rejected upon transplantation back into the patient. A methodology for generating induced pluripotent stem (iPS) cells from adult cells has recently been reported. It involves the forced activation of specific genes in the adult cells. This method is very recent: it was first reported in 2006 and only six groups, including our lab, have to date published work describing it. The published literature documents how remarkably similar to ES cells iPS cells are. However, they are not identical, and a major difference is that ES cells are derived from young embryonic cells, whereas iPS cells are derived from older differentiated cells. Furthermore, the method remains very inefficient, has been used with a limited number of adult cell types and needs to be improved in important ways. It will be essential to address the question of how the age or type of the original cell affects the resulting iPS cells. We propose to generate new iPS cells in order to test two hypotheses: 1) that younger cells may generate better quality iPS cells than older cells. If this hypothesis is true, it will be important to understand what are the critical factors that distinguish young versus old cells in their ability to generate iPS cells. From a practical point of view, it may be important to generate iPS cells at a young age and store them for future use, or to manipulate adult cells in additional ways to assure the quality and safety of the resulting iPS cells. 2) that a “memory” of the original cell may persist in iPS cells. If this hypothesis is true, it will be important to understand how that memory is maintained, and to investigate how it may affect the quality and/or safety of iPS cells. In practice, this may mean that certain cell types may be a better starting point for the generation of iPS cells. Our lab has developed improved methods for generation of iPS cells. We are using cells of different types and ages to generate iPS cells, and will be able to compare them. Our laboratory also has extensive experience with studies of the basic biology of pluripotent stem cells, and we will make use of that expertise in the proposed work. The proposed research is expected to provide the community of stem cell researchers with new pluripotent stem cells from diverse cell types, and to make important contributions towards the development of safe clinical applications of iPS cells.
Pluripotent stem cells hold the potential to revolutionize medicine and health care. Research on human pluripotent stem cells may provide new treatments for devastating and presently incurable conditions such as diabetes, Parkinson's disease, muscular dystrophies, spinal cord injuries, and many other diseases. In the case of diabetes alone, tens of billions of dollars per year are spent in California managing the disease. Recently, islet transplantation has provided an alternative path towards a cure for diabetes. However, this approach is severely limited by the short availability of cadaveric islets and the consequences of prolonged immunosuppression to avoid transplant rejection. If cells derived from the patients themselves could be turned into pancreatic beta cells in large numbers, both of these limitations would be overcome. A similar paradigm applies to many other diseases, where the ability to generate patient-matched pluripotent stem cells would provide a major new tool to study the disease in the lab, discover new drugs, or develop cells for transplantation. Recent results indicate that it may be possible to take cells from patients and induce them to become pluripotent stem cells. The method for doing this is very inefficient, and has been used with only a few cell types. Our proposal aims to understand how cells of different types and ages can be induced to become pluripotent stem cells. This research will pave the way for the development of safe clinical applications of human pluripotent stem cells. If we understand how the age and type of the original cells affects the pluripotent stem cells derived from them, we will be able to explore the use of this method to obtain cell types of therapeutic value, while avoiding unintended side-effects. The development of human pluripotent stem cell-based therapies will significantly increase the options available in the California health care system. These new therapies are expected to reduce the long-term health care costs to California by providing cures to diseases, like diabetes, that are currently chronic and require expensive periodic treatment. Our research is also expected to stimulate the development of biotechnology industry focused on drug screening on and clinical applications of human pluripotent stem cells. Such development will be of great benefit to California by attracting high-skill jobs and tax revenues, and by making the State a leader in a field that is poised to be the economic engine of the future. The State of California will also stand to benefit from the intellectual property generated by this research.
The goal of this proposal is to examine the effects of age and source of starting somatic cells on the quality of cells produced by induced pluripotent stem (iPS) cell generation. Placental, fetal, newborn, and adult skin fibroblasts will be used for iPS cell generation, and the pluripotency of resulting stem cells will be evaluated both in vitro and in vivo. Tests to examine whether a “memory” of the cells’ original state persists in iPS cells will involve microarray analysis to compare gene expression patterns in cells of different origins and ages. These experiments should yield new cell lines and provide important insights into mechanisms underlying the induction of pluripotency.
This is a proposal of high significance. The project focuses on important practical aspects of iPS cell generation and addresses a major gap in current knowledge of iPS cells. The results should prove significant for the field and will likely have substantial impact on subsequent strategies for generating and utilizing iPS cells.
Reviewers viewed the proposed investigation as highly feasible. The approach is clearly laid out with a set of appropriate questions and a cohesive research plan to answer them. Potential problems are anticipated and reasonable alternative strategies are provided.
The PI has a proven track record in the analysis of gene expression and recently has demonstrated high productivity in stem cell research. Named collaborators have excellent and appropriate expertise. Sources are identified for all of the starting cells, and vectors and other required materials are readily available. The institutional setting is excellent and provides a collaborative and supportive environment for the proposed research.
Reviewer enthusiasm was diminished somewhat by the narrow scope of the proposal and its perceived limited novelty. Nevertheless, reviewers recognized the value of the study in providing useful information for the field and its importance for stimulating further progress.
The proposal is highly responsive to the RFA. New human pluripotent cell lines will be generated from a diverse set of donor cells. The knowledge gained in the study will support the future derivation of additional iPS cell lines. A strong plan to share novel lines and data is presented.
With respect to generating iPS cells there are two major variables. The first is the choice of reprogramming factors to be used. The second is the source of the somatic cell to be reprogrammed. In this proposal the PI asks a very simple but important question: does the age of the somatic cell matter? The hypothesis to be tested is that younger somatic cells will be better reprogrammed, and that older cells may retain an epigenetic memory of their previous state.
Reviewer One Comments
The goal of the proposal is to determine the effects of age of donors cells on iPS generation and also how age or the origin of the somatic cell affects the outcome in detailed expression analysis of iPS cells. These are important practical aspects of iPS generation.
The study is feasible as described. Data in the literature although meager supports the notion that the younger the age of the donor cells, the more efficient the iPS generation. The use of placental fibroblasts is likely to take advantage of this. The gene expression analysis is within the capability of the PI. If anything, this is a modest proposal which doesn’t put forward any novel ideas or methods. Nonetheless, the data to be gathered will be useful to the field.
Responsiveness to RFA:
Reviewer Two Comments
The PI presents a fairly straight-forward proposal to test this hypothesis. The results will be of significant interest and likely will impact subsequent strategies. For example, it might be important to generate iPS cells from young cells and bank these for future needs. Alternatively, the data could stimulate research into reducing any latent memory, to reprogram age as well as pluripotency.
There are two Aims. The first is to generate iPS from human cells of different ages, using 3 distinct sources:
i) 7 week placental fibroblasts. These are of extra-embryonic origin, actually serve well as hES feeders, and are routinely isolated by CVS for prenatal genetic testing.
ii) Skin fibroblasts from fetal, newborn, or adult sources.
iii) Adult neural precursor cells.
The PI has collaborators that can provide each of the cell sources, and 2-3 lines will be generated from each, for a total of 10-15. The PI has generated a putative line already from placental fibroblasts and has a lentiviral vector system established. Experiments will test the minimal factors needed, perhaps less than 4 for some cells, test the efficiency of reprogramming and test pluripotency by markers, differentiation in vitro, and teratomas. It seems likely that new lines will be generated.
The second Aim is to look for evidence of age or cell-type specific memories. This represents a fairly standard microarray-based transcriptional profiling approach. Using the published literature, the PI demonstrates that gene sets can be derived comparing FL or newborn fibroblast profiles to ES cells (there is a set of genes more different in newborn compared to fetal). A major caveat here, of which the PI is fully aware, is that we do not yet know the variability among similar iPS cells from a common source. If this is significant it will make the analysis very difficult. The PI has a number of additional possible approaches, such as evaluating miRNA patterns. This Aim is essentially an extension of the marker expression that will be carried out in Aim 1, and should be feasible. Other approaches such as evaluating methylation status are not suggested.
Responsiveness to RFA:
Based on this proposal, multiple new human pluripotent cell lines will be established, from a diverse set of donor fetal or adult cells. The PI trained with Doug Melton and was a faculty fellow at UCSF from 2003, and a new Asst. Professor since 2007. He is funded by JDRF and CIRM and has published or submitted several papers using iPS technology. He has recruited appropriate colleagues to provide donor cells and is in a highly interactive and productive stem cell community. Overall this is a very well written and well thought out proposal that is responsive, focused, and not overly ambitious.
Reviewer Three Comments
This proposal seeks to generate new iPS cells from somatic cells to test whether younger as opposed to older somatic cells generate a higher quality iPS cell, and to test whether the memory of the original somatic cell lingers after induction of pluripotency. This proposal addresses a major gap in our current knowledge of iPS and thus enjoys a very high significance.
There are two specific aims. The first is to induce human cells of different ages and types to pluripotency. The second aim is to determine how the age or memory of somatic cells is reflected in the corresponding iPS cells.
This proposal is well written with a clearly laid out set of questions and cohesive research plan to answer them. The aims are not over-ambitious and should be feasible as currently written. The second aim poses some difficulty, as acknowledged by the authors in the proposal, given that variability between transcriptional profiles will make for a difficult statistical analysis. Other statistical tools, however, are proposed should this become a problem.
Dr. Ramalho-Santos has a proven track record in the analyses of gene expression in mouse and human pluripotent cells with a high level of productivity.
Collaborators include Dr. Susan Fisher (epigenetic regulation of pluripotent stem cells), Drs. Robert Blelloch and Shinya Yamanaka (pluripotency and reprogramming), Dr. Arturo Alvarez-Buylla (neural precursor cells), Dr. Ruby Ghadially (skin fibroblasts), Dr. Michael McManus (lentiviral gene delivery approaches, RNA interference and RNA induced gene activation), Dr. Ru-Fang Yeh (statistical analysis of microarray data), Dr. Long-Cheng Li (RNAa), and Drs. Jeff Bluestone, Matthias Hebrok, and Ed Baetge (consortium with the PI to derive clinical grade pancreatic beta cells for human ES and iPS).
The facilities at UCSF are excellent, and will provide a perfect setting for the accomplishment of the aims of this grant.
Responsiveness to RFA:
This proposal satisfies the aims of the RFA, including the derivation of new hESC lines and a strong plan to share lines and data with colleagues.