Basic Biology II
Adult human cells such as skin cells can be turn into “reprogrammed” stem cells that acquire properties of pluripotency - the ability to become multiple types of specialized cells of the body. These reprogrammed stem cells are called induced pluripotent stem cells (iPSCs). Generation of iPSCs may enable the development of new therapies for many human diseases such as Alzheimer's diseases, spinal cord injury, stroke, burns, heart disease, type I diabetes, osteoarthritis, and rheumatoid arthritis. For example, type I diabetes occurs when the pancreatic beta cells that produce insulin are damaged or die. The only current cure is a pancreatic transplant from a recently deceased donor, but the demand for transplants far outweighs the supply. If reprogramming becomes possible, skin cells can be easily taken from a patient with type I diabetes and converted to iPSCs which can then be grown into the insulin-producing beta cells. These beta cells genetically match the patient’s cells and can be transplanted back into the patient’s body without any risk of rejection. This would potentially cure the disease. Therefore, the reprogramming technology has enormous therapeutic potential. The most widely used method for reprogramming is delivering certain stem cell-associated genes or proteins called reprogramming factors (RFs) into adult human cells. The major drawback of this type of approach is the low efficiency of reprogramming, which compromises the clinical use of this powerful approach. Recently, it has been found that stimulation of cells with a protein called Wnt3a increases efficiency of the conversion of adult cells to iPSCs, but the efficiency is still too low for clinical use. However, adult cells containing less amount of a protein called p53 has higher efficiency of reprogramming. These observations suggest existence of barriers in the cell to restrict reprogramming. Our proposed study is to determine whether p120-catenin serves as a barrier to the reprogramming. To achieve this goal, we will use skin cells (keratinocytes) in which p120-catenin expression is deleted or reduced. We will then reprogram these cells by introducing RFs and/or Wnt, and examine the reprogramming efficiency. If the cells lacking p120-catenin or with low level of p120-catenin are more easily reprogrammed than are cells with normal level of p120-catenin, is would suggest that p120-catenin serves as barrier to the reprogramming. We will further determine how p120-catenin functions as a barrier by examining the interaction between p120-catenin and Wnt regulated events. Skin cells which are relatively easy to obtain and reprogram would be an ideal source of iPSCs. Successful completion of this study will provide information that helps develop new strategies such as inhibition of p120-catenin to improve the reprogramming efficiency. This would overcome existing hurdles that hamper the application of the technology useful in transplantation therapy of many human diseases.
Statement of Benefit to California:
Chronic degenerative diseases, such as Alzheimer's diseases, type I diabetes, heart disease, osteoarthritis, rheumatoid arthritis, etc., afflict a significant number of California’s population. Today, donated organs or tissues which match host’s immune system are often used to replace ailing or destroyed tissue of the patient, but the need for transplantable tissues and organs far outweighs the available supply. Right now, over 21,000 patients in Californian are waiting for life-saving transplants. That's 21 percent of the more than 100,000 people waiting across our country. One third of them will die before receiving a transplant. Stem cells can give rise to a variety of cell types, which offer the possibility of a renewable source of replacement cells and tissues to treat degenerative diseases. Adult somatic cells can be converted to induced pluripotent stem cells (iPSCs). This process is called reprogramming. The reprogrammed iPSCs have the potential to become any type of cells in the body. The iPSCs are remarkably similar to embryonic stem cells and would offer all the benefits of embryonic stem cells without the controversial use of embryos. For example, it may become possible to generate healthy heart muscle cells from patient’s own tissue in the laboratory and then transplant those cells into patients with chronic heart disease without risk of immune rejection. However, the low efficiency of the reprogramming limited clinical application of this powerful method. Our study is aimed at determining whether p120-catenin serves as barrier to prevent somatic cells from dedifferentiation (reprogramming). This study will uncover the role for p120-catenin in regulating somatic reprogramming and provide information for developing strategy about how to make reprogramming more efficient so that iPSCs can be generated on a large scale for transplantation, which is beneficial to the patients with degenerative diseases in California. The improved reprogramming technology can be commercialized by the biotech industry in California to generate revenue and create new job opportunities.
EXECUTIVE SUMMARY The goal of this proposal is to investigate the role of a signaling protein, p120-catenin, in the reprogramming of somatic cells to induced pluripotent stem cells (iPSCs). The central hypothesis of the proposal is that p120-catenin acts as a barrier to reprogramming by inhibiting the Wnt signaling pathway. The applicant proposes to test this hypothesis in three specific aims. In Aim 1, the applicant proposes to determine if depletion of p120-catenin enhances the efficiency of reprogramming of mouse and human keratinocytes. In Aim 2, the applicant proposes to investigate signaling mechanisms downstream of p120-catenin that may contribute to effects on reprogramming. Finally, in Aim 3, the applicant proposes to determine if p120-catenin signaling is itself regulated by any of the traditional reprogramming factors. Reviewers were not convinced that this proposal would have a major impact on the field of stem cell biology. They noted that the guiding hypothesis is highly speculative. If successful, the project would provide some insight into mechanisms of reprogramming, however keratinocytes can already be effectively reprogrammed so the overall impact would be limited. Reviewers also questioned whether the mechanisms of reprogramming would be fully illuminated by the proposed research plan. They were not convinced that the proposed pluripotency analysis would be sufficient to determine which aspects of p120-catenin signaling are essential for reprogramming. Aim 3, in particular, is highly descriptive and would benefit from experiments involving perturbation of downstream effectors that could provide greater mechanistic insight. Reviewers found the experimental design to be reasonable and logical, but cited the dependence of Aims 2 and 3 on Aim 1 as a major weakness. This is especially problematic, since Aim 1, to test the proposal’s guiding hypothesis, is not supported by proof-of-concept data. If the hypothesis is incorrect then little or no useful data will result from this proposal. Alternative plans, should this be the case, are not presented, thus the grant rests entirely upon a successful outcome for Aim 1. Reviewers presented several other minor criticisms of the plan. They noted that the use of both a genetic mouse model and gene knockdown approaches is somewhat redundant. Furthermore, in the absence of relevant preliminary data, it is not clear that the strategy for quantifying reprogramming efficiencies under various conditions will be sufficiently sensitive to detect subtle differences. Reviewers suggested that reprogrammed iPSCs be compared to bona fide human embryonic stem cells to evaluate the extent of reprogramming. They were also concerned about the lack of preliminary data related to establishment or analysis of iPSCs. The reviewers noted that while the applicant has an established track record in keratinocyte biology and p120-catenin signaling, s/he does not have any publications in stem cell biology and very few as a senior author. They recommended that the applicant enlist a collaborator with expertise in reprogramming or pluripotency. Overall, reviewers raised a number of significant concerns about this proposal, including that it relies heavily on a speculative hypothesis that is not supported by convincing preliminary data.