New Cell Lines
Pluripotent stem cells can by definition give rise to all cells of an organism and also self-renew. These properties have long been evident in embryonic stem cells isolated from early stage embryos of many species, and then maintained in culture with the aid of exogenous factors that promote proliferation in the absence of differentiation. Advances in isolating and propagating human embryonic stem cell lines have spurred enthusiasm for applying these cells to studies of disease mechanisms, to screening of more effective drugs, and to development of new therapies for disease or and injury. However, progress has been hindered by continuing ethical debates over the theoretical fates of such embryos. Therefore, there is high interest in alternative technology to reprogram differentiated somatic cells to a pluripotent-like state that has the potential to ultimately circumvent dependence on embryos. This has already been demonstrated to varying degrees of efficacy, but each of the current strategies has significant limitations. The objective of this proposal is to establish robust, efficient and high-throughput processes for reprogramming donor somatic cells into pluripotent stem-like cells. An important component of this effort will be to extend these processes through cell derivation and maintenance in a way that could expedite the banking of pluripotent cell lines that may have future clinical utility. Our approaches are both pragmatic and novel. Thus, we will focus on optimizing, comparing and then extending the capabilities of several established reprogramming strategies. Our goals are to (1) test the hypothesis that providing a qualitatively and quantitatively more potent reprogramming stimulus can improve reprogramming efficiency; (2) test the hypothesis that optimizing the source of primary human somatic cells can improve reprogramming efficiency; (3) test the hypothesis that the efficiency of derivation and recovery of pluripotent cell lines can be improved by providing a more permissive and supportive environment for cells during the nuclear remodeling and reprogramming process; ( 4) lay the initial foundation for establishing a large homozygous pluripotent stem cell bank that can meet most potential clinical needs by incorporating the optimized methods into a cGMP-compliant process at the earliest opportunity.
Statement of Benefit to California:
Severe injuries and degenerative diseases are leading contributors to healthcare-related costs that affect all Californians. The potential to derive new types of therapeutics from pluripotent stem cells could revolutionize healthcare by providing more effective and perhaps curative treatments. California is a major hub for research and development on stem cells. In recent years, thought-leaders in this field from around the country have relocated to California universities and biotech companies. Major investments in early-stage companies by venture capital firms and pharmaceutical companies have been announced to rapidly exploit and develop discoveries made by academic researchers. If CIRM-funded research continues to spur progress, this area of biotechnology has the potential to grow substantially. This will help to fulfill CIRM’s goals of increasing the availability of new stem cell-based therapies and diagnostics to citizens, create new jobs and keep the state at the forefront of the biotech industry.
Executive Summary The principal investigator (PI) for this application proposes to establish a robust, efficient and high-throughput process for reprogramming somatic cells into induced pluripotent stem (iPS) cells. First, the PI proposes to test several reprogramming strategies that involve the fusion of fibroblasts with extracts prepared from normal or genetically manipulated pluripotent cells. Second, the applicant proposes that some cell types may be easier to reprogram than others and intends to examine sources other than fibroblasts to optimize iPS cell generation. Additionally, the applicant proposes that current culture conditions for the derivation of iPS cell lines limit the efficiency of the process, thus the team will use a culture system they have developed for human embryonic stem cells (hESC) to improve iPS cell generation. Finally, the proposal is to move these optimized iPS cell generation methods into a process that is compliant with current good manufacturing practices (cGMP), with the goal of generating autologous iPS cell lines or establishing a HLA-homozygous pluripotent stem cell bank for clinical needs. Reviewers were in agreement that improving somatic cell reprogramming is an important goal. Reviewers also agreed that the most novel aspect of the proposal is the use of a strategy that avoids the presence of viral vectors and transgenic DNA in reprogrammed cells. However, reviewers expressed serious concern that this strategy is very risky, and the applicant does not provide preliminary data supporting the feasibility of the proposed work. The PI should have taken into consideration the failed attempts by others and tried to address them. Furthermore, there is no supporting rationale for the use of cell types other than fibroblasts for the reprogramming protocol. For one of the aims, the PI proposes to derive pluripotent cells from the rare population of cell donors that are homozygous for HLA types. A reviewer criticized that the applicant did not identify the source of such cells. There is no letter of support from any other group or organization supporting this endeavor. Finally, reviewers discussed the lack of information on how the proposed processes will be made cGMP-compliant, and the lack of the PI’s expertise with hESC and iPS cells. Taking into account these shortcomings, the overall enthusiasm for this proposal was low. Reviewer Synopsis: This proposal calls for large-scale reprogramming of somatic cells (iPS). The goal is to establish more robust, efficient, high-throughput procedures and techniques that will greatly increase the efficiency and derivation of reprogrammed cells. These proposed new methods will encompass all aspects of initial cell derivation, maintenance of pluripotency during culture, and banking (for subsequent clinical use). The strategies proposed are cell fusion, introduction of cell extracts (reprogramming material) into cells to be reprogrammed, and over-expression of pluripotency factors. Reviewer One Comments Significance: Improved iPS reprogramming is an important goal. The most novel aspect of the proposal is the use of the “surrogate induction pluripotency (SiP) strategy” that avoids presence of viral vectors and transgenic DNA in the reprogrammed cells. Feasibility: The principal novel aspect of the proposal is reliance on cell fusion. Cell extracts will be fused into fibroblasts to reprogram cells. They will then measure levels of reprogramming transcription factors and any deficiencies will be corrected by adding lentiviruses containing the relevant cDNA to the hESC or EC (to be used for obtaining extracts) and then these cells will be enucleated and used in fusion or extract preparation. This is a potential ingenious strategy but it is very risky. Although cell fusion will reprogram somatic cells to a pluripotent state, the process requires continuous production of the reprogramming material for an extended time. In the method proposed here, it is unclear whether the reprogramming factors in extracts will be present at a high enough concentration for a sufficient time to achieve stable iPS generation. It is likely that some targets will be activated but the process takes at least 7 days in the mouse and probably about twice as long in human cell reprogramming. This reviewer rates the likelihood of this approach working as described very low. The remaining aims of the application are straightforward. But if the goal is to improve reprogramming, aim 1 is the most important and the success of this work rises or falls on it. Responsiveness to RFA: Responsive Reviewer Two Comments Feasibility: Strengths: • The experimental design in strategy number 3, over expression of pluripotency-inducing factors in the cell line that will be used for the production of cell free extract, is somehow novel. • Use of a novel culture system based on factors produced by MEF-free hES Cells Weaknesses: • Aim 1. There is no preliminary data supporting the work. This aim lacks novelty. The PI should have taken into consideration the failed attempts by others and try to address them. • Aim 2. The PI proposes to use primary muscle cells, CD34+ cells and fibroblasts for reprogramming experiments. His hypothesis unfortunately has no supporting rationale. • Aim 3. This is not really an aim but an alternative to aim 1 and 2. • Aim 4. PI proposes to derive pluripotent cells from the rare population of cell donors that are homozygous for HLA types. Where are the cells coming from? There is no letter of support from any other group or organization supporting this endeavor. This is a naive proposition. • The PI was unable to find any pitfall in any of the 4 aims. Responsiveness to RFA: yes Reviewer Three Comments Significance: Significance and Innovation: The authors suggest that existing methods to generate ES-like stem cells will not be sufficient for clinical applications. He suggests that a more potent reprogramming stimulus will improve reprogramming efficiency. He proposes in Aim 1 to produce cytoplasts from enucleated hES or EC cell lines which will provide the reprogramming factors which can then be fused with somatic cells to generate iPS-like cell lines. Resultant cell lines will be profiled for gene expression and any pluripotent transcription factors (Oct ¾, Sox2, cMYC, Klf4, Nanog or Lin28) which are not expressed at appropriate levels could be supplemented by the transfection of lentiviral constructs of the deficient gene. In aim 2 he proposes that some cell types may be easier to reprogram than others (as has been shown with nuclear transfer experiments) so that the choice of the cell to be reprogrammed will be important. Thus, he proposes to examine the efficiency of iPS line generation following fusion of the reprogramming cytoplasts described in aim 1 with more stem like cells such as hematopoietic and myoblast stem cells in addition to regular dermal fibroblasts. Reprogrammed cells will be identified with antibodies to stem cell markers including SSEA 3 or 4 and separated from mixed cultures with magnetic beads. In aim 3 he proposes that current culture conditions for the derivation of iPS cell lines limit the efficiency of the process, thus the team at ACT will use a culture system they have developed for hESC under GLP conditions. Cell lines are used to produce conditioned media that contains growth factors such as IGF-2, TGFb1, and pleiotrophin. Preliminary (unpublished) studies suggest that the CM from these lines may be beneficial for the propagation of hESC growth. In a 4th aim, they will take all of these advances and generate large numbers of ES-like cell lines under GMP conditions. Responsiveness to RFA: Poor. I believe that it is unlikely that they will generate pluripotent cell lines. Reviewer Four Comments Significance: The proposal calls for large-scale reprogramming of somatic cells (iPS). The goal is to establish more robust, efficient, high-throughput processes that will greatly increase the efficiency for derivation of reprogrammed iPS cells. These methods described in this application will encompass all aspects of initial cell derivation, maintenance, and banking (for clinical use). Successful achievement of all of the aims in the proposal would greatly enhance the stem cell field, leading to the creation of a large bank of cells that, in theory, could be used for research and therapeutic treatments. Feasibility: They want to optimize, compare, and extend established reprogramming methods. They hypothesize that using more potent stimuli beyond the published factors will lead to more efficient reprogramming. However, minimal preliminary data were presented that this concept was correct, or that they would even be able to identify what these more potent stimuli would be. • They also propose to evaluate various starting sources of somatic cells (cord blood, bone marrow, or other adult tissue), suggesting that different cell types may be easier to reprogram. While this is a reasonable hypothesis no preliminary data evaluating this concept were presented. • They also propose to improve the efficiency or reprogramming by providing a better supportive environment for the derivation of the cells, however again almost no preliminary data to support this theory was included in the proposal. Assuming all of the above can be accomplished and “better” methods for generating iPS can be established, they propose to create a “homozygous” stem cell bank, incorporating their optimized methods into a GMP-compliant process for clinical use. Minimal description was given as to how these proposed methods would be made GMP compliant. Myoblasts were indicated in a collaboration letter with Dr. Dinsmore (but these are not listed under human tissue). These are stated as already being GMP compliant, but no documentation was provided that this was in fact the case. Weaknesses: This is an overly ambitious proposal, attempting to evaluate a very large number of different methodological options for generating iPS, with no general focus on a specific approach. There were insufficient preliminary data to support that the investigators could accomplish their goals. Further, it appears that much of the expertise regarding the proposed methods are based in Mass., and it was unclear that transfer of the technology to the Calif. company could be easily and rapidly accomplished. The PI has a limited publication record, suggests very little direct current experience with hESC or iPS. Dr. Dinsmore will be “unpaid”, but listed in budget as 5% effort (no salary). This raises a concern regarding how much time will be available to support this proposal. No other consultants were indicated. Responsiveness to RFA: This proposal would be responsive to the RFA, in that, if successful, larger numbers of iPS cell lines could be established.