Early Translational I
Transplantation of somatic stem cells (SCs) can be beneficial for patients with a multitude of degenerative conditions. However, the use of somatic stem cells has several disadvantages: 1) incompatibility of donor and host cells; 2) restricted differentiation potential; and 3) only limited quantities can be obtained from adult donors or neonatal tissues or by in vitro expansion. In contrast, embryonic stem cells (ESCs) are capable of developing into all tissues in the body and can be expanded ad infinitum. Thus, ESCs may serve as an excellent, alternative source of transplantable cells for regenerative medicine. However, similar to SCs, the use of ESCs produced conventionally using in vitro fertilization will be limited by host-graft rejection. Experimental approaches designed to avoid this outcome involve the use of retroviral vectors and the introduction of exogenous DNA (induced pluripotent stem cells, iPSCs). Another option is the use of somatic cell nuclear transfer (SCNT) to generate patient specific, histocompatible ESCs in a process referred to as therapeutic cloning (TC). While TC has not yet been accomplished in humans, recent success has been achieved in a preclinical model using a novel protocol for SCNT. Here, we propose to adapt this novel patented technology with a current efficiency of 10% in preclinical models to the generation of human, patient-specific, SCNT-ESCs and to compare their therapeutic potential to genetically related iPSCs.
Statement of Benefit to California:
The concept of stem cell based therapy implies that damaged tissues can be repaired by tissue-specific stem cells that generate mature, functionally active progeny. However despite obvious progress in the somatic stem cell transplantation approach, there are obstacles in effective stem cell-based therapy, namely a shortage of HLA-matched donors and technical limits on the in vitro expansion of somatic stem cells. In addition, the use of immunosuppressive drugs often results in severe site effects in these patients. Thus, alternative sources for transplantable cells are needed, and human histocompatible, pluripotent stem cells (PSCs) generated by somatic cell nuclear transfer (SCNT) theoretically represent an excellent source. Recently, a novel protocol for SCNT in large animal models was established with a 10% efficiency rate. An important aspect of this technology is that it has been patented and licensed by our California based company, thus providing a basis for further commercial development. Importantly, we will use the same donor fibroblasts to generate inducible PSCs (iPSCs) using viral-based and non-viral-based methods. Thus, we will create triads of genetically related PSCs, but derived by different methods to compare their therapeutic potential. These genetically-related triads of PSCs will be subjected to genetic, cytogenetic, epigenetic and potency analyses setting the stage for preclinical evaluation. Eventually benefits generated by the use of such cells in regenerative medicine should benefit all people but Californians will also profit from the generation of novel products (i.e. cell lines and media kits) commercially available to clinical, academic and for-profit research laboratories and from the regional experience and expertise in regenerative medicine.
This project is focused on defining the best cell types to use for regenerative medicine by targeting the bottleneck for the safe and stable generation of patient-specific, immunologically-tolerated human pluripotent stem cell lines. The applicants propose three different means of accomplishing this goal: 1) somatic cell nuclear transfer (SCNT); 2) viral-mediated direct reprogramming of donor cells, or induced pluripotent stem cells (iPS); and 3) reprogramming using non-viral methods. Cell lines derived by these different methods (“triads”) will be compared to one another using genetic, molecular and epigenetic analyses, transcriptional profiling, followed by differentiation into neural lineages and subsequent transplantation of these cells into experimental animals for functional analysis. This project is directed towards the generation of histocompatible stem cells using both SCNT with human oocytes and iPS cells from fibroblasts of Alzheimer’s disease patients and normal controls. Reviewers generally approved of this application’s objective aimed at establishing patient-specific pluripotent cell lines without the use of insertional viral-mediated reprogramming. Despite this good intention, the reviewers had many reservations regarding the applicant’s methodology and rationale. The applicant proposes to generate cloned pluripotent cells using SCNT with human oocytes, or non-viral-mediated genetic modification of donor cells compared to the standard virus-mediated approaches currently available. The general hypothesis is that SCNT is probably better than iPS, and that if using iPS, non-viral vector derivatives will be safer. This is not a very controversial stance, and the PI is probably correct that SCNT might make the “best” ES cell lines for regenerative purposes. Of course, this has not been possible to test, since human SCNT has not been documented to generate ES cell lines. As is suggested by the applicants, a direct molecular and genetic comparison of identical donor cells reprogrammed by the ooctye compared to those created by induced pluripotency (iPS) is needed, as it is still uncertain to what extent iPS cells are analogous to human ES cells derived from embryos. However, one reviewer thought the assumption that there was a bottleneck to cell-based therapies and the use of pluripotent cell lines for toxicology or drug discovery was unfounded. The reviewer suggested that there were a very large number of both genetically normal and disease-specific human ES cell lines available for fundamental research, and iPS cells created by viral means were still useful for basic research. Further, the translational question is, “What can each cell do?” and if the cells perform well and safely, the biological comparison is interesting but not a bottleneck. The reviewer further stated it was naïve to think cloned human pluripotent cell lines could be created for an individual for each envisaged cell therapy. Additionally, although the applicants proposed to generate iPS cells using non-viral means, they offered no concrete means of how they would achieve this other than citing previous literature demonstrating work with mouse cells. The applicants also presented preliminary data from their collaborators based on large animal SCNT, where they suggested the efficiency of that particular SCNT and subsequent generation of cloned cell lines could be increased to 10%. The hope that the preclinical model protocols for SCNT will transfer is reasonable and seems to be based on a strong scientific basis, but it is very difficult to evaluate the likelihood of success and hence, the potential impact. To date, no group (including this research team) has successfully generated cloned human ES cell lines, and many have tried, although there are a number of reports of successful nuclear transfer and progression to the blastocyst stage. Therefore, for the applicants to predict that they will be able to generate such cell lines in the first year of this project was considered to be over-stated. In addition, one reviewer suggested that the issue of viral or non-viral lines was perhaps moot, since considerable progress had been made using chemical induction or other non-integrating strategies, such that progress in this area makes that comparison a less compelling priority. One reviewer expressed concern that with the exception of the SCNT experiments, almost all of the methodology in the proposal was descriptive and lacking in significant details. For example, there were no time lines for the transplantation studies or metrics on how the human cells would be identified and characterized post-implantation. The preliminary data presented on neural differentiation described a protocol that utilized cell sorting to enrich for neural cells yet there was no quantification of the purity of the sorted cells. This reviewer opined that it was not clear how successful and reproducible this protocol would be. Another reviewer considered it impossible to comment on feasibility, since the entire project relied on success of human SCNT, which has not yet occurred. From a human subjects protection standpoint, it would have been useful for the PI to more clearly describe the oocyte numbers required for these studies. The reviewer was astonished at the enormous task of comparing, with all the required assays, all 108 cell lines generated from this study and recommended that it would be useful to prioritize. (Even if they were successful in generating 1% SCNT efficiency, a major feat, the numbers of oocytes required are huge.) Only if SCNT works efficiently, could Aim 1 be achieved. The experiments described in Aims 2 and 3 are then entirely dependent on the co-investigator’s lab (an expert with iPS lines and neural differentiation assays), but the level of commitment is unclear since 0% effort or salary is allotted by the co-investigator. The principal investigator is the Chief Scientific Officer of a company which has exclusive rights to the preclinical model SCNT technology, licensed from an out of state university. The applicant will devote 25% effort to the project. Funding for two research associates and four technicians is requested, but there is very little justification or description of the role for any of these posts. One reviewer noted that the applicants were clearly successful in primate SCNT and should have as good a chance as any of success. In summary, the applicant proposes to create and compare patient-specific human pluripotent cell lines. The application contains several deficiencies in the rationale and significant problems with feasibility, which categorically weaken its seemingly novel attempt to address a bottleneck in generating SCNT for cell-based therapies.