Establishing cell lines that can reproducibly generate cell types of interest is critical to translational use of stem cells, both for cell replacement therapy and for generating human disease models. Generation of pluripotent stem cells produces induced pluripotent (iPSC) and embryonic stem cell (ESC) lines that differ in their capacity to produce specific differentiated cell types. We have established a set of "fully pluripotent" mouse iPSCs that can reproducibly generate all cell types in an adult animal and have identified a novel set of potential biomarkers that distinguish these cells from less potent cell lines. We hypothesize that these biomarkers will identify human iPSCs and ESCs that can generate the widest variety of clinically-relevant cell types. We base this hypothesis on our preliminary studies and evidence for similarities in core regulators of pluripotency and self-renewal between mouse and human cells. We will exploit our unique set of cell lines and apply state-of-the-art proteomic and genetic analyses to discover new biomarkers of fully pluripotent human stem cells. Results of these experiments will remove a bottleneck in stem cell research that currently adds uncertainty to the validity of in vitro disease models and increases the cost of establishing clinically suitable cell lines and differentiation protocols. Identifying novel biomarkers of pluripotency should impact stem cell research across disciplines and establish needed molecular standards for cell banking and maintenance.
Statement of Benefit to California:
Pluripotent stem cells derived from embryos (ESCs) or from differentiated cells ( iPSCS) offer a unique source of human specific cell types for drug screening and cell replacement therapy. The most useful cell lines are those which are capable of generating all the different cell types useful for therapy, such as neurons for treatments of Alzheimer's, Parkinson's disease and spinal cord injury, pancreatic beta cells for treatment of diabetes or heart cells for cardiovascular defects. Some ESCs and iPSCs are better than others at generating particular cell types. Current tests to identify such cells are costly, difficult, time-consuming and not optimally effective. This proposal aims to identify simple and cost effective methods to identify the most useful ESCs and iPSCs by discovering new markers found on the cells that generate the most different cell types. These studies will reduce the costs of establishing and maintaining pluripotent cell lines in many laboratories and should help to standardize stem cell- based clinical trials. This proposal will benefit California by reducing research costs, accelerating the time to therapies and helping to establish the most useful banks of stem cells for future research and clinical applications.
This proposal is focused on the discovery of novel biomarkers of fully pluripotent embryonic and induced pluripotent stem cells (iPSCs and ESCs). The applicant identifies as translational bottlenecks the heterogeneity of pluripotency in human stem cell lines and the lack of efficient assays to assess this heterogeneity. There are two Specific Aims: (1) to identify biomarkers of fully pluripotent mouse ESCs and iPSCs by analyzing gene and protein expression; and (2) to validate these biomarkers in a large number of human ESCs and iPSCs.
While the reviewers agreed that the identification of biomarkers of fully pluripotent ESCs and iPSCs is an important goal, they were not convinced that this project would advance a significant translational bottleneck. Reviewers noted that the primary impact would be a greater understanding of the basic mechanisms of pluripotency. Although they conceded that the identification of pluripotency biomarkers could potentially reduce the need for analysis and screening of cell lines, they considered this outcome to be of moderate impact. Reviewers also questioned whether only fully pluripotent stem cell lines would be clinically useful, noting that cells biased toward certain lineages could be well suited or even preferable for specific applications.
Reviewers acknowledged the technical merits of Aim 1, with its goal of identifying pluripotency biomarkers in mouse ESCs and iPSCs, but were less enthusiastic about Aim 2, the translation of these findings to human cells. The major flaw they identified was that biomarkers identified in mouse may not inform human studies, a risk acknowledged by the applicant. Reviewers questioned the rationale for using a stringent murine pluripotency assay to identify biomarkers for human cells, given the many known differences between ESCs in these two species. They also noted that an alternative approach, identified but rejected by the applicant, of comparing human iPSC lines with different lineage biases could yield more therapeutically useful data. The reviewers praised the strong preliminary data in support of Aim 1, including the identification of several potential pluripotency biomarkers, but were disappointed that their relevance to human cells was not elaborated. They found Aim 2 to be vague, and specifically cited a lack of detail describing differentiation methods and characterization. Finally, reviewers noted that while some risks and pitfalls were acknowledged, alternative approaches were inadequately described.
The reviewers described the Principal Investigator (PI) as exceptionally well qualified to lead Aim 1 of the study. They noted that the PI has had excellent training and has published several seminal papers. Reviewers praised the impressive assembled team of collaborators, but were concerned that the project’s human stem cell expertise resides with a collaborating investigator devoting only 1% effort and receiving no salary support. This investigator will assist with differentiation studies proposed in Aim 2, but these studies aren’t mentioned in the accompanying letter of support. Reviewers felt that additional expertise with human pluripotent stem cells would increase the likelihood of success of Aim 2.
Overall, while reviewers found the basic research aspects of this proposal to be strong, they were not convinced that translating this work to human cells would be feasible or likely to have a major impact on the field of regenerative medicine.