Regenerative medicine using human pluripotent stem cells (hPSCs) holds a great promise in developing therapies for treating developmental abnormalities, degenerative disorders and aging-related illness. However, today’s hPSC collection faces two major challenges: shortage in supply and uncertain safety. Recently, the development of human induced pluripotent stem cells (hiPSCs) has solved the shortage problem by manually reprogramming resourceful somatic cells to hiPSCs. Scientists have been using these hiPSCs to conduct research and develop cures on multiple fronts, including testing drugs, developing therapies, devising tests for earlier diagnoses, and generating tissues/organs for transplantation. To further advance these research fronts, we need pure hiPSCs to prevent uncertain safety. As multiple factor-induced hiPSCs often present cell heterogeneity that leads to uncertain safety, this proposed project adopts a single factor – miR-302 induction method for hiPSC derivation. MiR-302 is a small regulatory microRNA that can replace all previously defined reprogramming factors for inducing homogeneous hiPSC formation. This microRNA-induced hiPSC technology is developed in California and has a higher reprogramming efficiency than previous Yamanaka’s methods using multiple factors. With these advantages, the hiPSCs provided by this project can serve as a better resource to the research and drug development community for advancing the progress of modern regenerative medicine.
Statement of Benefit to California:
The goal of CIRM is to establish a solid foundation for developing the future regenerative medicine industry in California. In order to achieve this goal, California needs its own pluripotent stem cell resource for further development. However, the legal right of using human embryonic stem cells (hESCs) belongs to WiCell Research Institute in Wisconsin, USA. Alternatively, OSKM-induced hiPSCs may replace hESCs but this technology is owned by Kyoto University in Japan. To solve this issue, PD has devised a new miR-302-induced hiPSC technology in California and is willing to contribute his right and efforts to achieve the same goal of CIRM. With this novel technology, California can freely develop its own regenerative medicine technologies to compete with other leading groups in the world. Since miR-302 also functions as a tumor suppressor in humans, the hiPSCs generated by PD’s method has an advantage in safety and hence may have a better chance to pass FDA approval. In light of these advantages, California has actually possessed all expertise, technologies, legal rights and resources required for the development of a top notch regenerative medicine industry in the world. To achieve this goal, we just need to coordinate these experts, technologies, legal rights and resources into one collaborative system. As the regenerative medicine market is expected to reach $1.4 billion dollars in 2012, a fruitful reward of this California-based system is highly foreseeable.
The applicant proposes to generate 9000 human induced pluripotent stem cell (hiPSC) lines from 3000 individual donors using a manual process. This is a collaborative effort in which two groups, the applicant and a collaborator, will generate the hiPSC lines, while hiPSC characterization will be subcontracted to a commercial entity. An additional collaborator will contribute to hiPSC characterization. The non-profit applicant institution is located in California. The applicant proposes to use plucked hair or skin biopsies as a source of primary cells for the derivation of hiPSC. Cells will be reprogrammed using a single microRNA family, miR-302, rather than the standard reprogramming factors. miR-302 will be introduced using two different genome integration-free methods, one as a backup that is compatible with low primary cell numbers. The characterization of derived hiPSC lines will involve marker analysis, both at the pluripotent stage and following differentiation into embryoid bodies, and also teratoma formation. Chromosomal integrity will be assessed using the classic karyotyping method and a molecular assay will be used to verify hiPSC line identity to the primary cells. The applicant argues that miR-302-mediated reprogramming leads to hiPSC cultures of such homogeneity that hiPSC characterization can be limited to one line per tissue donor. For cell line tracking, the applicant proposes to use the laboratory information management system (LIMS) already in place at the applicant institution, with modifications if necessary to integrate with the LIMS of the RFA 12-04 Award recipient. Protocols - Reviewers did not support the applicant’s claim that characterization of derived hiPSC can be limited to one line per tissue donor. To assess hiPSC quality, each line will need to be analyzed for pluripotency and genomic integrity. - The proposed pluripotency assays have drawbacks; the applicant intends to analyze a limited number of markers while larger panels of suitable markers are available. Furthermore, due to its large expense and long duration, the use of the teratoma assay is not justified in this program. - The cell identity assay will be limited to the analysis of a single locus when there are systems available for comparisons at multiple loci; analysis of only one locus introduces the potential for error. - The emphasis the applicant placed on issues related to maintaining the viability of the donated tissues during transfer from the Tissue Collectors raised concern amongst reviewers that there are limitations to the proposed derivation process. - The proposed miR-302-based derivation method seems to be an elegant way to undertake hiPSC generation. The protocols, although not in common use in the research community, have been established and published. - The backup method for miR-302 delivery is achievable but clearly a more labor intensive and slower method that may impact productivity depending on its frequency of use. Documentation and Quality Control - Very few details on Documentation and Quality Control are provided in the application. - While a complete Quality Management System is not required for the hiPSC derivation effort, it is necessary to have a system in place that tracks activities and testing results using a complete spectrum of documents; this is not readily apparent as being currently set up or in development. Feasibility and Resources - The proposed effort requires that multiple institutions work together to produce the hiPSC lines. There is an advantage to outsourcing hiPSC characterization, as it is an effective strategy for addressing lack of capacity by the applicant. However, the overall diffuse approach across several laboratories raises feasibility concerns, especially in light of the fact that a clearly laid out plan detailing individuals’ responsibilities and their timeline is lacking. - The subcontractor for hiPSC characterization has established methods for the proposed assays and should be able to accommodate the required pace of hiPSC line development. - The available resources and the relatively low start-up costs are a plus as the applicant group intends to utilize their current facility, supplementing equipment and space as needed. However, the applicant considers re-location in order to achieve the objectives of the proposal, which may impact the timeline. - The applicant does not define a historical hiPSC derivation success rate or redo rate. This is of particular concern as the proposed derivation method has not been widely used. - The proposed workload per laboratory staff is high. No mention is made of how the applicant intends to maintain efficiency or deal with productivity lapses in the context of the overall number of hires planned. Project Director (PD) and Team - The PD is clearly accomplished in the field as evidenced by his/her publication record and patent portfolio. - The PD lacks experience in managing a large-scale hiPSC generation program such as the one proposed. - The PD’s 50% effort commitment to the project is significant. - Key members of the team have expertise in the activities outlined in the proposal, and have recently held positions at very prestigious institutions. However, there are many positions that still need to be filled and this has the potential to strain the timeline. Budget - Due to high cost, the applicant intends to limit analysis of genomic integrity to a subset of derived hiPSC lines. However, karyotype data can be obtained more economically than by the source used in the proposal. - A large proportion of the total cost for generating and characterizing each hiPSC line is allocated to the teratoma assay. That cannot be justified when much more cost-effective assays based on gene expression arrays are now available. - Personnel funds are appropriately placed with a majority going towards active lab staff. Funds should also be allotted for administration and information technology.