The purpose of this project is to develop a commercially viable process to reproducibly grow billions of hESCs for the production of clinically important replacement cells. When completed, it will represent an important FOUNDATION for numerous commercial-scale human embryonic stem cell (hESC)-based treatments.
Tremendous inroads have been made in the past few years in developing cell therapies for many different human diseases from hESCs. For example, scientists have established laboratory-scale procedures for directing hESCs through the developmental pathways to insulin-producing pancreatic beta cells, and these hESC-derived pancreatic cells have been used to correct diabetes in mice. However, in virtually every case, significant TRANSLATIONAL work lies ahead to transform discoveries in the laboratory into real, viable, treatments in the clinic. Two major widely-recognized translational hurdles are reliability and scale-up. Manufacturing processes to generate enough stem cells to constitute a clinically relevant dose, and subsequently a product, will be orders of magnitude greater in scale than those that are currently available. Further, hESC-derived cell products must be manufactured reliably and consistently in order to be safe.
Current estimates indicate that a single dose of cell therapy to treat Type 1 Diabetes could be on the order of billions of cells. The format typically used today for expanding hESCs is adherent static culture. Continuing with this format will literally mean that "football fields" of cells will need to be generated in order to produce significant numbers of human doses of cell therapy. While remarkably the cells have the potential to grow to that scale, the biopharmaceutical industry cannot economically grow hESC in that format.
The proposed project will develop and qualify an improved, efficient, and cost-effective approach -- growth of hESC in suspension culture. Although suspension culture has been used extensively in the biopharmaceutical industry, it has not been well developed for hESCs. The final step of the proposed project is to qualify the use of this new scale-up approach in the manufacturing process for an hESC-derived Type 1 Diabetes product. However, success here will likely be applicable to any hESC-derived cell therapy product.
Statement of Benefit to California:
The proposed research will establish manufacturing procedures that will apply to virtually all human embryonic stem cell (hESC)-based cell therapies. One of the appealing aspects of hESCs is that their numbers can be rapidly expanded in culture. Thus, the number of cells required for pre-clinical testing in animals, clinical testing in human subjects, and eventual commercial manufacture of a cell therapy product can theoretically be generated in a matter of a few weeks. However, the expansion of hESC is limited by current procedures for their cultivation, and a lack of understanding of how to scale hESCs from the laboratory bench to the commercial fermentor. This has led to speculation that hESC-derived products are not practical or commercially viable. Currently, hESC are typically grown in adherent format on plastic surfaces. The proposed research will investigate and develop protocols for growing hESC in a suspension culture format, which is the common practice for most commercially available products made in mammalian cells. This will alleviate the spatial and physical restrictions of the adherent format, allowing for much higher cell yields; the approach also benefits from years of development by the biopharmaceutical industry. This work will benefit the state of California and its citizens in multiple ways. The technology will make hESC-derived cell therapy products more commercially viable, and therefore will allow the numerous research-level approaches to cell therapy to be TRANSLATED into commercial products. Since many of the research-level approaches are being developed in California, all of those laboratories and institutions can benefit from clinical and commercial translation. Having this technology developed in California will continue to attract new talented individuals and their innovations and intellectual property to California. And most importantly, making it feasible for hESCs to actually be TRANSLATED into real treatments for the numerous devastating diseases for which they hold such promise will benefit immeasurably the countless Californians affected directly and indirectly by those conditons.
The focus of this application is development of improved methods for the reproducible expansion of human embryonic stem cell (hESC) lines compatible with clinical use and commercial production. Currently, hESC are typically grown in static adherent culture, conditions incompatible with efficient and cost effective production of the number of cells required for clinical use and for commercialization for many disease indications. The applicant proposes the development and optimization of a scalable process by adaptation of hESC for cell suspension-based growth. The Principal Investigator (PI) proposes to: 1) establish a protocol to reliably make hESC aggregates and expand them in suspension culture; 2) optimize conditions for stable serial passaging of hESC in suspension culture and 3) demonstrate suspension culture expansion to a target level and then differentiation to target cell type in high purity.
Reviewers noted that the development of suspension culture conditions for expansion of hESC, compatible with GMP production and maintenance of stemness, would be an important benefit to the field of regenerative medicine. Such a technology could accelerate the pace of translation into clinical testing. However, reviewers considered the project risky given the stated strategy of developing and optimizing expansion of controlled-size aggregates in light of the well-known tendency of hESCs to aggregate and, usually, to initiate differentiation upon aggregation. This characteristic behavior, coupled with the reviewer assessment of inadequate assays for evaluation of stemness, lessened the reviewers’ enthusiasm for the proposal.
The reviewers found the proposal to be generally well-written. The rationale was clear, aims were defined and milestones seemed achievable within the designated time frames. The reviewers also agreed that there was appropriate emphasis on and measures for robustness and reproducibility to ensure development of a reliable process. However, the reviewers had several concerns. One concern was that, given the tendency of hESC to aggregate, it was unclear how conditions would be controlled to prevent larger aggregate-to- aggregate formation. As ESC aggregation usually initiates a differentiation program, reviewers were not convinced that the basic method (propagation as aggregates) will maintain hESC stemness. This concern also highlights the other key concern of reviewers -that the assays proposed for assessment of maintenance of the undifferentiated phenotype of hESC during expansion were inadequate. The only real analysis proposed for “stemness” was Oct 4 expression assessed by immunostaining and flow cytometry. Oct4 alone is insufficient as a marker of the undifferentiated state for many reasons, including the observation that Oct 4 is expressed for several days during differentiation. More markers and assays are required to verify whether the expanded cells are still indifferentiated hESCs. One reviewer considered that in vivo teratoma formation was a critical gold standard for assessing stemness. Another reviewer commented that the target criterion for karyotype stability was also not adequately described. Finally, it was not clear to the reviewers to what degree FDA requirements and compliance with GMP were taken into consideration as part of the development process.
The PI and team were viewed as having the requisite expertise and experience to conduct the proposed research. The PI has extensive experience with cell culture including hESC cell culture. The PI and the team are experienced in industrial production of cells as well as the growth and differentiation of hESCs. Preliminary experiments also supported the capability of the team to perform the proposed experiments.
The reviewers commented on the budget although this was not part of the scientific evaluation. They noted that although the overall budget was generally appropriate, the distribution of funds was not clearly justified. All reviewers commented on the number of co-investigators for a project that seemed most appropriately done by one or two investigators supported by technicians.
Overall, the reviewers considered the objective significant but were less enthusiastic about the proposed research plan