Tools and Technologies II
Human pluripotent stem cells offer unique advantages for cell replacement therapies and drug discovery. Because of their proliferative capacity and their ability to differentiate into different cell types, these cells may provide a cell source for cell replacement for many different diseases & for drug discovery & toxicology screening. However, development of these applications is hindered by a lack of scalable manufacturing methods to provide the quantity of differentiated cells required for clinical trials or drug screening. Current hESC/iPSC cell culture methods are inefficient and not readily scalable. The development of suspension culture systems for expansion & differentiation of hESC/iPSC would significantly advance the devolvement of hESC/iPSC as screening tools and therapies. Suspension culture systems are commonly used for many manufactured cell products & provide increased efficiency, yield and decreased costs. We have successfully demonstrated a scalable cGMP-compatible suspension culture system using several hESC lines and have achieve over 1million fold expansion with viability, pluripotency & overall expansion equivalent to adherent culture systems. While this is a significant technical advancement for expansion and banking of parental hESC lines, it is not clear how broadly applicable our system will be for hESC or iPSC lines. In addition, hESC/iPSC are not in themselves clinical products and development of a suspension culture system for production of differentiated cells types is needed. We intend to assess the broad utility of our system for banking multiple hESC/iPSC lines, and adapt adherent culture differentiation processes for production of cardiomyocytes (CM) and dopaminergic precursors (DP) to our suspension culture system. We will assess the general utility of the suspension differentiation processes with multiple hESC/iPSC lines. The ability to reproducibly generate large numbers of DP & CM will allow the use of these cells for cell replacement therapy in patients with Parkinson’s disease & heart disease and will provide cells for research, drug discovery & toxicology testing. Although we have demonstrated it is possible to generate a high percentage of dopaminergic neurons from hESC/iPSC in both adherent and suspension culture at laboratory scale, these methods maintain the cells at low density making scale-up impractical & costly. Similarly, the reproducible generation of sufficient quantities of CM has been challenging with adherent methods being impractical for scale up and providing only modest yields. Furthermore, the generation of DP or CM from iPSCs from donors with known genetic mutations causing heart or neurologic diseases will advance the study of these diseases & the development of disease-specific drugs. In addition, as significant percentage of drugs fail in clinical trials due to cardiac or other toxicities cultured CM, and DP, may prove to be predictive toxicology screening tools.
Statement of Benefit to California:
Advantages of human embryonic stem cells (hESC) and induced pluripotent stem cells (iPSC) in research & drug development are their ability to self-renew indefinitely & to differentiate into clinically useful cell types. hESC/iPSC provide an inexhaustible source of well-defined human cells & represent an important source of material for therapies in regenerative medicine, drug screening & many other areas of biomedical research. As hESC/iPSC based therapies developed in California near clinical trials & interest in using these cells to screen potential new drug candidates increases, there is a pressing need for scalable manufacturing methods that efficiently produce parental hESC/iPSC lines as well their differentiated cell products. Current procedures for the culture of these cells are limited to small scale methods developed in research laboratories that focus on research rather than large scale production. While we and others have produced hESC/iPSC cell banks using laboratory methods, the methods are neither practical nor amenable to the large scale production required for clinical development and drug screening. Further, once hESC/iPSC are expanded to sufficient numbers for banking, they must be differentiated into a desired cell type for each clinical application or drug screening activity. These differentiation processes are also currently based on small scale research laboratory cell culture techniques & are not amenable to large scale production. Our group has developed suspension culture technology that allows hESC/iPSC to be efficiently grown in large scale. These methods are compatible with industrial-scale production and can provide near limitless numbers of hESC/iPSC. We propose to extend our current systems to include differentiation of hESC/iPSC into relevant cell types. We will adapt differentiation processes developed by our team for the generation of neural progenitor cells and cardiomyocytes to large scale suspension culture and show the broad utility of our system by demonstrating scalable differentiation of multiple hESC/iPSC lines into these to important cell types. This proposal brings together nationally recognized leaders in hESC/iPSC culture and cGMP manufacturing to develop a complete and scalable differentiated cell product manufacturing process for two important cell types using suspension culture conditions. These processes will be compatible with industrial scale production and will provide methods for producing the numbers of cells required for clinical studies and large scale drug screening activities. These processes will be available to all CIRM investigators and will be a key resource for all investigators in the state of California. The unprecedented cGMP cell banking facility along with the collective expertise of the assembled team, offers a unique opportunity to advance the hESC/iPSC field by establishing large scale suspension production techniques not fundable under current NIH practices.
The goal of this proposal is to develop methods compatible with current Good Manufacturing Practices (cGMP) for scalable differentiation of dopaminergic neurons (DN) and cardiomyocytes (CM) from human pluripotent stem cells (hPSCs) in three dimensional (3D) suspension culture conditions. In the first Aim, the applicant will broaden the utility of current hPSC suspension culture system for expanding and banking up to 8 additional human embryonic stem cell (hESC) and induced pluripotent stem cell (iPSC) lines. Standard Operating Procedures (SOP) will be developed to assess and optimize cell growth conditions. The second Aim will focus on the development of a panel of marker assays to characterize hPSC-derived cell products. In the third Aim, the applicant will adapt existing differentiation procedures to a suspension culture system for scale up production CM and DN. The resulting cell products will be characterized in vitro and assessed for functionality in vivo. While reviewers agreed that the development of GMP-compatible suspension culture techniques for hPSC growth and differentiation are important for drug discovery and regenerative medicine applications, they were not convinced that the proposed research would have significant impact on this translational bottleneck. The level of innovation was considered low, as much of the proposed work relates to adapting or optimizing existing protocols rather than devising novel methodologies or techniques. Furthermore, the proposed targeted range for scale-up was considered modest as a primary objective. Reviewers found the proposal to be logical and well written, with convincing preliminary data supporting the capabilities of the team in all key aspects of the project. Reviewers particularly appreciated the inclusion of in vivo functional assays in Aim 3 for hPSC-derived CM and DN characterization, although use of a more highly immunocompromised model for the cardiac injection studies was recommended. However, some reviewers felt the proposal would have been benefited from a narrower focus. Much of the first two aims, though potentially enabling for the third, appeared rather tangential to the primary objective. Very few specific details were provided regarding the range or number of experimental conditions to be tested, making it difficult to estimate the impact of these studies. Furthermore, pitfalls and potential solutions were only superficially described. Reviewers discussed the possibility that transport and diffusion limitations posed by cell aggregates in 3D culture might necessitate increased concentrations of neuro- and/or cardio-inductive factors to achieve similar effectiveness compared to 2D culture conditions. Should this be the case, the increased cost of the new approaches may prove to be a significant barrier to the overall, long-term objectives of this effort. Finally, reviewers were concerned that use of a specific pathway inhibitor in the culture system would raise issues with regulatory authorities. Reviewers described the applicant team and institution as first class. The principal investigator (PI) has a good track record of grant funding and publications in the field, while both co-investigators were well regarded for their expertise in CM and DN, having made important contributions to the preliminary work that forms the basis of Aims 2 and 3, respectively. A key consultant was described as a leading hESC scientist, and the budget was judged to be reasonable. Overall, reviewers appreciated the strength of the investigators and found the research plan to be feasible. However, due its limited innovation and modest outcomes, reviewers did not the proposed research would have significant impact on a specific translational bottleneck.