Funding opportunities

Funding Type: 
Tools and Technologies I
Grant Number: 
Principle Investigator: 
Funds requested: 
$882 929
Funding Recommendations: 
Grant approved: 
Public Abstract: 

hESCs represent an important source of cell therapies in regenerative medicine and the study of early human development. A number of hESC-based therapies are nearing clinical trials. To bring these to clinical trials requires the scale-up production, or “banking”, of large numbers of the desired hESC cell. The current lack of large scale hESC culture methods presents a serious challenge to ensuring progression of new therapies into clinical testing. In addition, current characterization methods are inadequate to monitor genetic and epigenetic changes that may occur during the long term culture required for banking. Finally, the lack of well characterized hESC research banks limits the comparability of research between laboratories. We propose to address these issues by adapting three representative cell lines to scalable suspensions culture, develop epigenetic and genetic “fingerprinting” methods and generate well characterized Master Cell Banks of the three hESC lines for use by all CIRM investigators.

Procedures typically used in adherent hESC cell banking involve feeder cell layers, undefined media and/or mechanical manipulation. Banking of cell lines for anticipated clinical studies with existing technology is impractical, expensive and time consuming. The development of robust large scale banking technology will accelerate the speed of development of hESC therapeutics. In addition, monitoring pluripotency of hESC cell lines during culture adaptation and banking processes is critical. While the ultimate measure of hESC pluripotency is their ability to form teratomas in animal models, this method is insensitive, time consuming and costly, and is not feasible as an in-process or final product test method. Improved in-process characterization methods with demonstrated correlation to pluripotency are needed. While phenotypic and gene expression parameters have been defined for a number of lines, little is known about the fundamental genetic and epigenetic characteristics of these cells as they are maintained in culture. It is becoming apparent that the self-renewal and differentiation potential of hESCs may be impacted by the genetic and epigenetic status of the cells. Further, it is likely that the genetic and epigenetic status of the cells will be important predictors of safety for clinic studies. Correlating a genetic and epigenetic “fingerprint” of hESC lines during long term cell culture with pluripotency as measured by teratoma formation will provide a novel method of predicative in-process monitoring of cultures during banking and would facilitate comparison of processes between various laboratories.

The unprecedented cGMP cell banking facilities along with the collective expertise of the assembled team, offers an opportunity to advance the hESC field by establishing suspension adaptation techniques, epigenetic fingerprinting, cGMP scale-up processes & cGLP banking of hESC lines not fundable under current NIH rules and policies.

Statement of Benefit to California: 

An advantage of human embryonic stem cells (hESCs) in research is their ability to self-renew indefinitely. hESCs can provide an inexhaustible source of well-defined human cells and represent an important source of material for therapies in regenerative medicine, for the study of early human development and for many other areas of biomedical research. For the first time, it will be possible to grow large quantities of hESC that can meet the requirements of the FDA. As a number of hESC-based therapies developed in California near clinical trials, there is a pressing need for a source of high quality, well defined hESCs. However, translating bench science into clinical reality involves large scale cell banking and predictive cell characterization and release testing that correlate well with the pluripotent properties of these cells. Current procedures for the culture of these cells are limited to work from research laboratories where the focus is on research and not on methods for large scale production or the Good Manufacturing Practices (cGMP) required to meet FDA standards. While several groups have produced small scale cell banks using current technologies, the methods are neither practical or cost effective, and are not amendable to large the scale expansion and banking required for clinical development.

One of the major limitations to growing large amounts of hESCs is that current procedures require manual isolation of cell colonies –a time consuming process. A second limitation is that hESC grow only when adhered to surfaces with “feeder” cells and/or specialized coatings. As different laboratories employ their own methods, each may bias cells to be slightly different from the parent cell line. Lack of standardization of cell lines used by researchers is a concern in comparing research across the field. We propose to address these issues by 1. developing procedures to adapt three hESC lines to suspension culture, 2. create well characterized cell banks of these lines, using GMP processes suitable for preclinical and clinical use and 3. develop epigenetic and genetic fingerprinting for cell bank testing and monitoring of cell lines.

This proposal brings together nationally recognized leaders in cell culture, assay development and cGMP banking to develop hESC culture conditions that can be scaled up, to design and implement novel assays for characterizing cells obtained at each stage of production, and to create Master Cell Banks of important hESC lines. These cell banks and assay methodologies 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 an opportunity to advance the hESC field by establishing suspension adaptation techniques, epigenetic fingerprinting, cGMP processes & cGLP banking of hESC lines not funded nor fundable under current NIH policies.

Review Summary: 

The goal of this proposal is to develop human embryonic stem cell (hESC) lines that are compatible with suspension-based growth for scaled up culture and banking with cGMP compliance. Initially, the applicants will adapt 3 hESC lines for adherent-free growth with the objectives of eliminating use of feeder cells, more precisely defining media and matrix components, and minimizing the need for mechanical manipulation. The cells will be extensively evaluated throughout these procedures to ensure retention of pluripotency and stability of the genome. In the next phase of this effort, the applicants propose to optimize large-scale expansion technologies for establishing master cell banks of hESC. In addition to evaluation of several scale-up technologies for adaptation of adherent ES Cultures to suspension, the applicants will perform number of assays to demonstrate that the suspension-grown cells are comparable to the parental, adherent lines. The applicants propose to additionally test the cells using novel genetic and epigenetic fingerprinting assays, which will be evaluated as potential tools for final product release and in-culture monitoring. They also will attempt to correlate these fingerprints with the capacity for teratoma and pluripotency.

The reviewers were very impressed with the scope, breadth, and potential impact of this proposal. The research plan was well conceived, clearly conveyed, and extremely comprehensive. The collaborating team of investigators is excellent and well suited to this effort. The reviewers expressed some concerns about the choice of cell lines to be used and felt that more attention should have been devoted to providing alternative strategies for potential pitfalls. Despite these concerns, the reviewers agreed that the potential impact and utility of the resulting technology would more than adequately justify the risk of this approach.

The impact that the proposed technology offers is potentially very high and directly addresses a roadblock in stem cell biology; the inability to effectively scale and maintain functional, stable hESC lines. Such tools would be of tremendous value for regenerative medicine and could accelerate the translation of these studies into the clinic. Several reviewers expressed concern about the cell lines that are to be used in this study. It appears that one, or perhaps two of the three proposed lines were maintained on murine feeder cells. As these would not be optimal for clinical use, the impact of this effort would be lower. However, if the culturing techniques, assays and other proposed tools could be applied to other cell lines, the impact would remain significant.

Despite the complex and ambitious scope of this proposal, the reviewers were generally optimistic of its feasibility due to the logical, thorough research plan and the supreme qualifications of the applicants. The proposal was well conceived and clearly written, although the reviewers were disappointed in the paucity of details that were provided for alternative strategies in the event of failure. Other concerns that were raised alluded mainly to the general risk of the endeavor. The success of the proposal rests on the completion of the first aim, which is expected to take 9 months to 1 year to complete. In addition, the applicants offered too few details on several assays to judge their merit such as sensitivity of the assays.

The reviewers found the principal investigator and collaborators to have extensive experience and relevant expertise in all aspects of this proposal. The co-investigators have excellent track records in the areas in which they will contribute. The private/public partnership was considered one of the major assets in this proposal. The research environments were also judged to be outstanding.

The budget for this effort was judged to be reasonable, although some reviewers felt that the distribution of funds was too heavily weighted towards investigators with overlapping responsibilities.

Overall, this is an ambitious but highly interesting proposal that addresses a key roadblock in the field. The comprehensive approach and strong research team were deemed sound and more than adequate to justify the risk.