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CL1-00504-1: Translational Human Embryonic Stem Cell Shared Research Facility

Laboratory Recommendation: Not recommended for funding

Course Recommendation: Not recommended for funding

Public Abstract (provided by applicant)

The intent of the proposed shared research facility is to provide a state-wide resource for qualified scientists in California to study human embryonic stem cells (hESC) without federal restrictions. The shared facility will encourage a spirit of collaboration and include laboratories for investigators to culture, collect, store, and analyze hESC, provide necessary services that will be cost-effective and assist with research productivity, and ensure an environment that will facilitate the essential interactions among scientists. This approach will advance the use of hESC for regenerative medicine purposes and aid in developing new technologies and therapies for the treatment of human disease. Using established methods that have proven successful for other collaborative and service-based structures, this facility will encourage scientists to work together and provide the necessary resources to ensure their success. Investigators new to hESC research will benefit greatly by having this facility available because it will have a centralized supportive structure where experienced personnel will provide the necessary assistance and guidance. For those investigators with hESC research experience, new opportunities will be available to work with cell lines that can be obtained but not be used in laboratories that are supported by federal funding. This will greatly expand research programs that are focused, for example, on studying ways to differentiate hESC towards blood cells and vessels for the treatment of disorders such as sickle cell disease and vascular abnormalities associated with heart disease. In addition, regeneration of damaged organs such as the heart, lung, liver, or kidney may require methods to reconstruct these tissues using scaffolds on which to grow the cells. These approaches require the ideas of cell biologists, engineers, biomedical researchers, and clinicians working together, and testing these ideas to ensure the procedures are safe before considering treatments of human patients. Techniques such as those that focus on ways to monitor cells once they are injected into the body will provide a powerful tool to study the outcome of these therapies.

A techniques course will be offered to scientists, students, and staff which will result in more laboratories in California working with hESC. The cells are difficult to grow and specialized training is required by personnel that are highly skilled and can provide the necessary information and direction to ensure success. The course is designed to provide qualified applicants the training experiences that will reinforce the basics, and ensure they are able to establish these techniques in their laboratories. The training course will be offered 4 times during the calendar year and include presentations and ‘hands-on’ experiences. Continuing education through the facility will ensure trainees have the necessary support when they return to their respective laboratories.

Statement of Benefit to California (provided by applicant)

The Translational Human Embryonic Stem Cell Shared Research Facility will serve the state and its citizens by providing unparalleled opportunities to investigators, and establish a model for the manner in which researchers throughout California can work together to advance the use of cellular therapies for the treatment of human disease. This facility will remove barriers preventing the transfer of promising stem cell therapies to human patients by connecting people with expertise and new ideas with the resources necessary to develop and to evaluate new technologies and therapies under the necessary conditions before they are assessed in humans. The California community will benefit from the results of this collaborative environment because it will facilitate and advance research findings, promote a culture of sharing, and educate and train a new generation of scientists in human embryonic stem cell research. This approach, the infrastructure and goals of the proposed facility, and the plans for the techniques course will all provide opportunities for scientists, students, fellows, and staff, and increase the number of qualified scientific and medical personnel that will be able to make new discoveries and ultimately improve health care for patients.

Scientific Review Summary of Part One Application

SHARED LABORATORY

SYNOPSIS OF PROPOSAL: The applicants propose the establishment and maintenance of a non-federally funded shared research facility for the culture of human embryonic stem cells (hESCs), flow cytometry and cell sorting, quantitative polymerase chain reaction (qPCR), immunohistochemistry, controlled rate cryopreservation, and essential services for the state-wide hESC research community. There is currently no such space on the home campus. This space will include three fully-equipped cell culture labs. The applicants are proposing to use hESC lines from five sources in these studies. Half of the work could have been funded by the National Institutes of Health (NIH); however, establishment of new lines and derivation is part of the endeavor of the center which is not covered by federal funds.

QUALITY AND IMPACT OF THE SCIENCE: This application is for 3 years of funding to establish a shared research laboratory off campus near the home institution. It is predicted that the facility will provide services for 32 principal investigators (PIs) – 18 from the home institution, and 14 from neighboring and other institutions. A broad range of scientific questions are being addressed by the 32 investigators, and this proposal aims to form a laboratory that is not focused on a few areas but rather covers the entire spectrum of differential lineages providing support for this large number of investigators.

The basic biological studies performed in this facility have four aims:

1. the description of molecular pathways underlying epigenetic silencing in hESCs;
2. studies on changes in chromatin structure that occurs during differentiation of hESCs;
3. use of chemical biology to guide hESCs toward differentiation into specific pathways;
4. the “potential” for establishing new lines. The two first aims address epigenetic control of hESCs, which one reviewer feels is interesting but not of the highest priority in the hESC field or for CIRM. The third is sound, but it will be difficult for this institution to compete with other public and private universities all converging toward this goal with larger numbers of investigators and bigger collections of chemical compounds. Regarding the fourth aim, the meaning of “potential” is unclear in this context. The PI proposed for this work is currently on sabbatical, and upon return apparently will bring this technology to the home institution. Nevertheless, many of the studies proposed will require the use of hESC lines that are not approved for federal funding. It is not exactly clear how this facility will interact with some of the neighboring investigators. The likely draw for these PIs may be their collaborations with the program director (PD), who is a highly productive and helpful colleague. Finally, the applicants suggest the use of certain large animal models to facilitate the development of new cellular therapies for human disease. An important potential strength is the presence of these models, thus positioning this proposal closer to clinical applications, yet one reviewer notes potential complications with using these particular models.

APPROPRIATENESS OF SPACE AND EQUIPMENT TO SCOPE OF PLAN: The shared research facility, which will be located about 1/2 mile off the main campus, will be ~2,200 sq ft of laboratory area which will house cell culture laboratories along with facilities for fluorescence activated cell sorting (FACS) analysis and cell sorting, a molecular core laboratory, cryopreservation and cell storage facilities. The facility that has been designed is excellent, a nice floor plan is provided, and the work proposed with hESC is appropriate for the proposed facilities. There will be three cell culture rooms. Two will have one biosafety cabinet, and the third will have two biosafety cabinets. The third and largest cell culture room would also be used for the stem cell techniques course proposed. There will be a total of 12 incubators. Each lab will have one Zeiss Axiovert microscope with camera, a refrigerated centrifuge, and a microfuge. In addition, there will be two separate labs where investigators can work – one for histology with a cryostat and one for molecular work. The FACS sorting facility is directly adjacent to the cell culture rooms. Equipment proposed for this facility is adequate, which in addition to the equipment mentioned above includes carbon dioxide incubators, reverse-transcriptase PCR systems, -80 and -20 freezers, cryotanks, and an irradiator for mouse embryonic fibroblast feeders. Space might be limiting for the amount of equipment and number of rooms suggested, especially if the first year follows as predicted by the university regarding its popularity, but the applicants have not made an effort to justify the size based on the number of proposed users or the types of hESC research that will be performed.

QUALITY OF MANAGEMENT PLAN: One of the major strengths of this proposal is how well the laboratory will be managed. This facility will be overseen by a PD who has demonstrated expertise in multidisciplinary collaborations, and in the direction of core facilities focused on stem cells. The PD has a lot of experience running similarly complex cores that require multiple employees, quality control, federal and state oversight, and security. Well-trained and experienced technicians and administrators will assist the PD in the management of this facility. The PD currently serves as the chair of the institution’s Stem Cell Research Oversight Committee (SCRO) and the Institutional Animal Care and Use Committee (IACUC). The issue of conflict of interest or at least the appearance of conflict of interest will need to be dealt with accordingly, as the head of this facility cannot be both head of the scientific facility and head of IACUC and SCRO. Reviewers are confident that this would be resolved. The PD has amassed an impressive number of supporting letters, yet one reviewer feels that a rather bureaucratic system has been developed for California investigators wishing to gain access to this facility. This process will require the preparation of an “Investigator Request for Access” form documenting signed Material Transfer Agreements, necessary approvals from campus committees (Institutional Review Board, SCRO, IACUC, and biological use authorization), evidence of campus training in biological and chemical safety, bloodborne pathogens, and other required training and/or screening on an annual basis; evidence for prior laboratory experiences relevant to hESC research; and the completion of facility training requirements for all users. Once approved for access, investigators will be given key card access and user identification for online booking of the facility’s equipment and requests for reagents and supplies. This website will also include a cost structure for use of this facility (with hourly rates posted where applicable). This level of administration could act as a deterrent to scientists interested in this field; on the other hand, until the federal government loosens its restrictions on studies with hESC, this level of assurance of compliance is likely very important.

DISCUSSION: This institution has been approved for a good number of SEED grants, thus there is clearly a great deal of interest on the part of this institution in stem cell research. The goals of the center are straightforward, and the faculty possesses the requisite expertise for this kind of work. Reviewers generally believe they will do a good job. However, the science as presented is quite vague. The third aim, which is to look for compounds that change the epigenetic differentiation properties, is less well-developed than others in terms of the basic number of compounds to screen and how to follow up on hits. One reviewer noted that the chemical biology program is not as well developed compared to other institutions. The derivation of new lines in aim four is problematic in that one of the shared lab investigators is being trained in the technology while on sabbatical, but relevant issues for line derivation such as the origin/availability of embryos, patient consent requirements, and any connection to an IVF clinic are not described in the application. Progress in the research will depend on the availability of embryos.

There is a strong emphasis in the proposal on the fact that access to certain large animal models will be located adjacent to the hESC lab, but the relationship of the animal model center and the shared lab is unclear. One would assume the use of these large animal models in facilitating cell therapy development, but the downstream translational uses were not described in the application. Panel members generally believe that the field is not quite ready to use these models. Four investigators were included based on their intention to use these models, but one reviewer had trouble judging the science based on the description provided. The PD is a very good collaborator but the rationale for their inclusion as users is unclear. Another reviewer mentioned that the imaging proposed would be critical in using these models. This reviewer noted that the imaging instrument mentioned may only be suitable for smaller animal models. Another discussant countered, stating that s/he believed this technology could be used here. Finally, one discussant expressed mild concern over the location of the facility being off the main campus. Another discussant pointed out that this facility is easy to reach by a 15 minute drive, where traffic is not an issue.

PROGRAMMATIC REVIEW: A motion was made to recommend this Shared Research Laboratory application for funding. Panel members cited two main problems: 1) there is a lack of clarity around the relationship with and utility of the large animal models and 2) the style of this plan (including the location off the main campus and the location of the collaborators) is not adequate to justify funding a core lab. One panel member noted even though this application has flaws, this institution is trying to ramp up work in this area and so it should be funded. Another panel member stated that this represents CIRM’s only proposal that has anything to do with the specific large animal models, and asked whether this is an important resource to build now. A discussant responded that the role of these models and what they contribute in the context of the shared lab were not well articulated. A panel member cited the availability of imaging resources once again, but more discussion of how they would be used in the context of their contribution to a unique shared lab resource was needed. The motion to recommend this Shared Research Laboratory application for funding failed.

TECHNIQUES COURSE

QUALITY OF THE PROPOSED TECHNIQUES COURSE: This application proposes a technique course to be conducted four times a year, each made of a two-day “intensive” period. The proposed course is over-ambitious in proposing that students will learn about the growth of hESC in this short amount of time. The cell cycle of a human embryonic stem cell is 24 hours; moreover, the generation of embryoid bodies without analysis requires several days. During these two days, students are to be trained on feeders and hESC culture, morphological analysis and characterization, thawing cells (both feeders and hESCs), cell staining and flow cytometry, PCR and immunohistochemistry (of both differentiated and undifferentiated cells). On day two, the students are meant to learn passaging and cryopreservation, establishing cell banks, formation of embryoid bodies, and preparation of cells for karyotyping. In addition to the experiments of these two days, students would have a total of 4.5 hours on day one and 4 hours on day two of theoretical teaching, which includes issues from general techniques to discussions of MTAs, ESCRO and IRBs. This scheduling, in addition to a lack of expertise among the resident faculty as compared to other California institutions, dampened the enthusiasm of reviewers.

QUALIFICATIONS OF THE INSTITUTION: The institution is excellent. The qualifications of specific trainers are also excellent as the applicants propose to bring in well-known experts from other institutions, including one who has extensive experience in teaching hESC culture courses, and another who has worked on differentiating hESC down pulmonary epithelial lineages. The resident faculty require more expertise in hESCs.

DISCUSSION: There was no further discussion following reviewers’ comments.

The following Grants Working Group members had a conflict of interest with this application and were therefore recused from participating in review of, discussion of, and voting on the application:

• Feit, Marcy
• Lansing, Sherry