Human Embryonic Stem (hES) cells have the capacity to become all of the cell types of the human body. However, clinical applications of these cells require an understanding of how to isolate the downstream embryonic progenitors (EPs) of specific tissues. The CPMCRI Shared Research Laboratory (SRL) proposes to utilize a new two-step single cell technique to rapidly isolate a large number of potentially useful EPs, expand the number of those cells, test them for quality, and then share those purified cell types with other medical researchers throughout California. Specifically, the SRL will: 1. Isolate uniform populations of novel EPs that can become different tissues directly from discarded human IVF embryos. Using the two-step technology, ~ 1000 novel cell lines will be isolated and analyzed for gene expression and quality, banking, and distribution. 2. Isolate uniform populations of novel EPs that can become different tissues from hES cells derived from discarded IVF embryos carrying disease genes. In collaboration with Pacific Fertility Center, embryos carrying disease genes will be used to generate hES cells or caused to change into cells that can become different tissues using the two-step technology. Novel disease-bearing EP lines will be provided to California researchers in parallel to the normal lines for drug discovery and inherited disease research. To this end a 1500 sq/ft laboratory at CPMCRI will be renovated and dedicated as the SRL for stem cell research not sanctioned by the Federal Government. The facility will contain 3 cell culture hoods, 6 incubators, a cryopreservation system, microscopes, centrifuges, a robotics cell culture platform, and various equipment items necessary for cell culture. The Principal Investigator will serve as the Director of the facility. A Facilities Manager will be responsible for the day-to-day supervision of the facility. Core personnel will be responsible for the culture, cryopreservation, and distribution of the cells to researchers at CPMCRI, neighboring institutions, and institutions throughout California. CPMCRI researchers have projects that include neurological disease (ALS and nerve cell regeneration), cancer (breast cancer stem cells and cell migration), cardiovascular disease (myocyte regeneration), and inherited disease-related organ repair (cystic fibrosis and sickle cell disease).
Statement of Benefit to California:
The proposed research at the CPMCRI SRL benefits the people of California by providing medical researchers in the state novel human cell lines useful in both basic scientific study and potentially for use in the treatment of numerous degenerative diseases. The CPMCRI SRL will provide its collaborators with human embryo-derived cells that are more differentiated than human embryonic stem cells, are closer in their characteristics to the actual tissue that they are intended to repair, and therefore are closer to therapeutic application. The role of the SRL at CPMCRI will be to generate, characterize, test, and distribute these cell lines derived from normal human embryos through a process of direct differentiation wherein human embryonic stem cell lines are not utilized. In addition, the CPMCRI SRL will generate EPs from embryos carrying inherited disease gene alleles. The novel method of deriving these cells and the unique genetic properties of those generated with abnormal genetic backgrounds, offer a unique service to California researchers, useful in understanding the biology of disease and early human development, cell based drug discovery, and for the cell-based treatment of degenerative disease.
SHARED LABORATORY SYNOPSIS OF PROPOSAL: The stated goal of this application addresses establishing a Shared Research Lab (SRL) focused on an important area of stem cell research - the isolation and expansion of novel, clonal, stable, partially differentiated embryonic progenitor (EP) cells isolated from two sources: 1) direct differentiation of discarded IVF embryos; 2) the direct differentiation from embryos or human embryonic stem cell lines from discarded embryos diagnosed with genetic diseases. This work will be performed in collaboration with the Pacific Fertility Center. All of these embryonic progenitor lines would be made available to investigators to allow them to have access to uniformly committed lines of cells for terminal differentiation, thus avoiding some of the early pitfalls. To carry out this goal, the applicant proposes the renovation and equipping of 1500 square feet of laboratory space for an SRL for stem cell research not sanctioned by the Federal Government. A lab manager and core personnel will isolate EP lines using “two step clonal isolation technology”, culture, cryopreserve, test and distribute lines internally and externally to institutions throughout California. The institution’s investigators conduct research in neurological disease, cancer, cardiovascular disease and inherited disease organ repair. QUALITY AND IMPACT OF THE SCIENCE: The SRL would provide a source of new human embryonic progenitor (EP) cell lines with varied and early differentiation properties, but with still robust proliferation capacity. A list of PIs that would presumably be users of the lab is given, but no documentation of the nature, extent or purpose of their use is provided. Indeed, the implication is that the SRL would be dedicated to the generation of lines by the lab manager, a technician and the director. This grant proposes the establishment of an SRL for the generation of human embryo-derived cells. As such, these goals fall outside of NIH guidelines, yet do not involve the derivation of hESCs. No research is proposed. The proposed SRL will be used to isolate hundreds of diverse and scalable EPs from two novel sources – direct differentiation from discarded embryos from IVF and from embryos or cell lines derived from discarded embryos carrying disease alleles using a “two-step clonal isolation technology”. Approximately 1000 novel endodermal, mesodermal and ectodermal embryonic progenitor clones will be isolated, tested for quality, expanded, banked, and shared with qualified California researchers. A large bank of clonal hEP cell lines with a wide variety of primitive differentiation properties would, in principle, be a valuable resource for stem cell biologists to screen for a renewable cell source for a wide variety of therapeutic, toxicologic, and pharmacologic applications. More critical evaluation of cell lines and the nature of those analyses should be an important consideration prior to their release to other researchers. No research on adult or nonhuman stem cells will be performed. The qualifications and productivity of users of this cell-resource will be determined by who requests cells. Fifteen investigators are listed in the application: 10 are from the applicant institution, 1 is from Blood Systems Research Institute, 1 is from Advanced Cell Technology, 1 is from UC Berkeley and Advanced Cell Technology, 1 is from UCSF and 1 is from USF. The description of preliminary cell culture studies does not clearly delineate the experience of the PD, Dr. Gruenert, nor the experience of Dr. Chu, based on the cited references. In fact, Dr. Gruenert has extensive experience with cell culture derivation of human airway epithelial cells and Dr. Chu has derived hESC progenitor cell lines at Advanced Cell Technology. It was unclear to the reviewers what the research plans were for use of the proposed space by most of the investigators. Although the application contains bio-information about many highly qualified individuals and letters of support; it lacks sufficient information to understand what work will actually be performed by these investigators as a result of establishing the SRL. There were four figures included without discussion that appear to illustrate current research, but the PI associated with the figures is not stated. Although eight other PIs from CPMCRI are listed (besides Dr. Guernert and Dr. Chu), there are no roles or projects described for them, no indication that they will use the space or equipment, and none have prior experience with stem cells. This lack of relevant information severely limits enthusiasm for this application. The goal to create a resource of quality controlled early embryonic progenitors is valuable and important, but the relationship of this goal to the listed investigators is not provided, nor are the areas of research and plans/goals for the SRL clearly stated. APPROPRIATENESS OF SPACE AND EQUIPMENT TO SCOPE OF PLAN: Fifteen hundred square feet of laboratory space is to be renovated and dedicated as a non-federal shared research laboratory. The laboratory would be equipped with 3 cell culture hoods, 6 incubators, a cryopreservation system, microscopes, centrifuges, robotics cell culture platform, and "various items of cell culture equipment." The size and scale of the lab, equipment and personnel are appropriate for its single dedicated purpose of providing a large, unique source of human EP cell lines. A biosafety hood is available for other users, if needed. The project is not designed to meet the needs for NIH-free research space, training, or direct collaboration with other investigators of the host or neighboring institutions. It seems that this space and equipment is largely to be used as a facility for the aforementioned EP generation, and then as a shared research laboratory only as an afterthought. While these attempts at EP generation may be laudable, this is not the appropriate forum for funding of this grant application. Documentation of the need and projected use by other investigators would greatly increase the enthusiasm for this SRL application. Much NIH-free space is available at CPMCRI, and the intent of this application is not to provide access for researchers to equipment and space for work with cells not in the NIH-registry. Rather, the purpose is to equip space already available to apply a specific technology to create a thousand new stem cell lines. QUALITY OF MANAGEMENT PLAN: The quality of the plan to support, manage and maintain the lab and equipment is adequate, because a single dedicated project for one investigator and the absence of other committed users from within or without the host institution means little management is necessary. The ambiguity regarding other users needs clarification. The program director has long-term expertise in developing human non-stem cell lines, including hematopoietic multipotent progenitor cells, but has no publications demonstrating expertise with hESCs or generating stem cell lines. The qualifications of Dr. Chu to operate the ACTCellerate are likely unsurpassed, because she has been hired from ACT where her duties involved generating these kinds of embryonic progenitor lines, presumably using the ACTCellerate system, but that was not explicitly stated. In addition, preliminary studies with data presented suggest that useful partly differentiated cell clones can be obtained. However, it is not clear whether the data is derived from work with an ACTCellerate at CPMCRI, at ACT, or is information provided by ACT. Dr. Gruenert lists only 15% effort on this project, and Dr. Chu and the "to be named" technician will each devote 75% effort to this project. It is questionable whether this is sufficient personnel to derive, expand, characterize and bank the stated 1000 embryonic progenitor cell lines in three years. Although there are no descriptions for work in the SRL by any other than the technician who oversees the ACTCellerate and robotics instrumentation, scheduling is proposed to be on a signup first-come, first-serve basis with a web-based calendar. There is a clear institutional commitment to provide space for the Shared Lab. The oversight committee is advisory with emphasis on the technology and technology transfer. It is composed of comprised of highly knowledgeable, capable individuals from the Pacific Fertility Center, Advanced Cell Technology, the CPMCRI Tech Transfer Office, the director and assoc. director of CPMCRI, and the program director, who is also the chair of the CMPCRI SCRO. DISCUSSION: All the reviewers agreed that the proposed work – to derive 1000 lines from discarded and PGD embryos and then partially differentiate them, store and make available for research use - is conceptually good. However, significant parts of the proposal were missing such as the research of other proposed users and how this facility would enable such research.