Derivation of scalable clinical grade human embryonic stem cells under current Good Manufacturing Practice conditions using conventional and single blastomere biopsy protocols
New Cell Lines
New human embryonic stem cell lines for the clinical repair of damaged or missing tissues. Human embryonic stem cells have great therapeutic potential because of their ability to grow indefinitely and their potential to produce almost any cell type and tissue in the body. This makes it theoretically possibly to produce an inexhaustible supply of cells, tissues, and organs to repair damaged or diseased tissues in patients. However, several problems exist that must be resolved before human embryonic stem cells can be used for human therapies. Almost all the currently available human embryonic stem cell lines have been grown under conditions using animal products. This is problematic because the human embryonic stem cells may have been contaminated with animal viruses, prions or other animal pathogens making them unsuitable or, at least, suboptimal for therapeutic use in humans. We propose to develop a cell culture system to grow human embryonic stem cells that does not use animal products. Furthermore, in conventional methods used to develop new embryonic stem cell lines, the embryo is typically sacrificed, which raises an ethical concern about the destruction of the embryo. We propose to develop new human embryonic stem cell lines by removing a single cell from the embryo to develop a new embryonic stem cell line. Since human embryonic stem cell can divide indefinitely, we can expand that single cell into a new embryonic stem cell line. By removing only a single cell from the embryo, the embryo will remain alive, obviating the ethical concerns about sacrificing embryos to produce new human embryonic stem cell lines. Finally, many cell types in the body do not divide (duplicate themselves) well when grown in tissue culture plates, thus, making it difficult to produce enough cells for use in patients. We propose to develop new tissue culture systems to grow human embryonic stem cell in mass cultures so that they can be expanded in sufficient quantities and then converted into the desired cell type for use in patients.
Statement of Benefit to California:
California, like much of the United States, is facing a staggering challenge to its health care system. The large investments made in recent decades by the National Institutes of Health (NIH) have largely ignored the problems of age-related degenerative disease. As a result, increasingly physicians are treating the chronic, debilitating, and therefore expensive diseases associated with aging. This is made all the worse by the demographic wave caused by the entry of the Baby Boomers into retirement. It is estimated that by the year 2010, the Baby Boomers will be 25 percent of the population of California. By 2020 they will be approaching 64 years of age. As a result, the percentage of the elderly in California is expected to grow from 14 percent in 1990 to 22 percent in 2030. (Source: California Department of Finance, Population Projections 1993). Many of the chronic devastating diseases of an aging population are the degenerative diseases. Generally speaking, degenerative diseases are those diseases caused by the loss or dysfunction of cells. Examples include osteoarthritis (loss of cartilage cells that protect the ends of the bones), Parkinson’s disease (the loss of dopaminergic neurons), osteoporosis (dysfunction of osteoblasts), macular degeneration (dysfunction of retinal pigment cells) and so on. More significantly, the loss or dysfunction of cells in the heart (or the vessels that supply the heart with blood) results in heart disease, the most frequent cause of death in California. In 2001 (the most recent year data is available) heart disease caused 68,234 deaths (29% of all the deaths in the state). Stroke is also a vascular disease and the third leading cause of death in California. In 2001, stroke caused 18,088 deaths (8% of all of the deaths in the state). Regenerative medicine represents the effort of cell biologists to invent a new approach to the problem of degenerative disease. Human embryonic stem (hES) cells have the potential to become all of the cells in the human body, and their unique properties give researchers the hope that from these primitive cells new therapies can result that may be available in time for the looming health care crisis. It is estimated that are over 200 cell-types in the adult human and hES cells are capable of making all of these. However, to turn this new technology into actual therapies that can alleviate human suffering, researchers need new tools to generate large numbers of purified cell types. This proposal describes a project to derive new embryonic stem cells lines that are clinical grade and suitable for therapeutic use in regenerative medicine.
Executive Summary The primary goal of this proposal is to produce clinical grade human embryonic stem cell (hESC) lines under current good manufacturing practices (cGMP) conditions using conventional and single blastomere biopsy protocols, the latter having been developed at the applicant’s institution. In aim one, the applicant proposes to develop animal product free (APF) hESC culture media and cell substrates under cGMP conditions. The rationale for this goal is based on the fact that most existing hESC lines have been in contact with animal products during their derivation or propagation, thereby making them suboptimal for therapeutic applications. In the second aim, the applicant proposes to derive new cell lines from single blastomeres using the media and culture conditions derived in aim one. This technology has the advantage of leaving the embryos unharmed. In the third aim, the applicant plans to develop GMP procedures to expand cells en masse for differentiation of the cell lines to useful cell types, using a process developed by the applicant’s institution. The significance of this proposal lies in the development and optimization of an APF hESC derivation protocol, and the derivation of new hESC under cGMP conditions. However, reviewers questioned whether the proposed approach was truly APF, since some of the proposed components are known to contain animal products, others were not described in sufficient detail for the reviewers to assess their APF status, and the applicant makes no mention of the fact that the embryos themselves are not collected in a cGMP-compliant manner. So it was unclear to what extent cGMP-compliant hESC lines will be produced. Furthermore, reviewers felt that preliminary data were insufficient to assess the potential for a successful large scale expansion of the cell lines. Although one reviewer felt that the proposed derivation of hESC from single blastomeres is a useful goal, others criticized the use of this technology in the present proposal. While the single blastomere technique has the potential to yield hESC lines without destroying the embryo, thereby avoiding a major ethical concern, this holds little significance for the present proposal. The embryos used in this proposal are surplus embryos supplied by an in vitro fertilization (IVF) bank. It appears that these embryos will be consented for donation for research purposes and are not expected to be returned to the fertility clinics for implantation. There does not seem to be a shortage of surplus IVF embryos available for this project and others like it, as indicated by the letter of support submitted with the proposal. The researchers will most likely have better success rates using more traditional hESC derivation techniques, i.e. isolation and culture of the entire inner cell mass. Moreover, although some of the co-investigators have strong, relevant backgrounds in hESC research, it was unclear from the proposal whether the applicant actually has sufficient expertise to accomplish the proposed work. The most relevant experience with stem cell research in the applicant’s organization resides with a collaborator at a location outside of California, and the extent to which the collaborator will directly interact with the research team in California is not clear. Additional concern was raised with regard to the proposed time line, which was viewed as unrealistic. Overall, the enthusiasm for the proposal was not very high. Reviewer Synopsis This proposal calls for the derivation of clinical grade hESC lines. Use of the original NIH-approved hESC lines for therapy is problematic, because of possible exposure to animal products and other pathogens. Most of the current established hESC lines were not derived under GMP-compliant conditions, and there will be a need for a larger bank of “clinically” useful hESC to address tissue typing concerns for many of the possible applications of regenerative medicine. Reviewer One Comments Significance: The authors of this application propose to scale-up hES cell line culture using traditional methods and a new platform developed at Adv Cell Tech which they claim improves cell growth. In aim1 they propose to make cell lines for feeders from human fetal fibroblasts which are produced under GMP conditions and in the absence of animal products. In aim 1 b they propose to improve media for hES cell cultures through the use of animal product free media. The authors will test commercially available media and make improvements as necessary by the addition of commercially available growth factors or inhibitors of apoptosis (ROCK). They expect to find at least one that will maintain hESC in an undifferentiated state. In aim 2 they propose to derive new cell lines employing the single blastomere technology using the media and culture conditions derived in aim 1. Some scientists at ACT have a joint publication with outside scientists concerning the derivation of cell lines from single blastomeres. Experiments proposed in aim 3 will develop GMP procedures to expand cells en masse for differentiation of the cell lines to useful cell types. Here they propose to use a process they call ACTCellerate, a process by which cell conditioned media from specific cell lines can be used to scale-up cultures to the point where sufficient numbers of cells could be produced for regenerative medicine or other applications. Feasibility: The development of feeder layers from human cells is not particularly innovative. Although the goal was to produce animal product free media formulations, many of the formulations proposed in the application seem to include KOSR and or matrigel, both of which are derived from animal products. The goal to derive cell lines from single blastomeres seems to be useful. Once other GMP procedures were in place to create the lines under cGMP conditions, the lines generated in these experiments could be widely useful perhaps even for therapeutic procedures. The authors suggest that they will be able to generate approximately 30 such lines. In aim 3 they propose to use their ACTCellerate procedure to greatly expand the hES cells in culture. While little data is shown the authors claim great success with this approach. Several commercially available culture options were proposed for this aim. While all of the experiments proposed may not be particularly innovative, the group has the capacity to produce cells and conditioned media under cGMP level conditions. The blastomere technology is the strongest of the application. However, it is difficult to tell exactly where that technology resides within the ACT organization. The work cited was done in collaboration with outside university labs. Both Dr. Lanza and Klimanskaya from the Worcester facility were authors on the original paper and they have 5% effort on this application, but with out salary support. It is not clear that the technology has been transferred to the facility in CA. However, it is likely that at least some cell lines could be created by the single blastomere technology through concerted efforts between the 2 ACT campuses. If these can be propagated on human feeder layers with other culture or media formulations that may be developed, the result could be hES cell lines that might meet the expectation of the new cell lines RFA. The direction of the company research is definitely more in the applied area. Given the relative lack of innovation by the group, expectations for great success are not high. However it is likely that at least some useful cell lines will be created by these processes. PI and Personnel: Dr. James Murai received his Ph.D. in 1975 from UCLA in biochemistry. He did postdoctoral work at UCLA and U. Colorado and was an assistant research endocrinologist at UCSF from 1982-1993, when he moved to research positions at Geron, Immunotech and now Advanced Cell Technology. He lists 38 publications. From the publication record of the other team members there does not seem to be a lot of experience with stem cell-related research. The most direct experience seems to reside with Drs. Lanza and Klimanskaya from the Worcester facility. The extent to which they will directly interact with the research team in CA is not clear. Overall Evaluation: The project proposed here is mainly an applied research program which relies on established techniques to develop methods, media formulations and culture conditions which, if effective, will result in hES cell lines which are produced under cGMP conditions. Although the confidence level is not high, it is likely that at least some useful cell lines will be created by this group. Responsiveness to RFA: Adequate Reviewer Two Comments Significance: In this proposal from Advanced Cell Technology, Inc. (ACT) the PI Dr. Murai proposes to derive clinically relevant hESC lines in an animal-free system. Specifically, he will create an animal product free (APF) system for the derivation of human embryonic stem cell lines. He then proposes to combine this APF system with ACT’s single blastomere biopsy protocol to derive many new hESC lines in a cGMP manner. Lastly, he will apply ACT’s ACTCellerate platform to identify hESC-derived differentiated progeny for the further improvement of the APF system created in the first Specific Aim. While the single blastomere technique has the definite potential to yield hESC lines without destroying the embryo, thereby avoiding a major ethical point of conflict for the field of human ESC research, this holds no significance for the present proposal. The embryos used in this proposal are surplus IVF embryos supplied by the Pacific Fertility Center and the California Cryobank. It appears that these embryos will be consented for donation for research purposes and are not expected to be returned to the fertility clinics for implantation. The single blastomere technique is innovative and attractive; however, it is not ideally suited for the current application. There does not seem to be a shortage of surplus IVF embryos available for this project and others like it, as indicated by the letter of support submitted with the proposal. The researchers will most likely have better success rates using more traditional hESC derivation techniques – i.e. isolation and culture of the entire inner cell mass. While not particularly innovative, the real significance of this proposal lies in the development and optimization of the APF system and in the new hESC lines derived under cGMP conditions using this new system. These cell lines have the potential to match a wide sample of potential patients and serve as a valuable resource for the field of regenerative medicine. Feasibility: The design of the experiments is straightforward and well thought out. The researchers will begin in Aim 1 by developing an APF system using two established hESC lines. They will test published media formulations and matrices. The criticism of this approach is in the author’s misleading use of the terminology “animal product free.” There is a distinct difference between animal-free and xeno-free. The author seems to be confusing this distinction. The assumed intended meaning is xeno-free, since many of the proposed components contain human products – e.g. TeSR contains human serum albumin, HFF’s are derived from human fetuses, human serum will be used to culture the HFF’s, etc. In Aim 3 the PI proposes to use ACT’s platform to screen for a cell type that is able to produce a conditioned medium for the support and growth of hESC lines. While there is a great deal of emphasis on “animal-free” throughout the proposal, there is no mention of a chemically defined system. In their use of human components and cells for conditioning growth media, the researchers are introducing many undefined human components into the culture system. Since “defined” does not seem to be an issue in this proposal, in order to increase their chances of success, the researchers should consider using a condition equivalent to traditional growth medium with 15-20% human serum and/or 5-10% Plasmanate without FBS or KOSR. The last point relating to animal-free is the source of the enzymes the researchers will use to passage the hESC lines. At one point there is mention of using trypsin, but no reference given to the source. If it is standard porcine trypsin the researchers will be contaminating their xeno-free system. In Aim 2 the researchers propose to derive cGMP lines under cGMP conditions. The use of “current Good Manufacturing Practices” is confusing. The authors do not provide information as to whether or not the materials and components they propose to use in this project – media, sera, supplements, other cells - are cGMP-compliant products. Additionally, no mention is made of the fact that the embryos themselves are not cGMP-compliant. While it would be a step forward for the field if we were to have a well written, high-yield SOP for the derivation of hESC lines, we should be careful with the terminology. Finally, one must remember that a cell line need not be cGMP-derived, or even xeno-free, in order to be transferred to a GMP facility for production and use in clinical trials. The strength of this proposal lies in the experience and track record of ACT and its scientists. There is little doubt that the team will be able to derive new hESC lines. While little detail is given of the characterization and testing for pluripotency, the researchers cite references and most likely will apply the appropriate assays. The end result of this project will most likely be many new hESC lines derived under near xeno-free conditions. While new cell lines by themselves won’t advance the field very much, if they are derived from diverse genetic backgrounds using a xeno-free system these lines may represent a step forward. The cell culture system that will be developed may also help to advance the field and aid in future derivations. Responsiveness to RFA: This proposal is responsive to the RFA and is not fundable by the NIH. Reviewer Three Comments Significance: This proposal calls for the derivation of large numbers of clinical grade and GMP-compliant hESC lines. Use of the current existing hESC lines for therapy is problematic, because of possible exposure to animal products and other pathogens during their derivation and expansion. Most of the current established hESC lines were not derived under GMP-compliant conditions, and thus will be difficult and expensive to qualify for therapeutic use. Furthermore, there will be a need for a larger bank of "clinically" useful hESC with different HLA genotypes, to address tissue typing concerns for many of the possible applications of regenerative medicine. This proposal addresses many of those concerns, using established and newer approaches to derive hESC, and attempting to develop methods and reagents that will be easier to qualify for GMP manufacturing. Feasibility: The investigators propose to develop new hESC substrates and culture media that are clinically compliant. They plan to use single blastomeres obtained from IVFembryos to address ethical concerns of embryo destruction. They plan to develop a mechanical method that avoids animal-derived products for the initial selection, and have improved expansion of hESC using their ACTCellerate program. The starting embryos will be supplied by California Cryobank. There is some concern regarding the single blastomere biopsy approach. - Does this really eliminate all ethical issues? - Some of the proposed experiments utilize disrupted morulae? The proposed improved harvest technique also raises concerns. - Their preliminary results do not completely address GMP-compliance (co-culture with GFP hESC?, laminin, trypsin). Expansion - They plan to avoid the use of Matrigel (animal derived product). However, the proposal to use all human derived/recombinant matrix proteins may not prove effective and is likely to be too expensive ACTCellerate System - The novel concept is to use existing hESC lines as substrates and to provide conditioned media to support derivation of new hESC. However, there is a need to first develop qualified clinical-grade hESC or other lines to be used for the subsequent feeder layers and source of conditioned media. - They propose to use gene expression profiling to screen potential candidate lines (IGF, bFGF, Activin for conditioned media: laminins, collagen, fibronectin for substrate). - Lot-to-lot QA by Elisa and quant-PCR. - Explore bioreactor approaches for scale-up. - Human fetal fibroblasts, prepared animal-product free, for feeder cells (both derivation and expansion) - Obtained from aborted fetuses (which raises new ethical issues). - The proposal states that the fibroblasts may be fixed with glutaraldehyde for use as support substrate. Again, it is not clear how this will work, and how this can be done in a GMP-compliant manner. - They plan to conduct experiments to optimize the basal media - Supplement with other reagents reported to improve self-renewal (nothing novel proposed). Characterize new hESC - Standard assays, very minimal detail provided HLA matching - Create a bank of broad diversity MHC-typed lines - Estimate 150 will cover a majority of population Some of the co-investigators have strong, relevant backgrounds in hESC. Concerns: This is an overly ambitious proposal. The investigators are proposing to conduct a very large series of studies looking at all aspects of hESC derivation, but almost no preliminary data were given to support the proposed studies. The proposed timeline is overly aggressive, only 6 months are proposed to complete training of research associates in single blastomere isolation (in Worcester), develop and finalize GMP-compliant culture media and cell substrates, and to begin single blastomere experiments. There were no consultants listed (they plan to utilize the ACT team from Worcester. MA). Responsiveness to RFA: Generation of large numbers of clinically compliant hESC lines would be very responsive to the RFA. It will be very important to show that the newly derived lines meet the definition of pluripotency. The investigators are proposing to provide the hESC lines to the general research community for a "nominal" fee.