Funding opportunities

Optimization of Human Embryonic Stem Cell Derivation Techniques and Production/Distribution of GMP-Grade Lines

Funding Type: 
New Cell Lines
Grant Number: 
RL1-00648
Principle Investigator: 
Funds requested: 
$1 383 419
Funding Recommendations: 
Recommended
Grant approved: 
Yes
Public Abstract: 
The government has strict rules for producing cells that will be transplanted into patients. For example, these regulations discourage the use of animal products that could transmit diseases to humans. In this context, the high-quality and tightly regulated procedures that govern other cell-based therapies, e.g., bone marrow transplants, will be applied to regenerative-type clinical applications that employ human embryonic stem cells (hESCs). We need to produce these cells now so that they will be ready to use when research findings are translated into patient therapies. Our goal is to supply researchers in California and outside the state with the highest-quality hESCs. To achieve this goal, we will build on our previous work, published in the scientific literature, which includes deriving hESCs from intact embryos and their single-cell components. We also study the basic properties of embryos and hESCs so that we can formulate theories about how to improve the derivation process, which we then test in our laboratory. For example, adult humans need very precise levels of oxygen. Our work shows that the same is true for embryos and hESCs. We have also developed novel culture conditions that use defined components such as those that are required by the governmental agencies that set the standards for the production of cells used in therapeutic applications. Accordingly, we propose a two-phase approach. During the first two years, guided by advances made by our group and others, we will derive hESCs from embryos in a biologically relevant oxygen environment using defined, high-quality materials. We will also derive hESCs from single cells removed by biopsy from embryos at specific stages of development and/or from particular regions. We think that these lines might have more predictable properties than hESCs that arise randomly, the current practice. Thus, at the end of the first phase we will have produced and banked the next generation of lines, which will be derived under defined conditions that more closely comply with government regulations regarding the production of clinical-grade cells. In the second phase, year 3 of the project, we will use the conditions that best support hESC derivation/propagation to produce lines that can be transplanted into patients. Our efforts will benefit greatly from the infrastructure of our institution, which includes a government-approved facility for doing this work, and from colleagues with the requisite specialized expertise. With the resources provided, we think that we can generate and bank 12 to 20 cell lines; one-third will be produced in a manner that complies with government regulations pertaining to cell-based therapies. All will be widely distributed, as we believe that the pace of translational and clinical research depends on the availability of the highest-quality hESCs and on the important information about their fundamental properties that will emerge from this work.
Statement of Benefit to California: 
California citizens were overwhelmingly in favor of proposition 71, due in large part to the public's belief that laboratory scientists working together with their clinical colleagues could develop therapeutic approaches that utilize human embryonic stem cells and their derivatives in regenerative medicine applications. The research teams are equally excited about cell replacement strategies for treating a variety of medical conditions, as in many cases a cure might be feasible. However, we know from the collective experience of the biotechnology industry that filling the pipeline that leads from basic research to clinical applications inevitably takes time. How do we start this process and shorten the timelines for delivering cell-based therapies to patients? Much of the work in the individual pipelines that focus on specific diseases/conditions can happen in a nonlinear fashion. For example, we need not wait until safe and robust strategies are devised for differentiating human embryonic stem cells into specific cell types to develop the lines that meet government regulations regarding the production of cells for transplantation into patients. There are many reasons that deriving these human embryonic stem cell lines now is crucial. For example, it is likely that production of the cells that will eventually be used in clinical applications will be an iterative process. That is, we will continue to make key discoveries about the fundamental properties of human embryonic stem cells, about which basic information is still needed to improve conditions for growing and deriving lines. This is particularly relevant to the production of clinical-grade cells, as it will be much easier to meet Food and Drug Administration requirements if cells are produced using defined materials that contain only human and recombinant components. It will also be important to know if clinical-grade cells, which for regulatory reasons must be derived under streamlined conditions, have the same properties as other human embryonic cell lines that are used for research purposes. Thus, extensive preclinical testing will be required before these cells are approved by the Food and Drug Administration for use in humans. Thus, accomplishing the major goals set forth in this application will be of enormous benefit to California's citizens. We envision that production of the next generation of human embryonic stem cells that can be used in clinical applications will speed the delivery of therapeutic applications to patients. Along the way these cells will have many other valuable applications. For example, they can be used to screen pharmacologically active compounds for both beneficial and detrimental effects. They will also be valuable tools for understanding the molecular etiology of disease processes. Accordingly, accomplishing the goals of this project will greatly benefit the people’s health and California’s economy.
Review Summary: 
Executive Summary The overall objective of this proposal is to generate new human embryonic stem (hES) cell lines either from intact human embryos, or from individual blastomeres from cleavage stage embryos. The principal investigator (PI) proposes to optimize techniques and culture conditions to improve derivation of hES cell lines. The PI and his/her group have considerable expertise with hES cell derivation. In addition they have spent considerable effort in developing new culture technology, including the use of defined substrates and serum-free, chemically defined, culture medium. Previous data from this lab suggest that already existent cell lines can be cultured using these methods with pluripotency retained for at least 20 passages. In this application, the applicants would like to extend this work by attempting to generate new cell lines using these techniques. The PI also proposes to generate cell lines from single blastomeres. Previous work from the PI’s laboratory shows morphological heterogeneity among blastomeres which may contribute to functional heterogeneity observed in stem cell line derivatives obtained from these blastomeres. If successful, the proposed work may be useful for the derivation of more homogeneous hES cell lines. In the later part of the project the PI plans to use the optimized culture conditions to generate current Good Manufacturing Practices (cGMP)-grade hES cell lines destined for clinical use. The PI proposes to generate and bank a total of 12 to 20 new cell lines; one third will be produced under cGMP conditions. There was general agreement among the reviewers on the validity of the scientific rationale, and reviewers believed that the approach is sound and well developed. The PI’s institution has established an embryo bank with hundreds of embryos available for the derivation of new cell lines. However, it was pointed out during the discussion that these embryos were not collected under cGMP conditions, and therefore, cGMP production of cell lines proposed in this application will require further release testing required by the FDA. Reviewers concurred that the PI is qualified to lead this program. The PI is an established investigator in the stem cell field and collaborators listed in the application all have excellent credentials and scientific backgrounds to facilitate the derivation of cell lines under cGMP conditions. Overall, reviewers thought this to be an excellent application from a very competent group. Research proposed here will likely enhance our understanding of ES cell biology and differentiation. Research grade and cGMP grade new cell lines produced under this grant will be available to other investigators. Reviewer One Comments Significance: Significance and Innovation The author proposes to establish a GMP level facility for the generation of new hES lines from human embryos discarded following IVF procedures. This is not a proposal to apply the lines to specific diseases or disorders, but simply to function as a resource laboratory to generate new hES lines. The author proposes to develop techniques and culture conditions which will improve derivation of hES lines while still meeting the requirements expected by the FDA for transplant grade cells. The authors rely on the work of others as well as a large amount of work completed by their group on methods to improve hES cell line growth, differentiation and derivation. This group was the first to report the derivation of hES lines on human placental mesenchymal stromal cell feeders. They and others have reported that low oxygen, 6%, might more accurately reflect the in vivo environment of the developing embryo and therefore might be beneficial to hES cell line derivation, proliferation and differentiation. They have also identified a growth factor, GRO-alpha, which is dramatically upregulated by conditions that enhance hES self renewal. Thus these modifications make up the basis for what they suggest is a simple and more robust method for feeder free cell growth. They will use excess banked embryos which have been obtained appropriately. Lines will be generated by mechanical disruption rather than immunosurgery, using laser pulses to open the zona pellucida. Several media formulations have been devised to increase growth of established lines including the addition of 5mM lactate. Addition of human serum or serum albumin are also planned. In separate studies the authors suggest that some of the diversity in the growth and differentiation characteristics of different hES lines might come from heterogeneity among ICM blastomeres. They have good evidence in mouse embryos that tight junction formation and polarization occurs in cells of the outer surface of the embryo, cells that are allocated to the trophoblast lineage. Thus cells will be taken from different areas of the ICM and single blastomeres will be propagated. While arduous, this method may prove to be useful for the derivation of more homogeneous hES lines with predictable characteristics. Next, when appropriate (year 3) cell lines will be generated under strict cGMP standards with the goal to establish lines that could be used in clinical applications. A final aim will be to fully characterize the hES lines generated above. It seems that only normal embryos will function as source material for this group. No hES lines are proposed to be derived from embryos with genetic defects. Feasibility: The application is surprisingly detailed with proposed methods for each aim. The source of embryos for the research is the UCSF gamete and embryo bank. This bank is established and seemingly has more than 300 embryos available for derivation of the lines. The banking procedures comply with the UCSF IRB and the CIRM guidelines. The authors have derived hES lines previously and many of the methods proposed in the application spring from their own research. They have identified conditions which favor hES cell growth and assume that these conditions will also help them derive new lines. The experiments are well planned. The authors propose 2 full years of basic research to optimize the derivation and growth conditions of the hES lines prior to establishing the protocols for deriving lines under GMP conditions. That suggestion is not seen to be unreasonable. More progress is likely to be made in this area if more basic research is completed prior to implementing final plans for cGMP derivation of cell lines. PI and personnel. The PI, Susan Fisher, is Professor of Cell and Tissue Biology UCSF. She received her Ph.D in anatomy in 1977 from Univ. Kentucky. She has well respected research programs in placental development and function as well as in stem cell biology. She is generously funded in several areas including proteomics of normal and cancer cells and stem cells as well as more stem cell and developmental biology projects. She is a co-investigator on a CIRM funded program to establish a Non-federal human embryonic stem cell resource and teaching laboratory at UCSF and the PI on a training grant for students and fellows. She is also PI on another CIRM funded grant to “harness and standardize the process of developing new hES lines which was awarded in 2007 and runs through 2011. From the proposal it appears that there could be significant overlap between these two applications, especially in the first 2 years of this application when the PI does not propose to generate cGMP cell lines. Several other researchers are listed on the application and all have excellent credentials and backgrounds. Dr. Fisher enlists the help of Drs. Jeff Bluestone and Elizabeth Read to facilitate the derivation of cell lines under cGMP conditions. Their help will be critical to a successful project. Facilities/Environment: Excellent. The proposal indicates that the GMP laboratory at UCSF will be used for the proposed research. This facility is currently in operation and is being used to produce human islets for transplantation. Other non GMP level research can be conducted in a nonfederal laboratory which is fully stocked. Overall Evaluation: This is a very competent group. They have proposed modifications of existing protocols which will likely favor the derivation, growth and differentiation of new hES lines. The improved protocols will then be used to generate lines under cGMP conditions. Even if the proposed modifications do not improve cell line derivation they will still likely generate useful lines under cGMP conditions, albeit fewer. Research proposed here will likely enhance our understanding on ES biology and differentiation. Responsiveness to RFA: Good, lines will be characterized and shared Reviewer Two Comments Significance: The overall goal of this project is to generate new human embryonic stem cell lines either from intact human IVF embryos, or from individual blastomeres from cleavage stage embryos. Moreover, these applicants propose to generate these cell lines using highly defined culture conditions, including use of low oxygen to improve efficiency. The applicants have already displayed considerable effort developing more refined and xeno free culture conditions that support pluripotency of differentiated cell lines. The applicants would like to use this work to generate new cell lines using similar culture conditions. Feasibility: The overall aim of this application is to establish new human ES cell lines from both intact IVF embryos as well as cell lines derived from individual blastomeres. The applicants have considerable expertise in human embryos and previous experience with hES cell derivation. In addition they have spent considerable effort in developing new culture technology, including the use of defined substrates and serum free chemically defined culture medium. Previous data from this lab suggest that already existent cell lines can be cultured under these methods with pluripotency retained for at least 20 passages. In this application, the applicants would like to extend this work by attempting to generate new cell lines using these techniques and, in the last year, to extend this to the generation of cell lines made under GMP conditions. The PI on this application would appear to be already overly committed in terms of percent effort (more than 100%). Responsiveness to RFA: The applicants are experienced human embryonic stem cell biologists and the cell lines derived in this project will be subjected to standard tests of pluripotency. Cell lines that are derived in this work will be deposited in the CIRM Stem Cell Bank, but cells will also be made available to researchers at cost from the applicant’s research lab.
Conflicts: 

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