Executive Summary The overall aim of this project is the derivation of clinical grade HLA-homozygous human stem cell lines using parthenogenetic activation of oocytes. With the proper selection of oocyte donors with regard to human leukocyte antigen (HLA) haplotypes, it should be possible to generate a bank of human parthenogenetic HLA-homozygous stem cell (hpSC-Hhom) lines with different haplotypes. Significance of the creation of hpSC-Hhom lines lies in the possibility of enhancing immune-matching with higher proportions of the population, thereby potentially facilitating the transplantation of cellular derivatives of such lines by reducing the potential for rejection by immune-mediated mechanisms. The principal investigator (PI) and his/her team are experts in deriving human parthenogenetic stem cell lines as evidenced by their publications. They propose to derive two or three hpSC-Hhom lines carrying different common HLA haplotypes every year over the period of three years. These lines will be derived from normal oocytes from donors undergoing in vitro fertilization (IVF). The investigators propose to carry out derivation, expansion and storage of HLA-homozygous stem cells under current Good Manufacturing Practices (cGMP) conditions. The cell lines will be characterized for embryonic stem cell markers. Their differentiation ability will be assessed by formation of embryoid bodies and their teratoma formation ability will be assessed in immunodeficient mice. However, reviewers expressed concern that based on murine studies the differentiation potential of parthenogenetic cells is rather limited, and the analyses proposed in the application to assess cell line differentiation potential were considered rudimentary. Reviewers felt that the PI overestimated the value of HLA matching in avoiding graft rejection. The PI has not considered the contribution of minor histocompatibility antigens in tissue rejection. For example, it was pointed out that even genotypically HLA identical bone marrow transplants are rejected due to the presence of multiple minor histoincompatibilities between the donor and recipient unless immunosuppression is given. Moreover, the HLA complex is extremely polymorphic and even the most common haplotypes are shared by only a small fraction of the population. The applicant did not propose any proof of concept studies in the proposal to test if cell lines derived from parthenogenetic embryos will overcome problems associated with HLA mismatch. From a feasibility point of view there was also a concern whether the PI will be able to consent enough volunteers needed to screen for common HLA haplotypes and who would have sufficient excess oocytes for the study. Finally, there was concern that inadequate resources were allocated to the most labor intensive aspects of the proposal, line derivation and cell culture. Because of these concerns, overall enthusiasm for this proposal was low. Reviewer Synopsis The major aim of this project is to generate HLA homozygous embryonic stem cell lines derived by parthenogenetic activation of unfertilized human eggs. The goal of this research is to generate a bank of clinical grade cell lines that encompass common HLA haplotypes found within the US population. Reviewer One Comments Significance: The investigator proposes to generate HLA homozygous pluripotent human stem cells parthenogenetically from human oocytes. The investigator claims that this would provide a source of stem cells that would then be used to generate cells, tissues and organs that could be given to a large fraction of the population without rejection. However, the universality of such cell lines with respect to immune rejection is somewhat exaggerated for a number of reasons. First, even genotypically HLA identical transplants are rejected due to the presence of multiple minor histoincompatibilities between the donor and recipient unless immunosuppression is given. Minor histocompatibility antigens are not considered or discussed in the application. Secondly, the HLA complex is extremely polymorphic and even the commonest haplotypes are shared by only a small fraction of the population. The commonest haplotype for HLA-A,B,C,DRB1 and DQB1 covers only 7.4% of the population, and the second and third commonest cover an additional 3.5 and 2.4%, respectively. These data do not take into account HLA-DP, which has significant immunogenicity and is known not to be in strong linkage disequilibrium with DQ. Thus, the cell lines and their progeny would likely be subject to rejection responses by “matched” recipients and the claim that they would match a significant fraction of the population is exaggerated. Moreover, the ability to generate human parthenogenetically derived stem cell lines reliably is questionable and the degree of function of such cell lines derived in the murine system have been somewhat limited. Thus, even if such lines could be generated, the differentiation potential might be limited. The proposal is only moderately innovative, as no novel strategies for deriving improved stem cell lines are described. Feasibility: The investigators propose to derive two or three hpSC-Hhom lines carrying different common haplotypes every year over the period of three years. These will be derived from normal oocyte donors undergoing IVF after screening of potential donors and their parents. It is not made clear why it is necessary to test the patients’ parents. They expect to screen more than 150 people each year in order to identify those with common HLA haplotypes. Only women from whom more than 11 oocytes are harvested will be used as oocyte donors. The consent procedures and methods of activating the oocytes to undergo parthenogenesis seem feasible. The investigators will try to eliminate the utilization of animal-derived components when generating hpSC-Hhom lines in order to make then appealing for clinical application. The cells will then be characterized for embryonic stem cell markers. Affymetrix SNP microarray analysis will also be used to characterize the lines. HLA typing will be performed. Differentiation ability will be assessed by formation of embryoid bodies, which will be cultured in suspension for 14 days followed by either plating for outgrowth development or additional culturing in suspension for one week. Differentiated cells will be investigated for ectodermal, mesodermal and endodermal markers. Teratoma formation in immunodeficient mice will be assessed. These studies will provide only rudimentary information on differentiation potential. The investigators have already derived four human stem cells lines, three of which are HLA homozygous, using this procedure. Thus, the studies seem feasible. The Principal Investigator received her MD degree at the 1st Moscow Medical Institute in 1962, a PhD degree in Oncology in 1968 and a Doctor of Science degree in Oncology in 1982 at the Academy of Medical Science of USSR. She is currently Chief Scientific Officer of International Stem Cell Corporation, a role she has served in since 2001. Prior to that, she was a Senior Research Scientist at Keller Facial Surgery Clinic in Santa Barbara. She held various senior research positions in Moscow, the last of which was Head of Department of Experimental Models, Cancer Research Center at the Russian Academy of Medical Science, from 1992-1997. Thus, she has considerable leadership experience. Her publications include two in the stem cell field, both reporting on patient-specific stem cell lines derived from human parthenogenetic blastocysts and published in Cloning and Stem Cells. Her previous publications were in the tumor biology/therapy field and were published in the Russian literature. Thus, she is relatively new to the stem cell field. Her collaborators include technical and scientific staff at ISC and the Southern California Reproductive Center/ART Reproductive Center, where human oocytes will be donated under an IRB-approved collaborative research agreement. Dr. Hans Keirstead at Reeve-Irvine Research Center, UC Irvine, will provide animal and molecular biology facilities and provide advice and assistance with stem cell characterization. Overall, the needed facilities, expertise and technology are available for this project. Responsiveness to RFA: The studies aim to generate pluripotent stem cells, but further differentiation beyond the basic ability to form teratomas and ectoderm, mesoderm and endoderm will not be explored. In chimeric mice generated from parthenogenetic and fertilized embryos, the parthenogenetic cells showed very restricted differentiation. Thus, it is unclear whether these cells could be of clinical utility. The applicant indicates her intention to share the new cell lines under rules “stipulated by CIRM”. Reviewer Two Comments Significance: An efficient methodology to generate HLA homozygous human pluripotent ES lines by means of parthenogenesis would have major implications for the field of stem cell research. In one sense this circumvents an important ethical issue for many members of the community in which the creation of ES cells from fertilized eggs is viewed as destruction of life. In the context of the present application, however, the significance of the creation of HLA homozygous hESC lines lies in the possibility of enhancing immune-matching with higher proportions of the population, thereby potentially facilitating the transplantation of cellular derivatives of such lines by reducing the potential for rejection by immune mediated mechanisms. In potentially providing a solution to what is commonly regarded as one of the most significant barriers to the therapeutic application of hES cells, success in the current project would represent a very significant step forward for ES cell based cellular therapies. Feasibility: • This is a very timely application from the PI, Dr Revazova who is currently CSO at International Stem Cell Corp. She has enlisted the collaboration of a number of colleagues from ISC in addition to others such as Dr Keirstead. This will ensure adequate progress is made in achieving the stated aims. • The specific aims are well formulated. The corresponding experimental plan is very concise but somewhat lacking in methodological details that would allow for a more in depth review. • There appears little doubt as to the feasibility of the derivation of HLA homozygous cell lines via parthenogenesis as evidenced by the recent publications from Dr Revazova which strongly attest to her capacity to perform the necessary experimental manipulations. • The portion of the experimental plan devoted to characterizing the properties of the resulting stem cell lines is somewhat lacking in detail. The assays that will be employed to analyze differentiation into ectodem, endoderm and mesoderm derivatives are not described. Dr Keirstead’s involvement is presumed to be key in these experiments and their interpretation. • A shortcoming is the absence of any evidence in support of the notion that HLA homozygous lines generated in the manner proposed will be less susceptible to immune rejection. The argument in terms of greater possibility of HLA matching with the broader population is appreciated but functional data in a transplant setting is lacking. For example, transplantation of HLA-heterozygous vs. HLA-homozygous parthenote ES derivatives into NOD/SCID mice previously engrafted with HLA-matched vs. mis-matched cord blood. Such data would significantly enhance the application. • A strength of the application is the focus on deriving cell lines under cGMP compliant conditions thereby establishing a fast-track to therapeutic application if the cell lines exhibit the anticipated properties in relation to their transplantability. Responsiveness to RFA: This proposal will likely yield pluripotent human ES cell lines. The applicant states that ISC will share the lines with the broader academic community. Reviewer Three Comments Significance: The overall aim of this project is to generate a bank of human parthenogenetic HLA homozygous human embryonic stem cell lines. These cell lines could be used to establish a repository of cells that encode common HLA haplotypes found within the US population. Feasibility: The overall research methodology within this project is fairly straightforward. This group has previously derived parthenogenetic human embryonic stem cell lines, several of which display HLA homozygosity. Therefore the protocols for deriving such cell lines are already well established within this group. One slight concern is how human oocytes will be obtained and whether or not any financial inducement will be used to obtain the eggs. Once the cell lines are established, standard assessment of hES cell lines will be employed as well as a full assessment of HLA genotype and differentiation of the cells into a variety of germ layer specific lineages. Major criticisms of this application include the overall salary requested, including considerable funding for members of the company without clear defined roles in the project and the embryologist supplying the oocytes; the lack of justification of much of the budget including comsummable costings, and ethical considerations regarding the procurement of oocytes. Responsiveness to RFA: The proposed means of demonstrating pluripotency of these cell lines are well established in the applicant’s lab as they have already established hES like cell lines from parthogenetic activation. In regards to making the cell lines available to other researchers, there is little if any documentation within the application as to how this will be handled. Reviewer Four Comments Significance: The investigators propose to derive a bank of HLA homozygous parthenogenetic human embryonic stem cells. This bank may be of clinical utility because only 20 lines carrying common haplotypes would match 1/4 of the US population. Deriving such lines in cGMP and defined conditions is important for the cells and their derivaties to be used clinically. Feasibility: The PI and her team are expert in deriving human parthenogenetic hES cells - they have published 2 reports. Facilities are adequate, as is the collaboration with an IVF center to recruit egg donors. The derivation methods as well as the characterization of the cell lines are straightforward. Only 2 personnel (only 10% effort each) are listed as doing the derivation and cell culture - this reflects a small part of the total personnel cost, yet should represent the majority of the work. Supply costs include HLA testing on oocyte donors - does it include any of the IVF treatment cost or remuneration to donors? Although the investigators will follow the FDA’s Eligibility Determination for Donors of Human Cells, Tissues, and Cellular and Tissue-Based Products (Guidance for Industry, DHHS, 2007) and will have oversight by an Institutional Review Board (IRB) as required by California and Federal regulations, more detail on egg procurement should be provided. 2-3 hpSC-Hhom lines will be derived every year over the period of three years, for which they estimate 150 women per year will need to be screened for HLA type. How will women be recruited? Can they assure that enough women will agree to be screened to donate oocytes to this project? Responsiveness to RFA: Pluripotent human stem cell lines will be generated and they will shared with researchers under the rules stipulated by CIRM. These lines may have clinical utility, though that is not specifically addressed in this proposal.