Human embryonic stem cells (hESCs) hold significant promise for regenerative medicine. In this application our goal is to derive hESC lines from pre-implantation embryos to generate a source of low passage lines that can be used in research and to develop the procedures required to generate a clinic grade cell-based product. In this application we will develop the procedures for deriving, expanding and banking using current good manufacturing practices (cGMP), which are necessary for producing a clinic grade line. We aim to do this using chemically defined media and surfaces in consultation with the Director of Regulatory Compliance at our Institution. Secondly, we aim to derive hESC lines from embryos that have genetic disorders. These genetic aberrations are identified following pre implantation genetic diagnosis (PGD) or pre implantation genetic screening (PGS) of consented embryos from couples undergoing in vitro fertilization (IVF). Deriving hESC lines from these embryos will be essential for understanding the underlying causes of spontaneous miscarriage, or as a tool to improve the quality of life of individuals born with these chromosomal abnormalities including Trisomy 21 (Down Syndrome), or Monosomy X (Turner Syndrome).
Human embryonic stem cells could revolutionize modern medicine if used in cell-based therapies. However, the translational use of hESCs will not be realized unless an appropriate repository of low passage cells is derived and made available. This resource will solidify California as a recognized world leader in ESC research through the generation of a centralized and comprehensive resource of hESC lines and the creation of jobs to support this effort, and validate the decision of California voters who ensured the passage of Proposition 71. Our proposal works toward generating lines that make treatment strategies from hESCs one-step closer.
This proposal is a request for funds to generate new human embryonic stem cell (hESC) lines from both normal and genetically abnormal early embryos. One main goal is to develop consistent and reproducible standard operating procedures (SOPs) for the derivation, expansion and banking of clinical grade hESC lines using current Good Manufacturing Practices (cGMP). The second goal is to generate and bank lines from embryos rejected after pre-implantation genetic diagnosis (PGD). The applicant expects to have access to approximately 100 such embryos per year. The chromosomal abnormalities to be targeted for hESC derivation are trisomy 21, monosomy X, and additional aneuploidies.
This is a fairly straight-forward proposal. Although the level of innovation is not great, the experiments have the potential to yield highly significant results. The availability of a high quality, efficient protocol for the derivation of hESC lines under GMP conditions is essential for developing hESC for clinical trials and eventually cellular therapies, and would represent a step forward for the field. The project represents a collaboration between the applicant, who has access to a derivation lab within the cGMP suite at the applicant’s institution, and a prominent stem cell researcher from a nearby institution with extensive expertise in hESC derivation. The GMP facility is established and functioning, and has been used for FDA-approved clinical studies. The authors clearly intend to share the new lines and have considered mechanisms to facilitate that.
The derivation of hESC without immunosurgery and without feeder cell layers is a laudable goal, but the proposed solutions carry their own significant difficulties with FDA approval. Furthermore, the principal investigator (PI) proposes to systematically remove the xeno and undefined components of the system, but makes no mention of the actual in vitro fertilization (IVF) procedures, which include animal and undefined components for culturing and freezing embryos. Also, commercially available media, whose use is proposed in this application, may be chemically defined, but this is not the same as GMP grade. Thus, a reviewer was unsure whether the PI understands all GMP issues, and whether the new lines will be truly GMP-compliant. Furthermore, derivation of hESC in feeder cell-free conditions has proven to be difficult in the past, and a reviewer pointed out that preliminary data showing that the investigators would be able to accomplish this goal was lacking. Overall, though, the reviewers felt that based on past expertise within the applicant’s group, it seems likely that new cell lines can be derived.
The derivation of hESC lines from genetically diseased embryos is also an important goal, since it can lead to insights and potential treatments of genetic diseases. Members of the research team have performed several successful hESC derivations from normal and PGD embryos, including hESC lines from aneuploid embryos. There is little doubt that the group will succeed with this project. Appropriate characterization of all the cell lines for pluripotency and other attributes was planned, although one reviewer felt that not enough detail was provided. Furthermore, a genetic identity assay for each hESC line, although not proposed, will need to be developed, since the likelihood of cross-contamination is high when deriving and banking multiple cell lines. Additionally, one potential benefit of the availability of many newly derived hESC lines is the breadth of the population they may represent. Therefore, each line should be subjected to genetic profiling in order to record the unique “DNA fingerprint” of that line.
The PI is a recently appointed, promising young Assistant Professor. S/he does not yet have significant outside funding nor has s/he published a senior or first author paper since 2005. Therefore, a reviewer expressed some concern regarding how much effort s/he can apply at this stage of his/her career for this project, which is primarily an important banking project, rather than a defined scientific investigation. Furthermore, one reviewer pointed out that despite the ambitious nature of the proposal, the PI is only devoting a 10% effort to it, and co-investigators are also listed with low effort, so it was somewhat unclear who would be doing all of the proposed work. Concern was raised about the commitment of a prominent collaborator, who was judged to be critical to the success of this proposal, but did not request salary on the application, and has only a part time appointment at a nearby institution. On the other hand, another reviewer felt that key personnel with significant experience in developmental and stem cell biology and regulatory affairs were identified and strengthen the application.
This application constitutes a well written proposal from a strong group of stem cell researchers. The topic is important and the cell lines to be created during this award period will likely be quite useful for regenerative medicine and basic research.
Reviewer One Comments
Significance and Innovation
The author proposes to generate GMP level hESC 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 hESC lines. The author proposes to develop SOP which will allow them to generate the new lines without animal feeder layers. The lines will be produced in a GMP facility and the goal is to establish lines that could be used in clinical applications. A second aim of the application is to generate hESC lines with certain genetic defects identified by preimplantation genetic diagnosis and screening procedures. The third aim will be to fully characterize the hESC lines generated in aims 1 and 2.
Design and Feasibility of the Research Plan.
The ground work appears to be in place for the proposed work. The GMP facility is established and functioning. The authors have derived hESC lines, presumably some of them in this facility. The authors claim a 10-20% success rate on deriving hESC lines from previous attempts. This work is a collaboration between UCLA and Cedars Sinai Med Ctr. (CSMC). Both sites have IRB approved protocols for hESC derivation from human embryos and UCLA has an IRB approved procedure for hESC maintenance, banking, and intramural distribution which will be the framework for the extramural distribution of hESC lines generated through this protocol. The authors clearly intend to share the lines and have considered mechanisms to facilitate that.
The lines will be derived without immunosurgery (without using guinea pig complement) via laser surgery. Thus development of techniques will be required. Initially cell lines derived by these techniques would be grown on mouse feeder layers to learn the techniques. Subsequent steps would eliminate the need for xeno feeders. Proposals include human feeder layers and self assembled monolayers (SAM) that the authors have shown to support hESC growth in the absence of additional matrix (not tested on derivation yet). They will attempt to clone hESC from single cells.
The second aim will extend the derivation of new lines to those with identified genetic defects. The authors suggest that approximately 100 such embryos will be available per year for these studies. The most common genetic defects they have encountered were Fragile X syndrome, cystic fibrosis, Bloom syndrome, muscular dystrophies and Huntington’s disease. Other genetic abnormalities to be derived are those from trisomy 21, monosomy x, and aneuploidy on 9-12 other chromosomes.
Appropriate characterization of all of the cell lines was planned. At least 10 new hESC lines have been derived by the authors of this application, so some success with the more advanced techniques is likely.
PI and Personnel:
Amander Clark is an assistant professor in the Dept of Molec Cell and Dev Biol at UCLA and received a Ph.D. in 1999 from Univ. Melbourne which was followed by postdoctoral work at Baylor, Houston TX. The CV lists 12 selected publications all relevant to the proposed application and research support from start up and foundation sources. While young and relatively inexperienced, the PI seems quite capable. Additional support from Nissim Benvenisty, Co-Director of the CSMC stem cell institute and a highly respected stem cell researcher will be critical to a successful completion of the proposed research. Dr. Benvenisty is in fact so well known that he does not need to list the titles of his publications on the CV included in this application. Additional personnel with significant experience in developmental and stem cell biology and regulatory affairs were identified as key personnel and strengthen the application.
Facilities and environment are appropriate. The clean rooms for the GMP production of the cell lines is established and running. This is critical to the success of the application.
Overall evaluation: This application is a well written proposal from a strong group of stem cell researchers. The topic is important and the cell lines to be created during this award period will likely be quite useful for regenerative medicine and basic research. The lines with genetic abnormalities could provide additional insight into the nature and correction of the defects they carry.
Responsiveness to RFA:
Lines will be characterized appropriately.
Reviewer Two Comments
The proposal calls for the generation of hESC, with a focus on development of cGMP-compliant production. There is also the generation of hESC as disease models, possible utility for cancer predisposition testing, and to screen potential therapeutic agents.
Design and Feasibility
- This is an equal partnership between UCLA and Cedars Sinai, with CS providing the hESC for disease model focus
- They are proposing new culture strategies, in which they will be able to derive the new hESC in chemically defined media and culture surfaces that will enhance GMP-compliance and FDA approval for clinical studies.
- They hypothesize that relying on established hESC to predict optimal conditions for derivation may be problematic. They are proposing to derive the new hESC in feeder cell-free conditions, but this has proven to be difficult in the past, and preliminary data that they would be able to accomplish this goal was lacking.
- They plan to derive new hESC from embryos carrying a genetic disorder, using single cell biopsy from IVF for screening. 10 hESC lines have already been developed from aneuploid embryos, and they claim success with creating Lesch-Nyhan syndrome by homologous recombination in hESC. They propose starting with genetically screened and “defective” embryos, and using FISH to detect chromosomal abnormalities. They will characterize the new hESC using standard assays.
- HLA haplotype
PI has a small amount of funding
- Function of ESC genes in testicular cancer
- Career development award (0%) for molecular mechanisms in transformation of stem cells.
- In vivo imaging of germ line cancer stem cells (collaborator).
- Epigenetic signature of cancer
- Role of BMI in tumorigenesis
- Epigenetic Regulation of Germ cell derivation from hESC
- Functional genetic analysis of germ cell development (submitted)
- Already approved SCRO, IRB, and IACUC. Allows access to medical records of donors, important prerequisite for FDA.
- Good mix of investigators with demonstrated expertise working with hESC, both derivation, immunogenicity, tumorigenicity.
- Screening for aneuploid embryos already in place.
- GMP facility at UCLA, experience with previous FDA approved clinical studies.
- Access to 100 embryos per year, many pre-screened.
- Despite the ambitious nature of the proposal, PI is only on for 10%. Co-investigators are also low effort 10%, 1% (Zack listed as RA), 10%, and research associate 5%, 50% (Dr. Kim), 20%. There are no trainees, so it was a little unclear who would be doing all of the proposed work.
- There is a $150,000 subcontract with Cedar-Sinai, but no % effort listed?
- The derivation of hESC without immunosurgery is a laudable goal, but the proposed solutions carry their own significant difficulties with FDA approval (laser dissection, self-assembled monolayers, chemicals such as ROCK inhibitor and neurotrophins). They are clearly intending to rely on others such as Drs. Zack and Quan for their GMP experience, the proposal as written suggests they may not clearly completely understand requirements for clinical usage. For example, they describe the use of commercial media (e.g. StemPro) which may be chemically defined, but this is not the same as GMP grade.
Responsiveness to RFA:
Clearly responsive to the RFA
Reviewer Three Comments
This proposal is a collaboration between UCLA and Cedars Sinai Medical Center. The PI is Dr. Amander Clark from UCLA. Two promising future uses of hESC lines are 1. clinical application of the cells in cell replacement therapies and ; 2. creating in vitro disease models to study the mechanisms of disease progression in a controlled environment. The current proposal aims to address both of these. In Aim 1 the PI proposes to devise SOP’s for the derivation of new clinically relevant hESC lines under cGMP conditions using chemically defined growth media and surfaces. In Aim 2, the team proposes to derive new hESC lines from diseased embryos, as diagnosed by pre-implantation genetic testing.
The innovation in this proposal is not great; however, the experiments have the potential to yield highly significant results. The availability of a high quality, efficient protocol for the derivation of hESC lines under GMP conditions would represent a step forward for the field. The hESC lines derived under such a protocol would have the potential to be used in future clinical trials. Additionally, the hESC lines derived from diseased embryos can lead to insights and potential treatments for genetic diseases.
The design of the experiments is not complicated. In Aim 1 the research team proposes to derive new hESC lines with the goal of generating an SOP for cGMP derivation, expansion, and banking. They systematically will attempt to remove the xeno and undefined components of the system, beginning with removal of the ICM from the blastocyst. It must be pointed out that there is no mention of the actual IVF procedures, the culturing of the embryo to the blastocyst stage, or of the freezing of the embryo. IVF clinics traditionally use animal and undefined components during these stages.
The feeder cells, serum and other undefined components will then be removed from the derivation and culture system. Several media formulations will be tested and compared to their performance with established hESC lines. The researchers are careful to asses the media and matrices by four criteria – commercially available, chemically defined, feeder-free, and serum free. However, they do not seem to score for xeno-free. For example, both commercially available mTeSR and StemPro contain bovine serum albumin.
Aim 2 is the derivation of hESC lines from genetically diseased embryos. Members of the research team – e.g. Nissim Benvenisty – have performed several successful hESC derivations from normal and PGD embryos. There is little doubt that the group will succeed. Dr. Benvenisty has also succeeded in creating a model of a human disease by means of homologous recombination using a hESC line. The question arises, therefore, as to why the team proposes only new derivations from diseased embryos and not the creation of new disease models using homologous recombination.
Aim 3 of the proposal is the molecular characterization of the newly derived hESC lines. The characterization is crucial to the understanding of the cell lines and the disease models. However, not enough detail was provided. The PI overlooked the fact that some sort of identity assay will be necessary. When deriving and banking multiple cell lines, the likelihood of cross-contamination is high. Each line should be subjected to at least STR profiling as early as possible in order to record the unique “DNA fingerprint” of that line. Additionally, one potential benefit of the availability of many newly derived hESC lines is the breadth of the population they may represent. However, without SNP genotyping this information will be lost. So, in addition to the few assays proposed by the PI, STR and SNP analysis should be added. Dr. Benvenisty is a member of the International Stem Cell Initiative and has been involved in characterizing many hESC lines. He is very familiar with the STR and SNP assays, as well as others that were not proposed.
The proposed team of researchers seems to have the expertise necessary for success. They have performed similar work in the past and have access to excellent facilities. The Preliminary Data presented is encouraging.
While the researchers state that they will make the cell lines available to other researchers, no mention is made of sharing the SOP they will develop in Aim 1. Since Specific Aim 1 is to generate an SOP for cGMP hESC derivations, it should be shared and made available to other researchers just as the cell lines will be.
Responsiveness to RFA:
This proposal is responsive to the RFA and is not fundable by the NIH.
Reviewer Four Comments
This is a fairly straight-forward request for funds to generate new hESC lines from both normal and genetically abnormal early embryos. One main goal is to develop consistent and reproducible standard operating procedures (SOPs) to generate hESC lines using current Good Manufacturing Practices (cGMP) to derive, expand and bank clinical grade cell lines. This will of course be essential to develop the use of ES cells for clinical trials and eventually cellular therapies. The second goal is to generate and bank lines from discarded PGD embryos. This is also an important goal, although at this point it can also be accomplished using more standard procedures.
The project represents a collaboration among UCLA and Cedars Sinai stem cell researchers, taking advantage of the expertise at hESC derivation (CS) and the availability of a derivation lab within the cGMP suite (UCLA).
1) Derivation of clinical grade hESC lines. In order to avoid the use of guinea pig complement, the PI proposes to isolate ICM using laser dissection. These efforts will start using MEFs to optimize, with the MEFs eventually replaced by defined media and support. Some data is presented for specific media that will support serum free survival. The cultures will be adapted to self-assembled monolayers (SAM) that permit growth in the absence of common applied matrix (eg. Matrigel which is derived from rat sarcomas). The ES cells will be passaged as single cells using ROCK inhibitors, neurotrophins, or other additives.
2) Generate a repository of disease-specific hESC lines. The clinic performs 1000 IVF cycles per year, and screens 9-12 chromosomes by FISH. It is expected that several Downs and Turners lines can be derived, but others will also be collected depending on the PGD.
3) Characterize these lines (both normal and genetic variants). This will include karyotype, evaluation of pluripotency via markers, EBs, teratoma formation, transcript profiling. In addition the growth properties of the cGMP lines will be compared to evaluate consistency.
Regarding Aim 1, it is certainly feasible to try these and other approaches to culture cells in defined media and under clinical grade conditions. Of course this might not work. In this case, the PI will try to use a human feeder layer, and then adapt the cells off the feeder layer. Based on past expertise within the group, it seems likely that new lines can be derived including genetic variants from PGD.
The PI, Dr. Clark, is a promising young Asst. Professor with an interest in cancer stem cells. Her postdoctoral training was with Monica Justice at Baylor and then with Dr. Reijo Pera at UCSF, before being appointed as Asst. Professor at UCLA in 2006. She does not yet have significant outside funding nor has she published a senior or first author paper since 2005. Therefore, there could be some concern regarding how much effort she can apply at this stage of her career (10% is indicated), for this project, which is primarily an important banking project, rather than a defined scientific investigation. The project will rely upon direction of technical assistance from Dr. Benvenisty’s program at CS and the ESC derivation core at UCLA.
Responsiveness to RFA:
The application clearly responds to the RFA in an appropriate and broad manner. Based on this project, new hESC lines will likely be generated, banked, and made available for distribution. Strengths are the track record at CS for deriving hESC lines (at least 10 so far) and the established protocols for cGMP and distribution at UCLA. Of the two essentially independent “projects” that are outlined, both are important and would generate significant and valuable reagents. It is less clear why they were both included together on this single application.