Funding opportunities

Comparison of New Lines from Multiple Sources

Funding Type: 
New Cell Lines
Grant Number: 
RL1-00654
Funds requested: 
$1 335 252
Funding Recommendations: 
Not recommended
Grant approved: 
No
Public Abstract: 
Human embryonic stem cells (hESCs) have been made and kept alive using many different methods. Researchers throughout the world have noticed that different stem cells have different qualities, but no-one has conducted a systematic comparison of multiple hESCs that have been made or kept alive using different methods. In this study, we will generate and compare hESCs using 4 different methods. 3 of these methods will result in the generation of patient-specific hESC lines, which would not be rejected if ever administered to a patient as a treatment. All of the methods will use techniques that do not involve rodent reagents and are FDA-compliant, such that all of the resulting products will be clinical grade. In addition, to generate some of our new lines, we will use starting material from genetically-diseased individuals so as to generate cellular models of human disease. Such disease lines will be very useful to study disease, and generate drugs that may counteract disease progression. Our systematic characterization and comparison of various lines that we generate will allow us to better define the appropriate uses for each line in research and therapy. Our laboratory is uniquely positioned to attain these goals, given our lengthy experience with hESCs, our experience in making new hESC lines, our published differentiation protocols, our IRB approval for all proposed studies, and our uniquely large supply of starting material from which to make the lines.
Statement of Benefit to California: 
The proposed research will benefit California by preserving and strengthening the State’s position as a leader in the field of stem cell tools and therapeutics. Through the passage of Proposition 71 and subsequent establishment of the California Institute for Regenerative Medicine, the voters of California have identified stem cell research as a key area of focus for the State, with anticipated positive impacts including the creation of biotechnology jobs, attraction of leading researchers to California universities, creation of valuable intellectual property, and advancement of therapeutics beneficial to California residents. The studies outlined in this proposal will lead to the development of new technologies that will form valuable intellectual property. Commercial interest in these technologies could then lead to biotechnology jobs. If successful in the generation of patient-specific stem cell lines, these studies will make a significant contribution to the development of patient therapies. Given the number of diseases that have the potential to be addressed with patient-specific cell lines, the emotional and financial benefit to Californians could be considerable.
Review Summary: 
Executive Summary This project is designed to create new human embryonic stem cell (hESC) lines using a variety of different approaches. These include: 1) Spare morulae and blastocysts from in vitro fertilization embryos and from embryos rejected following preimplantation genetic diagnosis (PGD); 2) Stem cells derived from parthenotes; 3) Stem cell lines obtained using somatic cell nuclear transfer (SCNT); 4) Stem cell lines derived using different induced pluripotency methods (iPS cell lines). Once generated, all the cell lines will be assessed and compared to one another using standard genetic, molecular, histological, and teratoma formation assays. The plan is to assess the differentiation biases and molecular correlates between the various lines with a focus on the derivation method. Consensus among reviewers was that the project is overly ambitious. This research project is designed to generate hESC lines using a wide variety of different approaches. Although technologically feasible, the amount of work required to generate the number of lines from the different sources and technologies as outlined in the proposal seems unrealistic. The principal investigator (PI) is only listed at 10% effort, with one co-investigator at 8% and several consultants at only 2%. Other key personnel are a research associate, a graduate student and a post-doc, and reviewers commented that it is not likely that they could manage the large scope of this project without additional senior management effort. Although it is clear that there is a need for new hESC lines derived from healthy embryos and disease-specific cell lines from either PGD embryos or embryos created by SCNT, this proposal lacks sufficient detail to determine the extent to which these cell lines can be made and which specific genetic disorders the researchers are interested in studying using these disease-specific cell lines. In addition, reviewers were unconvinced that hESC lines from such a wide variety of different embryo sources would be useful. It is not apparent, for instance, what advantage cell lines from parthenotes would have over cell lines from other sources. The major strength of the proposal is the PI, who is very accomplished and shows clear evidence of success. The laboratory has demonstrated ability to create hESC lines. In addition, the applicant has access to required starting material including oocytes required for aims 2 and 3. The reviewers were, however, skeptical about the number of oocytes the applicant claimed access to, particularly given public knowledge about the difficulties that other institutions have had in obtaining oocytes. The required facilities and equipment are all in place. The proposed characterization of all the cell lines is adequate and relies on standard biochemical, histological, molecular, and teratoma formation assays. Reviewers raised a few specific concerns. Although one reviewer stated that the preliminary data shows progress on most of the proposed aims, another reviewer pointed out that in terms of differentiation capacity of the lines, with the exception of differentiation into oligodendrocytic and neuronal populations, little evidence of multilineage differentiation of hESC is presented in this application. With regard to the iPS methods of generating cell lines, the applicants propose to use adenoviral vectors, which, although they do not integrate into the genome, are also diluted out by proliferating cells. It may be that the genes required for induction of pluripotency may have to be maintained within the cells for at least a few cell divisions for full reprogramming to occur. An additional concern is that although the applicant states in the proposal that they have received training from a well-known expert in the field of SCNT, there is no evidence of collaboration between these groups and no letter of collaboration is included in the application, so the extent to which the lab can successfully carry this out is unclear. The applicant mentioned that the cell lines would be generated in a “clinical-grade manner” to enhance possible translation to therapeutic use, although very little detail was provided as to how the various starting cells, materials, reagents, and equipment would be qualified for good manufacturing practices (GMP) use. The applicant states that s/he has access to diverse starting tissues in large numbers although as noted above there was reviewer skepticism regarding access to some starting materials in the amounts claimed. Reviewer One Comments Significance: Significance The proposal calls for extensive comparison of new lines from multiple sources. They plan to use 4 methods to produce the lines (both normal and disease-specific) 1. standard hESC 2. parthenogenesis 3. nuclear transfer 4. iPS a. Non-inserting “gutless” Adeno (Four separate vectors-it is not clear how expression will be controlled to ensure adequate levels in individual cells). b. Fusion proteins delivery The plan is to assess the differentiation biases and molecular correlates between the various lines – with a focus on the derivation method, using a panel of genetic, molecular, histological, and in vivo assays. Feasibility: Design and Feasibility The proposal calls for the production of the new lines by each of the 4 methods. There was no indication of how many lines might be generated with each of the different approaches. The applicant did mention that the cell lines would be generated in a “clinical-grade manner” to enhance possible translation to therapeutic use, although very little detail was provided as to how the various starting cells, materials, reagents, and equipment would be qualified for GMP use. They claim to have access to diverse starting tissues in large numbers, and have already produced 5 parthenogenesis derived lines. They are proposing to use fusion protein delivery for the iPS, which they state may be repeated if necessary to maintain reprogramming. There was no preliminary data, so this falls into the category of high risk/high reward. They are planning to compare properties of the new lines using state-of-the-art molecular profiling. They propose 11 different measures (of note) 1. miRNA expression 2. SNP (disease specific analysis if appropriate) They also plan to differentiate neurons from the various lines, and compare the properties of those cells. The PI is well-funded, including the following areas: 1. CIRM hESC neurons for treatment of spinal cord injury 2. Implant retinal layers for vision regeneration 3. Characterization of neurons from hESC 4. hESC derivatives for macular degeneration 5. Chemokines and demyelination 6. hESC for spinal cord injury (Geron) Strengths: 1. PI is very accomplished, clear evidence of success 2. Demonstrated ability to create hESC, especially by parthenogenesis. 3. Access to required starting material 4. Already SCRO, IRB, IACUC approved 5. Preliminary data shows progress on most of the proposed aims 6. Facilities and equipment all in place Other concerns: This is an overly ambitious proposal, it does not seem realistic to expect to accomplish all of the specific aims in the proposed timeframe. It woud be much better to focus on one or two methods rather than trying to run the gamut of options. The PI is only listed at 10% effort, with one co-investigator at 8% and several consultants at only 2%. Other key personnel are research associate, graduate student, and post-doc level, it is not likely they could manage the large scope of this project without additional senior management effort. Responsiveness to RFA: This proposal is clearly responsive to the RFA, it includes derivation of multiple types of pluripotent stem cells from both normal and disease-specific specimens, and possible GMP clinical use. Reviewer Two Comments Feasibility: Strengths: 1- The PI has clearly identified a need for the development of new standardized cell lines from fertilized embryos (aim 1). Weaknesses: 1- Overly ambitious proposal with very limited preliminary data. 2- Aim 1. This aim is devoid of novelty, although this reviewer recognizes the need of the work proposed. 3- Aim 2. Feasibility. It is not clear how the PI will manage to obtain 5000 (five thousand!) oocytes per month. It is publicly known that other groups (Harvard) have been unable to recruit ANY oocyte donors that will provide oocytes after one year of trying. How will the PI obtain 60,000 in a year? How come there is not a letter from at least 1 (one) IVF clinic that assures that the supply of oocytes will be warranted? Other than culturing and characterizing the cell lines produced by others, there is no preliminary data demonstrating the capacity of the PI to produce parthenogenetic embryos or cell lines from any species. 4- Aim 3. Feasibility. PI is proposing to perform human SCNT using the oocytes described above. There is no preliminary data that shows the capability of the PI to produce SCNT embryos of any kind in his laboratory. A short training provided by a collaborator (Dr Jerry Yang, an expert indeed) will not suffice to transfer the techniques to the PI’s laboratory. 5- Aim 4. Feasibility. PI is proposing to produce iPS cells using Gutless adenoviral vectors and TAT-fusion proteins. Both approaches are in a drawing board phase, no preliminary data is shown. Responsiveness to RFA: yes Reviewer Three Comments Significance: This research project is designed to generate human ES cell lines using a wide variety of different approaches. Although it is clear that there is a need for new human embryonic stem cell lines derived from healthy embryos and disease specific cell lines from either PGD embryos or embryos created by SCNT, this proposal lacks sufficient detail to determine the extent to which these cell lines can be made and which specific genetic disorders the researchers are interested in using to obtain disease-specific cell lines. Feasibility: This research project is designed to generate human ES cell lines using a wide variety of different approaches. Overall the project is highly ambitious and although technologically feasible, the amount of work required to generate the number of lines outlined in the proposal would seem to be unrealistic. In particular, it is uncertain and not rationalized in the proposal the extent to which one needs to be making hES cell lines from such a wide variety of different embryo sources. In particular, it is not apparent as to why the applicants want to make stem cell lines from parthenotes and what advantage these cell lines would have if generated. Regarding the use of stem cell lines using SCNT, little information is presented as to what exact types of diseases the applicants are proposing to study. An additional concern about this is that although the applicant states in the proposal that they have received training from a well known expert in the field of SCNT, there is no evidence of collaboration between these groups and no letter of collaboration is included in the application, so the extent to which the lab can successfully carry this out is unclear. The proposed characterization of all the cell lines is adequate and relies on standard biochemical, histological, molecular, and teratoma-related assays. The evidence that this group can successfully derive hES cells is weak and no real data regarding the pluripotency of the purported cell line is presented in the application. In terms of differentiation, with the exception of differentiation into oligodendrocytic populations, little evidence of multilineage differentiation with hES cells is presented in this application. Thirdly in regards to the iPS methods of generating cell lines, the applicants propose to use adenoviral vectors, which although they don’t integrate into the genome, are also diluted out by proliferating cells. It may be that the genes required for induction of pluripotency may have to be maintained within the cells for at least a few cell divisions for full reprogramming to occur. Responsiveness to RFA: This application fits squarely within the call and an adequate tacit agreement to make the stem cell lines available to the other groups through an MTA through the University of California Irvine is proposed. Tests of pluripotency are sufficient though not exhaustive.
Conflicts: 

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