New Cell Lines
Human embryonic stem cells (hESC) have great potential for the treatment of disease and injury because they are pluripotent in their capability to form most cell types in the body. They will also be of great utility for screening new drug candidates, and for understanding the molecular mechanisms of human development and disease. However, a big challenge in the field of stem cell research is to develop patient-specific, immunologically matched hESC. Recently, it has been shown that both fetal and adult human somatic cells can be reprogrammed to induce a pluripotent state via expression of specific sets of transcription factors. Thus, a future therapeutic option would be to obtain a patient’s own skin cell, convert it to a cell resembling an embryonic stem cell, called an induced pluripotent stem (iPS) cell, and then use that cell for therapy. However, the iPS cells generated thus far are unsuitable for therapies because retroviral DNAs that integrate into the genome were used, which may cause mutations by inserting into a gene and altering it in a negative way (some cancers are caused by this mechanism). There is a critical need in the field to generate iPS cells using non-integrative reprogramming methods. We have obtained preliminary data suggesting that retinal pigment epithelial (RPE) cells can be reprogrammed to resemble hESC using a method that avoids the danger of integration of DNA into the genome. This application proposes to further develop this new technology to allow generation of iPS cells from RPE, which are easily obtained from biopsies. A second goal is to obtain iPS cells that differentiate into eye cells treatment of eye disease. The new cell lines we generate will be carefully characterized with respect to their capabilities for forming different useful cell types. They will be compared to hESC, which remain the gold standard for pluripotent stem cells, and other iPS cells. The propensity for differentiation into RPE and other ocular cells will be explored. If successful, these experiments will provide a method to obtain patient-specific cells that might be useful in therapies for a variety of maladies, including eye diseases such as age-related macular degeneration.
Statement of Benefit to California:
The State of California, like the rest of the nation, faces immense challenges to its health care system, with soaring medical costs due in part to continuing care of our aging population. The percentage of elderly in California is expected to grow from what was 14 percent in 1990 to 22 percent in 2030. Chronic degenerative diseases such as Alzheimer’s disease, Parkinson’s disease, age-related macular degeneration, cancer, diabetes, cardiovascular disease, osteoarthritis, and osteoporosis afflict a growing number of individuals in California. Major innovative approaches are now, more than ever, an imperative. Human embryonic stem cells (hESC) have great potential for the treatment of disease and injury because they are pluripotent in their capability to form most cell types in the body. They will also be of great utility for screening new drug candidates, and for understanding the molecular mechanisms of human development and disease. However, a big challenge in the field of stem cell research is to develop patient-specific, immunologically matched hESC. Recently, it has been shown that both fetal and adult human somatic cells can be reprogrammed to induce a pluripotent state via expression of specific sets of transcription factors. Our proposed research will develop new methods for generation of pluripotent stem cells from adult somatic cells, and identify new cell types for the treatment of eye diseases such as age-related macular degeneration. If successful, this work will be a great benefit to the state by providing useful new technology that addresses a critical need in the field of stem cell research. In addition, it provide new approaches for therapies to treat degenerative conditions that afflict millions of Californians.
Executive Summary The goal of this proposal is to develop a novel gene transfection technology that avoids the use of viruses for generation of induced pluripotent stem (iPS) cells. The proposed approach employs a novel, particle-based transient transfection system for the delivery of reprogramming genes. The method will be developed with retinal pigmented epithelial (RPE) cells, yielding a valuable tool for the study and treatment of macular degeneration. Experiments are proposed to refine and optimize methods for non-integrative reprogramming of adult RPE cells which are readily obtained from biopsies. This proposal is highly significant, because if successful, it could eliminate the need for retroviral vectors and their associated potential for mutagenesis. The use of the proposed technology for delivery of reprogramming genes is innovative, and the value of this approach will be heightened if the method is shown to be generally applicable to a variety of starting adult cell types. Preliminary data suggesting the generation of iPS cells from human embryonic fibroblasts via the novel transfection method appear promising. However, reviewers viewed these results as extremely preliminary and had strong reservations about the use of proposed particle transfection system. Aspects of serious concern included the non-degradable nature of particles and possible secondary effects from their persistence in cells, the lack of information about gene dosage or degree of DNA conjugation with the particles, and questions about the efficiency and reproducibility of the novel transfection system. The proposal describes a well designed and rigorous methodology to evaluate the pluripotency of derived cells. Reviewers were concerned about proposed alternative strategies which were viewed as too vague and not convincingly feasible. The PI and named co-investigators have appropriate expertise and experience to perform the proposed experiments. The planned collaboration with a biotech company strengthened the proposal. The low percent effort committed by a co-investigator who is a leading stem cell expert raised reviewers’ concerns as to whether he/she would be significantly involved in the project. The proposal is responsive to the RFA. It could provide a valuable approach for generation of cell lines and yield clinically useful new lines. The full identities and potential of derived cells and cell lines that might be produced by this technique remain to be determined, and the lack of sufficient preliminary analysis decreased reviewer enthusiasm for the proposal. The plan to deposit and share new cell lines appeared adequate. Reviewer Synopsis This grant addresses a major problem of using viruses to create pluripotent iPS cells – the presence of more than 20 viral insertions within the cells that are likely to seriously restrict their validity for translational and clinical use. Clearly, transient expression of the necessary transcription factors would be better provided the cells maintain a pluripotent state when factors are no longer expressed. The aim of this grant is to develop a novel transfection technology using carbon nanotubes (SWNTs) to re-program retinal pigment epithelium (RPE) cells, thus both proving a new technology suitable for widespread use and creating a clinically valuable cell line that would have utility in the treatment of patients with the dry form of age related macular degeneration (AMD) where degeneration of the RPE appears to cause a secondary photo receptor abnormality. Reviewer One Comments Significance: This proposed study aims to develop iPS cells from human retinal pigment epithelial (RPE) cells reprogrammed by non-integrative transfection method using carbon single walled carbon nanotubes (SWNT), and to explore the possible differentiation of iPS-RPE cells into ocular phenotypes for treatment of eye diseases. If successful, this study will provide a new method to generate patient-specific iPS cells. It might also be applicable to generating iPS cells from other human cells. The use of non-viral nanoparticles to transfect human cells for reprogramming is innovative. Feasibility: Aim 1 is to reprogram fetal RPE cells by SWNT-mediated transfection and characterize the resulting iPS cells. Transfection protocol will be refined to improve reprogramming efficiency. Aim 2 is to reprogram human adult RPE cells by both lentiviral and transient transfection protocols and characterize the resulting iPS cells. Aim 3 is to redifferentiate iPS-RPE cells to their original phenotype, hypothesizing that the iPS-RPE cells retain epigenetic marks and thus have a tendency to return to their original phenotype. The PI and three co-investigators are established developmental biologists. Recruiting James Thomson significantly strengthen the stem cell expertise. The new UCSB Laboratory for Stem Cell Biology will provide facility support to this proposed study. There is preliminary data showing the generation of cell lines by lentivirus-infection of fetal RPE cells, and ability to transfect human placental fibroblasts using SWNT-mediated transfection. However, the full identities of these derived cells remain to be characterized. Weaknesses: The use of SWNTs as a non-viral transfection reagent is not sufficiently justified. SWNTs are not degradable as the PI claimed. Rare studies have been reported using SWNT for pcDNA delivery. The DNA-SWNT conjugate was not characterized. The efficiency of SWNT-mediated transfection was not quantitatively characterized either. This makes it difficult to reproduce the results and to correlate the reprogramming efficiency with the transfection protocol. The reprogramming efficiency (page 3, Aim 1) is also poorly defined. The transfection protocol is highly dependent upon cell type, particularly for non-viral vehicles. The optimized protocol in Aim 1 for fetal RPE cells might not be applicable to adult RPE cells. The effect of SWNTs on cell line stability and toxicity should also be discussed, given that they are not degradable. The isolation of iPS cells by picking a few colonies is too crude of a method. Drug selection may be a better method. The hypothesis or logic for Aim 3 seems contradictory to that of Aims 1 and 2. It seems more of an alternative strategy for Aim 2. If these cells do retain epigenetic markers of mature RPE cells, then these cells can not be an equivalent of iPS cells. The PI has not carefully considered the alternative tactics. The alternative tactic discussed in Aim 1 (page 5) on conjugating transcriptional factors to SWNTs is premature. Feasibility of conjugating transcriptional factors to cell penetrating peptides also lacks supporting evidence. The alternative tactics discussed in Aim 2 (page 6) are too vague and not feasible. The proposed study is not well supported by available data, therefore remain risky. Responsiveness to RFA: The full identities of the derived cell lines from fetal and adult RPE cells, including those by retroviral protocol and SWNT protocol, remain to be characterized. The project is risky at its current stage. The PI has provided an adequate plan to deposit and share new cell lines if they are successfully developed. Reviewer Two Comments Significance: This proposal aims at the generation of induced pluripotent stem cells (iPS) by reprogramming adult retinal pigment epithelial cells (RPE). Currently, successfully generated iPS are unsuitable for therapies due to the use of retroviruses that integrate into the genome, and thus may give rise to insertional mutagenesis. The goals of this study are the development of non-integrative reprogramming technology and the generation of iPS cells that can differentiate into ocular phenotypes for the treatment of eye disease. The significance of this proposal is high given the problems posed by the use of retroviruses to translational and clinical use. Using non-viral nanoparticles for transfection represents this proposal’s key innovation. Feasibility: This proposal includes three specific aims. The first is to reprogram fetal RPE cells by transient transfection of transcription factor expression vectors coupled to SWNTs and characterize resulting iPS – fetal RPE cell lines. The second aim is to reprogram adult RPE cells by lentiviral and transient transfection protocols and characterize resulting iPS - adult RPE cell lines. The third aim will test the hypothesis that iPS-RPE cells have an increased propensity to redifferentiate to their original phenotype. Dr. Clegg has a decent publication track record, and will be devoting 29% effort (It is unclear how this fits with the budget justification of 100% “summer salary” over three months). In addition to the co-investigator, Dr. Lincoln Johnson (UCSB Center for the Study of Macular Degeneration - 10%), Dr. James Thomson (WiCell and satellite lab at UCSB- 1%), Dr. John Sundsmo (PrimeGen Biotech, 1%), a staff research associate and graduate student researcher are to be included in the project, but have yet to be named. The inclusion of Dr. Thomson greatly strengthens this proposal, but an effort greater than 1% would be more convincing. The facilities at UC Santa Barbara are adequate for the aims of this proposal, including a CIRM-funded shared stem cell lab, which will provide expertise and reagents for growing hESCs. Preliminary results include the generation of iPS-fetal RPE lines using lentivirus infection, the generation of putative iPS lines using transient transfection, and redifferentiation of putative iPS-fetal RPE (lenti) into cuboidal epithelial like cells. These preliminary results remain immature, and do not strongly support the proposed study. Responsiveness to RFA: Given that the proposed iPS lines have not been characterized, it is impossible to ascertain whether they will have the ability to differentiate into all three germ layers. The plan to share these new derived lines with researchers is adequate. Reviewer Three Comments Significance: Significance This is an important and interesting study. I think that the applicants have identified the key issues with the re-programming technology, and have found an attractive approach which may overcome the problems of viruses and may enable the transcription factors to be delivered. The important question is whether this will produce stable IPS cells, but this seems quite possible given the preliminary evidence provided and the likelihood that not all viral insertions will maintain expression in the long term. The rigorous methodology to be used to confirm the pluripotency is excellent, and the authors suggest a number of optimization strategies should initial attempts not be successful. The plan to address the question of whether cells prefer to differentiate back to their original cell type also adds to the significance of this study, as this is clearly a key issue for all approaches using the re-programming technology. Feasibility: The experimental plan is as follows: 1) Transcription factors will be coupled to SWNTs which are then added to fetal RPE cells. Clones with a human embryonic stem cell like morphology will be isolated and characterized using the methods described by the Thomson laboratory in their recent paper (and Thomson is a listed adviser on the grant). This is very thorough and includes karyotype, transgene insertion analysis, growth rate, RT-PCR for human embryonic stem cell markers, telomerase activity, immunohistochemistry, genome profiling, epigenetic and differentiation studies. 2) Adult RPE cells will be re-programmed, first using viruses to see if extra factors are required to generate pluripotent cells from these adult cells (as was found to be the case in the recent Daley lab study published in Nature) and then using SWNTs. Validation will be as before. 3) The applicants will then test the interesting idea that re-programmed cells retain a memory for their original fate by comparing differentiation of re-programmed cells with those of human embryonic stem cells in a previously developed protocol for RPE differentiation. Feasibility A number of factors make the feasibility of this study high. 1) The preliminary data presented shows that RPE cells can be re-programmed (as judged by morphology) using viral vectors to deliver the transcription factors, and that placental fibroblasts can be re-programmed using the SWNT technology (as judged by gene expression of oct2, nanog and sox2 as well as SSEA1 immunoreactivity). Additionally ES cell-like colonies are seen when SWNTs are applied to fetal RPE cells, although this data is very preliminary. 2) The applicants have the necessary expertise in RPE and human ES cell culture 3) The partnership with PrimeGene Biotech (who developed the SWNT technology) does ensure access to the technology and the collaboration with the Thomson lab ensures that effective tools for validation as well as sources of advice are available if needed. Responsiveness to RFA: RPE iPS cells without viral integrations would be valuable, so this proposal fits the call very well. In particular, they might have significant clinical utility in transplantation based approaches for AMD. Furthermore generation of cells using the technology described would provide a very important proof of principal for the field in validating the use of transient expression technologies for generating IPS cells. The long term benefits of this in providing clinically usable cell lines will be very considerable.