Generation and Testing of Induced Pluripotent Stem Cell Lines for Understanding Human Oligodendrocyte Development and Disease
New Cell Lines
Many human neurological disorders, as diverse as inherited leukodystrophies, periventricular leucomalacia, cerebral palsy, and multiple sclerosis, are characterized by the selective loss of oligodendrocytes, the myelin-forming cells in the central nervous system. In affected patients, an appropriate way to restore function may be to provide them with the relevant replacement cells, the oligodendrocytes. Scientists have already been working on methods to generate replacement oligodendrocytes from human embryonic stem cells (hESCs) or to generate accurate in vitro models of these human neurological diseases using hESCs. A recent breakthrough in stem cell research is the success of generating induced pluripotent stem cells (iPS cells) by in vitro reprogramming of human somatic cells back to a pluripotent state using defined factors. We will build upon this amazing technology to generate new iPS cells directly from adult cells from patients with myelin disorders either by using defined genetic transcription factors or by using small-molecule drugs/defined soluble factors, and test the utilities of these new cells in experimental models in the lab. We will compare the behaviors of the oligodendrocytes derived from these iP cells versus hESCs. Oligodendrocytes are the cell type that is diseased in patients with myelin disorders, so it would be ideal to derive iP cells directly from human oligodendrocytes (“dedifferentiation”) and then to “redifferentiate” them into better in vitro models to study oligodendroglial diseases. Such a “de- and re-differentiation” (hence making a complete “Cellular U-Turn”) approach represents an ideal model of “disease in a dish". Currently very few treatments for any neurological diseases exist, in part because of the lack of suitable in vitro models with which to test therapeutics. The proposal will lead to generation of new cell lines that have important research and clinical application. In vitro reprogramming could provide a way to generate patient-specific stem cells that, in culture, could be turned into the type of cell or tissue needed to cure the patient’s disease or injury. Moreover, genetic defects can be repaired in reprogrammed cells before being transplanted back into the patient’s body. Although both hESC or iPS cell-based therapeutic strategies that can promote remyelination will likely have significant clinical impact, one important advantage of patient-specific self-transplants is that they obviate the need for immunosuppression.
Statement of Benefit to California:
We plan to develop methods to make a complete “Cellular U-Turn” involving “dedifferentiation and redifferentiation” of specialized cell types to study “disease in a dish”. In addition to being integral to understanding the mechanism by which the stem cell program can be reinstated in specialized cells, if successful, this work will provide an important milestone for regenerative medicine. The significance of accomplishing this work is thus at the highest possible level currently for the field. The proposal addresses fundamental biological processes in reprogramming, i.e. reverting specialized cell types back to a more immature cell that can act like a stem cell, and may also pave the way for autologous stem cell-based therapies. We believe the proposed research will benefit Californian in many ways. It will result in development of novel technologies that will be broadly applicable to study stem cells and development of stem cell-based therapies, and will help position us and other Californian scientists at the forefront of stem cell research and medicine. We will make newly generated cell lines readily and freely available to other investigators and companies in the hope of accelerating the pace of discovery. The research relies on products and tools manufactured and sold in the state of California. If successful, research will require a scaled-up version of protocols designed in the proposed studies. This could attract new biotechnology companies in the state, boosting the tax revenue in the state. This in turn will provide new jobs for California residents. This research will increase experience and knowledge of stem cells among residents of California. Establishment of successful cellular therapeutics in California will encourage institutions of higher education to promote science education to fill the jobs created by stem cell research. This will retain California students in the state that are interested in biomedical research and medical careers. It could also attract out-of-state students seeking degrees that will allow them access to careers in stem cell research. This research will contribute to the California education and health care systems by training undergraduate, graduate and postdoctoral students into highly skilled stem cell biologists. It is also envisioned that this will trickle down to the K-12 levels and provide funding to promote science education at all levels.
Executive Summary This grant focuses on diseases of the oligodendrocyte, the myelin-forming cells of the central nervous system. The rationale behind this project is to take oligodendrocytes, reprogram them into induced pluripotent stem (iPS) cells, and then redifferentiate them, a process the applicant refers to as a cellular u-turn. Three sets of experiments are proposed: 1) Fibroblasts from patients with a specific form of leukodystrophy will be reprogrammed using existing technologies for producing iPS cells. These cell lines will then be compared with existing human embryonic stem cells (hESCs). 2) Normal human oligodendrocytes (obtained commercially) will be reprogrammed using viruses to express the transcription factors. Additionally, the effect of a previously identified small molecule, used in conjunction with growth factors known to inhibit oligodendrocyte differentiation, will be tested for their reprogramming activity. 3) These iPS cells will be differentiated into oligodendrocytes using an embryoid body-based approach already established in the lab, and tested in mouse models of periventricular leukomalacia. Cells will be transplanted either into the subventricular zone (SVZ) or intravenously, and their differentiation, survival, axon growth, behavior and migration will be assessed using magnetic resonance imaging (MRI)-detectable markers. The applicant proposes to use the reprogramming technology to develop a number of cell lines that would advance our understanding of glial diseases of the central nervous system. Stem cells derived from patients with inherited myelin diseases such as the leukodystrophies will be a very valuable resource and represent an excellent way forward for studies of the pathogenesis of these diseases. Although there is great merit in the target and the application is put forward by an impressive investigator, the reviewers disagreed with the applicant regarding the value of using a cellular u-turn method. In addition, they felt that the proposal suffers from a lack of defined focus in terms of the patient population and the relevant animal model to study cellular therapy. The applicant has begun to develop protocols for generating oligodendrocytes and for studying reprogramming factors, but at this preliminary stage the project remains unfocused. The novelty of this application is the development of cell lines from two human leukodystrophies, and the reprogramming of human oligodendrocytes into iPS cells. However, the applicant appears somewhat uninformed about myelin diseases in general. For example, s/he states that Canavan’s disease is a dysmyelinating disorder, which is not correct. The primary pathology is the vacuolation of white matter. S/he also cites periventricular leukomalacia (PVL) and cerebral palsy (CP) as glial-based diseases. A reviewer commented that this is not generally accepted, especially in the case of CP – there is white matter involvement, but the pathology is much more complex and heterogeneous. It is not entirely clear why the applicant chose the two diseases cited to generate iPS cells, nor what s/he will do with them if s/he succeeds. The second aim of this project was the most criticized by reviewers. In this aim, the applicant proposes to derive iPS cells from oligodendrocytes. Reviewers commented that there is no evidence that oligodendrocyte-derived iPS cells would be any better for studying myelin disease than fibroblast-derived iPS cells from the same patient. This argument presupposes some sort of cellular memory that ensures preferred differentiation back to their previous state. While there is some suggestion that this might be the case, it is a very important research question and not something that can be assumed. In addition, the use of small molecules to induce oligodendrocyte dedifferentiation is very challenging, and the applicant presented no preliminary data to suggest its feasibility. Overall, therefore, reviewers disputed the feasibility of some parts of this aim, and they didn’t feel that cells generated from oligodendrocytes (the novel aspect of the work) will necessarily be any better than cells from other tissues. For the first aim, the applicant presents good preliminary data on differentiation of cells into oligodendrocytes, and although s/he has not yet generated iPS cells, the review panel was not too concerned about feasibility. The final part of the grant uses straightforward techniques – transplantation into mouse models to analyze oligodendrocyte biology – and most reviewers weren’t concerned with feasibility. However, one reviewer commented that the experiments are not considered in sufficient detail. For example, intravenous delivery requires that the question of whether the cells enter the central nervous system at all be addressed, and the use of MRI to track migrating cells requires validation and proof. This same reviewer wondered what marker would be used in these experiments, as it was not discussed. Other reviewers commented that although the applicant is not an expert, the team described has the experience to do these experiments. The applicant is a newly-appointed Assistant Professor at a top research institution, and is collaborating with two well-regarded co-investigators and several Research Scientists. This proposal is responsive to the RFA, as the proposed research should generate pluripotent human stem cell lines. The cells will be made available to others, as required. Reviewer One Comments Feasibility: Strengths: 1- The team. The PI is well qualified to perform the proposed experiments and has assembled a group of collaborators that have been working for years in the areas proposed i.e. Dr Li – viral vectors, Drs David and Sam Pleasure – Oligodendrocytes. 2- The environment. Davis has a strong commitment to regenerative medicine as demonstrated by the Stem Cell Program put in place. 3- Experience on oligodendrocyte differentiation from hES cells. Weaknesses: 1- Aim one is still on the drawing board. The PI has obtained the vectors (commercially available) and some cell lines but no iPS have been produced in his laboratory yet. 2- Feasibility of Aim 2. Knowing that the efficiency of generation of iPS cells is very low and that the PI has not yet started the experiments, not only aim 2 depends on the success of aim 1 but also, the PI will use human oligodendrocytes, a cell line of unknown efficiency to produce iPS cells. 3- In Aim 2 the PI proposes to use reversine, among other ‘unknown’ molecules to dedifferentiate oligodendrocytes. This strategy is somehow naive. PI mentions that a combination of reversine and BMP4 can induce dedifferentiation of oligodendrocytes, at least partially, but no data is shown in the preliminary results. Responsiveness to RFA: The application is responsive to the RFA. Reviewer Two Comments Significance: Stem cells derived from patients with inherited myelin diseases such as the leukodystrophies will be a very valuable resource and represent an excellent way forward for studies on the pathogenesis of these diseases. The first part of the study is therefore of great merit. I have concerns over the logic of the next part. Why should oligodendrocyte-derived iPS cells be any better at studying myelin disease than fibroblast-derived iPS cells from the same patient? This presupposes some sort of cellular memory that ensures preferred differentiation back to their previous state. While there may be some suggestion that this might be the case, it is a very important research question and not something that can be assumed. Overall, therefore, I don’t see that the cells generated from oligodendrocytes (the novel aspect of the work) will necessarily be any better than cells from other tissues. The final part of the grant uses straightforward techniques – transplantation into mouse models to analyze oligodendrocyte biology. Although the use of iPS-derived oligodendrocytes is novel, many others will be pursuing a similar line of work. Feasibility: Three sets of experiments are proposed. 1) Fibroblasts from patients with the myelin disease metachromatic leukodystrophy will be reprogrammed using the iPS technologies expressing transcription factors in viral vectors. These cell lines will then be compared with existing human ES cells. 2) Normal human oligodendrocytes (obtained commercially) will be reprogrammed using viruses to express the transcription factors. Additionally, the effect of a previously identified small molecule called reversine, used in conjunction with growth factors known to inhibit oligodendrocyte differentiation, will be tested for their reprogramming activity. 3) These iPS cells will be differentiated into oligodendrocytes using an embryoid body-based approach already established in the lab, and tested in mouse models of periventricular leukomalacia. Cells will be transplanted either into the subventricular zone or intravenously and subsequently differentiation, survival, axon growth, behavior and migration using MRI-detectable markers analyzed. Preliminary data shows the ability of the lab to culture human ES cells and use doxycycline-inducible expression, differentiate oligodendrocytes from mouse ES cells using a protocol devised largely by the Brustle lab and generate a model of periventricular leukomalacia in the mouse. There is a lack of detail in the experimental description throughout the grant that makes me worry about feasibility. The methods to be used to reprogram oligodendrocytes are an example – we are told just that “We will further characterize the cellular effects of defined molecules as well as identify the molecular basis for their activity”. The transplantation experiments are also not considered in sufficient detail. For example, intravenous delivery requires that the question of whether the cells enter the central nervous system at all be addressed, and the use of MRI to track migrating cells requires validation and proof. We are not told what marker would be used in these experiments. Regarding the basic aspect of proving pluripotency, only the most superficial details are provided as to how this will be done. Certainly, the preliminary data do not include transplantation into the mouse model, quantification of the extent of oligodendrocyte differentiation into ES cells (although convincing MBP expression is shown) or show any preliminary efforts to use the Yamanaka protocols to generate iPS cells. So, while the Principal Investigator has good collaborators who are experts in oligodendrocyte and ES cell biology, there must be concerns about feasibility that are not allayed by the publication record of the applicant. Responsiveness to RFA: This is satisfactory – the proposed research should generate pluripotent human stem cell lines as required. Reviewer Three Comments Significance: The applicant seeks to generate oligodendrocytes (OLs) from patients with human myelin disorders and from normal human tissue, from hESCs or from reprogramming human oligodendrocytes (OLs). He will transplant normal OLs into a mouse model of perivascular leukomalacia and study the effect on function of the mouse and related tissue repair. The novelty here will be to develop cell lines from two human disorders, metachromatic leukodystrophy and Canavan’s disease, and the reprogramming of human OLs to iPS cells. Feasibility: Unfortunately, this proposal lacks good justification for some of the aims and focus of effort. The applicant appears somewhat uninformed about myelin disease in general. For example, he states that Canavan’s disease is a demyelinating disorder which is not correct. The primary pathology is the vacuolation of white matter. He also cites PVL and cerebral palsy as glial based diseases. However I don’t think this is generally accepted especially cerebral palsy – there is white matter involvement, but the pathology is much more complex and heterogeneous. It is not entirely clear why he chose the two diseases he cites, to generate iPS cells from patient fibroblasts, nor what he will do with them if he succeeds. In the second Specific Aim, he gives no rationale for trying to generate iPS cells from human OLs given that he provides quite good evidence of success in generating OLs from hESCs. The human cell line he plans to use is not well known nor is it detailed in the Preliminary Results. In Specific Aim 3 he plans to differentiate the iPS cells from Specific Aim 2, and transplant them into the hypoxic/ischemic mouse model. Why not just use the OLs he has detailed in the Preliminary Results from hESCs? In short, his so called “Cellular U-Turn” seems somewhat redundant. He proposes to inject cells intravenously as an alternative to direct brain transplants, but this approach with neural stem cells in a mouse model of MS, led to very few cells getting into the brain parenchyma (Pluchino et al Nature 2005:436,266). He does not provide any statement on how he will test the pluripotency of the iPS cells. Probably the greatest strength of the proposal is in the Preliminary Results. He shows convincing evidence of OL production (no note however of the percentage). Also Figure 3 does provide a nice picture of myelin deficiency with axon survival in his hypoxic lesion. The PI is a young investigator who is well funded and has a good track record. Some of his collaborations seem appropriate, but why include Dr. S. Pleasure as a provider of cells when this is being done in the PI’s lab? Also, there seems to be a major gap in Dr. D. Pleasure’s career, no CV for Dr. Tarantal and no letter of support from Dr. Rowitch. Responsiveness to RFA: No statement on testing the pluripotency of the cells. The cells will be made available to others. Reviewer Four Comments Significance: This project seeks to develop cellular therapies to treat myelin diseases, caused by alterations in oligodendrocyte function. OL are CNS glial cells that produce myelin, and defective myelination is at the root of debilitating diseases including multiple sclerosis and cerebral palsy. Certainly this is a problem of considerable significance. The ability to repair or replace defective OL would provide a very significant advance. A major problem now is the lack of good animal models to test cellular therapies. While the project should generate interesting cell lines for in vitro study, it is less clear how it will impact advances toward cellular therapies. Feasibility: The first aim is to generate iPS cells from patients with myelin disease. Unfortunately, right at this point the rationale and focus of the proposal is not well described. The PI will use one cell line isolated from a patient with metachromatic leukodystrophy (MLD) which represents a defect in the lysosomal enzyme ARSA. It is unclear why this is the best disease to study or how it will relate to subsequent aims (eg. the animal model). How many lines will be generated? Either the Thomson or the Yamanaka cocktail of factors will be used, and the line(s) established will be compared to original iPS lines for karyotype, morphology, proliferation and pluripotency. The establishment of lines should be feasible, although it’s unclear why focus on this particular disease and not others. Experiments in the second Aim will seek to generate iPS from OL. It is suggested that this might be better than using fibroblasts for OL cellular therapies, although there is no evidence that this is the case, if they both generate iPS. One OL line has been purchased and will be used. However, it is not known if this “line” is of normal karyotype or representative of “normal” OL. A general idea is outlined using various combinations of iPS genes +/- small molecules such as reversine or GFs such as BMP4, FGF, EGF, etc. to try and de-differentiate these OL into a more primitive or iPS state. It should be interesting to compare iPS lines derived from different cell types, although this may be less relevant for the stated goal. Finally, the PI proposes to differentiate OL from the various iPS lines and use this in a mouse model. A strategy is outlined (EBs generate NP cells, EGF+PDGF to generate OLP cells, and T3 to generate OL), although it would have helped to show some preliminary data, since the PI has not published in this field. The animal model is a hypoxic/ischemic brain injury mouse model. Cells will be put into the injured pups in the lateral ventricle, injured cortex, or the vasculature. Subsequent histology will be evaluated. How this model recapitulates normal myelin-based diseases is not described, nor how it will be used in the context of the patient-specific iPS lines. The PI is a new Asst. Professor (since 2006) at UC Davis and is well funded by the NIH to study GluR in OL (2 R01s). He has recruited his colleague Dr. Ron Li with expertise in hESC biology. Responsiveness to RFA: The application proposes to generate new pluripotent cell lines as iPS from either fibroblasts or OL. The proposal suffers from a lack of defined focus in terms of the patient population and the relevant animal model to study cellular therapy. The PI has begun to develop protocols for generating OL and for studying reprogramming factors, although at this preliminary stage the focus is not well defined.