DESIGN AND ENGINEERING OF NEURAL STEM CELLS DERIVED FROM HUMAN EMBRYONIC STEM CELLS FOR REGENERATION AND REPAIR OF MULTIPLE SCLEROSIS
The goal of this proposal is to design and engineer neural stem cells that have been derived from several lines of human embryonic stem cells. We will use both lines established on mouse cell feeders (NIH approved) and on human feeders (non-NIH approved). The cells will be specifically designed to serve as prototypes for the treatment of multiple sclerosis (MS). MS is a disease of young adults that is chronic and due to the destruction of white matter and the nerves that course through it. The destruction is due to the inflammatory response characterized by cells and substances. Some of these substances called cytokines and chemokines are destructive and some are helpful in repairing the disease. We plan to engineer cells to either secrete the positive substances or to inhibit the negative influences that have been shown to affect stem cells. We will transplant these cells into different models of MS that have similar clinical course, immunology and pathology as the human disease.
Statement of Benefit to California:
We believe that very soon human stem cell lines that have been grown under GMP conditions without contamination from other species and meet FDA specifications for human use will become available. One of the diseases that we think will be an early target of stem cell treatment will be multiple sclerosis (MS). All treatments of MS use a preclinical model of murine experimental allergic encephalomyelitis prior to Phase 1 human trials. The studies proposed here will have addressed the preclinical issues such as which cell to use (more or less differentiated), survival within a hostile inflammatory environment, efficacy in repair and regeneration, allelic variability in the host, whether murine or human feeders are factors in the ability of donor cells to repair MS damage and the questions related to tumor formation, transdifferentiation and rejection. Laboratories both in California industries and universities will require this information before considering Phase 1-2 studies in humans. This data will place them in an advanced position for manufacturing the cells for a specific disease.
SYNOPSIS: This application is based on the hypothesis that simple cell replacement, using human neural stem cells (hNSCs) and oligodendroglial precursor cells (OPC), is unlikely to be a successful therapy for multiple sclerosis (MS) because endogenous stem cells of these types are present in MS lesions, but are unable to reverse the disease course, presumably due to the robust inflammatory conditions present in the lesion. The applicant proposes to address this problem by using hNSCs derived from hESCs in vitro and genetically engineered to maximize their ability to survive and reverse MS pathology. Thus the aim is to determine the “optimal design” of hESCs and hNSCs to treat MS. Specific Aims 1 and 2 will use gene transfer technology to engineer hNSCs derived from both NIH (mouse feeder) and non-NIH (human feeder) lines. GFP-hESCs will be constructed, and transplanted into the myelin oligodendrocyte glycoprotein (MOG)- induced EAE (experimental autoimmune encephalitis) mouse model of MS. hNSC derivatives of these cells that are constructed to over-express a variety of chemokines and cytokines that positively influence hNSC proliferation and migration, as well as hNSCs in which expression of receptors for molecules associated with destruction of OPCs will be reduced in “knockdown” experiments. Specific Aim 3 will address the question of allelic variability by examining by testing modified hNSC lines that are successful with MOG-induced EAE in other EAE models using different genetic backgrounds. IMPACT & SIGNIFICANCE: MS, a debilitating neurological disorder that strikes individuals in the prime of their life with an estimated prevalence of 1 in a 1000, is thought to be an autoimmune disorder in which the patients develop an inflammatory response in the CNS that is directed against components of the myelin sheath, produced by oligodendrocytes, producing demyelination and axonal loss. Therapies that result in enhanced remyelination will thus likely provide significant benefit to MS patients. Failure to remyelinate does not appear to be the result of an inadequate supply of endogenous oligodendrocyte precursor cells (OPCs) in the CNS, as these are prevalent in the lesion. The key idea in this proposal is that cell replacement therapy for MS must do more than increase the number of oligodendrocytes. The aim of the proposal is thus to design stem cells that are optimized for survival and differentiation into both oligodendrocytes and astrocytes in the inflammatory environment. The concept that stem cells must do more than produce oligodendroglial precursors to remyelinate the central nervous system is interesting and important. The idea of designing hNSCs that may be more resistant to the harmful inflammatory environment in MS patients is particularly interesting, as is the proposed approach of getting the hNSCs to express various factors that enhance proliferation and differentiation. The proposal could provide important information about the fate of hNSC derived from hESC, following transplantation into the central nervous system (CNS). In particular, the study will follow these cells in EAE, a mouse model of MS. All reviewers agreed that if a cell therapy could be developed for MS, based on the principles outlined in the proposal, it would be an important step forward. QUALITY OF THE RESEARCH PLAN: There are some serious concerns with the design of the research plan that substantially lower enthusiasm for this application. First, the proposal fails to clearly articulate the goals and the anticipated mechanisms of action of the transplanted cells. For example, it is unclear whether the embryonic stem cell line will be used primarily as a carrier for the delivery of immunomodulatory cues, such as the selected chemokines and cytokines, or whether the transplanted cells themselves are likely to selectively or preferentially differentiate into OPCs, myelinating oligodendrocytes or astrocytes, and repair the lesions. The proposal appears to be built on the basis that these things are going to happen yet there is no data in the application or in the literature to support either supposition. Second, the applicant fails to provide a compelling rationale for the use of the selected cell lines and many of the studies testing efficacy are likely to be difficult to interpret. There is no evidence that the expression of the selected cytokines will render the cells refractory to the disease and so these studies seem highly speculative. Finally, the research plan is considered to have a critical weakness in that it does not at all discuss immunosuppression, which will be necessary for the transplanted human cells to survive in the mice. Since the disease model depends on the immune response, use of immunosuppression may alter the disease course. These concerns are not identified as either conceptual or experimental problems, nor are they addressed in any way. The reviewers had an additional concern that while the work proposed is novel and has the potential to provide important information relevant to the use of stem cells in the treatment of MS, the large amount of work proposed may mean that the aims of the proposal cannot be achieved. STRENGTHS: The problem to be addressed is of substantial medical importance and one that appears well-suited for a stem cell approach. The concept of using hESC lines to enhance repair in MS is extremely attractive. It seems likely that this disease will be among the most amenable of neurological diseases to cell therapy, and thus reviewers consider the overall goal of the proposal a strength. The PI is a well-respected clinician-scientist with considerable experience in MS research, the EAE model and with genetic manipulation of stem cells. Moreover, the PI has assembled a group of talented and excellent collaborators that will assist in the studies, including many collaborators at USC in the area of microfluidics, and also with John Rossi at the City of Hope, who is experienced in the design of shRNAs and their expression from lentiviruses and Outi Hovatta who will be providing human ESC cells grown solely on human feeder cells. . The preliminary data demonstrates the ability of the investigators to grow hESCs and to develop substantial numbers of cells that express oligodendrocytes lineage markers by manipulating environmental conditions. The evidence that these cells can generate myelinating cells in vivo is, however, weak. WEAKNESSES: A major weakness of the proposal is the lack of clear cut hypotheses relating to the function of the transplanted cells in the treatment of MS. This translates into a very confused research plan in which the fate of cells is being traced with GFP, using promoters that are only on at certain times. Cytokine expression is dependent on promoter activities that are uncharacterized in the context of a lesion and it is not clear if the transplanted hESCs are intended to contribute directly to the repair of the lesion or if they are intended to promote repair through activation of endogenous cell populations. The design of the experiments in either case is quite different. There also is a methodological concern that quantitation of repair/protection in the different experimental paradigms will be complex and no clear description of how this will be done is presented in the application. A second major weakness of the proposal is that it does not discuss how it is that the human cells would not be rejected when transplanted into mice. If immunosuppression will be used, how will it affect the EAE disease process and the transplanted ESC cells? One reviewer described this omission as a fundamental and fatal flaw in the proposal. A third weakness is the very large amount of work that is proposed. There are approximately ten different hNSC lines that will be engineered for use in in vivo studies. The analysis of each line (in vitro, as well as in vivo in the described EAE model) will represent a substantial amount of work. Without preliminary data demonstrating the feasibility of such an ambitious approach, there is concern that the proposal is unrealistic and overly ambitious. The use of human stem cells in a mouse model may generate misleading results. There is a varying degree of species specificity in the growth factor and cytokine interactions with their receptors. The species specificity of these interactions is well documented in the literature, but not addressed in the proposal. MOG-induced EAE results in inflammation and demyelination in the spinal cord, yet the investigator proposes to inject the stem cells into the brain. The rationale behind this peculiar choice is not developed. Also, it is not clear why the stem cells will be injected so late in the disease course (6-8 days after disease onset). A better explanation behind the choice of this time point would have been helpful. The investigator proposes to generate separate stem cell lines that will express fluorescent markers under the regulation of neuronal, astrocytic and oligodendrocytic transcriptional control regions to follow the fate of the injected stem cells. It is unclear why a common transcriptional control region is not used in combination with antibodies specific to cell-specific markers. The first specific aim appears to be poorly developed. In this aim the applicant proposes to drive a reporter construct off developmentally-specific promoters to examine the distribution, fate and survival of transplanted hESC cells. Since the reporter will presumably only be expressed when the cells are either immature (nestin) turned into astrocytes (GFAP) or oligodendrocytes (MBP) these studies will be impossible to interpret. A loss of labeled cells will not distinguish between divergent fate and cell death. Furthermore, since there is no evidence that the transplanted cells will develop into GFAP+ astrocytes in the demyelinating environment, the ability of these cells to deliver exogenous growth factors as proposed in aim 2 is unclear. A quantitative assessment of the fate of the different cell lines would also be useful, but this requires the ability to trace all progeny of transplanted cells, not simply those that express a specific temporally and spatially-restricted promoter. Reviewers suggest that for an improved proposal, the plan must address the issue of immune rejection in animals models that have xenografts. In addition, several other groups have shown the restorative activity with different populations of cells transplanted into animals with EAE, and inclusion of a comparison of the efficacies between different cell lines would be an important addition to the field. DISCUSSION: Discussants agree that the PI is a well-respected clinician-scientist in this field. A major flaw of this proposal is that there is no apparent recognition or discussion of the requirement for, and difficulties with, immunosuppression in using cell transplantation in the EAE model. Additional concerns included the lack of a suitable rationale or supporting evidence for the experiments proposed, the lack of a plan to track the cells, the unrealistic scope of the proposal. and the site of cell injection.