Strokes that affect the nerves cells, i.e., “gray matter”, consistently receive the most attention. However, the kind of strokes that affecting the “wiring” of the brain, i.e., “white matter”, cause nearly as much disability. The most severe disability is caused when the stroke is in the wiring (axons) that connect the brain and spinal cord; as many as 150,000 patients are disabled per year in the US from this type of stroke. Although oligodendrocytes (“oligos”) are the white matter cells that produce the lipid rich axonal insulator called myelin) are preferentially damaged during these events, stem cell-derived oligos have not been tested for their efficacy in preclinical (animal) trials. These same white matter tracts (located underneath the gray matter, called subcortical) are also the primary sites of injury in MS, where multifocal inflammatory attack is responsible for stripping the insulating myelin sheaths from axons resulting in axonal dysfunction and degeneration. Attempts to treat MS-like lesions in animals using undifferentiated stem cell transplants are promising, but most evidence suggests that these approaches work by changing the inflammation response (immunomodulation) rather than myelin regeneration. While immunomodulation is unlikely to be sufficient to treat the disease completely, MS may not be amenable to localized oligo transplantation since it is such a multifocal process. This has led to new emphasis on approaches designed to maximize the response of endogenous oligo precursors that may be able to regenerate myelin if stimulated. We hypothesize that by exploiting novel features of oligo differentiation in vitro (that we have discovered and that are described in our preliminary data) that we will be able to improve our ability to generate oligo lineage cells from human embryonic stem cells and neural stem cells for transplantation, and also to develop approaches to maximize oligo development from endogenous precursors at the site of injury in the brain. This proposal will build on our recent successes in driving oligo precursor production from multipotential mouse neural stem cells by expressing regulatory transcription factors, and apply this approach to human embryonic and neural stem cells to produce cells that will be tested for their ability to ameliorate brain damage in rodent models of human stroke. Furthermore, we hope to develop approaches that may facilitate endogenous recruitment of oligo precursors to produce mature oligos, which may prove a viable regenerative approach to treat a variety of white matter diseases including MS and stroke.
Diseases associated with disruption of oligodendrocyte function and integrity (such as subcortical ischemic stroke and multiple sclerosis) are major causes of morbidity and mortality. Stroke is the third leading cause of death and the leading cause of permanent disability in the United States, costing over $50 billion dollars annually, as approximately 150,000 chronic stroke patients survive the acute event and are left with permanent, severe motor and/or sensory deficits. While much less common, multiple sclerosis (MS) is the primary non-traumatic cause of neurologic disability in young adults. Most patients are diagnosed in their 20s-40s and live for many decades after diagnosis with increasing needs for expensive services, medications and ultimately long-term care. Existing strategies for stem cell based therapies include both strategies to replace lost cells and to augment regeneration after injury, but most of these efforts have emphasized the role of undifferentiated stem cells in treatment despite the realization that the main nexus of injury in both diseases is frequently a differentiated cell type – the oligodendrocyte. This project will use new insights into the development of oligodendrocytes from the laboratories of the investigators to find ways to improve production of oligodendrocytes from human ES cells and human neural stem cells, test whether these cells can improve the clinical outcome in rodent models of stroke and MS after transplantation and search for new molecular treatments that would augment the regeneration of oligodendrocytes from resident brain stem cells after injury. This is the first step to translating the basic fundamental understanding of oligodendrocyte development into viable therapies for important human diseases that are major burdens on the citizens of California.
SYNOPSIS: This project will look at Sox factors and other soluble growth and differentiation factors to help expand and differentiate large numbers of oligodendroctye precursors from hES cells. After the factor searching and in vitro studies have yielded large numbers of such cells, in vivo grafting studies will assess their survival in transplant models of stroke and demyelination, and ultimately behavioral studies will determine any improvements that appear in the myelin-deficient rodent models.
IMPACT AND SIGNIFICANCE: There is a pressing need to develop better, more efficient and specific methods for generating large numbers of oligodendrocytes from hES cells. Oligodendrocytes are the myelinating cell of the central nervous system (CNS), and their viability and function are believed to be compromised in a number of debilitating neurological conditions. The myelin sheath, which is produced by oligodendrocytes in the CNS, is critical for proper neurological function, such that disorders that disrupt this structure, either directly or indirectly through the perturbation of oligodendrocytes, are very devastating. Failure to remyelinate does not appear to be the result of an inadequate supply of endogenous oligodendrocyte precursor cells (OPCs) in the CNS. These cells appear prevalent. Nevertheless, the endogenous OPCs appear incapable of mounting a significant remyelination response. Thus, the design of stem cell approaches that will be more likely to produce myelinating oligodendrocytes is critically important. This proposal is designed to develop and test approaches that will allow for the manipulation of hESC, such that they will preferentially differentiate into oligodendrocytes. These cells have translational potential for stroke, and other demyelinating diseases. Therapeutic strategies that promote remyelination will thus likely have significant clinical impact.
Initially, in this proposal, the potential of hESCs to differentiate into oligodendrocyte precursor cells (OPCs) and then to oligodendrocytes will be explored. It will be determined if this differentiation path can be preferentially driven in hESCs using intrinsic as well as extrinsic factors. The hypothesis is that a major transcriptional regulatory pathway in the genesis of oligodendrocyte lineage cells is the activation of the Sox transcription factors. It is likely that identification of major regulatory pathways for the differentiation of distinct populations of neural cells will provide useful information in the development of novel therapies for insults involving the loss of oligodendrocytes.
Second, the applicant proposes that transplantation of hESCs that have been “instructed” along the oligodendrocyte lineage will facilitate recovery in both Multiple Sclerosis (MS) and stroke. The development of effective therapeutic treatments for either MS or stroke would have enormous impact in clinical neuroscience and thus this application has substantial potential significance. One concern, however, is that it is not currently clear what cell populations will be most effective as therapeutics for either MS or stroke. If oligodendrocytes are key, this effort could prove to be exceptional valuable in the development of therapeutic strategies for the treatment of demyelinating disorders.
QUALITY OF THE RESEARCH PLAN: Overall, this is a strong proposal with significant potential. The problem to be examined is particularly important and the approach to be used is relatively novel. Four aims are proposed that should produce novel and meaningful results in the area of propagating and defining growth conditions for oligodendrocytes from hESC (approved and non-approved lines), and then testing their survivability and differentiation in stroke and myelin-deficient rodent models. The first aim will determine if directed expression of SoxE factors or soluble differentiation factors, discovered through preliminary studies defining the regulatory network downstream of SoxE factors, regulate the efficiency of oligodendrocyte development from hESC and human neural precursor cells (hNPC). Responses will be compared in several different lines. One reviewer noted that the Sox10 studies rapidly became diffuse with the addition of combinations of other transcription factors, array analyses of responders and non-responders as well involvement of human neural precursor cells. While the ideas were good, each aspect lacked sufficient rationale or validation.
The second aim is to develop novel methods for differentiating hESC, (using available federally approved and nonapproved lines) toward oligodendrocyte lineages using a combination of published protocols and the novel factors defined in the first aim. The goal of these studies is to generate pure populations of myelinating cells. Presumably the rationale behind these studies is that the more pure the cells the more effective the repair will be post transplantation. A reviewer noted that there is no evidence that this will in fact be true and it might well be that “pure” oligo lines will be worse that mixed cultures and felt that, at the very least the justification for this aim should be clearly articulated.
The third aim is to assess the survival, proliferation, migration, maturation, and function of hESC-derived oligodendrocytes following injection into rodent stroke models. Several variations on timing and dosage are proposed that should cover the most effective interventional period. One reviewer commented that there were some curious omissions in the description of the study, not least of which was that it is unclear how the cells will be delivered (iv, intraventricle injection?). Based on preliminary data it seems likely to be iv, but it is unclear whether hESC derived OPCs will get to the brain under such conditions.
The final aim is to repeat these studies in stroke with the addition of a behavioral endpoint and extend these studies to a dysmyelination model. One major concern with these studies is how the efficacy of myelination of the different cell populations will be quantified. A second concern is the selection of the host animal. While shiverer animals allow for a reasonably robust readout with a low background of endogenous myelin, the parameters in this dysmyelination system are quite different from that in demyelination such as it occurs in MS. As a result it is unclear how valid the data from these studies will be for directing therapeutic development.
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 and other demyelinating disorders. Nevertheless, there is concern with some of the proposed studies and with regard to one of the disease models chosen.
STRENGTHS: The Principle Investigator is a well respected scientist with considerable experience and expertise in developmental neuroscience. The PI has demonstrated experience with both the manipulation of embryonic stem cells and the factors that influence the differentiation of OPCs and has extensive expertise in the technologies and approaches to be used here, i.e., they have recently shown that SoxE (Sox8.9, and 1) transcription factors in mouse neural precursor cells helps turn multipotential neural stem/progenitor cells into lineage-restricted oligodendrocyte precursor cells. Their rodent data suggests that these cells then can be further differentiated with extremely high efficiency into mature oligodendrocytes. This provides a high level of confidence that the current studies will lead to even better oligodendrocyte generation following the discovery of key regulatory network morphogenetic factors.
The problem to be addressed is of substantial medical importance and one that appears well-suited for a stem cell approach. The approach to be used is novel: instead of just implanting stem cells into the CNS to determine their potential to remyelinate axons, the investigator will manipulate the stem cells in an effort to increase their potential to differentiate into OPCs. It is a great idea to compare the indigenous NPC population with hES-derived OPCs to see which of the starting cell populations is most amenable to molecular manipulation to facilitate their expansion, differentiation into mature oligos, and their myelinating prowess that might lead to improved behavioral outcomes in transplant and drug studies of stroke and demyelination.
The molecular studies designed to dissect the role of transcription factors in the development of human oligodendrocytes are potentially very useful. The studies examining the effect of novel regulators of oligodendrocytes development, identified through a logical screen, are strong. Focusing on Sox10 seems like a good approach, since Sox10 has been shown to interact with the MBP promoter and its expression.
The PI has already generated a lenti-GFP-Sox10 vector that should reliably mark OPCs for monitoring their differentiation (w.f. looking at O4, PDGF-alpha, and NG2 expression) and determine if Sox10 overexpression affects different hES lines in different ways. The expression profiling and ChIP on ChIP on mouse NPC expression Sox10 have already revealed cytokine receptors (e.g. ErbB3 and the neuregulin receptor) as potential targets for manipulation in the prosed NPC and hES cell studies. ChiIP on ChIP will be followed by probing on a promoter array to find direct targets in the mouse genome. Trying to develop purification protocols for OPCs and OLs, using gradient centrifugation and immunomagnetic separation is a reasonable approach, and there are surface markers (e.g. A2B5) and maybe even better ones that can be tried to help this cause. The battery of behavioral tests seems reasonable as well - After tMCAO motor and cognitive functions will be examined in the rodents, first in the open field, followed by foot fault and asymmetry tests, then by object recognition test, then on the rotor-rod, and finally in the water maze.
WEAKNESSES: The first aim is diffuse and contains a large number of permutations on experiments with little or no ranking of priorities. The search for novel soluble factors that positively affect hESC or hNPC cultures is a bit of a fishing expedition, but the SoxE associated network is a reasonable focal point. This is a weakness, since it is possible that factors might not be discovered that support oligodendrogliogenesis, but unlikely taken the approaches laid out here. The second aim needs to be hypothesis driven. For example, the overall application could be improved if the studies were designed to examine in detail different populations of hESCs/NPC or OPCs in a logical fashion. Aims 3 and 4 do depend on success of Aims 1 and 2.
What happens if cultures and grafts cannot be completely homogeneous? Will the neurons or astrocytes complicate or help the cause? It is not currently clear what cell populations will be most effective as therapeutics for either MS or stroke. The proposal discusses the merits of transplanting neural precursor cells or mobilization of endogenous precursors, but does not discuss the advantages of transplanting cells that are primarily committed to an oligodendrocyte, astrocyte or neuronal lineage. Indeed, it is unclear from published data which cell types might be most effective therapeutically and “induced” oligodendrocytes might be worse than less induced cells. This is not considered in the proposal which is somewhat worrisome.
A reviewer noted that the hEPC (human endothelial precursor cell) or HUVEC grafts and their accumulation in ischemic nude rat cortex is interesting, but it is a bit confusing if this is part of the proposed studies (and if it should be) or just pilot data from another study.
The studies to apply the developed cell lines for in models for therapeutic purposes was recognized as critical however there was concern about the models. 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. One reviewer found the stroke studies rather confusing and repetitious and the use of the shiverer model not particularly applicable to MS. The shiverer studies should either be articulated differently (not as a remyelination model) or a different MS model used.
Another reviewer noted that the use of an ischemic brain injury model to test the remyelinating potential of hESCs does not seem like the best model of choice. Models that display a clear loss of oligodendrocytes and myelin would likely provide a more appropriate model for examining this potential of hESCs. Little detail is provided for how the ischemic and dysmyelinated models will be examined following stem cell implantation. How will the success of the transplantations be monitored? In order to interpret cell transplantation studies, clear endpoints are required and it is surely not unreasonable to expect that histology and behavior be correlated on the same animals rather than on different animals. It might be worthwhile to try and image and track the grafted cells using MRI-detectable markers.
There was some difference in interpretation among the reviewers as to the relative experience of the principle investigator and his assembled team with one noting that they appeared to be relative novices in the study of oligodendrocytes, demyelinating disorders and the manipulation of stem cells. A reviewer believed that the budget requested is inflated.
This is a reasonably strong application from excellent investigators. It seems likely that some interesting information on the regulation of human oligodendrocyte development will be generated during the course of the work. It would benefit from refinement and the rationale for a number of the experiments could be substantially strengthened.
DISCUSSION: The first part of application is strong. There is good supportive data that the PI’s focus on the role of the Sox family in oligodendrogenesis is reasonable. There was some question about the focus strictly on oligodendrocytes as the way to address demyelinating diseases with the comment made that may require different approaches for different demyelinating diseases. There was concern about the choice of models for the in vivo studies with the model's relevance to human disease questionable. The stroke model is not commonly used; not clear how much demyelination is involved; the shiverer model is not simply a remyelination model, but remyelination against a mutant background. There was a belief that PI was a wonderful investigator but not very sophisticated about the myelin field and demyelinating disease.
The question was raised as to how the proposed studies to make oligodendrocytes differed from Kierstad’s methods. The response was that Kierstad’s method uses a very poorly defined media to generate oligodendrocyte precursors whereas the proposed studies are more mechanistically focused and could potentially lead to advances in generating these cells.