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RC1-00134-1: Immunology of neural stem cell fate and function

Recommendation: Recommended for funding
Scientific Score: 79

First Year Funds Requested: $612,775.00
Total Funds Requested: $2,501,125.00

Public Abstract (provided by applicant)

One of the most difficult yet ultimately rewarding goals in stem cell research is to repair damaged neural systems with newly generated neurons. Our work examining neuronal integration and survival in the postnatal and adult brain shows that incoming neurons are uniquely and exquisitely sensitive to the immune response and inflammation that is always present when cells are transplanted into the injured or diseased brain or spinal cord. Here we propose to: 1) further refine our understanding of the molecular mechanisms that promote or inhibit new neuron integration; 2) evaluate pharmacological or biological methods for enhancing transplant efficiency and 3) test the developed techniques in a model of stem cell therapy for treating children who suffer neurological damage due to treatment for brain cancer. Future studies anticipate the use of these interventions to improve stem cell therapies for a variety of neurological injuries and diseases.

Statement of Benefit to California (provided by applicant)

The proposed studies focus on a critical yet poorly characterized aspect of stem cell therapy in the brain and spinal cord. Advances in these studies would not only stimulate new clinical studies but would also create novel biotechnologies, bringing new commercial opportunities to the state of California. The emphasis on immunological aspects of cellular transplant in this proposal are not only applicable in a neurological context but may be broadly beneficial across many therapies where stem cell-derived transplants are contemplated.

Review

SYNOPSIS: In this interesting proposal to better understand how an injured human brain might better respond to a cellular transplant, there are experiments described in three aims to: 1) further refine our understanding of the molecular mechanisms that promote or inhibit new neuron functional integration in the brain; 2) evaluate pharmacological or biological strategies to make cells more resistant to hostile environments and enhance the efficiency of new neuron survival and integration and 3) test these novel strategies in a model of embryonic stem cell transplant therapy where human embryonic stem cell (hESC)-derived neural progenitor cells (NPC) are used to reconstitute native progenitor cell pools that are depleted during treatments for brain cancer.

IMPACT AND SIGNIFICANCE: Transplantation of hESC-engineered derived cells and tissues will require their sustained engraftment in vivo, which in turn requires that these cells/tissues not be attacked and rejected by elements of the host immune system, either adaptive or innate. The proposal explores important issues of stem cell biology related to embryonic stem cell differentiation towards the neuronal lineage and how the immune system modulates the survival and integration of neural precursors and their derivatives into different central nervous system (CNS) regions. The proposing of controllable cellular therapies that can be improved by more effectively dealing with both innate and immune processes is potentially very high impact and significant to the field of restorative neurology. If some of the experiments are successful and if the Principal Investigator (PI)can determine which cellular elements and pathways are responsible for rejection reactions and then identify pharmacologic inhibitors, this would be an important contribution to cell transplantation therapies, both for neuronal and other cells. The focus here on cell replacement following cancer therapies that reduce numbers of stem and progenitor cells in the CNS and other areas is very important.

The hypotheses are straightforward and the aims are reasonable, though the rationale for using hESC derived cells in immunocompromised mice to address these immunological questions is problematic. The general ideas put forward in this proposal have been explored previously in a variety of cell types. What is new here is the systematic exploration of how the immune system modulates neural stem cell differentiation and grafting.

QUALITY OF THE RESEARCH PLAN: The overall quality of this proposal is very good. Some of the experiments are excellent and will produce meaningful results. Most of the experiments are self explanatory and easy to follow. While there was agreement among the reviewers that meaningful results can be obtained in the proposed timetable there was disagreement as to whether all of the proposed research could be accomplished in the funding period.

The focus is on identifying the pro-neurogenic signals that are affected by immune signaling, and then developing interventions that might improve engraftment and other outcomes in the injured or diseased brain. Three aims are proposed: 1) Define mechanisms and potential interventions against immune/inflammatory disruption of neuron survival, maturation and functional integration. It is hypothesized that immune signaling alters neuronal progenitor behaviors directly and indirectly. Techniques(s) are proposed that are innovative and where multiple molecules can be screened efficiently. Comparing native versus hESC-derived neural progenitors is important because it can reveal whether there are similarities in response. Also, this will yield results to build upon. 2)Determine the specific effects of MHC or species mismatch on hESC-derived grafts in mice to look at the immune response to allogeneic or xenogeneic cells in the immune-privileged context of the brain. This aim will help with the technical aspects of improving hESC grafting into rodents. 3) Develop pre-clinical models of neural stem cell reconstitution in rodent models of delayed radiation injury. The proposed pre-clinical model of neural stem cell reconstitution in rodent CNS after radiation injury is interesting and unique. Here it is hypothesized that neural stem/progenitor cell transplants in combination with “immunomodulatory interventions” might block chronic degenerative processes that follow therapeutic cranial irradiation during treatments of early childhood brain cancers.

The research plan is interesting and intriguing. Although the approaches proposed may not immediately nail down specific cells or mechanisms, the experiments may well yield intriguing results worthy of future study. Another reviewer noted that the main concern with this application is that its focus is about rodent neural stem cells and is not mechanism oriented.

STRENGTHS: The PI is an established leader in the area of understanding and controlling immune modulation of stem cell engraftment. The PI has published interesting work in the field of the adult neural stem cells and is a committed and imaginative experimentalist. The PI and colleagues have established excellent ex vivo culture systems and in vivo transplant systems, complete with relevant functional assays, for the study of neural progenitors. This research thus will benefit from the application of both cutting edge tissue engineering and in vivo transplantation.

The PI has identified, insightfully, pro-inflammatory pathways as important barriers to successful engraftment of neuronal tissues. The PI is probably correct with the hypothesis that there are two independent mechanisms that will influence neuron replacement and augmentation therapies: a proinflammatory response that is active in injured or degenerating brain, and, adaptive immune response that is triggered by allogeneic or xenogeneic graft mismatch. There are few laboratories pursuing this important line of research, this lab is at the forefront of this research.

The proposed study will carefully identify both immune and inflammatory barriers to cell transplantation. There have been mainly rumors and anecdotal information on which stem cells elicit the most immune or least immune response. The studies proposed by the PI will directly test and examine hESC-derived precursor transplants in appropriate animal models.

Molecular, pharmacological and behavioral studies, all will be studied to understand how to achieve better outcomes due to the unfriendly environments both in vitro and following transplants in mice. This is incredibly important for helping cell genesis following irradiation or even chemotherapeutic protocols that deplete stem/progenitor cell populations in the CNS and other tissues. The PI pioneered the study of NSAIDs (non-steroidal anti-inflammatory drugs) and related compounds as countermeasure drugs for cell and tissue damage to neurogenic regions of the adult brain following therapeutic radiation exposure for cancers.

This is a very straightforward paradigm where synthetic and cellular microenvironments will be generated to characterize specific influences of immune cells on newly-generated neurons, their survival and their differentiation. The potential applicability of results from this research—e.g. to neurological impairment following cranial radiation therapy is extremely high, and potentially very direct.

WEAKNESSES: The biggest weakness of this important proposal is the large number of somewhat open-ended studies that are proposed; this is true over-ambitiousness, and should be appreciated by the PI, but still does not significantly reduce the level of enthusiasm for this work. There are many combinatorial series of projects and reagents combined in assaying the performance of NPCs. All of the in vitro studies are extremely time-consuming; data analyses, for example quantifying all of the markers and lineage diversity from NPCs in this study, are even more demanding. In a second assay system, the cell interaction studies alone could occupy most of the attention of the investigators on this project for probably more than 4 years. Again, the PI knows this and has presumably laid out a series of milestone projects that will certainly present their own timetables for completion as the work proceeds.

Another weakness cited was that many of the proposed studies were descriptive rather than mechanistic in their design. Thus, in aim 1, the second co-culture assay is interesting but is rather descriptive. There also seems to be a third assay with cytokines and co-culture with immune cells. These experiments are also rather descriptive and seem more like a fishing expedition. Similarly, aim 2 is rather technical and lacks experiments for learning about the mechanisms of immune response to allogeneic transplants in CNS or about the immune characteristics of hESC NPC derived cells. It is difficult to extrapolate using NPC from adult or embryo versus NPC derived from hESC unless several basic immunological and biochemical characteristics are well defined before grafting. Also, no experiments are suggested to explain negative results. Again this aim seems rather descriptive and not much will be learned about the basic immune responses to hESC transplants unless some more basic immunology experiments are included in this proposal. Finally, aim 3 is rather descriptive and the experiments are not designed to understand basic mechanisms of any positive behavior. Perhaps the authors should focus more and provide more mechanistic approaches to their experiments.

Other concerns addressed the model(s) and what could be extrapolated from the proposed studies. Concerns were expressed about aim 2 hypothesis testing for the role of innate versus adaptive immunity in neural progenitor transplantation in the brain. One reviewer believed that the use of a xenotransplatation model to inquire how human NPCs will behave under different immunomodulatory signals is flawed. The responses to xenotransplantation are qualitatively different from the allogeneic responses. Xenotransplantation experiments are not relevant to biochemical and physiological processes of a species. The only way in which different immunomodulatory signals could be assessed is by comparing NPC autografts v. allografts. A suggestion was made to use a “Human-SCID mouse model” if the PI wants to make any sound conclusions about the role of the immune system in hESC grafts. This will be more relevant to the hESC transplantation experiments because will be providing an allogeneic like environment to study immune responses. No specific studies of, e.g. TLR antagonists or activators, costimulatory agonists or antagonists are proposed. That is, the PI’s immunological approaches will remain superficial. This will limit, unnecessarily, what could be learned from this research.

The broad spectrum pharmacologic agent have direct effects on most cells, and therefore could influence not only inflammatory cells but several aspects of neuronal maturation. Therefore, specific observations cannot be assigned only to inflammatory responses. This observation applies to the in vivo experiments involving the use of these agents.

In humans, the underlying pathogenesis after irradiation is rather complicated. Having a neurogenesis “centric view” of this pathological condition is limiting.

Finally, there was a lack of data about the hESC lines from the collaborator’s laboratory. There is no description of some characteristics of these lines. Are the investigators planning to use all the lines to compare NPC differentiation?

DISCUSSION: All of the reviewers acknowledged the importance of the problem of finding ways to mitigate the radiation damage to the pediatric brain subsequent to cancer treatment that can result in severe quality of life issues later. They also acknowledged the qualifications of the PI in the proposed field of study. There was some discussion of the hypothesis that specific immunomodulatory interventions could block degeneration and allow neural restoration. It was noted that the PI had an important publication on the role on NSAIDS in mitigating neural damage due to radiation therapy. The reviewers however had differing responses to the approaches proposed by the PI to test the hypotheses as outlined in this application. One reviewer, while regarding the proposed work as overly ambitious was still very enthusiastic about the proposal given the potential of the work to lead to important new insights into treating the compromised pediatric brain following radiation- or drug-induced altered neurogenesis. The other reviewers felt differently noting that there are more sophisticated and powerful ways to approach the role of immunity in transplantation.

The question was raised about effect of proposed research on clinical practice. A reviewer noted that the work had real potential for generating results that could have real time feedback impacting clinical practice and equipment (e.g., pharmacologic, shielding etc) in radiation therapy of children with cancer.

The following Working Group members had a conflict of interest with this application and were therefore recused from participating in review of, discussion of, and voting on the application:

• Wagers, Amy