Our understanding of the effect of immunosuppressive agents on stem cell proliferation and differentiation in the central nervous system is limited. Indeed, even the necessity for long-term immunosuppression to promote the survival of stem cells grafted into the “immunoprivileged” central nervous system (CNS) is unknown. Grafting multipotent stem cells into the injured CNS often results in a failure of the cells to survive. If the cells survive, often they differentiate into astrocytes, a cell-type not considered beneficial. We recently grafted human stem cells (hCNS-SC) into spinal injured mice and observed behavioral improvements coupled with differentiation of these human cells into neurons and oligodendrocytes. We also observed mouse-human synapse formation and remyelination. The mice we used lacked a functional immune system, enabling us to grafting human cells into the mice without the use of immunosuppressants. When these same cells were grafted into spinal injured rats with a normal immune system, we had to immunosuppress the animals. Exposure of these human stem cells to immunosuppressive drugs resulted poor cell survival. The cells that did survive predominantly differentiated into astrocytes. Did the immunosuppressive drugs we used alter the ability of the human stem cells to differentiate into useful cells?
All cell-based therapeutic approaches are dependent upon either immunosuppression in an otherwise normal animal or testing for proof of principal in an immunodeficient animal model. This has quite significant implications for animal experiments or human trials, where continuous immunosuppression is required to obtain successful graft survival. No one knows if there are direct effects of immunosuppressant drugs on neural stem cells.
Stem cells may also respond differently to immunosuppression depending on their “ontogenetic” age (embryonic vs. fetal vs. adult). There is a common perception that “young” ES cells will have greater potential than “older” stem cells. Stem cells isolated at different ontogenetic stages might respond differently to immunosuppression.
We predict that the immunosuppressive drugs will exert direct effects on stem cell proliferation, gene expression, and fate determination, both in cell culture and when grafted into animals with spinal cord injury. We will also test if “ontogenetic” age alters the responsiveness of stem cells.
The California Institute for Regenerative Medicine (CIRM) recognizes that the field of stem cell biology is in its infancy. CIRM has requested a broad range of research to fill in key gaps in our understanding of basic stem cell biology and the possible use of these cells as therapeutics. Grants are to be judged on impact (extent the proposed research addresses an important problem; significantly moves the field forward scientifically; moves the research closer to therapy; and changes the thinking or experimental practice in the field), quality (is proposed research planned carefully to give a meaningful result; are possible difficulties are acknowledge; does the timetable allows for achieving significant research) and innovation (to what extent the research approach is original, breaks new ground, and brings novel ideas to bear on an important problem).
We believe that the projects proposed here target several of the areas CIRM cites as beneficial to the State of California. This proposal addresses the critical area of immunosuppression and stem cell survival in animal transplantation models. Future therapies using human stem cells will have to surmount the possible rejection by the host of cells derived from another source. If traditional immunosuppressive drugs are to be used, we will need to understand whether these drugs have a direct effect on stem cell proliferation and fate determination (or differentiation). Furthermore, these projects will allow for a direct comparison of stem cells from different ontogenetic stages and the ability to improve functional outcome after spinal cord injury. Thus we may gain insight into whether embryonic derived stem cells are more useful than adult derived stem cells as a therapeutic tool.
SYNOPSIS: This proposal will explore the effect of immunosuppressive agents on stem cell proliferation and survival in order to understand better the effects of such therapy following the transplantation of stem cells into the CNS. Earlier the PI observed that exposure of hESCs resulted in poor cell survival, and the surviving cells differentiated into mostly astrocytes. Here, the applicant will explore three drugs: Cyclosporine A (CSA), Tacrolimus (FK506) and Rapamycin. He will explore the effect on these drugs on hESCs, human neural stem cells and amniotic cells by looking at division (with BrdU) and genes expressed that are associated with division (with microarrays). Secondly, he will explore the effect of immunosuppression on these cells after their differentiation into neural stem cells (in the case of hESCs and amniotic cells) and into spinal cord injury in the NOD-scid mouse. These mice will be studied for behavioral recovery and histologic evaluation for stem cell differentiation.
SIGNIFICANCE AND INNOVATION: This proposal seeks to systematically explore immunosuppressive agents that are likely to be used in human clinical trials of stem cell transplants and their effects on stem cell proliferation and differentiation. This is an important issue in the field with great practical significance, and the ideas behind this work are quite novel. The idea of using the NOD-scid mouse as a model in which xenografts survive to then test the effect of immunosuppressive agents on the grafted cells is interesting as an in-vivo model system. A note of caution here however, is that the first human trials will almost certainly involve allografts (as acknowledged by the applicant on page 5) and hence the xenograft data derived here might not be directly applicable.
STRENGTHS: This is a novel area of investigation that is important for clinical translation. The PI has generated useful information already and has published in the field. Here, the PI proposes feasible experiments that need to be done, and presents a detailed and careful experimental design. The PI has an excellent infrastructure and academic environment at UC-Irvine with support from a core Stem Cell Center, and has lined up excellent collaborators in Aileen Anderson, and Michele Musacchio.
WEAKNESSES: The study does not really answer the main question of whether immune suppression by calcineurin inhibitors or other factors are responsible for the poor survival of cells when the hESCs are transplanted to rat spinal cords. NOD and other immune-deficient animals may have mechanisms other than the absence of immune responses that favor survival of hESCs. More importantly, there is a need for studies of autografts or HLA-antigen matched cells without calcineurin inhibitors. There is a general lack of acknowledgement of data on immunosuppression from the Parkinson’s disease field where considerable information on neural transplants and CSA therapy has been published.
With respect to the experimental design, reviewers are concerned about why amniotic cells are included. The Zhang protocol for neural stem cell development only referred to hESC differentiation. In addition, microarray work, as always, may be viewed as a giant ‘screen’ that is expensive and may lead to an untestable list of genes. There are also discrepancies in 1) the PI's data on the outcome of transplanting human neural stem cells in the SCI model and 2) the results of H. Keirstead’s study with hESC-derived oligodendrocytes. This might have been mentioned.
DISCUSSION: In the field, there is conflicting data on the effects of immunosuppressive drugs on hESC and neural stem cell function. Here the PI will sort out these long-standing questions by studying cell division, differentiation, and arrays in-vitro. The applicant will look at the effects of drugs on division and differentiation in 3 types of cells: hESCs, human neural stem cells, and amniotic cells. This work follows up on studies published by the PI in PNAS where mice showed behavioral responses to drug-induced changes in oligodendrocyte and neural differentiation. The applicant is certainly qualified, and the key strength of the proposal is its application to clinical translation.
The real weakness of this proposal is in how relevant the xenograft data will be to human trials. One reviewer believes that mouse/rat work will not give useful information on how to protect transplanted cells. Questions arose over the use of the NOD mouse model to address direct effects of immunosuppressive drugs on stem cells. Reviewers note that the NOD mouse is not a "normal" mouse, and it is unclear whether the response to injury in the NOD mouse reflects the "normal" response. Since allogeneic responses will be very different from xenogeneic responses, reviewers felt that the proposed studies were unlikely to give relevant data. The direct toxicity of immunosuppressives could also be assessed in-vitro.
The inclusion of NSCs derived from amniotic stem cells weakens the proposal since the published protocol cited for the differentiation of hESC to hNSC never explored amniotic stem cells as a starting material for differentiation. Also, the amniotic cells will be obtained from a company. How well defined and consistent are they going to be? Reviewers also consider the use of microarrays for profiling the effects of immunosuppresive drugs on cells as a bit of "fishing".
There was a lengthy discussion regarding the body of literature on the direct toxic effects of these drugs on cells. While the PI states in the proposal that there is no knowledge of the effects of these agents in clinical trials, one reviewer points out that this isn't exactly true since there is data on Cyclosporine A in Parkinson's Disease patients. Nevertheless, within the proposal the discussion of the mechanisms of the drugs was thought to be well described and there was supporting preliminary data included in the proposal. Another reviewer wondered why the PI didn't think about/discuss the mechanism of CSA and rapamycin, and why they're not looking at calcineurin expression. This reviewer recommends looking at calcineurin expression by targeting the downstream effects of inhibiting calcineurin in Aim 1, which would tighten this work.
The PI presents an excellent timetable, but there were minor issues in the proposal itself. The PI does not mention the federal funding situation and went over the limit on all three sections, which reviewers found annoying. The PI also was over-confident in stating that there would be "no problem with any part of work".