schemia-induced paraplegia often combined with a qualitatively defined increase in muscle tone (i.e. spasticity and rigidity) is a serious complication associated with a temporary aortic cross-clamping ( a surgical procedure to repair an aortic aneurysm). In addition to spinal ischemic injury-induced spasticity and rigidity a significant population of patients with traumatic spinal injury develop a comparable qualitative deficit i.e. debilitating muscle spasticity. At present there are no effective treatment which would lead to a permanent amelioration of spasticity and rigidity and corresponding improvement in ambulatory function. In recent studies, by using rat model of spinal ischemic injury we have demonstrated that spinal transplantation of rat or human neurons leads to a clinically relevant improvement in motor function and correlates with a long term survival and maturation of grafted cells. More recently we have demonstrated a comparable maturation of human spinal precursors grafted spinally in immunosupressed minipig. In the proposed set of experiments we wish to characterize a therapeutical potential of human blastocyst-derived neuronal precursors when grafted into previously ischemia- injured rat or minipig spinal cord. Defining the potency of spinally grafted hESC-derived neuronal precursors in two in vivo models of spinal ischemic injury serves to delineate the differences and/or uniformity in the cell maturation when cells are transplanted in 2 different animals species and can provide an important data set for future implications of such a therapies in human patients.
Traumatic or ischemic spinal cord injury affect a significant number of people and in majority of cases can lead to a variable degree of motor dysfunction (such as paraparesis or paraplegia) and often combined with increased muscle tone (i.e. spasticity and rigidity). In contrast to other organ systems the central nervous system and spinal cord in particular has minimal or no neuron-regenerative capacity and therefore if a significant population of spinal cord neurons or fibers is lost the resulting deficit is permanent and irreversible. At present there is no effective therapy which would lead to a clinically relevant neurological improvement in patients with ischemia or trauma-induced paraplegia. Initial experimental data using paraplegic rats show that spinal grafting of rat or human neuronal precursors can provide a significant amelioration of spasticity and lead to improved ambulatory function. In the proposed set of experiments we wish to characterize a therapeutical potential of human blastocyst-derived neuronal precursors when grafted into previously ischemia- injured rat or minipig spinal cord. If proven effective such a treatment can potentially be used in patients with spinal ischemic paraplegia or in patients with other spinal injury-related dysfunction associated with a region-specific neuronal loss.
SYNOPSIS: Weakness, muscle atrophy, paraplegia and spasticity that results from spinal ischemia is a significant neurological complication of surgery. Previously work has suggested that injury associated with ischemic events involves largely inhibitory GABAergic interneurons. The investigator has limited data that suggest that engraftment of either rat or human spinal neuron precursors, in a spinal injury models, can lead to a significant functional improvement in these models. Furthermore, analysis has suggested that theses cells differentiate into GABAergic neurons and form inhibitory synaptic connections to local neurons. In this proposal, the investigators will use two models: a rat model and a mini pig model of spinal ischemic paraplegia to validate use of several human embryonic stem cell (hESC) lines in hopes of finding relevant engraftable cell based therapies. Overall the investigators will use a systematic approach of optimizing cellular delivery, timing, and volumes, and cell number.
IMPACT AND SIGNIFICANCE: During cross clamping of the thoracic aorta to repair aneurysms, the distal spinal cord is subjected to ischemic conditions with the potential for significant injury. Ischemic paraplegia is a significant complication of surgery, with no effective therapy. The lack of neuronal proliferation in the adult spinal cord after traumatic or ischemic injury indicates that cell replacement therapy is necessary for long-term therapeutic effects. These experiments should test the effectiveness of hESC derived neuronal progenitors in rodent and large preclinical models of ischemia induced paraplegia and lay a possible framework for translation into humans. The selective replacement of these inhibitory interneurons has the potential to alleviate this disabling condition and would be an important new strategy for helping repair the injured spinal cord.
QUALITY OF THE RESEARCH PLAN: This proposal is well thought-out and has the necessary expertise in place through long standing collaborations across two continents. The preclinical model systems are mature and are complementary being in two different species, one being a large animal. The experiments are feasible and focused to test specific questions as well as functional recovery. The experimental plan includes appropriate measures and quantification of differentiation as well as a limited attempt to track newly generated neurons- employing pre-labeling with viral tags. There are appropriate plans for monitoring histological outcome and functional outcome, with the ultimate plan of choosing the best cell line and conditions for evaluation in a mini pig model.
The experiments are logically described although tend to be dependent on the success of the first aim, specifically that at least one of the cell lines to be tested will differentiate into a significant number of inhibitory interneurons and be responsible for the functional improvements to be examined in the rat model. But in vitro data provided in Fig. 4 suggest that this can occur for either the H1 or H9 cell line (not clear which is shown in Fig. 4D-F) although whether the same number (70%) differentiate after being transplanted still needs to be determined.
STRENGTHS: The proposal addresses an important clinical problem for which treatment options are limited and for which a selective cell replacement therapy may be effective. The expertise of the applicant and the research team indicates that they will be able to complete all of the proposed studies. The applicant proposes an analysis of a number of existing hESC lines for the potential to differentiate into inhibitory interneurons following transplantation to the chronically injured spinal cord, a number of direct anatomical and functional assays to determine the efficacy of the transplant, and two well-defined animal models of ischemic paraplegia to assay long-term recovery and possible translation to the human condition.
The proposal presents an organized approach for deliver of different cell concentrations and injection time points. There is an extensive use of markers to identify outcomes for the use of multiple behavior outcome measures. Applicants have the techniques in place to assess the presence and degree of motor dysfunction and spasticity by using behavioral and quantitative electrophysiological tests, to obtain neuron-specific labeling with lentiviral reporter of neuronal precursors derived from hESCs and to distinguish human from rat or pig cells immunohistochemically. The applicants also have an immunosuppression protocol for both rat and pig such that the animals survive for at least 6 months (potential for teratomas will be followed for at least 6 months in the long term studies).
WEAKNESSES: The success of the last two aims is dependent on the successful completion of Aim 1. While some preliminary data is presented demonstrating that one of the cell lines to be tested can differentiate into inhibitory interneurons in vitro, it remains a possibility that none will effectively differentiate in vivo. If this is the case, it is not clear what alternative approaches the applicant will take. Furthermore, the proposal is somewhat redundant with previous published studies using other neuronal cell lines. Consequently, it is not particularly innovative.
Several technical weaknesses were noted. First, no control cells (sham or lysed) are proposed. Second, it’s not clear that utilizing a viral neuronal marker will help extinguish the differentiation of these cells into astrocytes (i.e., it’s likely that many of these cells will differentiations to astrocytes). Third, it’s not clear that this approach would fare better than including aims with focused differentiation of these cells into GABAergic interneurons rather then using early progenitors.
This proposal requires an extensive amount of histology that the investigator does not seem to adequately have within their facility and is relying extensively on foreign collaborators. The ability of this proposal be adequately carried out in a timeline using foreign collaborators who are not being funded, seems to be questionable. Overall, at least 50% of this proposal needs to be done on foreign soil. The investigator has extensive experience with models of injury but has very limited true hands on experience with utilization of stem cells, in terms of engraftment studies or extensive histology, required to truly evaluate almost 50% of this study. This dramatically lowers confidence that this study can be completed in the planned timeline.
DISCUSSION: This proposal aims to generate appropriate cells, follow their outcome and look at their function once they are engrafted. There are two well-developed animal models proposed and the applicant proposes to check both functional and histological outcomes. This is a clean system for looking at repair of the spinal cord and the researchers have the required expertise. Ischemic paraplegia is an underappreciated problem which, unlike traumatic spinal cord injury, involves a specific loss of inhibitory interneurons. Concern was expressed that the PI needs to examine four cell lines to generate the inhibitory interneurons, but this is an elegant proposal nonetheless.