Final Report Summary - HUMAN IPS IN SCI (Human iPS cell therapy for spinal cord injury)
Summary description of the project objectives:
The overall aim of our research is to develop a clinically safe and effective cell therapy, combining novel biomaterials and derivatives of neural stem cells to improve functional outcome after spinal cord injury (SCI). We evaluate and characterize human neural cells derived from fetal neural stem/progenitor cells (fNPCS) and induced pluripotent stem cells (iPS). Furthermore, we study bioresorbable hydrogels and whether they in addition to neural cell therapy offer neurorescue.
In the present project we analyze the immunocompetence and immunomodulatory potential of human neural donor cells and how neural cell therapy affect the host spinal cord after injury. Specifically, we evaluate the degree of host spinal cord cell death and survival, degree of host inflammatory and microglial reaction, and finally sensory-motor function after an injury with and without intervention.
Description of the work performed since the beginning of the project,
During the two year Marie Curie funded post doctoral project, we have expanded and further developed our methods concerning the evaluation of human NPCs and their immunocompetence in in vitro and in vivo models of SCI.
We have evaluated the immunogenicity and immunomodulatory properties of fNPC in naïve condition, under inflammatory stimuli, and in co-culture with human peripheral blood mononuclear cells (PBMCs), and non activated microglia.
Briefly, unstimulated human fNPCs secrete significant amounts of transforming growth factor-beta1 and beta 2 and hinder an alloreaction between non compatible human PBMCs. Furthermore, the fNPCs up-regulate CD4+CD25+forkhead box P3+ T cells in vitro, regarded as an important pool of T-regulatory cells that has the ability to induce tolerance. Finally, human fNPCs did not trigger human PBMC alloreaction in vitro (Liu J et al. 2013). When co-cultured with human microglia, human fNPC increased microglial proliferation, phagocytic activity, expression of HLA-II and CD206. Vice versa, microglia promoted human fNPC proliferation, and inhibited fNPC maturation. Moreover, in co-cultures, expression of CD200R and CD200 increased in microglia and human fNPC respectively. Finally we observed an increased TGF-beta2 release in co-cultures, and an increased TGF-beta 1 expression in fNPC microglia co-cultures. All together, these data support the capability of fNPC to modulate human peripheral leukocytes (PBMCs) and activated microglia, limiting microglial inflammation properties (Liu J et al. 2013).
To further investigate immunomodulatory and neuroprotective properties of human fNPC, we have developed organotypic slice cultures from brain stem and spinal cord of human and rodent tissues as model system of SCI. This allows both xenogeneic and human allogeneic studies.
In human organotypic cultures, mechanical SCI, with and without cell therapy (fNPCs) was performed. After 2 weeks post injury, we observed a significant increased host apoptotic cells and activated microglia in injured slices, while in presence of transplanted fNPC, no differences in apoptotic or activated microglia amounts were observed compared to control non-injured nervous tissue slices. So far, donor cells survived at least 14 days without host astrogliosis. These data support the neuroprotective and immunomodulatory properties we observed in our previous in vitro studies (Calzarossa et al. Manuscript).
These in vitro observations were further tested in in vivo experiments. Neuroprotective and immunomodulatory properties of fNPC were studied in animal models with a contusion SCI. Briefly, fNPCs at different passages (P0 and P5) were grafted at different time points post injury, and host spinal cord rejection responses and microglial activation were observed. Interestingly, fNPCs grafted at low passage (P0) showed lower rejection-ratios compared to fNPCs at P5 in the xenograft model. Host microglial response was also affected by fNPC graft. Indeed, in all animals moderately injured and transplanted with fNPCs (P5), we observed a reduction of microglial activation at injury epicenter. In another set of studies in both contusion and compression SCI, an increased functional outcome and a neuroprotective effect with increased number of surviving host neurons was observed by acute or subacutely grafted human fNPCs.
We also have through a valuable collaboration with Prof Koistinaho (University of Eastern Finland, Finland) and Prof Hovatta (Karolinska Institutet, Sweden) expanded human iPS cells in our laboratory. Regarding iPS, we were able to derive neural cells so called neurospheres from 2 out of 4 iPS lines (supported by Prof Koistinaho and his team in Kuopio University). Characterization analysis showed that human iPS-derived neurospheres were mainly composed by neural progenitor cells (nestin), beta Tubulin III and GFAP positive cells. Moreover, the pluripotent subpopulation was completely lost and no expression of OCT-4, NANOG and SSEA-4 was detected. We have performed a pilot study grafting human allogeneic iPS-NPCs into human organotypic slices cultures of injured and control spinal cord tissue. So far iPS-NPC survived up to 5 days, and no alterations in proliferation, apoptosis, or activated microglia were observed in grafted cases compared to controls at 1 or 5 days post transplantation.
The significance of our findings is that human neural cells both in the xenogeneic (transplantation in between species) and allogeneic (transplantation from human to human individuals) transplantation situation have been proven to reduce the inflammatory microglial activity and act neuroprotectively, in the injured spinal cord. These findings may not only have significance for the SCI field but also for development of neural cell therapy for other nervous system lesions and neurodegnerative disorders.
The overall aim of our research is to develop a clinically safe and effective cell therapy, combining novel biomaterials and derivatives of neural stem cells to improve functional outcome after spinal cord injury (SCI). We evaluate and characterize human neural cells derived from fetal neural stem/progenitor cells (fNPCS) and induced pluripotent stem cells (iPS). Furthermore, we study bioresorbable hydrogels and whether they in addition to neural cell therapy offer neurorescue.
In the present project we analyze the immunocompetence and immunomodulatory potential of human neural donor cells and how neural cell therapy affect the host spinal cord after injury. Specifically, we evaluate the degree of host spinal cord cell death and survival, degree of host inflammatory and microglial reaction, and finally sensory-motor function after an injury with and without intervention.
Description of the work performed since the beginning of the project,
During the two year Marie Curie funded post doctoral project, we have expanded and further developed our methods concerning the evaluation of human NPCs and their immunocompetence in in vitro and in vivo models of SCI.
We have evaluated the immunogenicity and immunomodulatory properties of fNPC in naïve condition, under inflammatory stimuli, and in co-culture with human peripheral blood mononuclear cells (PBMCs), and non activated microglia.
Briefly, unstimulated human fNPCs secrete significant amounts of transforming growth factor-beta1 and beta 2 and hinder an alloreaction between non compatible human PBMCs. Furthermore, the fNPCs up-regulate CD4+CD25+forkhead box P3+ T cells in vitro, regarded as an important pool of T-regulatory cells that has the ability to induce tolerance. Finally, human fNPCs did not trigger human PBMC alloreaction in vitro (Liu J et al. 2013). When co-cultured with human microglia, human fNPC increased microglial proliferation, phagocytic activity, expression of HLA-II and CD206. Vice versa, microglia promoted human fNPC proliferation, and inhibited fNPC maturation. Moreover, in co-cultures, expression of CD200R and CD200 increased in microglia and human fNPC respectively. Finally we observed an increased TGF-beta2 release in co-cultures, and an increased TGF-beta 1 expression in fNPC microglia co-cultures. All together, these data support the capability of fNPC to modulate human peripheral leukocytes (PBMCs) and activated microglia, limiting microglial inflammation properties (Liu J et al. 2013).
To further investigate immunomodulatory and neuroprotective properties of human fNPC, we have developed organotypic slice cultures from brain stem and spinal cord of human and rodent tissues as model system of SCI. This allows both xenogeneic and human allogeneic studies.
In human organotypic cultures, mechanical SCI, with and without cell therapy (fNPCs) was performed. After 2 weeks post injury, we observed a significant increased host apoptotic cells and activated microglia in injured slices, while in presence of transplanted fNPC, no differences in apoptotic or activated microglia amounts were observed compared to control non-injured nervous tissue slices. So far, donor cells survived at least 14 days without host astrogliosis. These data support the neuroprotective and immunomodulatory properties we observed in our previous in vitro studies (Calzarossa et al. Manuscript).
These in vitro observations were further tested in in vivo experiments. Neuroprotective and immunomodulatory properties of fNPC were studied in animal models with a contusion SCI. Briefly, fNPCs at different passages (P0 and P5) were grafted at different time points post injury, and host spinal cord rejection responses and microglial activation were observed. Interestingly, fNPCs grafted at low passage (P0) showed lower rejection-ratios compared to fNPCs at P5 in the xenograft model. Host microglial response was also affected by fNPC graft. Indeed, in all animals moderately injured and transplanted with fNPCs (P5), we observed a reduction of microglial activation at injury epicenter. In another set of studies in both contusion and compression SCI, an increased functional outcome and a neuroprotective effect with increased number of surviving host neurons was observed by acute or subacutely grafted human fNPCs.
We also have through a valuable collaboration with Prof Koistinaho (University of Eastern Finland, Finland) and Prof Hovatta (Karolinska Institutet, Sweden) expanded human iPS cells in our laboratory. Regarding iPS, we were able to derive neural cells so called neurospheres from 2 out of 4 iPS lines (supported by Prof Koistinaho and his team in Kuopio University). Characterization analysis showed that human iPS-derived neurospheres were mainly composed by neural progenitor cells (nestin), beta Tubulin III and GFAP positive cells. Moreover, the pluripotent subpopulation was completely lost and no expression of OCT-4, NANOG and SSEA-4 was detected. We have performed a pilot study grafting human allogeneic iPS-NPCs into human organotypic slices cultures of injured and control spinal cord tissue. So far iPS-NPC survived up to 5 days, and no alterations in proliferation, apoptosis, or activated microglia were observed in grafted cases compared to controls at 1 or 5 days post transplantation.
The significance of our findings is that human neural cells both in the xenogeneic (transplantation in between species) and allogeneic (transplantation from human to human individuals) transplantation situation have been proven to reduce the inflammatory microglial activity and act neuroprotectively, in the injured spinal cord. These findings may not only have significance for the SCI field but also for development of neural cell therapy for other nervous system lesions and neurodegnerative disorders.