Human spinal cord models for development of spinal cord injury repair strategies

There is still no available cure for human spinal cord injury (SCI). After a SCI, neural cell loss, excitotoxicity, neuroinflammation, astrocytic and oligodendrocytic derived inhibiting factors contribute to a non-permissive spinal cord environment hindering neuroregeneration with little, if any, endogeneous functional improvement. Experimental studies based on nonhuman cells and tissues as well as in vivo models have significantly increased our understanding of the pathophysiological processes and repair mechanisms after SCI. However, this gained knowledge may not always be applicable in the human setting, due to species specific differences. Therefore, having access to relevant human models to supplement existing animal models would be beneficial. In vitro cellular and ex vivo tissue cultures may allow us to mimic, at least to some degree, in vivo conditions, and to further understand human-specific spinal cord development, pathological conditions as well as repair mechanisms.

Therefore, in this thesis, human spinal cord models have been further developed to offer additional tools in the translation of treatment strategies from the experimental to clinical setting.

In Paper I, we developed an ex vivo human spinal cord model referred to as human organotypic cultures (hOCs). Cellular, molecular and functional characteristics of the hOCs as well as human spinal cord-derived neural stem/progenitor cell (hNPC) allogeneic cell therapy were studied. The hOCs presented a relatively intact cytoarchitecture and functional viability and could to a certain level replicate the in situ human spinal cord microenvironment. We conclude that the hOC model has potential for future structural and functional studies of human spinal cord development or injury, and can be used as a platform for the study and further development of human SCI treatment strategies.

To counteract the non-permissive spinal cord milieu after SCI, support host neural regeneration, modify the microenvironment, or offer drug or cell delivery, the application of biomaterials as part of a composite treatment strategy for SCI has been proposed. In the present thesis two different types of biomaterials with potential to serve as scaffolds for hNPC therapy was applied – hyaluronic acid (HA) hydrogels (bulk and granular HA hydrogels in Paper II and III) and recombinant spidroins (NT2RepCT and VN-NT2RepCT in Paper II).

In Paper II, an in vitro model to, relatively resource effectively, evaluate the immunocompatibility of the above-mentioned biomaterials with human peripheral blood mononuclear cells (host) and/or allogenic hNPCs. The VN-NT2RepCT recombinant spidroin activated human B cells, CD4+ T cells and NK cells. We therefore suggest efforts to develop recombinant spidroin with reduced endotoxin contamination. The HA hydrogels with/without hNPCs did on the other hand not stimulate lymphocyte activation or proliferation, indicating that HA hydrogels may be a suitable scaffold for hNPC therapy in SCI. We suggest that this model can be used to evaluate human compatibility of novel biomaterials resource effectively early in the production process and when necessary, make alterations to minimize the risk of rejection.

In Paper III, a 3D culture system was applied aiming to mimic the extracelular matrix (ECM) and support hNPC survival and differentiation utilizing HA hydrogels combined with laminin and brain-derived neurotrophic factor (BDNF). The granular HA hydrogel together with laminin and BDNF presented in preliminary studies beneficial features compared to bulk HA hydrogel in the context of physical property and the capability to support hNPC survival and differentiation. Human neuronal differentiation was observed to be supported up to 14 days in vitro in the granular HA hydrogel, which is promising for future clinical applications after SCI.

In conclusion, the human spinal cord-derived in vitro model systems presented here have potential to supplement existing animal models for the study of human SCI, human biocompatibility, and for the translation of promising experimental SCI treatments to the clinic.

List of scientific papers

I. Chenhong Lin*, Cinzia Calzarossa*, Teresa Fernandez-Zafra, Jia Liu, Xiaofei Li, Åsa Ekblad-Nordberg, Erika Vazquez-Juarez, Simone Codeluppi, Lena Holmberg, Maria Lindskog, Per Uhlén and Elisabet Åkesson. Human ex vivo spinal cord slice culture as a useful model of neural development, lesion and allogeneic neural cell therapy. Stem Cell Research and Therapy. 11(1):320, 2020. *Shared first co-authorship.
https://doi.org/10.1186/s13287-020-01771-y

II. Chenhong Lin*, Åsa Ekblad-Nordberg*, Jakob Michaëlsson, Cecilia Götherström, Chia-Chen Hsu, Hua Ye, Jan Johansson, Anna Rising, Erik Sundström and Elisabet Åkesson. In vitro Study of Human Immune Responses to Hyaluronic Acid Hydrogels, Recombinant Spidroins and Human Neural Progenitor Cells of Relevance to Spinal Cord Injury Repair. Cells. Jul 6;10(7):1713, 2021. *Shared first co-authorship.
https://doi.org/10.3390/cells10071713

III. Chenhong Lin, Chia-Chen Hsu, Zhanna Alekseenko, Hua Ye, Åsa Ekblad, Xiaofei Li, Erik Sundström and Elisabet Åkesson. Granular hyaluronic acid hydrogel as a potential scaffold for human neural progenitor cells. [Manuscript]