Differentiation of neurosphere after transplantation into the damaged spinal cord

This study aimed to compare the differentiation and survival of human neural stem/progenitor cells of various origins in vitro and after transplantation into the injured spinal cord of laboratory animals. Rats with simulated spinal cord injury were transplanted with neurosphere cells obtained by directed differentiation of HUES6 cell lines. Fluorescence microscopy was used to visualize the obtained results. HUES6#1 and iPSC#1 neurospheres showed a wide range of markers associated with glial differentiation. The expression of the proliferation marker Ki67 did not exceed 25%, both in the lines of early and late neurospheres. Although neurospheres did not fully differentiate into astrocytes in vitro, they massively approached the GFAP+ astrocyte phenotype when exposed to the transplanted environment. PSC-derived neurospheres transplanted into the site of SM injury without additional growth factors showed only moderate survival, a significant degree of differentiation into astrocytes, and moderate differentiation into neurons. The difference in the survival and differentiation of HUES6#1 and iPSC#1 neurospheres, both in vitro and in vivo, can be explained by the difference in the regulatory behavior of signaling molecules corresponding to the source of origin of PSCs. Derivatives of human PSCs of various origins obtained according to the described differentiation protocol did not mature into astrocytic populations, nor did the glycogenic transition of PSC-derived NSCs occur in vitro. The study demonstrated the impact of the injured spinal cord microenvironment on the differentiation of transplanted HUES6#1 and iPSC#1 into astrocytes. The results showed that HUES6-derived neurospheres generated 90% of GFAP+ astrocytes and 5-10% of early neurons, while iPSC-derived neurospheres generated an average of 74% GFAP+ astrocytes and 5% of early neurons in vivo.