Traumatic spinal cord injury (SCI) initiates a cascade of events, including persistent inflammation, which contributes to secondary injury. At a molecular level, the lesion is characterized by an altered microenvironment with changes in extracellular matrix (ECM) composition and organization, identified as a potential obstacle for effective stem cell-based cell therapies. We investigated the interactions between decellularized intact and injured rat spinal cords and rat embryonic (RESCs) and neural stem cells (NSCs) at 2 and 47 days post-lesion (dpl). Decellularized ECM was used to generate 2D coating and 3D gel in vitro platforms for cell seeding. Results showed that the 2dpl 2D coating exerted a significant negative effect on the viability of both cell types, while the 47dpl 2D coating maintained RESC pluripotency. NSCs cultured on the 2dpl 2D coating for seven days showed a severe impairment in cell growth, while maintaining a cluster formation potential and differentiation marker expression comparable to normal ECM for astrocytic and oligodendroglial lineages. Notably, when NSCs are grown in 47dpl 3D gel, the lineage turns dramatically toward an astroglial lineage. These results clearly show the detrimental effects of the SCI ECM microenvironment on stem cells, advancing the understanding of potential timings suitable for effective SCI cell-based therapies.