Harnessing developmental dynamics of spinal cord extracellular matrix improves regenerative potential of spinal cord organoids

Neonatal spinal cord tissues exhibit remarkable regenerative capabilities as compared to adult spinal cord tissues after injury, but the role of extracellular matrix (ECM) in this process has remained elusive. Here, we found that early developmental spinal cord had higher levels of ECMproteins associated with neural development and axon growth, but fewer inhibitory proteoglycans, compared to those of adult spinal cord. Decellularized spinal cord ECM from neonatal (DNSCM) and adult (DASCM) rabbits preserved these differences. DNSCM promoted proliferation, migration, and neuronal differentiation of neural progenitor cells (NPCs) and facilitated axonal outgrowth and regeneration of spinal cord organoids more effectively than DASCM. Pleiotrophin (PTN) and Tenascin (TNC) inDNSCMwere identified as contributors tothese abilities. Furthermore,DNSCMdemonstrated superior performance as a delivery vehicle forNPCs and organoids in spinal cord injury (SCI)models. This suggests that ECMcues from early development stages might significantly contribute to the prominent regeneration ability in spinal cord. To investigate the different effects of DNSCM and DASCM on the cell fate differentiation of spinal cord neural precursor cells (scNPC), we cultured scNPC in DNSCM and DASCM. RNA-seq was performed after 7 and 14 days of 3D culture for differential analysis of gene expression. In addition, RNA-seq and differential analysis were performed on spinal motor neuron organoids(scMN-Organs) cultured in DNS CM, DASCM and Matrigel for 21 days.