Abstract

Aims Ischemic stroke (IS) is a harmful neurological disorder, yet current therapies fail to achieve effective functional neural and vascular restoration. Neural stem cell (NSC) transplantation offers high potential for neuronal replenishment and vascular reconstruction. However, its clinical application is limited by inconsistent in vivo cell fate, unclear therapeutic mechanisms, and variability in differentiation protocols. To address these limitations, we aimed to identify a neural lineage-biased NSC suitable for neuronal and vascular recovery after IS. Methods We assessed two strategies for deriving transplantable NSCs: one involving embryoid body (EB) formation, and the other employing a direct differentiation protocol bypassing EB formation. We compared the cell fate of NSCs derived via both protocols in vitro and following transplantation into mice subjected to IS induced by transient middle cerebral artery occlusion (tMCAO). RNA sequencing and immunofluorescence were used to assess cell fate. Cerebral blood flow (CBF) and behavioral tests were conducted to evaluate functional recovery. Results Comparative analyses demonstrated that nEB-NSCs exhibit elevated neural stemness marker expression, improved neuronal differentiation with reduced astrocytic commitment, and accelerated neurovascular repair kinetics, contributing to considerable motor recovery. Conclusions nEB-NSCs represent a promising cell source for enhancing neurovascular repair and functional recovery following IS.