Human induced neural progenitor cells generated from three-dimensional aggregate-based culture significantly improve post-stroke recovery in tMCAO mice.

Abstract

Background: Despite the high prevalence of cerebral ischemic stroke, effective clinical treatments remain limited. With the development of regenerative medicine, induced neural progenitor cells (iNPCs) demonstrate ideal potential and good availability for autologous transplantation therapy. However, current differentiation protocols for iNPCs still have room for improvement in terms of purity, reproducibility, scalability and differentiation potential.

Methods: We aimed to develop a scalable, stable, and efficient 3D aggregate-based method for iNPC production in suspension culture, avoiding detrimental cell dissociation and replating processes. We evaluated the therapeutic potential of iNPCs in the chronic phase of a transient middle cerebral artery occlusion (tMCAO) mouse model and explored iNPC subpopulations via single-cell RNA sequencing to elucidate their pleiotropic therapeutic potentials.

Results: iNPCs generated from three iPSC lines displayed high NPC marker expression and an average 176-fold cell expansion over the 12-day culture period. These iNPCs could spontaneously differentiate into both neurons and glial cells in vitro. In the tMCAO model, transplanted iNPCs remodeled the microenvironment by alleviating neuroinflammation, inhibiting chronic microgliosis and astrogliosis, promoting M2 polarization of microglia, and preserving astrocytic morphology in the ischemic penumbra. Mechanistically, iNPCs can be divided into four subpopulations, with neuroepithelia being the most abundant and capable of rapidly replenishing damaged cells and mitigating microenvironmental deterioration.

Conclusions: We developed a simple and efficient 3D aggregate-based method for iNPC differentiation. These iNPCs showed excellent potential for post-stroke recovery and represent a valuable tool for clinical translation.