PROJECT SUMMARY Current stem cell-based treatments for central nervous system (CNS) injuries such as spinal cord injury (SCI), are severely hampered by poor stem cell survival rates, inefficient integration, loss of neural plasticity, and uncontrollable differentiation of implanted cells, all of which are caused by the highly inhibitory and inflammatory microenvironment at disease or injury sites. Specifically, gliosis at the injury site causes the secretion of inhibitory factors leading to poor axon regeneration and sprouting of surviving neuronal populations, resulting in the intrinsic limitations of the CNS to regenerate after the initial injury. Therefore, there is an urgent need for effective strategies to generate a robust population of functional neurons derived from patient-derived stem cells and re- establish the damaged neural circuitry. To this end, we propose to integrate several fields of research, including nanotechnology, biomaterials, neuroscience, and stem cell biology, to develop a novel nanoscaffold-based stem cell assembly platform that allows for the generation of favorable microenvironments during stem cell implantation and the control of stem cell fate in vivo for potential clinical applications. To address the fundamental impediment of regeneration associated with CNS injuries and diseases, we propose to develop injectable 3D-Hybrid SMART neuro-spheroids for enhanced stem cell therapy and effective treatment of SCI in vivo. The 3D-Hybrid SMART neuro-spheroids are assembled from biodegradable scaffold nanomaterials enriched with natural neural ECM to promote neural stem cell (NSC) survival and differentiation. The SMART neuro-spheroids also permit the loading of a bioactive molecule (i.e., Notch inhibitor), resulting in the synergy between suppressing neuroinhibitory signaling and promoting neural stem cell (NSC) survival and differentiation. This novel technology platform will be further integrated into two clinically advanced models: i) an inflammatory CNS organoid model incorporated with microglia, and ii) a spinal cord injury animal model. This multidisciplinary study will provide a next-generation platform for research and cell therapy in neuro-regenerative medicine from the perspective of developing a new 3D spheroid assembly method for enhanced stem cell survival and suppression of inhibitory environment after CNS injuries. We propose to verify our central hypothesis and achieve our objectives by addressing the following specific aims: AIM #1 – Develop bioactive and biodegradable-nanoscaffold-based injectable 3D-Hybrid SMART spheroids; AIM #2 – Investigate deep drug (Notch-i) delivery in SMART spheroids and study neuronal differentiation of stem cells and axonal growth under neuroinhibitory and immune microenvironments in vitro; AIM #3 – Determine the therapeutic effects of 3D-Hybrid SMART spheroids on the modulation of neuroinhibitory microenvironments and the enhancement of SCI functional recovery in vivo. Collectively, we anticipate that our proposed studies will provide an innovative, highly effective, and robust method for developing therapeutic interventions for neurological disorders.

PROJECT NARRATIVE Our proposed studies are relevant to public health, as developing an injectable, biocompatible, and biodegradable method to form therapeutic 3D spheroids to promote stem cell therapy will facilitate CNS repair and modulate neuroinhibitory microenvironments after CNS injury. Therefore, the proposed research is relevant to the part of NIH’s mission that relates to treating human illness and disability by investigating how innovative technology can safely translate to clinics.