{"doi":"10.5281/zenodo.6935779","title":"Growth and Connectivity of Neural Stem Cells after Severe Spinal Cord Injury","abstract":"Neural stem cells (NSCs) expressing GFP were embedded into fibrin matrices containing growth factor cocktails and grafted to sites of severe spinal cord injury. Grafted cells differentiated into multiple cellular phenotypes, including neurons, which extended large numbers of axons over remarkable distances. Extending axons formed abundant synapses with host cells. Axonal growth was partially dependent on mammalian target of rapamycin (mTOR), but not Nogo signaling. Grafted neurons supported formation of electrophysiological relays across sites of complete spinal transection, resulting in functional recovery. Two human stem cell lines (566RSC and HUES7) embedded in growth-factor-containing fibrin exhibited similar growth, and 566RSC cells supported functional recovery. Thus, properties intrinsic to early-stage neurons can overcome the inhibitory milieu of the injured adult spinal cord to mount remarkable axonal growth, resulting in formation of new relay circuits that significantly improve function. These therapeutic properties extend across stem cell sources and species. <strong>Survival, Filling, and Differentiation of Neural Stem Cell Grafts</strong> Rapamycin injection did not alter neural stem cell graft survival or differentiation. These findings demonstrate mTOR signaling as a mechanism contributing to the ability of early-stage neurons to extend axons in the adult lesioned CNS. Importantly, host axons also penetrated neural stem cell grafts in spinal cord lesion sites, including biotin dextran amine (BDA)-labeled reticulospinal axons and 5-hydroxytryptamine (HT)-labeled serotonergic (raphespinal) axons. Host inputs into grafts were both locally and supraspinally derived. Although host supraspinal axons regenerated into grafts, in no case did they regenerate beyond the graft into host spinal cord parenchyma caudal to the lesion site. Like graft-derived axons, host axons penetrating grafts colocalized with the synaptic marker synaptophysin, establishing a mechanism for host-to-graft connectivity. Early-stage neurons grafted in a fibrin matrix containing a growth factor cocktail extend large numbers of axons over long distances in the lesioned spinal cord and form neuronal relays that significantly improve electrophysiological and functional outcomes. The magnitude of functional effect substantially exceeds those previously reported in studies of fetal or stem cell grafts to the injured spinal cord, possibly due to enhanced graft survival generated by coimplantation with fibrin matrices containing growth factors. These results suggest strong translational possibilities. We report an extensive number, distance, and functional impact of axonal outgrowth from neural stem cells to sites of severe spinal cord injury. These findings indicate that properties intrinsic to the early-stage neuron are sufficient to overcome barriers to growth in the adult CNS. There are important potential clinical implications; treatments were delayed for a clinically practical time frame of at least 1 week postinjury, resulted in significant functional improvement, and were achieved by using a human neural stem source already employed in clinical trials for amyotrophic lateral sclerosis (ALS) Cellular differentiation was determined by counting individual cells labeled for NeuN, GFAP, or APC within the graft, divided by the total number of cells per sample box labeled with DAPI. Two randomly selected fields corresponding to the graft epicenter were counted in each subject for each label, and all analyses were conducted in a blinded manner. An average of 170, 150, and 92 NeuN-, APC-, and GFAP-labeled cells were counted in each of six grafted animals, respectively, and divided by the mean number of DAPI-labeled nuclei in the sampled field. The number of GFP-labeled axons emerging from a typical graft placed at the T3 transection site was quantified by using Stereo Investigator. Briefly, in every sixth sagittal section, a dorsoventral line was drawn 50","journal":"Zenodo (CERN European Organization for Nuclear Research)","year":2022,"id":310315,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":0,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9532,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2022-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1005608,"name":"Mingyong Gao","orcid":"0000-0002-8824-7600","position":1,"is_corresponding":false},{"id":247029,"name":"Mark H. Tuszynski","orcid":"0000-0003-4182-1481","position":2,"is_corresponding":false},{"id":1006187,"name":"Yaozhi Wang","orcid":null,"position":0,"is_corresponding":true}],"reference_count":0,"raw_metadata":null,"created_at":"2026-07-19T00:33:19.712967Z","pmid":null,"pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}