Human cranial bone-derived mesenchymal stem cells cultured under microgravity can improve cerebral infarction in rats

Background Transplantation of human cranial bone-derived mesenchymal stem cells (hcMSCs) in patients with central nervous system disorders may improve motor function through high neurotrophic factor expression. However, the effects of hcMSCs cultured under microgravity (MG) conditions on cerebral infarction remain unclear. Thus, this study investigated the transplantation effects of hcMSCs cultured in a simulated MG environment on cerebral infarction model rats. Methods For immunohistological analysis and neurological function evaluation, hcMSCs cultured in normal gravity (1G) or MG environment were transplanted in rats 1 day after inducing cerebral infarction. The expression of endogenous neurotrophic; axonal, neuronal, and synaptogenic; angiogenic; and apoptosis-related factors in infarcted rat brain tissue was examined by real-time polymerase chain reaction (PCR) and western blotting analyses 35 days after stroke induction. MicroRNAs (miRNAs) of hcMSCs cultured under 1G or MG environments were sequenced. Results Neurological function was significantly improved after transplantation of hcMSCs from the MG group compared with that from the 1G group. Protein expressions of nerve growth factor, fibroblast growth factor 2, and synaptophysin were significantly higher in the MG group than in the 1G group, whereas sortilin 1 expression was significantly lower. MiRNA analysis revealed that genes related to cell proliferation, angiogenesis, neurotrophy, anti-apoptosis, neural and synaptic organization, and cell differentiation inhibition were significantly upregulated in the MG group. In contrast, genes promoting microtubule and extracellular matrix formation and cell adhesion, signaling, and differentiation were downregulated. Conclusions These results indicate that hcMSCs cultured in an MG environment may be a useful source of stem cells for the recovery of neurological function in cerebral infarction patients. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement Part of this research was supported by a Grant-in-Aid for Scientific Research from the Japanese Society for the Promotion of Science (Issue No. 20K09348). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: All experimental protocols were approved by the Institutional Review Board of Hiroshima University (approval number: E2010-0379). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Data will be provided on request. * 1G : normal gravity ANGPT1 : angiopoietin 1 BDNF : brain-derived neurotrophic factor bMSCs : bone marrow-derived mesenchymal stem cells BSA : bovine serum albumin cMSCs : cranial bone-derived mesenchymal stem cells CNS : central nervous system CTSS : cathepsin S DCTN1 : dynactin subunit 1 DWI : diffusion-weighted image EPGN : epithelial mitogen FBN2 : fibrillin 2 FGF-7 : fibroblast growth factor 7 Fgf2 : fibroblast growth factor 2 Gap43 : growth-associated protein 43 GDNF : glial cell-derived neurotrophic factor GO : gene ontology hbMSCs : human bone marrow-derived mesenchymal stem cells hcMSCs : human cranial bone-derived mesenchymal stem cells HE : hematoxylin-eosin IBZ : ischemic border zone ISS : International Space Station ITGB3 : integrin subunit beta 3 LAMA1 : laminin subunit alpha 1 MCAO : middle cerebral artery occlusion MG : microgravity miRNA : microRNA mNSS : modified neurological severity score MRI : magnetic resonance imaging MSCs : mesenchymal stem cells Ngf : nerve growth factor Ntrk2 : neurotrophic tyrosine kinase receptor type 2 PBS : phosphate-buffered saline PCR : polymerase chain reaction PFA : paraformaldehyde Sort1 : sortilin 1 Act-b : β-Actin Syp : synaptophysin T2WI : T2-weighted image VEGF-A : vascular endothelial growth factor-A VEGF : vascular endothelial growth factor