Neural precursor cells (NSCs) hold great potential to treat a variety of neurodegenerative diseases and injuries to the spinal cord. However, current delivery techniques require an invasive approach in which an injection needle is advanced into the spinal parenchyma to deliver cells of interest. As such, this approach is associated with an inherent risk of spinal injury, as well as a limited delivery of cells into multiple spinal segments. Here, we characterize the use of a novel cell delivery technique that employs single bolus cell injections into the spinal subpial space. In immunodeficient rats, two subpial injections of human NSCs were performed in the cervical and lumbar spinal cord, respectively. The survival, distribution, and phenotype of transplanted cells were assessed 6-8 months after injection. Immunofluorescence staining and mRNA sequencing analysis demonstrated a near-complete occupation of the spinal cord by injected cells, in which transplanted human NSCs (hNSCs) preferentially acquired glial phenotypes, expressing oligodendrocyte (Olig2, APC) or astrocyte (GFAP) markers. In the outermost layer of the spinal cord, injected hNSCs differentiated into glia limitans-forming astrocytes and expressed human-specific superoxide dismutase and laminin. All animals showed normal neurological function for the duration of the analysis. These data show that the subpial cell delivery technique is highly effective in populating the entire spinal cord with injected NSCs, and has a potential for clinical use in cell replacement therapies for the treatment of ALS, multiple sclerosis, or spinal cord injury.

Department of Anesthesia University of Ryukyus Okinawa Japan

Department of Biomaterials and Bioanalogous Systems Institute of Macromolecular Chemistry Czech Academy of Sciences Prague Czech Republic

Department of Neurosurgery University of California San Diego La Jolla California

Gene Expression Laboratory Howard Hughes Medical Institute Salk Institute for Biological Studies La Jolla California

Institute of Animal Physiology and Genetics Czech Academy of Sciences Libechov Czech Republic

Neuralstem Inc Germantown Maryland

Neuroregeneration Laboratory Department of Anesthesiology University of California San Diego La Jolla California

Zobrazit více v PubMed

Usvald D, Vodicka P, Hlucilova J, et al. Analysis of dosing regimen and reproducibility of intraspinal grafting of human spinal stem cells in immunosuppressed minipigs. Cell Transplant. 2010;19:1103‐1122. PubMed

Lu P, Wang Y, Graham L, et al. Long‐distance growth and connectivity of neural stem cells after severe spinal cord injury. Cell. 2012;150:1264‐1273. PubMed PMC

Lepore AC, Rauck B, Dejea C, et al. Focal transplantation‐based astrocyte replacement is neuroprotective in a model of motor neuron disease. Nat Neurosci. 2008;11:1294‐1301. PubMed PMC

Gutierrez J, Lamanna JJ, Grin N, et al. Preclinical validation of multilevel intraparenchymal stem cell therapy in the porcine spinal cord. Neurosurgery. 2015;77:604‐612. discussion 612. PubMed

Hefferan MP, Galik J, Kakinohana O, et al. Human neural stem cell replacement therapy for amyotrophic lateral sclerosis by spinal transplantation. PLoS One. 2012;7:e42614. PubMed PMC

van Gorp S, Leerink M, Kakinohana O, et al. Amelioration of motor/sensory dysfunction and spasticity in a rat model of acute lumbar spinal cord injury by human neural stem cell transplantation. Stem Cell Res Ther. 2013;4:57. PubMed PMC

Lepore AC, Bakshi A, Swanger SA, Rao MS, Fischer I. Neural precursor cells can be delivered into the injured cervical spinal cord by intrathecal injection at the lumbar cord. Brain Res. 2005;1045:206‐216. PubMed

Curtis E, Martin JR, Gabel B, et al. A first‐in‐human, phase I study of neural stem cell transplantation for chronic spinal cord injury. Cell Stem Cell. 2018;22:941‐950.e946. PubMed

Glass JD, Hertzberg VS, Boulis NM, et al. Transplantation of spinal cord‐derived neural stem cells for ALS: analysis of phase 1 and 2 trials. Neurology. 2016;87:392‐400. PubMed PMC

Priest CA, Manley NC, Denham J, Wirth ED III, Lebkowski JS. Preclinical safety of human embryonic stem cell‐derived oligodendrocyte progenitors supporting clinical trials in spinal cord injury. Regen Med. 2015;10:939‐958. PubMed

Curtis E, Gabel BC, Marsala M, Ciacci JD. 172 A phase I, open‐label, single‐site, safety study of human spinal cord‐derived neural stem cell transplantation for the treatment of chronic spinal cord injury. Neurosurgery. 2016;63(Suppl 1):168‐169.

Miyanohara A, Kamizato K, Juhas S, et al. Potent spinal parenchymal AAV9‐mediated gene delivery by subpial injection in adult rats and pigs. Mol Ther Methods Clin Dev. 2016;3:16046. PubMed PMC

Tadokoro T, Miyanohara A, Navarro M, et al. Subpial adeno‐associated virus 9 (AAV9) vector delivery in adult mice. J Vis Exp. 2017;13(125):e55770. PubMed PMC

Kakinohana O, Cizkova D, Tomori Z, et al. Region‐specific cell grafting into cervical and lumbar spinal cord in rat: a qualitative and quantitative stereological study. Exp Neurol. 2004;190:122‐132. PubMed

McGinley LM, Sims E, Lunn JS, et al. Human cortical neural stem cells expressing insulin‐like growth factor‐I: a novel cellular therapy for Alzheimer's disease. Stem Cells Translational Medicine. 2016;5:379‐391. PubMed PMC

Basso DM, Beattie MS, Bresnahan JC. A sensitive and reliable locomotor rating scale for open field testing in rats. J Neurotrauma. 1995;12:1‐21. PubMed

Bohaciakova D, Hruska‐Plochan M, Tsunemoto R, et al. A scalable solution for isolating human multipotent clinical‐grade neural stem cells from ES precursors. Stem Cell Res Ther. 2019;10:83. PubMed PMC

Sofroniew MV. Astrocyte barriers to neurotoxic inflammation. Nat Rev Neurosci. 2015;16:249‐263. PubMed PMC

Choi BH. Role of the basement membrane in neurogenesis and repair of injury in the central nervous system. Microsc Res Tech. 1994;28:193‐203. PubMed

Peluffo H, Acarin L, Faiz M, Castellano B, Gonzalez B. Cu/Zn superoxide dismutase expression in the postnatal rat brain following an excitotoxic injury. J Neuroinflammation. 2005;2:12. PubMed PMC

Abnet K, Fawcett JW, Dunnett SB. Interactions between meningeal cells and astrocytes in vivo and in vitro. Brain Res Dev Brain Res. 1991;59:187‐196. PubMed

Danbolt NC. Glutamate uptake. Prog Neurobiol. 2001;65:1‐105. PubMed

Trotti D, Aoki M, Pasinelli P, et al. Amyotrophic lateral sclerosis‐linked glutamate transporter mutant has impaired glutamate clearance capacity. J Biol Chem. 2001;276:576‐582. PubMed

Maragakis NJ, Rao MS, Llado J, et al. Glial restricted precursors protect against chronic glutamate neurotoxicity of motor neurons in vitro. Glia. 2005;50:145‐159. PubMed

Basso DM, Beattie MS, Bresnahan JC. Graded histological and locomotor outcomes after spinal cord contusion using the NYU weight‐drop device versus transection. Exp Neurol. 1996;139:244‐256. PubMed

Barnabe‐Heider F, Goritz C, Sabelstrom H, et al. Origin of new glial cells in intact and injured adult spinal cord. Cell Stem Cell. 2010;7:470‐482. PubMed

West TW. Transverse myelitis—a review of the presentation, diagnosis, and initial management. Discov Med. 2013;16:167‐177. PubMed

Beh SC, Greenberg BM, Frohman T, Frohman EM. Transverse myelitis. Neurol Clin. 2013;31:79‐138. PubMed PMC

Siddiqi F, Wolfe JH. Stem cell therapy for the central nervous system in lysosomal storage diseases. Hum Gene Ther. 2016;27:749‐757. PubMed PMC

Kim SU. Lysosomal storage diseases: stem cell‐based cell‐ and gene‐therapy. Cell Transplant. 2014. 10.3727/096368914X681946. PubMed DOI

Fandel TM, Trivedi A, Nicholas CR, et al. Transplanted human stem cell‐derived interneuron precursors mitigate mouse bladder dysfunction and central neuropathic pain after spinal cord injury. Cell Stem Cell. 2016;19:544‐557. PubMed

Braz JM, Wang X, Guan Z, et al. Transplant‐mediated enhancement of spinal cord GABAergic inhibition reverses paclitaxel‐induced mechanical and heat hypersensitivity. Pain. 2015;156:1084‐1091. PubMed PMC

Eaton MJ, Berrocal Y, Wolfe SQ. Potential for cell‐transplant therapy with human neuronal precursors to treat neuropathic pain in models of PNS and CNS injury: comparison of hNT2.17 and hNT2.19 cell lines. Pain Res Treat. 2012;2012:1‐31. PubMed PMC

Expandable Sendai-Virus-Reprogrammed Human iPSC-Neuronal Precursors: In Vivo Post-Grafting Safety Characterization in Rats and Adult Pig