The in vitro derivation of Schwann cells from human bone marrow stromal cells (hBMSCs) opens avenues for autologous transplantation to achieve remyelination therapy for post-traumatic neural regeneration. Towards this end, we exploited human induced pluripotent stem-cell-derived sensory neurons to direct Schwann-cell-like cells derived from among the hBMSC-neurosphere cells into lineage-committed Schwann cells (hBMSC-dSCs). These cells were seeded into synthetic conduits for bridging critical gaps in a rat model of sciatic nerve injury. With improvement in gait by 12-week post-bridging, evoked signals were also detectable across the bridged nerve. Confocal microscopy revealed axially aligned axons in association with MBP-positive myelin layers across the bridge in contrast to null in non-seeded controls. Myelinating hBMSC-dSCs within the conduit were positive for both MBP and human nucleus marker HuN. We then implanted hBMSC-dSCs into the contused thoracic cord of rats. By 12-week post-implantation, significant improvement in hindlimb motor function was detectable if chondroitinase ABC was co-delivered to the injured site; such cord segments showed axons myelinated by hBMSC-dSCs. Results support translation into a protocol by which lineage-committed hBMSC-dSCs become available for motor function recovery after traumatic injury to both peripheral and central nervous systems.

Transplantation of oligodendrocyte precursors (OPs) is potentially therapeutic for myelin disorders but a safe and accessible cell source remains to be identified. Here we report a two-step protocol for derivation of highly enriched populations of OPs from bone marrow stromal cells of young adult rats (aMSCs). Neural progenitors among the aMSCs were expanded in non-adherent sphere-forming cultures and subsequently directed along the OP lineage with the use of glial-inducing growth factors. Immunocytochemical and flow cytometric analyses of these cells confirmed OP-like expression of Olig2, PDGFRα, NG2, and Sox10. OPs so derived formed compact myelin both in vitro, as in co-culture with purified neurons, and in vivo, following transplantation into the corpus callosum of neonatal shiverer mice. Not only did the density of myelinated axons in the corpus callosum of recipient shiverer mice reach levels comparable to those in age-matched wild-type mice, but the mean lifespan of recipient shiverer mice also far exceeded those of non-recipient shiverer mice. Our results thus promise progress in harnessing the OP-generating potential of aMSCs towards cell therapy for myelin disorders.

/ SUMMARY: Oligodendrocytes (OLs) are the only myelinating glia in the central nervous system (CNS). In congenital myelin disorders, OL dysfunction or death results in loss of myelin. This causes progressive and irreversible impairment to motor and cognitive functions, and is amongst the most disabling neurological disorder. Neonatal engraftment by glial progenitor cells (GPCs) allows the robust myelination of congenitally dysmyelinated brain, thereby preserving brain function and quality of life of patients. However, endogenous sources of glial progenitors are hard to obtain without causing secondary injury, while use of exogenous sources such as embryonic stem cells and induced-pluripotent stem cells face considerable ethical and safety issues. To circumvent such hurdles, we asked whether NG2 ⁺ cells in the bone marrow could be a potential cell source for GPCs. We successfully generated glial progenitor cells (GPCs) from human bone marrow stromal cells (hBMSCs) from 3 donors using a 14- day induction protocol. The generated hBMSC-GPCs were highly enriched in OPC marker expression, including OLIG2, PDGFRα, NG2, SOX10 and O4, and showed efficient differentiation into myelinogenic oligodendrocytes when transplanted into postnatal day 7 (P7) myelin-deficient shiverer mice. Remyelination of the shiverer mouse brain significantly extended lifespan and improved motor function. The novel induction protocol described here provides a method for fast, simple and effective glial therapy for myelin disorders, overcoming existent hurdles of cell source restriction and time frame requirement. Graphical Highlights: - Human bone marrow stromal cells (BMSCs) can be programmed to myelinating glia (GPCs, glial progenitor cells), via a novel 14-day in vitro induction protocol - Transplantation of these hBM-GPCs robustly remyelinated myelin deficient shiverer mice. - hBM-GPC transplant significantly extended lifespan, increased body weight and improved motor function

Loss of myelinating oligodendrocytes and myelin impairs motor and cognitive functions. Transplantation of autologous oligodendrocyte precursors (OPCs) holds promise for treatment of such diseases, but a protocol to derive human OPCs from a safe, ethical and accessible cell source with the rapidity required to catch the therapeutic window remains to be found. Although we previously generated myelinating glia from rat bone marrow stromal cells (BMSCs), it remains unknown if clinically sourced human BMSCs (hBMSCs) share the same potential. Moreover, whether the multipotency of BMSCs results from diverse progenitors preexisting in the bone marrow or from a single multipotent progenitor population remains unaddressed. Single-cell RNA sequencing data revealed a CD90^hiEGFR⁺PDGFRA⁺ pre-OPC-like subpopulation within hBMSCs. With a small-molecule-based (virus-free and supporting-cell-free) two-step induction protocol designed to expand this pre-OPC population, we generated functional OPCs with high purity in eight days. These derived OPCs showed phenotypic transcriptomes and immunoprofiles. They were also capable of myelinating naked axons when transplanted into myelin-deficient shiverer mice. Results highlight how targeted enrichment and maturation of specific progenitor subpopulations within hBMSCs allows rapid induction of desired cell types. These results place hBMSCs as a robust source of OPCs, unlocking the possibility for cell transplantation therapy for myelin deficiency in the central nervous system.

CNS oligodendrocytes generate myelin, an RNA-containing proteolipid substance that enhances axonal transmission. In multiple sclerosis (MS), myelin debris is phagocytosed by microglia (MG), and prior studies have detected myelin-derived mRNA in MG nuclei, suggesting a retrograde transport pathway. We report myelin basic protein (MBP) is a nucleic acid binding and trafficking protein. We found that retro-transport of myelin RNA into the MG nucleus was phagocytosis and importin dependent. Transcriptomic and proteomic analyses of MG nuclei revealed enrichment of myelin mRNAs and proteins, with MBP singularly detected in soluble and chromatin associated fractions. MBP bound mRNA with high affinity (Kd ≈ 0.30 nM) and was sufficient to facilitate MG RNA nuclear import in vitro and in vivo. Functionally, MBP mediated the delivery of small interfering RNAs for targeted knockdown of toll-like receptor 4. These findings indicate MBP as an RNA binding protein capable of MG nuclear import, providing insight into neuroinflammatory pathology of MS.