Functional myelination of neurons

Abstract

Hair follicle bulge region/LLP region CD34(+) MeSCs can be isolated from mammalian skin bearing hair follicles. These cells are multipotent and retain the ability to differentiate into cells of neural crest lineage, including glia-like cells that express the glial marker Gfap, and are able to express myelin basic protein, and to remyelinate naked (unmyelinated or demyelinated) neuronal processes with a functional, dense myelin sheath. These cells of neural crest lineage can be used to produce a dense myelin sheath on neurons which lack myelin due to genetic defect, trauma, toxin, infection, or disease process. Therefore, embodiments of the invention provide methods for preparing such cells, the cells themselves and compositions containing the cells, as well as methods for using the cells.

1 . A method of isolating melanocyte stem cells from the hair follicle of mammalian skin to yield CD34(+) multipotent neural crest progenitor cells that express the marker Gfap and express myelin basic protein, comprising the steps of:
 (a) obtaining a suspension of skin cells that includes melanocyte stem cells from the bulge region, the lower permanent portion, or both, of the hair follicle;   (b) separating CD34(+) melanocyte stem cells from said single cell suspension; and   (c) exposing said CD34(+) melanocyte stem cells to conditions that promote neural crest progenitor formation,   to form CD34(+) multipotent neural crest progenitor cells that express at least the marker Gfap and myelin basic protein.   
     
     
         2 . The method of  claim 1 , wherein said conditions that promote neural crest progenitor formation comprise culturing said CD34(+) MeSCs in neural crest differentiation medium. 
     
     
         3 . Isolated CD34(+) multipotent neural crest progenitor cells made according to the method of  claim 1 . 
     
     
         4 . The isolated CD34(+) multipotent neural crest progenitor cells of  claim 3 , which are at least 80% pure, at least 85% pure, at least 90% pure, at least 95% pure, at least 97% pure, at least 98% pure, at least 99% pure, at least 99.5% pure, or at least 99.9% pure CD34(+) multipotent neural crest progenitor cells. 
     
     
         5 . The isolated CD34(+) multipotent neural crest progenitor cells of  claim 3 , which are human cells. 
     
     
         6 . The isolated CD34(+) multipotent neural crest progenitor cells of  claim 3 , which are mouse cells. 
     
     
         7 . The isolated CD34(+) multipotent neural crest progenitor cells of  claim 3 , wherein the mouse is a Dct-H2BGFP mouse. 
     
     
         8 . A composition comprising an acceptable carrier and the isolated CD34(+) multipotent neural crest progenitor cells of  claim 3 . 
     
     
         9 . Substantially pure isolated CD34(+) multipotent neural crest progenitor cells from mammalian hair follicle bulge/LPP and lower permanent portion of the hair follicle, which express at least the cell marker Gfap and myelin basic protein. 
     
     
         10 . A method of producing a dense myelin sheath around an axon comprising contacting said axon with the isolated CD34(+) multipotent neural crest progenitor cells of  claim 3 . 
     
     
         11 . The method of  claim 10 , wherein said contacting results in myelination. 
     
     
         12 . The method of  claim 10 , wherein said contacting is performed under conditions comprising culturing said CD34(+) MeSCs in Poly D Lysine and Laminin coated chambers in the presence of neural crest differentiation medium said medium comprising ascorbic acid. 
     
     
         13 . A method of producing a functional myelin sheath on a axon which lacks a functional myelin sheath or has become demyelinated, comprising contacting said axon with the isolated CD34(+) multipotent neural crest progenitor cells of  claim 3 . 
     
     
         14 . The method of  claim 13 , wherein said contacting is administration by direct injection into the area of said axon, or by intrathecal injection, or intravenously, or by stereotaxic injection. 
     
     
         15 . The method of  claim 13 , wherein the area of said axon is in the central nervous system. 
     
     
         16 . The method of  claim 13 , wherein the area of said axon is in the peripheral nervous system. 
     
     
         17 . The method of  claim 13 , wherein the demyelination of said axon is immune-mediated, auto-antibody mediated, or caused by a demyelinating disease, trauma, toxin, bacterial infection, viral infection, parasitic infection, or genetic defect. 
     
     
         18 . The method of  claim 17 , wherein said demyelinating disease is selected from the group consisting of: experimental allergic encephalomyelitis, acute disseminated encephalomyelopathy, acute hemorrhagic encephalomyelopathy, experimental allergic neuritis, amoebic meningoencephalitis, Guillain-Barre syndrome, multiple sclerosis, stroke, traumatic brain injury, and traumatic peripheral nerve injury, Devic's disease (otherwise known as neuromyelitis optica (NMO)), NMO spectrum disorder, progressive multifocal leukoencephalopathy, central pontine myelinolysis, Tabes dorsalis, optic neuritis, transverse myelitis, progressive inflammatory neuropathy, myelopathy, chronic inflammatory demyelinating polyneuropathy, Charcot-Marie-Tooth disease, visna, and the like.