BONE MARROW-DERIVED NEURONAL CELLS
Bone marrow stromal cells (BMSC) differentiate into neuron-like phenotypes in vitro and in vivo, engrafted into normal or denervated rat striatum. The BMSC administered into the ventricle did not remain localized to the site of the graft, but migrated throughout the brain and integrated into specific brain regions in various architectonic patterns. The most orderly integration of BMSC was in the laminar distribution of cerebellar Purkinje cells, where the BMSC-derived cells took on the Purkinje phenotype. The BMSC exhibited site-dependent differentiation and expressed several neuronal markers including neuron-specific nuclear protein, tyrosine hydroxylase and calbindin. Treated BMSC implanted intrastriatally stayed in the cortex and the striatum, produced tyrosine hydroxylase which produces the dopamine needed in Parkinson's disease.
1 . A mixture of human mesenchymal stem cells and about 1% to about 4% differentiated neurons, glia and fibroblasts, the latter three cell types staining for NeuN, GFAP and TH but not CD40.
2 . A method of preparing neuronal cells from bone marrow stromal cells, the method comprising
a. providing a solution of disaggregated bone marrow cells in buffer;
b. suspending the bone marrow cell solution in additional medium;
c. centrifuging the diluted bone marrow cells to a pellet;
d. collecting from the pellet the interface mononuclear cells;
e. washing twice with medium;
f. suspending the pellet in culture and culturing in polystyrene flasks for more than one week;
g. lifting the confluent, adherent cells;
h. replating the cells in the presence of serum-free medium for 5-7 days until 70-80% confluent to produce nestin+ cells.
3 . The method of claim 2 , further comprising the step
i. replating the Nestin+ cells onto polylysine-coated cell culture slides in N2 medium containing 0.5 microM all-trans retinoic acid (RA) and brain-derived neurotrophic factor (BDNF).
3 . A cell line of neuronal cells arising from bone marrow stromal cells having been incubated with retinoic acid A and BDNF for a time sufficient to modify at least a portion to neuronal cells, wherein the neuronal cells have the ability to migrate and localize to specific neuroanatomical regions where the neuronal cells differentiate into neurons typical of the region and integrate in characteristic architectonic patterns.
4 . A kit for neuronal cell auto-transplant comprising a) a syringe suitable for obtaining bone marrow, b) a plastic flask with dehydrated culture medium, c) bone marrow stem cell, and d) a mixture of Retinoic Acid A and BDNF.
5 . A method of treating a neurodegenerative disorder comprising
a. providing the cells of claim 3 ; and
b. administering a sufficient quantity of the cells 3 to treat an individual with the neurodegenerative disorder.
6 . The method of claim 5 , wherein the disorder is Parkinson's Disease.