Bone marrow-derived mesenchymal stem cells as a source of neural progenitors
Methods are provided for treating and/or reducing the severity of multiple sclerosis in a human, by administering autologous mesenchymal stem cell-derived neural precursors. Also described is an in vitro method for differentiating mesenchymal stem-cell derived neural precursor oligodendroglial and neuronal cell types.
1. A method for the in vitro differentiation of mesenchymal stem cells and selection of neural precursors derived therefrom, the method comprising the steps of:
(a) isolating mesenchymal stem cells from a human,
(b) expanding the isolated mesenchymal stem cells,
(c) culturing a portion of said expanded mesenchymal stem cells in a neural progenitor basal medium (NPBM) supplemented with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and neural cell survival factor-1 (NSF-1),
(d) collecting floating cell clusters from the NPBM-cultured cells,
(e) measuring, in a test portion of the collected cells, expression of nestin, glial fibrillary acidic protein (GFAP), neurofilament-M (NF-M), and alpha smooth muscle (αSM) actin, relative to expression of nestin, GFAP, NF-M, and αSM actin in a test portion of the mesenchymal stem cells expanded in step (b), and
(f) selecting the remaining portion of the collected cells as neural precursors for further use based on increased expression of nestin, GFAP, and NF-M, and decreased expression of αSM actin in said test portion of the collected cells, relative to the expression of nestin, GFAP, NF-M, and αSM actin in said test portion of mesenchymal stem cells.
2. The method of claim 1 , wherein
the mesenchymal stem cells are expanded in step (b) in mesenchymal stem cell basal medium comprising autologous serum.
3. The method of claim 1 , wherein the culturing of step (c) is performed for 10-15days.
4. The method of claim 1 , further comprising:
(g) culturing the cells selected in step (f) in a medium comprising basic fibroblast growth factor, thereby further differentiating mesenchymal stem cell-derived neuronal precursors.
5. The method of claim 4 , wherein the medium comprising basic fibroblast growth factor for the culturing of step (g) comprises 100ng/ml basic fibroblast growth factor.
6. The method of claim 4 , wherein the culturing of step (g) comprises changing the medium every 2-3 days for 21 days.
7. The method of claim 6 , wherein the human has multiple sclerosis.
8. The method of claim 1 , wherein if the test portion of the collected cells do not show increased expression of nestin, GFAP, and NF-M, and decreased expression of αSM actin in said test portion of the collected cells, relative to the expression of nestin, GFAP, NF-M, and αSM actin in said test portion of mesenchymal stem cells, steps (c) through (d) are repeated using another portion of mesenchymal stem cells expanded in step (b).