Neuron-like differentiation of bone marrow-derived mesenchymal stem cells

Abstract

Purpose: Mesenchymal stem cells (MSCs) are multipotent and give rise to distinctly differentiated cells from all three germ layers. Neuronal differentiation of MSC has great potential for cellular therapy. We examined whether the cluster of mechanically made, not neurosphere, could be differentiated into neuron-like cells by growth factors, such as epidermal growth factor (EGF), hepatocyte growth factor (HGF), and vascular endothelial growth factor (VEGF).

Materials and methods: BMSCs grown confluent were mechanically separated with cell scrapers and masses of separated cells were cultured to form cluster BMSCs. As described here cluster of BMSCs were differentiated into neuron-like cells by EGF, HGF, and VEGF. Differentiated cells were analyzed by means of phase-contrast inverted microscopy, reverse transcriptase-polymerase chain reaction (RT-PCR), immunofluorescence, and immunocytochemistry to identify the expression of neural specific markers.

Results: For the group with growth factors, the shapes of neuron-like cells was observable a week later, and two weeks later, most cells were similar in shape to neuron-like cells. Particularly, in the group with chemical addition, various shapes of filament structures were seen among the cells. These culture conditions induced MSCs to exhibit a neural cell phenotype, expressing several neuro-glial specific markers.

Conclusion: bone marrow-derived mesenchymal stem cells (BMSCs) could be easily induced to form clusters using mechanical scraping, not neurospheres, which in turn could differentiate further into neuron-like cells and might open an attractive possibility for clinical cell therapy for neurodegenerative diseases. In the future, we consider that neuron-like cells differentiated from clusters of BMSCs are needed to be compared and analyzed on a physiological and molecular biological level with preexisting neuronal cells, and studies on the possibility of their transplantation and differentiation capability in animal models are further required.

Fig. 1

Mesenchymal stem cell culture (×100). (A) After 7 days of culturing. (B) After 14 days of culturing. (C) Subculture (4 times). (D) Cluster of BMSCs.

Fig. 2

Fluorescence Activated Cell Sorting (FACS) analysis.

Fig. 3

Possibility of mesenchymal stem cells' differentiation to osteoblasts (×100). (A) Alkaline phosphatese staining. (B) Silver nitrate staining.

Fig. 4

Cultured cells in chondrogenic medium for 3 weeks (×100). TGF, tumor growth factor.

Fig. 5

Oil red-O staining of adipocytes. MSC, mesenchymal stem cell.

Fig. 6

Differentiation into bone marrow-derived neuron-like cells (×200). (A) Mesenchymal stem cells. (B) Growth factor group (1 week). (C) Growth factor group (2 weeks). (D) Chemical group (2 weeks).

Fig. 7

Immunochemical staining (×200). (A) NSE, neuron specific enolase. (B) NeuN, neuronal nuclei. (C) GFAP, glial fibrillary acidic protein.

Fig. 8

Immunofluorescence staining (×200). (A and B) GFAP, glial fibrillary acidic protein. (C and D) NeuN, neuronal nuclei. (E and F) MAP2, microtubule-associated protein 2. (G and H) Gal C, galactocerebroside.

Fig. 9

Reverse transcriptase-Polymerase Chain Reaction. GFAP, glial fibrillary acidic protein; NSE, Neuron Specific Enolase; MAP2, Microtubule-associated protein 2; NF-M, Neurofilament-Middle; GAP 43, Growth Associated Protein 43.

Fig. 10

Western blotting. GFAP, glial fibrillary acidic protein; NSE, neuron specific enolase; NeuN, neuronal nuclei; Gal C, galactocerebroside.