Evaluation of Differential Gene Expression during Transdifferentiation of Bone Marrow Stromal Cells to Glial Phenotype in the Presence of Cerebrospinal Fluid

Abstract

Background: The present study assessed the alteration of gene expression during transdifferentiation of Bone Marrow Stromal Cells (BMSCs) into oligodendrocyte in the presence of Cerebrospinal Fluid (CSF).

Methods: BMSCs were collected from female Sprague-Dawley rats and were cultured in DMEM/F12 medium supplemented with Retinoic Acid (RA), basic Fibroblast Growth Factor (bFGF), and Epidermal Growth Factor (EGF). CSF was added daily to the culture media. The oligoprogenitor and oligodendrocyte generation was assessed by immunocytochemistry for Oligo 2, A2B5, CNP and MBP markers.

Results: The mean percentages of immunopositive cells for Olig2 and A2B5 were 52.1±1.74 and 56.34±2.55%, respectively. The number of immunopositive cells for glial markers CNP and MBP were 48.8±3.12 and 40.5±8.92%, respectively. Alteration of gene expression of Oct4, Olig 2, PDGFR-α and PLP were examined by real time PCR during transdifferentiation of BMSC to oligodendrocyte. Immunocytochemical results indicate that oligoprogenitor cells were immunopositive for Oligo2 and A2B5 markers. Also, oligodendrocytes expressed the mature glial markers of CNP and MBP indicating successful differentiation.

Conclusion: In conclusion, CSF promotes the transdifferentiation of BMSC into mature oligodendrocyte via providing an appropriate niche for glial maturation.

Keywords: Bone marrow stromal cells; Cells; Cerebrospinal fluid; Oligodendrocyte; Oligoprogenitor.

Figure 1.

The morphological appearance of cultured BMSC. A) Primary culture of BMSC after 4 hr culture, B) The cell population after the 4th passage culture. Scale bars 10 μm.

Figure 2.

Flow cytometric analysis of BMSC (Passage 4). Isolated bone marrow cells are strongly positive for MSC surface markers but didn’t express CD33 and CD34.

Figure 3.

Multilineage differentiation of BMSC, A) Adipogenic differentiation of BMSC (Oil Red O stain of lipid droplets). B) Osteogenic differentiation of BMSC; (Alizarin Red Stain for calcium). Scale bars 10 μm.

Figure 4.

Scheme showing the protocol for differentiation of BMSC to oligodendrocyte. For glial cells, undifferentiated BMSCs A) were plated in media that induce OPC generation B). Further cultivation in the presence of CSF was used to complete the maturation of OPC to Oligodendrocyte C). Scale bars 10 μm.

Figure 5.

Immunocytochemistry analysis of differentiated bone marrow stromal cells into oligodendrocyte like cells. Fluorescence images of oligoprogenitor markers (Olig2 and A2B5) and mature oligodendrocyte markers; (CNP and MBP) in experimental group (A-D); Negative Control 1 (E-H) Control 2 (I-L), and Control 3. (M-P). Scale bars 10 μm.

Figure 6.

Ratio of gene expression of Oct4, Olig2, PDGF-α in oligoprogenitor cells compared to BMSC. *: Significant increase or decrease. OPC: Oligoprogenitor cells. BMSC: Bone marrow stromal cells. Data are shown as mean±SEM from three independent experiments.

Figure 7.

Ratio of gene expression of Olig2, PDGF-α and PLP in oligodendrocyte compared to oligoprogenitor cells. *: Significant increase or decrease. OPC: Oligoprogenitor cells. OLC: Oligodendrocyte. Data are shown as mean ±SEM from three independent experiments.

Figure 8.

Comparison of gene expression of Oct 4, Olig2, PDGF-α in experimental group compared to Control 2 and Control 3. *: Significant decrease with experimental group. Data are shown as mean± SEM from three independent experiments.