Expression of OCT-4 and SOX-2 in Bone Marrow-Derived Human Mesenchymal Stem Cells during Osteogenic Differentiation

Abstract

Aim: Determine the levels of expression of pluripotency genes OCT-4 and SOX-2 before and after osteogenic differentiation of human mesenchymal stem cells (hMSCs).

Methods: Human MSCs were derived from the bone marrow and differentiated into osteoblasts. The analyses were performed on days 0 and 14 of the cell culture. In vitro differentiation was evaluated due to bone markers - alkaline phosphatase (AP) activity and the messenger RNA (mRNA) expression of AP and bone sialoprotein (BSP). The OCT-4 and SOX-2 expression was evaluated at mRNA level by real-time qPCR and at protein level by immunocytochemistry.

Results: In vitro cultures on day 14 showed an increase in AP activity and upregulation of AP and BSP gene expression. OCT-4 and SOX-2 in undifferentiated hMSCs on day 0 is detectable and very low compared to tumor cell lines as a positive control. Immunocytochemistry detected OCT-4 in the cell nuclei prior (day 0) and post differentiation (day 14). On the same time points, cultures were negative for SOX-2 protein.

Conclusion: Messenger RNA for pluripotency markers OCT-4 and SOX-2 isolated from hMSCs was less present, while OCT-4 protein was detected in cell nuclei prior and post differentiation into osteoblast lineage.

Keywords: human mesenchymal stem cells; osteodifferentiation; pluripotency markers.

Figure 1

High alkaline phosphatase activity is an indication of successful differentiation of hMSCs into osteoblast after 14 days of osteoinduction. The alkaline phosphatase (AP) activity was measured by using p-nitrophenyl phosphate as a substrate. This quantitative method has shown higher alkaline phosphatase activity in differentiated hMSC cultures (Day 14) than in undifferentiated cultures (Day 0) (A). This result has been confirmed by AP cytochemical staining (B, C). Red dye deposits indicate sites of alkaline phosphatase activity in hMSC cultures on day 14 and just a few undifferentiated hMSCs were red. The data are expressed as the means + SD of triplicate determination (*P<0.05).

Figure 2

Relative mRNA expressions of osteogenic markers detected by real-time quantitative PCR on day 0 and day 14. The relative gene expression of BSP and AP was analyzed by ΔΔ cycle threshold method and the values were normalized to β-actin expression. Those values were then normalized to hMSCs after 14 day of osteoinduction. Data are represented as an average of three independent patient samples and error bars represent mean value + SD (*P<0.05). Abbreviations: BSP, bone sialoprotein; AP, alkaline phosphatase; hMSCs, human Mesenchymal Stem Cells.

Figure 3

Expression levels of OCT-4 and SOX-2 genes in hMSCs after 14 days of osteoinduction, undifferentiated hMSCs and two human cancer cell lines A1235 and HepG2 detected by real-time qPCR. OCT-4 is expressed in undifferentiated hMSC (Day 0) but expression of OC-T4 gene does not go down after osteoinduction on day 14. Relative mRNA expression of OCT-4 in Hep G2 cell line was 9.9 x more expressed than in undifferentiated hMSCs (A). Transcription factor SOX-2 is expressed in undifferentiated hMSCs and its expression goes down after osteoinduction on day 14. Relative mRNA expression of SOX-2 in Hep G2 cell line was 5321.9 x more expressed than in undifferentiated hMSCs while in glioblastoma A1235 cell line was 3169.8 x respectively (B). The quantity of gene expression was normalized to β-actin to determine the quantitative differences. Data are represented as an average of three independent patient samples and error bars represent mean value + SD. In the columns marked with asterisk * the mean values are significantly different then control (Hep G2) (A) or A1235 and Hep G2 (B), respectively, according to Duncan test (*P<0.05).

Figure 4

Immunostaining for OCT-4 did not show any changes in OCT-4 protein level in osteo-differentiated hMSC cultures compared to undifferentiated. Osteoblast and undifferentiated hMSCs were both positive for OCT-4 expression and had a nuclear localization of OCT-4 (A). Immunofluorescence staining for SOX-2 did not confirm levels of SOX-2 protein in osteo-differentiated hMSCs (Day 14) nor in undifferentiated hMSCs (Day 0) (B). Liver cell cancer line HepG2 has been used as positive control for OCT-4 expression. Glioblastoma A1235 cell line has been used as positive control for SOX-2 expression and for no primary antibody control (-ctrl) as well. Nuclei were stained with Hoechst (200x, CCD camera on Olympus BX51). Abbreviations: ICC, immunocytochemistry; BF, bright field; d0, the day when differentiation medium was added; d14, 14th day of osteogenic differentiation.

Figure 5

Fluorescence microscopy image of undifferentiated hMSCs immunostained for OCT-4. Immunofluorescence staining for Hoechst (blue) and OCT-4 (green) indicates a nuclear localization of OCT-4 protein (20x, CCD camera on Olympus BX51).