Human Amnion-Derived Mesenchymal Stem Cells Promote Osteogenic Differentiation in Human Bone Marrow Mesenchymal Stem Cells by Influencing the ERK1/2 Signaling Pathway

Abstract

Human amnion-derived mesenchymal stem cells (HAMSCs) are considered to be an important resource in the field of tissue engineering because of their anti-inflammatory properties and fewer ethical issues associated with their use compared with other sources of stem cells. HAMSCs can be obtained from human amniotic membranes, a readily available and abundant tissue. However, the potential of HAMSCs as seed cells for treating bone deficiency is unknown. In this study, HAMSCs were used to promote proliferation and osteoblastic differentiation in human bone marrow mesenchymal stem cells (HBMSCs) in a Transwell coculture system. Proliferation levels were investigated by flow cytometry and immunofluorescence staining of 5-ethynyl-2'-deoxyuridine (EdU). Osteoblastic differentiation and mineralization were evaluated in chromogenic alkaline phosphatase (ALP) activity substrate assays, Alizarin red S staining, and RT-PCR analysis of early HBMSCs osteogenic marker expression. We demonstrated that HAMSCs stimulated increased alkaline phosphatase (ALP) activity, mRNA expression of osteogenic marker genes, and mineralized matrix deposition. Moreover, the effect of HAMSCs was significantly inhibited by U0126, a highly selective inhibitor of extracellular signaling-regulated kinase 1/2 (ERK1/2) signaling. We demonstrate that HAMSCs promote osteogenic differentiation in HBMSCs by influencing the ERK1/2 signaling pathway. These observations confirm the potential of HAMSCs as a seed cell for the treatment of bone deficiency.

Figure 1

Characterization of third-passage adherent amniotic cells: cells were stained with monoclonal antibodies specific for human CD105, CD90, CD79b, CD44, and CD29 and analyzed by flow cytometry.

Figure 2

The effect of HAMSCs on HBMSC proliferation was measured by flow cytometry and immunofluorescence staining of 5-ethynyl-2′-deoxyuridine (EdU). (a) Cell cycle fractions (G0, G1, S, and G2 M phases) at 3, 6, and 12 d. (b) Immunofluorescence staining of EdU at 12 d. (c) Area of stained EdU-positive nodules relative to the total culture surface at 12 d was measured by Image-Pro Plus (IPP) analysis. Scale bar: 100 μm, ^∗ P < 0.05, and ^∗∗ P < 0.01. Control: HBMSCs cultured without HAMSCs; HBMSCs cocultured with HAMSCs; H1: HBMSC : HAMSC = 1 : 1; H2: HBMSC : HAMSC = 1 : 2; H3: HBMSC : HAMSC = 1 : 3.

Figure 3

RT-PCR analysis of HBMSCs cultured with or without HAMSCs. Transwells containing HAMSCs were moved into the correlating wells containing HBMSCs in regular medium after cells were attached. After the cells approached confluence in regular medium, both cell types were cultured in OS medium for 7 days. The mRNA expression of ALP, Runx2, and OC was analyzed by RT-PCR. GAPDH was used as the internal control. (a) Alkaline phosphatase (ALP); (b) runt-related transcription factor 2 (Runx2). (c) Osteocalcin (OC). ^∗ P < 0.05; ^∗∗ P < 0.01. Control: HBMSCs cultured without HAMSCs; HBMSCs cocultured with HAMSCs; H1: HBMSC : HAMSC = 1 : 1; H2: HBMSC : HAMSC = 1 : 2; H3: HBMSC : HAMSC = 1 : 3.

Figure 4

ALP activity and mineralized matrix deposition in HBMSCs cultured with or without HAMSCs. Transwells containing HAMSCs were moved into the correlating wells containing HBMSCs in regular medium after cells were attached. After the cells approached confluence in regular medium, both cell types were cultured in OS medium for 7 days and 14 days. (a) ALP activity was measured at 7 d using p-nitrophenyl phosphate as the substrate. (b) Mineralized matrix deposition was measured at 14 d by Alizarin red S staining and Image-Pro Plus (IPP) analysis. The relative intensity is expressed as a percentage of the stained extracellular matrix area relative to the total culture surface area. (c) Micrographs of mineralized matrix deposition, scale bar: 100 μm. ^∗ P < 0.05; ^∗∗ P < 0.01. Control: HBMSCs cultured without HAMSCs; HBMSCs cocultured with HAMSCs; H1: HBMSC : HAMSC = 1 : 1; H2: HBMSC : HAMSC = 1 : 2; H3: HBMSC : HAMSC = 1 : 3.

Figure 5

Expression of p-ERK1/2, ERK1/2, and Runx2 proteins in HBMSCs cultured with or without HAMSCs. Transwells containing HAMSCs were moved into the correlating wells containing HBMSCs in regular medium after cells were attached. After the cells approached confluence in regular medium, both cell types were cultured in OS medium for 7 days. (a) Cells were treated without OS for 7 days. Densitometric measures of the band intensity are expressed as the signal ratios indicating the level of ERK phosphorylation (p-ERK1/2/ERK1/2). (b) Cells were treated with OS for 7 days. Densitometric measures of the band intensity are expressed as the signal ratios indicating the level of ERK phosphorylation (p-ERK1/2/ERK1/2). ^∗ P < 0.05; ^∗∗ P < 0.01. Control: HBMSCs cultured without HAMSCs; HBMSCs cocultured with HAMSCs; H1: HBMSC : HAMSC = 1 : 1; H2: HBMSC : HAMSC = 1 : 2; H3: HBMSC : HAMSC = 1 : 3.

Figure 6

U0126 inhibited the expression of p-ERK1/2, ERK1/2, and Runx2 proteins in HBMSCs cultured with or without HAMSCs. Transwells containing HAMSCs were moved into the correlating wells containing HBMSCs in regular medium after cells were attached. After the cells approached confluence in regular medium, both cell types were cultured in OS medium for 7 days, followed by treatment with and without U0126 for a further 24 h. Densitometric measures of the band intensity are expressed as the signal ratios indicating the level of ERK phosphorylation (p-ERK1/2/ERK1/2), ^∗ P < 0.05; ^∗∗ P < 0.01. Control: HBMSCs cultured without HAMSCs; HBMSCs cocultured with HAMSCs; H1: HBMSC : HAMSC = 1 : 1; H2: HBMSC : HAMSC = 1 : 2; H3: HBMSC : HAMSC = 1 : 3.

Figure 7

U0126 downregulated the mRNA expression of ALP, Runx2, and OC genes in HBMSCs cultured with or without HAMSCs. Transwells containing HAMSCs were moved into the correlating wells containing HBMSCs in regular medium after cells were attached. After the cells approached confluence in regular medium, both cell types were cultured in OS medium for 7 days, followed by treatment with and without U0126 for a further 24 h. GAPDH was used as the internal control. (a) ALP, (b) Runx2, and (c) OC. ^∗ P < 0.05; ^∗∗ P < 0.01. Control: HBMSCs cultured without HAMSCs; HBMSCs cocultured with HAMSCs; H1: HBMSC : HAMSC = 1 : 1; H2: HBMSC : HAMSC = 1 : 2; H3: HBMSC : HAMSC = 1 : 3.

Figure 8

U0126 inhibited the ALP activity and mineralized matrix deposition in HBMSCs cultured with or without HAMSCs. Transwells containing HAMSCs were moved into the correlating wells containing HBMSCs in regular medium after cells were attached. After the cells approached confluence in regular medium, both cell types were cultured in OS medium for 7 days, followed by treatment with and without U0126 for a further 24 h. (a) ALP activity was measured at 7 d using p-nitrophenyl phosphate as the substrate. (b) Mineralized matrix deposition was measured at 14 d by Alizarin red S staining and Image-Pro Plus (IPP) analysis. (c) Micrographs of mineralized matrix deposition, scale bar: 100 μm. ^∗ P < 0.05; ^∗∗ P < 0.01. Control: HBMSCs cultured without HAMSCs; HBMSCs cocultured with HAMSCs; H1: HBMSC : HAMSC = 1 : 1; H2: HBMSC : HAMSC = 1 : 2; H3: HBMSC : HAMSC = 1 : 3.