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. 2017 Oct 11;12(10):e0186253.
doi: 10.1371/journal.pone.0186253. eCollection 2017.

Human amnion-derived mesenchymal stem cells promote osteogenic and angiogenic differentiation of human adipose-derived stem cells

Chunli Zhang  1 , Lidong Yu  2 , Songjian Liu  3 , Yuli Wang  4
Affiliations

Human amnion-derived mesenchymal stem cells promote osteogenic and angiogenic differentiation of human adipose-derived stem cells

Chunli Zhang et al. PLoS One. .

Abstract

Tissue engineering using suitable mesenchymal stem cells (MSCs) shows great potential to regenerate bone defects. Our previous studies have indicated that human amnion-derived mesenchymal stem cells (HAMSCs) could promote the osteogenic differentiation of human bone marrow mesenchymal stem cells (HBMSCs). Human adipose-derived stem cells (HASCs), obtained from adipose tissue in abundance, are capable of multi-lineage differentiation. In this study, the effects of HAMSCs on osteogenic and angiogenic differentiation of HASCs were systematically investigated. Proliferation levels were measured by flow cytometry. Osteoblastic differentiation and mineralization were investigated using chromogenic alkaline phosphatase activity (ALP) activity substrate assays, Alizarin red S staining, real-time polymerase chain reaction (real-time PCR) analysis of osteogenic marker expression, and Western blotting. We found that HAMSCs increased the proliferation and osteoblastic differentiation of HASCs. Moreover, enzyme-linked immunosorbent assay (ELISA) and human umbilical vein endothelial cells (HUVECs) tube formation suggested HAMSCs enhanced angiogenic potential of HASCs via secretion of increased vascular endothelial growth factor (VEGF). Thus, we conclude that HAMSC might be a valuable therapeutic approach to promote HASCs-involved bone regeneration.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. HAMSCs promoted HASCs proliferation.
The cell cycle fractions (G0, G1, S, and G2 M phases) of HASCs cultured with or without HAMSCs were determined by flow cytometry at 1, 3 and 5 d.
Fig 2
Fig 2. ALP activity and mineralized matrix deposition in HAMSCs, HASCs, and HASCs/HAMSCs groups.
(A): ALP activity in HAMSCs and HASCs groups was measured at 7 and 14 d using ALP assay kit. (B): Mineralized matrix deposition in HAMSCs and HASCs groups was measured at 21 d by Alizarin red S. (C): ALP activity in HASCs and HASCs/HAMSCs groups was measured at 7 and 14 d using ALP assay kit. (D): Mineralized matrix deposition in HASCs and HASCs/HAMSCs groups was measured at 21 d by Alizarin red S. Scale bar: 300 μm.*P < 0.05 and **P < 0.01 in contrast to the HAMSCs or HASCs groups.
Fig 3
Fig 3. Expression of relative genes and proteins in HASCs cultured with or without HAMSCs.
(A, B, C and D): The mRNA expression of RUNX2, OCN, COL1, and VEGF were analyzed by real-time PCR at 14 d. GAPDH were used as the internal control. (E): Protein expression of RUNX2, OCN, COL1, VEGFR1, and Angiogenin were determined by Western blotting at 14 d, β-actin served as an internal control. *P < 0.05 and **P < 0.01 in contrast to the HASCs groups.
Fig 4
Fig 4. HAMSCs promoted angiogenesis and ERK1/2 phosphorylation in HASCs.
(A): The VEGF level in culture supernatant from HASCs and HASCs/HAMSCs groups was measured by VEGF ELISA assay on day 14. (B): Tube formation from HUVECs was detected at 24 h after culture. (C): Protein expression of p-ERK, ERK, p-p38, p38, p-JNK, and JNK were determined by Western blotting at 14 d, β-actin served as an internal control. Scale bar: 300 μm.*P < 0.05 and **P < 0.01 in contrast to the HASCs groups.
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This study was supported by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD; 2014-37). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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