Comparative investigation of human amniotic epithelial cells and mesenchymal stem cells for application in bone tissue engineering

Abstract

Emerging evidence suggests amniotic epithelial cells (AECs) as a promising source of progenitor cells in regenerative medicine and bone tissue engineering. However, investigations comparing the regenerative properties of AECs with other sources of stem cells are particularly needed before the feasibility of AECs in bone tissue engineering can be determined. This study aimed to compare human amniotic epithelial cells (hAECs), human bone marrow mesenchymal stem cells (hBMSCs), and human amniotic fluid derived mesenchymal stem cells (hAFMSCs) in terms of their morphology, proliferation, immunophenotype profile, and osteogenic capacity in vitro and in vivo. Not only greatly distinguished by cell morphology and proliferation, hAECs, hAFMSCs, and hBMSCs exhibited remarkably different signature regarding immunophenotypical profile. Microarray analysis revealed a different expression profile of genes involved in ossification along the three cell sources, highlighting the impact of different anatomical origin and molecular response to osteogenic induction on the final tissue-forming potential. Furthermore, our data indicated a potential role of FOXC2 in early osteogenic commitment.

Figures

Figure 1

hAECs show a different phenotype compared to hBMSCs and hAFMSCs. (a) hAECs exhibited an epithelial-like morphology under the light microscope and scanning electron microscope, while both hAFMSCs and hBMSCs showed a spindle-shaped fibroblast morphology. (b) CCK-8 results at days 0, 2, 4, 6, 8, 10, and 12 after cell seeding revealed a significant higher proliferation activity of hAECs at day 8, 10, and 12 compared to the other two cells (∗: P < 0.05). Flow cytometry analysis for the basic surface makers demonstrated that all cell sources were positive for the MSC markers CD44, CD90, CD105, and lacked the expression of hematopoietic makers CD45, CD34. Moreover, hAECs expressed a higher level of CD326 and SSEA4, while hBMSCs barely expressed these markers. (c) After culturing with or without 10 ng/mL IFN-γ for 5 days, expression patterns of immunologic markers in all cell types are presented as heat maps of the percentage of cells in the total population expressing the marker (see color legend). (d) Flow cytometry results for HLA-DR, HLA-G, HLA-E, PD-L1, PD-L2, and Fas were selectively shown. Nonspecific fluorescence was gated by using respective isotype-matched monoclonal-antibody controls.

Figure 2

hAECs show a confirmed though relative lower osteoblastic capacity in vitro. (a, b) Progressively increased cellular ALP and ARS staining after osteogenic induction were observed in all cell types. (c) Immunofluorescence labeling of Runx2 (FITC, green), OPN (Cy3, red), and Nucleus (DAPI, blue) in hAECs, hBMSCs, and hAFMSCs following the 10-day osteogenic induction exhibited a more intense fluorescence of OPN and clear nuclei-localization of RUNX2 (small images at the corner of the merged images indicated the merged images of control group). (d) Further semiquantification of ALP activity and extracellular mineralization showed the lowest cellular ALP activity and mineral producing efficiency in hAECs group while hAFMSCs exhibited the highest level of extracellular mineralization (c; ∗: P < 0.05).

Figure 3

hAECs, hBMSCs, and hAFMSCs show a different molecular response to osteogenic induction in vitro. (a) Hierarchical cluster analysis of the differentially expressed genes involved in ossification. (b) Protein-protein interaction network analysis of the genes upregulated in hAECs, hBMSCs, and hAFMSCs after the 7-day osteogenic induction. (c) RUNX2, OSX, COLI, ALP, OPN, BMP6, FOXO1, and FOXC1 in osteogenic groups were gradually upregulated with time compared to those in the control group, while BMP4 and FOXC2 were only significantly upregulated in hAECs and hAFMSCs but not hBMSCs (∗: P < 0.05).

Figure 4

Verification of the upregulation of FOXC2 and ectopic osteogenesis of hAECs, hBMSCs, and hAFMSCs. (a) Expression of FOXC2 in all three cell types was significantly increased in a BMP2-dependent manner. (b, c) Both real-time PCR and western blot revealed that BMP2 significantly promoted the expression of FOXC2 in all the three cell sources. The western blot study also demonstrated that undifferentiated hBMSCs exhibited a higher expression level of FOXC2 than the other two cell sources. (d) Ectopic osteogenesis of hAECs, hBMSCs, and hAFMSCs in nude mice. β-TCP scaffolds carrying hAECs, hAFMSCs, and hBMSCs or alone were implanted subcutaneously for 4 weeks. HE staining showed no well-mineralized islands in either experimental groups or control group. Immunohistochemical staining showed that all cell types were viable as indicated by the positive expression of GFP. Moreover, OPN and OCN were evident in the experimental groups but not in the control group.