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. 2022 Mar 4;23(5):2830.
doi: 10.3390/ijms23052830.

Human Amnion Epithelial Cells: A Potential Cell Source for Pulp Regeneration?

Cristina Bucchi  1 , Ella Ohlsson  2 , Josep Maria de Anta  3 , Melanie Woelflick  2 , Kerstin Galler  4 , María Cristina Manzanares-Cespedes  3 , Matthias Widbiller  2
Affiliations

Human Amnion Epithelial Cells: A Potential Cell Source for Pulp Regeneration?

Cristina Bucchi et al. Int J Mol Sci. .

Abstract

The aim of this study was to analyze the suitability of pluripotent stem cells derived from the amnion (hAECs) as a potential cell source for revitalization in vitro. hAECs were isolated from human placentas, and dental pulp stem cells (hDPSCs) and dentin matrix proteins (eDMPs) were obtained from human teeth. Both hAECs and hDPSCs were cultured with 10% FBS, eDMPs and an osteogenic differentiation medium (StemPro). Viability was assessed by MTT and cell adherence to dentin was evaluated by scanning electron microscopy. Furthermore, the expression of mineralization-, odontogenic differentiation- and epithelial-mesenchymal transition-associated genes was analyzed by quantitative real-time PCR, and mineralization was evaluated through Alizarin Red staining. The viability of hAECs was significantly lower compared with hDPSCs in all groups and at all time points. Both hAECs and hDPSCs adhered to dentin and were homogeneously distributed. The regulation of odontoblast differentiation- and mineralization-associated genes showed the lack of transition of hAECs into an odontoblastic phenotype; however, genes associated with epithelial-mesenchymal transition were significantly upregulated in hAECs. hAECs showed small amounts of calcium deposition after osteogenic differentiation with StemPro. Pluripotent hAECs adhere on dentin and possess the capacity to mineralize. However, they presented an unfavorable proliferation behavior and failed to undergo odontoblastic transition.

Keywords: dental pulp stem cells; dentin matrix proteins; human amnion epithelial cells; odontoblastic differentiation; revitalization.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Amnion staining and expression profile of hAECs. Amnion before digestion (A) and after the first (B) and second digestion (C). The hAECs were attached to a collagen membrane forming a monolayer of columnar/cuboidal cells (hematoxylin and eosin; scale bars: 100 µm). Expression profile of hAECs determined by flow cytometry analysis (D). The hAECs in culture expressed both mesenchymal markers (CD44 and CD105) as well as epithelial markers (CD49f and CD326) (D,E).
Figure 2
Figure 2
Viability and morphology of hAECs and hDPSCs. Cell viability of hAECs and hDPSCs cultured with eDMP and StemPro after 2, 4 and 8 days (A). Median values and 25–75% percentiles were calculated from three independent experiments performed in triplicate (n = 9). Fluorescence microscopy of hAECs and hDPSCs cultured with different media after 7 days and stained with DAPI and phalloidin (B–G). Cells were cultured in DMEM with 10% FBS (B,E), with eDMP (C,F) and with StemPro (D,G). hAECs exhibit a cobblestone-like morphology (B–D) while hDPSCs exhibit a mesenchymal stem cell phenotype (E–G). (Scale bars: 50 µm).
Figure 3
Figure 3
Adhesion of hDPSC and hAECs onto dentin surface. Representative SEM images of dentin surface with hDPSC (A,B) and hAECs (C,D) after 48 h (cells marked by asterisks). Cell adhesion and spreading on the surface of dentin was evident with (B,D) and without (A,C) EDTA conditioning. Some cytoplasmic processes (arrowheads) were evident in both cell types. (Scale bars: 20 µm).
Figure 4
Figure 4
Expression of odontogenic and mineralization-associated genes. Effect of eDMPs and StemPro on expression of odontogenic and mineralization-associated marker genes (COL1A1, BMP4, IBSP, IGFBP-2, NES, TGFB1 and BGLAP) in hAECs and hDPSCs at days 1, 7 and 14 (A). Genes indicative of epithelial–mesenchymal transition (S100A4) and protection against oxidative damage (GPX3) are also depicted (A). Target gene expressions are depicted relative to the untreated control (hDPSCs with 10% FBS at day 1) and median values were calculated from two independent experiments in duplicated samples (n = 4). Non-significant differences between hAECs and hDPSCs for each medium and follow-up point are marked with lowercase letters (a, b, c). The effect of eDMPs and StemPro on mineralization of hAECs (B–D) and hDPSCs (E–G) using Alizarin Red staining assay. Calcium deposits were evident in hAECs cultured with StemPro (D) and hDPCS cultured with eDMPs (F) and StemPro (G). (Scale bars: 50 µm).
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