Publication: Transcriptional profiling of shed-epithelial differentiation in co-culture model
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Date
2025-02
Authors
Hashim, Siti Nurnasihah Md
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Abstract
Regenerating epithelial tissue through stem cell differentiation gives new hope for wound healing. However, stem cell’s origin and potency need to be considered to achieve high efficiency epithelial differentiation, as it is a crucial step in wound healing. In this study, ectodermal stem cells, known as stem cells from human exfoliated deciduous teeth (SHED), were used because they arose from a similar origin to the skin. The aim of this study was to investigate the transcriptional profiling of SHED-epithelial differentiation in a co-culture model. Here, SHED were grown in the epithelial-like microenvironment along with human epidermal keratinocytes (HEK001) cultured in the induction medium, keratinocyte serum free medium (KSFM), to promote epithelial differentiation. SHED cultured in the basal medium, minimum essential medium alpha (α-MEM) (SA) and KSFM (SK), and HEK001 cultured in KSFM (HK) were used as controls. The treatment consisted of co-cultured SHED cultured in the KSFM (CC SK) on days 1, 3, 5, 7, 10, and 14. The epithelial gene expression markers were screened using reverse transcriptase-polymerase chain reaction (RT-PCR). The subsequent experiment used microarray gene expression, subjected to bioinformatics analysis. Then, microarray validation was done using reverse transcriptase-qualitative real-time PCR (RT-qPCR). The bioinformatics analysis suggested that the TGFβ signalling pathway is involved in SHED-epithelium differentiation. Therefore, a downstream TGFβ pathway inhibition study was conducted using A83-01 (1 μM) to inhibit TGFβR1 that blocks SMAD2/3 activity.
The inhibitory effects were analysed using RT-qPCR, flow cytometry, and immunocytochemistry. RT-PCR analysis showed that the genes involved in the early epithelial formation like, CK18 and AQP3, were expressed in SHED with or without co-cultured. Notably, only ΔNp63 were expressed in co-cultured SHED. Bioinformatics analysis has determined that SUSD2, CXCL1, and GGT5 were found to be highly regulated differentially expressed genes (DEGs). The overall result of the bioinformatics analysis suggested that the DEGs on days 5 and 7 were mainly associated with cell cycle activity and epithelial differentiation. In addition, the high enrichment of the DEGs related to the TGFβ signalling pathway marked its involvement in regulating SHED-epithelial differentiation. The gene expression analysis through RT-qPCR confirmed the involvement of the TGFβ pathway in regulating the process due to the high expression of markers associated with this pathway, TGFβ3 and BMP8A. Captivatingly, the result demonstrated that the TGFβ inhibition by A83-01 inhibitor regulated glandular epithelial-like differentiation in the co-cultured SHED, which was supported by the morphological change and expression of gene markers associated with glandular epithelial cells, such as SUSD2, GGT5, PPARG, and COL6A2, and protein marker, such as Muc 1. This study proved the regulatory role of the TGFβ pathway in the SHED early differentiation into epithelial-like cells. Nevertheless, the TGFβ inhibition by A83-01 has been suggested could manipulate this pathway to regulate glandular epithelial-like cells. Our co-cultured SHED model demonstrated epithelial differentiation, which holds promise in future studies for application in epithelial tissue regeneration, especially in wound healing.