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Hyaluronic Acid Induction Promotes the Differentiation of Human Neural Crest-like Cells into Periodontal Ligament Stem-like Cells

ABSTRACT: Periodontal ligament (PDL) stem-like cells (PDLSCs) are considered to be promising for the regeneration of periodontium because they demonstrate multipotency, high proliferative capacity, and potential to regenerate bone, cementum, and PDL tissue. However, the transplantation of autologous PDLSCs is restricted by limited availability and the need for tooth extraction to isolate these cells. PDLSCs are derived from neural crest cells (NCs) in early vertebrate development and NCs persist in adult PDL tissue. Therefore, we devised to promote the regeneration of periodontium by activating NCs to differentiate into PDLSCs and increasing their total number. SK-N-SH cells cultured on the extracellular matrix derived of human PDL cells (SK-PDLSCs) strongly expressed PDL-related marker genes and highly differentiated into mesenchymal lineage cells compared to untreated SK-N-SH cells. Expression levels of various hyaluronic acid (HA)-related genes were upregulated in induced pluripotent stem cells (iPSCs)-derived PDLSCs and SK-PDLSCs compared to iPSCs-derived NCs and SK-N-SH cells, respectively. Magnetic-activated cell sorting-assisted CD44-positive enrichment population of SK-N-SH cells showed higher expression of PDL-related marker genes after SK-PDLSCs induction than the CD44-negative population. Knockdown of CD44 in SK-N-SH cells significantly inhibited their ability to differentiate into SK-PDLSCs, while lower molecular HA (LHA) induction enhanced SK-PDLSC differentiation. LHA-containing electrospun nanofibrous membranes also promoted their SK-PDLSC differentiation. Our findings suggest that SK-N-SH cells are applied as a new model to induce the differentiation of NCs into PDLSCs. The LHA-CD44 relation is important for the differentiation of NCs into PDLSCs. LHA-containing electrospun nanofibrous membranes would promote the regeneration of periodontium by activating NCs in PDL tissue and increasing the total number of PDLSCs.

ORGANISM(S): Homo sapiens

PROVIDER: GSE235864 | GEO | 2023/06/27

REPOSITORIES: GEO

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Project description:Background: SH-SY5Y cells exhibit a neuronal phenotype when treated with all-trans retinoic acid (RA), but the molecular mechanism of activation in the signaling pathway mediated by phosphatidylinositol 3-kinase (PI3K) is not sufficiently understood. To shed new light on the mechanism, we comprehensively compared the gene expression profiles between SK-N-SH cells and two subtypes of SH-SY5Y cells (SH-SY5Y-A and SH-SY5Y-E), each of which showed a different phenotype during RA-mediated differentiation. Results: SH-SY5Y-A cells differentiated in the presence of RA, whereas RA-treated SH-SY5Y-E cells required additional treatment with brain-derived neurotrophic factor (BDNF) for full differentiation. In combination with perturbation using a PI3K inhibitor, LY294002, we identified 386 genes and categorized them into two clusters dependent on the PI3K signaling pathway during RA-mediated differentiation in SH-SY5Y-A cells. Transcriptional regulation of the gene cluster was greatly reduced in SK-N-SH cells or partially impaired in SH-SY5Y-E cells in coincidence with a defect in the neuronal phenotype of these cell lines. Additional stimulation with BDNF induced a set of neural genes which were down-regulated in RA-treated SH-SY5Y-E cells but were abundant in the differentiated SH-SY5Y-A cells. Conclusions: We identified the gene clusters controlled by PI3K- and TRKB-mediated signaling pathways during differentiation in two subtypes of SH-SY5Y cells. TRKB-mediated bypass pathway compensates for the impaired neural functions generated by defects in several signaling pathways including PI3K in SH-SY5Y-E cells. The expression profiling data are useful for further studies to elucidate the signal transduction-transcriptional network including PI3K and/or TRKB. Experiment Overall Design: Human neuroblastomas, SK-N-SH (HTB-11) and SH-SY5Y-A cells (CRL-2266) were obtained from the American Type Culture Collection (ATCC). We also obtained SH-SY5Y-E cells (EC94030304) from the European Collection of Cell Cultures (ECACC). Tissue culture cells were maintained in D-MEM/F12 1:1 mixture supplemented with 15% FBS (Fetal Bovine Serum) and 1% NEAA (Non-essential amino acid) in a 5% CO2 humidified incubator at 37oC. The culture medium was changed twice a week. For the RA-inducible experiment, random culture cells from two clone subtypes of SH-SY5Y and SK-N-SH were seeded in laminin coated culture dishes (BioCoat Laminin Cellware; BD Biosciences, Billerica, MA, USA) for 1 day and then transferred to a medium containing 10 μM of RA in the presence or the absence of LY294002 (10μM) for five days. For BDNF-induced sequential differentiation of the SH-SY5Y-E strain, cells were washed with D-MEM/F12 twice after five days in the presence of RA and then incubated with 50 ng/ml of BDNF in D-MEM/F12 without serum for three days.

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Hyaluronic Acid Induction Promotes the Differentiation of Human Neural Crest-like Cells into Periodontal Ligament Stem-like Cells

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