Nunthawan Nowwarote

Basic fibroblast growth factor inhibits mineralization but induces neuronal differentiation by human dental pulp stem cells through a FGFR and PLCγ signaling pathway

Basic fibroblast growth factor (basic FGF) has pivotal roles in the function of various cell types. Here, we report the effects of basic FGF in the regulation of dental pulp stem cell (DPSC) behaviors including maintaining stemness and directing differentiation. Cells isolated from human dental pulp tissues exhibited stem cell properties including the expression of mRNA markers for embryonic and mesenchymal stem cells, the expression of Stro‐1, and the multipotential differentiation. Basic FGF stimulated colony‐forming units of DPSCs and up‐regulated the expression of the embryonic stem cell markers; Oct4, Rex‐1, and Nanog. Moreover, osteogenic medium containing basic FGF inhibited alkaline phosphatase enzymatic activity and mineralization of DPSCs. On the contrary, basic FGF appeared to be an influential growth factor in the neurogenic differentiation of DPSCs. In the presence of basic FGF, increased DPSCs neurosphere size and the up‐regulation of neurogenic markers were noted. Inhibitors of FGFR or PLCγ were able to ablate the basic FGF‐induced neuronal differentiation of DPSCs. Taken together, these results suggest basic FGF may be involved in the mechanisms controlling DPSCs cell fate decisions. J. Cell. Biochem. 112: 1807–1816, 2011.

Surface‐bound orientated Jagged‐1 enhances osteogenic differentiation of human periodontal ligament‐derived mesenchymal stem cells

Notch signaling plays critical roles in various cell types by regulating cell fate determination and differentiation. Here, we investigated the ability to control differentiation of human periodontal ligament derived mesenchymal stem cells using modified surfaces containing the affinity immobilized Notch ligand, Jagged-1. After seeding human periodontal ligament derived mesenchymal stem cells (HPDLs) on Jagged-1 modified surfaces, expression of Notch signaling target genes, Hes-1 and Hey-1, was higher than those exposed to soluble Jagged-1 or control surfaces. Upregulation of Notch signaling target genes was attenuated after treatment with the γ secretase inhibitor. Upon seeding the cells on Jagged-1 immobilized surface and maintained in osteogenic medium, alkaline phosphatase enzymatic activity and mineralization as well as mRNA expression of alkaline phosphatase (ALP), collagen type I (COL I) and osteopontin (OPN) were significantly increased compared to those of controls. However, osteocalcin (OCN) mRNA expression level was decreased when cells were exposed to Jagged-1 modified surfaces. HPDLs on Jagged-1 modified surfaces expressed lower TWIST2 mRNA levels than the control, suggesting that the mechanism whereby Jagged-1 enhances osteogenic differentiation of HPDLs may occur through Notch signaling and TWIST regulation. In summary, an alteration of biomaterial interface using Notch ligands illustrates a promising system to control HPDLs differentiation toward osteogenic lineage.

Neurogenic differentiation of human dental pulp stem cells using different induction protocols

OBJECTIVE An investigation on neuronal differentiation capacity of human dental pulp stem cells (DPSCs) was still lacking. In this study, two different neuronal induction protocols were investigated and compared. METHODS The neuronal differentiation was induced using chemical or growth factor induction protocol. The differentiation was confirmed by the neurogenic mRNA and protein expression using polymerase chain reaction and immunocytochemistry, respectively. RESULTS Chemical-induced neuronal differentiation protocol promoted morphological change and β3-TUBULIN protein expression. Though, SOX2, SOX9, and β3-TUBULIN mRNA levels were not different compared with the control, indicating a defective differentiation. For growth factor induction protocol, the cells were exhibited neurite-like cellular process and positively stained with β3-TUBULIN. In addition, the increase in intracellular calcium was noted upon NMDA stimulation, implying the neuronal function. A dramatic increased mRNA expression of neurogenic markers [SOX2, SOX9, β3-TUBULIN, and gamma-aminobutyric acid (GABA receptors)] was noted as compared to the control. In addition, a remarkable increased expression of Notch signaling target gene, HEY1, was observed in growth factor-induced DPSCs derived neuronal-like cells compared with the control. CONCLUSION These data indicate that growth factor induction method is a preferable protocol for neuronal differentiation by DPSCs.

Notch Signaling Is Involved in Neurogenic Commitment of Human Periodontal Ligament-Derived Mesenchymal Stem Cells

Notch signaling plays critical roles in stem cells by regulating cell fate determination and differentiation. The aim of this study was to evaluate the participation of Notch signaling in neurogenic commitment of human periodontal ligament-derived mesenchymal stem cells (hPDLSCs) and to examine the ability to control differentiation of these cells using modified surfaces containing affinity immobilized Notch ligands. Neurogenic induction of hPDLSCs was performed via neurosphere formation. Cells were aggregated and form spheres as early 1 day in culture. In addition, the induced cells exhibited increased mRNA and protein expression of neuronal markers that is, β3-tubulin and neurofilament. During neuronal differentiation, a significant increase of Hes1 and Hey1 mRNA expression was noted. Using pharmacological inhibition (γ-secretase inhibitor) or genetic manipulation (overexpression of dominant negative mastermind-like transcription co-activators), neurosphere formation was attenuated and a marked decrease in neurogenic mRNA expression was observed. To confirm the role of Notch signaling in neuronal differentiation of hPDLSCs, the Notch ligand, Jagged-1, is bound to the surface using an affinity immobilization technique. The hPDLSC cultured on a Jagged-1-modified surface had increased expression of Notch signaling target genes, Hes-1 and Hey-1, confirming the activity and potency of surface-bound Jagged-1. Further, hPDLSC on surface-bound Jagged-1 under serum-free conditions showed multiple long and thin neurite-like extensions, and an increase in the expression of neurogenic mRNA markers was observed. Pretreatment of the cells with γ-secretase inhibitor, DAPT, before seeding on the Jagged-1-modified surface blocked development of the neurite-like morphology. Together, the results in this study suggest the involvement of Notch signaling in neurogenic commitment of hPDLSCs.

bFGF and JAGGED1 regulate alkaline phosphatase expression and mineralization in dental tissue‐derived mesenchymal stem cells

Basic fibroblast growth factor (bFGF) and Notch signaling play critical roles in various cell behaviors. Here, we investigated the influence of bFGF and Notch signaling in alkaline phosphatase (ALP) expression and mineralization process in human periodontal ligament‐derived mesenchymal stem cells (PDLSCs) and stem cells isolated from human exfoliated deciduous teeth (SHEDs). PDLSCs and SHEDs were cultured in osteogenic medium supplemented with bFGF or on the immobilized Notch ligands, JAGGED1. The ALP mRNA and protein expression were measured by quantitative reverse transcriptase polymerase chain reaction and enzymatic activity assay, respectively. Mineral deposition was determined using alizarin red S staining. The results showed that the addition of bFGF resulted in the decrease of ALP mRNA expression and enzymatic activity. In addition, the attenuation of mineralization was noted. These phenomenons were blocked by the addition of a fibroblast growth factor receptor inhibitor (SU5402) or a MEK inhibitor (PD98059). Interestingly, bFGF supplementation also decreased the Notch signaling component mRNA levels. Thus, to evaluate effect of Notch signaling in mineralization process, PDLSCs and SHEDs were exposed to JAGGED1 modified surface. The ALP mRNA and protein expression were significantly upregulated and the mineral deposition was markedly increased. These results could be reversed by the addition of a γ‐secretase inhibitor. In addition, bFGF could attenuate the Notch‐signaling‐induced mineralization in both PDLSCs and SHEDs. These results suggest that mineralization was enhanced by Notch signaling but attenuated by bFGF signaling. This knowledge can be further utilized to control PDLSCs and SHEDs mineralization for tissue regeneration purpose. J. Cell. Biochem. 114: 2551–2561, 2013.

The efficacy of polycaprolactone/hydroxyapatite scaffold in combination with mesenchymal stem cells for bone tissue engineering.

Major drawbacks of using an autograft are the possibilities of insufficient bony source and patients morbidity after operation. Bone tissue engineering technology, therefore, has been applied for repairing bony defects. Previous study showed that a novel fabricated 3D-Polycaprolactone/Hydroxyapatite (PCL/HAp) scaffold possessed a good biocompatibility for bone cells. This study aimed to determine the ability of PCL/HAp for supporting cell growth, gene expression, and osteogenic differentiation in three types of mesenchymal stem cells, including bone marrow-derived mesenchymal stem cells (BMSCs), dental pulp stem cells (DPSCs), and adiposed-derived mesenchymal stem cells (ADSCs). These were assessed by cell viability assay (MTT), reverse-transcription polymerase chain reaction (RT-PCR) analysis, alkaline phosphatase activity, and osteogenic differentiation by alizarin red-S staining. The results showed that PCL/HAp scaffold could support growth of all three types of mesenchymal stem cells. In addition, DPSCs with PCL/HAp showed the highest level of calcium deposition compared to other groups. In conclusion, DPSCs exhibited a better compatibility with these scaffolds compared to BMSCs and ADSCs. However, the PCL/HAp could be a good candidate scaffold for all tested mesenchymal stem cells in bone tissue engineering.

Asiaticoside Induces Type I Collagen Synthesis and Osteogenic Differentiation in Human Periodontal Ligament Cells

Asiaticoside, an active ingredient extracted from Centella asiatica, has been widely used to promote wound healing. In this study, the effects of asiaticoside on proliferation, protein synthesis, and osteogenic differentiation in human periodontal ligament cells (HPDLs) were investigated. HPDLs were treated with asiaticoside at concentrations of 25, 50, and 100 µg/mL. Cell number was determined by MTT assay. The mRNA expression was analyzed by reverse transcription‐polymerase chain reaction. Western blot analysis and immunocytochemistry were used to confirm protein synthesis. Osteogenic differentiation was determined by alkaline phosphatase activity, osteoblast marker gene expression, and in vitro mineralization. The results showed that asiaticoside treatment, ranging from 25 to 100 mg/mL, had no effect on cytotoxicity or cell proliferation. When HPDLs were treated with asiaticoside in serum‐free medium, dose‐dependent increases in the levels of fibronectin and collagen type I mRNA and protein were observed at 72 h. Moreover, asiaticoside attenuated matrix metalloproteinase‐1 but enhanced tissue inhibitor of metalloproteinase‐1 mRNA expression. The addition of asiaticoside to osteogenic medium resulted in an increase in alkaline phosphatase enzymatic activity, up‐regulation of osteoblast marker gene mRNA expression, and enhancement of mineralization by HPDLs. These results suggest the potential application of asiaticoside for enhancing periodontal tissue healing. Copyright

The responses of human adipose-derived mesenchymal stem cells on polycaprolactone-based scaffolds: an in vitro study

Polycaprolactone (PCL) has been investigated as an alternative synthetic polymeric scaffold for tissue engineering application. In this study, the biological responses of human adipose-derived mesenchymal stem cells (hADSCs) on PCL-based scaffolds were investigated in vitro. The hADSCs were isolated and characterized. Solvent casting and particulate leaching method was employed as the fabrication method for PCL-based scaffolds. Here, we illustrated that the isolated hADSCs exhibited fibroblast-like morphology, formed colonies in culture, and expressed several stem cell markers. Moreover, the differentiation potency toward adipogenic, neurogenic and osteogenic lineage was noted when cultured in the specific conditions. Polycaprolactone/hydroxyapatite composite scaffold (PCL/HA) supported hADSCs attachment better than PCL scaffolds. Moreover, the alkaline phosphatase enzymatic activity and mineral deposition were greater on PCL/HA than PCL. Together, this present study illustrates the potential utilization of PCL/HA and hADSC for bone tissue engineering.

A feasibility study of an in vitro differentiation potential toward insulin-producing cells by dental tissue-derived mesenchymal stem cells

Dental tissue-derived mesenchymal stem cells have been proposed as an alternative source for mesenchymal stem cells. Here, we investigated the differentiation ability toward insulin producing cells (IPCs) of human dental pulp stem cells (hDPSCs) and human periodontal ligament stem cells (hPDLSCs). These cells expressed mesenchymal stem cell surface markers and were able to differentiate toward osteogenic and adipogenic lineages. Upon 3 step-IPCs induction, hDPSCs exhibited more colony number than hPDLSCs. The mRNA upregulation of pancreatic endoderm/islet markers was noted. However, the significant increase was noted only for PDX-1, NGN-3, and INSULIN mRNA expression of hDPSCs. The hDPSCs-derived IPCs expressed PRO-INSULIN and released C-PEPTIDE upon glucose stimulation in dose-dependent manner. After IPCs induction, the Notch target, HES-1 and HEY-1, mRNA expression was markedly noted. Notch inhibition during the last induction step or throughout the protocol disturbed the ability of C-PEPTIDE release upon glucose stimulation. The results suggested that hDPSCs had better differentiation potential toward IPCs than hPDLSCs. In addition, the Notch signalling might involve in the differentiation regulation of hDPSCs into IPCs.

Expression and influence of Notch signaling in oral squamous cell carcinoma.

Notch signaling dysregulation plays an important role in altering cancer cell behaviors; however, its role in oral squamous cell carcinoma (OSCC) remains controversial. This study aimed to investigate the role of Notch signaling related genes in human OSCC using a meta-analysis of Gene Expression Omnibus database (GEO-publicly available gene expression microarray data) and to examine the role of Notch signaling in OSCC behaviors. The meta-analysis included 13 GEO datasets and was performed by combining effect sizes in a random effect model. The results demonstrated that in OSCC dysregulated genes participated in the metabolic process and protein binding as determined by gene ontology analysis. Enriched pathway analysis demonstrated the majority of the dysregulated genes were involved in pathway categories as follow; pathway in cancers, small cell lung cancer, extracellular matrix-receptor interaction, focal adhesion, and cell cycle progression. Interestingly, the enriched pathway analysis also demonstrated that OSCC samples exhibited an upregulation of genes in Notch signaling pathway, namely JAG1, JAG2, ADAM17, NCSTN, PSEN1, NCOR2, NUMB, DVL3, HDAC1, and HDAC2. Furthermore, Notch signaling inhibition by a γ-secretase inhibitor significantly decreased OSCC cell proliferation in vitro, corresponding with a decrease in C-FOS mRNA expression. The study demonstrated that Notch signaling is dysregulated in human OSCC and plays a role in cell proliferation. (J Oral Sci 58, 283-294, 2016).