Thanaphum Osathanon

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 signalling inhibits the adipogenic differentiation of single-cell-derived mesenchymal stem cell clones isolated from human adipose tissue.

ADSCs (adipose‐derived mesenchymal stem cells) are candidate adult stem cells for regenerative medicine. Notch signalling participates in the differentiation of a heterogeneous ADSC population. We have isolated, human adipose tissue‐derived single‐cell clones using a cloning ring technique and characterized for their stem cell characteristics. The role of Notch signalling in the differentiation capacity of these adipose‐derived single‐cell‐clones has also been investigated. All 14 clones expressed embryonic and mesenchymal stem cell marker genes. These clones could differentiate into both osteogenic and adipogenic lineages. However, the differentiation potential of each clone was different. Low adipogenic clones had significantly higher mRNA expression levels of Notch 2, 3 and 4, Jagged1, as well as Delta1, compared with those of high adipogenic clones. In contrast, no changes in expression of Notch signalling component mRNA between low and high osteogenic clones was found. Notch receptor mRNA expression decreased with the adipogenic differentiation of both low and high adipogenic clones. The γ‐secretase inhibitor, DAPT (N‐[N‐(3,5‐difluorophenacetyl)‐l‐alanyl]‐(S)‐phenylglycine t‐butyl ester), enhanced adipogenic differentiation. Correspondingly, cells seeded on a Notch ligand (Jagged1) bound surface showed lower intracellular lipid accumulation. These results were noted in both low and high adipogenic clones, indicating that Notch signalling inhibited the adipogenic differentiation of adipose ADSC clones, and could be used to identify an adipogenic susceptible subpopulation for soft‐tissue augmentation application.

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.

Cobalt chloride supplementation induces stem-cell marker expression and inhibits osteoblastic differentiation in human periodontal ligament cells

OBJECTIVE Low oxygen tension is one of the crucial factors of the stem-cell niche. However, the long-term hypoxic culture of stem cells is difficult and requires special equipment. In this study, we investigated whether mimicking hypoxia using cobalt chloride (CoCl2) could maintain human periodontal ligament (HPDL) cell stemness. METHODS HPDL cells were treated with either 50 or 100 μM CoCl2. Cell proliferation was determined by an MTT assay. The mRNA expression of stem-cell marker and osteogenic associated genes were analyzed by RT-PCR and Real-time PCR. Osteogenic differentiation was determined by assaying alkaline phosphatase activity and in vitro mineralization. RESULTS The results showed that the CoCl2 supplementation had no effect on cell proliferation. CoCl2 treatment increased the mRNA expression of the embryonic stem-cell markers REX1 and OCT4. Culturing HDPL cells in osteogenic medium containing CoCl2 resulted in a decrease in alkaline phosphatase activity, down-regulation of osteogenic associated gene expression, and suppression of mineralization. The use of Apigenin, an HIF-1α inhibitor, indicated that CoCl2 might inhibit osteogenic differentiation through an HIF-1α- dependent mechanism. CONCLUSION This study shows that CoCl2 treatment can induce stem-cell marker expression and inhibit the osteoblastic differentiation of HPDL cells. These findings suggest the potential application of CoCl2 for maintaining the stem-cell state in the laboratory.

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.