Effects of noni on cellular viability and osteogenic differentiation of gingiva-derived stem cells demonstrated by RNA sequencing and quantitative PCR.

Noni fruit (Morinda citrifolia) has been widely used in traditional medicine across tropical and subtropical regions, and is now being paid more attention in Western medicine. The present study aimed to investigate the effects of noni extract on the change in the cellular morphology, maintenance of cellular viability and enhancement of osteogenic differentiation of stem cells. Stem cells obtained from gingiva were cultured where noni extracts existed at concentrations ranging from 10-200 ng/ml. Evaluations of cell morphology and cellular viability were performed. Alkaline phosphatase activity assays were performed to assess the osteogenic differentiation. Alizarin Red S staining was performed to evaluate the calcium deposits in the culture, with the addition of noni extract. Global gene expression was analyzed via next-generation mRNA sequencing. Gene ontology and pathway analyses were performed to determine the associated mechanisms. Validation procedures were performed via quantitative (q)PCR analysis. The addition of noni at concentrations ranging from 10-200 ng/ml did not produce significant morphological changes. There were significantly higher values of cellular viability, with the highest value at 100 ng/ml compared with the control (P<0.05). Furthermore, significantly higher values of alkaline phosphatase activity was noted in the 10 and 100 ng/ml groups compared with the 0 ng/ml group on day 7 (P<0.05). Alizarin Red S staining revealed calcium deposits in each group. In addition, the highest value for Alizarin Red S staining was observed at 100 ng/ml compared with the unloaded control (P<0.05). qPCR analysis demonstrated that the mRNA expression levels of RUNX2, BSP, OCN and COL1A1 increased following treatment with noni. Taken together, the results of the present study suggest that noni extract has enhancing effects on gingiva-derived mesenchymal stem cells, by enhancing cellular viability and osteogenic differentiation.

NELL-1 Increased the Osteogenic Differentiation and mRNA Expression of Spheroids Composed of Stem Cells.

Background and objectives: NELL-1 is a competent growth factor and it reported to target cells committed to the osteochondral lineage. The secreted, osteoinductive glycoproteins are reported to rheostatically control skeletal ossification. This study was performed to determine the effects of NELL-1 on spheroid morphology and cell viability and the promotion of osteogenic differentiation of stem cell spheroids. Materials and Methods: Cultures of stem cell spheroids of gingiva-derived stem cells were grown in the presence of NELL-1 at concentrations of 1, 10, 100, and 500 ng/mL. Evaluations of cell morphology were performed using a microscope, and cell viability was assessed using a two-color assay and Cell Counting Kit-8. Evaluation of the activity of alkaline phosphatase and calcium deposition assays involved anthraquinone dye assay to determine the level of osteogenic differentiation of cell spheroids treated with NELL-1. Real-time quantitative polymerase chain reaction (qPCR) was used to evaluate the expressions of RUNX2, BSP, OCN, COL1A1, and ß-actin mRNAs. Results: The applied stem cells produced well-formed spheroids, and the addition of NELL-1 at tested concentrations did not show any apparent changes in spheroid shape. There were no significant changes in diameter with addition of NELL-1 at 0, 1, 10, 100, and 500 ng/mL concentrations. The quantitative cell viability results derived on Days 1, 3, and 7 did not show significant disparities among groups (p > 0.05). There was statistically higher alkaline phosphatase activity in the 10 ng/mL group compared with the unloaded control on Day 7 (p < 0.05). A significant increase in anthraquinone dye staining was observed with the addition of NELL-1, and the highest value was noted at 10 ng/mL (p < 0.05). qPCR results demonstrated that the mRNA expression levels of RUNX2 and BSP were significantly increased when NELL-1 was added to the culture. Conclusions: Based on these findings, we conclude that NELL-1 can be applied for increased osteogenic differentiation of stem cell spheroids.

Insulin-like growth factor 2-enhanced osteogenic differentiation of stem cell spheroids by regulation of Runx2 and Col1 expression.

Insulin-like growth factor 2 (IGF-2) is a growth factor that is involved in various functions of cells, including stem cells. The effects of IGF-2 on the cellular viability and osteogenic differentiation of stem cell spheroids were investigated in the present study. Stem cell spheroids were formed using concave microwells in the presence of IGF-2 at final concentrations of 0, 10 and 100 ng/ml. Cellular viability was measured qualitatively using a microscope and quantitatively using an assay kit based on water-soluble tetrazolium salt. The level of alkaline phosphatase activity, and an anthraquinone dye assay for calcium deposit evaluation, were used to assess osteogenic differentiation. A quantitative PCR analysis was conducted to evaluate the expression of Runx2 and Col1. Spheroid formation was noticed on day 1 in the microwells, and the spheroidal shape was maintained up to day 7. The cell viability assay values for IGF-2 at 0, 10 and 100 ng/ml at day 1 were 0.193±0.002, 0.191±0.002 and 0.201±0.006, respectively (P>0.05). The absorbance values at 405 nm for the alkaline phosphatase activity assays on day 21 were 0.221±0.006, 0.375±0.010 and 0.280±0.015 for IGF-2 at 0, 10 and 100 ng/ml, respectively. There were significantly higher values for IGF-2 in the 10 and 100 ng/ml groups when compared with the control (P<0.05). Significantly higher Alizarin red staining was noted for IGF-2 in the 10 ng/ml group when compared with the unloaded control at day 21 (P<0.05). Quantitative PCR revealed that mRNA levels of Runx2 and Col1 were significantly higher at 100 ng/ml on day 7. Conclusively, the present study demonstrated that the application of IGF-2 increased alkaline phosphatase activity, Alizarin red staining, and Runx2 and Col1 expression of stem cell spheroids.

Effects of platelet-derived growth factor-BB on cellular morphology and cellular viability of stem cell spheroids composed of bone-marrow-derived stem cells.

Platelet-derived growth factor-BB (PDGF-BB) is a potent mitogenic, angiogenic and chemoattractant, and is one of the most abundant growth factors in platelet-derived products. The goal of the present study was to examine the effects of PDGF-BB on cellular morphology and cellular viability using 3D stem cell cultures. On day 1, spheroids formed well in silicon-elastomer-based concave microwells. The addition of 10 or 100 ng/ml PDGF-BB did not affect the morphology of the cell spheroids. During longer periods of incubation, the cell spheroids maintained their shape without noticeable alterations. The majority of cells in the spheroids exhibited green fluorescence when analyzed using a live/dead assay, indicative of live cells. On day 1, the Cell Counting Kit-8 (CCK-8) assay values for PDGF-BB at 0, 10 and 100 ng/ml were 0.241±0.003, 0.227±0.001 and 0.241±0.004, respectively; on day 3, the CCK-8 assay values for PDGF-BB were 0.233±0.005, 0.278±0.001 and 0.194±0.003, respectively; and on day 7, they were 0.248±0.014, 0.293±0.031 and 0.346±0.034, respectively. The 100 ng/ml group showed significantly higher values compared with the control group on day 7. Together, the results of the present study showed that the addition of 10 and 100 ng/ml PDGF-BB increased cellular viability, suggesting that PDGF-BB may be usable in cell therapy.

Bone morphogenetic protein 2-enhanced osteogenic differentiation of stem cell spheres by regulation of Runx2 expression.

Bone morphogenetic protein 2 (BMP-2) is a growth factor that is used to induce osteogenic differentiation in stem cells. The present study assessed the effects of BMP-2 on stem cell spheroid morphology, viability and osteogenic differentiation. Stem cell spheres were constructed and treated with BMP-2 at predetermined concentrations (0-100 ng/ml) using concave microwells. Cell viability was qualitatively and quantitatively analyzed via microscopy and a water-soluble tetrazolium salt assay kit, respectively. Alkaline phosphatase activity was assessed and an anthraquinone dye for calcium deposit evaluation was performed to determine osteogenic differentiation. The expressions of (runt-related transcription factor 2) and collagen 1 were also determined via quantitative PCR. Spherical shapes were formed using concave microwells on day 1, which were maintained up to day 21. On day 1, the relative cell viability of 0, 10 and 100 ng/ml BMP-2 treated cells was 100.0±1.9, 97.3±4.4 and 101.3±2.6%, respectively. Significantly higher values for alkaline phosphatase activity were determined in the 100 ng/ml treated group when compared with the control group. Additionally, Runx2 mRNA levels were significantly higher in the 100 ng/ml BMP-2 group compared with the control group, as determined via quantitative PCR. The results of the present study indicated that BMP-2 enhanced the differentiation of stem cell spheres, which was demonstrated by increased alkaline phosphatase activity and Runx2 expression.

The Effects of Morinda citrifolia (Noni) on the Cellular Viability and Osteogenesis of Stem Cell Spheroids.

Background and objectives: Morinda citrifolia (Noni) has been widely used in herbal remedies to treat and prevent various kinds of diseases. We conducted this study to evaluate the effects of Noni extract on the maintenance of morphology, the improvement of cellular viability, and the enhancement of osteogenesis of stem cell spheroids. Materials and Methods: We cultured stem cell spheroids made with gingiva-derived stem cells in the presence of Noni extract at concentrations of 10, 100 and 200 ng/mL. We performed analysis of the cell morphology and changes in the cellular viability. We conducted alkaline phosphatase activity assays using a kit, and mineralization assays using an anthraquinone dye to evaluate the osteogenesis of stem cell spheroids with the addition of Noni extract. Results: The applied cells formed spheroids well, and the addition of Noni at 10, 100 and 200 ng/mL concentrations did not produce significant morphological changes. The quantitative values for cellular viability on Day 3 showed that the absorbance values at 450 nm were 0.314 ± 0.013, 0.318 ± 0.008, 0.304 ± 0.000 and 0.300 ± 0.011 for Noni at 0, 10, 100 and 200 ng/mL concentrations, respectively. The results of alkaline phosphatase activity with absorbance values at 405 nm were 0.189 ± 0.019, 0.174 ± 0.023, 0.192 ± 0.014 and 0.210 ± 0.062 for Noni at 0, 10, 100 and 200 ng/mL concentrations, respectively, on Day 4. There were significantly higher values of Alizarin Red S staining for Noni in the 10, 100 and 200 ng/mL groups, with the highest value at 100 ng/mL when compared with the unloaded control on Day 14. Conclusions: Based on these findings, we concluded that Noni extract might be applied for the enhanced osteogenic differentiation of stem cell spheroids.

Fibroblast growth factor-4 maintains cellular viability while enhancing osteogenic differentiation of stem cell spheroids in part by regulating RUNX2 and BGLAP expression.

Fibroblast growth factors (FGFs) are growth factors that were initially identified as proteins that stimulate fibroblast proliferation. The aim of the present study was to examine the effects of FGF-4 on the morphology, cellular viability and osteogenic differentiation of stem cell spheroids. Stem cell spheroids were generated using concave microwells in the presence of FGF-4 at concentrations of 0, 50, 100 and 200 ng/ml. Cellular viability was qualitatively assessed by a fluorometric live/dead assay using a microscope and quantitatively determined by using Cell Counting Kit-8. Furthermore, alkaline phosphatase activity and calcium deposition were determined to assess osteogenic differentiation. Reverse transcription-quantitative PCR (RT-qPCR) was performed to evaluate the mRNA expression levels of Runt-related transcription factor 2 (RUNX2) and bone γ-carboxyglutamate protein (BGLAP). Spheroidal shapes were achieved in the microwells on day 1 and a significant increase in the spheroid diameter was observed in the 200 ng/ml FGF-4 group compared with the control group on day 1 (P<0.05). The results regarding viability using Cell Counting Kit-8 in the presence of FGF-4 at 50, 100 and 200 ng/ml at day 1 were 98.0±2.5, 106.2±17.6 and 99.5±6.0%, respectively, when normalized to the control group (P>0.05). Furthermore, the alkaline phosphatase activity was significantly elevated in the 200 ng/ml group, when compared with the control group. The RT-qPCR results demonstrated that the mRNA expression levels of RUNX2 and BGLAP were significantly increased at 200 ng/ml. Therefore, the present results suggested that the application of FGF-4 maintained cellular viability while enhancing the osteogenic differentiation of stem cell spheroids, at least partially by regulating RUNX2 and BGLAP expression levels.

Bone morphogenetic protein-7 upregulates genes associated with osteoblast differentiation, including collagen I, Sp7 and IBSP in gingiva-derived stem cells.

The present study was performed to evaluate the effects of short-term application of bone morphogenetic protein-7 (BMP-7) on human gingiva-derived mesenchymal stem cells with next-generation sequencing. Human gingiva-derived stem cells were treated with a final concentration of 100 ng/ml BMP-7 and the same concentration of a vehicle control. mRNA sequencing and data analysis were performed along using gene ontology and pathway analysis. RT-qPCR of mRNA of collagen I, Sp7, IBSP and western blot analysis of collagen I, osterix and bone sialoprotein was also performed. A total of 25,737 mRNAs were identified to be differentially expressed. Regarding osteoblast differentiation, 14 mRNAs were upregulated and 10 were downregulated when the results of the BMP-7 at 3 h were compared with the control at 3 h. The expression of collagen I was increased following the application of BMP-7 at 3 h, and this increase was also observed following western blot analysis. The effects of BMP-7 on stem cells were evaluated with mRNA sequencing, and the expression was validated with RT-qPCR and western blot analysis. The short-term application of BMP-7 produced an increased expression of collagen I, which was associated with target genes selected for osteoblast differentiation. This study may provide novel insights into the role of BMP-7 using mRNA sequencing.

A Study of the Effects of Doxorubicin-Containing Liposomes on Osteogenesis of 3D Stem Cell Spheroids Derived from Gingiva.

The objective of the present investigation is to determine the effects of neutral, anionic, and cationic liposomes loaded with doxorubicin with thin-lipid-film-hydration method on the cellular viability and osteogenesis of stem cell spheroids. Spheroid formation and morphology of the three-dimensional spheroid were noted with an inverted microscope. Quantitative cellular viability was assessed using a commercially available kit. Osteogenic potential was evaluated by applying alkaline phosphatase activity and anthraquinone dye of Alizarin Red S. Western blot analysis was performed using collagen I expression. Spheroids were formed in each silicon elastomer-based concave microwell on Day 1. Noticeable changes of the spheroid were seen with a higher concentration of doxorubicin, especially in the cationic liposome group at Days 5 and 7. We found that the application of doxorubicin for 5 days significantly reduced the cellular viability. A higher concentration of doxorubicin produced a significant decrease in alkaline phosphatase activity. Alizarin Red S staining showed that extracellular calcium deposits were evenly noted in each group. An increase of calcium deposits was noted on Day 14 when compared to Day 7. The morphology of the groups with higher concentrations of doxorubicin showed to be more dispersed. We noticed that doxorubicin-loaded cationic liposomes resulted in the highest uptake of the examined cell spheroids and that doxorubicin-loaded liposomes affected the osteogenic differentiation. The implication of this study is that the type of liposome should be selected based on the purpose of the application.

Effects of demographic factors on adipogenic and chondrogenic differentiation in bone marrow-derived stem cells.

Stem cells have the characteristics of long-term self-renewal and plasticity and the ability to differentiate into specialized cells. Stem cells are widely recognized as potential tools for use in the development of novel therapeutic strategies. The aim of the current study was to investigate the effect of demographic factors on adipogenic and chondrogenic differentiation in bone marrow-derived stem cell (BMSC) spheroids. Age- and gender-associated alterations in the adipogenic and chondrogenic differentiation potential of BMSCs were examined. Human BMSCs were isolated from male and female participants in their 20s, 30s and 50s. Cell morphology and relative values of adipogenesis and chondrogenesis were examined by measuring the relative intensity of oil red O and Alcian blue staining, respectively. Cell morphology alterations in BMSCs isolated from male and female participants in their 20s, 30s and 50s and grown in adipogenic media were very similar. In addition, there were no significant differences in the relative values of adipogenesis in BMSCs for the 20s, 30s and 50s age groups on day 8 and 16. Similarly, no significant differences were observed in the relative values of adipogenesis in BMSCs for the male and female groups on day 8 and 16. Cell morphology changes in BMSCs isolated from male and female participants in their 20s, 30s and 50s and grown in chondrogenic media were very similar. In addition, there were no significant differences in the relative values of chondrogenesis in BMSCs for the 20s, 30s and 50s age groups on day 8, however there was a significant difference observed in the relative values of chondrogenesis in BMSCs on day 16 for the 30s and 50s age groups, compared with the 20s age group. Furthermore, no significant differences were observed in the relative values of adipogenesis in BMSCs for the male and female groups on day 8 and 16. The current study demonstrated that there were no significant differences in the adipogenic and chondrogenic differentiation potential of BMSCs isolated from healthy male donors vs. healthy female donors. Similarly, no significant differences were observed in the adipogenic differentiation potential of BMSCs isolated from different age groups on day 8. However, there was a significant increase in the chondrogenic differentiation potential of BMSCs isolated from participants in their 30s and 50s, compared with BMSCs isolated from participants in their 20s on day 16.