scholarly journals The Effects of Bone Morphogenetic Protein-4 on Cellular Viability, Osteogenic Potential, and Global Gene Expression on Gingiva-Derived Stem Cell Spheroids

Coatings ◽  
2020 ◽  
Vol 10 (11) ◽  
pp. 1055
Author(s):  
Jae-Yong Tae ◽  
Yoon-Hee Park ◽  
Youngkyung Ko ◽  
Jun-Beom Park
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Alkaline Phosphatase ◽  
Mesenchymal Stem Cells ◽  
Stem Cell ◽  
Global Gene Expression ◽  
Bone Morphogenetic Protein 4 ◽  
Cellular Viability ◽  
Cell Spheroids ◽  
Morphogenetic Protein

Bone morphogenetic protein-4 (BMP-4) is engaged in the migration ability of mesenchymal stem cells and the transition of mesenchymal stem cells into osteogenic and adipocytic lines. The aim of this study was to evaluate the effects of BMP-4 on the cellular viability, osteogenic differentiation, and genome-wide mRNA levels using three-dimensional cell spheroids composed of stem cells. Stem cell spheroids were formed using concave microwells in the presence of BMP-4 with final concentrations of 0, 2, 6, and 10 ng/mL. Cellular viability was measured qualitatively using a microscope and quantitatively using an assay kit based on water-soluble tetrazolium salt. Osteogenic differentiation was assessed by measuring the level of alkaline phosphatase activity. Global gene expression was assessed using next-generation mRNA sequencing and performing gene ontology and pathway analyses. Spheroids were well-maintained with the addition of BMP-4 up to Day 7. No significant differences were observed in cell viability between each group. There were significantly higher alkaline phosphatase values in the 2 ng/mL BMP-4 groups when compared with the control (p < 0.05). A total of 25,737 mRNAs were differentially expressed. Expression of β-catenin (CTNNB1) was increased with higher dosages of BMP-4. The expression of runt-related transcription factor 2 (RUNX2) was increased up to 6 ng/mL. The phosphoinositide-3-kinase–protein kinase B/Akt signaling pathway was associated with the target genes. This study demonstrates that the application of BMP-4 enhanced alkaline phosphatase activity and the expression of CTNNB1 and RUNX2 without affecting cellular viability.

scholarly journals Induction of Osteogenic Differentiation of Mesenchymal Stem Cells by Bioceramic Root Repair Material

Materials ◽  
2019 ◽  
Vol 12 (14) ◽  
pp. 2311 ◽  
Author(s):  
Hadeel Y. Edrees ◽  
Sawsan T.H. Abu Zeid ◽  
Hazem M. Atta ◽  
Mehal A. AlQriqri
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Alkaline Phosphatase ◽  
Mesenchymal Stem Cells ◽  
Cell Viability ◽  
Osteogenic Differentiation ◽  
Bone Sialoprotein ◽  
Bone Morphogenetic Protein 2 ◽  
Repair Material ◽  
Morphogenetic Protein

This study aimed to evaluate the osteogenic activity of Endosequence Root Repair Material (ERRM) putty using rat mesenchymal stem cells (MSCs). The extract of set ERRM and ProRoot-mineral trioxide aggregate (MTA) (control) was cocultured with rat MSCs and incubated for one, three, and seven days. The cell viability and proliferation were assessed. A quantitative real-time polymerase chain reaction for bone morphogenetic protein-2 (BMP-2), alkaline phosphatase, bone sialoprotein, and osteocalcin gene expression was performed. Both materials enhanced cell viability and proliferation, which increased over time. On day seven, the cells treated with either material exhibited significantly greater cell viability compared with control untreated cells. MSCs treated with either material showed deeper alkaline phosphatase staining after three days compared to control untreated cells. Treated MSCs also exhibited upregulation of the gene expression of bone morphogenetic protein-2, alkaline phosphatase, bone sialoprotein, and osteocalcin. Both ERRM and ProRoot-MTA enhance the osteogenic differentiation of MSCs.

3D bioprinting of mesenchymal stem cells and endothelial cells in an alginate-gelatin-based bioink

2021 ◽  
Author(s):  
Sindhuja D Eswaramoorthy ◽  
Nandini Dhiman ◽  
Akshay Joshi ◽  
Subha N Rath
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Alkaline Phosphatase ◽  
Endothelial Cells ◽  
Mesenchymal Stem Cells ◽  
Osteogenic Differentiation ◽  
Alkaline Phosphatase Activity ◽  
Histochemical Staining ◽  
Vital Role ◽  
3D Bioprinting

Aim: Bioink is one of the essential factors in 3D bioprinting that determines the fate of cells, in our case, umbilical cord-derived mesenchymal stem cells (UMSC). The aim was to determine if the presence of the osteoinductive factors in the bioink enhances osteodifferentiation as compared with adding them postprinting and if the UMSC and endothelial cells (EC) coculture result in better osteodifferentiation. Materials & methods: Alginate-gelatin along with UMSC–EC were bioprinted using an extrusion 3D bioprinter. Results & conclusion: The UMSC–EC interaction, as well as intrinsic addition of the differentiation components in the bioink, were observed to play a vital role in increasing the osteogenic differentiation as shown by the histochemical staining, alkaline phosphatase activity and gene expression of osteogenic markers.

scholarly journals The The Influence of Mesenchymal Stem Cell Wharton Jelly toward Prostaglandin E2 Gene Expression on Synoviocyte Cell Osteoarthritis

2019 ◽  
Vol 7 (8) ◽  
pp. 1252-1258 ◽  
Author(s):  
Vivi Sofia ◽  
Moch Saiful Bachri ◽  
Rizki Rahmadian
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Stem Cell ◽  
Mesenchymal Stem Cell ◽  
Prostaglandin E2 ◽  
Expression Analysis ◽  
Control Group ◽  
E2 Gene

BACKGROUND: Pharmacological therapy in the management of OA causes many new health problems due to side effects caused by long-term use of drugs, such as long-term use of Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) will cause gastric ulcers and impaired kidney function. In OA pathogenesis, PGE2 gene is involved in the inflammation process. AIM: This study aims to identify the influence of Wharton Jelly Mesenchymal Stem Cell (MSC-WJ) on PGE2 expression gene in synoviocyte by in vitro. MATERIAL AND METHODS: The method used in this study is the co-culture method of primary cells and stem cells in the appropriate media. This research is pure experimental research. The sample used came from synovial tissue of osteoarthritis patients who underwent Total Knee Replacement (TKR) surgery. This study was divided into 6 groups treated with 4 replications. The expression analysis of the Prostaglandin E2 gene was done using qPCR (Real-Time Polymerase Chain Reaction). The expression analysis of the Prostaglandin E2 gene was carried out before and after the co-culture with Wharton's Jelly and continued with the analysis of statistical data processing using the SPSS.15 program. PGE2 gene expression data were processed using the Kruskal-Wallis test and continued with the Mann-Whitney test with a 95% confidence level. RESULTS: The results showed that Mesenchymal Stem Cells Wharton Jelly could reduce the expression of Prostaglandin E2 gene after co-culture for 24 hours and 48 hours in synoviocyte cells osteoarthritis significantly compared with the control group. The administration of Mesenchymal Stem Cells for 24 hours reduced the expression level of PGE2 gene by 0.61 times compared to the control group (p < 0.05) and the administration of Mesenchymal Stem Cells for 48 hours decreased the expression level of PGE2 gene by 0, 47 times compared to the control group (p < 0.05). CONCLUSION: This study concluded that MSC-WJ in OA synoviocyte significantly reduced the expression of the PGE2 gene (p < 0.05).

Murine spinal fusion induced by engineered mesenchymal stem cells that conditionally express bone morphogenetic protein—2

2005 ◽  
Vol 3 (1) ◽  
pp. 47-52 ◽  
Author(s):  
Amir Hasharoni ◽  
Yoram Zilberman ◽  
Gadi Turgeman ◽  
Gregory A. Helm ◽  
Meir Liebergall ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Spinal Fusion ◽  
Genetically Engineered ◽  
Bone Morphogenetic Protein 2 ◽  
Content Type ◽  
Histological Studies ◽  
Morphogenetic Protein ◽  
Fine Print

Object. The authors hypothesized that spinal fusion can be achieved and monitored by using cell-mediated gene therapy. Mesenchymal stem cells (MSCs) genetically engineered to express recombinant human bone morphogenetic protein—2 (rhBMP-2) conditionally, were implanted into the paraspinal muscles of mice to establish spinal fusion. The goal was to demonstrate an MSC-based gene therapy platform in which controlled gene expression is used to obtain spinal fusion in a murine model. Methods. Mesenchymal stem cells expressing the rhBMP-2 gene were injected into the paravertebral muscle in mice. Bone formation in the paraspinal region was longitudinally followed by performing micro—computerized tomography scanning, histological studies, and an analysis of osteocalcin expression to demonstrate the presence of engrafted engineered MSCs. The minimal period of rhBMP-2 expression by the engineered MSCs required to induce fusion was determined. The results of this study demonstrate that genetically engineered MSCs induce bone formation in areas adjacent to and touching the posterior elements of the spine. This newly formed bone fuses the spine, as demonstrated by radiological and histological studies. The authors demonstrate that injected cells induce active osteogenesis at the site of implantation for up to 4 weeks postinjection. They found that a 7-day induction of rhBMP-2 expression in genetically engineered MSCs was sufficient to form new bone tissue, although the quantity of this bone increased as longer expression periods were implemented. Conclusions. After their injection genetically engineered MSCs can efficiently form new bone in the paraspinal muscle of the mouse to obtain spinal fusion. The extent and quantity of this newly formed bone can be monitored by controlling the duration of rhBMP-2 gene expression.

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

Medicina ◽  
2020 ◽  
Vol 56 (8) ◽  
pp. 389
Author(s):  
Sae Kyung Min ◽  
Jaekwen Oh ◽  
Jun-Beom Park
Keyword(s):  
Alkaline Phosphatase ◽  
Stem Cell ◽  
Phosphatase Activity ◽  
Alkaline Phosphatase Activity ◽  
Morinda Citrifolia ◽  
Herbal Remedies ◽  
Cellular Viability ◽  
Alizarin Red ◽  
Anthraquinone Dye ◽  
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.

Vitrification affects global gene expression of rhesus macaque mesenchymal stem cells

Cryobiology ◽  
2018 ◽  
Vol 80 ◽  
pp. 183-184
Author(s):  
Xufeng Fu ◽  
Yaping Yan ◽  
Shanshan Li ◽  
Yanchao Duan ◽  
Bingrong Zheng ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Rhesus Macaque ◽  
Global Gene Expression

128 Next-generation RNA sequencing of horse adipose and endometrial mesenchymal stem cells from the same donors unveils striking differences in their transcriptomic pattern

2019 ◽  
Vol 31 (1) ◽  
pp. 189
Author(s):  
F. Navarrete ◽  
E. Mellisho ◽  
Y. Wang ◽  
J. Cabezas ◽  
L. L. Rodriguez-Alvarez ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Gene Ontology ◽  
Mesenchymal Stem Cells ◽  
Stem Cell ◽  
Cell Types ◽  
Biological Traits ◽  
Next Generation ◽  
Pca Analysis ◽  
Heat Map

Earlier we successfully isolated and characterised endometrial (eMSC) and adipose (aMSC) mesenchymal stem cells from the same donors. Mesenchymal stem cells share biological traits but display different surface marker phenotype and migration ability. Here we extended our research to their mRNA signature using next-generation sequencing. The RNA from cells (3 biological replicates from each cell type and 3 technical replicates) at 90% confluence was extracted using a total RNA extraction kit and sent for mRNA-Seq (Norgen, Ontario, Canada; Illumina Sequencing Platform NextSEqn 500). Raw 76-bp single-end reads were aligned against the EquCab3 genome using RNA-STAR aligner. Counts were filtrated at a minimum of 5. Pairwise comparisons between the cell types were the input for gene ontology enrichment analysis. Only genes differentially expressed (DE) with 5 folds change (FC; P&lt;0.05) were analysed. For DE analysis, eMSC were set as control and compared with aMSC. Unsupervised hierarchical clustering of the global gene expression signatures was done to compare the samples from each line using principal component analysis (PCA) and EdgeR: v3.20.9. Gene expression was normalized using FPKM. The heat map was built using R studio with G-plot package. A total of 14,896 transcripts with at least 5 reads were found; of these, 1598 were DE: 627 up-regulated (FC range: 2 to 236×) and 971 down-regulated (FC range: 2 to 464×) in eMSC. There was a marked dispersion in the FC of up- and down-regulated genes (&gt;50×: 8 and 13; &gt;20×, &lt;50×: 9 and 17; &gt;10×, &lt;20×: 29 and 63; &gt;5×, &lt;10×: 91 and 130 and &gt;2×, &lt;5×: 490 and 748, respectively). Only genes DE with FC at least 5× were used for gene ontology and PCA analysis. Though 14,058 genes were common to both cell types, specific set of genes were found only in eMSC (n=162) or aMSC (n=676). Among the top 50 genes overexpressed in eMSC, several genes key for stem cell growth, immune response, migration and angiogenesis were found: TRIL, CXCL8, PDGF-D, SEMA5A, PTGS2, FGD, LAMA2, IL36G. In the top 50 down-regulated genes, some pivotal for osteoblast, adipogenic and neural differentiation were dramatically down-regulated (GPM6B, SCARA5 and NOTCH3 or NEFM, respectively), but no genes involved in immune rejection or stem cell proliferation were found. In gene ontology, the categories represented the most were cellular, developmental, metabolic, and immune system processes, as well as biological regulation, response to stimuli, organellar biogenesis, locomotion, localization and biological adhesion. Heat map and PCA analysis showed that one individual cell line from each type diverged markedly from the shared pattern. Individual variability of the donors may impinge upon the results; nevertheless, striking differences in the mRNA portfolio of eMSC and aMSC were detected. The importance and potential biological role of several of the genes and processes named above will be discussed in detail elsewhere. This work was supported by grant FONDECYT REGULAR 1150757 and the Government of Chile.

scholarly journals The Effect of Blood-Derived Products on the Chondrogenic and Osteogenic Differentiation Potential of Adipose-Derived Mesenchymal Stem Cells Originated from Three Different Locations

2019 ◽  
Vol 2019 ◽  
pp. 1-20 ◽  
Author(s):  
Markus Neubauer ◽  
Olga Kuten ◽  
Christoph Stotter ◽  
Karina Kramer ◽  
Andrea De Luna ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Stem Cell ◽  
Regenerative Medicine ◽  
Blood Product ◽  
Blood Products ◽  
Fat Tissue ◽  
Differentiation Potential ◽  
Lower Blood

Background. Adipose-derived mesenchymal stem cells (AD-MSCs) from fat tissue considered “surgical waste” during joint surgery may provide a potent source for regenerative medicine. Intra-articular, homologous fat tissue (Hoffa’s fat pad, pouch fat) might possess a superior chondrogenic and osteogenic differentiation potential in comparison to extra-articular, nonhomologous fat. Blood products might further enhance this potential. Methods. AD-MSCs were isolated from fat tissue of 3 donors from 3 locations each, during total knee replacement. Isolated cells were analyzed via flow cytometry. Cells were supplemented with blood products: two types of platelet-rich plasma (EPRP—PRP prepared in the presence of EDTA; CPRP—PRP prepared in the presence of citrate), hyperacute serum (hypACT), and standard fetal calf serum (FCS) as a positive control. The viability of the cells was determined by XTT assay, and the progress of differentiation was tested via histological staining and monitoring of specific gene expression. Results. Blood products enhance ex vivo cell metabolism. Chondrogenesis is enhanced by EDTA-PRP and osteogenesis by citrate PRP, whereas hyperacute serum enhances both differentiations comparably. This finding was consistent in histological analysis as well as in gene expression. Lower blood product concentrations and shorter differentiation periods lead to superior histological results for chondrogenesis. Both PRP types had a different biological effect depending upon concentration, whereas hyperacute serum seemed to have a more consistent effect, independent of the used concentration. Conclusion. (i) Blood product preparation method, (ii) type of anticoagulant, (iii) differentiation time, and (iv) blood product concentration have a significant influence on stem cell viability and the differentiation potential, favouring no use of anticoagulation, shorter differentiation time, and lower blood product concentrations. Cell-free blood products like hyperacute serum may be considered as an alternative supplementation in regenerative medicine, especially for stem cell therapies.

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