scholarly journals Differentiation of Rat Adipose-Derived Stem Cells into Parathyroid-Like Cells

2020 ◽  
Vol 2020 ◽  
pp. 1-6
Author(s):  
Ping Zhang ◽  
Hao Zhang ◽  
Wenwu Dong ◽  
Zhihong Wang ◽  
Yuan Qin ◽  
...  
Keyword(s):  
Stem Cells ◽  
Parathyroid Hormone ◽  
Osteogenic Differentiation ◽  
Hormone Secretion ◽  
Activin A ◽  
High Price ◽  
Dependent Manner ◽  
Adipose Derived Stem Cells ◽  
Alizarin Red ◽  
Postoperative Hypoparathyroidism

Background. The current treatment for postoperative hypoparathyroidism has shortcomings, such as repeated blood monitoring for dosage adjustment, uncertain long-term efficacy, and the high price of recombinant parathyroid hormone therapy. Adipose-derived stem cells can undergo adipogenic and osteogenic differentiation in vitro and are considered a novel source of parathyroid-like cells, but the idea lacks theoretical basis and feasibility. We aimed at establishing a protocol for differentiating adipose-derived stem cells into parathyroid-like cells for treating hypoparathyroidism. Materials/Methods. Adipose-derived stem cells were isolated and purified from the inguinal adipose tissue of Sprague Dawley rats. Adipogenic differentiation and osteogenic differentiation of the cells were identified by oil red O and alizarin red S staining, respectively. The adipose-derived stem cells were stimulated by sonic hedgehog (SHH) and activin A. The differentiation of the adipose-derived stem cells to parathyroid-like cells was confirmed by the detection of parathyroid hormone and the related parathyroid markers. Results. Adipose-derived stem cells were successfully isolated and purified from the rat adipocytes. The adipogenic and osteogenic differentiation capabilities of the adipose-derived stem cells were determined. SHH and activin A stimulated parathyroid hormone secretion by the adipose-derived stem cells and significantly increased the expression of calcium-sensing receptor (CaSR), parathyroid hormone, and glial cells missing homolog 2 (GCM2) in the cells in a time- and concentration-dependent manner. Conclusion. We successfully differentiated rat adipose-derived stem cells into parathyroid-like cells, which will pave a new route to curing hypoparathyroidism.

scholarly journals Actin Polymerization State Regulates Osteogenic Differentiation in Human Adipose-derived Stem Cells

2020 ◽  
Author(s):  
Bing Sun ◽  
Xin Jiang ◽  
Rongmei Qu ◽  
Tingyu Fan ◽  
Yuchao Yang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Proliferation ◽  
Osteogenic Differentiation ◽  
Focal Adhesion ◽  
Actin Polymerization ◽  
Dependent Manner ◽  
Adipose Derived Stem Cells ◽  
Alizarin Red ◽  
20 Nm ◽  
Actin Polymerization State

Abstract Background:Actin is an essential cellular protein that assembles into microfilaments and regulates numerous processes such as cell migration, maintenance of cell shape, and material transport. In this study, we explored the effect of actin polymerization state on the osteogenic differentiation of human adipose-derived stem cells (hASCs). Methods:The hASCs were treated with different concentrations (0, 1, 5, 10, 20, and 50 nM)of jasplakinolide (JAS), a reagent that directly polymerizes F-actin.The effects ofthe actin polymerization state on cell proliferation, apoptosis, migration, and the maturity of focal adhesion-related proteins were assessed. In addition, western blotting and alizarin red staining assays were performed to assess osteogenic differentiation. Results: These results revealed that cell proliferation and migration in the JAS (0, 1, 5, 10, and 20 nM) groupswashigher than that inthe control group andthe JAS (50 nM) group.The protein expressionof focal adhesion kinase, vinculin, paxillin, and talinwere highest in the JAS (20 nM) group, whilezyxin expression was highestinthe JAS (50 nM) group.Western blottingshowed thatosteogenic differentiation in theJAS (0, 1, 5, 10, 20, and 50 nM) groupswas enhanced compared with that in thecontrol group, and was strongest inthe JAS (50 nM) group.Conclusions: Our data suggest thatthe actinpolymerization state may promote the osteogenic differentiation of hASCs by regulating the protein expression of focal adhesion-associated proteins in a concentration-dependent manner. Our findings provide valuable information for exploring the mechanism of osteogenic differentiationin hASCs.

scholarly journals Yunnan Baiyao Conditioned Medium Promotes the Odonto/Osteogenic Capacity of Stem Cells from Apical Papilla via Nuclear Factor Kappa B Signaling Pathway

2019 ◽  
Vol 2019 ◽  
pp. 1-11 ◽  
Author(s):  
Xiyao Pang ◽  
Yanqiu Wang ◽  
Jintao Wu ◽  
Zhou Zhou ◽  
Tao Xu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Alkaline Phosphatase ◽  
Osteogenic Differentiation ◽  
Conditioned Medium ◽  
Signaling Pathway ◽  
Cell Counting ◽  
Dependent Manner ◽  
Alizarin Red ◽  
Apical Papilla ◽  
Yunnan Baiyao

Yunnan Baiyao is a traditional Chinese herbal remedy that has long been used for its characteristics of wound healing, bone regeneration, and anti-inflammation. However, the effects of Yunnan Baiyao on the odonto/osteogenic differentiation of stem cells from apical papilla (SCAPs) and the potential mechanisms remain unclear. The aim of this study was to investigate the odonto/osteogenic differentiation effects of Yunnan Baiyao on SCAPs and the underlying mechanisms involved. SCAPs were isolated and cocultured with Yunnan Baiyao conditioned media. The proliferation ability was determined by cell counting kit 8 and flow cytometry. The differentiation capacity and the involvement of NF-κB pathway were investigated by alkaline phosphatase assay, alizarin red staining, immunofluorescence assay, real-time RT-PCR, and western blot analyses. Yunnan Baiyao conditioned medium at the concentration of 50 μg/mL upregulated alkaline phosphatase activity, induced more mineralized nodules, and increased the expression of odonto/osteogenic genes/proteins (e.g., OCN/OCN, OPN/OPN, OSX/OSX, RUNX2/RUNX2, ALP/ALP, COL-I/COL-I, DMP1, DSP/DSPP) of SCAPs. In addition, the expression of cytoplasmic phos-IκBα, phos-P65, and nuclear P65 was significantly increased in Yunnan Baiyao conditioned medium treated SCAPs in a time-dependent manner. Conversely, the differentiation of Yunnan Baiyao conditioned medium treated SCAPs was obviously inhibited when these stem cells were cocultured with the specific NF-κB inhibitor BMS345541. Yunnan Baiyao can promote the odonto/osteogenic differentiation of SCAPs via the NF-κB signaling pathway.

scholarly journals Composite Hydrogel of Methacrylated Hyaluronic Acid and Fragmented Polycaprolactone Nanofiber for Osteogenic Differentiation of Adipose-Derived Stem Cells

Pharmaceutics ◽  
2020 ◽  
Vol 12 (9) ◽  
pp. 902
Author(s):  
Madhumita Patel ◽  
Won-Gun Koh
Keyword(s):  
Tissue Engineering ◽  
Stem Cells ◽  
Hyaluronic Acid ◽  
Bone Tissue Engineering ◽  
Osteogenic Differentiation ◽  
Bone Tissue ◽  
Composite Hydrogel ◽  
Adipose Derived Stem Cells ◽  
Alizarin Red ◽  
Composite Hydrogels

Composite hydrogels with electrospun nanofibers (NFs) have recently been used to mimic the native extracellular matrix. In this study, composite hydrogels of methacrylated hyaluronic acid containing fragmented polycaprolactone NFs were used for bone tissue engineering. The composite (NF/hydrogel) was crosslinked under ultraviolet (UV) light. The incorporation of fragmented polycaprolactone NFs increased the compression modulus from 1762.5 to 3122.5 Pa. Subsequently, adipose-derived stem cells incorporated into the composite hydrogel exhibited a more stretched and elongated morphology and osteogenic differentiation in the absence of external factors. The mRNA expressions of osteogenic biomarkers, including collagen 1 (Col1), alkaline phosphatase, and runt-related transcription factor 2, were 3–5-fold higher in the composite hydrogel than in the hydrogel alone. In addition, results of the protein expression of Col1 and alizarin red staining confirmed osteogenic differentiation. These findings suggest that our composite hydrogel provides a suitable microenvironment for bone tissue engineering.

scholarly journals MiR-204-5p/FOXC1/GDF7 Axis Regulates Osteogenic Differentiation of Human Adipose-Derived Stem Cells Via the AKT and p38 Signaling Pathways

2020 ◽  
Author(s):  
You Zhou ◽  
Siyu Liu ◽  
Wei Wang ◽  
Qiang Sun ◽  
Mengzhu Lv ◽  
...  
Keyword(s):  
Stem Cells ◽  
Osteogenic Differentiation ◽  
Signaling Pathways ◽  
Regulatory Mechanism ◽  
Adipose Derived Stem Cells ◽  
Alizarin Red ◽  
Protein Levels ◽  
Forkhead Box ◽  
Mineral Deposition ◽  
Chip Experiment

Abstract Background: Human adipose-derived stem cells (hADSCs) are stem cells with the potential to differentiate in multiple directions. MiR-204-5p is poorly expressed during osteogenic differentiation of hADSCs, and its specific regulatory mechanism remains unclear. Here, we aimed to explore the function and possible molecular mechanism of miR-204-5p involved in the osteogenic differentiation of hADSCs. Methods: The expression pattern of miR-204-5p, Runx2, Alkaline phosphatase (ALP), Osteocalcin (OCN), and Forkhead box C1(FOXC1) and growth differentiation factor 7(GDF7) in hADSCs during osteogenesis were detected by qRT-PCR. Then, ALP and alizarin red staining (ARS) were used to detect the activity of osteoblasts and mineral deposition. Western blot was conducted to confirm the protein levels. The regulation relationship among miR-204-5p, FOXC1 and GDF7 was verified by double luciferase activity and CHIP experiment.Results: First, miR-204-5p expression was down-regulated and overexpressed miR-204-5p suppressed the osteogenic differentiation. Furthermore, the levels of FOXC1 and GDF7 were decreased in the miR-204-5p mimics group, which indicate that overexpressed miR-204-5p would suppress the expression of FOXC1 and GDF7 through binding the 3’UTR region each. Overexpression of FOXC1 or GDF7 could improve the inhibition of osteogenic differentiation of hADSCs induced by the miR-204-5p mimics. Moreover, FOXC1 could bind to the promoter of miR-204-5p and GDF7, promote the deacetylation of miR-204-5p and reduced the expression of miR-204-5p, thus promoting the expression of GDF7 during osteogenic differentiation. GDF7 could induce hADSCs osteogenesis differentiation by activating the AKT and P38 signaling pathways. Conclusions: Our results demonstrated that miR-204-5p/FOXC1/GDF7 axis regulates osteogenic differentiation of hADSCs via the AKT and p38 signaling pathways. This study further understood the regulatory mechanism of hADSCs differentiation balance from the perspective of miRNAs regulation.

scholarly journals The miR-204-5p/FOXC1/GDF7 axis regulates the osteogenic differentiation of human adipose-derived stem cells via the AKT and p38 signalling pathways

2020 ◽  
Author(s):  
You Zhou ◽  
Siyu Liu ◽  
Wei Wang ◽  
Qiang Sun ◽  
Mengzhu Lv ◽  
...  
Keyword(s):  
Stem Cells ◽  
Osteogenic Differentiation ◽  
Regulatory Mechanism ◽  
Expression Patterns ◽  
Signalling Pathways ◽  
Adipose Derived Stem Cells ◽  
Mirna Regulation ◽  
Regulatory Relationship ◽  
Alizarin Red ◽  
Protein Levels

Abstract Background: Human adipose-derived stem cells (hADSCs) are stem cells with the potential to differentiate in multiple directions. MiR-204-5p is expressed at low levels during the osteogenic differentiation of hADSCs, and its specific regulatory mechanism remains unclear. Here, we aimed to explore the function and possible molecular mechanism of miR-204-5p in the osteogenic differentiation of hADSCs.Methods: The expression patterns of miR-204-5p, Runx2, alkaline phosphatase (ALP), osteocalcin (OCN), forkhead box C1 (FOXC1) and growth differentiation factor 7 (GDF7) in hADSCs during osteogenesis were detected by qRT-PCR. Then, ALP and alizarin red staining (ARS) were used to detect osteoblast activities and mineral deposition. Western blotting was conducted to confirm the protein levels. The regulatory relationship among miR-204-5p, FOXC1 and GDF7 was verified by dual-luciferase activity and chromatin immunoprecipitation (ChIP) assays.Results: MiR-204-5p expression was downregulated in hADSC osteogenesis, and overexpression of miR-204-5p suppressed osteogenic differentiation. Furthermore, the levels of FOXC1 and GDF7 were decreased in the miR-204-5p mimics group, which indicates that miR-204-5p overexpression suppresses the expression of FOXC1 and GDF7 by binding to their 3'-untranslated regions (UTRs). Overexpression of FOXC1 or GDF7 improved the inhibition of osteogenic differentiation of hADSCs induced by the miR-204-5p mimics. Moreover, FOXC1 was found to bind to the promoter of miR-204-5p and GDF7, promote the deacetylation of miR-204-5p and reduce the expression of miR-204-5p, thus promoting the expression of GDF7 during osteogenic differentiation. GDF7 induced hADSC osteogenesis differentiation by activating the AKT and P38 signalling pathways.Conclusions: Our results demonstrated that the miR-204-5p/FOXC1/GDF7 axis regulates the osteogenic differentiation of hADSCs via the AKT and p38 signalling pathways. This study further revealed the regulatory mechanism of hADSC differentiation from the perspective of miRNA regulation.

scholarly journals The miR-204-5p/FOXC1/GDF7 axis regulates the osteogenic differentiation of human adipose-derived stem cells via the AKT and p38 signalling pathways

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
You Zhou ◽  
Siyu Liu ◽  
Wei Wang ◽  
Qiang Sun ◽  
Mengzhu Lv ◽  
...  
Keyword(s):  
Stem Cells ◽  
Osteogenic Differentiation ◽  
Regulatory Mechanism ◽  
Expression Patterns ◽  
Signalling Pathways ◽  
Adipose Derived Stem Cells ◽  
Mirna Regulation ◽  
Regulatory Relationship ◽  
Alizarin Red ◽  
Protein Levels

Abstract Background Human adipose-derived stem cells (hADSCs) are stem cells with the potential to differentiate in multiple directions. miR-204-5p is expressed at low levels during the osteogenic differentiation of hADSCs, and its specific regulatory mechanism remains unclear. Here, we aimed to explore the function and possible molecular mechanism of miR-204-5p in the osteogenic differentiation of hADSCs. Methods The expression patterns of miR-204-5p, Runx2, alkaline phosphatase (ALP), osteocalcin (OCN), forkhead box C1 (FOXC1) and growth differentiation factor 7 (GDF7) in hADSCs during osteogenesis were detected by qRT-PCR. Then, ALP and alizarin red staining (ARS) were used to detect osteoblast activities and mineral deposition. Western blotting was conducted to confirm the protein levels. The regulatory relationship among miR-204-5p, FOXC1 and GDF7 was verified by dual-luciferase activity and chromatin immunoprecipitation (ChIP) assays. Results miR-204-5p expression was downregulated in hADSC osteogenesis, and overexpression of miR-204-5p suppressed osteogenic differentiation. Furthermore, the levels of FOXC1 and GDF7 were decreased in the miR-204-5p mimics group, which indicates that miR-204-5p overexpression suppresses the expression of FOXC1 and GDF7 by binding to their 3′-untranslated regions (UTRs). Overexpression of FOXC1 or GDF7 improved the inhibition of osteogenic differentiation of hADSCs induced by the miR-204-5p mimics. Moreover, FOXC1 was found to bind to the promoter of miR-204-5p and GDF7, promote the deacetylation of miR-204-5p and reduce the expression of miR-204-5p, thus promoting the expression of GDF7 during osteogenic differentiation. GDF7 induced hADSC osteogenesis differentiation by activating the AKT and P38 signalling pathways. Conclusions Our results demonstrated that the miR-204-5p/FOXC1/GDF7 axis regulates the osteogenic differentiation of hADSCs via the AKT and p38 signalling pathways. This study further revealed the regulatory mechanism of hADSC differentiation from the perspective of miRNA regulation.

scholarly journals Intermittent Administration of Parathyroid Hormone Enhances Odonto/Osteogenic Differentiation of Stem Cells from the Apical Papilla via JNK and P38 MAPK Pathways

2020 ◽  
Vol 2020 ◽  
pp. 1-13 ◽  
Author(s):  
Xiyao Pang ◽  
Ying Zhuang ◽  
Zehan Li ◽  
Shuanglin Jing ◽  
Qin Cai ◽  
...  
Keyword(s):  
Stem Cells ◽  
Parathyroid Hormone ◽  
Osteogenic Differentiation ◽  
P38 Mapk ◽  
Cell Counting ◽  
Mrna Levels ◽  
Alizarin Red ◽  
Alp Activity ◽  
Mapk Pathways ◽  
Apical Papilla

Objective. Parathyroid hormone (PTH) is considered to be essential during the tooth development. Stem cells from the apical papilla (SCAPs) are responsible for dentine formation. However, the interaction between PTH and SCAPs remains unclear. This study was aimed at investigating the effects of PTH on odonto/osteogenic differentiation capacity of SCAPs and elucidating the underlying molecular mechanisms. Materials and Methods. Here, SCAPs were isolated and identified in vitro. Effects of PTH on the proliferation of SCAPs were determined by Cell Counting Kit-8 (CCK-8), flow cytometry (FCM), and EdU. Alkaline phosphatase (ALP) activity, alizarin red staining, Western blot, and RT-PCR were carried out to detect the odonto/osteogenic differentiation of PTH-treated SCAPs as well as the participation of the MAPK signaling pathway. Results. An ALP activity assay determined that 10-8 mol/L PTH was the optimal concentration for the induction of SCAPs with no significant influence on the proliferation of SCAPs as indicated by CCK-8, FCM, and EdU. The expression of odonto/osteogenic markers was significantly upregulated in mRNA levels and protein levels. Moreover, intermittent treatment of PTH also increased phosphorylation of JNK and P38, and the differentiation was suppressed following the inhibition of JNK and P38 MAPK pathways. Conclusion. PTH can regulate the odonto/osteogenic differentiation of SCAPs via JNK and P38 MAPK pathways.

scholarly journals Parathyroid Hormone Secretion and Receptor Expression Determine the Age-Related Degree of Osteogenic Differentiation in Dental Pulp Stem Cells

2021 ◽  
Vol 11 (5) ◽  
pp. 349
Author(s):  
Shilpa Bhandi ◽  
Ahmed Alkahtani ◽  
Rodolfo Reda ◽  
Mohammed Mashyakhy ◽  
Nezar Boreak ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Parathyroid Hormone ◽  
Osteogenic Differentiation ◽  
Dental Pulp ◽  
Hormone Secretion ◽  
Age Groups ◽  
Dental Pulp Stem Cells ◽  
Before And After ◽  
Osteogenic Induction

Objective: To demonstrate the levels of parathyroid hormone secretion and genetic expressions of parathyroid hormone (PTH) and PTH1 receptor (PTH1R) genes in the dental pulp stem cells (DPSCs) from different age groups before and after induction of osteogenic differentiation. In addition, we also wanted to check their correlation with the degree of osteogenic differentiation. Methods: Human primary DPSCs from three age groups (milk tooth (SHEDs), 7–12 years old; young DPSCs (yDPSCs), 20–40 years old; old DPSCs (oDPSCs), 60+ years old) were characterized for mesenchymal stem cell (MSC) markers. DPSCs were subjected to osteogenic differentiation and functional staining. Gene expression levels were analyzed by qRT-PCR. Surface receptor analysis was done by flow cytometry. Comparative protein levels were evaluated by ELISA. Results: All SHEDs, yDPSCs, and oDPSCs were found to be expressing mesenchymal stem cell markers. SHEDs showed more mineralization than yDPSCs and oDPSCs after osteogenic induction. SHEDs exhibited higher expression of PTH and PTH1R before and after osteogenic induction, and after osteogenic induction, SHEDs showed more expression for RUNX2, ALPL, and OCN. Higher levels of PTH were observed in SHEDs and yDPSCs, and the number of PTH1R positive cells was relatively lower in yDPSCs and oDPSCs than in SHEDs. After osteogenic induction, SHEDs were superior in the secretion of OPG, and the secretions of ALPL and PTH and the number of PTH1R positive cells were relatively low in the oDPSCs. Conclusions: The therapeutic quality of dental pulp stem cells is largely based on their ability to retain their stemness characteristics. This study emphasizes the criterion of aging, which affects the secretion of PTH by these cells, which in turn attenuates their osteogenic potential.

scholarly journals Role of Fzd6 in Regulating the Osteogenic Differentiation of Adipose-derived Stem Cells in Osteoporosis

2021 ◽  
Author(s):  
Tianli Wu ◽  
Zhihao Yao ◽  
Gang Tao ◽  
Fangzhi Lou ◽  
Hui Tang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Osteogenic Differentiation ◽  
Wnt Signaling Pathway ◽  
Osteogenic Potential ◽  
Adipose Derived Stem Cells ◽  
Alizarin Red ◽  
Differentiation Potential

Abstract Objective: Although it has been demonstrated that adipose-derived stem cells (ASCs) from osteoporosis mice (OP-ASCs) exhibit impaired osteogenic differentiation potential, the molecular mechanism has not yet been elucidated. We found that Fzd6 was decreased in OP-ASCs compared with ASCs. This study investigates the effects and underlying mechanisms of Fzd6 in the osteogenic potential of OP-ASCs. Methods: Fzd6 expression in ASCs and OP-ASCs was measured by PCR gene chip. Fzd6 overexpression and silencing lentiviruses were used to evaluate the role of Fzd6 in the osteogenic differentiation of OP-ASCs. Real-time PCR (qPCR) and western blotting (WB) was performed to detect the expression of Fzd6 and bone-related molecules, including runt-related transcription factor 2 (Runx2) and osteopontin (Opn). Alizarin red staining and Alkaline phosphatase (ALP) staining was performed following osteogenic induction. Microscopic CT (Micro-CT), hematoxylin and eosin staining (H&E) staining, and Masson staining were used to assess the role of Fzd6 in osteogenic differentiation of osteoporosis (OP) mice in vivo.Results: Expression of Fzd6 was decreased significantly in OP-ASCs. Fzd6 silencing down-regulated the osteogenic ability of OP-ASCs in vitro. Overexpression of Fzd6 rescued the impaired osteogenic capacity in OP-ASCs in vitro. We obtained similar results in vivo.Conclusions: Fzd6 plays an important role in regulating the osteogenic ability of OP-ASCs both in vivo and in vitro. Overexpression of Fzd6 associated with the Wnt signaling pathway promotes the osteogenic ability of OP-ASCs, which provides new insights for the prevention and treatment of OP.

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