Biochanin A Promotes Osteogenic but Inhibits Adipogenic Differentiation: Evidence with Primary Adipose-Derived Stem Cells

Biochanin A has promising effects on bone formationin vivo, although the underlying mechanism remains unclear yet. This study therefore aimed to investigate whether biochanin A regulates osteogenic and adipogenic differentiation using primary adipose-derived stem cells. The effects of biochanin A (at a physiologically relevant concentration of 0.1–1 μM) were assessedin vitrousing various approaches, including Oil red O staining, Nile red staining, alizarin red S staining, alkaline phosphatase (ALP) activity, flow cytometry, RT-PCR, and western blotting. The results showed that biochanin A significantly suppressed adipocyte differentiation, as demonstrated by the inhibition of cytoplasmic lipid droplet accumulation, along with the inhibition of peroxisome proliferator-activated receptor gamma (PPARγ), lipoprotein lipase (LPL), and leptin and osteopontin (OPN) mRNA expression, in a dose-dependent manner. On the other hand, treatment of cells with 0.3 μM biochanin A increased the mineralization and ALP activity, and stimulated the expression of the osteogenic marker genes ALP and osteocalcin (OCN). Furthermore, biochanin A induced the expression of runt-related transcription factor 2 (Runx2), osteoprotegerin (OPG), and Ras homolog gene family, member A (RhoA) proteins. These observations suggest that biochanin A prevents adipogenesis, enhances osteoblast differentiation in mesenchymal stem cells, and has beneficial regulatory effects in bone formation.

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Effects of melatonin on the proliferation and differentiation of rat adipose-derived stem cells

Indian Journal of Plastic Surgery ◽

10.1055/s-0039-1699220 ◽

2008 ◽

Vol 41 (01) ◽

pp. 08-14 ◽

Cited By ~ 1

Author(s):

Arash Zaminy ◽

Iraj Ragerdi Kashani ◽

Mohammad Barbarestani ◽

Azim Hedayatpour ◽

Reza Mahmoudi ◽

...

Keyword(s):

Stem Cells ◽

Flow Cytometry ◽

Culture Media ◽

Adipose Derived Stem Cells ◽

Osteogenic Medium ◽

Adult Rats ◽

Alizarin Red ◽

Alp Activity ◽

Proliferation And Differentiation

ABSTRACT Background: Osteogenesis driven by adipose-derived stem cells (ADSCs) is regulated by physiological and pathological factors. Accumulating evidence from in vitro and in vivo experiments suggests that melatonin may have an influence on bone formation. However, little is known about the effects of melatonin on osteogenesis, which thus remains to be elucidated. This study was performed to determine whether melatonin at physiological concentrations (0.01-10 nM) could affect the in vitro proliferation and osteogenic differentiation of rat ADSCs.Materials and Methods: ADSCs were isolated from the fat of adult rats. After cell expansion in culture media and through three passages, osteogenesis was induced in a monolayer culture using osteogenic medium with or without melatonin at physiological concentrations (0.01-10 nM). After four weeks, the cultures were examined for mineralization by Alizarin Red S and von Kossa staining and for alkaline phosphatase (ALP) activity using an ALP kit. Cell viability and apoptosis were also assayed by 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTT) assay and flow cytometry, respectively.Results: The results indicated that at physiological concentrations, melatonin suppressed proliferation and differentiation of ADSCs. These data indicate that ADSCs exposed to melatonin, had a lower ALP activity in contrast to the cells exposed to osteogenic medium alone. Similarly, mineral deposition (calcium level) also decreased in the presence of melatonin. Flow cytometry confirmed that cell growth had decreased and that the numbers of apoptotic cells had increased.Conclusion: These results suggest that the physiological concentration of melatonin has a negative effect on ADSC osteogenesis.

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

International Journal of Endocrinology ◽

10.1155/2020/1860842 ◽

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.

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

10.21203/rs.3.rs-33693/v1 ◽

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.

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Rg1 promotes the proliferation and adipogenic differentiation of human adipose-derived stem cells via FXR1/Lnc-GAS5-AS1 pathway

Current Stem Cell Research & Therapy ◽

10.2174/1574888x16666211129121414 ◽

2021 ◽

Vol 16 ◽

Author(s):

Fang-Tian Xu ◽

Yin-Li Xu ◽

Yong-Xian Rong ◽

Dong-Lin Huang ◽

Zhong-Hong Lai ◽

...

Keyword(s):

Stem Cells ◽

Regenerative Medicine ◽

Signaling Pathway ◽

Regulatory Network ◽

Transcriptional Regulatory Network ◽

Adipogenic Differentiation ◽

Cell Counting ◽

Adipose Derived Stem Cells ◽

Alizarin Red ◽

Medicine Research

Background: Human adipose-derived stem cells (hASCs) play an important role in regenerative medicine. Objective: Exploring the mechanism of Rg1 in the promotion of the proliferation and adipogenic differentiation of hASCs is important in regenerative medicine research. Methods: In order to observe ginsenoside Rg1 in promoting the proliferation and adipogenic differentiation of hASCs, Rg1 medium at different concentrations was established and tested using the cell counting kit-8 (CCK-8) assay, oil red O staining, alizarin red, and alcian blue. Compared to the control, differentially expressed genes (DEGs) were screened via DEG analysis, which were carried out in the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. To explore the relationship among mRNA, long non-coding RNA (lncRNA) and microRNA (miRNA), we constructed a competing endogenous RNA (ceRNA) network. Results: In this study, Rg1 was observed to promote the proliferation and adipogenic differentiation of hASCs. Additionally, enriched BPs and KEGG pathways may be involved in the promotion process, where FXR1 and Lnc-GAS5-AS1 were found to be regulatory factors. The regulatory network suggested that Rg1 could regulate the adipocytokine signaling pathway and IL−17 signaling pathway via FXR1 and Lnc-GAS5-AS1, which served as the mechanism encompassing the promotion of Rg1 on the proliferation and adipogenic differentiation of hASCs. Conclusion: A comprehensive transcriptional regulatory network related to the promotion ability of Rg1 was constructed, revealing mechanisms regarding Rg1’s promotion of the proliferation and adipogenic differentiation of hASCs. The present study provides a theoretical basis in optimizing the function of hASCs.

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Ligustrum Japonicum fructus Induces Anti-adipogenetic and Pro-osteoblastogenic Activities in Human Bone Marrow-derived Mensencymal Stem Cells (P06-016-19)

Current Developments in Nutrition ◽

10.1093/cdn/nzz031.p06-016-19 ◽

2019 ◽

Vol 3 (Supplement_1) ◽

Author(s):

JungHwan Oh ◽

Fatih Karadeniz ◽

JungIm Lee ◽

Youngwan Seo ◽

Chang-Suk Kong

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Lipid Accumulation ◽

Osteoblast Differentiation ◽

Adipogenic Differentiation ◽

Dependent Manner ◽

Cellular Lipid ◽

Protein Levels ◽

Alp Activity ◽

Ligustrum Japonicum

Abstract Objectives Masenchymal stem cells (MSCs) have pluripotent differentiation properties that confirmed to differentiate into myocyte, adipocyte, osteoblast, neuron and chondrocyte when specific culture conditions and stimuli are applied. In bone, both adipocytes and osteoblasts are derived from bone marrow MSCs (BMSCs) and production of these cells has been reported as reciprocal processes. The bone mass disequilibrium causes osteoporosis, as a result of elevated adipogenic differentiation accompanied by reducing bone formation. Therefore, in this study the effect of Ligustrum japonicum fructus (Waxleaf privet) on the adipogenesis and osteoblast differentiation was investigated in BMSCs. Methods The fruits of L. japonicum were extracted with dichloromethane and methanol, and the combined extracts were concentrated. Differentiation of BMSCs was performed by changing the medium into adipocyte and osteoblast differentiation supplied by Promocell GmbH. The cellular lipid was stained with Oil Red O and the alkaline phosphatase (ALP) activity was measured using a colormetric assay kit (Biovision, Inc.). The relative protein levels were measured by immunoblotting assay. Results Presense of L. japonicum fructus extract (LJE) inhibited the cellular lipid accumulation in a dose-dependent manner. Consistent with the effects on lipid accumulation, the adipocyte specific genes including PPARγ, C/EBPα and SREBP1c was down regulated by treatment with LJE. Moreover, treatment with LJE enhanced osteoblast differentiation observed as increased ALP activity and upregulated the proteALP, BMP-2 and osteocalcine protein levels. Conclusions The results indicated that LJE may prevent bone loss by inhibiting adipogenesis while activating the osteogenic differentiation in BMSCs. Therefore, LJE possess the potential to be utilized as a source of nutraceutical agents against osteoporosis. Funding Sources This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP).

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Human Dental Pulp Stem Cells via the NF-κB Pathway

Cellular Physiology and Biochemistry ◽

10.1159/000430145 ◽

2015 ◽

Vol 36 (5) ◽

pp. 1725-1734 ◽

Cited By ~ 5

Author(s):

Shensheng Gu ◽

Shujun Ran ◽

Feng Qin ◽

Dong Cao ◽

Jia Wang ◽

...

Keyword(s):

Stem Cells ◽

Dental Pulp ◽

Dental Pulp Stem Cells ◽

Marker Genes ◽

Nodule Formation ◽

Dependent Manner ◽

Odontoblastic Differentiation ◽

Alp Activity ◽

Odontogenic Differentiation ◽

Human Dental Pulp

Background/Aims: Odontogenic differentiation of human dental pulp stem cells (HDPSCs) is regulated by multiple factors and signaling molecules. However, their regulatory mechanisms are not completely understood. In this study, we investigated the role of Zinc finger and BTB domain-containing 20 (ZBTB20) in odontoblastic differentiation of HDPSCs. Methods: HDPSCs were obtained from human third molars and ZBTB20 expression was examined by qRT-PCR and western blot. Their osteo/odontogenic differentiation and the involvement of NF-κB pathway were subsequently investigated. Results: The expression of ZBTB20 is upregulated in a time-dependent manner during odontogenic differentiation of hDPSCs. Inhibition of ZBTB20 reduced osteogenic medium (OM)-induced odontogenic differentiation, reflected in decreased alkaline phosphatase (ALP) activity, mineralized nodule formation and mRNA expression of odonto/osteogenic marker genes. In contrast, overexpression of ZBTB20 enhanced ALP activity, mineralization and the expression of differentiation marker genes. Furthermore, the expression of IκBa was increased by ZBTB20 silencing in HDPSCs, whereas ZBTB20 overexpression decreased IκBa and enhanced nuclear NF-κB p65. Inhibition of the NF-κB pathway significantly suppressed the odontogenic differentiation of HDPSCs induced by ZBTB20. Conclusion: This study shows for the first time that ZBTB20 plays an important role during odontoblastic differentiation of HDPSCs and may have clinical implications for regenerative endodontics.

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Thrombin-activated platelet rich plasma (PRP) enhanced osteogenic differentiation of bone marrow mesenchymal stem cells by activing autophagy through miR-140-3p/SPRED2 axis

10.21203/rs.3.rs-136527/v1 ◽

2020 ◽

Author(s):

Shuting Jiang ◽

Hongyan Liu ◽

Weiyan Zhu ◽

Hui Yan ◽

Beizhan Yan

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Cell Proliferation ◽

Mesenchymal Stem Cells ◽

Osteogenic Differentiation ◽

Luciferase Reporter ◽

Dependent Manner ◽

Alizarin Red ◽

Alp Activity ◽

Marrow Mesenchymal Stem Cells

Abstract Background Mesenchymal stem cells transplantation gradually become a potential treatment for bone defect in clinic practice. This study aimed to investigate the molecular mechanism of PRP and autophagy for osteogenic differentiation in bone marrow mesenchymal stem cells (BMSCs). Methods Thrombin activated PRP was prepared and the BMSCs were treated with activated PRP with different concentration and transfected with miR-140-3p vector (mimics or inhibitor), si-SPRED2 or co-transfected with miR-140-3p inhibitor and si-SPRED2, respectively. qRT-PCR and Western blotting were used to determine the mRNA expression and protein expression. A luciferase reporter assay was conducted to identified the targeting relationship between iR-140-3p and SPRED2 Subsequently, cell proliferation was detected by MTT and ALP activity was also determined. Alizarin red staining was used for the evaluating the formation of calcium nodules. Results MiR-140-3p expression was found to be inhibited by PRP in a dose-dependent manner, besides, cell proliferation, ALP activity, the expression of COL-I, OPN, Runx2 and OCN, and the formation of calcium nodules related to osteogenic differentiation were enhanced by PRP. Subsequently, we found that PRP activated autophagy and up-regulated SPRED2 expression in BMSCs through suppressing miR-140-3p expression. Moreover, we confirmed that miR-140-3p targeted SPRED2 and negatively regulation its expression. Finally, the findings showed that inhibition of miR-140-3p enhanced cell proliferation, osteogenic differentiation and autophagy of BMSCs by negatively regulating SPRED2 expression. Conclusion Thrombin activated PRP accelerated osteogenic differentiation of BMSCs by activing autophagy through miR-140-3p/SPRED2 axis.

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Impacts of Angelica Polysaccharide on Proliferation and Differentiation of Mesenchymal Stem Cells of Rat Bone Marrow

Journal of Healthcare Engineering ◽

10.1155/2022/5277356 ◽

2022 ◽

Vol 2022 ◽

pp. 1-11

Author(s):

Shimao Yang ◽

Fei Gao ◽

Min Li ◽

Zhennan Gao

Keyword(s):

Adipogenic Differentiation ◽

Angelica Sinensis ◽

Dependent Manner ◽

Isolation And Identification ◽

Alizarin Red ◽

Alp Activity ◽

Through Flow ◽

Proliferation And Differentiation ◽

The Impact

In literature, antiosteoporotic effects of Angelica sinensis root have been confirmed, but the impact of Angelica sinensis polysaccharide (ASP) on osteoblastic or adipogenic distinction of BMSCs is limited. This paper aimed to explore the role of ASP on proliferation and differentiation of rat BMSCs. Rat BMSCs were subjected to isolation and identification through flow cytometry. The proliferation of rat BMSCs under ASP was performed by CCK-8 kit. Measures of osteogenesis under different concentrations of ASP were detected by using alizarin red staining for mesenchymal cells differentiation and ALP activity assay to identify ALP activity. Quantitative RT-PCR was selected to identify osteoblastic or adipogenic biomarkers from a genetic perspective. Likewise, we have evaluated measures of indicators of Wnt/β-catenin signal. ASP significantly promoted the proliferation, increased osteogenesis, and decreased adipogenesis of rat BMSCs within the limit of 20–60 mg/L in a dose-dependent manner but was suppressed at 80 mg/L. The expression of cyclin D1 and ß-catenin showed a considerable rise over the course of ASP induced osteogenesis. Dickkopf 1 (DKK1) suppressed the regulation of rat BMSCs differentiation through the mediation of ASP. We have observed that ASP upregulated the osteogenic but downregulated adipogenic differentiation of BMSCs, and our findings help to contribute to effective solutions for treating bone disorders.

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197 THE EFFECT OF ZINC ON THE DIFFERENTIATION OF ADIPOSE-DERIVED STEM CELLS INTO OSTEOBLASTS

Reproduction Fertility and Development ◽

10.1071/rdv29n1ab197 ◽

2017 ◽

Vol 29 (1) ◽

pp. 207 ◽

Cited By ~ 1

Author(s):

J. C. Bertels ◽

M. Rubessa ◽

S.R. Schreiber ◽

M. B. Wheeler

Keyword(s):

Stem Cells ◽

Bone Formation ◽

Negative Control ◽

Nodule Formation ◽

Adipose Derived Stem Cells ◽

Osteogenic Medium ◽

Alizarin Red ◽

The Media ◽

Positive Effect

The aim of this project was to evaluate the effects of zinc in osteogenic media and its effect on the differentiation of adipose-derived stem cells (ASC) into osteoblasts. Zinc has a stimulatory effect on bone formation and mineralization in vivo and vitro (Seo et al. 2010 Nutr. Res. Pract. 4, 356–361). Our hypothesis was that the presence of zinc in the osteogenic media would positively influence both the speed of formation and the number of osteoblastic nodules formed. Swine ASC were isolated as described (Monaco et al. 2009 Open Tissue Eng. Regen. Med. J. 2, 20–33). The ASC were divided in 8 different treatments: 6 different concentrations of zinc in the osteogenic medium (8, 4, 0.8, 0.4, 0.08, and 0.04 mM) plus 2 control treatments (osteogenic medium without zinc and a negative control, DMEM). The media was changed twice a week for 4 weeks. The experiment was replicated 4 times. At the end of the culture period, cells were stained with Alizarin Red S. In each well, we counted the nodules and divided them in 2 categories: formed and forming nodules. The second evaluation that we did was to evaluate the diameter of the largest nodules (2/well) in each group. Data were analysed by ANOVA using the Generalized Linear Model procedure (SPSS, IBM Corp., Armonk, NY, USA). Bonferroni’s post-hoc test was used to perform statistical multiple comparison. The α-level was set at 0.05. The results showed that the doses of zinc of both 4 and 8 mM were toxic to the whole cell populations in this treatment, which was indicated by cell death, whereas the concentrations of 0.8 and 0.4 mM were not cytotoxic but no nodules formed. Here we report the results that are greater than zero in Table 1. There is a positive effect on nodule formation when the zinc is added to the media. It is clear that the total number of nodules is different between the 0.08 mM zinc group and the control (P < 0.003). When we evaluated nodule diameter we found a direct correlation between the zinc concentration and the diameter of the nodules: 292.7 (±136.6) v. 366.8 (±218.7) v. 423.7 (±267.7) µm for the control, 0.04 mM zinc, and 0.08 mM zinc, respectively. The largest nodule was found in the 0.08 mM zinc treatment at 886.6 µm. These results confirmed the positive effect of this mineral on bone formation. This preliminary experiment is the first step towards the analysis of the behaviour of ASC on scaffolds with zinc incorporated into their matrix. Table 1. The average number (SD in parentheses) of formed and forming osteoblast nodules compared between treatment groups

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miR-452-3p inhibited osteoblast differentiation by targeting Smad4

PeerJ ◽

10.7717/peerj.12228 ◽

2021 ◽

Vol 9 ◽

pp. e12228

Author(s):

Ming Wu ◽

Hongyan Wang ◽

Dece Kong ◽

Jin Shao ◽

Chao Song ◽

...

Keyword(s):

Bone Formation ◽

Osteoblast Differentiation ◽

Cell Model ◽

Pcr Analysis ◽

Marker Genes ◽

Alizarin Red ◽

Alp Activity ◽

Complex Process ◽

Collagen Type 1

Osteoblast differentiation is a complex process that is essential for normal bone formation. A growing number of studies have shown that microRNAs (miRNAs) are key regulators in a variety of physiological and pathological processes, including osteogenesis. In this study, BMP2 was used to induce MC3T3-E1 cells to construct osteoblast differentiation cell model. Then, we investigated the effect of miR-452-3p on osteoblast differentiation and the related molecular mechanism by RT-PCR analysis, Western blot analysis, ALP activity, and Alizarin Red Staining. We found that miR-452-3p was significantly downregulated in osteoblast differentiation. Overexpression miR-452-3p (miR-452-3p mimic) significantly inhibited the expression of osteoblast marker genes RUNX2, osteopontin (OPN), and collagen type 1 a1 chain (Col1A1), and decreased the number of calcium nodules and ALP activity. In contrast, knockdown miR-452-3p (miR-452-3p inhibitor) produced the opposite effect. In terms of mechanism, we found that Smad4 may be the target of miR-452-3p, and knockdown Smad4 (si-Smad4) partially inhibited the osteoblast differentiation enhanced by miR-452-3p. Our results suggested that miR-452-3p plays an important role in osteoblast differentiation by targeting Smad4. Therefore, miR-452-3p is expected to be used in the treatment of bone formation and regeneration.

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