Effect of GARP on osteogenic differentiation of bone marrow mesenchymal stem cells via the regulation of TGFβ1 in vitro

Mesenchymal stem cells (MSCs), which have multipotential differentiation and self-renewal potential, are possible cells for tissue engineering. Transforming growth factor β1 (TGFβ1) can be produced by MSCs in an inactive form, and the activation of TGFβ1 functions as an important regulator of osteogenic differentiation in MSCs. Recently, studies showed that Glycoprotein A repetitions predominant (GARP) participated in the activation of latent TGFβ1, but the interaction between GARP and TGFβ1 is still undefined. In our study, we successfully isolated the MSCs from bone marrow of rats, and showed that GARP was detected in bone mesenchymal stem cells (BMSCs). During the osteogenic differentiation of BMSCs, GARP expression was increased over time. To elucidate the interaction between GARP and TGFβ1, we downregulated GARP expression in BMSCs to examine the level of active TGFβ1. We then verified that the downregulation of GARP decreased the secretion of active TGFβ1. Furthermore, osteogenic differentiation experiments, alkaline phosphatase (ALP) activity analyses and Alizarin Red S staining experiments were performed to evaluate the osteogenic capacity. After the downregulation of GARP, ALP activity and Alizarin Red S staining significantly declined and the osteogenic indicators, ALP, Runx2, and OPN, also decreased, both at the mRNA and protein levels. These results demonstrated that downregulated GARP expression resulted in the reduction of TGFβ1 and the attenuation of osteoblast differentiation of BMSCs in vitro.

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miRNA-126 Inhibits Osteogenic Differentiation of Rat Bone Marrow Mesenchymal Stem Cells (BMSCs)

Journal of Biomaterials and Tissue Engineering ◽

10.1166/jbt.2022.2943 ◽

2022 ◽

Vol 12 (4) ◽

pp. 794-799

Author(s):

Le Chang ◽

Wei Duan ◽

Chuang Wang ◽

Jian Zhang

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Osteogenic Differentiation ◽

Mrna Level ◽

Alizarin Red ◽

Runx2 Expression ◽

Alp Activity ◽

Smad4 Protein ◽

Marrow Mesenchymal Stem Cells

This study was to determine whether microRNA (miRNA)-126 regulates osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). Rat BMSCs were extracted and stimulated for osteogenic differentiation. Functional experiments were conducted to assess miR-126’s impact on BMSCs differentiation. Western blot and RT-qPCR determined miR-126 expression. ALP activity detection and alizarin red staining detection were also performed. After osteogenic differentiation of BMSCs, miR-126 expression was gradually decreased over time. Overexpression of miR-26 decreased ALP activity, Notch signaling activity as well as declined Runx2 expression and calcium Salt nodules after treatment. Importantly, we found that Smad4 serves as a target of miR-126 while upregulation of the miRNA was accompanied with the decreased Smad4 protein expression without affecting the Smad4 mRNA level. In conclusion, miR-126 restrains osteogenic differentiation through inhibition of SMAD4 signaling, providing a novel insight into the mechanism.

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Polypeptides IGF-1C and P24 synergistically promote osteogenic differentiation of bone marrow mesenchymal stem cells in vitro through the p38 and JNK signaling pathways

10.1101/2021.06.09.447738 ◽

2021 ◽

Author(s):

Gaoying Ran ◽

Wei Fang ◽

Lifang Zhang ◽

Yuting Peng ◽

Jiatong Li ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Osteogenic Differentiation ◽

Bone Morphogenetic Protein 2 ◽

Cell Counting ◽

Signal Pathways ◽

Alizarin Red ◽

Alp Activity ◽

Marrow Mesenchymal Stem Cells

Objectives: Insulin-like growth factor-1 (IGF-1) and bone morphogenetic protein 2 (BMP-2) both promote osteogenesis of bone marrow mesenchymal stem cells (BMSCs). IGF-1C, the C domain peptide of IGF-1, and P24, a BMP-2-derived peptide, both have similar biological activities as their parent growth factors. This study aimed to investigate the effects and their mechanisms of polypeptides IGF-1C and P24 on the osteogenic differentiation of BMSCs. Methods: The optimum concentrations of IGF-IC and P24 were explored. The effects of the two polypeptides on the proliferation and osteogenic differentiation of BMSCs were examined using the Cell Counting Kit-8 (CCK-8), Alkaline phosphatase (ALP) staining, ALP activity assay, alizarin red S staining, qPCR, and western blotting. In addition, specific pathway inhibitors were utilized to explore whether p38 and JNK pathways were involved in this process. Results: The optimal concentrations of action were both 50 g/ml. IGF-1C and P24 synergistically promoted the proliferation of BMSCs, increased ALP activity and the formation of calcified nodules and upregulated the mRNA and protein levels of osterix (Osx), runt-related transcription factor 2 (Runx2), and osteocalcin (Ocn), phosphorylation level of p38 and JNK proteins also improved. Inhibition of the pathways significantly reduced the activation of p38 and JNK, blocked the expression of Runx2 while inhibiting ALP activity and the formation of calcified nodules. Conclusions: These findings suggest IGF-1C and P24 synergistically promote the osteogenesis of BMSCs through activation of p38 and JNK signal pathways.

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Dihydrotestosterone and 17-Estradiol Enhancement of In Vitro Osteogenic Differentiation of Castrated Male Rat Bone Marrow Mesenchymal Stem Cells (rBMMSCs)

International Journal of Hematology-Oncology and Stem Cell Research ◽

10.18502/ijhoscr.v13i4.1897 ◽

2019 ◽

Author(s):

FAM Abo-Aziza ◽

AA Zaki ◽

AS Amer ◽

RA Lotfy

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Osteogenic Differentiation ◽

Population Doubling ◽

Calcium Deposition ◽

Alp Activity ◽

Marrow Mesenchymal Stem Cells ◽

Rat Bone

Background: In vitro impact of dihydrotestosterone (DHT) and 17-estradiol (E2) in osteogenic differentiation of castrated rat bone marrow mesenchymal stem cells (rBMMSC) still need to be clarified. Materials and Methods: The viability, proliferation and density of cultured rBMMSC isolated from sham operated (Sham) and castrated (Cast) male rats were evaluated. rBMMSC were cultured with osteogenic differentiating medium (ODM) in the presence of DHT (5,10 nM) and E2 (10,100 nM). Osteogenesis was evaluated by alizarin red staining and measurement of calcium deposition and bone alkaline phosphatase (BALP) activity. Results: Population doubling (PD) of rBMMSC isolated from Cast rats was significantly lower (P<0.05) compared to that isolated from Sham rats. rBMMSC from Cast rats showed low scattered calcified nodule after culturing in ODM and did not cause a significant increase in calcium deposition and B-ALP activity compared to rBMMSCs from Sham rats. Exposure of rBMMSC isolated from Cast rats to DHT (5 nM) or E2 (10 nM) in ODM showed medium scattered calcified nodules with significantly higher (P<0.05) calcium deposition and B-ALP activity. Moreover, exposure of rBMMSC to DHT (10 nM) or E2 (100 nM) showed high scattered calcified nodules with higher (P<0.01) calcium deposition and B-ALP activity Conclusion: These results indicated that the presence of testes might participate in controlling the in vitro proliferation and osteogenic differentiation capacity of rBMMSCs. DHT and E2 can enhance the osteogenic capacity of rBMMSCs in a dose-dependent manner. Based on these observations, optimum usage of DHT and E2 can overcome the limitations of MSCs and advance the therapeutic bone regeneration potential in the future.

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Impaired Osteogenic Differentiation of Mesenchymal Stem Cells Derived From Multiple Myeloma Patients Is Associated with a Failure In the Deactivation of the NOTCH Pathway

Blood ◽

10.1182/blood.v116.21.1894.1894 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1894-1894

Author(s):

Song Xu ◽

Jinsong Hu ◽

Dehui Xu ◽

Isabelle Vande Broek ◽

Xavier Leleu ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Osteogenic Differentiation ◽

Notch Pathway ◽

Notch Signalling ◽

Matrix Mineralization ◽

Alp Activity ◽

Hes1 Expression

Abstract Abstract 1894 Mesenchymal stem cells (MSCs) give rise to bone marrow (BM) stromal cells and play an essential role in the formation and function of the MM microenvironment. Some recent studies revealed that MSCs from myeloma patients (MM-hMSCs) show an enhanced spontaneous and myeloma cell-induced production of cytokines and a distinctive gene expression profile, when compared to MSCs from normal donors (ND-hMSCs). However, regarding the osteogenic differentiation ability of MM-hMSCs conflicting observations were reported. In this study, we observed that MM-hMSCs, especially for those from MM patients with bone lesions, exhibited in the presence of osteogenic differentiation (OD) medium, significantly decreased alkaline phosphatase (ALP) activity, reduced expression of specific osteogenic markers (OPN, BMP2, OTX and BSP) and impaired matrix mineralization, compared to ND-hMSCs. However, MGUS-hMSCs, did not show a significantly impaired osteogenesis ability. Primary CFU-ALP assay from BM samples of diseased mice in the 5T33MM model also confirmed that the osteogenic differentiation ability of MSCs was impaired. Previous reports indicated that MM cells can suppress MSCs osteogenesis by HGF and DKK1 as observed in vitro (Giuliani et al, Cancer Res. 2007; Standal et al, Blood. 2007). Since MM-hMSCs have been cultured in vitro for several weeks and without any stimulation of MM cells, we believe that the impaired osteogenic differentiation of MM-hMSCs was due to an intrinsic abnormality. Several reports suggested that NOTCH signalling can maintain bone marrow mesenchymal progenitors in a more undifferentiated state by suppressing osteoblast differentiation (Hilton et al, Nat Med. 2008; Zanotti et al, Endocrinology. 2008). Therefore, we postulate that impaired osteogenic ability of MM-hMSCs might be (at least partly) related to abnormal NOTCH activity during osteogenesis. We found by quantitative real time PCR that NOTCH1, NOTCH2, Dll-1, Jagged-1, and NOTCH pathway downstream genes hes1, hey1, hey2, heyL were considerably decreased in ND-hMSCs after shifting them from normal culture medium to OD medium, indicating that NOTCH signalling was gradually suppressed during MSC osteogenesis. However, it was observed that the expression of NOTCH1, Jagged-1, Hes1 and Hes5 in MM-hMSCs did not decrease to the level of ND-hMSC with statistical difference. This implicates that the NOTCH signaling pathway remains in MM-hMSCs over-activated even in the presence of osteogenesis inducing signals. When the NOTCH signalling inhibitor DAPT was added to MM-hMSCs in OD medium, we found that hes1 expression was suppressed while, RUNX2 expression, a key transcription factor for osteoblastogenesis, as well as ALP activity, osteogenic genes expression and mineralization deposition were all increased. In conclusion our data indicate that MM-hMSCs exhibit in vitro lower osteogenic differentiation ability compared to ND-hMSCs, and that this impairement is associated with an inappropriate NOTCH pathway deactivation during the osteogenesis process. Targeting hMSCs in vivo by NOTCH inhibitors might have therapeutical potential to control bone disease in MM patients. Disclosures: No relevant conflicts of interest to declare.

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Osteogenic differentiation of rat bone mesenchymal stem cells cultured on poly (hydroxybutyrate-co-hydroxyvalerate), poly (ε-caprolactone) scaffolds

Journal of Materials Science Materials in Medicine ◽

10.1007/s10856-021-06615-6 ◽

2021 ◽

Vol 32 (11) ◽

Author(s):

Ana A. Rodrigues ◽

Nilza A. Batista ◽

Sônia M. Malmonge ◽

Suzan A. Casarin ◽

José Augusto M. Agnelli ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Osteogenic Differentiation ◽

Mtt Assay ◽

Vero Cells ◽

Sem Analysis ◽

Bone Mesenchymal Stem Cells ◽

Alizarin Red ◽

Cytochemical Study ◽

Alp Activity

AbstractBioresorbable biomaterials can fill bone defects and act as temporary scaffold to recruit MSCs to stimulate their differentiation. Among the different bioresorbable polymers studied, this work focuses on poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) and poly(ε-caprolactone) (PCL). Were prepared blends of PHBV and PCL to obtain PHBV based biomaterials with good tenacity, important for bone tissue repair, associated with biocompatible properties of PCL. This study assesses the viability of Vero cells on scaffolds of PHBV, PCL, and their blends and the osteogenic differentiation of mesenchymal stem cells (MSCs). Materials were characterized in surface morphology, DSC and Impact Strength (IS). Vero cells and MSCs were assessed by MTT assay, cytochemical and SEM analysis. MSC osteogenic differentiation was evaluated through alizarin red staining and ALP activity. We found some roughness onto surface materials. DSC showed that the blends presented two distinct melting peaks, characteristic of immiscible blends. IS test confirmed that PHBV-PCL blends is an alternative for increase the tenacity of PHBV. MTT assay showed cells with high metabolic activities on extract toxicity test, but with low activity in the direct contact test. SEM analysis showed spreading cells with irregular and flattened morphology on different substrates. Cytochemical study revealed that MSCs maintained their morphology, although in smaller number for MSCs. The development of nodules of mineralized organic matrix in MSC cultures was identified by alizarin red staining and osteogenic differentiation was confirmed by the quantification of ALP activity. Thus, our scaffolds did not interfere on viability of Vero cells or the osteogenic differentiation of MSCs.

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CHNQD-00603 Promotes Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells by the miR-452-3p-Mediated Autophagy Pathway

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.779287 ◽

2021 ◽

Vol 9 ◽

Author(s):

Shanshan Xin ◽

Shao-Ming Li ◽

Ling Gao ◽

Jing-Jing Zheng ◽

Yan-Wei Wu ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Osteogenic Differentiation ◽

Bone Structure ◽

Alizarin Red S ◽

Hydroxyl Group ◽

Alizarin Red ◽

Qrt Pcr ◽

Marrow Mesenchymal Stem Cells

Background: Periodontitis is a chronic and progressive disease accompanied by bone loss. It is still a challenge to restore the bone structure. The osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) plays a decisive role in bone restoration and regeneration. Marine natural products (MNPs) have multiple biological activities, including anti-tumor and anti-inflammatory properties. However, the exploration of MNPs in osteogenesis is far from sufficient.Methods: We obtained a series of derivatives through structural optimization from 4-phenyl-3,4-dihydroquinolin-2(1H)-one alkaloid isolated from Scopulariopsis sp. Some preliminary cytological experiments showed that CHNQD-00603, obtained by adding a methoxy group to the position C3 and a hydroxyl group to the position C4 of 4-phenyl-3,4-dihydroquinolin-2(1H)-one, might promote the osteogenic differentiation of BMSCs. To further investigate the effects of CHNQD-00603 on BMSCs, we performed a CCK-8 assay and qRT-PCR, alkaline phosphatase staining (ALP), and alizarin red S staining to assess the cytotoxicity and the ability of osteogenic differentiation of CHNQD-00603. The autophagy level was assessed and validated by WB, qRT-PCR, and transmission electron microscopy. Then, 3-methyladenine (3-MA) was added to further examine the role of autophagy. Based on the expression of autophagy-related genes, we predicted and examined the potential miRNAs by bioinformatics.Results: CCK-8 assay showed that CHNQD-00603 at 1 µg/ml did not influence BMSCs activity. However, the proliferation rate decreased from the seventh day. qRT-PCR, ALP staining, ALP activity assay, and Alizarin red S staining showed that the best concentration of CHNQD-00603 to promote osteogenic differentiation was 1 µg/ml. Further investigations indicated that CHNQD-00603 activated autophagy, and the inhibition of autophagy by 3-MA attenuated CHNQD-00603-enhanced osteogenic differentiation. Subsequently, the findings from bioinformatics and qRT-PCR indicated that miR-452-3p might be a regulator of autophagy and osteogenesis. Furthermore, we transfected BMSCs with miR-452-3p NC and mimics separately to further determine the function of miR-452-3p. The data showed that the overexpression of miR-452-3p moderated the level of autophagy and osteogenic differentiation of CHNQD-00603-treated BMSCs.Conclusion: Our data suggested that CHNQD-00603 promoted the osteogenic differentiation of BMSCs by enhancing autophagy. Meanwhile, miR-452-3p played a regulatory role in this process.

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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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Acute Lymphoblastic Leukemia Cells Inhibit the Differentiation of Bone Mesenchymal Stem Cells into Osteoblasts In Vitro by Activating Notch Signaling

Stem Cells International ◽

10.1155/2015/162410 ◽

2015 ◽

Vol 2015 ◽

pp. 1-11 ◽

Cited By ~ 9

Author(s):

Gui-Cun Yang ◽

You-Hua Xu ◽

Hong-Xia Chen ◽

Xiao-Jing Wang

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Acute Lymphoblastic Leukemia ◽

Notch Signaling ◽

Osteogenic Differentiation ◽

Lymphoblastic Leukemia ◽

Induction Medium ◽

Bone Mesenchymal Stem Cells ◽

Alizarin Red ◽

Alp Activity

The disruption of normal hematopoiesis has been observed in leukemia, but the mechanism is unclear. Osteoblasts originate from bone mesenchymal stem cells (BMSCs) and can maintain normal hematopoiesis. To investigate how leukemic cells inhibit the osteogenic differentiation of BMSCs and the role of Notch signaling in this process, we cocultured BMSCs with acute lymphoblastic leukemia (ALL) cells in osteogenic induction medium. The expression levels of Notch1, Hes1, and the osteogenic markers Runx2, Osteopontin (OPN), and Osteocalcin (OCN) were assessed by real-time RT-PCR and western blotting on day 3. Alkaline phosphatase (ALP) activity was analyzed using an ALP kit, and mineralization deposits were detected by Alizarin red S staining on day 14. And then we treated BMSCs with Jagged1 and anti-Jagged1 neutralizing Ab. The expression of Notch1, Hes1, and the abovementioned osteogenic differentiation markers was measured. Inhibition of the expression of Runx2, OPN, and OCN and reduction of ALP activity and mineralization deposits were observed in BMSCs cocultured with ALL cells, while Notch signal inhibiting rescued these effects. All these results indicated that ALL cells could inhibit the osteogenic differentiation of BMSCs by activating Notch signaling, resulting in a decreased number of osteoblastic cells, which may impair normal hematopoiesis.

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Activation of Cannabinoid Receptor 2 Enhances Osteogenic Differentiation of Bone Marrow Derived Mesenchymal Stem Cells

BioMed Research International ◽

10.1155/2015/874982 ◽

2015 ◽

Vol 2015 ◽

pp. 1-8 ◽

Cited By ~ 2

Author(s):

Yong-Xin Sun ◽

Ai-Hua Xu ◽

Yang Yang ◽

Jia-Xing Zhang ◽

Ai-Wen Yu

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Osteogenic Differentiation ◽

Cannabinoid Receptor ◽

P38 Map Kinase ◽

Immunofluorescent Staining ◽

Alizarin Red ◽

Small Interference Rna ◽

Alp Activity

Bone marrow derived mesenchymal stem cells (BM-MSCs) are considered as the most promising cells source for bone engineering. Cannabinoid (CB) receptors play important roles in bone mass turnover. The aim of this study is to test if activation of CB2receptor by chemical agonist could enhance the osteogenic differentiation and mineralization in bone BM-MSCs. Alkaline phosphatase (ALP) activity staining and real time PCR were performed to test the osteogenic differentiation. Alizarin red staining was carried out to examine the mineralization. Small interference RNA (siRNA) was used to study the role of CB2receptor in osteogenic differentiation. Results showed activation of CB2receptor increased ALP activity, promoted expression of osteogenic genes, and enhanced deposition of calcium in extracellular matrix. Knockdown of CB2receptor by siRNA inhibited ALP activity and mineralization. Results of immunofluorescent staining showed that phosphorylation of p38 MAP kinase is reduced by knocking down of CB2receptor. Finally, bone marrow samples demonstrated that expression of CB2receptor is much lower in osteoporotic patients than in healthy donors. Taken together, data from this study suggested that activation of CB2receptor plays important role in osteogenic differentiation of BM-MSCs. Lack of CB2receptor may be related to osteoporosis.

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