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Wang, Jing, Junyi Liao, Fugui Zhang, Dongzhe Song, Minpeng Lu, Jianxiang Liu, Qiang Wei et al. „NEL-Like Molecule-1 (Nell1) Is Regulated by Bone Morphogenetic Protein 9 (BMP9) and Potentiates BMP9-Induced Osteogenic Differentiation at the Expense of Adipogenesis in Mesenchymal Stem Cells“. Cellular Physiology and Biochemistry 41, Nr. 2 (2017): 484–500. http://dx.doi.org/10.1159/000456885.
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Background: BMP9 induces both osteogenic and adipogenic differentiation of mesenchymal stem cells (MSCs). Nell1 is a secretory glycoprotein with osteoinductive and anti-adipogenic activities. We investigated the role of Nell1 in BMP9-induced osteogenesis and adipogenesis in MSCs. Methods: Previously characterized MSCs iMEFs were used. Overexpression of BMP9 and NELL1 or silencing of mouse Nell1 was mediated by adenoviral vectors. Early and late osteogenic and adipogenic markers were assessed by staining techniques and qPCR analysis. In vivo activity was assessed in an ectopic bone formation model of athymic mice. Results: We demonstrate that Nell1 expression was up-regulated by BMP9. Exogenous Nell1 potentiated BMP9-induced late stage osteogenic differentiation while inhibiting the early osteogenic marker. Forced Nell1 expression enhanced BMP9-induced osteogenic regulators/markers and inhibited BMP9-upregulated expression of adipogenic regulators/markers in MSCs. In vivo ectopic bone formation assay showed that exogenous Nell1 expression enhanced mineralization and maturity of BMP9-induced bone formation, while inhibiting BMP9-induced adipogenesis. Conversely, silencing Nell1 expression in BMP9-stimulated MSCs led to forming immature chondroid-like matrix. Conclusion: Our findings indicate that Nell1 can be up-regulated by BMP9, which in turn accelerates and augments BMP9-induced osteogenesis. Exogenous Nell1 may be exploited to enhance BMP9-induced bone formation while overcoming BMP9-induced adipogenesis in regenerative medicine.
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Choi, Somang, Sung Hyun Noh, Chae Ouk Lim, Hak-Jun Kim, Han-Saem Jo, Ji Seon Min, Kyeongsoon Park und Sung Eun Kim. „Icariin-Functionalized Nanodiamonds to Enhance Osteogenic Capacity In Vitro“. Nanomaterials 10, Nr. 10 (20.10.2020): 2071. http://dx.doi.org/10.3390/nano10102071.
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Nanodiamonds (NDs) have been used as drug delivery vehicles due to their low toxicity and biocompatibility. Recently, it has been reported that NDs have also osteogenic differentiation capacity. However, their capacity using NDs alone is not enough. To significantly improve their osteogenic activity, we developed icariin (ICA)-functionalized NDs (ICA-NDs) and evaluated whether ICA-NDs enhance their in vitro osteogenic capacity. Unmodified NDs and ICA-NDs showed nanosized particles that were spherical in shape. The ICA-NDs achieved a prolonged ICA release for up to 4 weeks. The osteogenic capacities of NDs, ICA (10 μg)-NDs, and ICA (50 μg)-NDs were demonstrated by alkaline phosphatase (ALP) activity; calcium content; and mRNA gene levels of osteogenic-related markers, including ALP, runt-related transcript factor 2 (RUNX2), collagen type I alpha 1 (COL1A1), and osteopontin (OPN). In vitro cell studies revealed that ICA (50 μg)-ND-treated MC3T3-E1 cells greatly increased osteogenic markers, including ALP, calcium content, and mRNA gene levels of osteogenic-related markers, including ALP, RUNX2, COL1A1, and OPN compared to ICA (10 μg)-NDs or ND-treated cells. These our data suggest that ICA-NDs can promote osteogenic capacity.
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Zhang, Fei, Zehua Zhang, Dong Sun, Shiwu Dong, Jianzhong Xu und Fei Dai. „Periostin: A Downstream Mediator of EphB4-Induced Osteogenic Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells“. Stem Cells International 2016 (2016): 1–11. http://dx.doi.org/10.1155/2016/7241829.
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Erythropoietin-producing hepatocyte B4 (EphB4) has been reported to be a key molecular switch in the regulation of bone homeostasis, but the underlying mechanism remains poorly understood. In this study, we investigated the role of EphB4 in regulating the expression of periostin (POSTN) within bone marrow-derived mesenchymal stem cells (MSCs) and assessed its effect and molecular mechanism of osteogenic induction in vitro. Treatment with ephrinB2-FC significantly increased the expression of POSTN in MSCs, and the inhibition of EphB4 could abrogate this effect. In addition, osteogenic markers were upregulated especially in MSCs overexpressing EphB4. To elucidate the underlying mechanism of cross talk between EphB4 and the Wnt pathway, we detected the change in protein expression of phosphorylated-glycogen synthase kinase 3β-serine 9 (p-GSK-3β-Ser9) andβ-catenin, as well as the osteogenic markers Runx2 and COL1. The results showed that GSK-3βactivation and osteogenic marker expression levels were downregulated by ephrinB2-FC treatment, but these effects were inhibited by blocking integrinαvβ3 in MSCs. Our findings demonstrate that EphB4 can promote osteogenic differentiation of MSCs via upregulation of POSTN expression. It not only helps to reveal the interaction mechanism between EphB4 and Wnt pathway but also brings a better understanding of EphB4/ephrinB2 signaling in bone homeostasis.
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Di Pietro, Lorena, Marta Barba, Chiara Prampolini, Sabrina Ceccariglia, Paolo Frassanito, Alessia Vita, Enrico Guadagni et al. „GLI1 and AXIN2 Are Distinctive Markers of Human Calvarial Mesenchymal Stromal Cells in Nonsyndromic Craniosynostosis“. International Journal of Molecular Sciences 21, Nr. 12 (19.06.2020): 4356. http://dx.doi.org/10.3390/ijms21124356.
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All skeletal bones house osteogenic stem cell niches, in which mesenchymal stromal cells (MSC) provide progenitors for tissue growth and regeneration. They have been widely studied in long bones formed through endochondral ossification. Limited information is available on the composition of the osteogenic niche in flat bones (i.e., skull vault bones) that develop through direct membranous ossification. Craniosynostosis (CS) is a congenital craniofacial defect due to the excessive and premature ossification of skull vault sutures. This study aimed at analysing the expression of GLI1, AXIN2 and THY1 in the context of the human skull vault, using nonsyndromic forms of CS (NCS) as a model to test their functional implication in the aberrant osteogenic process. The expression of selected markers was studied in NCS patients’ calvarial bone specimens, to assess the in vivo location of cells, and in MSC isolated thereof. The marker expression profile was analysed during in vitro osteogenic differentiation to validate the functional implication. Our results show that GLI1 and AXIN2 are expressed in periosteal and endosteal locations within the osteogenic niche of human calvarial bones. Their expression is higher in MSC isolated from calvarial bones than in those isolated from long bones and tends to decrease upon osteogenic commitment and differentiation. In particular, AXIN2 expression was lower in cells isolated from prematurely fused sutures than in those derived from patent sutures of NCS patients. This suggests that AXIN2 could reasonably represent a marker for the stem cell population that undergoes depletion during the premature ossification process occurring in CS.
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Chen, Kai, Xianqi Li, Ni Li, Hongwei Dong, Yiming Zhang, Michiko Yoshizawa und Hideaki Kagami. „Spontaneously Formed Spheroids from Mouse Compact Bone-Derived Cells Retain Highly Potent Stem Cells with Enhanced Differentiation Capability“. Stem Cells International 2019 (05.05.2019): 1–13. http://dx.doi.org/10.1155/2019/8469012.
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The results from our recent study showed the presence of two distinct spheroid-forming mechanisms, i.e., spontaneous and mechanical. In this study, we focused on the spontaneously formed spheroids, and the character of spontaneously formed spheroids from mouse compact bone-derived cells (CBDCs) was explored. Cells from (C57BL/6J) mouse leg bones were isolated, and compact bone-derived cells were cultured after enzymatic digestion. Spontaneous spheroid formation was achieved on a culture plate with specific water contact angle as reported. The expression levels of embryonic stem cell markers were analyzed using immunofluorescence and quantitative reverse transcription polymerase chain reaction. Then, the cells from spheroids were induced into osteogenic and neurogenic lineages. The spontaneously formed spheroids from CBDCs were positive for ES cell markers such as SSEA1, Sox2, Oct4, and Nanog. Additionally, the expressions of fucosyltransferase 4/FUT4 (SSEA1), Sox2, and Nanog were significantly higher than those in monolayer cultured cells. The gene expression of mesenchymal stem cell markers was almost identical in both spheroids and monolayer-cultured cells, but the expression of Sca-1 was higher in spheroids. Spheroid-derived cells showed significantly higher osteogenic and neurogenic marker expression than monolayer-cultured cells after induction. Spontaneously formed spheroids expressed stem cell markers and showed enhanced osteogenic and neurogenic differentiation capabilities than cells from the conventional monolayer culture, which supports the superior stemness.
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Gu, Mingyong, und Runquan Zheng. „Apolipoprotein E Inhibits Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells by Inhibiting β-Catenin Expression“. Journal of Biomaterials and Tissue Engineering 9, Nr. 12 (01.12.2019): 1739–44. http://dx.doi.org/10.1166/jbt.2019.2194.
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Bone marrow mesenchymal stem cells (BMSCs) can differentiate into adipocytes, osteoblasts. Apolipoprotein E (ApoE) is closely related to bone metabolism and its effect on bone marrow mesenchymal stem cells is unclear. Therefore, this study investigated ApoE's effect on BMSCs osteogenic differentiation. BMSCs were isolated from ApoE – and WT mouse and cultured to induce osteogenic induction followed by analysis of expression of osteogenic differentiation marker genes by Real-time PCR, calcium nodules formation by ARS staining, ALP activity and -catenin protein level by Western blot. The number of bone differentiation markers, ALP activity and calcium nodules formation as well as β-catenin protein level in ApoE– group were significantly elevated compared with WT (P < 0 05). After treatment with DKK-1, β-catenin expression was significantly reduced (P < 0 05) without difference between ApoE– + DKK-1 group and WT group (P > 0 05). WT+ DKK1 group showed significantly reduced osteogenic differentiation marker expression, ALP activity and calcium nodule number compared to WT (P < 0 05) without difference between ApoE– + DKK1 group and WT group (P > 0 05). ApoE inhibits BMSCs osteogenic differentiation by inhibiting β-catenin expression.
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Li, Xiaoliang, Guofeng Xia, Hongmei Xin, Chunsheng Tao, Weiwei Lai und Peifeng Sun. „lncRNA MALAT1 Inhibits Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells by Down-Regulating WNT5A“. Journal of Biomaterials and Tissue Engineering 9, Nr. 11 (01.11.2019): 1520–27. http://dx.doi.org/10.1166/jbt.2019.2167.
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ncRNA involves in osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). WNT5A participates in the growth and development of osteogenic differentiation. This study aims to investigate whether lncRNA MALAT1 regulates BMSCs osteogenesis through WNT5A. qRT-PCR was done to detect the lncRNA MALAT1 level and osteogenic markers in osteoporosis patients and control groups. The above markers and WNT5A protein levels were detected by Western blot. The degree of osteogenic differentiation was detected by ALP activity assay and ALP staining. The differentiation ability of BMSCs after lncRNA MALAT1 overexpression was detected by ARS staining. The binding site of lncRNA MALAT1 to WNT5A was determined by dual luciferase reporter assay. lncRNA MALAT1 expression was higher in patients with osteoporosis, and decreased significantly with increased osteogenic induction. Overexpression of lncRNA MALAT1 in BMSCS reduced WNT5A level, while interference with lncRNA MALAT1 increased WNT5A levels. In cells with overexpression of lncRNA MALAT1, transfection of si-WNT5A can significantly downregulate the RUNX2, OSX, ALP, OCN, OPN, and COL1A1, thereby inhibiting osteogenic differentiation, interfering with the regulation of WNT signaling pathway and regulating BMSCs osteogenic differentiation. lncRNA MALAT1 and WNT5A can regulate BMSCs osteogenesis, thus accelerating the progression of osteoporosis.
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Kannan, Sangeetha, Jyotirmoy Ghosh und Sujoy K. Dhara. „Osteogenic differentiation potential of porcine bone marrow mesenchymal stem cell subpopulations selected in different basal media“. Biology Open 9, Nr. 10 (24.09.2020): bio053280. http://dx.doi.org/10.1242/bio.053280.
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ABSTRACTMultipotent porcine mesenchymal stem cells (pMSC) are invaluable for research and therapeutic use in regenerative medicine. Media used for derivation and expansion of pMSC may play an important role for the selection of MSC subpopulation at an early stage and thereby, the specific basal medium may also affect differentiation potential of these cells. The present study was undertaken to evaluate the effects of αMEM, aDMEM, M199, αMEM/M199, aDMEM/M199 and αMEM/aDMEM media on (1) porcine bone marrow MSC derivation; (2) expression of number of osteogenic markers (ALP, COL1A1, SPP1 and BGLAP) at 5th and 10th passage in pMSC before differentiation; and (3) differentiation of pMSC (at 5th passage) to osteogenic lineage. Morphological changes and matrix formation in osteogenic cells were evaluated by microscopic examination. Calcium deposits in osteocytes were confirmed by Alizarin Red S staining. Based on expression of different markers, it was evident that selection of bone marrow pMSC subpopulations was independent of basal media used. However, the differentiation of those pMSCs, specifically to osteogenic lineage, was dependent on the medium used for expansion of pMSC at the pre-differentiation stage. We demonstrated here that the pMSC grown in combined αMEM/aDMEM (1:1) medium expressed number of osteogenic markers and these pMSC underwent osteogenic differentiation most efficiently, in comparison to porcine mesenchymal stem cells grown in other media. In conclusion, osteogenic differentiation potential of pMSC maintained in αMEM/aDMEM medium was observed significantly higher compared to cells cultivated in other media and therefore, the combined medium αMEM/aDMEM (1:1) may preferentially be used for expansion of pMSC, if needed for osteogenic differentiation.
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Wang, Jian, Bo Xiang, Jixian Deng, Darren H. Freed, Rakesh C. Arora und Ganghong Tian. „Inhibition of Viability, Proliferation, Cytokines Secretion, Surface Antigen Expression, and Adipogenic and Osteogenic Differentiation of Adipose-Derived Stem Cells by Seven-Day Exposure to 0.5 T Static Magnetic Fields“. Stem Cells International 2016 (2016): 1–12. http://dx.doi.org/10.1155/2016/7168175.
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After seven-day exposure to 0.5-Tesla Static Magnetic Field (SMF), Adipose-derived Stem Cells (ASCs) and those labeled by superparamagnetic iron oxide (SPIO) nanoparticles were examined for viability by methyl thiazol tetrazolium (MTT) assay, proliferation by cell counting and bromodeoxyuridine (BrdU) incorporation, DNA integrity by single cell gel electrophoresis, surface antigen by flow cytometry analysis, and the expression of cytokines and genetic markers by reverse transcription-PCR and underwent adipogenic and osteogenic differentiation assessed by quantifying related specific genes expression. The SMF slightly reduced cell viability and proliferation and inhibited the expression of CD49d, CD54, and CD73 but did not damage DNA integrity. The SMF slightly downregulated the expression of cytokines including Vascular Endothelial Growth Factor (VEGF), Insulin-like Growth Factor-1 (IGF-1), Transforming Growth Factor Beta 1 (TGF-β1), genetic markers comprising Stem Cell Antigen-1 (Sca1), Octamer-4 (Oct-4), ATP-binding Cassette Subfamily B Member 1 (ABCB1), adipogenic marker genes containing Lipoprotein Lipase (LPL), Peroxisome Proliferator-Activated Receptor Gamma (PPAR-γ), and osteogenic marker genes including Secreted Phosphor-protein 1 (SPP1) and Osterix (OSX). Exposure to 0.5 T SMF for seven days inhibited viability, proliferation, surface antigen expression, cytokine secretion, stem cell genetic marker expression, and adipogenic and osteogenic differentiation but did not affect the DNA integrity in ASCs with or without SPIO labeling.
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Zhang, Hongyu, Li Li, Qian Dong, Yufeng Wang, Qiaoling Feng, Xinying Ou, Pengfei Zhou, Tongchuan He und Jinyong Luo. „Activation of PKA/CREB Signaling is Involved in BMP9-Induced Osteogenic Differentiation of Mesenchymal Stem Cells“. Cellular Physiology and Biochemistry 37, Nr. 2 (2015): 548–62. http://dx.doi.org/10.1159/000430376.
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Background/Aims: BMP9 is highly capable of promoting osteogenic differentiation of mesenchymal stem cells (MSCs) although the molecular mechanism involved is largely unknown. Here, we explored the detail role of PKA/CREB signaling in BMP9-induced osteogenic differentiation. Methods: Activation status of PKA/CREB signaling is assessed by nonradioactive assay and Western blot. Using PKA inhibitors and a dominant negative protein of CREB (A-CREB), we investigated the effect of PKA/CREB signaling on BMP9-induced osteogenic differentiation. Results: We found that BMP9 promotes PKA activity and enhances CREB phosphorylation in MSCs. BMP9 is shown to down-regulate protein kinase A inhibitor γ (PKIγ) expression. We demonstrated that PKA inhibitors suppress BMP9-induced early osteogenic marker alkaline phosphatase (ALP) activity in MSCs as well as late osteogenic markers osteopontin (OPN), osteocalcin (OCN) and matrix mineralization. We found that PKA inhibitor reduces BMP9-induced Runx2 activation and p38 phosphorylation in MSCs. Lastly, interference of CREB function by A-CREB decreased BMP9-induced osteogenic differentiation as well. Conclusion: Our results revealed that BMP9 may activate PKA/CREB signaling in MSCs through suppression of PKIγ expression. It is noteworthy that inhibition of PKA/CREB signaling may impair BMP9-induced osteogenic differentiation of MSCs, implying that activation of PKA/CREB signaling is required for BMP9 osteoinductive activity.
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Glemžaitė, Monika, und Rūta Navakauskienė. „Osteogenic Differentiation of Human Amniotic Fluid Mesenchymal Stem Cells Is Determined by Epigenetic Changes“. Stem Cells International 2016 (2016): 1–10. http://dx.doi.org/10.1155/2016/6465307.
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Osteogenic differentiation of human amniotic fluid derived mesenchymal stem cells (AF-MSCs) has been widely studiedin vitroandin vivoas a potential tool for regenerative medicine and tissue engineering. While most of the studies analyze changes in transcriptional profile during differentiation to date there is not much information regarding epigenetic changes in AF-MSCs during differentiation. The aim of our study was to evaluate epigenetic changes during osteogenic differentiation of AF-MS cells. Isolated AF-MSCs were characterized morphologically and osteogenic differentiation was confirmed by cell staining and determining expression of alkaline phosphatase and osteopontin by RT-qPCR. Variation in gene expression levels of pluripotency markers and specific microRNAs were also evaluated. Analysis of epigenetic changes revealed that levels of chromatin modifying enzymes such as Polycomb repressive complex 2 (PRC2) proteins (EZH2 and SUZ12), DNMT1, HDAC1, and HDAC2 were reduced after osteogenic differentiation of AF-MSCs. We demonstrated that the level of specific histone markers keeping active state of chromatin (H3K4me3, H3K9Ac, and others) increased and markers of repressed state of chromatin (H3K27me3) decreased. Our results show that osteogenic differentiation of AF-MSCs is conducted by various epigenetic alterations resulting in global chromatin remodeling and provide insights for further epigenetic investigations in human AF-MSCs.
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Rochira, Alessio, Luisa Siculella, Fabrizio Damiano, Andrea Palermo, Franco Ferrante, Maria Annunziata Carluccio, Nadia Calabriso, Laura Giannotti und Eleonora Stanca. „Concentrated Growth Factors (CGF) Induce Osteogenic Differentiation in Human Bone Marrow Stem Cells“. Biology 9, Nr. 11 (30.10.2020): 370. http://dx.doi.org/10.3390/biology9110370.
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Bone regeneration is a complex process regulated by several factors that control overlapping biological processes, coordinating interactions among distinct cell populations. There is a great interest in identifying new strategies for inducing osteogenesis in a safe and efficient manner. Concentrated Growth Factor (CGF) is an autologous blood derived product obtained by centrifugation of venous blood following the procedure set on the Silfradent device. In this study the effects of CGF on osteogenic differentiation of human Bone Marrow Stem Cells (hBMSC) in vitro have been investigated; hBMSC were cultured with CGF or osteogenic medium, for 21 days. The osteogenic differentiation was evaluated measuring alkaline phosphatase (ALP) enzyme activity, matrix mineralization by alizarin red staining and through mRNA and protein quantification of osteogenic differentiation markers by Real-time PCR and Western blotting, respectively. The treatment with CGF stimulated ALP activity and promoted matrix mineralization compared to control and seems to be more effective than osteogenic medium. Also, hBMSC lost mesenchymal markers and showed other osteogenic features. Our study showed for the first time that CGF alone is able to induce osteogenic differentiation in hBMSC. The application of CGF on hBMSC osteoinduction might offer new clinical and biotechnological strategies in the tissue regeneration field.
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Okajcekova, Terezia, Jan Strnadel, Michal Pokusa, Romana Zahumenska, Maria Janickova, Erika Halasova und Henrieta Skovierova. „A Comparative In Vitro Analysis of the Osteogenic Potential of Human Dental Pulp Stem Cells Using Various Differentiation Conditions“. International Journal of Molecular Sciences 21, Nr. 7 (26.03.2020): 2280. http://dx.doi.org/10.3390/ijms21072280.
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Dental pulp stem cells (DPSCs) have excellent proliferative properties, mineralization potential and can be easily obtained from third molar teeth. Recently, many studies have focused on isolation and differentiation of DPSCs. In our study, we focused on biological properties of non-differentiated DPSCs in comparison with osteogenic differentiated cells from DPSCs. We analyzed morphology as well as mineralization potential using three varied osteogenic differentiation media. After fifteen days of differentiation, calcium deposit production was observed in all three osteogenic differentiation media. However, only one osteogenic medium, without animal serum supplement, showed rapid and strong calcification—OsteoMAX-XF™ Differentiation Medium. Therefore, we examined specific surface markers, and gene and protein expression of cells differentiated in this osteogenic medium, and compared them to non-differentiated DPSCs. We proved a decrease in expression of CD9 and CD90 mesenchymal stem cell surface markers, as well as downregulation in the expression of pluripotency genes (NANOG and OCT-4) and increased levels of expression in osteogenic genes (ALP, BSP, OCN and RUNX2). Moreover, osteogenic proteins, such as BSP and OCN, were only produced in differentiated cells. Our findings confirm that carefully selected differentiation conditions for stem cells are essential for their translation into future clinical applications.
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Rezai Rad, Maryam, Mahbobeh Bohloli, Mahshid Akhavan Rahnama, Azadeh Anbarlou, Pantea Nazeman und Arash Khojasteh. „Impact of Tissue Harvesting Sites on the Cellular Behaviors of Adipose-Derived Stem Cells: Implication for Bone Tissue Engineering“. Stem Cells International 2017 (2017): 1–9. http://dx.doi.org/10.1155/2017/2156478.
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The advantages of adipose-derived stem cells (AdSCs) over bone marrow stem cells (BMSCs), such as being available as a medical waste and less discomfort during harvest, have made them a good alternative instead of BMSCs in tissue engineering. AdSCs from buccal fat pad (BFP), as an easily harvestable and accessible source, have gained interest to be used for bone regeneration in the maxillofacial region. Due to scarcity of data regarding comparative analysis of isolated AdSCs from different parts of the body, we aimed to quantitatively compare the proliferation and osteogenic capabilities of AdSCs from different harvesting sites. In this study, AdSCs were isolated from BFP (BFPdSCs), abdomen (abdomen-derived mesenchymal stem cells (AbdSCs)), and hip (hip-derived mesenchymal stem cells (HdSCs)) from one individual and were compared for surface marker expression, morphology, growth rate, and osteogenic differentiation capability. Among them, BFPdSCs demonstrated the highest proliferation rate with the shortest doubling time and also expressed vascular endothelial markers including CD34 and CD146. Moreover, the expression of osteogenic markers were significantly higher in BFPdSCs. The results of this study suggested that BFPdSCs as an encouraging source of mesenchymal stem cells are to be used for bone tissue engineering.
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Padial-Molina, Miguel, Juan G. de Buitrago, Raquel Sainz-Urruela, Dario Abril-Garcia, Per Anderson, Francisco O’Valle und Pablo Galindo-Moreno. „Expression of Musashi-1 During Osteogenic Differentiation of Oral MSC: An In Vitro Study“. International Journal of Molecular Sciences 20, Nr. 9 (02.05.2019): 2171. http://dx.doi.org/10.3390/ijms20092171.
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Background: Musashi-1 (MSI1) is a negative regulator of mesenchymal stromal cell (MSC) differentiation which in turn favors cell proliferation. However, little is known about its expression by MSC from the oral cavity and in the context of osteogenic differentiation. Aim: The aim of this study was to analyze the expression of MSI1 in the context of osteogenic differentiation of MSC derived from the oral cavity. Material/methods: For this in vitro study, MSC were isolated from six different origins of the oral cavity. They were extensively characterized in terms of proliferative and clonogenicity potential, expression of stemness genes (MYC, NANOG, POU5F1, and SOX2), expression of surface markers (CD73, CD90, CD105, CD14, CD31, CD34, and CD45) and adipo-, chondro- and osteogenic differentiation potential. Then, osteogenic differentiation was induced and the expression of MSI1 mRNA and other relevant markers of osteogenic differentiation, including RUNX2 and Periostin, were also evaluated. Results: Cell populations from the alveolar bone (pristine or previously grafted with xenograft), dental follicle, dental germ, dental pulp, and periodontal ligament were obtained. The analysis of proliferative and clonogenicity potential, expression of the stemness genes, expression of surface markers, and differentiation potential showed similar characteristics to those of previously published MSC from the umbilical cord. Under osteogenic differentiation conditions, all MSC populations formed calcium deposits and expressed higher SPARC. Over time, the expression of MSI1 followed different patterns for the different MSC populations. It was not significantly different than the expression of RUNX2. In contrast, the expression of MSI1 and POSTN and RUNX2 were statistically different in most MSC populations. Conclusion: In the current study, a similar expression pattern of MSI1 and RUNX2 during in vitro osteogenic differentiation was identified.
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Aenlle, Kristina K., Kevin M. Curtis, Bernard A. Roos und Guy A. Howard. „Hepatocyte Growth Factor and p38 Promote Osteogenic Differentiation of Human Mesenchymal Stem Cells“. Molecular Endocrinology 28, Nr. 5 (01.05.2014): 722–30. http://dx.doi.org/10.1210/me.2013-1286.
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Abstract Hepatocyte growth factor (HGF) is a paracrine factor involved in organogenesis, tissue repair, and wound healing. We report here that HGF promotes osteogenic differentiation through the transcription of key osteogenic markers, including osteocalcin, osterix, and osteoprotegerin in human mesenchymal stem cells and is a necessary component for the establishment of osteoblast mineralization. Blocking endogenous HGF using PHA665752, a c-Met inhibitor (the HGF receptor), or an HGF-neutralizing antibody attenuates mineralization, and PHA665752 markedly reduced alkaline phosphatase activity. Moreover, we report that HGF promotion of osteogenic differentiation involves the rapid phosphorylation of p38 and differential regulation of its isoforms, p38α and p38β. Western blot analysis revealed a significantly increased level of p38α and p38β protein, and reverse transcription quantitative PCR revealed that HGF increased the transcriptional level of both p38α and p38β. Using small interfering RNA to reduce the transcription of p38α and p38β, we saw differential roles for p38α and p38β on the HGF-induced expression of key osteogenic markers. In summary, our data demonstrate the importance of p38 signaling in HGF regulation of osteogenic differentiation.
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Gong, Caipan, Li Li, Chunmei Qin, Weihua Wu, Qi Liu, Ying Li, Linwang Gan und Santao Ou. „The Involvement of Notch1-RBP-Jk/Msx2 Signaling Pathway in Aortic Calcification of Diabetic Nephropathy Rats“. Journal of Diabetes Research 2017 (2017): 1–11. http://dx.doi.org/10.1155/2017/8968523.
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Background. This study explored the changes in expression of vascular smooth muscle cell (VSMC) markers and osteogenic markers, as well as the involvement of Notch1-RBP-Jk/Msx2 pathway in a rat model of diabetic nephropathy (DN) with vascular calcification. Methods. A rat model of DN with concomitant vascular calcification was created by intraperitoneal injection of streptozotocin followed by administration of vitamin D3 and nicotine. Biochemical analysis and histological examination of aortic tissue were performed. VSMC markers and osteogenic markers as well as target molecules in Notch1-RBP-Jk/Msx2 were determined by quantitative real-time polymerase chain reaction and immunohistochemical analysis. Results. Serum calcium and phosphorus levels were significantly increased in model rats as compared to that in normal controls. Diabetic rats with vascular calcification exhibited mineral deposits in aortic intima-media accompanied by decreased expression of VSMC markers and increased expression of osteogenic markers. Notch1, RBP-Jk, Msx2, Jagged1, and N1-ICD were barely expressed in the aortic wall of normal rats. In contrast, these were significantly increased in the model group at all time points (8, 12, and 16 weeks), as compared to that in the normal rats. Conclusion. Activation of the Notch1-RBP-Jk/Msx2 signaling pathway may be involved in the development and progression of vascular calcification in DN.
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Garna, Devy, Manmeet Kaur, Francis J. Hughes und Mandeep Ghuman. „Comparison of the Expression of Periodontal Markers in Dental and Bone Marrow-derived Mesenchymal Stem Cells.“ Open Dentistry Journal 14, Nr. 1 (23.05.2020): 196–202. http://dx.doi.org/10.2174/1874210602014010196.
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Background: Periodontal ligament stem cells are a source of mesenchymal stem cells, but it is unclear whether their phenotype is distinct from mesenchymal stem cells derived from different tissues, such as those derived from bone marrow. Objective: To investigate the expression of the putative PDL markers asporin, periostin, nestin and cementum protein 1, by periodontal ligament stem cells both constitutively and during osteogenic differentiation when compared to bone marrow-derived mesenchymal stem cells, and dental pulp stem cells. Methods: The primary human periodontal ligament, bone marrow, and dental pulp stem cells, and osteoblasts from different donors were cultured in vitro. The expression of periodontal marker associated genes during osteogenic induction was tested by qRT-PCR and immunofluorescence staining. Results: Asporin expression was detected in periodontal ligament stem cells and increased markedly during the time in culture (upregulated x53 fold at 21 days post-induction). During osteogenic differentiation, asporin expression significantly decreased in periodontal ligament cells whereas periostin significantly decreased in dental pulp cells. Periostin expression was absent in osteoblasts, but expression gradually increased in all other cells with time in culture. Nestin expression was mainly seen in the periodontal ligament and dental pulp cells and was largely absent in osteoblasts and bone marrow cells. Cementum protein-1 was most highly expressed in bone marrow cells and osteoblasts following osteogenic induction. Conclusions: The results provide further evidence that periodontal ligament-derived and bone marrow derived mesenchymal stem cells are phenotypically distinct. Periodontal markers are also expressed in dental pulp stem cells.
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Haddouti, El-Mustapha, Thomas M. Randau, Cäcilia Hilgers, Werner Masson, Robert Pflugmacher, Christof Burger, Sascha Gravius und Frank A. Schildberg. „Vertebral Bone Marrow-Derived Mesenchymal Stromal Cells from Osteoporotic and Healthy Patients Possess Similar Differentiation Properties In Vitro“. International Journal of Molecular Sciences 21, Nr. 21 (05.11.2020): 8309. http://dx.doi.org/10.3390/ijms21218309.
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Osteoporosis is a disease characterized by low bone mass and an increased risk of fractures. Although several cellular players leading to osteoporosis have been identified, the role of mesenchymal stromal cells (MSC) is still not fully elaborated. The aim of this study was, therefore, to isolate and characterize MSCs from vertebral body of healthy non-osteoporotic and osteoporotic patients, with a particular focus on their osteogenic differentiation potential. Isolated MSCs were characterized by their osteogenic, adipogenic, and chondrogenic differentiation, as well as surface marker expression, proliferation behavior, and immunomodulatory capacity. The mineralization process was confirmed using Alizarin Red S and alkaline phosphatase (ALP) stains and further evaluated by determining ALP activity, mineral deposition, and free phosphate ion release. MSCs from both healthy and osteoporotic patients showed common fibroblast-like morphology and similar proliferation behavior. They expressed the typical MSC surface markers and possessed immunomodulatory capacity. Both groups demonstrated solid trilineage differentiation potential; osteogenic differentiation was further confirmed by increased ALP activity, deposition of inorganic crystals, phosphate ion release, and expression of osteoblast marker genes. Overall, MSCs from osteoporotic and non-osteoporotic patients showed neither a difference in general MSC features nor in the detailed analysis regarding osteogenic differentiation. These data suggest that vertebral body MSCs from osteoporotic patients were not impaired; rather, they possessed full osteogenic potential compared to MSCs from non-osteoporotic patients.
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Wolint, Petra, Lukas Näf, Désirée Schibler, Nora Hild, Wendelin J. Stark, Pietro Giovanoli, Maurizio Calcagni und Johanna Buschmann. „Suspension of Amorphous Calcium Phosphate Nanoparticles Impact Commitment of Human Adipose-Derived Stem Cells In Vitro“. Biology 10, Nr. 7 (16.07.2021): 675. http://dx.doi.org/10.3390/biology10070675.
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Amorphous calcium phosphate (aCaP) nanoparticles may trigger the osteogenic commitment of adipose-derived stem cells (ASCs) in vitro. The ASCs of three human donors are investigated using basal culture medium DMEM to either 5 or 50 µg/mL aCaP nanoparticles suspension (control: no nanoparticles). After 7 or 14 days, stem cell marker genes, as well as endothelial, osteogenic, chondrogenic, and adipogenic genes, are analyzed by qPCR. Free calcium and phosphate ion concentrations are assessed in the cell culture supernatant. After one week and 5 µg/mL aCaP, downregulation of osteogenic markers ALP and Runx2 is found, and averaged across the three donors. Our results show that after two weeks, ALP is further downregulated, but Runx2 is upregulated. Endothelial cell marker genes, such as CD31 and CD34, are upregulated with 50 µg/mL aCaP and a 2-week exposure. Inter-donor variability is high: Two out of three donors show a significant upregulation of ALP and Runx2 at day 14 with 50 µg/mL aCaP compared to 5 µg/mL aCaP. Notably, all changes in stem cell commitment are obtained in the absence of an osteogenic medium. While the chemical composition of the culture medium and the saturation status towards calcium phosphate phases remain approximately the same for all conditions, gene expression of ASCs changes considerably. Hence, aCaP nanoparticles show the potential to trigger osteogenic and endothelial commitment in ASCs.
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Komrakova, Marina, Martina Blaschke, Maria Laura Ponce, Anne Klüver, Regine Köpp, Michael Hüfner, Matthias Schieker, Nicolai Miosge und Heide Siggelkow. „Decreased Expression of the Human Urea Transporter SLC14A1 in Bone is Induced by Cytokines and Stimulates Adipogenesis of Mesenchymal Progenitor Cells“. Experimental and Clinical Endocrinology & Diabetes 128, Nr. 09 (20.01.2020): 582–95. http://dx.doi.org/10.1055/a-1084-3888.
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AbstractThe human urea transporter SLC14A1 (HUT11/UT-B) has been suggested as a marker for the adipogenic differentiation of bone cells with a relevance for bone diseases. We investigated the function of SLC14A1 in different cells models from bone environment. SLC14A1 expression and cytokine production was investigated in bone cells obtained from patients with osteoporosis. Gene and protein expression of SLC14A1 was studied during adipogenic or osteogenic differentiation of human mesenchymal progenitor cells (hMSCs) and of the single-cell–derived hMSC line (SCP-1), as well as in osteoclasts and chondrocytes. Localization was determined by histochemical methods and functionality by urea transport experiments. Expression of SLC14A1 mRNA was lower in cells from patients with osteoporosis that produced high levels of cytokines. Accordingly, when adding a combination of cytokines to SCP-1 SLC14A1 mRNA expression decreased. SLC14A1 mRNA expression decreased after both osteogenic and more pronounced adipogenic stimulation of hMSCs and SCP-1 cells. The highest SLC14A1 expression was determined in undifferentiated cells, lowest in chondrocytes and osteoclasts. Downregulation of SLC14A1 by siRNA resulted in an increased expression of interleukin-6 and interleukin-1 beta as well as adipogenic markers. Urea influx through SLC14A1 increased expression of osteogenic markers, adipogenic markers were suppressed. SLC14A1 protein was localized in the cell membrane and the cytoplasm. Summarizing, the SLC14A1 urea transporter affects early differentiation of hMSCs by diminishing osteogenesis or by favoring adipogenesis, depending on its expression level. Therefore, SLC14A1 is not unequivocally an adipogenic marker in bone. Our findings suggest an involvement of SLC14A1 in bone metabolism and inflammatory processes and disease-dependent influences on its expression.
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Kim, Ah Young, Yongsun Kim, Seung Hoon Lee, Yongseok Yoon, Wan-Hee Kim und Oh-Kyeong Kweon. „Effect of Gelatin on Osteogenic Cell Sheet Formation Using Canine Adipose-Derived Mesenchymal Stem Cells“. Cell Transplantation 26, Nr. 1 (Januar 2017): 115–23. http://dx.doi.org/10.3727/096368916x693338.
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Osteogenically differentiated cell sheet techniques using mesenchymal stem cells (MSCs) are available to stimulate bone regeneration. The advantage of the cell sheet technique is delivering live cells effectively into the focal region. We developed a novel osteogenic cell sheet technique by adding gelatin to osteogenic cell medium. Gelatin-induced osteogenic cell sheets (GCSs) were compared to conventional osteogenic cell sheets (OCSs). Undifferentiated MSCs (UCs) were used as a control. The morphology of these cell sheets was evaluated microscopically and histologically. The time-dependent cell proliferation rate was estimated by DNA quantification. The expression of osteogenic gene markers and the number of calcium depositions were assessed by quantitative real-time polymerase chain reaction and Alizarin red S (ARS) staining, respectively. GCSs were thicker and stronger than OCSs. GCSs showed a significantly higher cell proliferation rate compared to OCSs ( p < 0.05). GCSs exhibited significantly higher upregulation of BMP-7 mRNA compared to OCSs ( p < 0.05). Both GCSs and OCSs showed negative ARS reactivity on day 10, but only GCSs showed positive ARS reactivity on day 21. With this technique, we observed active cell proliferation with abundant ECM and upregulation of osteogenic bone markers, and our results suggest that GCSs could be promising for therapeutic applications in bone regeneration.
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Bartolozzi, Alice, Federica Viti, Silvia De Stefano, Francesca Sbrana, Loredana Petecchia, Paola Gavazzo und Massimo Vassalli. „Development of label-free biophysical markers in osteogenic maturation“. Journal of the Mechanical Behavior of Biomedical Materials 103 (März 2020): 103581. http://dx.doi.org/10.1016/j.jmbbm.2019.103581.
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Umrath, Felix, Carla Thomalla, Simone Pöschel, Katja Schenke-Layland, Siegmar Reinert und Dorothea Alexander. „Comparative Study of MSCA-1 and CD146 Isolated Periosteal Cell Subpopulations“. Cellular Physiology and Biochemistry 51, Nr. 3 (2018): 1193–206. http://dx.doi.org/10.1159/000495497.
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Background/Aims: Periosteal tissue is a valuable source of multipotent stem cells for bone tissue engineering. To characterize these cells in detail, we generated an immortalized human cranial periosteal cell line and observed an increased MSCA-1 and CD146 expression, as well as an earlier and stronger mineralization compared to the parental cells. Further, we detected a higher osteogenic potential of MSCA-1high compared to MSCA-1low cranial periosteal cell (CPC) fractions. In the present study, a possible synergism of MSCA-1 and CD146 for periosteal cell mineralization was investigated. Methods: MSCA-1/CD146 positive and negative CPCs were magnetically isolated (MACS) or sorted by flow cytometry (FACS) and subjected to osteogenic differentiation. The expression of osteogenic marker genes in the four subpopulations was analyzed by quantitative real-time PCR. Furthermore, the co-expression of osteogenic markers/antigens was analyzed by multispectral imaging flow cytometry (ImageStream, AMNIS). The mineralization potential was assessed by the quantification of alizarin stainings. Results: While the total cell yield after separation was higher using MACS compared to the FACS approach, the isolation of MSCA-1+/- and CD146+/- subpopulations was more efficient with the FACS separation. The accuracy of the FACS separation of the four distinguished cell subpopulations was confirmed by multispectral imaging flow cytometry. Further, we detected increasing levels of MSCA-1 and CD146 during in vitro differentiation in all subpopulations. However, MSCA-1 expression was significantly higher in the MSCA-1+/CD146+ and MSCA-1+/ CD146- subpopulations, while CD146 expression remained clearly lower in these fractions. Significantly higher gene expression levels of osteogenic markers, ALP and RUNX2, were detected in MSCA-1+ compared to MSCA-1- CPCs at different time points during in vitro differentiation. Staining and quantification of calcium phosphate precipitates revealed a significantly higher mineralization potential of MACS separated MSCA-1+ and CD146- CPCs, compared to their respective counterparts. FACS sorted CPCs displayed earlier mineralization in both MSCA-1+ fractions (d13), while later (d28) only the CD146+/MSCA-1- fraction had a significantly lower calcium phosphate concentration compared to all other fractions. Conclusion: Our results demonstrate, that MSCA-1+ cells isolated from CPCs represent a subpopulation with a higher osteogenic potential. In contrast, we found a lower osteogenic potential in CD146+ CPCs. In conclusion, only MSCA-1, but not CD146, is a suitable marker for the isolation of osteoprogenitors from CPCs.
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Bhandi, Shilpa, Ahmed Alkahtani, Rodolfo Reda, Mohammed Mashyakhy, Nezar Boreak, Prabhadevi C. Maganur, Satish Vishwanathaiah et al. „Parathyroid Hormone Secretion and Receptor Expression Determine the Age-Related Degree of Osteogenic Differentiation in Dental Pulp Stem Cells“. Journal of Personalized Medicine 11, Nr. 5 (27.04.2021): 349. http://dx.doi.org/10.3390/jpm11050349.
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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.
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Marrazzo, Pasquale, Cristina Angeloni, Michela Freschi, Antonello Lorenzini, Cecilia Prata, Tullia Maraldi und Silvana Hrelia. „Combination of Epigallocatechin Gallate and Sulforaphane Counteracts In Vitro Oxidative Stress and Delays Stemness Loss of Amniotic Fluid Stem Cells“. Oxidative Medicine and Cellular Longevity 2018 (17.12.2018): 1–13. http://dx.doi.org/10.1155/2018/5263985.
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Amniotic fluid stem cells (AFSCs) are characterized in vivo by a unique niche guarantying their homeostatic role in the body. Maintaining the functionality of stem cells ex vivo for clinical applications requires a continuous improvement of cell culture conditions. Cellular redox status plays an important role in stem cell biology as long as reactive oxygen species (ROS) concentration is finely regulated and their adverse effects are excluded. The aim of this study was to investigate the protective effect of two antioxidants, sulforaphane (SF) and epigallocatechin gallate (EGCG), against in vitro oxidative stress due to hyperoxia and freeze-thawing cycles in AFSCs. Human AFSCs were isolated and characterized from healthy subjects. Assays of metabolic function and antioxidant activity were performed to investigate the effect of SF and EGCG cotreatment on AFSCs. Real-time PCR was used to investigate the effect of the cotreatment on pluripotency, senescence, osteogenic and adipogenic markers, and antioxidant enzymes. Alkaline phosphatase assays and Alizarin Red staining were used to confirm osteogenic differentiation. The cotreatment with SF and EGCG was effective in reducing ROS production, increasing GSH levels, and enhancing the endogenous antioxidant defences through the upregulation of glutathione reductase, NAD(P)H:quinone oxidoreductase-1, and thioredoxin reductase. Intriguingly, the cotreatment sustained the stemness state by upregulating pluripotency markers such as OCT4 and NANOG. Moreover, the cotreatment influenced senescence-associated gene markers in respect to untreated cells. The cotreatment upregulated osteogenic gene markers and promoted osteogenic differentiation in vitro. SF and EGCG can be used in combination in AFSC culture as a strategy to preserve stem cell functionality.
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Yang, Gui-Cun, You-Hua Xu, Hong-Xia Chen und Xiao-Jing Wang. „Acute Lymphoblastic Leukemia Cells Inhibit the Differentiation of Bone Mesenchymal Stem Cells into Osteoblasts In Vitro by Activating Notch Signaling“. Stem Cells International 2015 (2015): 1–11. http://dx.doi.org/10.1155/2015/162410.
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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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Yusop, Norhayati, Paul Battersby, Amr Alraies, Alastair J. Sloan, Ryan Moseley und Rachel J. Waddington. „Isolation and Characterisation of Mesenchymal Stem Cells from Rat Bone Marrow and the Endosteal Niche: A Comparative Study“. Stem Cells International 2018 (2018): 1–14. http://dx.doi.org/10.1155/2018/6869128.
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Within bone, mesenchymal stromal cells (MSCs) exist within the bone marrow stroma (BM-MSC) and the endosteal niche, as cells lining compact bone (CB-MSCs). This study isolated and characterised heterogeneous MSC populations from each niche and subsequently investigated the effects of extensive cell expansion, analysing population doublings (PDs)/cellular senescence, colony-forming efficiencies (CFEs), MSC cell marker expression, and osteogenic/adipogenic differentiation. CB-MSCs and BM-MSCs demonstrated similar morphologies and PDs, reaching 100 PDs. Both populations exhibited consistent telomere lengths (12–17 kb), minimal senescence, and positive telomerase expression. CB-MSCs (PD15) had significantly lower CFEs than PD50. CB-MSCs and BM-MSCs both expressed MSC (CD73/CD90/CD105); embryonic (Nanog) and osteogenic markers (Runx2, osteocalcin) but no hematopoietic markers (CD45). CB-MSCs (PD15) strongly expressed Oct4 and p16INK4A. At early PDs, CB-MSCs possessed a strong osteogenic potency and low potency for adipogenesis, whilst BM-MSCs possessed greater overall bipotentiality for osteogenesis and adipogenesis. At PD50, CB-MSCs demonstrated reduced potency for both osteogenesis and adipogenesis, compared to BM-MSCs at equivalent PDs. This study demonstrates similarities in proliferative and mesenchymal cell characteristics between CB-MSCs and BM-MSCs, but contrasting multipotentiality. Such findings support further comparisons of human CB-MSCs and BM-MSCs, facilitating selection of optimal MSC populations for regenerative medicine purposes.
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Gromolak, Sandra, Agnieszka Krawczenko, Agnieszka Antończyk, Krzysztof Buczak, Zdzisław Kiełbowicz und Aleksandra Klimczak. „Biological Characteristics and Osteogenic Differentiation of Ovine Bone Marrow Derived Mesenchymal Stem Cells Stimulated with FGF-2 and BMP-2“. International Journal of Molecular Sciences 21, Nr. 24 (20.12.2020): 9726. http://dx.doi.org/10.3390/ijms21249726.
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Cell-based therapies using mesenchymal stem cells (MSCs) are a promising tool in bone tissue engineering. Bone regeneration with MSCs involves a series of molecular processes leading to the activation of the osteoinductive cascade supported by bioactive factors, including fibroblast growth factor-2 (FGF-2) and bone morphogenetic protein-2 (BMP-2). In this study, we examined the biological characteristics and osteogenic differentiation potential of sheep bone marrow MSCs (BM-MSCs) treated with 20 ng/mL of FGF-2 and 100 ng/mL BMP-2 in vitro. The biological properties of osteogenic-induced BM-MSCs were investigated by assessing their morphology, proliferation, phenotype, and cytokine secretory profile. The osteogenic differentiation was characterized by Alizarin Red S staining, immunofluorescent staining of osteocalcin and collagen type I, and expression levels of genetic markers of osteogenesis. The results demonstrated that BM-MSCs treated with FGF-2 and BMP-2 maintained their primary MSC properties and improved their osteogenic differentiation capacity, as confirmed by increased expression of osteocalcin and collagen type I and upregulation of osteogenic-related gene markers BMP-2, Runx2, osterix, collagen type I, osteocalcin, and osteopontin. Furthermore, sheep BM-MSCs produced a variety of bioactive factors involved in osteogenesis, and supplementation of the culture medium with FGF-2 and BMP-2 affected the secretome profile of the cells. The results suggest that sheep osteogenic-induced BM-MSCs may be used as a cellular therapy to study bone repair in the preclinical large animal model.
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Chang, Chunkang, Chengming Fei, Youshan Zhao, Juan Guo und Xiao Li. „Impaired Osteogenic Differentiation Of Mesenchymal Stem Cells Derived From Bone Marrow Of Patients With Low-Risk Myelodysplastic Syndromes“. Blood 122, Nr. 21 (15.11.2013): 1579. http://dx.doi.org/10.1182/blood.v122.21.1579.1579.
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Abstract Background The pathogenesis of MDS has not been completely understood, and insufficiency of the hematopoietic microenvironment can be an important factor. MSCs and osteoblasts are key components of the hematopoietic microenvironment. Studying osteoblastic differentiation of MSCs quantitatively may help to understand the pathogenesis of MDS. Methods 38 patients with MDS and 15 normal donors were investigated in this study. Osteoblastic differentiation assays were performed in 16 MDS cases and 8 controls. The expression of osteogenic differentiation markers were measured by real-time PCR. Alkaline phosphatase staining was performed with Alkaline Phosphatase staining kit after 3,7,14 days of incubation. ALP activity was assessed at 3, 7, and 10 days after osteogenic differentiation. Mineralization analysis was performed at 7, 14 and 21 days of osteogenic induction. The areas of mineralization were measured by Image-Pro Plus 6.0 software. Results Both MDS-MSCs and normal cells displayed same fibroblast-like morphology and similar antigen expression. The expression level of RUNX2 was significantly decreased in MSCs from MDS, compaired with normal controls, especially in lower-risk MDS. After osteogenic induction, lower-risk MDS showed lower alkaline phosphatase activity, less intense alizarin red S staining, and lower gene expression of osteogenic differentiation markers, however, higher-risk MDS was normal. Conclusions We concluded that impaired osteogenic differentiation of MSCs was seen mainly in patients with lower-risk MDS. It may contribute to the ineffective hamatopoiesis of MDS. Disclosures: No relevant conflicts of interest to declare.
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Torreggiani, E., C. Bianchini, L. Penolazzi, E. Lambertini, R. Vecchiatini, A. Canella, R. Gambari et al. „Osteogenic potential of cells derived from nasal septum“. Rhinology journal 49, Nr. 2 (01.06.2011): 148–54. http://dx.doi.org/10.4193/rhino10.087.
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BACKGROUND: The research addressed to detect new molecular targets in the development of therapeutic strategies aimed to repair bone tissues. The AIM OF THIS STUDY was to determine the potential osteogenic activity of bone cells from the nasal septum and their use to perform accurate molecular analysis from a single sample. METHODOLOGY: The cells, after nasal septum surgery, were subjected to gene silencing, Reverse Transcriptase - Polymerase Chain reactions, immunocytochemistry and chromatin immunoprecipitation. RESULTS: Cells from the nasal septum can give rise to mature osteoblasts that express osteogenic markers (ALP, Runx2, Slug) and are able to mineralize. We demonstrated that Runx2, a transcription factor critical in early osteospecific differentiation, interacts in vivo with the promoter of the SLUG gene, a marker of osteoblast maturation. CONCLUSIONS: We demonstrated that nasal septum-derived osteoblasts represent an interesting alternative source for bone forming cells, and a promising material to be utilized in bone cellular therapy.
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Bianchi, Michele, Alessandra Pisciotta, Laura Bertoni, Matteo Berni, Alessandro Gambardella, Andrea Visani, Alessandro Russo, Anto de Pol und Gianluca Carnevale. „Osteogenic Differentiation of hDPSCs on Biogenic Bone Apatite Thin Films“. Stem Cells International 2017 (2017): 1–10. http://dx.doi.org/10.1155/2017/3579283.
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A previous study reported the structural characterization of biogenic apatite (BAp) thin films realized by a pulsed electron deposition system by ablation of deproteinized bovine bone. Thin films annealed at 400°C exhibited composition and crystallinity degree very close to those of biogenic apatite; this affinity is crucial for obtaining faster osseointegration compared to conventional, thick hydroxyapatite (HA) coatings, for both orthopedics and dentistry. Here, we investigated the adhesion, proliferation, and osteogenic differentiation of human dental pulp stem cells (hDPCS) on as-deposited and heat-treated BAp and stoichiometric HA. First, we showed that heat-treated BAp films can significantly promote hDPSC adhesion and proliferation. Moreover, hDPSCs, while initially maintaining the typical fibroblast-like morphology and stemness surface markers, later started expressing osteogenic markers such as Runx-2 and OSX. Noteworthy, when cultured in an osteogenic medium on annealed BAp films, hDPSCs were also able to reach a more mature and terminal commitment, with respect to HA and as-deposited films. Our findings suggest that annealed BAp films not only preserve the typical biological properties of stemness of, hDPSCs but also improve their ability of osteogenic commitment.
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Schmitt, Andreas, Sabrina Ehnert, Lilianna Schyschka, Peter Buschner, Andreas Kühnl, Stefan Döbele, Sebastian Siebenlist, Martin Lucke, Ulrich Stöckle und Andreas K. Nussler. „Monocytes Do Not Transdifferentiate into Proper Osteoblasts“. Scientific World Journal 2012 (2012): 1–11. http://dx.doi.org/10.1100/2012/384936.
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Recent publications suggested that monocytes might be an attractive cell type to transdifferentiate into various cellular phenotypes. Aim was, therefore, to evaluate the potential of blood monocytes to transdifferentiate into osteoblasts. Monocytes isolated from peripheral blood were subjected to two previously published treatments to obtain unique, multipotent cell fractions, named programmable cells of monocytic origin (PCMOs) and monocyte-derived mesenchymal progenitor cells (MOMPs). Subsequently, MOMPs and PCMOs were treated with osteogenic differentiation medium (including either vitamin D or dexamethasone) for 14 days. Regarding a variety of surface markers, no differences between MOMPs, PCMOs, and primary monocytes could be detected. The treatment with osteogenic medium neither resulted in loss of hematopoietic markers nor in adoption of mesenchymal phenotype in all cell types. No significant effect was observed regarding the expression of osteogenic transcription factors, bone-related genes, or production of mineralized matrix. Osteogenic medium resulted in activation of monocytes and appearance of osteoclasts. In conclusion, none of the investigated monocyte cell types showed any transdifferentiation characteristics under the tested circumstances. Based on our data, we rather see an activation and maturation of monocytes towards macrophages and osteoclasts.
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Youssef, Amer, und Victor K. M. Han. „Regulation of Osteogenic Differentiation of Placental-Derived Mesenchymal Stem Cells by Insulin-Like Growth Factors and Low Oxygen Tension“. Stem Cells International 2017 (2017): 1–17. http://dx.doi.org/10.1155/2017/4576327.
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Placental mesenchymal stem cells (PMSCs) are multipotent cells that can differentiate in vitro to multiple lineages, including bone. Insulin-like growth factors (IGFs, IGF-1 and IGF-2) participate in maintaining growth, survival, and differentiation of many stem cells, including osteoprogenitors. Low oxygen tension (PO2) can maintain stem cell multipotency and impede osteogenic differentiation. In this study, we investigated whether PMSC osteogenic differentiation is influenced by low PO2 and by IGFs. Our results indicated that low PO2 decreased osteogenic markers RUNX2 and OPN; however, re-exposure to higher oxygen tension (room air) restored differentiation. IGFs, especially IGF-1, triggered an earlier expression of RUNX2 and enhanced OPN and mineralization. RUNX2 was phosphorylated in room air and augmented by IGFs. IGF-1 receptor (IGF-1R) was increased in low PO2 and reduced by IGFs, while insulin receptor (IR) was increased in differentiating PMSCs and enhanced by IGF-1. Low PO2 and IGFs maintained higher IR-A which was switched to IR-B in room air. PI3K/AKT was required for osteogenic differentiation, while MEK/ERK was required to repress an RUNX2 and OPN increase in low PO2. Therefore, IGFs, specifically IGF-1, trigger the earlier onset of osteogenic differentiation in room air, whereas, reversibly, low PO2 impedes complete differentiation by maintaining higher multipotency and lower differentiation markers.
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Liang, Youde, Xin Liu, Ruiping Zhou, Dawei Song, Yi-Zhou Jiang und Weiwei Xue. „Chaetocin Promotes Osteogenic Differentiation via Modulating Wnt/Beta-Catenin Signaling in Mesenchymal Stem Cells“. Stem Cells International 2021 (06.02.2021): 1–6. http://dx.doi.org/10.1155/2021/8888416.
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Mesenchymal stemXin cells (MSCs) are a great cell source for bone regeneration. Although combining MSCs with growth factors and scaffolds provides a useful clinical strategy for bone tissue engineering, the efficiency of MSC osteogenic differentiation remains to be improved. Epigenetic modification is related to the differentiation ability of MSCs during osteogenic induction. In this study, we evaluate the effect of Chaetocin, an inhibitor of lysine-specific histone methyltransferases, on the differentiation of MSCs. We found that MSCs treated with Chaetocin demonstrated increased osteogenic ability and reduced adipogenic ability. The expression of osteogenic markers (Runx2 and OPN) was induced in MSCs by Chaetocin during osteogenic induction. Moveover, treatment of Chaetocin in MSCs improves Wnt/β-catenin signaling pathways and its downstream targets. Finally, we showed increased bone formation of MSC and Wnt/β-catenin signaling activity by treatment of Chaetocin using in vivo bone formation assays. Our data uncovered a critical role of Chaetocin in MSC osteogenic differentiation and provide new insights into bone tissue regeneration and repair.
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Li, Fang, Jianglin Chen, Mengjia Gong, Yang Bi, Chengchen Hu, Yuanyuan Zhang und Ming Li. „Isolation and Characterization of Human Synovial Fluid-Derived Mesenchymal Stromal Cells from Popliteal Cyst“. Stem Cells International 2020 (18.09.2020): 1–15. http://dx.doi.org/10.1155/2020/7416493.
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Mesenchymal stem cells (MSCs) are multipotent progenitor cells in adult tissues. The aim of this study is to isolate and identify synovial fluid-derived mesenchymal stromal cells (SF-MSCs) from the popliteal cyst fluid of pediatric patients. SF-MSCs were collected from the popliteal cyst fluid of pediatric patients during cystectomy surgery. After cyst fluid extraction and adherent culturing, in vitro morphology, growth curve, and cell cycle were observed. The expression of stem cell surface markers was analyzed by flow cytometry, and expression of cell marker protein was detected by immunofluorescence. SF-MSCs were cultured in osteogenic, adipogenic, and chondrogenic differentiation medium. The differentiation potential of SF-MSCs was analyzed by alkaline phosphatase (Alizarin Red), Oil Red O, and Alcian blue. Antibody detection of human angiogenesis-related proteins was performed compared with bone marrow mesenchymal stem cells (BM-MSCs). The results show that SF-MSCs from the popliteal cyst fluid of pediatric patients showed a shuttle appearance and logarithmic growth. Flow cytometry analysis revealed that SF-MSCs were negative for hematopoietic lineage markers (CD34, CD45) and positive for MSC markers (CD44, CD73, CD90, and CD105). Interstitial cell marker (vimentin) and myofibroblast-like cell marker alpha-smooth muscle actin (α-SMA) were positive. These cells could differentiate into osteogenic, adipogenic, and chondrogenic lineages, respectively. Several types of human angiogenesis-related proteins were detected in the cell secretory fluid. These results show that we successfully obtained SF-MSCs from the popliteal cyst fluid of pediatric patients, which have the potential to be a valuable source of MSCs.
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Fuchs, Bruno, Kunbo Zhang, Alex Schabel, Mark E. Bolander und Gobinda Sarkar. „Identification of twenty-two candidate markers for human osteogenic sarcoma“. Gene 278, Nr. 1-2 (Oktober 2001): 245–52. http://dx.doi.org/10.1016/s0378-1119(01)00731-4.
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Adamova, Eva, Eva Janeckova, Karel Kleparnik und Eva Matalova. „Caspases and osteogenic markers—in vitro screening of inhibition impact“. In Vitro Cellular & Developmental Biology - Animal 52, Nr. 2 (28.10.2015): 144–48. http://dx.doi.org/10.1007/s11626-015-9964-1.
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Adhikari, Roshan, Chongxiao Chen und Woo Kyun Kim. „Effect of 20(S)-Hydroxycholesterol on Multilineage Differentiation of Mesenchymal Stem Cells Isolated from Compact Bones in Chicken“. Genes 11, Nr. 11 (17.11.2020): 1360. http://dx.doi.org/10.3390/genes11111360.
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Bone health and body weight gain have significant economic and welfare importance in the poultry industry. Mesenchymal stem cells (MSCs) are common progenitors of different cell lineages such as osteoblasts, adipocytes, and myocytes. Specific oxysterols have shown to be pro-osteogenic and anti-adipogenic in mouse and human MSCs. To determine the effect of 20(S)-hydroxycholesterol (20S) on osteogenic, adipogenic, and myogenic differentiation in chicken, mesenchymal stem cells isolated from compact bones of broiler chickens (cBMSCs) were subjected to various doses of 20S, and markers of lineage-specific mRNA were analyzed using real-time PCR and cell cytochemistry. Further studies were conducted to evaluate the molecular mechanisms involved in lineage-specific differentiation pathways. Like human and mouse MSCs, 20S oxysterol expressed pro-osteogenic, pro-myogenic, and anti-adipogenic differentiation potential in cBMSCs. Moreover, 20(S)-Hydroxycholesterol induced markers of osteogenic genes and myogenic regulatory factors when exposed to cBMSCs treated with their specific medium. In contrast, 20S oxysterol suppressed expression of adipogenic marker genes when exposed to cBMSCs treated with OA, an adipogenic precursor of cBMSCs. To elucidate the molecular mechanism by which 20S exerts its differentiation potential in all three lineages, we focused on the hedgehog signaling pathway. The hedgehog inhibitor, cyclopamine, completely reversed the effect of 20S induced expression of osteogenic and anti-adipogenic mRNA. However, there was no change in the mRNA expression of myogenic genes. The results showed that 20S oxysterol promotes osteogenic and myogenic differentiation and decreases adipocyte differentiation of cBMSCs. This study also showed that the induction of osteogenesis and adipogenesis inhibition in cBMSCs by 20S is mediated through the hedgehog signaling mechanism. The results indicated that 20(S) could play an important role in the differentiation of chicken-derived MSCs and provided the theory basis on developing an intervention strategy to regulate skeletal, myogenic, and adipogenic differentiation in chicken, which will contribute to improving chicken bone health and meat quality. The current results provide the rationale for the further study of regulatory mechanisms of bioactive molecules on the differentiation of MSCs in chicken, which can help to address skeletal health problems in poultry.
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Quiroz, Felipe Garcia, Olga M. Posada, Daniel Gallego-Perez, Natalia Higuita-Castro, Carlos Sarassa, Derek J. Hansford, Piedad Agudelo-Florez und Luis E. López. „Housekeeping gene stability influences the quantification of osteogenic markers during stem cell differentiation to the osteogenic lineage“. Cytotechnology 62, Nr. 2 (April 2010): 109–20. http://dx.doi.org/10.1007/s10616-010-9265-1.
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Li, Linli, Yiqun He, Han Tang, Wei Mao, Haofei Ni, Feizhou Lyu und Youhai Dong. „Cerebrospinal Fluid Pulsation Stress Promotes the Angiogenesis of Tissue-Engineered Laminae“. Stem Cells International 2020 (02.07.2020): 1–12. http://dx.doi.org/10.1155/2020/8026362.
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Background. Angiogenesis is a prerequisite step to achieve the success of bone regeneration by tissue engineering technology. Previous studies have shown the role of cerebrospinal fluid pulsation (CSFP) stress in the reconstruction of tissue-engineered laminae. In this study, we investigated the role of CSFP stress in the angiogenesis of tissue-engineered laminae. Methods. For the in vitro study, a CSFP bioreactor was used to investigate the impact of CSFP stress on the osteogenic mesenchymal stem cells (MSCs). For the in vivo study, forty-eight New Zealand rabbits were randomly divided into the CSFP group and the Non-CSFP group. Tissue-engineered laminae (TEL) was made by hydroxyapatite-collagen I scaffold and osteogenic MSCs and then implanted into the lamina defect in the two groups. The angiogenic and osteogenic abilities of newborn laminae were examined with histological staining, qRT-PCR, and radiological analysis. Results. The in vitro study showed that CSFP stress could promote the vascular endothelial growth factor A (VEGF-A) expression levels of osteogenic MSCs. In the animal study, the expression levels of angiogenic markers in the CSFP group were higher than those in the Non-CSFP group; moreover, in the CSFP group, their expression levels on the dura mater surface, which are closer to the CSFP stress stimulation, were also higher than those on the paraspinal muscle surface. The expression levels of osteogenic markers in the CSFP group were also higher than those in the Non-CSFP group. Conclusion. CSFP stress could promote the angiogenic ability of osteogenic MSCs and thus promote the angiogenesis of tissue-engineered laminae. The pretreatment of osteogenic MSC with a CSFP bioreactor may have important implications for vertebral lamina reconstruction with a tissue engineering technique.
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Yamamoto, Maiko, Hidemi Nakata, Jia Hao, Joshua Chou, Shohei Kasugai und Shinji Kuroda. „Osteogenic Potential of Mouse Adipose-Derived Stem Cells Sorted for CD90 and CD105 In Vitro“. Stem Cells International 2014 (2014): 1–17. http://dx.doi.org/10.1155/2014/576358.
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Adipose tissue-derived stromal cells, termed ASCs, play an important role in regenerative applications. They resemble mesenchymal stem cells owing to their inexhaustibility, general differentiation potential, and plasticity and display a series of cell-specific and cluster-of-differentiation (CD) marker profiles similar to those of other somatic stem cells. Variations in phenotypes or differentiation are intimately associated with CD markers. The purpose of our study was to exhibit distinct populations of ASCs with differing characteristics for osteogenic differentiation. The primary cell batch of murine-derived ASCs was extracted from subcutaneous adipose tissue and the cells were sorted for the expression of the surface protein molecules CD90 and CD105 using flow cytometry. Each cell population sorted for CD90 and CD105 was analyzed for osteogenic potency after cell culture. The results suggested that ASCs exhibit distinct populations with differing characteristics for osteogenic differentiation: unsorted ASCs stimulated comparable mineralized nodule formation as bone marrow stromal cells (BMSCs) in osteogenic medium and viral transfection for BMP2 accelerated the formation of mineralized nodules in CD90 and/or CD105 positive ASCs with observation of decrease in CD105 expression after 14 days. Future studies assessing different immunophenotypes of ASCs should be undertaken to develop cell-based tissue engineering.
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Waddell, Shona J., María C. de Andrés, Penelope M. Tsimbouri, Enateri V. Alakpa, Maggie Cusack, Matthew J. Dalby und Richard OC Oreffo. „Biomimetic oyster shell–replicated topography alters the behaviour of human skeletal stem cells“. Journal of Tissue Engineering 9 (Januar 2018): 204173141879400. http://dx.doi.org/10.1177/2041731418794007.
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The regenerative potential of skeletal stem cells provides an attractive prospect to generate bone tissue needed for musculoskeletal reparation. A central issue remains efficacious, controlled cell differentiation strategies to aid progression of cell therapies to the clinic. The nacre surface from Pinctada maxima shells is known to enhance bone formation. However, to date, there is a paucity of information on the role of the topography of P. maxima surfaces, nacre and prism. To investigate this, nacre and prism topographical features were replicated onto polycaprolactone and skeletal stem cell behaviour on the surfaces studied. Skeletal stem cells on nacre surfaces exhibited an increase in cell area, increase in expression of osteogenic markers ALP ( p < 0.05) and OCN ( p < 0.01) and increased metabolite intensity ( p < 0.05), indicating a role of nacre surface to induce osteogenic differentiation, while on prism surfaces, skeletal stem cells did not show alterations in cell area or osteogenic marker expression and a decrease in metabolite intensity ( p < 0.05), demonstrating a distinct role for the prism surface, with the potential to maintain the skeletal stem cell phenotype.
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Mizerska-Kowalska, Magdalena, Adrianna Sławińska-Brych, Katarzyna Kaławaj, Aleksandra Żurek, Beata Pawińska, Wojciech Rzeski und Barbara Zdzisińska. „Betulin Promotes Differentiation of Human Osteoblasts In Vitro and Exerts an Osteoinductive Effect on the hFOB 1.19 Cell Line Through Activation of JNK, ERK1/2, and mTOR Kinases“. Molecules 24, Nr. 14 (19.07.2019): 2637. http://dx.doi.org/10.3390/molecules24142637.
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Although betulin (BET), a naturally occurring pentacyclic triterpene, has a variety of biological activities, its osteogenic potential has not been investigated so far. The aim of this study was to assess the effect of BET on differentiation of human osteoblasts (hFOB 1.19 and Saos-2 cells) in vitro in osteogenic (with ascorbic acid as an osteogenic supplement) and osteoinductive (without an additional osteogenic supplement) conditions. Osteoblast differentiation was evaluated based on the mRNA expression (RT-qPCR) of Runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), type I collagen-α1 (COL1A1), and osteopontin (OPN). Additionally, ALP activity and production of COL1A1 (western blot analysis) and OPN (ELISA) were evaluated. The level of mineralization (calcium accumulation) was determined with Alizarin red S staining. BET upregulated the mRNA level of RUNX2 and the expression of other osteoblast differentiation markers in both cell lines (except the influence of BET on ALP expression/activity in the Saos-2 cells). Moreover, it increased mineralization in both cell lines in the osteogenic conditions. BET also increased the mRNA level of osteoblast differentiation markers in both cell lines (except for ALP in the Saos-2 cells) in the osteoinductive conditions, which was accompanied with increased matrix mineralization. The osteoinductive activity of BET in the hFOB 1.19 cells was probably mediated via activation of MAPKs (JNK and ERK1/2) and mTOR, as the specific inhibitors of these kinases abolished the BET-induced osteoblast differentiation. Our results suggest that BET has the potential to enhance osteogenesis.
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Merceron, Christophe, Claire Vinatier, Sophie Portron, Martial Masson, Jérôme Amiaud, Lydie Guigand, Yan Chérel, Pierre Weiss und Jérôme Guicheux. „Differential effects of hypoxia on osteochondrogenic potential of human adipose-derived stem cells“. American Journal of Physiology-Cell Physiology 298, Nr. 2 (Februar 2010): C355—C364. http://dx.doi.org/10.1152/ajpcell.00398.2009.
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Human adipose tissue-derived stem cells (hATSC) have been contemplated as reparative cells for cartilage engineering. Chondrogenic differentiation of hATSC can be induced by an enriched culture medium and a three-dimensional environment. Given that bone is vascularized and cartilage is not, oxygen tension has been suggested as a regulatory factor for osteochondrogenic differentiation. Our work aimed at determining whether hypoxia affects the osteochondrogenic potential of hATSC. hATSC were cultured in chondrogenic or osteogenic medium for 28 days, in pellets or monolayers, and under 5% or 20% oxygen tension. Cell differentiation was monitored by real-time PCR (COL2A1, aggrecan, Runx2, and osteocalcin). The chondrogenic differentiation was further evaluated by Alcian blue and immunohistological staining for glycosaminoglycans (GAGs) and type II collagen, respectively. Osteogenic differentiation was also assessed by the staining of mineralized matrix (Alizarin Red) and measurement of alkaline phosphatase (ALP) activity. The expression of chondrogenic markers was upregulated when hATSC were exposed to hypoxia in chondrogenic medium. Conversely, osteocalcin expression, mineralization, and ALP activity were severely reduced under hypoxic conditions even in the presence of osteogenic medium. Our data strongly suggest that hypoxia favors the chondrogenic differentiation of hATSC as evidenced by the expression of the chondrogenic markers, whereas it could alter their osteogenic potential. Our results highlight the differential regulatory role of hypoxia on the chondrogenic and osteogenic differentiation processes of hATSC. These data could help us exploit the potential of tissue engineering and stem cells to replace or restore the function of osteoarticular tissues.
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Ishiy, Felipe Augusto Andre, Roberto Dalto Fanganiello, Karina Griesi-Oliveira, Angela May Suzuki, Gerson Shigeru Kobayashi, Andressa Gois Morales, Luciane Portas Capelo und Maria Rita Passos-Bueno. „Improvement ofIn VitroOsteogenic Potential through Differentiation of Induced Pluripotent Stem Cells from Human Exfoliated Dental Tissue towards Mesenchymal-Like Stem Cells“. Stem Cells International 2015 (2015): 1–9. http://dx.doi.org/10.1155/2015/249098.
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Constraints for the application of MSCs for bone reconstruction include restricted self-renewal and limited cell amounts. iPSC technology presents advantages over MSCs, providing homogeneous cellular populations with prolonged self-renewal and higher plasticity. However, it is unknown if the osteogenic potential of iPSCs differs from that of MSCs and if it depends on the iPSCs originating cellular source. Here, we compared thein vitroosteogenesis between stem cells from human deciduous teeth (SHED) and MSC-like cells from iPSCs from SHED (iPS-SHED) and from human dermal fibroblasts (iPS-FIB). MSC-like cells from iPS-SHED and iPS-FIB displayed fibroblast-like morphology, downregulation of pluripotency markers and upregulation of mesenchymal markers. Comparativein vitroosteogenesis analysis showed higher osteogenic potential in MSC-like cells from iPS-SHED followed by MSC-like cells from iPS-FIB and SHED. CD105 expression, reported to be inversely correlated with osteogenic potential in MSCs, did not display this pattern, considering that SHED presented lower CD105 expression. Higher osteogenic potential of MSC-like cells from iPS-SHED may be due to cellular homogeneity and/or to donor tissue epigenetic memory. Our findings strengthen the rationale for the use of iPSCs in bone bioengineering. Unveiling the molecular basis behind these differences is important for a thorough use of iPSCs in clinical scenarios.
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Zhang, Yingying, Yanghui Xing, Jian Li, Zhiqiang Zhang, Huiqin Luan, Zhaowei Chu, He Gong und Yubo Fan. „Osteogenesis-Related Behavior of MC3T3-E1 Cells on Substrates with Tunable Stiffness“. BioMed Research International 2018 (01.11.2018): 1–10. http://dx.doi.org/10.1155/2018/4025083.
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Osteogenic differentiation of cells has considerable clinical significance in bone defect treatment, and cell behavior is linked to extracellular matrix stiffness. This study aimed to determine how matrix stiffness affects cell morphology and subsequently regulates the osteogenic phenotype of osteogenesis precursor cells. Four PDMS substrates were prepared with stiffness corresponding to the elastic modulus ranging from 0.6 MPa to 2.7 MPa by altering the Sylgard 527 and Sylgard 184 concentrations. MC3T3-E1 cells were cultured on the matrices. Cell morphology, vinculin expression, and key osteogenic markers, Col I, OCN, OPN, and calcium nodule, were examined. The activity and expression level of Yes-associated protein (YAP) were evaluated. Results showed that cell spreading exhibited no correlation with the stiffness of matrix designed in this paper, but substratum stiffness did modulate MC3T3-E1 osteogenic differentiation. Col I, OPN, and OCN proteins were significantly increased in cells cultured on soft matrices compared with stiff matrices. Additionally, cells cultured on the 1:3 ratio matrices had more nodules than those on other matrices. Accordingly, cells on substrates with low stiffness showed enhanced expression of the osteogenic markers. Meanwhile, YAP expression was downregulated on soft substrates although the subcellular location was not affected. Our results provide evidence that matrix stiffness (elastic modulus ranging from 0.6 MPa to 2.7 MPa) affects the osteogenic differentiation of MC3T3-E1, but it is not that “the stiffer, the better” as showed in some of the previous studies. The optimal substrate stiffness may exist to promote osteoblast differentiation. Cell differentiation triggered by the changes in substrate stiffness may be independent of the YAP signal. This study has important implications for biomaterial design and stem cell-based tissue engineering.
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Li, Chen-Shuang, Zhong Zheng, Xiao-Xia Su, Fei Wang, Michelle Ling, Min Zou und Hong Zhou. „Activation of the Extracellular Signal-Regulated Kinase Signaling Is Critical for Human Umbilical Cord Mesenchymal Stem Cell Osteogenic Differentiation“. BioMed Research International 2016 (2016): 1–10. http://dx.doi.org/10.1155/2016/3764372.
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Human umbilical cord mesenchymal stem cells (hUCMSCs) are recognized as candidate progenitor cells for bone regeneration. However, the mechanism of hUCMSC osteogenesis remains unclear. In this study, we revealed that mitogen-activated protein kinases (MAPKs) signaling is involved in hUCMSC osteogenic differentiationin vitro. Particularly, the activation of c-Jun N-terminal kinases (JNK) and p38 signaling pathways maintained a consistent level in hUCMSCs through the entire 21-day osteogenic differentiation period. At the same time, the activation of extracellular signal-regulated kinases (ERK) signaling significantly increased from day 5, peaked at day 9, and declined thereafter. Moreover, gene profiling of osteogenic markers, alkaline phosphatase (ALP) activity measurement, and alizarin red staining demonstrated that the application of U0126, a specific inhibitor for ERK activation, completely prohibited hUCMSC osteogenic differentiation. However, when U0126 was removed from the culture at day 9, ERK activation and osteogenic differentiation of hUCMSCs were partially recovered. Together, these findings demonstrate that the activation of ERK signaling is essential for hUCMSC osteogenic differentiation, which points out the significance of ERK signaling pathway to regulate the osteogenic differentiation of hUCMSCs as an alternative cell source for bone tissue engineering.
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Agata, H., I. Asahina, Y. Yamazaki, M. Uchida, Y. Shinohara, M. J. Honda, H. Kagami und M. Ueda. „Effective Bone Engineering with Periosteum-derived Cells“. Journal of Dental Research 86, Nr. 1 (Januar 2007): 79–83. http://dx.doi.org/10.1177/154405910708600113.
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Bone augmentation via tissue engineering has generated significant interest. We hypothesized that periosteum-derived cells could be used in place of bone marrow stromal cells (which are widely used) in bone engineering, but the differences in osteogenic potential between these 2 cell types are unclear. Here, we compared the osteogenic potential of these cells, and investigated the optimal osteoinductive conditions for periosteum-derived cells. Both cell types were induced, via bFGF and BMP-2, to differentiate into osteoblasts. Periosteal cells proliferated faster than marrow stromal cells, and osteogenic markers indicated that bone marrow stromal cells were more osteogenic than periosteal cells. However, pre-treatment with bFGF made periosteal cells more sensitive to BMP-2 and more osteogenic. Transplants of periosteal cells treated with BMP-2 after pre-treatment with bFGF formed more new bone than did marrow stromal cells. Analysis of these data suggests that combined treatment with bFGF and BMP-2 can make periosteum a highly useful source of bone regeneration.
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Choi, Jung-Won, Sunhye Shin, Chang Youn Lee, Jiyun Lee, Hyang-Hee Seo, Soyeon Lim, Seahyoung Lee et al. „Rapid Induction of Osteogenic Markers in Mesenchymal Stem Cells by Adipose-Derived Stromal Vascular Fraction Cells“. Cellular Physiology and Biochemistry 44, Nr. 1 (2017): 53–65. http://dx.doi.org/10.1159/000484582.
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Background/Aims: Stromal vascular fraction (SVF) cells are a mixed cell population, and their regenerative capacity has been validated in various therapeutic models. The purpose of this study was to investigate the regenerative mechanisms utilized by implanted SVF cells. Using an in vitro co-culture system, we sought to determine whether SVF implantation into impaired tissue affects endogenous mesenchymal stem cell (MSC) differentiation; MSCs can differentiate into a variety of cell types, and they have a strong regenerative capacity despite their low numbers in impaired tissue. Methods: Adipose-derived SVF cells obtained from four donors were co-cultured with bone marrow-derived MSCs, and the differential expression of osteogenic markers and osteogenic differentiation inducers over time was analyzed in mono-cultured MSCs and MSCs co-cultured with SVF cells. Results: The co-cultivation of MSCs with SVF cells significantly and mutually induced the expression of osteogenic-specific markers via paracrine and/or autocrine regulation but did not induce adipocyte, chondrocyte or myoblast marker expression. More surprisingly, subsequent osteogenesis and/or comparable effects were rapidly induced within 48 h. Conclusion: To the best of our knowledge, this is the first study in which osteogenesis and/or comparable effects were rapidly induced in bone marrow-derived MSCs and adipose-derived SVF cells through co-cultivation. Our findings suggest that the positive effects of SVF implantation into impaired bone may be attributed to the rapid induction of MSC osteogenesis, and the transplantation of co-cultured and preconditioned SVF cells and/or MSCs may be more effective than the transplantation of untreated cells for the treatment of bone defects.