scholarly journals A NOTCH1/LSD1/BMP2 Co-Regulatory Network Mediated by miR-137 Negatively Regulates Osteogenesis of Human Adipose-Derived Stem Cells

2020 ◽  
Author(s):  
Cong Fan ◽  
Xiaohan Ma ◽  
Yuejun Wang ◽  
Longwei Lv ◽  
Yuan Zhu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Osteoblastic Differentiation ◽  
Bone Morphogenetic Protein 2 ◽  
Luciferase Reporter ◽  
Adipose Derived Stem Cells ◽  
Degree Relative ◽  
Matrix Mineralization ◽  
Differentiation Potential ◽  
Lineage Differentiation ◽  
Relative Expression Level

Abstract Background: MicroRNAs have been recognized as critical regulators for the osteoblastic lineage differentiation of human adipose-derived stem cells (hASCs). Previously, we have displayed that silencing of miR-137 enhances the osteoblastic differentiation potential of hASCs partly through the coordination of lysine-specific histone demethylase 1 (LSD1), bone morphogenetic protein 2 (BMP2), and mothers against decapentaplegic homolog 4 (SMAD4). However, still numerous molecules involved in the osteogenic regulation of miR-137 remain unknown. This study aimed to further elucidate the epigenetic mechanisms of miR-137 on the osteogenic differentiation of hASCs.Methods: hASCs transfected with miR-137 overexpression or knockdown lentiviruses were used to assess the osteogenic capacity by testing the alkaline phosphatase activity, matrix mineralization degree, relative expression level of osteogenesis-associated genes and ectopic osteogenesis in nude mice. Dual-luciferase reporter assay was performed to examine the targeting of the 3' untranslated region (3' UTR) of NOTCH1 by miR-137. Interrelationships of signaling pathways of NOTCH1-hairy and enhancer of split 1 (HES1), LSD1, and BMP2-SMAD4 were thoroughly investigated by separate knockdown of NOTCH1, LSD1, and BMP2.Results: We confirmed that miR-137 directly targeted the 3' UTR of NOTCH1 while positively regulated HES1. After knocking down NOTCH1 or BMP2 individually, we found that these two signals formed a positive feedback loop and activated LSD1. In addition, LSD1 knockdown induced the expression of NOTCH1 while suppressed HES1.Conclusions: Collectively, we proposed a NOTCH1/LSD1/BMP2 co-regulatory signaling network to elucidate the modulation of miR-137 on the osteoblastic differentiation of hASCs, thus providing mechanism-based rationale for miRNA-targeted therapy of bone defect.

scholarly journals A NOTCH1/LSD1/BMP2 co-regulatory network mediated by miR-137 negatively regulates osteogenesis of human adipose-derived stem cells

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Cong Fan ◽  
Xiaohan Ma ◽  
Yuejun Wang ◽  
Longwei Lv ◽  
Yuan Zhu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Osteoblastic Differentiation ◽  
Negative Control ◽  
Bone Morphogenetic Protein 2 ◽  
Luciferase Reporter ◽  
Adipose Derived Stem Cells ◽  
Differentiation Potential ◽  
Lineage Differentiation ◽  
Morphogenetic Protein ◽  
Genes Expression

Abstract Background MicroRNAs have been recognized as critical regulators for the osteoblastic lineage differentiation of human adipose-derived stem cells (hASCs). Previously, we have displayed that silencing of miR-137 enhances the osteoblastic differentiation potential of hASCs partly through the coordination of lysine-specific histone demethylase 1 (LSD1), bone morphogenetic protein 2 (BMP2), and mothers against decapentaplegic homolog 4 (SMAD4). However, still numerous molecules involved in the osteogenic regulation of miR-137 remain unknown. This study aimed to further elucidate the epigenetic mechanisms of miR-137 on the osteogenic differentiation of hASCs. Methods Dual-luciferase reporter assay was performed to validate the binding to the 3′ untranslated region (3′ UTR) of NOTCH1 by miR-137. To further identify the role of NOTCH1 in miR-137-modulated osteogenesis, tangeretin (an inhibitor of NOTCH1) was applied to treat hASCs which were transfected with miR-137 knockdown lentiviruses, then together with negative control (NC), miR-137 overexpression and miR-137 knockdown groups, the osteogenic capacity and possible downstream signals were examined. Interrelationships between signaling pathways of NOTCH1-hairy and enhancer of split 1 (HES1), LSD1 and BMP2-SMADs were thoroughly investigated with separate knockdown of NOTCH1, LSD1, BMP2, and HES1. Results We confirmed that miR-137 directly targeted the 3′ UTR of NOTCH1 while positively regulated HES1. Tangeretin reversed the effects of miR-137 knockdown on osteogenic promotion and downstream genes expression. After knocking down NOTCH1 or BMP2 individually, we found that these two signals formed a positive feedback loop as well as activated LSD1 and HES1. In addition, LSD1 knockdown induced NOTCH1 expression while suppressed HES1. Conclusions Collectively, we proposed a NOTCH1/LSD1/BMP2 co-regulatory signaling network to elucidate the modulation of miR-137 on the osteoblastic differentiation of hASCs, thus providing mechanism-based rationale for miRNA-targeted therapy of bone defect.

scholarly journals ZNF521 Represses Osteoblastic Differentiation in Human Adipose-Derived Stem Cells

2018 ◽  
Vol 19 (12) ◽  
pp. 4095 ◽  
Author(s):  
Emanuela Chiarella ◽  
Annamaria Aloisio ◽  
Stefania Scicchitano ◽  
Valeria Lucchino ◽  
Ylenia Montalcini ◽  
...  
Keyword(s):  
Stem Cells ◽  
Zinc Finger Protein ◽  
Lentiviral Vector ◽  
Adipogenic Differentiation ◽  
Bone Matrix ◽  
Osteoblastic Differentiation ◽  
Adipose Derived Stem Cells ◽  
Matrix Mineralization ◽  
Calcium Deposits ◽  
Vector Transduction

Human adipose-derived stem cells (hADSCs) are multipotent mesenchymal cells that can differentiate into adipocytes, chondrocytes, and osteocytes. During osteoblastogenesis, the osteoprogenitor cells differentiate into mature osteoblasts and synthesize bone matrix components. Zinc finger protein 521 (ZNF521/Zfp521) is a transcription co-factor implicated in the regulation of hematopoietic, neural, and mesenchymal stem cells, where it has been shown to inhibit adipogenic differentiation. The present study is aimed at determining the effects of ZNF521 on the osteoblastic differentiation of hADSCs to clarify whether it can influence their osteogenic commitment. The enforced expression or silencing of ZNF521 in hADSCs was achieved by lentiviral vector transduction. Cells were cultured in a commercial osteogenic medium for up to 20 days. The ZNF521 enforced expression significantly reduced osteoblast development as assessed by the morphological and molecular criteria, resulting in reduced levels of collagen I, alkaline phosphatase, osterix, osteopontin, and calcium deposits. Conversely, ZNF521 silencing, in response to osteoblastic stimuli, induced a significant increase in early molecular markers of osteogenesis and, at later stages, a remarkable enhancement of matrix mineralization. Together with our previous findings, these results show that ZNF521 inhibits both adipocytic and osteoblastic maturation in hADSCs and suggest that its expression may contribute to maintaining the immature properties of hADSCs.

Human adipose-derived stem cells isolated from young and elderly women: their differentiation potential and scaffold interaction during in vitro osteoblastic differentiation

Cytotherapy ◽  
2009 ◽  
Vol 11 (6) ◽  
pp. 793-803 ◽  
Author(s):  
Laura de Girolamo ◽  
Silvia Lopa ◽  
Elena Arrigoni ◽  
Matteo F. Sartori ◽  
Franz W. Baruffaldi Preis ◽  
...  
Keyword(s):  
Stem Cells ◽  
Elderly Women ◽  
Osteoblastic Differentiation ◽  
Adipose Derived Stem Cells ◽  
Differentiation Potential

scholarly journals miRNA-187-5p Regulates Osteoblastic Differentiation of Bone Marrow Mesenchymal Stem Cells in Mice by Targeting ICAM1

2020 ◽  
Vol 2020 ◽  
pp. 1-12
Author(s):  
Yi Sun ◽  
Xin Wang ◽  
Guanghua Chen ◽  
Chengchao Song ◽  
Xinnan Ma ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Bone Formation ◽  
Osteoblastic Differentiation ◽  
Luciferase Reporter ◽  
Heterotopic Bone ◽  
Heterotopic Bone Formation ◽  
Matrix Mineralization ◽  
Marrow Mesenchymal Stem Cells

Osteoporosis (OP) is a common bone metabolic disease, the process of which is fundamentally irreversible. Therefore, the investigation into osteoblastic differentiation of bone marrow mesenchymal stem cells (BMSCs) will provide more clues for OP treatment. In the present study, we found that microRNA-187-5p (miR-187-5p) played a key role on osteoblastic differentiation, which was significantly upregulated during osteogenic differentiation of BMSCs in mice. Moreover, overexpression of miR-187-5p suppressed osteoblastic differentiation of BMSCs through increasing alkaline phosphatase (ALP), matrix mineralization, and levels of Osterix (OSX), and osteopontin (OPN) as well as runt-related transcription factor 2 (Runx2) in vitro. The results in vivo indicated that the upregulation of miR-187-5p enhanced the efficacy of new bone formation in the heterotopic bone formation assay. Luciferase reporter assay and western blot analysis revealed that miR-187-5p was involved in osteogenesis by targeting intracellular adhesion molecule 1 (ICAM-1). Furthermore, ICAM-1 silence inhibited osteoblastic differentiation of BMSCs. Taken together, our results suggested for the first time that miR-187-5p may promote osteogenesis by targeting ICAM-1, and provided a possible therapeutic target for bone metabolic diseases.

scholarly journals Effect of Uniaxial Tensile Cyclic Loading Regimes on Matrix Organization and Tenogenic Differentiation of Adipose-Derived Stem Cells Encapsulated within 3D Collagen Scaffolds

2017 ◽  
Vol 2017 ◽  
pp. 1-16 ◽  
Author(s):  
Gayathri Subramanian ◽  
Alexander Stasuk ◽  
Mostafa Elsaadany ◽  
Eda Yildirim-Ayan
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Expression Profiles ◽  
Three Dimensional ◽  
Uniaxial Tensile ◽  
Adipose Derived Stem Cells ◽  
Collagen Scaffolds ◽  
Differentiation Potential ◽  
Tenogenic Differentiation ◽  
Matrix Organization

Adipose-derived mesenchymal stem cells have become a popular cell choice for tendon repair strategies due to their relative abundance, ease of isolation, and ability to differentiate into tenocytes. In this study, we investigated the solo effect of different uniaxial tensile strains and loading frequencies on the matrix directionality and tenogenic differentiation of adipose-derived stem cells encapsulated within three-dimensional collagen scaffolds. Samples loaded at 0%, 2%, 4%, and 6% strains and 0.1 Hz and 1 Hz frequencies for 2 hours/day over a 7-day period using a custom-built uniaxial tensile strain bioreactor were characterized in terms of matrix organization, cell viability, and musculoskeletal gene expression profiles. The results displayed that the collagen fibers of the loaded samples exhibited increased matrix directionality with an increase in strain values. Gene expression analyses demonstrated that ASC-encapsulated collagen scaffolds loaded at 2% strain and 0.1 Hz frequency showed significant increases in extracellular matrix genes and tenogenic differentiation markers. Importantly, no cross-differentiation potential to osteogenic, chondrogenic, and myogenic lineages was observed at 2% strain and 0.1 Hz frequency loading condition. Thus, 2% strain and 0.1 Hz frequency were identified as the appropriate mechanical loading regime to induce tenogenic differentiation of adipose-derived stem cells cultured in a three-dimensional environment.

scholarly journals High glucose induces early senescence in adipose-derived stem cells by accelerating p16 and mTOR

2019 ◽  
Vol 6 (6) ◽  
pp. 3213-3221
Author(s):  
Hieu Liem Pham ◽  
Phuc Van Pham
Keyword(s):  
Stem Cells ◽  
High Glucose ◽  
Glucose Concentration ◽  
Culture Medium ◽  
Diabetic Patients ◽  
Adipose Derived Stem Cells ◽  
Differentiation Potential ◽  
Normal Glucose

Introduction: The senescence of stem cells is the primary reason that causes aging of stem cell-containing tissues. Some hypotheses have suggested that high glucose concentration in diabetic patients is the main factor that causes senescence of cells in those patients. This study aimed to evaluate the effects of high glucose concentrations on the senescence of adipose-derived stem cells (ADSCs). Methods: ADSCs were isolated and expanded from human adipose tissues. They were characterized and confirmed as mesenchymal stem cells (MSCs) by expression of surface markers, their shape, and in vitro differentiation potential. They were then cultured in 3 different media- that contained 17.5 mM, 35 mM, or 55 mM of D-glucose. The senescent status of ADSCs was recorded by the expression of the enzyme beta-galactosidase, cell proliferation, and doubling time. Real-time RT-PCR was used to evaluate the expression of p16, p21, p53 and mTOR. Results: The results showed that high glucose concentrations (35 mM and 55 mM) in the culture medium induced senescence of human ADSCs. The ADSCs could progress to the senescent status quicker than those cultured in the lower glucose-containing medium (17.5 mM). The senescent state was related to the up-regulation of p16 and mTOR genes. Conclusion: These results suggest that high glucose in culture medium can trigger the expression of p16 and mTOR genes which cause early senescence in ADSCs. Therefore, ADSCs should be cultured in low glucose culture medium, or normal glucose concentration, to extend their life in vitro as well as in vivo.  

scholarly journals The Treasury of Wharton's Jelly

2021 ◽  
Author(s):  
Rebecca Guenther ◽  
Stephan Dreschers ◽  
Jessika Maassen ◽  
Daniel Reibert ◽  
Claudia Skazik-Voogt ◽  
...  
Keyword(s):  
Stem Cells ◽  
Umbilical Cord ◽  
Cultured Cells ◽  
Single Cells ◽  
Mesenchymal Progenitor Cells ◽  
Differentiation Potential ◽  
Lineage Differentiation ◽  
Umbilical Cord Tissue ◽  
The Common ◽  
Culture Supernatants

Abstract Background Postnatal umbilical cord tissue contains valuable mesenchymal progenitor cells of various differentiation stages. While mesenchymal stem cells are plastic-adherent and tend to differentiate into myofibroblastic phenotypes, some round cells detach, float above the adherent cells, and build up cell aggregates, or form spheroids spontaneously. Very small luminescent cells are always involved as single cells or within collective forms and resemble the common well-known very small embryonic-like cells (VSELs). In this study, we investigated these VSELs-like cells in terms of their pluripotency phenotype and tri-lineage differentiation potential. Methods VSELs-like cells were isolated from cell-culture supernatants by a process that combines filtering, up concentration, and centrifugation. To determine their pluripotency character, we measured the expression of Nanog, Sox-2, Oct-4, SSEA-1, CXCR4, SSEA-4 on gene and protein level. In addition, the cultured cells derived from UC tissue were examined regarding their potential to differentiate into three germ layers. Result The VSELs-like cells express all of the pluripotency-associated markers we investigated and are able to differentiate into meso- endo- and ectodermal precursor cells. Conclusions Umbilical cord tissue hosts highly potent VSELs-like stem cells. Graphical Abstract

scholarly journals Soft Substrate Maintains Proliferative and Adipogenic Differentiation Potential of human Mesenchymal Stem Cells on Long Term Expansion by Delaying Senescence

2018 ◽  
Author(s):  
Sanjay K. Kureel ◽  
Pankaj Mogha ◽  
Akshada Khadpekar ◽  
Vardhman Kumar ◽  
Rohit Joshi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Culture System ◽  
Human Mesenchymal Stem Cells ◽  
Population Doubling ◽  
Differentiation Potential ◽  
Lineage Differentiation ◽  
Poly Acrylamide

AbstractHuman mesenchymal stem cells (hMSCs), when cultured on tissue culture plate (TCP) for in vitro expansion, they spontaneously lose their proliferative capacity and multi-lineage differentiation potential. They also lose their distinct spindle morphology and become large and flat. After a certain number of population doubling, they enter into permanent cell cycle arrest, called senescence. This is a major roadblock for clinical use of hMSCs which demands large number of cells. A cell culture system is needed which can maintain the stemness of hMSCs over long term passages yet simple to use. In this study, we explore the role of substrate rigidity in maintaining stemness. hMSCs were serially passaged on TCP and 5 kPa poly-acrylamide gel for 20 population doubling. It was found that while on TCP, cell growth reached a plateau at cumulative population doubling (CPD) = 12.5, on 5 kPa gel, they continue to proliferate linearly till we monitored (CPD = 20). We also found that while on TCP, late passage MSCs lost their adipogenic potential, the same was maintained on soft gel. Cell surface markers related to MSCs were also unaltered. We demonstrated that this maintenance of stemness was correlated with delay in onset of senescence, which was confirmed by β-gal assay and by differential expression of vimentin, Lamin A and Lamin B. As preparation of poly-acrylamide gel is a simple, well established, and well standardized protocol, we believe that this system of cell expansion will be useful in therapeutic and research applications of hMSCs.One Sentence SummaryhMSCs retain their stemness when expanded in vitro on soft polyacrylamide gel coated with collagen by delaying senescence.Significance StatementFor clinical applications, mesenchymal stem cells (MSCs) are required in large numbers. As MSCs are available only in scarcity in vivo, to fulfill the need, extensive in vitro expansion is unavoidable. However, on expansion, they lose their replicative and multi-lineage differentiation potential and become senescent. A culture system that can maintain MSC stemness on long-term expansion, without compromising the stemness, is need of the hour. In this paper, we identified polyacrylamide (PAA) hydrogel of optimum stiffness that can be used to maintain stemness of MSCs during in vitro long term culture. Large quantity of MSCs thus grown can be used in regenerative medicine, cell therapy, and in treatment of inflammatory diseases.

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