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

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.

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

Biomedical Research and Therapy ◽

10.15419/bmrat.v6i6.548 ◽

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.

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Differentiation potential of rabbit CD90-positive cells sorted from adipose-derived stem cells in vitro

In Vitro Cellular & Developmental Biology - Animal ◽

10.1007/s11626-016-0081-6 ◽

2016 ◽

Vol 53 (1) ◽

pp. 77-82 ◽

Cited By ~ 2

Author(s):

Xinghui Song ◽

Chaoyang Hong ◽

Qingqing Zheng ◽

Hailan Zhao ◽

Kangping Song ◽

...

Keyword(s):

Stem Cells ◽

Adipose Derived Stem Cells ◽

Differentiation Potential

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In Vitro Studies on Nanoporous, Nanotubular and Nanosponge-Like Titania Coatings, with the Use of Adipose-Derived Stem Cells

Materials ◽

10.3390/ma13071574 ◽

2020 ◽

Vol 13 (7) ◽

pp. 1574 ◽

Cited By ~ 2

Author(s):

Michalina Ehlert ◽

Aleksandra Radtke ◽

Tomasz Jędrzejewski ◽

Katarzyna Roszek ◽

Michał Bartmański ◽

...

Keyword(s):

Stem Cells ◽

Cell Line ◽

Osteogenic Potential ◽

Biological Research ◽

Adipose Derived Stem Cells ◽

Differentiation Potential ◽

Nanoporous Surface ◽

Titania Coatings ◽

The Impact

In vitro biological research on a group of amorphous titania coatings of different nanoarchitectures (nanoporous, nanotubular, and nanosponge-like) produced on the surface of Ti6Al4V alloy samples have been carried out, aimed at assessing their ability to interact with adipose-derived mesenchymal stem cells (ADSCs) and affect their activity. The attention has been drawn to the influence of surface coating architecture and its physicochemical properties on the ADSCs proliferation. Moreover, in vitro co-cultures: (1) fibroblasts cell line L929/ADSCs and (2) osteoblasts cell line MG-63/ADSCs on nanoporous, nanotubular and nanosponge-like TiO2 coatings have been studied. This allowed for evaluating the impact of the surface properties, especially roughness and wettability, on the creation of the beneficial microenvironment for co-cultures and/or enhancing differentiation potential of stem cells. Obtained results showed that the nanoporous surface is favorable for ADSCs, has great biointegrative properties, and supports the growth of co-cultures with MG-63 osteoblasts and L929 fibroblasts. Additionally, the number of osteoblasts seeded and cultured with ADSCs on TNT5 surface raised after 72-h culture almost twice when compared with the unmodified scaffold and by 30% when compared with MG-63 cells growing alone. The alkaline phosphatase activity of MG-63 osteoblasts co-cultured with ADSCs increased, that indirectly confirmed our assumptions that TNT-modified scaffolds create the osteogenic niche and enhance osteogenic potential of ADSCs.

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202 IN VITRO DIFFERENTIATION OF ADULT PORCINE ADIPOSE-DERIVED STEM CELLS AFTER LABELING WITH PKH26 AND FLOW CYTOMETRY

Reproduction Fertility and Development ◽

10.1071/rdv18n2ab202 ◽

2006 ◽

Vol 18 (2) ◽

pp. 209

Author(s):

M. Mello ◽

A. Lima ◽

S. Malusky ◽

S. Lane ◽

M. Wheeler

Keyword(s):

Stem Cells ◽

Flow Cytometry ◽

Adipose Tissue ◽

Osteogenic Differentiation ◽

Fluorescent Dye ◽

Positive Staining ◽

Adipose Derived Stem Cells ◽

Differentiation Potential ◽

Oil Red O

The purpose of this study was to investigate the possible effects of the fluorescent dye PKH26 and flow cytometry on adult porcine adipose-derived stem cells (ADSCs) after exposing them to adipogenic and osteogenic differentiation conditions. Adipose tissue was isolated from swine (11 months of age) and digested with 0.075% collagenase at 37�C for 90 min. The digested adipose tissue was centrifuged at 200g for 10 min to obtain a cell pellet. The pellet was re-suspended with DMEM, and the ADSCs were plated onto 75 cm2 flasks (5000-10 000 cells per cm2) and cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% gentamicin. Passage 3 cells were labeled with fluorescent dye (PKH26 red fluorescent cell linker kit; Sigma Chemical, St. Louis, MO, USA) and sorted by flow cytometry. After labeling and sorting, the sorted and unsorted (control group) cells were replated and exposed to adipogenic (1 �M dexamethasone, 0.5 mM isobutylmethylxantine, 10 �M insulin and 200�M indomethacin) and osteogenic (0.1 �M dexamethasone, 10 mM �-glycerophosphate, and 50�M ascorbic acid) differentiation conditions when the cells were 90% confluent. Cells were evaluated based on morphology and specific staining properties. Adipogenic differentiation was confirmed by oil red O-positive staining of large lipid vacuoles, and osteogenic differentiation by Von Kossa staining 2 weeks after initiation of differentiation. The frequency of oil red O-positive colonies in both sorted and unsorted group was similar (15.0% vs. 13.2%, respectively). The number of osteogenic nodules, confirmed by the presence of calcium by Von Kossa staining, in the sorted and unsorted group was 17 and 184 per flask, respectively. In conclusion, this study demonstrates that adult porcine adipose-derived stem cells maintain their differentiation potential after labeling with fluorescent dye and sorting by flow cytometry. This should allow for more rapid evaluation of the differentiation potential of ADSCs in vitro. This work was partially supported by the Council for Food and Agricultural Research (C-FAR) Sentinel Program, University of Illinois and CNPq, Brazil (M. Mello).

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

10.21203/rs.3.rs-328417/v1 ◽

2021 ◽

Author(s):

Tianli Wu ◽

Zhihao Yao ◽

Gang Tao ◽

Fangzhi Lou ◽

Hui Tang ◽

...

Keyword(s):

Stem Cells ◽

Osteogenic Differentiation ◽

Wnt Signaling Pathway ◽

Osteogenic Potential ◽

Adipose Derived Stem Cells ◽

Alizarin Red ◽

Differentiation Potential

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

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Runx2 Overexpression Enhances Osteoblastic Differentiation and Mineralization in Adipose - Derived Stem Cells in vitro and in vivo

Calcified Tissue International ◽

10.1007/s00223-006-0083-6 ◽

2006 ◽

Vol 79 (3) ◽

pp. 169-178 ◽

Cited By ~ 111

Author(s):

X. Zhang ◽

M. Yang ◽

L. Lin ◽

P. Chen ◽

K. T. Ma ◽

...

Keyword(s):

Stem Cells ◽

Osteoblastic Differentiation ◽

Adipose Derived Stem Cells

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Effects mediated by the α7 nicotinic acetylcholine receptor on cell proliferation and migration in rat adipose-derived stem cells

European Journal of Histochemistry ◽

10.4081/ejh.2020.3159 ◽

2020 ◽

Vol 64 (s2) ◽

Author(s):

Marta Pernarella ◽

Roberta Piovesana ◽

Carlo Matera ◽

Alessandro Faroni ◽

Mario Fiore ◽

...

Keyword(s):

Stem Cells ◽

Nicotinic Receptors ◽

Adipose Derived Stem Cells ◽

Α7 Nicotinic Acetylcholine Receptor ◽

Differentiation Potential ◽

Physiological Processes ◽

Α7 Nachr ◽

And Migration ◽

Proliferation And Migration

Adipose-derived stem cells (ASCs) are an attractive source for regenerative medicine as they can be easily isolated, rapidly expandable in culture and show excellent in vitro differentiation potential. Acetylcholine (ACh), one of the main neurotransmitters in central and peripheral nervous systems, plays key roles in the control of several physiological processes also in non-neural tissues. As demonstrated in our previous studies, ACh can contribute to the rat ASCs physiology, negatively modulating ASCs proliferation and migration via M2 muscarinic receptor (mAChR) activation. In the present work we show that rat ASCs also express α7 nicotinic receptors (nAChRs). In particular, we have investigated the effects mediated by the selective activation of α7 nAChRs, which causes a reduction of ASC proliferation without affecting cell survival and morphology, and significantly promotes cell migration via upregulation of the CXCR4 expression. Interestingly, the activation of the α7 nAChR also upregulates the expression of M2 mAChR protein, indicating a cooperation between muscarinic and nicotinic receptors in the inhibition of ASC proliferation.

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

10.21203/rs.3.rs-70186/v1 ◽

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.

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