MicroRNA-150 Modulates Adipogenic Differentiation of Adipose-Derived Stem Cells by Targeting Notch3.
ABSTRACT: MicroRNAs (miRNAs) influence stem cell functions, including mobilization, proliferation, and differentiation. miR-150 is abundantly expressed in monocytes. Knockdown of miR-150 promotes bone marrow stem cell migration. The role of miR-150 in adipose-derived stem cells (ADSCs) is unclear. In this study, the effects of miR-150 on adipogenic differentiation and proliferation of ADSCs were investigated. ADSCs were isolated from the inguinal adipose tissue of wild-type (WT) and miR-150 knockout (KO) mice and were induced for adipogenic differentiation. The miR-150 level was detected by real-time PCR. ADSCs were transfected by miR-150 or small-interfering RNA (siRNA) of Notch3. MTT assay and colony formation assay were performed in miR-150 knockdown and control ADSCs. Real-time PCR showed that miR-150 was expressed in ADSCs. miR-150 knockdown significantly decreased the capacity of adipogenic differentiation of ADSCs, as compared with their counterparts from WT mice. It is intriguing that the overexpression of miR-150 significantly increased C/EBP? and PPAR-? expression and lipid formation in ADSCs with adipogenic induction. Overexpression of miR-150 significantly decreased Notch3 expression in ADSCs compared with the control groups. Furthermore, Notch3 inhibition promoted the adipogenic differentiation in ADSCs. miR-150 also suppressed proliferation potential and the expression of Nanog in ADSCs. In summary, this study demonstrates, for the first time, that miR-150 promotes adipogenic differentiation and inhibits proliferation of ADSCs. miR-150 regulates adipogenic differentiation of ADSCs, likely mediated by the downregulation of Notch3.
Project description:Klotho was originally discovered as an aging-suppressor gene. The purpose of this study was to investigate whether secreted Klotho (SKL) affects the proliferation and differentiation of adipose-derived stem cells (ADSCs). RT-PCR and Western blot analysis showed that short-form Klotho was expressed in mouse ADSCs. The Klotho gene mutation KL(-/-) significantly decreased proliferation of ADSCs and expression of pluripotent transcription factors (Nanog, Sox-2, and Oct-4) in mice. The adipogenic differentiation of ADSCs was also decreased in KL(-/-) mice. Incubation with Klotho-deficient medium decreased ADSC proliferation, pluripotent transcription factor levels, and adipogenic differentiation, which is similar to what was found in KL(-/-) mice. These results indicate that Klotho deficiency suppresses ADSC proliferation and differentiation. Interestingly, treatment with recombinant SKL protein rescued the Klotho deficiency-induced impairment in ADSC proliferation and adipogenic differentiation. SKL also regulated ADSCs' differentiation to other cell lineages (osteoblasts, myofibroblasts), indicating that SKL maintains stemness of ADSCs. It is intriguing that overexpression of SKL significantly increased PPAR-? expression and lipid formation in ADSCs following adipogenic induction, indicating enhanced adipogenic differentiation. Overexpression of SKL inhibited expression of TGF?1 and its downstream signaling mediator Smad2/3. This study demonstrates, for the first time, that SKL is essential to the maintenance of normal proliferation and differentiation in ADSCs. Klotho regulates adipogenic differentiation in ADSCs, likely via inhibition of TGF?1 and activation of PPAR-?. Stem Cells 2016;34:1615-1625.
Project description:OBJECTIVES:It is of profound significance for clinical bone regeneration to clarify the specific molecular mechanism from which we found that osteogenic differentiation of adipose-derived stem cells (ADSCs) will be probably promoted by exosomes. MATERIALS AND METHODS:By means of lentiviral transfection, miR-130a-3p overexpression and knockdown ADSCs were constructed. Alizarin Red S was used to detect the calcium deposits, and qPCR was used to detect osteogenesis-related genes, to verify the effect of miR-130a-3p on the osteogenic differentiation of ADSCs. CCK-8 was used to detect the effect of miR-130a-3p on the proliferation of ADSCs. The target binding between miR-130a-3p and SIRT7 was verified by dual-luciferase reporter gene assay. Furthermore, the role of Wnt signalling pathway in the regulation of ADSCs osteogenesis and differentiation by miR-130a-3p was further verified by detecting osteogenic-related genes and proteins and alkaline phosphatase activity. RESULTS:(a) Overexpression of miR-130a-3p can enhance the osteogenic differentiation of ADSCs while reducing protein and mRNA levels of SIRT7, a target of miR-130a-3p. (b) Our study further found that overexpression of miR-130a-3p leads to down-regulation of SIRT7 expression with up-regulation of Wnt signalling pathway-associated protein. (c) Overexpression of miR-130a-3p inhibited proliferation of ADSCs, while knockdown promoted it. CONCLUSIONS:The obtained findings indicate that exosomal miR-130a-3p can promote osteogenic differentiation of ADSCs partly by mediating SIRT7/Wnt/?-catenin axis, which will hence promote the application of exosomal microRNA in the field of bone regeneration.
Project description:BACKGROUND:White adipose tissue includes subcutaneous and visceral adipose tissue (SAT and VAT) with different metabolic features. SAT protects from metabolic disorders, while VAT promotes them. The proliferative and adipogenic potentials of adipose-derived stem cells (ADSCs) are critical for maintaining adipose tissue homeostasis through driving adipocyte hyperplasia and inhibiting pathological hypertrophy. However, it remains to be elucidated the critical molecules that regulate different potentials of subcutaneous and visceral ADSCs (S-ADSCs, V-ADSCs) and mediate distinct metabolic properties of SAT and VAT. CD90 is a glycosylphosphatidylinositol-anchored protein on various cells, which is also expressed on ADSCs. However, its expression patterns and differential regulation on S-ADSCs and V-ADSCs remain unclear. METHODS:S-ADSCs and V-ADSCs were detected for CD90 expression. Proliferation, colony formation, cell cycle, mitotic clonal expansion, and adipogenic differentiation were assayed in S-ADSCs, V-ADSCs, or CD90-silenced S-ADSCs. Glucose tolerance test and adipocyte hypertrophy were examined in mice after silencing of CD90 in SAT. CD90 expression and its association with CyclinD1 and Leptin were analyzed in adipose tissue from mice and humans. Regulation of AKT by CD90 was detected using a co-transfection system. RESULTS:Compared with V-ADSCs, S-ADSCs expressed high level of CD90 and showed increases in proliferation, mitotic clonal expansion, and adipogenic differentiation, together with AKT activation and G1-S phase transition. CD90 silencing inhibited AKT activation and S phase entry, thereby curbing proliferation and mitotic clonal expansion of S-ADSCs. In vivo CD90 silencing in SAT inhibited S-ADSC proliferation, which caused adipocyte hypertrophy and glucose intolerance in mice. Furthermore, CD90 was highly expressed in SAT rather than in VAT in human and mouse, which had positive correlation with CyclinD1 but negative correlation with Leptin. CD90 promoted AKT activation through recruiting its pleckstrin homology domain to plasma membrane. CONCLUSIONS:CD90 is differentially expressed on S-ADSCs and V-ADSCs, and plays critical roles in ADSC proliferation, mitotic clonal expansion, and hemostasis of adipose tissue and metabolism. These findings identify CD90 as a crucial modulator of S-ADSCs and V-ADSCs to mediate distinct metabolic features of SAT and VAT, thus proposing CD90 as a valuable biomarker or target for evaluating ADSC potentials, monitoring or treating obesity-associated metabolic disorders.
Project description:Adipose-derived stem cells (ADSCs) are an attractive cell source for bone tissue engineering and have great potential for bone regeneration and defect repair. The transcriptional coactivator with PDZ-binding motif (TAZ) has been demonstrated to modulate osteogenic and adipogenic differentiation of mesenchymal stem cells. However, its roles during ADSC differentiation and therapeutic potentials for bone regeneration have as yet not been well established.TAZ expression was measured during osteogenic differentiation of ADSCs in vitro. Both loss-of-function and gain-of-function approaches by TAZ knockdown or enforced overexpression were utilized to determine its functions during osteogenic differentiation of ADSCs. TM-25659, a chemical activator of TAZ, was used to determine whether pharmacological activation of TAZ in ADSCs enhanced osteogenic differentiation in vitro and bone formation in animal models. The molecular mechanisms underlying TAZ in promoting osteogenesis of ADSCs were also explored.Increased TAZ expression was observed during osteogenic differentiation of human ADSCs. TAZ knockdown resulted in compromised osteogenic differentiation and enhanced adipogenic differentiation of ADSCs. In contrast, enforced TAZ overexpression yielded increased osteogenic differentiation and bone regeneration in vivo, and impaired adipogenic differentiation of ADSCs. Pharmacological activation of TAZ by its chemical activator TM-25659 facilitated osteogenic differentiation of ADSCs. Noticeably, transient treatment of ADSCs with TM-25659 or intraperitoneal injection of TM-25659 significantly enhanced bone regeneration of ADSCs loaded with porous ?-TCP in vivo. Mechanistically, TM-25659 exposure significantly promoted TAZ phosphorylation and nuclear translocation, and potentiated the assembly of the TAZ-Runx2 complex. Subsequently, the TAZ-Runx2 complex was further recruited to the promoter of osteocalcin and in turn enhanced its transcription.Our findings indicate that TAZ is a key mediator that promotes ADSC commitment to the osteoblast lineage. Pharmacological activation of TAZ in ADSCs might become a feasible and promising approach to enhance bone regeneration and repair.
Project description:A better understanding of the molecular mechanisms that regulate adipose tissue-derived stromal cell (ADSC) differentiation could provide new insight into some adipose-tissue-related disease. The differentiation of ADSCs into adipocytes is a complex physiological process that includes clonal expansion, growth arrest, and terminal differentiation. Here the role of microRNA-143 (miR-143) during ADSC adipogenic differentiation was systematically investigated. We found that miR-143 expression was transiently decreased after adipogenic induction while increased from day 3 and peaked on day 7 after induction. We show for the first time that the role of miR-143 is not consistent in the differentiation process. The regulatory role depends on the differentiation stage that miR-143 acts on. When miR-143 is overexpressed during the clonal expansion stage, the adipogenic differentiation of ADSCs is inhibited, whereas the overexpression of miR-143 during the growth arrest stage or terminal differentiation stage promotes differentiation. Further we firstly demonstrate that miR-143 plays the modulational role by directly repressing MAP2K5, a key member of the MAPKK family in the MAPK signaling pathway. These findings suggest that miR-143 plays an important role in adipose tissue formation, with special implications for some metabolic disease in which the amount and/or function of adipose tissue is altered.
Project description:In this study, we performed an adipogenic differentiation of human adipose-derived stem cells (ADSCs) in vitro with different deuterium content (natural, low and high) in the culture medium during differentiation process with parallel analysis of the gene expression, metabolic activity and cell viability/toxicity. After ADSCs differentiation into adipocytes we have done the analysis of differentiation process efficiency and determined a type of resulting adipocytes (by morphology, gene expression, UCP1 protein detection and adipokine production analysis). We have found that high (5 × 105 ppm) deuterium content significantly inhibit in vitro adipogenic differentiation of human ADSCs compared to the groups with natural (150 ppm) and low (30 ppm) deuterium content. Importantly, protocol of differentiation used in our study leads to white adipocytes development in groups with natural (control) and high deuterium content, whereas deuterium-depleted differentiation medium leads to brown-like (beige) adipocytes formation. We have also remarked the direct impact of deuterium on the cellular survival and metabolic activity. Interesting, in deuterium depleted-medium, the cells had normal survival rate and high metabolic activity, whereas the inhibitory effect of deuterated medium on ADSCs differentiation at least was partly associated with deuterium cytotoxicity and inhibitory effect on metabolic activity. The inhibitory effect of deuterium on metabolic activity and the subsequent decrease in the effectiveness of adipogenic differentiation is probably associated with mitochondrial dysfunction. Thus, deuterium could be considered as an element that affects the substance chirality. These findings may be the basis for the development of new approaches in the treatment of obesity, metabolic syndrome and diabetes through the regulation of adipose-derived stem cell differentiation and adipocyte functions.
Project description:Despite many advances in tissue engineering, there are still significant challenges associated with restructuring, repairing, or replacing damaged tissue in the body. Currently, a major obstacle has been trying to develop a scaffold for cartilage tissue engineering that provides the correct mechanical properties to endure the loads associated with articular joints as well as promote cell-scaffold interactions to aid in extracellular matrix deposition. In addition, adipogenic tissue engineering is widely growing due to an increased need for more innovative reconstructive therapies following adipose tissue traumas and cosmetic surgeries. Recently, lipoaspirate tissue has been identified as a viable alternative source for mesenchymal stem cells because it contains a supportive stroma that can easily be isolated. Adipose derived stem cells (ADSCs) can differentiate into a variety of mesodermal lineages including the adipogenic and chondrogenic phenotypes. Biodegradable polymeric scaffolds have been shown to be a promising alternative and stem cells have been widely used to evaluate the compatibility, viability, and bioactivity of these materials. Polycaprolactone is a bioresorbable polymer, which has been widely used for biomedical and tissue engineering applications. The fundamental concept behind successful synthetic tissue-engineered scaffolds is to promote progenitor cell migration, adhesion, proliferation, and induce differentiation, extracellular matrix synthesis, and finally integration with host tissue. In this study, we investigated the adhesion, proliferation, and chondrogenic and adipogenic differentiation of ADSCs on nanowire surfaces. A solvent-free gravimetric template technique was used to fabricate polycaprolactone nanowires surfaces. The results indicated that during the growth period i.e., initial 7 days of culture, the nanowire surfaces (NW) supported adhesion and proliferation of the cells that had elongated morphologies. However, cell on surfaces without nanowires had non-elongated morphologies. Further, immunofluorescence imaging of marker proteins showed that the nanowires surfaces did not appear to support chondrogenic differentiation whereas supported adipogenic differentiation of ADSCs.
Project description:Macrodactyly is a congenital malformation characterized by enlargement of bone and soft tissues in limbs, typically with excessive accumulation of adipose tissues. Although gain-of-function mutation of PIK3CA has been identified in macrodactyly, the mechanism of PIK3CA mutation in adipose accumulation is poorly understood. In this study, we found that adipocytes from macrodactyly were more hypertrophic than those observed in polydactyly. PIK3CA (H1047R) activating mutation and enhanced activity of PI3K/AKT pathway were detected in macrodactylous adipose-derived stem cells (Mac-ADSCs). Compared to polydactyly-derived ADSCs (Pol-ADSCs), Mac-ADSCs had higher potential in adipogenic differentiation. Knockdown of PIK3CA or inhibition by BYL-719, a potent inhibitor of PIK3CA, impaired adipogenesis of Mac-ADSCs in vitro. In vivo study, either transient treatment of ADSCs or intragastrical gavage with BYL-719 inhibited the adipose formation in patient-derived xenograft (PDX). Furthermore, RNA-seq revealed that E2F1 was up-regulated in Mac-ADSCs and its knockdown blocked the PIK3CA-promoted adipogenesis. Our findings demonstrated that PIK3CA activating mutation promoted adipogenesis of ADSCs in macrodactyly, and that this effect was exerted by the up-regulation of E2F1. This study revealed a possible mechanism for adipose accumulation in macrodactyly and suggested BYL-719 as a potential therapeutic agent for macrodactyly treatment.
Project description:BACKGROUND: Previous studies on the effects of aging in human and mouse mesenchymal stem cells suggest that a decline in the number and differentiation potential of stem cells may contribute to aging and aging-related diseases. In this report, we used stromal cells isolated from adipose tissue (ADSCs) of young (8-10 weeks), adult (5 months), and old (21 months) mice to test the hypothesis that mechanical loading modifies aging-related changes in the self-renewal and osteogenic and adipogenic differentiation potential of these cells. RESULTS: We show that aging significantly reduced the proliferation and increased the adipogenesis of ADSCs, while the osteogenic potential is not significantly reduced by aging. Mechanical loading (10% cyclic stretching, 0.5 Hz, 48 h) increased the subsequent proliferation of ADSCs from mice of all ages. Although the number of osteogenic colonies with calcium deposition was increased in ADSCs subjected to pre-strain, it resulted from an increase in colony number rather than from an increase in osteogenic potential after strain. Pre-strain significantly reduced the number of oil droplets and the expression of adipogenic marker genes in adult and old ADSCs. Simultaneously subjecting ADSCs to mechanical loading and adipogenic induction resulted in a stronger inhibition of adipogenesis than that caused by pre-strain. The reduction of adipogenesis by mechanical strain was loading-magnitude dependent: loading with 2% strain only resulted in a partial inhibition, and loading with 0.5% strain could not inhibit adipogenesis in ADSCs. CONCLUSIONS: We demonstrate that mechanical stretching counteracts the loss of self-renewal in aging ADSCs by enhancing their proliferation and, at the same time, reduces the heightened adipogenesis of old cells. These findings are important for the further study of stem cell control and treatment for a variety of aging related diseases.
Project description:The stemness maintenance of adipose-derived stem cells (ADSCs) is important for adipose homeostasis and energy balance. Programmed cell death 4 (Pdcd4) has been demonstrated to be involved in the development of obesity, but its possible roles in ADSC function and adipogenic capacity remain unclear. In this study, we demonstrate that Pdcd4 is a key controller that limits the self-renewal and white-to-beige transdifferentiation of ADSCs. Pdcd4 deficiency in mice caused stemness enhancement of ADSCs as evidenced by increased expression of CD105, CD90, Nanog and Oct4 on ADSCs, together with enhanced in situ proliferation in adipose tissues. Pdcd4 deficiency promoted proliferation, colony formation of ADSCs and drove more ADSCs entering the S phase accompanied by AKT activation and cyclinD1 upregulation. Blockade of AKT signaling in Pdcd4-deficient ADSCs led to a marked decline in cyclinD1, S-phase entry and cell proliferation, revealing AKT as a target for repressing ADSC self-renewal by Pdcd4. Intriguingly, depletion of Pdcd4 promoted the transdifferentiation of ADSCs into beige adipocytes. A reduction in lipid contents and expression levels of white adipocyte markers including C/EBPα, PPAR-γ, adiponectin and αP2 was detected in Pdcd4-deficient ADSCs during white adipogenic differentiation, substituted by typical beige adipocyte characteristics including small, multilocular lipid droplets and UCP1 expression. More lactate produced by Pdcd4-deficient ADSCs might be an important contributor to the expression of UCP1 and white-to-beige transdifferentiation. In addition, an elevation of UCP1 expression was confirmed in white adipose tissues from Pdcd4-deficient mice upon high-fat diet, which displayed increased energy expenditure and resistance to obesity as compared with wild-type obese mice. These findings provide evidences that Pdcd4 produces unfavorable influences on ADSC stemness, which contribute to adipose dysfunction, obesity and metabolic syndromes, thereby proposing Pdcd4 as a potential intervening target for regulating ADSC function.