Effects of GSK3 inhibitors on in vitro expansion and differentiation of human adipose-derived stem cells into adipocytes

<p>Abstract</p> <p>Background</p> <p>Multipotent stem cells exist within adipose tissue throughout life. An abnormal recruitment of these adipose precursor cells could participate to hyperplasia of adipose tissue observed in severe obesity or to hypoplasia of adipose tissue observed in lipodystrophy. Therefore, pharmacological molecules that control the pool of stem cells in adipose tissue are of great interest. Glycogen Synthase Kinase (GSK) 3 has been previously described as involved in differentiation of preadipose cells and might be a potential therapeutic target to modulate proliferation and differentiation of adipocyte precursors. However, the impact of GSK3 inhibition on human adipose-derived stem cells remained to be investigated. The aim of this study was to investigate GSK3 as a possible target for pharmacological inhibition of stem cell adipogenesis. To reach this goal, we studied the effects of pharmacological inhibitors of GSK3, i.e. lithium chloride (LiCl) and BIO on proliferation and adipocyte differentiation of multipotent stem cells derived from human adipose tissue.</p> <p>Results</p> <p>Our results showed that GSK3 inhibitors inhibited proliferation and clonogenicity of human stem cells, strongly suggesting that GSK3 inhibitors could be potent regulators of the pool of adipocyte precursors in adipose tissue. The impact of GSK3 inhibition on differentiation of hMADS cells was also investigated. Adipogenic and osteogenic differentiations were inhibited upon hMADS treatment with BIO. Whereas a chronic treatment was required to inhibit osteogenesis, a treatment that was strictly restricted to the early step of differentiation was sufficient to inhibit adipogenesis.</p> <p>Conclusion</p> <p>These results demonstrated the feasibility of a pharmacological approach to regulate adipose-derived stem cell function and that GSK3 could represent a potential target for controlling adipocyte precursor pool under conditions where fat tissue formation is impaired.</p

Small RNA sequencing reveals miR-642a-3p as a novel adipocyte-specific microRNA and miR-30 as a key regulator of human adipogenesis

International audienceABSTRACT: BACKGROUND: In severe obesity, as well as in normal development, the growth of adipose tissue is the result of an increase in adipocyte size and numbers, which is underlain by the stimulation of adipogenic differentiation of precursor cells. A better knowledge of the pathways that regulate adipogenesis is therefore essential for an improved understanding of adipose tissue expansion. As microRNAs (miRNAs) have a critical role in many differentiation processes, our study aimed to identify the role of miRNA-mediated gene silencing in the regulation of adipogenic differentiation. RESULTS: We used deep sequencing to identify small RNAs that are differentially expressed during adipogenesis of adipose tissue-derived stem cells. This approach revealed the un-annotated miR-642a-3p as a highly adipocyte-specific miRNA. We then focused our study on the miR-30 family, which was also up-regulated during adipogenic differentiation and for which the role in adipogenesis had not yet been elucidated. Inhibition of the miR-30 family blocked adipogenesis, whilst over-expression of miR-30a and miR-30d stimulated this process. We additionally showed that both miR-30a and miR-30d target the transcription factor RUNX2, and stimulate adipogenesis via the modulation of this major regulator of osteogenesis. CONCLUSIONS: Overall, our data suggest that the miR-30 family plays a central role in adipocyte development. Moreover, as adipose tissue-derived stem cells can differentiate into either adipocytes or osteoblasts, the down-regulation of the osteogenesis regulator RUNX2 represents a plausible mechanism by which miR-30 miRNAs may contribute to adipogenic differentiation of adipose tissue-derived stem cells

Activin A Plays a Critical Role in Proliferation and Differentiation of Human Adipose Progenitors

International audienceAbstractObjective: Growth of white adipose tissue takes place in normal development and in obesity. A pool of adipose progenitors is responsible for the formation of new adipocytes and for the potential of this tissue to expand in response to chronic energy overload. However, factors controlling self-renewal of human adipose progenitors are largely unknown. We investigated the expression profile and the role of activin A in this process. Research Design and Methods: Expression of INHBA/activin A has been investigated in three types of human adipose progenitors. We then analyzed at the molecular level the function of activin A during human adipogenesis. We finally investigated the status of activin A in adipose tissues of lean and obese subjects and analyzed macrophage-induced regulation of its expression. Results: INHBA/activin A is expressed by adipose progenitors from various fat depots and its expression dramatically decreases as progenitors differentiate into adipocytes. Activin A regulates the number of undifferentiated progenitors. Sustained activation or inhibition of the activin A pathway impairs or promotes respectively adipocyte differentiation via C/EBPbeta-LAP and Smad2 pathway in an autocrine/paracrine manner. Activin A is expressed at higher levels in adipose tissue of obese patients compared to lean subjects. Indeed, activin A levels in adipose progenitors are dramatically increased by factors secreted by macrophages derived from obese adipose tissue. Conclusions: Altogether, our data show that activin A plays a significant role in human adipogenesis. We propose a model in which macrophages which are located in adipose tissue regulate adipose progenitor self-renewal through activin A

TGFbeta Family Members Are Key Mediators in the Induction of Myofibroblast Phenotype of Human Adipose Tissue Progenitor Cells by Macrophages

International audienceOBJECTIVE: The present study was undertaken to characterize the remodeling phenotype of human adipose tissue (AT) macrophages (ATM) and to analyze their paracrine effects on AT progenitor cells. RESEARCH DESIGN AND METHODS: The phenotype of ATM, immunoselected from subcutaneous (Sc) AT originating from subjects with wide range of body mass index and from paired biopsies of Sc and omental (Om) AT from obese subjects, was studied by gene expression analysis in the native and activated states. The paracrine effects of ScATM on the phenotype of human ScAT progenitor cells (CD34(+)CD31(-)) were investigated. RESULTS: Two main ATM phenotypes were distinguished based on gene expression profiles. For ScAT-derived ATM, obesity and adipocyte-derived factors favored a pro-fibrotic/remodeling phenotype whereas the OmAT location and hypoxic culture conditions favored a pro-angiogenic phenotype. Treatment of native human ScAT progenitor cells with ScATM-conditioned media induced the appearance of myofibroblast-like cells as shown by expression of both α-SMA and the transcription factor SNAIL, an effect mimicked by TGFβ1 and activinA. Immunohistochemical analyses showed the presence of double positive α-SMA and CD34 cells in the stroma of human ScAT. Moreover, the mRNA levels of SNAIL and SLUG in ScAT progenitor cells were higher in obese compared with lean subjects. CONCLUSIONS: Human ATM exhibit distinct pro-angiogenic and matrix remodeling/fibrotic phenotypes according to the adiposity and the location of AT, that may be related to AT microenvironment including hypoxia and adipokines. Moreover, human ScAT progenitor cells have been identified as target cells for ScATM-derived TGFβ and as a potential source of fibrosis through their induction of myofibroblast-like cells

Engraftment of human adipose derived stem cells delivered in a hyaluronic acid preparation in mice Implante de células tronco do tecido adiposo humano numa preparação de ácido hialurônico em camundongos

PURPOSE: To evaluate the implant of human adipose derived stem cells (ADSC) delivered in hyaluronic acid gel (HA), injected in the subcutaneous of athymic mice. METHODS: Control implants -HA plus culture media was injected in the subcutaneous of the left sub scapular area of 12 athymic mice. ADSC implants: HA plus ADSC suspended in culture media was injected in the subcutaneous, at the contra lateral area, of the same animals. With eight weeks, animals were sacrificed and the recovered implants were processed for extraction of genomic DNA, and histological study by hematoxilin-eosin staining and immunufluorescence using anti human vimentin and anti von Willebrand factor antibodies. RESULTS: Controls: Not visualized at the injection site. An amorphous substance was observed in hematoxilin-eosin stained sections. Human vimentin and anti von Willebrand factor were not detected. No human DNA was detected. ADSC implants - A plug was visible at the site of injection. Fusiform cells were observed in sections stained by hematoxilin- eosin and both human vimentin and anti von Willebrand factor were detected by immunofluorescence. The presence of human DNA was confirmed. CONCLUSION: The delivery of human adipose derived stem cells in preparations of hyaluronic acid assured cells engraftment at the site of injection.<br>OBJETIVO: Avaliar o implante de células tronco do tecido adiposo humano (CTTAH) em gel de ácido hialurônico (AH), injetados no tecido subcutâneo de camundongos atímicos. MÉTODOS: Implantes controle - HA com meio de cultura foram injetados no tecido subcutâneo da região infraescapular esquerda de 12 camundongos atímicos. Implantes de CTTAH: HA com CTTAH suspensas em meio de cultura foi injetado no subcutâneo da região contra lateral, dos mesmos animais. Com oito semanas, os animais foram sacrificados e os implantes recuperados foram processados para extração de DNA genômico, estudo histológico por coloração por hematoxilina eosina e imnuoflurescência utilizando anticorpos anti vimentina humana e anti fator de von Willebrand. RESULTADOS: Controles - implantes não visualizados no local da injeção. Uma substância amorfa foi observada nos cortes corados por hematoxilina eosina. Vimentina humana e fator anti von Willebrand não foram identificados. DNA humano não foi detectado. Implantes de CTTAH - Uma massa era visível no local da injeção. Células fusiformes foram observadas nos corte corados com hematoxilina eosina. Tanto vimentina humana quanto fator de von Willebrand foram identificados pela imunofluorescência. A presença de DNA humano foi confirmada. CONCLUSÃO: O implante de células tronco do tecido adiposo humano em veículo de ácido hialurônico gel assegurou a manutenção das células no local do implante

Characterization of Human Knee and Chin Adipose-Derived Stromal Cells

Animal study findings have revealed that individual fat depots are not functionally equivalent and have different embryonic origins depending on the anatomic location. Mouse bone regeneration studies have also shown that it is essential to match the Hox code of transplanted cells and host tissues to achieve correct repair. However, subcutaneous fat depots from any donor site are often used in autologous fat grafting. Our study was thus carried out to determine the embryonic origins of human facial (chin) and limb (knee) fat depots and whether they had similar features and molecular matching patterns. Paired chin and knee fat depots were harvested from 11 subjects and gene expression profiles were determined by DNA microarray analyses. Adipose-derived stromal cells (ASCs) from both sites were isolated and analyzed for their capacity to proliferate, form clones, and differentiate. Chin and knee fat depots expressed a different HOX code and could have different embryonic origins. ASCs displayed a different phenotype, with chin-ASCs having the potential to differentiate into brown-like adipocytes, whereas knee-ASCs differentiated into white adipocytes. These results highlighted different features for these two fat sites and indicated that donor site selection might be an important factor to be considered when applying adipose tissue in cell-based therapies

Effects of GSK3 inhibitors on in vitro expansion and differentiation of human adipose-derived stem cells into adipocytes-2

<p><b>Copyright information:</b></p><p>Taken from "Effects of GSK3 inhibitors on in vitro expansion and differentiation of human adipose-derived stem cells into adipocytes"</p><p>http://www.biomedcentral.com/1471-2121/9/11</p><p>BMC Cell Biology 2008;9():11-11.</p><p>Published online 13 Feb 2008</p><p>PMCID:PMC2257931.</p><p></p>er cell plating, 0.5 μM BIO was added to the culture medium. Fifteen days later, the number of colonies was scored. Bars represent means of 2 independent experiments. **: p < 0.01, ***: p < 0.00

Effects of GSK3 inhibitors on in vitro expansion and differentiation of human adipose-derived stem cells into adipocytes-3

<p><b>Copyright information:</b></p><p>Taken from "Effects of GSK3 inhibitors on in vitro expansion and differentiation of human adipose-derived stem cells into adipocytes"</p><p>http://www.biomedcentral.com/1471-2121/9/11</p><p>BMC Cell Biology 2008;9():11-11.</p><p>Published online 13 Feb 2008</p><p>PMCID:PMC2257931.</p><p></p>bsence (Ctr) or presence of 0.5 μM BIO or 0.5 μM MeBIO. Ten days later, cells were stained with Oil Red O for adipocytes or with Alizarin red for osteoblasts (left panel). Effects of the compounds on GPDH, expressed in adipocytes only, and ALP, expressed in osteoblasts only, enzymatic activities are shown (right panel). Bars are the means ± SE of 3 independent experiments. *: p < 0.05, **: p < 0.0