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Objective:To investigate the possible role of long non-coding RNA (LncRNA) 00602 in promoting browning in adipocytes induced by adenovirus type 36 (Ad36).Methods:According to Ad36 infection, adipose tissue samples of obese patients were divided into Ad36-negative group and Ad36-infected group. Realtime fluorescent quantitative PCR (qRT-PCR) was used to detect the changes in the expression of LncRNA00602 mRNA in omental adipose tissue of the two groups, and analyze the differences between the two groups. The correlation between waist-to-hip ratio, systolic blood pressure, diastolic blood pressure, fasting blood glucose, triacylglyceride and other indicators of the patients in the group with LncRNA00602 mRNA expression were analyzed. HE staining was used to detect the size of adipocytes in the omental adipose tissue of the Ad36 negative group and the Ad36 infection group. qRT-PCR and Western blotting were used to detect the mRNA and protein expression levels of uncoupling protein 1 (UCP1) and PR domain containing 16 (PRDM16) in omental adipose tissue of two groups of patients. Human adipose-derived stem cells (hADSC) were isolated and cultured, using Ad36 to induce differentiation, and divided into control group and LncRNA00602 knockdown group. On 0, 2, and 4 days after LncRNA00602 knockdown, fluoroboron dipyrrole (BODIPY) and mitochondrial red fluorescence (Mito-Tracker Red) were used to stain intracellular lipid droplets and mitochondria. At the same time, qRT-PCR and Western blotting were used to detect changes in the expression of UCP1 and PRDM16.Results:The expression of LncRNA00602 gene in the Ad36 infection group was higher than that in the Ad36 negative group (all P<0.05). The expression of LncRNA00602 in the Ad36 negative group was not significantly different from the above clinical indicators, while the expression of LncRNA00602 was negatively correlated with serum fasting blood glucose and triacylglyceride ( r=-0.522, -0.486, P<0.05) in the Ad36 infection group; HE staining showed that the average adipocyte area of the Ad36 infection group was smaller than that of the Ad36 negative group. At the same time, UCP1 and PRDM16 gene expression were higher than the negative group (all P<0.05). At the cellular level, on the 2nd and 4th days after knockdown of LncRNA00602, the lipid droplet area of adipocytes in the LncRNA00602 knockdown group was larger than that of the control group, the number of mitochondria decreased compared with the control group, and difference was statistically significant ( P<0.05 or P<0.01); Compared with the control group, there was significantly lower expression of the browning marker genes UCP1, PRDM16, and protein in the adipocytes in the LncRNA00602 knockdown group (all P<0.05). Conclusion:In Ad36-induced adipocyte differentiation, LncRNA00602 may positively regulate the expression of UCP1, PRDM16 and lipid droplet metabolism, and promote the browning of adipocytes.
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Objective: To explore the possible mechanism of human adipose-derived stem cells (hADSCs) promoting seawater immersion wound healing in vitro. Methods: Human epidermal cell line HaCaT cells and artificially simulated seawater were used to establish an in vitro model of cell damage induced by seawater immersion. hADSCs were isolated from human adipose tissues, and a co-culture system of HaCaT cells and hADSCs was established. The proliferation and migration abilities of HaCaT cells were detected by cell counting kit-8 (CCK-8), 5-ethynyl-2′-deoxyuridine (EdU) cell proliferation detection kit and cell scratch test. The activation levels of epidermal growth factor receptor (EGFR)/extracellular-regulated protein kinase (ERK) signaling pathway were detected by Western blotting and real-time quantitative PCR. Results: The proliferation of HaCaT cells cultured with the medium containing 10% artificial seawater was significantly inhibited compared with the cells cultured without artificial seawater (P0.05). The expression of EGFR/ERK signaling pathway in seawater-cultured HaCaT cells was significantly inhibited compared with the cells cultured without seawater and those co-cultured with hADSCs and seawater (P0.05). Conclusion: Seawater can block the activation of EGFR/ERK signaling pathway and inhibit the proliferation and migration of HaCaT cells. hADSCs can promote the activation of EGFR/ERK signaling pathway and reduce the inhibition effect of seawater against proliferation and migration of HaCaT cells.
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Objective To investigate the role of LncRNA ROR in Ad36-induced browning of human adipose-derived stem cells(hADSC). Methods After hADSC was induced by cocktail and Ad36 for 2,4,6,and 8 days,Oil red O staining was performed for observing the adipogenic status. The mRNA expressions of LncRNA ROR, uncoupling protein 1(UCP1),and PRDM16 were detected by real-time PCR and the protein expressions of UCP1 and PRDM16 were detected by Western-blot. After LncRNA ROR was knocked down by siRNA, UCP1 and PRDM16 mRNA and protein expression levels in the process of Ad36-induced adipocyte differentiation were detected by real-time PCR and Western-blot. Results Oil red O staining showed that fat droplets in the cocktail-induced group were larger than those in the Ad36-induced group. Compared with the cocktail group,the mRNA expressions of LncRNA ROR, UCP1 and PRDM16, and the protein expressions of UCP1 and PRDM16 in Ad36 group were significantly increased(P<0.05). The mRNA and protein expressions of UCP1 and PRDM16 in LncRNA ROR knockdown group were significantly lower than those in control group(P<0.05). Conclusion In the process of Ad36-induced hADSC differentiation,the up-regulation of LncRNA ROR may stimulate UCP1 and PRDM16 expression,and thus promote the browning of hADSC.
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Objective:To construct a novel biomimetic calcium phosphate (BioCaP) scaffold loaded with bone morphogenetic protein-2 (BMP-2),and to investigate its role in the osteogenesis of human adipose-derived stem cells (hASCs) in vitro and in vivo.Methods:The BioCaP scaffold coprecipitated with BMP-2 (BMP-2-BioCaP) was constructed in this study.Field emission scanning electron microscopy (SEM) was used to analyze the morphology of the surfaces.The release kinetics was measured to evaluate the slow-release characteristics in vitro.BMP-2-BioCaP was immersed in proliferation medium (PM) or osteogenic medium (OM),respectively.The supernatants were collected and used to culture hASCs in vitro.Cell numbers were determined using the cell-counting kit-8 (CCK-8) to assess the cell proliferation.After 7 and 14 days,alkaline phosphatase (ALP) staining and quantification were performed to test the activity of ALP.After 14 and 21 days,the calcification deposition was determined by alizarin red S (ARS) staining and quantification.The expressions of the osteoblast-related genes were tested on day 4 and day 14.In the in vivo study,6 nude mice were used and implanted subcutaneously into the back of the nude mice for 4 groups:(1) BioCaP scaffold only,(2) BioCaP scaffold + hASCs,(3) BMP-2-BioCaP scaffold,(4) BMP-2-BioCaP scaffold + hASCs (test group).After 4 weeks of implantation,hematoxylin-eosin (HE) staining was performed to evaluate the in vivo osteogenesis of hASCs.Results:SEM observations showed that BioCaP and BMP-2-BioCaP scaffold were entirely composed of straight,plate-like and sharp-edged crystal units,and the length of the crystal units varied between 5 and 10 μm.Release kinetics analysis demonstrated that BMP-2 incorporated with BioCaP could be released at certain concentration and last for more than 21 days,and the accumulative protein release could reach 20％.CCK-8 assays showed that cell proliferation was not significantly affected by BMP-2BioCaP.ALP activity was higher by the induction of OM + BMP-2-BioCaP than of the other groups (P ＜0.01).More mineralization deposition and more expressions of osteoblast-related genes such as Runt-related transcription factor 2 (RUNX2),ALP,osteopontin (OPN) and osteocalcin (OC) were determined in the OM + BMP-2-BioCaP group at different time points (P ＜0.01).HE staining showed that,in the test group and BMP-2-BioCaP scaffold group,the extracellular matrix (ECM) with eosinophilic staining were observed around hASCs,and newly-formed bone-like tissues could be found in ECM around the scaffold materials.Moreover,compared with the BMP-2-BioCaP scaffold group,more bone-like tissues could be observed in ECM with typical structure of bone tissue in the test groups.No obvious positive results were found in the other groups.Conclusion:BMP-2-BioCaP scaffold could achieve slow-release of BMP-2 and promote the osteogenic differentiation of hASCs in vitro and in vivo.The novel tissue-engineered bone composed of hASCs and BMP-2-BioCaPis promising for the repair of bone defect.
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BACKGROUND AND OBJECTIVES: Recent findings suggest that therapeutic potential of mesenchymal stem cells (MSCs) could be increased through aggregation into three-dimensional (3D) bodies, and different culture methods have been employed to obtain 3D spheroids of MSCs. In the current study we report accidentally encountered spontaneous formation of adipose-derived stem cell (ASC) bodies in standard ASC culture of a single donor. METHODS AND RESULTS: Human ASCs from passages 1 to 3, cultured in a medium containing 5% autologous serum (AS), spontaneously clustered and formed floating 3D bodies. After a transfer of floating ASC bodies onto new adherent plastic dish, they attached to the surface and gradual migration of spindle-shaped ASCs out of the bodies was detected. A substitution of AS with allogeneic sera did not hinder this ability, but commercial medium containing fetal bovine serum delayed the process. Substantial part of ASCs surrounding transferred ASC bodies showed alkaline phosphatase (AP) activity, while ASC aggregates were AP negative. Similar 3D bodies formed when ASCs were grown on an uncoated glass surface. These ASC aggregates as well as clusters of ASCs, where formation of the 3D bodies is initiated, expressed pluripotency marker NANOG, but the expression of OCT4A was not detected. CONCLUSIONS: Obtained results suggest that spontaneously formed ASC aggregates may represent a more primitive cell subpopulation within the individual ASC culture. The ability to form 3D aggregates, the expression of NANOG, and the lack of the AP activity may be used to enrich ASC cultures with potentially more primitive cells serving as an excellent basis for therapeutic applications.
Subject(s)
Humans , Alkaline Phosphatase , Glass , Mesenchymal Stem Cells , Plastics , Stem Cells , Tissue DonorsABSTRACT
Sirtuins (SIRTs) are involved in multiple cellular processes. And they are involved in cellular pathways related aging, cancer, and a variety of cellular functions including cell cycle, DNA repair and proliferation. Also they modulate life span. Stem cells have the ability to self-renew for unlimited proliferation and differentiate into various cell types. It has been a little known that the mutation of undifferentiated stem cells in tissue may result in the development of cancer cells by genotoxic carcinogens. Therefore, this study investigated whether some carcinogenic compounds can modulate the expression of sirtuin mRNA on human adipose-derived stem cells. Adipose-derived stem cells (ADSC) were exposed to genotoxic carcinogenic compound (2-nitrofluorene, 2NF) and non-genotoxic carcinogenic compound (clonidine, CND) for 24 hours, 48 hours, 72 hours and 96 hours. The expression of SIRT1 mRNA increased on 72 hours. Expressions of SIRT2 and SIRT7 mRNA increased robustly on 48 hours. But all of SIRTs decreased to a level before a treatment of genotoxic compound on adipose-derived stem cells. These results demonstrated that a treatment of genotoxic compound induced the expression of SIRT mRNA only in the short time. But their level returned to untreated cells on 96 hours. They suggest that the possibility that the sirtuins can retard the carcinogenesis of adipose derived stem cells.
Subject(s)
Humans , Aging , Carcinogenesis , Carcinogens , Cell Cycle , Clonidine , DNA Repair , RNA, Messenger , Sirtuins , Stem CellsABSTRACT
Objective To study the effect of the exogenous recombinant human FGF-basic with different concentrations upon inducing human adipose-derived stem cells (hASCs) to differentiate into adipose cells,and the optimum concentration of exogenous rh-bFGF by experimental research.Methods hASCs were isolated and extracted by enzymatic digestion from the liposuction aspirate.hASCs using adipogenic supplement were divided into experimental group and blank group:the experimental group of adipogenic supplement was divided into adding the exogenous rh-bFGF 10 ng/ml,20 ng/ml and 40 ng/ml,the blank group of adipogenic supplement was cultured without exogenous rh-bFGF.MTT method was used to detect the adipocytes proliferation.The oil red O staining was used in the qualitative analysis on the time of newly forming adipocyte cells.Western blot was used to detect the effects of rh-bFGF on the expression of lipid droplets surface protein CIDEC at different stages during the culture.Results The experimental group could obviously shorten the period of inducing hASCs to differentiate into adioicytes,and promote the proliferation of adipocytes.The formation rate and the proliferation of adipocytes in the group adding 40 ng/ml rh-bFGF were superior to those in the experimental group else and blank group.The average time of the newly formed lipid droplets by adding 40 ng/ml rh-bFGFwas (11.5±1.9)h.The average absorbance of cell proliferation by adding 40ml rh-bFGF was 0.52 ±0.10.The CIDEC expression quantity of adding 40 ng/ml rh-bFGF group was also superior to that in the experimental group and blank group.Conclusions rh-bFGF in hASCs adipogenic supplement could promote the proliferation of adipocytes and dramatically accelerates the program of hASCs differentiating to adipocytes,in which the optimum concentration of rh-bFGF is 40ng/ml.
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PURPOSE: This study aims to establish a new strategy that provides for the rapid establishment of human clonal adipose derived stem cell (hADSC) lines with aspirated adipose tissue and to characterize newly generated hMSC lines for their cell phenotype, differentiation potential, lineage-specific gene expression. METHODS: Human adipose tissue-derived stem cells (hADSCs) were isolated from subcutaneous adipose tissue based on standard protocols. After incubation for 2 h, only the cell culture supernatant was transferred to a new dish. This process was repeated several times with 30 h incubations. RESULTS: We confirmed the difference in growth rate, however, differences were not seen in the differentiation capabilities and stemness of the each cell lines. CONCLUSION: It is necessary to establish cell lines via single cell level for application to disease specific tissue engineering.