Knockdown of LAP2α inhibits adipogenesis of human adipose-derived stem cells and ameliorates high-fat diet-induced obesity.

Adipogenesis, a pivotal cellular process involving the differentiation of mesenchymal stem cells (MSCs) to mature adipocytes, plays a significant role in various physiological functions. Dysregulation of adipogenesis is implicated in conditions such as obesity. However, the complete molecular understanding of adipogenesis remains elusive. This study aimed to uncover the novel role of lamina-associated polypeptide 2 alpha (LAP2α) in human adipose-derived stem cells (hASCs) adipogenesis and its impact on high-fat diet (HFD)-induced obesity and associated metabolic disturbances. LAP2α expression was assessed during the adipogenic differentiation of hASCs using RT-qPCR and western blotting. The functional role of LAP2α in adipogenesis was explored both in vitro and in vivo through loss- and gain-of-function studies. Moreover, mice with HFD-induced obesity received lentivirus injection to assess the effect of LAP2α knockdown on fat accumulation. Molecular mechanisms underlying LAP2α in adipogenic differentiation were investigated using RT-qPCR, Western blotting, immunofluorescence staining, and Oil Red O staining. LAP2α expression was upregulated during hASCs adipogenic differentiation. LAP2α knockdown hindered adipogenesis, while LAP2α overexpression promoted adipogenic differentiation. Notably, LAP2α deficiency resisted HFD-induced obesity, improved glucose intolerance, mitigated insulin resistance, and prevented fatty liver development. Mechanistically, LAP2α knockdown attenuated signal transducer and activator of transcription 3 (STAT3) activation by reducing the protein level of phosphorylated STAT3. A STAT3 activator (Colivelin) counteracted the negative impact of LAP2α deficiency on hASCs adipogenic differentiation. Taken together, our current study established LAP2α as a crucial regulator of hASCs adipogenic differentiation, unveiling a new therapeutic target for obesity prevention.

Exosomes derived from human adipose-derived stem cells alleviate hepatic ischemia-reperfusion (I/R) injury through the miR-183/ALOX5 axis.

Ischemia-reperfusion (I/R) injury is a crucial factor causing liver injury in the clinic. Recent research has confirmed that human adipose-derived stem cells (ADSCs) can differentiate into functional hepatocytes. However, the mechanism of the effects of ADSCs in the treatment of liver injury remains unclear. The characteristics of ADSCs were first identified, and exosome-derived ADSCs were isolated and characterized. The function and mechanism of action of miR-183 and arachidonate 5-lipoxygenase (ALOX5) were investigated by functional experiments in HL-7702 cells with I/R injury and in I/R rats. Our data disclosed that exosome release from ADSCs induced proliferation and inhibited apoptosis in HL-7702 cells with I/R injury. The effect of miR-183 was similar to that of exosomes derived from ADSCs. In addition, ALOX5, as a target gene of miR-183, was involved in the related functions of miR-183. Moreover, in vivo experiments confirmed that miR-183 and exosomes from ADSCs could improve liver injury in rats and inhibit the MAPK and NF-κB pathways. All of these findings demonstrate that exosomes derived from ADSCs have a significant protective effect on hepatic I/R injury by regulating the miR-183/ALOX5 axis, which might provide a therapeutic strategy for liver injury.

Skin rejuvenation and photoaging protection using adipose-derived stem cell extracellular vesicles loaded with exogenous cargos.

BACKGROUND: Small extracellular vesicles from adipose-derived stem cells (ASC-sEVs) have gained remarkable attention for their regenerative and protective properties against skin aging. However, the use of ASC-sEVs to further encapsulate certain natural anti-aging compounds for synergistic effects has not been actively explored. For large-scale production in skincare industry, it is also crucial to standardize cost-effective methods to produce highly pure ASC-sEVs. METHODS: Human ASCs were expanded in serum-free media with different compositions to first optimize the sEV production. ASC-sEVs from different batches were then purified using tangential flow filtration and sucrose cushion ultracentrifugation, followed by extensive characterization for identity and content profiling including proteomics, lipidomics and miRNA sequencing. ASC-sEVs were further loaded with nicotinamide riboside (NR) and resveratrol by sonication-incubation method. The therapeutic effect of ASC-sEVs and loaded ASC-sEVs was tested on human keratinocyte cell line HaCaT exposed to UVB by measuring reactive oxygen species (ROS). The loaded ASC-sEVs were later applied on the hand skin of three volunteers once a day for 8 weeks and skin analysis was performed every 2 weeks. RESULTS: Our standardized workflow produced ASC-sEVs with high yield, high purity and with stable characteristics and consistent biocargo among different batches. The most abundant subpopulations in ASC-sEVs were CD63+ (∼30%) and CD81+ -CD63+ (∼35%). Purified ASC-sEVs could be loaded with NR and resveratrol at the optimized loading efficiency of ∼20%. In UVB-exposed HaCaT cells, loaded ASC-sEVs could reduce ROS by 38.3%, higher than the sEVs (13.3%) or compounds (18.5%) individually. In human trial, application of loaded ASC-sEVs after 8 weeks substantially improved skin texture, increased skin hydration and elasticity by 104% and reduced mean pore volume by 51%. CONCLUSIONS: This study demonstrated a robust protocol to produce ASC-sEVs and exogenously load them with natural compounds. The loaded ASC-sEVs exhibited synergistic effects of both sEVs and anti-aging compounds in photoaging protection and skin rejuvenation.

[Ubiquitin-specific protease 42 regulates osteogenic differentiation of human adipose-derived stem cells].

OBJECTIVE: To explore the effect of ubiquitin-specific protease 42 (USP42) on osteogenic differentiation of human adipose-derived stem cells (hASCs) in vivo and in vitro. METHODS: A combination of experiments was carried out with genetic depletion of USP42 using a lentiviral strategy. Alkaline phosphatase (ALP) staining and quantification, alizarin red S (ARS) staining and quantification were used to determine the osteogenic differentiation ability of hASCs under osteogenic induction between the experimental group (knockdown group and overexpression group) and the control group. Quantitative reverse transcription PCR (qRT-PCR) was used to detect the expression levels of osteogenesis related genes in the experimental group and control group, and Western blotting was used to detect the expression levels of osteogenesis related proteins in the experimental group and control group. Nude mice ectopic implantation experiment was used to evaluate the effect of USP42 on the osteogenic differentiation of hASCs in vivo. RESULTS: The mRNA and protein expressions of USP42 in knockdown group were significantly lower than those in control group, and those in overexpression group were significantly higher than those in control group. After 7 days of osteogenic induction, the ALP activity in the knockdown group was significantly higher than that in the control group, and ALP activity in overexpression group was significantly lower than that in control group. After 14 days of osteogenic induction, ARS staining was significantly deeper in the knockdown group than in the control group, and significantly lighter in overexpression group than in the control group. The results of qRT-PCR showed that the mRNA expression levels of ALP, osterix (OSX) and collagen type Ⅰ (COLⅠ) in the knockdown group were significantly higher than those in the control group after 14 days of osteogenic induction, and those in overexpression group were significantly lower than those in control group. The results of Western blotting showed that the expression levels of runt-related transcription factor 2 (RUNX2), OSX and COLⅠ in the knockout group were significantly higher than those in the control group at 14 days after osteogenic induction, while the expression levels of RUNX2, OSX and COLⅠ in the overexpression group were significantly lower than those in the control group. Hematoxylin-eosin staining of subcutaneous grafts in nude mice showed that the percentage of osteoid area in the knockdown group was significantly higher than that in the control group. CONCLUSION: Knockdown of USP42 can significantly promote the osteogenic differentiation of hASCs in vitro and in vivo, and overexpression of USP42 significantly inhibits in vivo osteogenic differentiation of hASCs, and USP42 can provide a potential therapeutic target for bone tissue engineering.

Small extracellular vesicles derived from human adipose-derived stem cells regulate energetic metabolism through the activation of YAP/TAZ pathway facilitating angiogenesis.

Recent studies have found small extracellular vesicles (sEVs) that are secreted from human adipose tissue-derived stem cells (hADSCs-sEVs) and contribute to angiogenesis. Glycolysis, the primary energetic pathway of vascular endothelial cells, plays a key role in the process of angiogenesis. However, hADSCs-sEVs' effects on energy metabolism within endothelial cells remain unclear. In our study, we found that hADSCs-sEVs restored glycolytic metabolism suppressed by 3-(pyridinyl)-1-(4-pyridinyl)-2-propen-1-one(3PO), a unique glycolytic inhibitor increasing the extracellular acidification rate (ECAR), oxygen consumption rate (OCR), glycolytic gene expression as well as pyruvate, lactate, and ATP production in HUVEC cells. In contrast, hADSCs-sEVs decreased PDH-E1α expression and acetyl-CoA production. The above results indicate that hADSCs-sEVs promote HUVEC angiogenesis via enhancing glycolysis and suppressing mitochondrial oxidative phosphorylation. Furthermore, we found that the YAP/TAZ pathway may play a key role in the effects hADSCs-sEVs have on HUVECs, thus, providing a promising approach for pro-angiogenesis-related regeneration.

Fabrication and multiscale modeling of polycaprolactone/amniotic membrane electrospun nanofiber scaffolds for wound healing.

BACKGROUND: Enhancing the efficiency of cell-based skin tissue engineering (TE) approaches is possible via designing electrospun scaffolds possessing natural materials like amniotic membrane (AM) with wound healing characteristics. Concentrating on this aim, we fabricated innovative polycaprolactone (PCL)/AM scaffolds through the electrospinning process. METHODS: The manufactured structures were characterized by employing scanning electron microscope (SEM), attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy, tensile testing, Bradford protein assay, etc. In addition, the mechanical properties of scaffolds were simulated by the multiscale modeling method. RESULTS: As a result of conducting various tests, it was concluded that the uniformity and distribution of fibers decreased with an increase in the amniotic content. Furthermore, PCL-AM scaffolds contained amniotic and PCL characteristic bands. In the case of protein release, greater content of AM led to the release of higher amounts of collagen. Tensile testing revealed that scaffolds' ultimate strength increased when the AM content augmented. The multiscale modeling demonstrated that the scaffold had elastoplastic behavior. In order to assess cellular attachment, viability, and differentiation, human adipose-derived stem cells (ASCs) were seeded on the scaffolds. In this regard, SEM and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assays showed significant cellular proliferation and viability on the proposed scaffolds, and these analyses illustrated that higher cell survival and adhesion could be achieved when scaffolds possessed a larger amount of AM. After 21 days of cultivation, particular keratinocyte markers, such as keratin I and involucrin, were identified through utilizing immunofluorescence and real-time polymerase chain reaction (PCR) tests. The markers' expressions were higher in the PCL-AM scaffold with a ratio of 90:10 v v-1 compared with the PCL-epidermal growth factor (EGF) structure. Moreover, the presence of AM in the scaffolds resulted in the keratinogenic differentiation of ASCs even without employing EGF. Consequently, this state-of-the-art experiment suggests that the PCL-AM scaffold can be a promising candidate in skin bioengineering. CONCLUSION: This study showed that mixing AM with PCL, a widely used polymer, in different concentrations can overcome PCL disadvantages such as high hydrophobicity and low cellular compatibility.

Down-regulation of hsa-circ-0107593 promotes osteogenic differentiation of hADSCs via miR-20a-5p/SMAD6 signaling.

OBJECTIVES: Increasing evidence indicated circRNAs were involved in stem cells osteogenesis differentiation. Herein, we aimed to clarify the role of hsa-circ-0107593 during the osteogenesis process of human adipose-derived stem cells (hADSCs) and the underlying mechanisms. METHODS: The ring structure of hsa-circ-0107593 was confirmed using RNase R treatment and Sanger sequencing. Nucleoplasmic separation and fluorescence in situ hybridization detected hsa-circ-0107593 distribution. Lentivirus and siRNA were used to modulate the expression of hsa-circ-0107593, and the binding relationship between hsa-circ-0107593 and miR-20a-5p was verified by luciferase assay and RNA immunoprecipitation. We detected the osteogenic activity of hADSCs through alkaline phosphatase staining, alizarin red S staining, real-time polymerase chain reaction (RT-PCR), western blot, and cellular immunofluorescence experiment. In vivo, micro-computed tomography was performed to analyze bone formation around skull defect. RESULTS: RT-PCR results exhibited that hsa-circ-0107593 was downregulated while miR-20a-5p was upregulated during hADSCs osteogenesis. In vivo and in vitro experiments results indicated that knocking down hsa-circ-0107593 promoted the osteogenic differentiation of hADSCs, while overexpression of hsa-circ-0107593 showed an inhibitory effect on hADSCs osteogenic differentiation. In vitro experiment results showed hsa-circ-0107593 acted as a hADSCs osteogenic differentiation negative factor for it inhibited the suppressing effect of miR-20a-5p on SMAD6. CONCLUSION: Knocking down hsa-circ-0107593 acts as a positive factor of the osteogenic differentiation of hADSCs via miR-20a-5p/SMAD6 signaling.

Netrin-1 promotes the vasculogenic capacity of human adipose-derived stem cells.

Adipose derived stem cells (ADSCs) have been increasingly explored for use in cell-based therapy against ischemic diseases. However, unsatisfactory angiogenesis limits the therapeutic efficacy. Netrin-1, a known axon guidance molecule, improves neovascularization in the ischemic region. Thus, our study was performed to evaluate the potential effect of Netrin-1 on the angiogenic behaviors of human ADSCs (hADSCs). hADSCs acquired from human abdominal adipose tissue were modified by liposome transfection of Netrin-1 plasmid, and the proliferation of hADSCs was determined by Cell Counting Kit-8 (CCK-8) assay. The transcript levels of pro-invasive proteins such as matrix metalloproteinase 2 (MMP2) and matrix metalloproteinase 9 (MMP-9), were measured to test migratory and invasive capabilities, and the levels of vascular endothelial growth factors were assayed to monitor angiogenic activity. Our results showed that Netrin-1 overexpression enhanced the proliferation of hADSCs, and promoted the migration and invasion of hADSCs, as indicated by increased levels of MMP-2 and MMP-9. Furthermore, Netrin-1 overexpression increased the expression of vascular endothelial growth factor and placental growth factor in hADSCs. Our results highlighted the possibility that genetic modification of hADSCs by Netrin-1 overexpression might be beneficial for cell transplantation therapy against ischemic diseases.

3D-Cultured Adipose-Derived Stem Cell Spheres Using Calcium-Alginate Scaffolds for Osteoarthritis Treatment in a Mono-Iodoacetate-Induced Rat Model.

Osteoarthritis (OA) is a degenerative disease that causes pain, cartilage deformation, and joint inflammation. Mesenchymal stem cells (MSCs) are potential therapeutic agents for OA treatment. However, the 2D culture of MSCs could potentially affect their characteristics and functionality. In this study, calcium-alginate (Ca-Ag) scaffolds were prepared for human adipose-derived stem cell (hADSC) proliferation with a homemade functionally closed process bioreactor system; the feasibility of cultured hADSC spheres in heterologous stem cell therapy for OA treatment was then evaluated. hADSC spheres were collected from Ca-Ag scaffolds by removing calcium ions via ethylenediaminetetraacetic acid (EDTA) chelation. In this study, 2D-cultured individual hADSCs or hADSC spheres were evaluated for treatment efficacy in a monosodium iodoacetate (MIA)-induced OA rat model. The results of gait analysis and histological sectioning showed that hADSC spheres were more effective at relieving arthritis degeneration. The results of serological and blood element analyses of hADSC-treated rats indicated that the hADSC spheres were a safe treatment in vivo. This study demonstrates that hADSC spheres are a promising treatment for OA and can be applied to other stem cell therapies or regenerative medical treatments.

Oxytocin Modulates Osteogenic Commitment in Human Adipose-Derived Stem Cells.

Human adipose-derived stem cells (hASCs) are commonly harvested in minimally invasive contexts with few ethical concerns, and exhibit self-renewal, multi-lineage differentiation, and trophic signaling that make them attractive candidates for cell therapy approaches. The identification of natural molecules that can modulate their biological properties is a challenge for many researchers. Oxytocin (OXT) is a neurohypophyseal hormone that plays a pivotal role in the regulation of mammalian behavior, and is involved in health and well-being processes. Here, we investigated the role of OXT on hASC proliferation, migratory ability, senescence, and autophagy after a treatment of 72 h; OXT did not affect hASC proliferation and migratory ability. Moreover, we observed an increase in SA-ß-galactosidase activity, probably related to the promotion of the autophagic process. In addition, the effects of OXT were evaluated on the hASC differentiation ability; OXT promoted osteogenic differentiation in a dose-dependent manner, as demonstrated by Alizarin red staining and gene/protein expression analysis, while it did not affect or reduce adipogenic differentiation. We also observed an increase in the expression of autophagy marker genes at the beginning of the osteogenic process in OXT-treated hASCs, leading us to hypothesize that OXT could promote osteogenesis in hASCs by modulating the autophagic process.

A Rare Olive Compound Oleacein Improves Lipid and Glucose Metabolism, and Inflammatory Functions: A Comprehensive Whole-Genome Transcriptomics Analysis in Adipocytes Differentiated from Healthy and Diabetic Adipose Stem Cells.

Oleacein (OLE), a rare natural compound found in unfiltered extra virgin olive oil, has been shown to have anti-inflammatory and anti-obesity properties. However, little is known regarding the mechanisms by which OLE influences metabolic processes linked to disease targets, particularly in the context of lipid metabolism. In the present study, we conducted whole-genome DNA microarray analyses in adipocytes differentiated from human adipose-derived stem cells (hASCs) and diabetic hASCs (d-hASCs) to examine the effects of OLE on modulating metabolic pathways. We found that OLE significantly inhibited lipid formation in adipocytes differentiated from both sources. In addition, microarray analysis demonstrated that OLE treatment could significantly downregulate lipid-metabolism-related genes and modulate glucose metabolism in both adipocyte groups. Transcription factor enrichment and protein-protein interaction (PPI) analyses identified potential regulatory gene targets. We also found that OLE treatment enhanced the anti-inflammatory properties in adipocytes. Our study findings suggest that OLE exhibits potential benefits in improving lipid and glucose metabolism, thus holding promise for its application in the management of metabolic disorders.

CD146 expression regulates osteochondrogenic differentiation of human adipose-derived stem cells.

Tissue engineering aims to develop innovative approaches to repair tissue defects. The use of adipose-derived stem cells (ASCs) in tissue regeneration was extensively investigated for osteochondrogenesis. Among the ASC population, ASCs expressing the CD146 were demonstrated to be multipotent and considered as perivascular stem cells, although the functional role of CD146 expression in these cells remains unclear. Herein, we investigated the influence of CD146 expression on osteochondrogenic differentiation of ASCs. Our results showed that, in two-dimensional culture systems, sorted CD146+ ASCs proliferated less and displayed higher adipogenic and chondrogenic potential than CD146- ASCs. The latter demonstrated a higher osteogenic capacity. Besides this, CD146+ ASCs in three-dimensional Matrigel/endothelial growth medium (EGM) cultures showed the highest angiogenic capability. When cultured in three-dimensional collagen scaffolds, CD146+ ASCs showed a spontaneous chondrogenic differentiation, further enhanced by the EGM medium's addition. Finally, CD146- ASCs seeded on hexafluoroisopropanol silk scaffolds displayed a greater spontaneous osteogenetic capacity. Altogether, these findings demonstrated a functional and relevant influence of CD146 expression in ASC properties and osteochondrogenic commitment. Exploiting the combination of specific differentiation properties of ASC subpopulations and appropriate culture systems could represent a promising strategy to improve the efficacy of new regenerative therapies.

A new hydrogel with fluorapatite nanoparticles for osteogenic differentiation of human adipose-derived stem cells in tissue engineering field.

Since scaffolds are engineered to support functional tissue formation, their design and materials play an essential role in medical fields by providing different mechanical function. The aim of this study was to investigate the synthesis and structural characterization of collagen-gelatin (COL-GEL) composite scaffolds containing fluorapatite (FA) nanoparticles as well as evaluation of the osteogenic differentiation of human adipose-derived stem cells (hADSCs). First, the composite scaffolds were evaluated using Fourier transform infrared spectroscopy, scanning electron microscopy, and X-ray diffraction. The cytotoxicity of scaffolds and various concentrations of FA nanoparticles was studied through MTT assay and acridine orange/ethidium bromide staining. Next, the differentiated hADSCs were analyzed using Alizarin red and von Kossa staining, calcium content assay, alkaline phosphatase (ALP) activity, real-time RT-PCR, and immunocytochemical analyses. According to the characterization analyses, the composite scaffolds were properly integrated. The results also illustrated that COL-GEL composite scaffolds in the presence of FA nanoparticles not only showed no cytotoxicity but also increased ALP activity and calcium deposition as well as the expression of osteogenic genes, including Runx2, Col-I, ALP, and osteocalcin and the synthesis of proteins such as osteocalcin and osteopontin in vitro. The obtained data were confirmed by Alizarin red and von Kossa staining. These results are very promising for further tissue engineering experiments, in which FA nanoparticle incorporation into COL-GEL composite scaffolds is a novel approach that improves the surface COL-GEL composite scaffolds for tissue engineering application in vitro.

Circ_0011129 Encapsulated by the Small Extracellular Vesicles Derived from Human Stem Cells Ameliorate Skin Photoaging.

Photoaging is not only the main cause of skin aging caused by exogenous factors, it is also related to a variety of skin diseases and even malignant tumors. Excessive and repeated exposure to ultraviolet radiation, especially UVA induces oxidative stress, DNA damage, inflammation, and collagen and elastin degeneration, ultimately leads to skin photoaging, manifested by skin redness, coarse wrinkles, and pigmentation even skin cancer. There has been a large demand of effective prevention and medications but approaches in the current management of photoaging are very limited. In the previous study, we found that a non-coding circular RNA circ_0011129 acts as a miR-6732-5p adsorption sponge to inhibit the reduction of type I collagen and the denaturation and accumulation of elastin in UVA-induced HDF cells photoaging model. However, in vivo instability and efficient delivery to the target cell of circRNA is a major challenge for its clinical application. Therefore, improving its stability and delivery efficiency are desired. In this study, we proposed a strategy of delivering circ_0011129 with small extracellular vesicles (sEVs) from human adipose-derived stem cells (hADSCs) to intervene in the photoaging process. The results showed that sEVs from hADSCs in 3D bioreactor culture (3D-sEVs) can prevent photoaging. Consequently, by overexpressing circ_0011129 in hADSCs, we successfully loaded it into 3D-sEVs (3D-circ-sEVs) and its protective effect was better. Our studies provide a novel approach to preventing skin photoaging, which has important clinical significance and application value for the development of non-coding RNA drugs to treat skin photoaging. We first screened out hADSCs-derived sEVs with excellent anti-oxidant effects. We then compared the sEVs collected from traditional 2D culture with 3D bioreactor culture. By miRNA-seq and GEO data analysis, we found that miRNAs in 3D-sEVs were enriched in cell activities related to apoptosis, cellular senescence, and inflammation. Subsequently, we prepared circ_0011129-loaded 3D-sEVs (3D-circ-sEVs) by overexpressing it in hADSCs for the treatment of photoaging in vitro. We proved that 3D-circ-sEVs can interfere with the process of cell photoaging and protect cells from UVA radiation damage, as well as in a H2O2-induced oxidative stress model.

Knock-down of circular RNA H19 induces human adipose-derived stem cells adipogenic differentiation via a mechanism involving the polypyrimidine tract-binding protein 1.

PURPOSE: The metabolic syndrome (MetS) is characterized of a cluster of medical disorders. Altered function of adipose tissue has a significant impact on whole-body metabolism and represents a key driver for MetS. In this study, we aim to explore the function of human circular RNA H19 (hsa_circH19) in human adipose-derived stem cells (hADSCs). METHODS: The blood samples from MetS patients and normal subjects were used to determine the expression level of the hsa_circH19. After knock-down of hsa_circH19 in hADSCs, we measured the expression of adipogenic genes. Oil red O, Nile red staining assay and triglyceride assessment were performed to examine the role of hsa_circH19 in hADSCs differentiation. Then, RNA Pull-down and RIP assays were conducted to explore the related RNA binding protein of hsa_circH19. IF was performed to determine the potential molecular regulatory mechanism. RESULTS: After accounting for confounding factors, high levels of hsa_circH19 remained an independent risk factor for MetS. Furthermore, the knockdown of hsa_circH19 significantly increased the expression of adipogenic genes and the formation of lipid droplets. Bioinformatics analyses revealed that has_circH19 shared multiple binding sites with polypyrimidine tract-binding protein 1 (PTBP1) and their interaction was validated by circRNA pull-down and RIP assays. Mechanistically, depletion of hsa_circH19 triggered translocation of sterol-regulatory element binding proteins (SREBP1) from cytoplasm to nucleus in the presence of PTBP1. CONCLUSION: Our experiments suggest that knockdown of hsa_circH19 promotes hADCSs adipogenic differentiation via targeting of PTBP1. In consequence, the expression of hsa_circH19 might correlated to lipid metabolism in adipose tissue from MetS.

Combination of 5-azaytidine and hanging drop culture convert fat cell into cardiac cell.

One of the promising approaches for the treatment of cardiac disease is stem cell therapy. In this study, we compared the cardiomyogenic differentiation rate, from human adipose-derived stem cells (hADSCs) in a three-dimensional (3D) hanging drop (HD) spheroid culture system, versus a two-dimensional (2D) culture condition at different concentrations of 5-azacytidine (5-Aza). 5-Azaytidine (5-Aza) is a pyrimidine nucleoside analogue of cytidine that initiates cell differentiation programs through DNA demethylation. The hADSCs were isolated and cultured both in 2D and 3D HD conditions, with either 10 or 50 µM concentrations of 5-Aza. Then DNA content, gene expression, and protein content were analyzed. 3D HD culture resulted in a higher percentage of cells in G0/G1 and S phase in the cell division cycle, whereas 2D culture led to a greater percentage of cells in the G2/M phase. A significantly higher gene expression rate of HAND1, HAND2, cTnI, Cx43, ßMHC, GATA4, NKX2.5, and MLC2V was observed in HD treated with 50 µM 5-Aza. This was confirmed by immunocytochemistry. These findings suggest that 50 µM concentration of 5-Aza can induce hADSCs to differentiate into cardiomyocytes. The differentiation rate was significantly higher when accompanied by the 3D HD culture system. This work provides a new culture system for cell differentiation for cardiovascular tissue engineering.

Actin polymerization state regulates osteogenic differentiation in human adipose-derived stem cells.

BACKGROUND: Actin is an essential cellular protein that assembles into microfilaments and regulates numerous processes such as cell migration, maintenance of cell shape, and material transport. METHODS: In this study, we explored the effect of actin polymerization state on the osteogenic differentiation of human adipose-derived stem cells (hASCs). The hASCs were treated for 7 days with different concentrations (0, 1, 5, 10, 20, and 50 nM) of jasplakinolide (JAS), a reagent that directly polymerizes F-actin. The effects of the actin polymerization state on cell proliferation, apoptosis, migration, and the maturity of focal adhesion-related proteins were assessed. In addition, western blotting and alizarin red staining assays were performed to assess osteogenic differentiation. RESULTS: Cell proliferation and migration in the JAS (0, 1, 5, 10, and 20 nM) groups were higher than in the control group and the JAS (50 nM) group. The FAK, vinculin, paxillin, and talin protein expression levels were highest in the JAS (20 nM) group, while zyxin expression was highest in the JAS (50 nM) group. Western blotting showed that osteogenic differentiation in the JAS (0, 1, 5, 10, 20, and 50 nM) group was enhanced compared with that in the control group, and was strongest in the JAS (50 nM) group. CONCLUSIONS: In summary, our data suggest that the actin polymerization state may promote the osteogenic differentiation of hASCs by regulating the protein expression of focal adhesion-associated proteins in a concentration-dependent manner. Our findings provide valuable information for exploring the mechanism of osteogenic differentiation in hASCs.

Exosomes as Part of the Human Adipose-Derived Stem Cells Secretome- Opening New Perspectives for Cell-Free Regenerative Applications.

Human adipose-derived stem cells (hASCs) represent a great resource for regenerative medicine based on their accessibility, self-renewal potential, low immunogenicity, high proliferative rate and potential to differentiate on multiple lineages. Their secretome is rich in chemokines, cytokines and protein growth factors that are actively involved in regeneration processes. In addition, part of this secretome are also the exosomes (hASC-exos), which display high content in proteins, messenger RNAs (mRNAs) and non-coding RNAs (ncRNAs). Due to their content, exosomes promote tissue regeneration by different mechanisms, either by activating or inhibiting several signaling pathways involved in wound healing, extracellular matrix remodeling, immunomodulation, angiogenesis, anti-apoptotic activity and cell migration, proliferation and differentiation. The use of hASC-exos may provide an improved alternative to standard therapies used in regenerative medicine, as a cell-free new approach with multiple possibilities to be modulated according to the patient needs. This review offers an updated overview on the functions and applications of hASC-exos in all areas of tissue regeneration, aiming to highlight to the reader the benefits of using hASCs in modern tissue engineering and regenerative medicine applications.

Mixed oxide nanotubes in nanomedicine: A dead-end or a bridge to the future?

Nanomedicine has seen a significant rise in the development of new research tools and clinically functional devices. In this regard, significant advances and new commercial applications are expected in the pharmaceutical and orthopedic industries. For advanced orthopedic implant technologies, appropriate nanoscale surface modifications are highly effective strategies and are widely studied in the literature for improving implant performance. It is well-established that implants with nanotubular surfaces show a drastic improvement in new bone creation and gene expression compared to implants without nanotopography. Nevertheless, the scientific and clinical understanding of mixed oxide nanotubes (MONs) and their potential applications, especially in biomedical applications are still in the early stages of development. This review aims to establish a credible platform for the current and future roles of MONs in nanomedicine, particularly in advanced orthopedic implants. We first introduce the concept of MONs and then discuss the preparation strategies. This is followed by a review of the recent advancement of MONs in biomedical applications, including mineralization abilities, biocompatibility, antibacterial activity, cell culture, and animal testing, as well as clinical possibilities. To conclude, we propose that the combination of nanotubular surface modification with incorporating sensor allows clinicians to precisely record patient data as a critical contributor to evidence-based medicine.

Osteogenically-induced exosomes stimulate osteogenesis of human adipose-derived stem cells.

Exosomes exhibit great therapeutic potential in bone tissue engineering. The study aimed to investigate whether the exosomes derived from human adipose-derived stem cells (hADSCs-Exos) during different time-span of osteogenic differentiation could promote osteogenesis. The appropriate concentrations of hADSCs-Exos to enhance the proliferation, migration and osteogenesis of hADSCs-Exos were also examined. PKH67 labelled hADSCs-Exos was used to detect the internalization ability of hADSCs. The osteogenic differentiation abilities of hADSCs after treatment with hADSCs-Exos was evaluated by Alizarin red staining (ARS). The proliferation and migration of hADSCs was examined by cell counting kit-8 and wound healing assay, respectively. The expression of exosomal surface markers and osteoblast-related protein of hADSCs was assessed by Western blot. PKH67-labelled exosomes were internalized by hADSCs after 4 h incubation. ARS showed that the amount of mineralized nodules in Exo1-14d group was significantly higher than that in Exo15-28d group. hADSCs-Exos could promote the proliferation and migration capacity of hADSCs. Western blot analysis showed that after hADSCs-Exos treatment, ALP and RUNX2 were significantly enhanced. Specially, the Exo1-14d group of 15 µg/mL significantly upregulated the expression of RUNX2 than the other exosomes treated groups. Our findings suggest that exosomes secreted by hADSCs during osteogenic induction for 1-14 days could be efficiently internalized by hADSCs and could induce osteogenic differentiation of hADSCs. Moreover, administration of Exo1-14d at 15 µg/mL promoted the proliferation and migration of hADSCs. In conclusion, our research confirmed that comprised of hADSCs-Exos and hADSCs may provide a new therapeutic paradigm for bone tissue engineering.