Extracellular vesicle activities regulating macrophage- and tissue-mediated injury and repair responses

Macrophages are typically identified as classically activated (M1) macrophages and alternatively activated (M2) macrophages, which respectively exhibit pro- and anti-inflammatory phenotypes, and the balance between these two subtypes plays a critical role in the regulation of tissue inflammation, injury, and repair processes. Recent studies indicate that tissue cells and macrophages interact

IcarisideII facilitates the differentiation of ADSCs to SCs via let-7i/STAT3 axis to preserve erectile function

BACKGROUND: IcarisideII (ICAII) could promote the differentiation of adipose tissue-derived stem cells (ADSCs) to Schwann cells (SCs), leading to improvement of erectile function (EF) and providing a realistic therapeutic option for the treatment of erectile dysfunction (ED). However, the underlying molecular mechanisms of ADSCs and ICAII in this process remain largely unclear. METHODS: ADSCs were treated with different concentrations of ICAII. Cell proliferation was determined by MTT assay. qRT-PCR and western blot were performed to detect expressions of SCs markers, signal transducer and activator of transcription-3 (STAT3), and microRNA-let-7i (let-7i). Luciferase reporter assay was conducted to verify the regulatory relationship between let-7i and STAT3. The detection of intracavernosal pressure (ICP) and the ratio of ICP/mean arterial pressure (MAP) were used to evaluate the EF in bilateral cavernous nerve injury (BCNI) rat models. RESULTS: ICAII promoted cell proliferation of ADSCs in a dose-dependent manner. The mRNA and protein levels of SCs markers were increased by ICAII treatment in a dose-dependent manner in ADSCs. Moreover, let-7i was significantly decreased in ICAII-treated ADSCs and upregulation of let-7i attenuated ICAII-induced promotion of SCs markers. In addition, STAT3 was a direct target of let-7i and upregulated in ICAII-treated ADSCs. Interestingly, overexpression of STAT3 abated the let-7i-mediated inhibition effect on differentiation of ADSCs to SCs and rescued the ICAII-mediated promotion effect on it. Besides, combination treatment of ADSCs and ICAII preserved the EF of BCNI rat models, which was undermined by let-7i overexpression. CONCLUSION: ICAII was effective for preserving EF by promoting the differentiation of ADSCs to SCs via modulating let-7i/STAT3 pathway.

Feasibility study of adipose-derived mesenchymal stem cells in repairing traum-atic femoral neck fracture / 中国免疫学杂志

Objective:To explore the feasibility of ADSCs in the clinical treatment of traumatic femoral neck fracture.Methods: After the intervention by ADSCs,femoral neck fracture repair was observed by hematoxylin-eosin staining.The vascular endothelial growth factor expression levels at the fracture segment were quantitatively measured by immunohistochemical staining,RT-qPCR was utilized to detect the mRNA formation in callus tissue.Results: In the study group,we observed less fracture repair than in the sham surgery group but more than in the blank group.Conclusion: ADSCs administration can promote osseous tissue and osteoblast repair,thereby contributing to the healing of traumatic femoral neck fracture in rats.

Construction of VEGF165 lentivirus vectors and expression in adipose tissue-derived stem cells after transfection / 中国药学杂志

OBJECTIVE: To clone target gene by RT-PCR method, construct VEGF165 lentivirus vectors, transfect adipose tissue derived stem cells (ADSCs) and verify the expression of VEGF165 in vitro and in vivo. METHODS: RT-PCR technology was employed to clone VEGF165 gene, and this gene was cloned to lentivirus vector pLVX-EF1α-IRES2-AcGFP1 to construct a lentiviral vector pLVX-EF1α-VEGF165-IRES2-AcGFP1. Bacterial colonies PCR and sequencing analysis were used for identification. Then, Lenti-X 293T cells were transfected with main vector pLVX-EF1α-VEGF165-IRES2-AcGFP1, packaging plasmid gag-pro, vpr-pol, Tet-Off™, tat-IRES-rev and coating plasmid env (VSV-G). Lentiviral vectors were packaged and the titer was determined. ADSCs were isolated by collagenase digestion method, then cultured, and identificated by morphology, immunofluorescence and multi-directional differentiation. ADSCs was transfected with packaged VEGF165 lentivirus. Immunofluorescence and ELISA were used to detect the expression of VEGF165 in vitro. ADSCs transfected with VEGF165 lentivirus were injected into nude mice. ELISA was used to detect the expression of VEGF165. RESULTS: The VEGF165 gene fragment was cloned successfully, and the lentiviral vector plasmid pLVX-EF1α-HVEGF165-IRES2-AcGFP1 was confirmed to contain VEGF165 gene fragment as shown by bacterial colonies PCR. DNA sequencing analysis confirmed that VEGF165 gene sequencing was exactly the same with that reported by Genbank. After transfection, a large number of Lenti-X 293T cells with green fluorescence were observed by fluorescent microscopy. The concentration of the virus titer was 1×108·TU·mL-1. ADSCs were identified by morphology, immunofluorescence and multi-directional differentiation methods, in line with the literature reported ADSCs characteristics. There were about 90% of ADSCs which could express VEGF165 after being transfected with the viruses by immunofluorescence detection, also, VEGF165 protein was proved by ELISA to express stably and efficiently in vitro and in vivo. CONCLUSION: Lentiviral vectors expressing VEGF165 are successfully constructed by cloning target gene with RT-PCR method. After transfection, ADSCs expressing VEGF165 protein stably in vitro and in vivo can be obtained.

The effects of PRF and its three growth factors on the proliferation and adhesion of rat ADSCs / 实用口腔医学杂志

Objective:To study the effects of PRF and recombinant hPDGF-AB,TGF-β1 and VEGF on the proliferation and adhe-sion of rat adipose tissue-derived stem cells(ADSCs)in vitro.Methods:ADSCs were cultured with PRF membrane and various do-ses of PDGF-AB,TGF-β1 and VEGF,cell adhesion was examined by adhesion assay after 2 culture,cell proliferation was examined by CCK-8 kit after 1 -7 d culture.Results:Cell adhesion assay showed that the adhesive numbers of rat ADSCs in PRF group were significantly higher than those in the negative group(P 0.05).The adhesive numbers of the ADSCs treated by VEGF or TGF-β1 at different concentrations showed significant difference(P <0.05).CCK-8 kit assay showed that at different time points, the A values of ADSCs in PRF group were significantly higher than those of the negative control group(P <0.05).The A values of ADSCs in VEGF or PDGF-AB groups at different concentrations showed significant difference(P <0.05).The A values of rat AD-SCs in TGF-β1 group at different concentrations were lower than those in the negative control group(P <0.05).Conclusion:PRF as a combination of growth factors may stimulate the proliferation and adhesion of rat ADSCs in vitro.PDGF-AB and VEGF may stim-ulate the proliferation of rat ADSCs.TGF-β1 and VEGF may stimulate the adhesion of rat ADSCs in a dose-response manner to some degree.

Advanced Properties of Urine Derived Stem Cells Compared to Adipose Tissue Derived Stem Cells in Terms of Cell Proliferation, Immune Modulation and Multi Differentiation

Adipose tissue stem cells (ADSCs) would be an attractive autologous cell source. However, ADSCs require invasive procedures, and has potential complications. Recently, urine stem cells (USCs) have been proposed as an alternative stem cell source. In this study, we compared USCs and ADSCs collected from the same patients on stem cell characteristics and capacity to differentiate into various cell lineages to provide a useful guideline for selecting the appropriate type of cell source for use in clinical application. The urine samples were collected via urethral catheterization, and adipose tissue was obtained from subcutaneous fat tissue during elective laparoscopic kidney surgery from the same patient (n = 10). Both cells were plated for primary culture. Cell proliferation, colony formation, cell surface markers, immune modulation, chromosome stability and multi-lineage differentiation were analyzed for each USCs and ADSCs at cell passage 3, 5, and 7. USCs showed high cell proliferation rate, enhanced colony forming ability, strong positive for stem cell markers expression, high efficiency for inhibition of immune cell activation compared to ADSCs at cell passage 3, 5, and 7. In chromosome stability analysis, both cells showed normal karyotype through all passages. In analysis of multi-lineage capability, USCs showed higher myogenic, neurogenic, and endogenic differentiation rate, and lower osteogenic, adipogenic, and chondrogenic differentiation rate compared to ADSCs. Therefore, we expect that USC can be an alternative autologous stem cell source for muscle, neuron and endothelial tissue reconstruction instead of ADSCs.

Therapeutic Effect of Adipose Derived Stem Cells versus Atorvastatin on Amiodarone Induced Lung Injury in Male Rat

BACKGROUND AND OBJECTIVES: Amiodarone (AM), a class 3 antiarrhythmic drug, has been associated with variety of adverse effects, the most serious of which is pulmonary toxicity. Ator (A) is a statin, known for their immunomodulatory and anti-inflammatory activities. Recent studies provide evidence of potential therapeutic effect of statins on lung injury. Adipose derived stem cells (ADSCs) have shown great promise in the repair of various tissues. The present study aimed at investigating and comparing the possible therapeutic effect of A and ADSCs on AM induced lung injury in albino rats. METHODS AND RESULTS: 34 adult male albino rats were divided into 5 groups: control group (Gp I), A group (Gp II) received 10 mg/kg of A orally 6 days (d)/week (w) for 4 weeks (ws), AM group (Gp III) received 30 mg/kg of AM orally 6 d/w for 4 ws, AM&A group (Gp IV) received AM for 4ws then A for other 4 ws and AM&SCs group (Gp V) received AM for 4 ws then injected with 0.5 ml ADSCs on 2 successive days intravenously (IV). Histological, histochemical, immunohistochemical and morphometric studies were performed. Group III displayed bronchiolitis obliterans, thickened interalveolar septa (IAS) and thickened vascular wall which were proven morphometrically. Increased area% of collagen fibers and apoptotic changes were recorded. All findings regressed on A administration and ADSCs therapy. CONCLUSION: Ator proved a definite ameliorating effect on the degenerative, inflammatory, apoptotic and fibrotic changes induced by AM. ADSCs administration denoted more remarkable therapeutic effect compared to A.

Characterization of Mesenchymal Stem Cells Derived from Rat Bone Marrow and Adipose Tissue: A Comparative Study

BACKGROUND AND OBJECTIVES: Stem cell technology offers a new hope for many chronic disorders patients. The types of stem cells are different with many differences existing between each type. Mesenchymal stem cells (MSCs) represent one type of adult stem cells that can be easily isolated, then re-transplanted to the patients. This offers potential for their future application in treating many disorders without fear of rejection possibility. MSCs can be isolated from different sources e.g. bone marrow (BMSCs) and adipose tissue (ADSCs). In the present study we compared BMSCs and ADSCs isolated from Sprague-Dawley rats. METHODS AND RESULTS: For this comparison, immunophenotyping, the analysis of growth rates, proliferation by colony forming unit-fibroblast assay, population doubling time, and trilineage differentiation assays were performed for both BMSCs and ADSCs. The findings revealed that despite no difference in immunphenotypic character between BMSC and ADSC, a better proliferative capacity was observed for ADSCs which would advocate their better use in regenerative applications. On the other hand, BMSCs showed more potential for osteogenic and chondrogenic differentiation. CONCLUSIONS: Our study showed that, despite many similarities between both types of cells, there are differences existing which can offer assistance on choosing type of cell to be used in specific diseases. Although ADSCs seem more promising for regenerative application generally, BMSCs may represent a better choice for treating bone disorders.

Effect of EGF on angiogenesis induced by adipose-derived stem cells / 中华医学美学美容杂志

Objective To explore the effect of epidermal growth factor (EGF) on tube formation of HUVEC induced by the secretion of angiogenesis factors of adipose-derived stem cells (ADSCs).Methods ADSCs were primarily cultured by enzyme digestion method.The flow cytomertry was performed to detect the expression of cell surface marker.ELISA was used to detect the expression of VEGF,HGF,and SDF-1 after given different doses of EGF.Tube formation assay was used to examine the effect of EGF on the tube formation induced by ADSCs.Results ADSCs were successfully isolated and cultured from human liposuction tissue and specific markers were expressed on ADSCs.EGF promoted the secretion of angiogenesis factors VEGF,HGF,and SDF-1,which were secreted by ADSCs.EGF pretreatment increased the ability of tube formation of HUVECs induced by ADSCs.Conclusions ADSCs induce the secretion of angiogenesis factors in vitro,and thus increase the ability of tube formation of HUVECs.EGF promotes the secretion ability of ADSCs,and the best concentration is 15 mg/L.

Effect of platelet-rich fibrin on proliferation and adipogenic differentiation of adipose-derived stem cells / 中华医学美学美容杂志

Objective To study the effect of autogeneic platelet-rich fibrin (PRF) on proliferation and adipogenic differentiation of human adipose-derived stem cells (ADSCs) in vitro.Methods ADSCs were isolated from adipose tissue obtained from donors undergoing liposuction and were cultured,and underwent identification.ADSCs at passage 3 were divided into three groups:test groups were cultured with 1PRFM and 2PRFM,and control group was cultured without PRF membrane.Then the growth of the cells was observed by inverted microscope.MTT method was used to observe cell proliferation activity at days 1,2,3,4,5,6 and 7 after culture.Adipogenic differentiation of ADSCs was observed and quantified by oil red O staining at days 3,5,7,9,11 and 14.Results Cell proliferation and adipogenic differentiation would be increased with the PRFM,There were significant differences among three groups.Conclusions PRF could significantly promote proliferation and adipogenic differentiation of ADSCs.

Effect on bone healing by the application of low intensity pulsed ultrasound after injection of adipose tissue-derived stem cells at the implantation of titanium implant in the tibia of diabetes-induced rat

INTRODUCTION: This study examined the effect of the application of low intensity pulsed ultrasound on bone healing after an injection of adipose tissue-derived stem cells (ADSCs) during the implantation of a titanium implant in the tibia of diabetes-induced rats. MATERIALS AND METHODS: Twelve Sprague-Dawely rats were used. After inducing diabetes, the ADSCs were injected into the hole for the implant. Customized screw type implants, 2.0 mm in diameter and 3.5 mm in length, were implanted in both the tibia of the diabetes-induced rats. After implantation, LIPUS was applied with parameters of 3 MHz, 40 mW/cm2 , and 10 minutes for 7 days to the left tibiae (experimental group) of the diabetes-induced rats. The right tibiae in each rat were used in the control group. At 1, 2 and 4 week rats were sacrificed, and the bone tissues of both tibia were harvested. The bone tissues of the three rats in each week were used for bone-to-implant contact (BIC) and bone area (BA) analyses and the bone tissues of one rat were used to make sagittal serial sections. RESULTS: In histomorphometric analyses, the BIC in the experimental and control group were respectively, 39.00+/-18.17% and 42.87+/-9.27% at 1 week, 43.74+/-6.83% and 32.27+/-6.00% at 2 weeks, and 32.62+/-11.02% and 47.10+/-9.77% at 4 weeks. The BA in experimental and control group were respectively, 37.28+/-3.68% and 31.90+/-2.84% at 1 week, 20.62+/-2.47% and 15.64+/-2.69% at 2 weeks, and 11.37+/-4.54% and 17.69+/-8.77% at 4 weeks. In immunohistochemistry analyses, Osteoprotegerin expression was strong at 1 and 2 weeks in the experimental group than the control group. Receptor activator of nuclear factor kB ligand expression showed similar staining at each week in the experimental and control group. CONCLUSION: These results suggest that the application of low intensity pulsed ultrasound after an injection of adipose tissue-derived stem cells during the implantation of titanium implants in the tibia of diabetes-induced rats provided some positive effect on bone regeneration at the early stage after implantation. On the other hand, this method is unable to increase the level of osseointegration and bone regeneration of the implant in an uncontrolled diabetic patient.

Role of Reactive Oxygen Species in the Adipogenesis of Adipose-derived Stem Cells

PURPOSE: Stem cells continue to receive research attention in the clinical fields, and adipose-derived stem cells(ADSCs) have been shown to be a good source raw material. Many plastic surgeons are researching the ADSC adipogenesis with a view of conducting clinical trials, and many attempts have been made to identify the factors that promote the adipogenesis of ADSCs, but comparatively few correlation studies have been undertaken to explore the relation between reactive oxygen species(ROS) and the ADSC adipogenesis. We undertook this study is to investigate the effects of ROS on ADSC adipogenesis. METHODS: ADSCs were isolated and cultured from abdominal adipose tissue, and cultured in different media; 1) DMEM(control), 2) adipogenesis induction culture medium, 3) adipogenesis induction culture medium with ROS(20 microM/50 microM H2O2), 4) adipogenesis induction culture medium containing ROS(20 microM/50 microM H2O2) and antioxidant(10 microM/20 microM Deferoxamine). We compared adipogenesis in these different media by taking absorbance measurements after Oil-Red O staining every 5 days. RESULTS: After culturing for 20 days, significant differences were observed between these various culture groups. Absorbance results showed significantly more adipogenesis had occurred in media containing adipogenesis induction culture medium and H2O2(in a H2O2 dose-dependently manner) than in media containing adipogenesis induction culture medium and no H2O2(p<0.001). Furthermore, in media containing adipogenesis induction culture medium, H2O2, and antioxidant, absorbance results were significantly lower than in adipogenesis induction culture medium and H2O2(p<0.001). CONCLUSION: These findings suggest that ROS promote the adipogenesis of ADSCs. We suggests that ROS could be used in the adipose tissue engineering to improve fat cell differentiation and implantable fat tissue organization.

Construction of Tissue Engineered Nucleus Pulposus with Adipose-derived Stromal Cells and Thermo-Sensitive Scaffold / 中国康复理论与实践

@#ObjectiveTo evaluate the feasibility of using injective chitosan scaffold and induced- adipose-derived stromal cells(ADSCs) to construct tissue engineered injectable nucleus pulposus (NP).MethodsADSCs were harvested from rabbits to culture 3 passage and induce 2 weeks to NP-like cells. The injective chitosan hydrogel scaffold was made of chitosan and disodium β-glycerophosphate. Its physical properties and gross condition were observed. The tissue engineered NP was constructed by compounding the scaffold and induced-ADSCs. Then, the viability of ADSCs in the scaffold was observed 2 days after compound culture and the growth condition of ADSCs on the scaffold was observed by scanning electron microscope (SEM) 14 days after compound culture. Expression of aggrecan and Col Ⅱ mRNA in ADSCs were analyzed by RT-PCR 14 days after inductive culture and compound culture.ResultsThe injective chitosan hydrogel was liquid at room temperature and solidified into gel at 37 ℃ (10～15 minutes) due to crosslinking reaction. Acridine orange/propidiumiodide staining showed that the viability rate of induced-ADSCs in chitosan scaffoldl was above 90%. Scanning electron microscope observation demonstrated that the ADSCs were distributed in the reticulate scaffold. RT-PCR results showed that the expression of Col Ⅱ and aggrecan mRNA in induced-ADSCs demonstrated differentiation of ADSCs to a phenotype which showed similarities to NP cells, and the co-culture NP-like cells with scaffold didn`t cause dedifferentiation.ConclusionWith good cellular compatibilities, C/Gp scaffold may be a potential NP cells carrier for tissue engineered NP.

Characterization and differentiation into adipocytes of mesenchymal stem cells (MSCs) from human adipose tissue and amniotic fluid / 대한산부인과학회지

OBJECTIVE: Mesenchymal stem cells (MSCs) are potentially very useful for regenerative and reparative medicine as well as therapeutic possibilities. The aim of this study is to examine the ability of ADSCs and AFSCs to be phenotypically and functionally differentiated into adipocyte and to determine the appropriate stem cell source and conditions for efficient adipocyte regeneration. METHODS: Adipogenic differentiation was induced by culturing confluent ADSCs and AFSCs in adipogenic medium for 2~4 weeks. During the differentiation inducing period, we evaluated the successful adipogenesis by performing immunocytochemistry and RT-PCR to detect the lipid producibility and several adipogenic gene expressions including lipoprotein lipase (LPL), peroxisome proliferator-activated receptor gamma2 (PPAR gamma2) and adiponectin. RESULTS: ADSCs and AFSCs are expanded easily in vitro and exhibited a fibroblast-like morphology as previously known in MSCs from bone marrow and a commercial source. Flow cytometric analysis showed that ADSCs and AFSCs expressed several CD marker antigens similar to those observed on bone marrow-derived MSCs. Adipogenic induction of ADSCs and AFSCs resulted in the extended cell morphology, intracellular staining of an established lipid dye Oil Red O, and expression of adipocyte-specific genes. CONCLUSION: Both ADSCs and AFSCs successfully differentiate in vitro into adipogenic cells in the presence of the lineage-specific induction factors although ADSC showed the greater capability. Therefore, the results suggest that ADSCs and AFSCs may be an excellent choice for many future tissue engineering strategies and cell-based therapies.

Adipose tissue derived stem cells transferred with TGF-?_1 gene and differentiated into chondrocytes / 中国矫形外科杂志

[Objective]To induce TGF-?1 gene which can increase ECM synthesis into adipose tissue derived stem cells(ADSCs) by employing gene transfer techniques and observe whether TGF-?1 gene could expresse continuously and whether type II collagen and aggrecan are synthesized in order to provide experimental data for constructing tissue-engineering cartilage. [Method]ADSCs were transferred with TGF-?1 gene ,TGF-?1,FN,ColⅡ and aggrecan were detected with RT-PCR and TGF-?1 protein was detected with Western-blot.[Result]RT-PCR demonstrated that the expression of TGF-?1,FN,ColⅡ and aggrecan in the TGF-?1 gene transferred groups increased more evidently than non-gene groups and control groups (P

The effect of long-term culture in vitro on biological characteristics of human adipose tissue derived stem cells / 中华创伤骨科杂志

Objective To observe the effect of lnng-term in vitro culture on the biological properties of adipose-derived stem cells(ADSCs)as seeding cells of tissue engineering.Methods The surface makers and apoptosis of primary and passaged human ADSCs were identified by flow cytometric analysis.Osteogenic differentiation of ADSCs at different passages were identified by alkaline phosphatase(ALP),Von Kossa staining and RT-PCR respectively.Results The surface marker expression of mesenchymal stem cells on ADSCs was high and did not change with passages of the cells.The early apoptosis rate of the cells was 1% to 2%,and increased insignificantly from passage one to passage nine.The osteogenic potential of ADSCs confirmed by ALP,Von Kossa staining and RT-PCR was maintained to as late as passage eight.Conclusion Since the biological properties of ADSCs are stable,they can be served as optimal seeding cells for tissue engineering and regenerative research.