Injection of freshly collected adipose tissue for the treatment complex cryptoglandular anal fistula: case report

Introduction: Perianal fistula is a common colorectal disease which is caused mainly by cryptoglandular disease. Although most cases are treated successfully by surgery, management of complex perianal fistulas (CPAF) remains a challenge with limited results in recurrence and sometimes associated with fecal incontinence. The CPAF treatment with autologous adipose-derived mesenchymal stem cells (ASCs) had become a research hotspot. The technique started to be used in the treatment of Crohn's disease (CD) fistulas, where the studies showed safe and goods result from the procedure. Cultured ASCs have been used but this approach requires the preceding collection of adipose tissue, time for isolation of ASCs and subsequent in vitro expansion, need for laboratory facilities, and expertise in cell culturing. These factors have been getting over by using the commercially available alternative, allogenic ASCs. Treatment with allogeneic ASCs has shown good results in patients with CD fistulas, however with the disadvantage of being expensive. Objective: To show that the injection with freshly collected adipose tissue is an alternative to treatment with autologous or allogenic ASCs with several advantages. Methods: In this case report, we show our first experience in the treatment of CPAF with the application of collected adipose tissue in a tertiary referral hospital from Belo Horizonte, Brazil. Results The patient had a good postoperative recuperation with a complete fistula healing after 8 months without adverse effects. Conclusion: Injection with freshly collected adipose tissue is a promising and apparently safe sphincter-sparing technique in the treatment of CPAF. (AU)

Roles of ten eleven translocation proteins family and 5-hydroxymethylcytosine in epigenetic regulation of stem cells and regenerative medicine / 北京大学学报(医学版)

The methylation of cytosine is one of the most fundamental epigenetic modifications in mammalian genomes, and is involved in multiple crucial processes including gene expression, cell differentiation, embryo development and oncogenesis. In the past, DNA methylation was thought to be an irreversible process, which could only be diluted passively through DNA replication. It is now becoming increa-singly obvious that DNA demethylation can be an active process and plays a crucial role in biological processes. Ten eleven translocation (TET) proteins are the key factors modulating DNA demethylation. This family contains three members: TET1, TET2 and TET3. Although three TET proteins have relatively conserved catalytic domains, their roles in organisms are not repeated, and their expression has significant cell/organ specificity. TET1 is mainly expressed in embryonic stem cells, TET2 is mainly expressed in hematopoietic system, and TET3 is widely expressed in cerebellum, cortex and hippocampus. This family catalyzes 5-methylcytosine to 5-hydroxymethylcytosine and other oxidative products, reactivates silenced-gene expression, in turn maintains stem cell pluripotency and regulates lineage specification. With the development of tissue engineering, organ transplantation, autologous tissue transplantation and artificial prosthesis have been widely used in clinical treatment, but these technologies have limitations. Regenerative medicine, which uses stem cells and stem cell related factors for treatment, may provide alternative therapeutic strategies for multiple diseases. Among all kinds of human stem cells, adipose-derived stem cells (ADSCs) are the most prospective stem cell lineage since they have no ethical issues and can be easily obtained with large quantities. To date, ADSCs have been shown to have strong proli-feration capacity, secrete numerous soluble factors and have multipotent differentiation ability. However, the underlying mechanism of the proliferation, secretion, acquired pluripotency, and lineage specific differentiation of ADSCs are still largely unknown. Some studies have explored the role of epigenetic regulation and TET protein in embryonic stem cells, but little is known about its role in ADSCs. By studying the roles of TET proteins and 5-hydroxymethylcytosine in ADSCs, we could provide new theoretical foundation for the clinical application of ADSCs and the stem cell-based therapy. In the future, combined with bioprinting technology, ADSCs may be used in tissue and organ regeneration, plastic surgery reconstruction and other broader fields.

Experimental study of effect and mechanism of cysteine rich protein 61 on survival of adipose tissues in rats after autologous fat grafting / 器官移植

Objective To evaluate the effect and mechanism of cysteine rich protein 61, namely CCN family member 1(CCN1) on the survival of adipose tissues in rats after autologous fat grafting. Methods At 1 week after the establishment of autologous fat grafting rat models, all animals were randomly divided into the CCN1 group (n=20) and control group (n=20). The survival of fat grafts, the morphology of fat graft tissues, the proportion of active adipocytes and the number of new blood vessels of rats were statistically compared between two groups. The levels of differential expressed messenger ribonucleic acid (mRNA) in the fat graft tissues of rats were compared between two groups by high-throughput sequencing and subsequently subject to cluster analysis. The expression levels of related proinflammatory cytokines of fat graft tissues of rats were statistically compared between two groups. Results The weight retention rate of adipose tissues in the CCN1 group was significantly higher than that in the control group (P < 0.05). In the CCN1 group, the integrity of adipocytes was considerably higher, the degree of vesiculation and vacuolation, the degree of inflammatory cell aggregation and the degree of fibrosis were significantly lower than those in the control group (all P < 0.000 1). Immunofluorescence staining demonstrated that the proportion of active adipocytes with uniform morphology was higher in the CCN1 group, whereas the proportion of active adipocytes was lower and the cells were observed in different sizes accompanied by vesiculation in the control group. Compared with the control group, the quantity of new blood vessels was significantly higher, and the expression levels of platelet derived growth factor (PDGF) and fibroblast growth factor (FGF) mRNA were remarkably higher in the CCN1 group (all P < 0.05). High-throughput sequencing analysis showed that the data at the transcriptome levels significantly differed between two groups. In the CCN1 group, the gene expression levels of cell surface markers, inflammatory cytokines and chemokines related to M1 macrophages tended to decline. Real-time fluorescent quantitative polymerase chain reaction (RT-qPCR) revealed that the mRNA expression levels of interleukin (IL)-8, IL-1 and Toll-like receptor (TLR) 2 in the CCN1 group were significantly lower than those in the control group (P < 0.01-0.05). Conclusions During autologous fat grafting, supplement of exogenous CCN1 may effectively promote the neovascularization of adipose tissues and improve the survival rate of fat graft probably by mediating the transformation of macrophages into M2 phenotype via down-regulating the TLR2 expression level.

Standardization and quality assessment for clinical grade mesenchymal stem cells from human adipose tissue

Abstract Background Mesenchymal stem cells have immense potential in stem cell-based therapies, however there is a pre-requisite to develop a curative cell dose. Adipose tissue-derived mesenchymal stem cells are promising mainly due to their potential abundance, immunomodulatory effect and remarkable differentiation potential. Nevertheless, senescence may develop during their in vitro expansion due to the incidence of genetic instability. Hence, it is important to attain an ideal balance between mesenchymal stem cell growth, quality and genetic integrity before their clinical use. Methods Stromal vascular fraction was obtained from omentum tissue of patients undergoing liposuction procedures for morbid obesity. This study standardized a closed system protocol which can be utilized for clinical grade stem cell derivation. Stages of cell growth and characterization of human adipose tissue-derived mesenchymal stem cells were also assessed along with the chromosomal stability in these in vitro cultures. Results Human adipose tissue-derived mesenchymal stem cells maintained their spindle-shaped morphology and were able to proliferate and renew, confirming their suitability for in vitro cultivation and generate clinical grade mesenchymal stem cells. Immunophenotyping indicates that the cells expressed cluster of differentiation (CD)73/CD90/CD105, mesenchymal stem-cell markers, while lacked CD34/CD45/ Human Leukocyte antigen-antigen D related (HLA-DR) expression (hematopoietic cell markers). A cell cycle study demonstrated growth kinetics under in vitro culture conditions. Human adipose tissue derived mesenchymal stem cells expressed normal cell karyotype by chromosomal G-banding indicating their genetic stability at Passage 5. Mesenchymal stem cells also demonstrated trilineage differentiation. Conclusions Availability of adipose tissue in abundance is a major advantage for clinical applications. Furthermore, detailed characterization of human adipose tissue-derived mesenchymal stem cells, their genomic stability and differentiation potential from stromal vascular fraction of human adipose tissue would help assist in tissue regeneration and repair.

Adipose-Derived Stem Cell Coculturing Stimulates Integrin-Mediated Extracellular Matrix Adhesion of Melanocytes by Upregulating Growth Factors

Coculture with adipose-derived stem cells (ADSCs) can stimulate proliferation and migration of melanocytes. To enhance outcomes of skin disorders caused by melanocyte loss or death, mixed transplantation with ADSCs has been suggested. However, role of cocultured ADSCs in proliferation and migration of melanocytes remains unclear. This study determined the effect of ADSCs on production of growth factors and expression levels of intergrins in primary culture of adult human melanocytes with or without ADSCs and in nude mice grafted with such melanocytes. Higher amounts of growth factors for melanocytes, such as bFGF and SCF were produced and released from ADSCs by coculturing with melanocytes. Relative levels of integrins β1, α5, and α6 as well as adhesion to fibronectin and laminin were increased in melanocytes cocultured with ADSCs. Such increases were inhibited by neutralization of bFGF or SCF. Relative levels of bFGF, SCF and integrins were increased in nude mice skin after grafting with melanocyte+ADSC cocultures. Collectively, these results indicate that ADSCs can stimulate proliferation and migration of melanocytes by increasing expression of integrins in melanocytes through upregulation of production/release of melanocyte growth factors such as bFGF and SCF.

Construction of adipose-derived stem cells line that can stably express brain-derived neurotrophic factor and neurotrophin-3 genes and its significance / 西安交通大学学报(医学版)

Objective: To construct adipose-derived stem cells (ADSCs) line that can stably express brain-derived neurotrophic factor (BDNF) and neurotrophin-3(NT-3) genes and elucidate its significance. Methods: ADSCs were obtained by collagenase digestion along with differential adhesion method. After 2 and 4 weeks of osteogenic induction, alkaline phosphatase staining and alizarin red staining were performed. The third generation of ADSCs were transfected with Lenti-BDNF-GFP and Lenti-NT-3-RFP recombinant lentivirus solution. The ADSCs line that stably expressed BDNF and NT-3 genes were obtained by the optimal infection value determined before. Results: The ADSCs isolated and cultured successfully had the potential to differentiate in varous directions. After being induced to osteogenesis, alkaline phosphatase and alizarin red staining both showed positive. The best infection value for Lenti-BDNF-GFP and Lenti-NT-3-RFP recombinant lentivirus transfection was 100 while the infection duration was 72 hours. Expressions of BDNF and NT-3 in co-transfection group remained stable and high at both gene and protein levels. Conclusion: Establishment of ADSCs with stable and over-expressed BDNF and NT-3 genes is of great significance for treatment of spinal cord injury (SCI). It can solve the problem of low amount of neurotrophin secreted and short half-life during the treatment of SCI by ADSCs transplantation, which has great significance for further studies on the repair mechanism of SCI.

Preparation and Characterization of Human Adipose Tissue-Derived Extracellular Matrix, Growth Factors, and Stem Cells: A Concise Review

BACKGROUND: Human adipose tissue is routinely discarded as medical waste. However, this tissue may have valuable clinical applications since methods have been devised to effectively isolate adipose-derived extracellular matrix (ECM), growth factors (GFs), and stem cells. In this review, we analyze the literature that devised these methods and then suggest an optimal method based on their characterization results. METHODS: Methods that we analyze in this article include: extraction of adipose tissue, decellularization, confirmation of decellularization, identification of residual active ingredients (ECM, GFs, and cells), removal of immunogens, and comparing structural/physiological/biochemical characteristics of active ingredients. RESULTS: Human adipose ECMs are composed of collagen type I–VII, laminin, fibronectin, elastin, and glycosaminoglycan (GAG). GFs immobilized in GAG include basic fibroblast growth factor (bFGF), transforming growth factor beta 1(TGF-b1), insulin like growth factor 1 (IGF-1), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), BMP4 (bone morphogenetic protein 4), nerve growth factor (NGF), hepatocyte growth factor (HGF), and epithermal growth factor (EGF). Stem cells in the stromal-vascular fraction display mesenchymal markers, self-renewal gene expression, and multi-differentiation potential. CONCLUSION: Depending on the preparation method, the volume, biological activity, and physical properties of ECM, GFs, and adipose tissue-derived cells can vary. Thus, the optimal preparation method is dependent on the intended application of the adipose tissue-derived products.

Anti-aging effect of adipose-derived stem cell conditioned medium on senescent human dermal fibroblasts / 中华整形外科杂志

Objective@#To study the protective effect of adipose-derived stem cell conditioned medium against ultraviolet radiation B (UVB)-induced photoaging and replicative senescence in human dermal fibroblasts (HDFs).@*Methods@#HDFs were cultured and passaged in vitro, and treated with ADSC-CM after being irradiated once with UVB. The mRNA expression of CollagenⅠ(Col1), CollagenⅢ(Col3) and Elastin were detected by real-time PCR to define the anti-aging effects of ADSC-CM. The protein expressions of phosphorylated c-Jun N-terminal kinase (p-JNK) and p-p38 were detected by Western blot.@*Results@#①Both successive passage and UVB irradiation reduced the expression of Col1, Col3 and Elastin in HDFs.②ADSC-CM inhibited the reduction of Col1, Col3 and Elastin protein expressions induced by successive passage and UVB. ③ADSC-CM activated p38 and JNK pathway. Downregulation of p38 MAPK by SB203580 decreased the mRNA expression of Col1, Col3, and Elastin. Inhibiting JNK by SP600125 did not induce significant ECM changes.@*Conclusions@#Both intrinsic and extrinsic stimuli can decrease the expression of collagen and elastin, common markers of skin aging in HDFs, and ADSC-CM can attenuate the above decreasing and promoting the expression of ECM by p38 signaling pathway.

The effect of adipose-derived stem cells on the lung colonization, TNF-α and IL-4 in rats with LPS-induced acute lung injury / 中华急诊医学杂志

Objective To explore the effect of 5-ethynyl-2'-deoxyuridine (EdU) -labeled adipose-derived stem cells (ADSCs) on lung colonization, TNF-α and IL-4 in rats induced by lipopolysaccharide (LPS) with acute lung injury. Methods Thirty male Sprague-Dawley (SD) rats were randomly divided into the normal control group (n=10), LPS model group (n=10), and LPS+ADSCs intervention group (n=10). The ALI model rats were intraperitoneally injected with 8 mg/kg LPS, rats in the normal control group were intraperitoneally injected with 4 mL/kg physiological saline, and rats in the LPS+ADSCs group were intravenously injected with 300 μL ADSCs by tail vein after 30 minutes for the ALI model establishment, and rats in the normal control group and LPS group were intravenously injected with 300μL physiological saline by tail vein. The time of death in rats was observed, lung tissue and blood from left ventricular were collected, and the serum tumor necrosis factor-α (TNF-α) and interleukin-10 (IL-4) were detected by Enzyme-linked immunosorbent assay (ELISA). Lung wet/dry weight (W/D) ratio was detected by thoracotomy, the pathological changes of lung tissue were observed under optical microscope, and the colonization of ADSCs in the lungs were observed under immunofluorescence microscopy. LSD-t method was used to compare between every two groups. Results There was no significant difference in mortality between the LPS group and LPS + ADSCs group (50％ vs. 70％, P> 0.05); EdU-labeled ADSCs were extensively colonized in the lungs by tail vein injection after 24 h; Compared with the normal control group, the lung injury of the LPS group was heavier, the ratio of lung W/D and TNF-α were significantly increased (all P< 0.01), and IL-4 level was significantly decreased (P< 0.01). Compared with the LPS model group, the degree of lung injury in the LPS + ADSCs group was significantly reduced, lung W/D ratio (5.57±0.27 vs. 5.98±0.28) and TNF-α level of blood [(41.51±4.14)ng/L vs. (45.52±3.74)ng/L] were significantly reduced (all P< 0.05), whereas the IL-4 levels were significantly increased [(7.01±1.11)pg/mL vs. (3.27±0.54)pg/mL, P< 0.05]. Conclusions EdU-labeled ADSCs could be colonized in the lungs of LPS-induced ALI rats, reduce the inflammatory response from TNF-α and improve the anti-inflammatory response from IL-4.

Nanotechnology-assisted adipose-derived stem cell (ADSC) therapy for erectile dysfunction of cavernous nerve injury: In vivo cell tracking, optimized injection dosage, and functional evaluation / 亚洲男科学杂志(英文版)

Stem cell therapy is a potentially promising option for erectile dysfunction; however, its risk of tumorigenicity is a clinical hurdle and the risk is positively related to the number of injected cells. Our previous study showed that nanotechnology improved adipose-derived stem cell (ADSC) therapy for erectile dysfunction of cavernous nerve injury (CNI) by attracting cells in the corpus cavernosum. These results indicated the possibility of using a reduced dosage of ADSCs for intracavernous injection. In this exploratory study, we used lower dosage (2 × 105 cells) of ADSCs for intracavernous injection (ICI) and the nanotechnology approach. Intracavernous pressure and mean arterial pressure were measured at day 28 to assess erectile function. The low-dose ADSC therapy group showed favorable treatment effects, and nanotechnology further improved these effects. In vivo imaging of ICI cells revealed that the fluorescein signals of NanoShuttle-bound ADSCs (NanoADSCs) were much stronger than those of ADSCs at days 0, 1, and 3. Both immunofluorescence and Western blot analysis showed a significant increase in smooth muscle, endothelium, and nerve tissue in the ADSC group compared to that in the CNI group; further improvement was achieved with assisted nanotechnology. These findings demonstrate that nanotechnology can be used to further improve the effect of small dosage of ADSCs to improve erectile function. Abundant NanoADSCs remain in the corpus cavernosum in vivo for at least 3 days. The mechanism of erectile function improvement may be related to the regeneration of the smooth muscle, endothelium, and nerve tissues.

The role of Wnt/β-catenin signaling pathway in the process of adipose-derived stem cells differentiating into neurons / 西安交通大学学报(医学版)

Objective To investigate the role of Wnt/β-catenin signaling pathway in the process of adipose-derived stem cells (ADSCs)differentiating into neurons.Methods The third generation of ADSCs were divided into three groups.Neural induction medium was used in induction group and DDK-1 was added into neural induction medium in inhibition group.Normal culture medium was used in control group.Ten days after culture,Real-time PCR and Western blot were used to detect the expressions of NSE,β-catenin and GSK-3βin each group.Results The expressions of NSE and β-catenin were high but the expression of GSK-3 was low in induction group.The expression ofβ-catenin was lower but GSK-3 was higher in inhibition group;the expression of NSE was much lower than that in induction group,but higher than control group.Conclusion The differentiation of ADSCs into neurons is related to activation of Wnt signaling pathway,but Wnt signal pathway is not the only pathway to regulate ADSCs differentiation which may be controlled by different signaling pathways.

Identification of biological characteristics of platelet-rich fibrin and its effects on proliferation and differentiation of adipose derived stem cells / 上海交通大学学报（医学版）

Objective · To explore the biological characteristics of platelet-rich fibrin (PRF) and its effects on proliferation and differentiation of adipose derived stem cells (ADSCs). Methods · The whole blood was collected from the forelimb vein of healthy beagles to prepare the PRF membrane, which were observed with optical microscope and scanning electron microscope. ADSCs were collected from the inguinaladipose tissue and were isolated and cultured. Identification of multi-directionaldifferentiation potential was performed. ADSCs were assigned to the PRF group and the control group, the former was treated with PRF in vitro. Cell proliferation wasmeasured with CCK-8. Osteogenesis induction was performed for two groups and the expression of genes associated with osteogenesis, including osteocalcin (OCN), osteopontin (OPN) and collagen I (Col- Ⅰ ), was measured with RT-PCR before induction and 4 and 7 days after induction. The alkaline phosphatase (ALP) activitywas measured 7 days after induction. Results · PRF is a milk white fibrin glue with elasticity and toughness. PRF can form loose and porous three dimensional network structure, which harbors lots of platelets and leucocytes. The cell proliferation activity was significantly higher in the PRF group than in the control group. After osteogenesis induction, the ALP activity and the mRNA levels of OCN, OPN, and Col- Ⅰ were significantly increased. Conclusion · PRF is a fibrin glue with three dimension network structure and contains lots of platelets, which can slowly release growth factors. PRF can promote the proliferation and osteogenic differentiation of ADSCs.

The role of Wnt/β-catenin signaling pathway in the process of adipose-derived stem cells differentiating into neurons / 西安交通大学学报(医学版)

Objective To investigate the role of Wnt/β-catenin signaling pathway in the process of adipose-derived stem cells (ADSCs)differentiating into neurons.Methods The third generation of ADSCs were divided into three groups.Neural induction medium was used in induction group and DDK-1 was added into neural induction medium in inhibition group.Normal culture medium was used in control group.Ten days after culture,Real-time PCR and Western blot were used to detect the expressions of NSE,β-catenin and GSK-3βin each group.Results The expressions of NSE and β-catenin were high but the expression of GSK-3 was low in induction group.The expression ofβ-catenin was lower but GSK-3 was higher in inhibition group;the expression of NSE was much lower than that in induction group,but higher than control group.Conclusion The differentiation of ADSCs into neurons is related to activation of Wnt signaling pathway,but Wnt signal pathway is not the only pathway to regulate ADSCs differentiation which may be controlled by different signaling pathways.

Cyclic Stretch Effects on Adipose-Derived Stem Cell Stiffness, Morphology and Smooth Muscle Cell Gene Expression

Recent investigations consider adipose-derived stemcells (ASCs) as a promising source of stemcells for clinical therapies. To obtain functional cells with enhanced cytoskeleton and aligned structure, mechanical stimuli are utilized during differentiation of stem cells to the target cells. Since function of muscle cells is associated with cytoskeleton, enhanced structure is especially essential for these cells when employed in tissue engineering. In this study by utilizing a custom-made device, effects of uniaxial tension (1Hz, 10% stretch) on cytoskeleton, cell alignment, cell elastic properties, and expression of smooth muscle cell (SMC) genes in ASCs are investigated.Due to proper availability ofASCs, results can be employed in cardiovascular engineeringwhen production of functional SMCs in arterial reconstruction is required. Results demonstrated that cells were oriented after 24 hours of cyclic stretch with aligned pseudo-podia. Staining of actin filaments confirmed enhanced polymerization and alignment of stress fibers. Such phenomenon resulted in stiffening of cell body which was quantified by atomic force microscopy (AFM). Expression of SM α-actin and SM22 α-actin as SMC associated genes were increased after cyclic stretch while GAPDH was considered as internal control gene. Finally, it was concluded that application of cyclic stretch on ASCs assists differentiation to SMC and enhances functionality of cells.

Effects of Uniaxial Cyclic Stretch Loading on Morphology of Adipose Derived Stem Cells

Adipose derived stem cells (ADSC) are good candidates for the replacement of bone marrow derived mesenchymal stem cells due to their abundance, multipotency property, and easier accessibility. In order to explore the behavior of these cells in response to mechanical stimulation, in this study we have investigated the effects of uniaxial dynamic mechanical loading on ADSC's morphology. Stem cells derived from the fat tissue of human and after an overnight culture were seeded on a silicone rubber strips. Afterwards, cells were subjected to a uniaxial dynamic loading in three different groups. Cell images were evaluated considering different morphological parameters. Fractal dimension decreased significantly after loading while in control groups there were a significant increase (p<0.05), approving that cyclic strain would lead to more aligned and organized cells. Cell orientation also increased significantly (p<0.05). Moreover cells' orientation angle, 24 hour after loading does not change compared to the observations immediately after loading, which attests to the practicality of the cyclic strain in functional tissue engineering. Cell width decreased and cell length increased which led to a significant increase in cell shape index (p<0.05). Results confirmed that uniaxial dynamic loading affects cell morphological parameters comparing their values before and after loading. In addition, the number of cycles are also an important factor since different number of cycles lead to different amounts of certain morphological parameters. Conclusively, cyclic strain can be a practical method in the field of functional tissue engineering.

The effect of progesterone and 17-β estradiol on membrane-bound HLA-G in adipose derived stem cells

Membrane-bound HLA-G (mHLA-G) discovery on adipose derived stem cells (ADSCs) as a tolerogenic and immunosuppressive molecule was very important. Many documents have shown that HLA-G expression can be controlled via some hormones such as progesterone (P4) and estradiol (E2). Therefore, this study was designed to evaluate progesterone and estradiol effects on mHLA-G in ADSCs at restricted and combination concentrations. Three independent cell lines were cultured in complete free phenol red DMEM and subcultured to achieve suffi cient cells. These cells were treated with P4, E2 and P4 plus E2 at physiologic and pregnancy concentrations for 3 days in cell culture conditions. The HLA-G positive ADSCs was measured via monoclonal anti HLA-G-FITC/MEMG-09 by means of flow cytometry in nine groups. Data were analyzed by one way ANOVA and Tukey's post hoc tests. There were no signifi cant values of the mean percentage of HLA-G positive cells in E2-treated and the combination of P4 plus E2-treated ADSCs compared to control cells (p value>0.05) but P4 had a signifi cant increase on mHLA-G in ADSCs (p value<0.05). High P4 concentration increased mHLA-G but E2 and the combination of P4 plus E2 could not change mHLA-G on ADSCs.

ADSCs promotes the proliferation and vascularization of HUVECs when co-cultured in vitro / 西安交通大学学报(医学版)

ABSTRACT:Objective For preparation of vascularized islets , to isolate and culture human adipose derived stem cells , investigate the role of adipose derived stem cells (ADSCs ) in promoting the proliferation and vascularization of human umbilical vein endothelial cells (HUVECs ) co‐cultured in vitro , and explore its mechanism .Methods ADSCs and HUVECs were isolated by collagenase digestion method ,then cultured ,and identified by morphology ,immunofluorescence or multi‐directional differentiation .The co‐culture system of ADSCs and HUVECs was established , HUVECs cultured alone were set up for control group . The proliferation , vascularization and concentration of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (b‐FGF)in the supernatant were compared between the two groups .Results The third generational ADSCs had uniform long spindle fiberous morphology and multi‐directional differentiational function . Immunofluorescence test of surface antigens on ADSCs revealed CD44/CD49d (+ ) ,CD31/CD34 (-) ,on HUVECs CD31/vWF (+ ) . High vascular density was found when co‐cultured in Matrigel of ADSCs and HUVECs than alone of HUVECs .Growth curve shown at days 3 , 4 and 5 of the logarithmic phase , HUVECs count in co‐culture group of ADSCs and HUVECs was (4 .52 ± 0 .31) × 104 ,(7 .18 ± 0 .45) × 104 ,and (8 .23 ± 0 .36) × 104 under indirect co‐culture condition , while that in individual HUVECs group was (2 .71 ± 0 .25) × 104 ,(4 .87 ± 0 .26) × 104 ,and (6 .86 ± 0 .33) × 104 ( P<0 .01) .Population doubling time of HUVECs was shorter in co‐culture group than in individual group .Also ,the OD value of HUVECs was higher in co‐culture group than in individual group when cultured at days 1 ,3 ,5 and 7 ( P<0 .01) .When cultured at days 3 ,7 and 13 ,the concentration of VEGF and b‐FGF in the supernatant was higher in co‐culture group than in individual group ( P< 0 .01 ) . Conclusion ADSCs can promote the proliferation and vascularization of HUVECs in vitro co‐culture conditions by secreting or increasing the HUVECs secretion of VEGF and b‐FGF .

Effect of adipose-derived stem cells on inflammatory factors expression in rats with lipopolysaccharide-induced acute lung injury / 中华危重病急救医学

Objective To explore the effect of adipose-derived stem cells (ADSCs) on inflammatory factors in rats with lipopolysaccharide (LPS)-induced acute lung injury (ALI) and the possible mechanism of anti-inflammatory. Methods Seventy male Sprague-Dawley (SD) rats were randomly divided into normal control group (n = 10), LPS model group (n = 30), and ADSCs intervention group (n = 30) by random number table. ALI model was reproduced by intraperitoneal injection of 8 mg/kg LPS, and the rats in ADSCs intervention group received tail vein injection of 300 μL ADSCs 30 minutes after the model reproduction, the samples of normal control group were harvested immediately without any intervention, and the specimens in remained two groups were taken at 6, 24, 72 hours respectively. Arterial partial pressure of oxygen (PaO2) and lactate level in femoral artery were determined. Enzyme-linked immunosorbent assay (ELISA) was used to detect the serum myeloperoxidase (MPO) and interleukin-10 (IL-10) in the blood of left ventricle. Lung wet/dry weight (W/D) ratio was detected by thoracotomy, and the pathological changes of lung tissue were observed under an optical microscope. Western Blot was used to detect the protein expression of nuclear factor-κB (NF-κB) in lung tissue of rats. Results Compared with the normal control group, the damage degree of lung tissue of LPS model group was significantly heavier from 6 hours, and lung W/D ratio, blood lactate, MPO, IL-10 and expression level of NF-κB in lung tissue were significantly increased respectively, while PaO2 was decreased significantly. Compared with LPS model group, the damage degree of lung tissue of ADSCs intervention group was significantly reduced from 6 hours, and lung W/D ratio, blood lactate, MPO, and NF-κB expression in lung tissue were significantly decreased, while PaO2 was increased significantly, and it became normal at 72 hours [lung W/D ratio: 5.33±0.29 vs. 5.77±0.42 at 6 hours, 5.14±0.46 vs. 5.43±0.38 at 72 hours; blood lactate (mmol/L): 3.6±1.0 vs. 5.7±1.1 at 6 hours, 3.1±1.0 vs. 3.8±1.2 at 72 hours; blood MPO (μg/L): 1.50±0.90 vs. 2.70±1.85 at 6 hours, 0.46±0.30 vs. 0.71±0.22 at 72 hours; NF-κB (gray value): 0.40±0.11 vs. 0.50±0.09 at 6 hours, 0.24±0.03 vs. 0.33±0.06; PaO2 (mmHg, 1 mmHg = 0.133 kPa): 78.0±4.1 vs. 74.5±3.2 at 6 hours, 89.3±9.4 vs. 81.9±3.4 at 72 hours; all P < 0.05]. The IL-10 level was significantly higher than that of LPS model group only at 24 hours (ng/L: 27.75±15.49 vs. 17.52±6.56, P < 0.05). Conclusion ADSCs can effectively relieve the inflammatory response of ALI induced by LPS, probably by inhibiting the expressions of NF-κB and blocking the release of inflammatory cytokines.

Impact of Cell Density on Differentiation Efficiency of Rat Adipose-derived Stem Cells into Schwann-like Cells

BACKGROUND AND OBJECTIVES: Schwann-like (SC-like) cells induced from adipose-derived stem cells (ASCs) may be one of the ideal alternative cell sources for obtaining Schwann cells (SCs). They can be used for treating peripheral nerve injuries. Co-culture with SCs or exposure to glial growth factors are commonly used for differentiation of ASCs to SC-like cells. However, the effect of initial cell density as an inductive factor on the differentiation potential of ASCs into the SC-like cells has not been yet investigated. METHODS AND RESULTS: ASCs were harvested from rat and characterized. The cells were seeded into the culture flasks at three different initial cell densities i.e. 2×10³, 4×10³ and 8×10³ cells/cm² an overnight and differentiated toward SC-like cells using glial growth factors. After two weeks, the differentiation rate of ASCs to SC-like cells at different densities was assessed by immunofluorescence, fluorescence-activated cell sorting analysis and real time RT-PCR. Expression of the typical SCs markers, S-100 proteins and glial fibrillary acidic protein (GFAP) protein, was observed in all cell densities groups although the number of S100-positive and GFAP-positive cells, and the expression of p75(NTR) mRNA, another SC marker, were significantly higher at the density of 8×10³ cells/cm² when compared with the other cell densities groups (p<0.001). CONCLUSIONS: The results suggest that the higher differentiation rate of ASCs to SC-like cells can be obtained at initial cell density of 8×10³ cells/cm², possibly via increased cell-cell interaction and cell density-dependent influence of glial growth factors.

Liver Regenerating Potential of the Secretome Obtained from Adipose-derived Stem Cells Cultured under the Hypoxic Environment / 대한이식학회지

BACKGROUND: The stem cell-derived secretome has received considerable attention as an alternative to stem cells for therapeutic applications. However, establishing optimal culture conditions is key to obtaining appropriate secretome contents. Here, the optimal culturing environment for achieving a high-efficiency secretome was determined via hypoxic preconditioning of human adipose-derived stem cells (ASC). METHODS: Normoxic conditioned media (NCM) and hypoxic conditioned media (HCM) were obtained after culturing human ASCs under normoxia (20% O2) or hypoxia (1% O2), respectively. Subsequently, both normal and thioacetamide-induced hepatotoxic hepatocytes were treated with NCM or HCM. In addition, partially hepatectomized mice were infused with control saline, NCM, and HCM. The effects on liver regeneration and serum transaminases levels were then compared. RESULTS: Hypoxic preconditioning significantly increased mRNA expression of proinflammatory cytokines (interleukin-6 and tumor necrosis factor-α) and growth factors (hepatocyte growth factor and vascular endothelial growth factor). In both normal and thioacetamide-induced hepatotoxic hepatocyte (alpha mouse liver 12 [AML12]) cell lines, HCM treatment resulted in the highest cell viability (122% and 95%, respectively), followed by NCM (111% and 78%, respectively). In addition, intravenous administration of HCM to partially hepatectomized mice resulted in substantially enhanced liver regeneration compared with the NCM group (P<0.05). CONCLUSIONS: Taken together, the secretome obtained from ASC with hypoxic preconditioning showed potential to alleviate liver damage both in vitro and in vivo. Hypoxic culture of ASC is expected to play an important role in regenerative medicine by inducing secretome production that is beneficial for improving liver regeneration.