Rat Adipose Tissue-derived Stem Cells Research Articles

A Dopamine-Modified Hyaluronic Acid-Based Mucus Carrying Phytoestrogen and Urinary Exosome for Thin Endometrium Repair.

Thin endometrium (TE) is closely associated with infertility in reproductive medicine. Estrogen therapy gains unsatisfactory outcomes. In this study, an artificial mucus based on dopamine (L-DOPA)-modified hyaluronic acid combining phytoestrogen cajaninstilbene acid and rat urinary exosomes (CUEHD) is constructed for TE treatment using a rat TE model. In the rat TE model, the dominant elastic behavior and adhesive properties of CUEHD guarantee adequate retention, rendering superior synergistic treatment efficacy and favorable biosafety characteristics. CUEHD treatment significantly increases endometrial thickness and promotes receptivity and fertility. Mechanistically, estrogen homeostasis, inflammation inhibition, and endometrial regeneration are achieved through the crosstalk between ER-NLRP3-IL1β and Wnt-β catenin-TGFβ-smad signaling pathways. Moreover, the therapeutic potential of exosomes from human urine and adipose tissue-derived stem cells (ADSCs) and rat ADSCs are also demonstrated, indicating extensive use of the artificial mucus system. Thus, this study illustrates a platform combining phytoestrogen and exosomes with promising implications for TE treatment.

The Effect of bisphenol A and Photobiomodulation Therapy on Autophagy-Related Genes Induction in Adipose Tissue-Derived Stem Cells.

Introduction: As adipose tissue-derived stem cells (ADSCs) can divide rapidly and be prepared non-invasively, they have extensively been used in regenerative medicine. On the other hand, a new method of therapy, known as photobiomodulation (PHT), has been used to treat many diseases, such as inflammatory conditions, wound healing and pain. Besides, exposure to chemical substances such as bisphenol A (BPA), at low levels, can lead to autophagy. This study investigated the effects of BPA and PHT on the expression of autophagy-related genes, including LC3, NRF2, P62, in rat ADSCs as a model. Methods: ADSCs isolation and purification were confirmed by immunocytochemistry (ICC). The cells were then treated with different concentrations of BPA and also subjected to PHT. Reverse transcription polymerase chain reaction (RT-PCR) was used for the evaluation of LC3, NRF2 and P62 gene expressions. Oil red O staining was used for adipogenic vacuole formation. Result: ICC showed that the isolated cells were CD 49-positive but CD 31 and CD 34-negative. The viability test indicated that the number of live cells after 24 hours in the BPA groups at concentrations of 0, 1, 50, 100 and 200 μM was 100%, 93%, 81%, 72%, and 43% respectively. The difference in cell viability between groups 50, 100 and 200 μM was significant as compared with the control groups (P < 0.05). Moreover, in the group with 1 μM concentration of BPA, the expressions of LC3, NRF2 and P62 genes were upregulated. However, in the treatment group at the concentration of 200 μM of BPA, the LC3 gene was expressed, but NRF2 and P62 genes were downregulated. Conclusion: BPA and PHT induce autophagy and adiposeness in ADSCs in a dose-dependent manner.

Valproic acid up-regulates the whole NO-citrulline cycle for potent iNOS-NO signaling to promote neuronal differentiation of adipose tissue-derived stem cells

Valproic acid (VPA) remarkably promotes the differentiation of adipose tissue-derived stem cells (ASCs) to mature neuronal cells through nitric oxide (NO) signaling due to up-regulated inducible NO synthase (iNOS) as early as within 3 days. Here, we investigated mechanisms of VPA-promoted neuronal differentiation of ASCs concerning the NO-citrulline cycle, the metabolic cycle producing NO. Cultured rat ASCs were differentiated to mature neuronal cells rich in dendrites and expressing a neuronal marker by treatments with VPA at 2 mM for 3 days and subsequently with the neuronal induction medium for 2 h. Inhibitor (α-methyl-d, l-aspartic acid, MDLA) of arginosuccinate synthase (ASS), a key enzyme of the NO-citrulline cycle, abolishes intracellular NO increase and VPA-promoted neuronal differentiation in ASCs. l-Arginine, the substrate of iNOS, restores the promotion effect of VPA, being against MDLA. Immunocytochemistry showed that ASS and iNOS were increased in ASCs expressing neurofilament medium polypeptide (NeFM), a neuronal marker, by VPA and NIM synergistically. Real-time RT-PCR analysis showed that mRNAs of Ass and arginosuccinate lyase (Asl) in the NO-citrulline cycle were increased by VPA. Chromatin immunoprecipitation assay indicated that Ass and Asl were up-regulated by VPA through the acetylation of their associated histone. From these results, it was considered that VPA up-regulated the whole NO-citrulline cycle, which enabled continuous NO production by iNOS in large amounts for potent iNOS-NO signaling to promote neuronal differentiation of ASCs. This may also indicate a mechanism enabling short-lived NO to function conveniently as a potent signaling molecule that can disappear quickly after its role.

The Effect of Various Surface Treatments of Ti6Al4V on the Growth and Osteogenic Differentiation of Adipose Tissue-Derived Stem Cells

The physical and chemical properties of the material surface, especially its roughness and wettability, have a crucial effect on the adhesion, proliferation, and differentiation of cells. The aim of this study is to select the most appropriate surface modifications of Ti6Al4V implants for pre-colonization of the implants with adipose tissue-derived stem cells (ASCs) in order to improve their osseointegration. We compared the adhesion, growth, and osteogenic differentiation of rat ASCs on Ti6Al4V samples modified by methods commonly used for preparing clinically used titanium-based implants, namely polishing (PL), coating with diamond-like carbon (DLC), brushing (BR), anodizing (AND), and blasting (BL). The material surface roughness, measured by the Ra and Rq parameters, increased in the following order: PL &lt; DLC ˂ BR ˂ AND ˂ BL. The water drop contact angle was in the range of 60–74°, with the exception of the DLC-coated samples, where it was only 38°. The cell number, morphology, mitochondrial activity, relative fluorescence intensity of osteogenic markers RUNX2, type 1 collagen, and osteopontin, the calcium consumption by the cells and the alkaline phosphatase activity depended on the surface roughness rather than on the surface wettability of the materials. Materials with a surface roughness of several tens of nanometers (Ra 60–70 nm), i.e., the BR and AND samples, supported a satisfactory level of cell proliferation. At the same time, they achieved the highest level of osteogenic cell differentiation. These surface modifications therefore seem to be most suitable for pre-colonization of Ti6Al4V implants with stem cells pre-differentiated toward osteoblasts, and then for implanting them into the bone tissue.

Cardioprotective effects of rat adipose‑derived stem cells differ under normoxic/physioxic conditions and areassociated with paracrine factor secretion.

Adipose tissue‑derived stem cells (ASCs) are beneficial for myocardial regeneration. The physiological oxygen content of human organs is estimated to range between 1 and 11%. However, in the majority of previous invitro studies with cultured ASCs, the O2 concentration was artificially set to 21%. The present study aimed to compare the protective effects of rat ASCs on neonatal rat ventricular myocytes (NRVMs) under normoxic (21%O2) and physioxic (5%O2) conditions. Rat NRVMs cultured under normoxia or physioxia were treated with H2O2 or left untreated, and further co‑cultured with ASCs in 21% or 5%O2. The apoptosis of NRVMs was evaluated by Annexin V staining and quantitating the protein levels of Bcl‑2 and Bax by western blotting. The oxidative stress of NRVMs was determined by a glutathione/oxidized glutathione assay kit. The concentrations of secreted vascular endothelium growth factor (VEGF), insulin like growth factor‑1 (IGF‑1) and basic fibroblast growth factor (bFGF) in the culture medium were quantified by enzyme‑linked immunosorbent assay. Under both normoxia and physioxia, co‑culture with ASCs protected H2O2‑exposed NRVMs from apoptosis and significantly alleviated the oxidative stress in NRVMs. The protective effects of ASCs were associated with increased secretion of VEGF, IGF‑1 and bFGF. ASCs cultured in 5% O2 exhibited certain cardioprotective effects against H2O2 stress. The results of the present study suggested that O2 concentrations influenced the cardioprotective effects of ASCs. VEGF, IGF‑1 and bFGF may serve a role in the myocardial regeneration mediated by transplanted ASCs.

Valproic acid promotes mature neuronal differentiation of adipose tissue-derived stem cells through iNOS–NO–sGC signaling pathway

Valproic acid (VPA) remarkably promotes the differentiation of adipose tissue-derived stem cells (ASCs) to mature neuronal cells, enabling neuronal induction within only three days. Here, we investigated the involvement of NO-signaling in the VPA-promoted neuronal differentiation of ASCs as a possible mechanism. Cultured rat ASCs were differentiated to matured neuronal cells rich in dendrites and expressing βIII-tubulin protein, a neuronal marker, by treatments with VPA at 2 mM for 3 days and subsequently with the neuronal induction medium (NIM) containing cAMP-elevating agents for 2 h. Increased intracellular NO was detected in neuronal cells differentiated from ASCs treated with VPA by a fluorescence NO-specific probe, diaminofluorescein-FM diacetate. However, a NO donor (NOC18) increased the incidence of neuronal cells only to a lesser extent than VPA, indicating the insufficiency of exogenous NO. RT-PCR analysis of ASCs treated with VPA showed increased mRNA expression of inducible nitric oxide synthase (iNOS) with the acetylation of its associated histone H3K9. iNOS inhibitors (1400 W and dexamethasone) or a soluble guanylate cyclase (sGC) inhibitor (ODQ) decreased the incidence of neuronal cells differentiated from ASCs treated with VPA. These inhibitors also decreased the mRNA expression of mature neuronal markers, neurofilament medium polypeptide (NeFM) and microtubule-associated protein 2 (MAP2), as well as βIII-tubulin (TUBB3), to various extents. It was considered from these results that VPA promoted mature neuronal differentiation of ASCs through the iNOS–NO–sGC signaling pathway. This provided insights into the regulated neuronal differentiation of ASCs in clinical applications.

Cell-free therapy with the secretome of adipose tissue-derived stem cells in rats\u2019 frozen-thawed ovarian grafts

The use of secretome may be a new strand of cell therapy, which is equal to or even superior to the injection of live cells, called cell-free therapy. In ovarian transplantation, this approach may be a therapeutic possibility for the ovarian graft in hypoxia. We designed the present study to evaluate whether the cell-free therapy with the secretome of adipose tissue-derived stem cells (ASCs) in rat frozen-thawed ovarian grafts could protect a graft against ischemic injury. A single dose of rat ASCs secretome or vehicle was injected into the bilateral frozen-thawed ovaries of 18 adult female rats immediately after an autologous transplant. Nine animals were used to control the cryopreservation protocol and were evaluated before and after the cryopreservation process. Daily vaginal smears were performed for estrous cycle evaluation until euthanasia on postoperative day 30. Follicle viability by trypan blue, graft morphology by HE, and apoptosis by TUNEL and cleaved-caspase-3 were assessed. No differences were found with respect to estrous cycle resumption and follicle viability (p > 0.05). However, compared with the vehicle-treated grafts, the morphology of the secretome-treated grafts was impaired, showing reduced follicular population and increased apoptosis (p < 0.05). ASC secretome impaired the rat frozen-thawed ovarian graft from ischemic injury. However, more studies are needed to evaluate the factors involved and the possibility of applying the secretome in scaffolds to optimize its use.

Curcumin Affects Adipose Tissue-Derived Mesenchymal Stem Cell Aging Through TERT Gene Expression

Aging and losing cell survival is one of the main problems in cell therapy. Aging of Mesenchymal Stem Cells (MSCs) is associated with a rise in intracellular reactive oxygen species, decrease in telomerase reverse transcriptase (TERT) expression and finally eroded telomere ends. Given that the production of free radicals in the aging process is effective, the use of antioxidants can help in scavenging free radicals and prevent the aging of cells. The aim of this study is to evaluate the effects of curcumin on proliferation, aging and TERT expression of rat adipose tissue-derived stem cells (rADSC). rADSCs were isolated from inguinal rat adipose tissue and their viabilities were assessed by MTT after exposure to different concentrations of curcumin. Flow-cytometry was performed for investigating the cell surface markers. Adipogenic and osteogenic differentiation were carried out to evaluate the pluripotency of rADSCs. Telomerase expression and percentage of senescent cells were evaluated using real-time PCR and senescence-associated β-galactosidase activity, respectively. The results demonstrated significant proliferation of rADSCs after 48 h treatment with 1 and 5 µM curcumin. Additionally, these concentrations could significantly reduce the population doubling time and aging of rADSCs at different passages. The findings of SA-ß-gal staining showed that curcumin significantly decreased the number of senescent cells in the 5 and 7 cell passages. Moreover, expression levels of TERT increased in the presence of 1 and 5 µM curcumin than control group (P<0.001). As a conclusion, curcumin may be a good candidate to improve lifespan of rADSCs through promoting TERT gene expression.

Effect of berberine on the viability of adipose tissue-derived mesenchymal stem cells in nutrients deficient condition

This study was designed to examine whether berberine protects rat adipose tissue-derived stem cells (ASCs) against glucose and serum deprivation (GSD)-induced cell death. ASCs were cultured for 24 h in GSD condition in the presence of berberine and then cell viability, apoptosis and generation of reactive oxygen species (ROS) were evaluated. The GSD condition significantly decreased ASCs viability and increased ROS generation and apoptosis. Incubation with 0.75–3 μM berberine partially increased cell viability and decreased ROS generation and apoptosis in GSD condition. In conclusion, berberine partially protects ASCs in nutrients deficient condition and may help ASCs to preserve their survival during cell therapy of ischemia.

137 Superparamagnetic Iron Oxide Nanoparticle Targeting of Adipose Tissue-Derived Stem Cells in Diabetes-Associated Erectile Dysfunction

To explore the effect of Adipose tissue-derived stem cells (ADSCs) labelled with superparamagnetic iron oxide nanoparticles (SPIONs) on improving the erectile function of streptozocin (STZ)-induced diabetic rats with an external magnetic field. Ten-week-old male Sprague Dawley rats were injected either with STZ to induce diabetes or citrate buffer as controls. Rat ADSCs were harvested and labelled with SPIONs, and then transplanted into corpus cavernosum of STZ-induced diabetic rats with or without an external magnetic field. At 28 days after transplantation, all rats were sacrificed for tracking of transplanted cells. Erectile response was assessed by cavernous nerve stimulation. Smooth muscle, endothelium and vascular endothelial growth factor (VEGF) expression in corpora cavernosum were assessed using immunohistochemistry. In vitro studies revealed that stem cell properties were minimally affected by SPION labeling. With magnetic targeting, SPION-labelled ADSCs were visibly attracted toward the magnet field. In vivo experiments, magnetic targeting of ADSCs contributed to long-term cell retention in corpus cavernosum and improved the erectile function of diabetic rats compared with ADSC injection alone. ADSCs were identified in the corpus cavernosums of diabetic rats at 28 days after transplantation, and they were negative for vWF and a-SMA, indicating that ADSCs did not differentiate into endothelial or smooth muscle cells. VEGF expression in the corpus cavernosum was significantly increased in both ADSC-treated groups compared with the PBS-treated group. The underlying mechanism of recovery appears to the paracrine mechanism of transplanted ADSCs.

Defocused low-energy shock wave activates adipose tissue-derived stem cells in vitro via multiple signaling pathways

Background aimsWe found defocused low-energy shock wave (DLSW) could be applied in regenerative medicine by activating mesenchymal stromal cells. However, the possible signaling pathways that participated in this process remain unknown. In the present study, DLSW was applied in cultured rat adipose tissue-derived stem cells (ADSCs) to explore its effect on ADSCs and the activated signaling pathways. MethodsAfter treating with DLSW, the cellular morphology and cytoskeleton of ADSCs were observed. The secretions of ADSCs were detected. The expressions of ADSC surface antigens were analyzed using flow cytometry. The expressions of proliferating cell nuclear antigen and Ki67 were analyzed using western blot. The expression of CXCR2 and the migrations of ADSCs in vitro and in vivo were detected. The phosphorylation of selected signaling pathways with or without inhibitors was also detected. ResultsDLSW did not change the morphology and phenotype of ADSCs, and could promote the secretion, proliferation and migration of ADSCs. The phosphorylation levels were significantly higher in mitogen-activated protein kinases (MAPK) pathway, phosphoinositide 3-kinase (PI-3K)/AKT pathway and nuclear factor-kappa B (NF-κB) signaling pathway but not in Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway. Furthermore, ADSCs were not activated by DLSW after adding the inhibitors of these pathways simultaneously. ConclusionsOur results demonstrated for the first time that DLSW could activate ADSCs through MAPK, PI-3K/AKT and NF-κB signaling pathways. Combination of DLSW and agonists targeting these pathways might improve the efficacy of ADSCs in regenerative medicine in the future.

Effect of adipose tissue-derived stem cell injection in a rat model of urethral fibrosis.

We sought to evaluate the therapeutic effect of adi-pose tissue-derived stem cells (ADSCs) in a rat model of urethral fibrosis. Eighteen (18) male Sprague-Dawley rats (300‒350 g) were divided into three groups: (1) sham (saline injection); (2) urethral fibrosis group (10 μg transforming growth factor beta 1 (TGF-β1) injection); and (3) ADSCs group (10 μg TGF-β1 injection plus 2 × 105 ADSCs). Rat ADSCs were harvested from rat inguinal fat pads. All study animals were euthanized at two weeks after urethral injection. Following euthanasia, rat urethral tissue was harvested for histologic evaluation. Type I and III collagen levels were quantitated by Western blot analysis. TGF-β1 injection induced significant urethral fibrosis and increased collagen type I and III expression (p<0.05). Significant decrease in submucosal fibrosis and collagen type I and III expression were noted in the ADSCs group compared with the urethral fibrosis group (p<0.05). TGF-β1 induced fibrotic changes were ameliorated by injection of ADSCs. Local injection of ADSCs in a rat model of urethral fibrosis significantly decreased collagen type I and III. These findings suggest that ADSC injection may prevent scar formation and potentially serve as an adjunct treatment to increase the success rate of primary treatment for urethral stricture disease. Further animal and clinical studies are needed to confirm these results.

Cytokines inflammatory genes profile in experimental frozen-thawed ovarian grafts treated with scaffold-base delivery of adipose tissue-derived stem cells

Event Abstract Back to Event Cytokines inflammatory genes profile in experimental frozen-thawed ovarian grafts treated with scaffold-base delivery of adipose tissue-derived stem cells Luciana Damous1, Juliana S. Nakamuta2, Ana E. Saturi De Carvalho2, Katia C. Carvalho1, Manuel J. Simões3, José M. Soares-Jr1, José E. Krieger2 and Edmund C. Baracat1 1 Faculdade de Medicina da Universidade de São Paulo, Obstetrics and Gynecology, Brazil 2 Instituto do coração, Faculdade de Medicina da Universidade de São Paulo, Laboratório de Cardiologia Genética e Molecular, Brazil 3 Universidade Federal de São Paulo, Morphology, Brazil Introduction: Previous works of our group demonstrated that cellular therapy with adipose tissue-derived stem cells (ASC) of frozen-thawed ovarian autografts preserves the graft morphology and didn’t enhance fibrosis[1]. However, new researchers are necessary to better evaluate the inflammatory response in this graft. Materials and Methods: Twelve 12-week-old adult female Wistar rats were use. Frozen-thawed ovarian grafts of adult female rats were treated with rat ASC delivery in an acellular matrix (Gelfoam) immediately after an autologous retroperitoneal transplant (n=6). Controls received Gelfoam with vehicle (DMEM low glucose, n=6). The ovarian grafts were retrieved 30 days after transplantation. Quantitative gene expression (qPCR) for inflammatory cytokines (84 genes) was evaluated by Real Time-Polymerase Chain Reaction (RT-PCR). All reactions were performed in triplicate and controlled by negative RT (no enzyme) and no-template controls and all gene expression levels were normalized to the mean of the internal genes. A Fold Regulation reference value of -2 and +2 was considered for expression analyses. Further analyses were done by Ingenuity Pathway Analysis (IPAÒ) software in order to determine the main canoninal pathways and the expression values of the top molecules. Results and Discussion: There were 36 genes up-regulated and three down-regulated. The ten top molecules up-regulated, in descending order, were CXCL6 (+13.1), IL21 (+10), LTA (+7.6), IL1A (+7.1), CXCL11 (+6.7), CCL19 (+6.4), CCR3 (+6.2), CCR6 (+5.4), LTB (+5.1) and CXCR2 (+5). The top canonical pathways involved were Differential Regulation of Cytokine Production in Macrophages and T Helper Cells (44%), Differential Regulation of Cytokine Production in Intestinal Epithelial Cells (43%), Role of Cytokines in Mediating Communication between Immune Cells (18%), Granulocyte Adhesion and Diapedesis (9%) and Agranulocyte Adhesion and Diapedesis (7%). The top diseases and biofunctions envolved were Inflammatory Response (38 molecules), Inflammatory Disease (37), Immunological Disease (37), Connective Tissue Disorders (31) and Skeletal and Muscular Disorders (31). Tox functions were little involved and represented mainly by hepatic mechanisms such as Increases Liver Damage (11 molecules), Hepatic Cholestasis (10), Hepatic Fibrosis (9), Liver Proliferation (9) and Increases Liver Hepatitis (6). Conclusion: ASC therapy based on scaffold base-delivery strategy in rat ovarian grafts induces upregulation in 36 genes involved in inflammatory cytokines pathway. New researchers are necessary to evaluate if these findings could compromise the graft quality. São Paulo Research Foundation (Process numbers 2010/17897-5 and 2012/09469-9); Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for post-doctoral scholarship

Gene Expression Analysis of Rat Adipose Tissue-Derived Stem Cells

Gene Expression Analysis of Rat Adipose Tissue-Derived Stem Cells

Differentiation of rat adipose tissue-derived stem cells into neuron-like cells by valproic acid, a histone deacetylase inhibitor.

Valproic acid (VPA) is a widely used antiepileptic drug, which has recently been reported to modulate the neuronal differentiation of adipose tissue-derived stem cells (ASCs) in humans and dogs. However, controversy exists as to whether VPA really acts as an inducer of neuronal differentiation of ASCs. The present study aimed to elucidate the effect of VPA in neuronal differentiation of rat ASCs. One or three days of pretreatment with VPA (2 mM) followed by neuronal induction enhanced the ratio of immature neuron marker βIII-tubulin-positive cells in a time-dependent manner, where the majority of cells also had a positive signal for neurofilament medium polypeptide (NEFM), a mature neuron marker. RT-PCR analysis revealed increases in the mRNA expression of microtubule-associated protein 2 (MAP2) and NEFM mature neuron markers, even without neuronal induction. Three-days pretreatment of VPA increased acetylation of histone H3 of ASCs as revealed by immunofluorescence staining. Chromatin immunoprecipitation assay also showed that the status of histone acetylation at H3K9 correlated with the gene expression of TUBB3 in ASCs by VPA. These results indicate that VPA significantly promotes the differentiation of rat ASCs into neuron-like cells through acetylation of histone H3, which suggests that VPA may serve as a useful tool for producing transplantable cells for future applications in clinical treatments.

Intratunical Injection of Genetically Modified Adipose Tissue-Derived Stem Cells with Human Interferon α-2b for Treatment of Erectile Dysfunction in a Rat Model of Tunica Albugineal Fibrosis.

Peyronie's disease (PD) has frequently been associated with erectile dysfunction (ED) and may further compromise coitus. To investigate the efficacy of intratunical injection of genetically modified rat adipose tissue-derived stem cells (ADSCs) expressing human interferon α-2b (ADSCs-IFN) in decreasing fibrosis and restoring erectile function in a rat model of tunica albugineal fibrosis (TAF). A total of 36 Sprague-Dawley rats (12 weeks old; 300-350 g) were randomly divided in six equal groups: (i) sham group (50 μL saline-injected into the tunica albuginea [TA]); (ii) TAF group (transforming growth factor [TGF]-β1 [0.5 μg/50 μL] injected into the TA); (iii) TGF-β1 plus 5 × 10(5) control ADSCs injected same day; (iv) TGF-β1 plus 5 × 10(5) ADSCs-IFN injected same day; (v) TGF-β1 plus 5 × 10(5) control ADSCs injected after 30 days; and (vi) TGF-β1 plus 5 × 10(5) ADSCs-IFN injected after 30 days. Rat allogeneic ADSCs were harvested from inguinal fat tissue. Forty-five days following the TGF-β1 injection, erectile function was assessed, and penile tissues were harvested for further evaluations. In the same-day injection groups, intratunical injection of ADSCs and ADSC-IFN improved erectile response observed upon stimulation of cavernous nerve compared with TAF group. Intratunical ADSC-IFN injection at day 30 improved erectile responses 3.1, 1.8, and 1.3 fold at voltages of 2.5, 5.0, and 7.0, respectively, when compared with TAF group. Furthermore, at voltages of 2.5 and 5.0, treatment on day 30 with ADSCs-IFN improved erectile responses 1.6- and 1.3-fold over treatment with ADSCs alone. Local injection of ADSCs or ADSCs-IFN reduced Peyronie's-like manifestations, and these effects might be associated with a decrease in the expression of tissue inhibitors of metalloproteinases. This study documents that transplantation of genetically modified ADSCs, with or without human IFN α-2b, attenuated Peyronie's-like changes and enhanced erectile function in a rat model of TAF.

Adipose tissue-derived stem cell therapy in rat cryopreserved ovarian grafts.

The preliminary results of ovarian transplantation in clinical practice are encouraging. However, the follicular depletion caused by ischemic injury is a main concern and is directly related to short-term graft survival. Cell therapy with adipose tissue-derived stem cells (ASCs) could be an alternative to induce early angiogenesis in the graft. This study aimed to evaluate ASCs therapy in rat cryopreserved ovarian grafts. A single dose of rat ASC (rASCs) or vehicle was injected into the bilateral cryopreserved ovaries of twelve adult female rats immediately after an autologous transplant. Daily vaginal smears were performed for estrous cycle evaluation until euthanasia on postoperative day 30. Follicle viability, graft morphology and apoptosis were assessed. No differences were found with respect to estrous cycle resumption and follicle viability (P > 0.05). However, compared with the vehicle-treated grafts, the morphology of the ASCs-treated grafts was impaired, with diffuse atrophy and increased apoptosis (P < 0.05). ASCs direct injected in the stroma of rat cryopreserved ovarian grafts impaired its morphology although may not interfere with the functional resumption on short-term. Further investigations are necessary to evaluated whether it could compromise their viability in the long-term.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-015-0068-3) contains supplementary material, which is available to authorized users.

Efficient and sustained IGF-1 expression in the adipose tissue-derived stem cells mediated via a lentiviral vector.

The adipose tissue-derived stem cells (ADSCs) represent a significant area of the cell therapy. Genetic modification of ADSCs may further improve their therapeutic potential. Here, we aimed to generate a lentiviral vector expressing insulin-like growth factor-I (IGF-1) and investigate the impact of IGF-1 transduction on the properties of cultured ADSCs. Isolated rat ADSCs were assessed by flow cytometric analysis. IGF-1 was cloned and inserted into the pLenO-DCE plasmid to acquire pLenO-DCE-IGF-1 plasmid. Lentivirus was enveloped with pRsv-REV, pMDlg-pRRE and pMD2G plasmids in 293T cells. The ADSCs were transfected with the vectors. And then IGF-1-induced anti-apoptosis was evaluated by annexin V-FITC. Besides, proliferation of cells was detected by MTT assay and EdU. Moreover, Akt phosphorylation was evaluated by Western blotting analysis. Stable expression of IGF-1 in ADSCs was confirmed. ADSCs were positive for CD90 and CD29, but negative for CD31, CD34 and CD45. The transduction of IGF-1 to the ADSCs caused a dramatic increase in P-Akt expression. Over-expression of IGF-1 in ADSCs could improve the paracine of IGF-1 in a time-dependent manner, but could not promote the proliferation of ADSCs. This study indicated that lentiviral vectors offered a promising mean of delivering IGF-1 to the ADSCs. Lentiviral-mediated over-expression of therapeutic IGF-1 gene in ADSCs could prolong the anti-apoptosis effect of IGF-1, which might be induced by the activation of the PI3K/Akt pathway. And our data would improve the efficacy of ADSC-based therapies.

Spatiotemporally limited BDNF and GDNF overexpression rescues motoneurons destined to die and induces elongative axon growth

Axonal injury close to cell bodies of motoneurons induces the death of the vast majority of affected cells. Neurotrophic factors, such as brain derived neurotrophic factor (BDNF) and glial cell derived neurotrophic factor (GDNF), delivered close to the damaged motor pool in a non-regulated manner induce good survival of injured motoneurons and sprouting of their axons but fail to induce functional reinnervation.To avoid these drawbacks of high levels of neurotrophic expression, we devised an ex vivo gene therapy system to induce transient expression of BDNF/GDNF in transfected rat adipose tissue-derived stem cells (rASCs) which were grafted around the reimplanted ventral root, embedded in collagen gel.Strong BDNF/GDNF expression was induced in vitro in the first days after transfection with a significant decline in expression 10–14 days following transfection. Numerous axons of injured motoneurons were able to enter the reimplanted root following reimplantation and BDNF or GDNF treatment (192±17 SEM vs 187±12 SEM, respectively) and produce morphological and functional reinnervation. Treatment with a combined cell population (BDNF+GDNF-transfected rASCs) induced slightly improved reinnervation (247±24 SEM). In contrast, only few motoneurons regenerated their axons in control animals (63±4 SEM) which received untransfected cells. The axons of surviving motoneurons showed elongative growth typical of regenerative axons, without aberrant growth or coil formation of sprouting axons.These findings provide evidence that damaged motoneurons require limited and spatially directed amounts of BDNF and GDNF to support their survival and regeneration. Moreover, neurotrophic support appears to be needed only for a critical period of time not longer than for two weeks after injury.

Histone deacetylase inhibitor sodium butyrate promotes the osteogenic differentiation of rat adipose‐derived stem cells

Adult stem cells hold great promise for use in tissue repair and regeneration. Recently, adipose tissue-derived stem cells (ADSCs) were found to be an appealing alternative to bone marrow stem cells (BMSCs) for bone tissue engineering. The main benefit of ADSCs is that they can be easily and abundantly available from adipose tissue. However, our prior study discovered an important phenomenon that BMSCs have greater osteogenic potential than ADSCs in vitro and epigenetic regulation plays a critical role in runt-related transcription factor 2 (Runx2) expression and thus osteogenesis. In this study, we aimed to improve the osteogenic potential of ADSCs by histone deacetylase inhibitor sodium butyrate (NaBu). We found that NaBu promoted rat ADSC osteogenic differentiation by altering the epigenetic modifications on the Runx2 promoter.