Co-culture Of Adipose-derived Stem Cells Research Articles

Co-culture of RhoA-overexpressed microtia chondrocytes and adipose-derived stem cells in the construction of tissue-engineered ear-shaped cartilage.

Microtia is a congenital auricle dysplasia with a high incidence and tissue engineering technology provides a promising strategy to reconstruct auricles. We previously described that the engineered cartilage constructed from microtia chondrocytes exhibited inferior levels of biochemical and biomechanical properties, which was proposed to be resulted of the decreased migration ability of microtia chondrocytes. In the current study, we found that Rho GTPase members were deficient in microtia chondrocytes. By overexpressing RhoA, Rac1, and CDC42, respectively, we further demonstrated that RhoA took great responsibility for the decreased migration ability of microtia chondrocytes. Moreover, we constructed PGA/PLA scaffold-based cartilages to verify the chondrogenic ability of RhoA overexpressed microtia chondrocytes, and the results showed that overexpressing RhoA was of limited help in improving the quality of microtia chondrocyte engineered cartilage. However, coculture of adipose-derived stem cells (ADSCs) significantly improved the biochemical and biomechanical properties of engineered cartilage. Especially, coculture of RhoA overexpressed microtia chondrocytes and ADSCs produced an excellent effect on the wet weight, cartilage-specific extracellular matrix, and biomechanical property of engineered cartilage. Furthermore, we presented that coculture of RhoA overexpressed microtia chondrocytes and ADSCs combined with human ear-shaped PGA/PLA scaffold and titanium alloy stent fabricated by CAD/CAM and 3D printing technology effectively constructed and maintained auricle structure in vivo. Collectively, our results provide evidence for the essential role of RhoA in microtia chondrocytes and a developed strategy for the construction of patient-specific tissue-engineered auricular cartilage.

O119: Investigating the Secretome of Adipose Derived Stem Cells on Extracellular Matrix Remodelling in Radiation-Induced Fibrosis

Abstract Background Radiation-induced fibrosis (RIF) is a debilitating complication following radiotherapy, characterised by fibroblast proliferation and excessive extracellular matrix (ECM) deposition, leading to functional and aesthetic impairments. Although autologous fat grafting (AFG) has shown promise in reversing RIF, the mechanisms, largely attributed to adipose-derived stem cells (ADSCs), remain elusive. This study aimed to investigate ADSCs’ and their secretome on ECM remodelling in RIF management. Methods The anti-fibrotic potential of the ADSC secretome on HDFs subjected to ionising radiation was assessed via a series of in vitro experiments. Control HDFs were compared with irradiated HDFs to assess any phenotypical changes. Irradiated HDFs were treated with ADSC pre-conditioned media (CM) or ADSC co-culture (CC). Gene expression (COL1A1, TGF-β, CCN-2, MMP-1, MMP-3, TIMP-1, ACTA2, TNC) was assessed via real-time quantitative PCR (RT-qPCR). ProCollagen1a1 and CCN2 protein concentrations were assessed by ELISA. Results Irradiated HDFs demonstrated changes in fibrotic gene expression. ADSC-CM treatment significantly reduced COL1A1 expression, although protein levels remained unchanged. TGF-β1, CCN2, MMP-3, TIMP-1 and ACTA-2 exhibited reduced expression post-ADSC-CM treatment whereas TNC expression was found to be significantly increased. Co-culture with ADSCs led to a general, but not statistically significant, decrease in pro-fibrotic gene expression. Notably, CCN-2 protein expression significantly increased. Conclusion This study demonstrates that ionising radiation results in a pathological pro-fibrotic phenotype in HDFs. The ADSC secretome potentially mitigates this, reducing pro-fibrotic gene expression. Unexpectedly, co-culture experiments highlighted a complex interaction with upregulation of fibrosis-associated genes. Further research is needed to clarify these interactions to improve therapeutic interventions for RIF.

Cartilage 3D bioprinting for rhinoplasty using adipose-derived stem cells as seed cells: Review and recent advances.

Nasal deformities due to various causes affect the aesthetics and use of the nose, in which case rhinoplasty is necessary. However, the lack of cartilage for grafting has been a major problem and tissue engineering seems to be a promising solution. 3D bioprinting has become one of the most advanced tissue engineering methods. To construct ideal cartilage, bio-ink, seed cells, growth factors and other methods to promote chondrogenesis should be considered and weighed carefully. With continuous progress in the field, bio-ink choices are becoming increasingly abundant, from a single hydrogel to a combination of hydrogels with various characteristics, and more 3D bioprinting methods are also emerging. Adipose-derived stem cells (ADSCs) have become one of the most popular seed cells in cartilage 3D bioprinting, owing to their abundance, excellent proliferative potential, minimal morbidity during harvest and lack of ethical considerations limitations. In addition, the co-culture of ADSCs and chondrocytes is commonly used to achieve better chondrogenesis. To promote chondrogenic differentiation of ADSCs and construct ideal highly bionic tissue-engineered cartilage, researchers have used a variety of methods, including adding appropriate growth factors, applying biomechanical stimuli and reducing oxygen tension. According to the process and sequence of cartilage 3D bioprinting, this review summarizes and discusses the selection of hydrogel and seed cells (centered on ADSCs), the design of printing, and methods for inducing the chondrogenesis of ADSCs.

MAPK/ERK-CBP-RFPL-3 Mediates Adipose-Derived Stem Cell-Induced Tumor Growth in Breast Cancer Cells by Activating Telomerase Reverse Transcriptase Expression.

Adipose-derived stem cells (ASCs) improve the self-renewal and survival of fat grafts in breast reconstruction after oncology surgery. However, ASCs have also been found to enhance breast cancer growth, and its role in tumor proliferation remains largely elusive. Here, we explored a novel mechanism that mediates hTERT reactivation during ASC-induced tumor growth in breast cancer cells. In this study, we found the proliferative ability of breast cancer cells markedly increased with ASC coculture. To explore the molecular mechanism, we treated cells with anibody/inhibitor and found that the activation of MEK-ERK pathway was triggered in breast cancer cells by SCF secreted from ASCs, leading to the nuclear recruitment of CBP. As a coactivator of hTERT, CBP subsequently coordinated with RFPL-3 upregulated hTERT transcription and telomerase activity. The inhibition of CBP and RFPL-3 abrogated the activation of hTERT transcription and the promotion of proliferation in breast cancer cells with cocultured ASCs in vitro and in vivo. Collectively, our study findings indicated that CBP coordination with RFPL-3 promotes ASC-induced breast cancer cell proliferation by anchoring to the hTERT promoter and upregulating telomerase activity, which is activated by the MAPK/ERK pathway.

Adipose-derived stem cells ameliorate atopic dermatitis by suppressing the IL-17 expression of Th17 cells in an ovalbumin-induced mouse model

BackgroundMesenchymal stem cells (MSCs) have therapeutic potential for atopic dermatitis (AD) owing to their immunoregulatory effects. However, the underlying mechanisms associated with the therapeutic efficacy of MSCs on AD are diverse and related to both cell type and delivery method.ObjectivesThis study investigated the therapeutic effect and mechanisms of adipose-derived stem cells (ADSCs) on AD using an ovalbumin (OVA)-induced AD mouse model.MethodsAD mice were subcutaneously injected with mouse ADSCs, cortisone, or PBS, and the therapeutic effects were determined by gross and histological examinations and serum IgE levels. Additionally, qPCR, RNA-sequencing analyses of skin samples and co-culture of ADSCs and Th17 cells were conducted to explore the underlying therapeutic mechanisms.ResultsADSCs treatment attenuated the AD pathology, decreased the serum IgE levels, and decreased mast cells infiltration in the skin of the model mice. Moreover, tissue levels of IL-4R and Th17-relevant products (IL-17A, CCL20, and MMP12) were suppressed in the ADSC- and cortisone-treated groups. Genomics and bioinformatics analyses demonstrated significant enrichment of inflammation-related pathways in the downregulated genes of the ADSC- and cortisone-treated groups, specifically the IL-17 signaling pathway. Co-culture experiments revealed that ADSCs significantly suppressed the proliferation of Th17 cells and the expression of proinflammatory cytokines (IL-17A and RORγT). Furthermore, expression levels of PD-L1, TGF-β, and PGE2 were significantly upregulated in co-cultured ADSCs relative to those in monocultured ADSCs.ConclusionADSCs ameliorate OVA-induced AD in mice mainly by downregulating IL-17 secretion of Th17 cells.

Effects of Tissue-engineered Bone by Coculture of Adipose-derived Stem Cells and Vascular Endothelial Cells on Host Immune Status.

The study aimed to explore the effects of tissue-engineered bone constructed with partially deproteinized biologic bone (PDPBB) and coculture of adipose-derived stem cells (ADSCs) and vascular endothelial cells (VECs) on host immune status, providing a very useful clue for the future development of bone engineering. Tissue-engineered bones constructed by PDPBB and ADSCs, VECs or coculture of them were implanted into the muscle bag of bilateral femurs of Sprague-Dawley rats. Partially deproteinized biologic bone alone and blank control were also implanted. After transplantation, the proliferation of implanted seed cells in tissue-engineered bones was labeled by bromodeoxyuridine staining. Moreover, the changes of T-lymphocyte subpopulations, including CD3 + CD4+ and CD3 + CD8+ in peripheral blood were then detected using flow cytometry to analyze the immune rejection of tissue-engineered bone implantation based on peripheral blood CD4/CD8 ratios. After transplantation, the proliferation of implanted seed cells was observed in tissue-engineered bones of different groups. At different time points after transplantation, the CD4+/CD8+ ratio in peripheral blood of PDPBB + ADSCs, PDPBB + coculture, and blank control groups did not exhibit significant change. Although the CD4+/CD8+ ratio in peripheral blood of PDPBB + VECs group was significantly higher than other group at 1 week after transplantation, that of PDPBB + VECs and PDPBB + coculture group was significantly decreased at 8 week after transplantation compared with that of blank control group. Our results indicated that there was no significant immune rejection after transplantation of tissue-engineered bone constructed with PDPBB and coculture of ADSCs and VECs as seed cells.

Induction of Apoptosis in the U937 Cell Line Co-cultured with Adipose-derived Stem Cells Secreting Bone Morphogenetic Protein-4.

Transforming growth factor-beta (TGF-β) plays a significant role in tumorigenesis. MiR-181b is a multifunctional miRNA involved in numerous cellular processes, such as cell fate and cell invasion. This study aimed to examine whether the co-culture of adipose-derived stem cells (ADSCs), highly expressing bone morphogenetic protein-4, with the U937 cell line, which is a human myeloid leukemia cell line, is able to induce cell death in this cancer cell line, considering the potential ability of ADSCs to migrate from tumor sites. Cell surface markers, namely CD73 and CD105, were analyzed to verify the identity of mesenchymal stem cells isolated from adipose tissue. Besides, the osteogenic and adipogenic differentiation potentials of ADSCs were evaluated. The induction of cell death and apoptosis in the U937 cell line was assessed using MTT and annexin V/ PI assays, respectively. The expression levels of miR-181 and TGF-β were determined in the co-culture system using real-time PCR. The results of MTT and annexin V/ PI assays showed that BMP4-expressing ADSCs could inhibit cell viability and induce apoptosis in U937 cells in the co-culture system. The co-culture of ADSCs, highly expressing BMP-4, with the U937 cell line led to the downregulation of miR-181 and TGF-β genes in the human cancer cell line. ADSCs may further be studied as a candidate for the treatment of hematological cancers.

The Application of Decellularized Adipose Tissue Promotes Wound Healing.

Due to adipose-derived stem cells (ASCs) being easy to obtain, their rapid proliferation rate, and their multidirectional differentiation capabilities, they have been widely used in the field of regenerative medicine. With the progress of decellularized adipose tissue (DAT) and adipose tissue engineering research, the role of DAT in promoting angiogenesis has gradually been emphasized. We examined the biological characteristics and biosafety of DAT and evaluated the stem cell maintenance ability and promotion of growth factor secretion through conducting in vitro and in vivo studies. The tested ASCs showed high rat:es of proliferation and adhered well to DAT. The expression levels of essential genes for cell stem maintenance, including OCT4, SOX2, and Nanog were low at 2-24h and much higher at 48 and 96h. The Adipogenic expression level of markers for ASCs proliferation including PPARγ, C/EPBα, and LPL increased from 2 to 96h. Co-culture of ASCs and DAT increased the secretion of local growth factors, such as VEGF, PDGF-bb, bFGF, HGF, EGF, and FDGF-bb, and secretion gradually increased from 0 to 48h. A model of full-thickness skin defects on the back of nude mice was established, and the co-culture of ASCs and DAT showed the best in vivo treatment effect. The application of DAT promotes wound healing, and DAT combined with ASCs may be a promising material in adipose tissue engineering and regenerative medicine.

Adipose-Derived Stem Cells Promote Proliferation and Invasion in Cervical Cancer by Targeting the HGF/c-MET Pathway.

BackgroundCervical cancer is a serious female malignancy affecting women’s health worldwide. The HGF/c-MET signaling pathway is activated in cervical cancer. Adipose-derived stem cells (ADSCs) with multipotential differentiation can carry out paracrine secretion of hepatocyte growth factor (HGF). Here, we investigated the effect and underlying mechanism of ADSCs on the promotion and invasion of cervical cancer in vitro and in vivo.Materials and MethodsADSCs were isolated, identified, and co-cultured with cervical cancer cells. HGF was detected using ELISA, and the HGF and c-MET signaling pathway was assessed with Western blot. The proliferation and invasion of human cervical cancer cell lines (HeLa and CaSki cells) were measured using CCK-8 and transwell assays. A HeLa xenograft mouse model was established to determine the effect of ADSCs on tumor growth in vivo.ResultsADSCs secreted a high level of HGF into the supernatant, while co-culture of ADSCs and cervical cancer cells increased the supernatant level of HGF. The HGF/c-MET pathway was activated in HeLa and CaSki cells co-cultured with ADSCs. Both co-culture with ADSCs and use of ADSC-derived conditioned medium (ADSCs-CM) significantly promoted the proliferation and invasion of cervical cancer cells in vitro, an effect that was reduced by inhibiting tumor cell c-MET expression. Furthermore, ADSCs-CM promoted HeLa cervical tumor growth in vivo, which could be suppressed by intratumoral c-MET siRNA injection.ConclusionADSCs promote cervical cancer growth and invasion through paracrine secretion of HGF and involvement of the HGF/c-MET signaling pathway.

Co-Culture of Adipose-Derived Stem Cells and Chondrocytes With Transforming Growth Factor-Beta 3 Promotes Chondrogenic Differentiation.

Tissue engineering cartilage is a promising strategy to reconstruct the craniofacial cartilaginous defects. It demands plenty of chondrocytes to generate human-sized craniofacial frameworks. Partly replacement of chondrocytes by adipose-derived stem cells (ADSCs) can be an alternative strategy.The study aimed at evaluating the chondrogenic outcome of ADSCs and chondrocytes in direct co-culture with transforming growth factor-beta (TGF-β3). Porcine ADSCs and chondrocytes were obtained from abdominal wall and external ears. Four groups: ADSCs or chondrocytes monocultured in medium added with TGF-β3; ADSCs and ACs co-cultured with or without TGF-β3. Cell growth rate was performed to evaluate the cell proliferation. Morphological, histologic and real-time polymerase chain reaction analysis were performed to characterize the chondrogenic outcome of pellets. ADSCs had favorable multi-lineage differentiation potential. Further, when ADSCs were co-cultured with chondrocytes in medium added with TGF-β3, the cell proliferation was promoted and the chondrogenic differentiation of ADSCs was enhanced. We demonstrate that pellet co-culture of ADSCs and chondrocyte with TGF-β3 could construct high quantity cartilages. It suggests that this strategy might be useful in future cartilage repair.

Bone tissue engineering using adipose-derived stem cells and endothelial cells: Effects of the cell ratio.

The microvascular endothelial network is essential for bone formation and regeneration. In this context, endothelial cells not only support vascularization but also influence bone physiology via cell contact‐dependent mechanisms. In order to improve vascularization and osteogenesis in tissue engineering applications, several strategies have been developed. One promising approach is the coapplication of endothelial and adipose derived stem cells (ADSCs). In this study, we aimed at investigating the best ratio of human umbilical vein endothelial cells (HUVECs) and osteogenic differentiated ADSCs with regard to proliferation, apoptosis, osteogenesis and angiogenesis. For this purpose, cocultures of ADSCs and HUVECs with ratios of 25%:75%, 50%:50% and 75%:25% were performed. We were able to prove that cocultivation supports proliferation whereas apoptosis was unidirectional decreased in cocultured HUVECs mediated by a p‐BAD‐dependent mechanism. Moreover, coculturing ADSCs and HUVECs stimulated matrix mineralization and the activity of alkaline phosphatase (ALP). Increased gene expression of the proangiogenic markers eNOS, Flt, Ang2 and MMP3 as well as sprouting phenomena in matrigel assays proved the angiogenic potential of the coculture. In summary, coculturing ADSCs and HUVECs stimulates proliferation, cell survival, osteogenesis and angiogenesis particularly in the 50%:50% coculture.

Role of Vascular Endothelial Growth Factor and Human Umbilical Vein Endothelial Cells in Designing An In Vitro Vascular-Muscle Cellular Model Using Adipose-Derived Stem Cells.

Objective Researchers have been interested in the creation of a favorable cellular model for use in vascular-muscle tissue engineering. The main objective of this study is to determine the myogenic effects of vascular endothelial growth factor (VEGF) and human umbilical vein endothelial cells (HUVECs) on adipose-derived stem cells (ADSCs) to achieve an in vitro vascular-muscle cellular model. Materials and Methods The present experimental research was conducted on two primary groups, namely ADSCs monoculture and ADSCs/HUVECs co-culture that were divided into control, horse serum (HS), and HS/VEGF differentiation subgroups. HUVECs were co-cultured by ADSC in a ratio of 1:1. The myogenic differentiation was evaluated using the reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence in different experimental groups. The interaction between ADSCs and HUVECs, as well as the role of ADSCs conditional medium, was investigated for endothelial tube formation assay. Results Immunofluorescence staining indicated that Tropomyosin was positive in ADSCs and ADSCs and HUVECs co-culture groups on HS and HS/VEGF culture medium. Furthermore, the MyHC2 gene expression significantly increased in HS and HS/VEGF groups in comparison with the control group (P<0.001). More importantly, there was a significant difference in the mRNA expression of this gene between ADSCs and ADSCs and HUVECs co-culture groups on HS/VEGF culture medium (P<0.05). Current data revealed that the co-culture of ADSCs and HUVECs could develop endothelial network formation in the VEGF-loaded group. Also, the ADSCs-conditioned medium improved the viability and formation of the endothelial tube in the HS and VEGF groups, respectively. ConclusionIt was concluded that ADSCs/HUVECs co-culture and dual effects of VEGF can lead to the formation of differentiated myoblasts in proximity to endothelial network formations. These in vitro cellular models could be potentially used in vascular-muscle tissue engineering implanted into organ defects where muscle tissue and vascular regeneration were required.

Microribbon-hydrogel composite scaffold accelerates cartilage regeneration in vivo with enhanced mechanical properties using mixed stem cells and chondrocytes

Juvenile chondrocytes are robust in regenerating articular cartilage, but their clinical application is hindered by donor scarcity. Stem cells offer an abundant autologous cell source but are limited by slow cartilage deposition with poor mechanical properties. Using 3D co-culture models, mixing stem cells and chondrocytes can induce synergistic cartilage regeneration. However, the resulting cartilage tissue still suffers from poor mechanical properties after prolonged culture. Here we report a microribbon/hydrogel composite scaffold that supports synergistic interactions using co-culture of adipose-derived stem cells (ADSCs) and neonatal chondrocytes (NChons). The composite scaffold is comprised of a macroporous, gelatin microribbon (μRB) scaffolds filled with degradable nanoporous chondroitin sulfate (CS) hydrogel. We identified an optimal CS concentration (6%) that best supported co-culture synergy in vitro. Furthermore, 7 days of TGF-β3 exposure was sufficient to induce catalyzed cartilage formation. When implanted in vivo, μRB/CS composite scaffold supported over a 40-fold increase in compressive moduli of cartilage produced by mixed ADSCs/NChons to ~330 kPa, which surpassed even the quality of cartilage produced by 100% NChons. Together, these results validate μRB/CS composite as a promising scaffold for cartilage regeneration using mixed populations of stem cells and chondrocytes.

Adipose-derived stem cells promote survival, growth, and maturation of early-stage murine follicles

BackgroundPremature ovarian insufficiency is a common complication of anticancer treatments in young women and girls. The ovary is a complex, highly regulated reproductive organ, whose proper function is contingent upon the bidirectional endocrine, paracrine, and autocrine signaling. These factors facilitate the development of the follicles, the functional units of the ovary, to progress from the gonadotropin-independent, paracrine-controlled early stage to the gonadotropin-dependent, endocrine-controlled later stage. We hypothesized that the low survival rate of individually cultured early-stage follicles could be improved with co-culture of adipose-derived stem cells (ADSCs) that secrete survival- and growth-promoting factors.Materials and methodsOvarian follicles ranging from 85 to 115 μm in diameter, from 10- to 12-day-old B6CBAF1 mice were mechanically isolated and co-encapsulated with ADSCs within alginate-based 3D culture system. The follicles were cultured for 14 days, imaged using light microscopy every 2 days, and matured at the end. Follicle media were changed every 2 days and collected for hormone measurements. Follicle diameter, morphology, number of transzonal projections, and survival and maturation rates were recorded. Statistical analyses using one- and two-way ANOVA were performed to compare hormone levels, survival of the follicles and ADSCs, oocyte maturation rates, and follicle growth.ResultsThe co-encapsulation of the follicles with ADSCs increased follicle survival, ranging from 42.4% for the 86–95 μm to 86.2% for the 106–115-μm follicle size group. Co-culture also improved the follicle growth, the rate of antrum formation and oocyte maturation compared to the follicles cultured alone. The levels of androstenedione, estradiol, and progesterone of co-encapsulated follicles increased progressively with time in culture.ConclusionsTo our knowledge, this is the first report of an in vitro system utilizing mouse adipose-derived stem cells to support the development of the mouse follicles. Our findings suggest that co-encapsulation of ADSCs with early-stage follicles supports follicular development, through secretion of cytokines that promote follicular survival, antrum formation, and meiotic competence. The unique 3D culture system that supports the survival of both cell types has translational implications, as ADSCs could be used as an autologous source for in vitro maturation of early-stage human follicles.

Co-culture of adipose-derived stem cells and chondrocytes on three-dimensionally printed bioscaffolds for craniofacial cartilage engineering.

Reconstruction of craniofacial cartilagenous defects are among the most challenging surgical procedures in facial plastic surgery. Bioengineered craniofacial cartilage holds immense potential to surpass current reconstructive options, but limitations to clinical translation exist. We endeavored to determine the viability of utilizing adipose-derived stem cell-chondrocyte co-culture and three-dimensional (3D) printing to produce 3D bioscaffolds for cartilage tissue engineering. We describe a feasibility study revealing a novel approach for cartilage tissue engineering with in vitro and in vivo animal data. Porcine adipose-derived stem cells and chondrocytes were isolated and co-seeded at 1:1, 2:1, 5:1, 10:1, and 0:1 experimental ratios in a hyaluronic acid/collagen hydrogel in the pores of 3D-printed polycaprolactone scaffolds to form 3D bioscaffolds for cartilage tissue engineering. Bioscaffolds were cultured in vitro without growth factors for 4 weeks and then implanted into the subcutaneous tissue of athymic rats for an additional 4 weeks before sacrifice. Bioscaffolds were subjected to histologic, immunohistochemical, and biochemical analysis. Successful production of cartilage was achieved using a co-culture model of adipose-derived stem cells and chondrocytes without the use of exogenous growth factors. Histology demonstrated cartilage growth for all experimental ratios at the post-in vivo time point confirmed with type II collagen immunohistochemistry. There was no difference in sulfated-glycosaminoglycan production between experimental groups. Tissue-engineered cartilage was successfully produced on 3D-printed bioresorbable scaffolds using an adipose-derived stem cell and chondrocyte co-culture technique. This potentiates co-culture as a solution for several key barriers to a clinically translatable cartilage tissue engineering process. NA. Laryngoscope, 128:E251-E257, 2018.

The role of fibrinolysis inhibition in engineered vascular networks derived from endothelial cells and adipose-derived stem cells

BackgroundCo-cultures of endothelial cells with mesenchymal stem cells currently represent one of the most promising approaches in providing oxygen and nutrient supply for microvascular tissue engineering. Still, to translate this model into clinics several in vitro parameters including growth medium and scaffold degradation need to be fine-tuned.MethodsWe recently described the co-culture of adipose-derived stem cells with endothelial cells in fibrin, resulting in capillary formation in vitro as well as their perfusion in vivo. Here, we aimed to further characterise microvascular tube formation in fibrin by determining the role of scaffold degradation, thrombin concentration and culture conditions on vascularisation.ResultsWe observed that inhibition of cell-mediated fibrin degradation by the commonly used inhibitor aprotinin resulted in impaired vascular network formation. Aprotinin had no effect on laminin and collagen type IV deposition or formation of tube-like structures in scaffold-free co-culture, indicating that poor vascularisation of fibrin clots is primarily caused by inhibition of plasminogen-driven fibrinolysis. Co-culture in plasminogen- and factor XIII-depleted fibrin did not result in different vascular network density compared to controls. Furthermore, we demonstrate that thrombin negatively affects vascular network density at high concentrations. However, only transient activation of incorporated endothelial cells by thrombin could be observed, thus excluding a long-term inflammatory response in tissue-engineered micro-capillaries. Finally, we show that vascularisation of fibrin scaffolds in basal medium is undermined because of increased fibrinolytic activity leading to scaffold destabilisation without aprotinin.ConclusionsTaken together, our data reveal a critical role of fibrinolysis inhibition in in vitro cell-mediated vascularisation of fibrin scaffolds.

SOST Gene Inhibits Osteogenesis from Adipose-Derived Mesenchymal Stem Cells by Inducing Th17 Cell Differentiation

Background/Aims: Postmenopausal osteoporosis is considered to be an autoimmune and inflammatory process, and IL-17 plays important roles in the loss of bone mass. Sclerostin (SOST) acts as a negative regulator of bone formation by inhibiting the Wnt signaling pathway. It also is a mediator of the crosstalk between the skeletal and immune systems. However, few studies have examined the role of SOST gene in the differentiation of T helper 17 (Th17) cells. Methods: Adipose-derived stem cells (ADSCs) were isolated and transfected with pcDNA3-SOST or shSOST, and then co-cultured with CD4+ T cells isolated from peripheral blood mononuclear cells. The differentiation, adipogenesis, and osteogenesis of Th17 and regulatory T (Treg) cells were examined by western blot, intracellular and intranuclear staining, ELISA, and real-time quantitative PCR in this co-culture model. Results: The SOST gene promoted the secretion of IL-6 and TGF-β in ADSCs. After co-culture of ADSCs with CD4+ T cells, the SOST gene increased the number of CD4+IL-17+ cells and the levels of IL-17 and RORγ. However, the number of CD4+CD25+Foxp3+ cells was decreased, which was accompanied with a reduction of IL-10 and Foxp3 expression. In the meantime, the SOST gene inhibited the expression of COL1, OCN, and OPN, reduced the activity of alkaline phosphatase, and increased the expression of LPL and PPARγ. Furthermore, IL-17 promoted SOST gene-induced adipogenesis and increased the inhibition of osteogenesis. Conclusions: SOST promoted the differentiation of Th17 cells and reduced the differentiation of Treg cells, which exacerbated the SOST gene-induced inhibition of osteogenesis from ADSCs.

Tendon Tissue Engineering: Mechanism and Effects of Human Tenocyte Coculture With Adipose-Derived Stem Cells.

Adipose-derived stem cells (ASCs) are a potential candidate for cell-based therapy targeting tendon injury; however, their therapeutic benefit relies on their ability to interact with native tenocytes. This study examines the mechanism and effects of coculturing human tenocytes and ASCs. Tenocytes (T) were directly cocultured with either ASCs (A) or fibroblasts (F) (negative control) in the following ratios: 50% T/50% A or F; 25% T/75% A or F; and 75% T/25% A or F. Cells were indirectly cocultured using a transwell insert that allowed for exchange of soluble factors only. Proliferation and collagen I production were measured and compared with monoculture controls. Synergy was quantified using the interaction index (II), which normalizes measured values by the expected values assuming no interaction (no synergy when II= 1). The ability of ASCs to elicit tenocyte migration was examined invitro using a transwell migration assay and exvivo using decellularized human flexor tendon explants. Compared with monoculture controls, II of proliferation was greater than 1 for all tenocyte and ASC direct coculture ratios, but not for tenocyte and fibroblast direct coculture ratios or for tenocyte and ASC indirect coculture. The ASCs elicited greater tenocyte migration invitro and exvivo. The II of collagen I production was greater than 1 for direct coculture groups with 25% T/75% A and 75% T/25% A. Direct coculture of ASCs and tenocytes demonstrated synergistic proliferation and collagen I production, and ASCs elicited tenocyte migration invitro and exvivo. These interactions play a key role in tendon healing and were absent when ASCs were replaced with fibroblasts, supporting the use of ASCs for cell-based therapy targeting tendon injuries. When ASCs are delivered for cell-based therapy, they directly interact with native tenocytes to increase cell proliferation, collagen I production, and tenocyte migration, which may enhance tendon healing.

Inhibition of Pathological Phenotype of Hypertrophic Scar Fibroblasts Via Coculture with Adipose-Derived Stem Cells.

Hypertrophic scar (HS) is a dermal fibroproliferative disease characterized by fibroblast over-proliferation, overproduction, and deposition of the extracellular matrix. Growing evidence demonstrated that adipose-derived stem cells (ASCs) secrete a plethora of trophic and antifibrotic factors, which suppress inflammation and ameliorate fibrosis of different tissues. However, few studies investigate their effect on repressing HS activity. This study evaluated the suppressing effect of ASCs on HS fibroblast bioactivity and the possible mechanism via a coculture model. HS-derived fibroblasts (HSFs) and ASCs were isolated from individual patients. HSFs or HSFs treated with transforming growth factor-β1 (TGF-β1) were cocultured with ASCs and the change of HSF cellular behaviors, such as cell proliferation, migration, contractility, and gene/protein expression of scar-related molecules, were evaluated by cell counting assay, cell cycle analysis, scratch wound assay, fibroblast-populated collagen lattice (FPCL) contractility assay, real-time quantitative polymerase chain reaction, ELISA, and western blotting assay. After 5 days of ASC coculture treatment, the expression levels of collagen I (Col 1), collagen III (Col 3), fibronectin (FN), TGF-β1, interleukin-6 (IL-6), interleukin-8 (IL-8), connective tissue growth factor (CTGF), and alpha-smooth muscle actin (α-SMA) in HSFs decreased significantly while the expression levels of decorin (DCN) and MMP-1/TIMP-1 (matrix metalloproteinase/tissue inhibitor of MMP) ratio increased significantly. Besides, after 5 days of exogenous TGF-β1 stimulation, the expression levels of Col 1, FN, TGF-β1, IL-6, CTGF, and α-SMA in HSFs increased significantly. Impressively, all these increased gene expression levels were reversed by 5 days of ASCs coculture treatment. Additionally, the proliferation, migration, and contractility of HSFs were all significantly reduced by ASC coculture treatment. Furthermore, the protein levels of TGF-β1 and intracellular signal pathway-related molecules, such as p-smad2, p-smad3, p-Stat3, and p-ERK, were downregulated significantly in HSFs after 5 days of ASCs coculture treatment. This study demonstrated that coculture of HSFs with ASCs not only inhibited proliferation, migration, and contractility of HSFs but also decreased the expression levels of HSF-related or TGF-β1-induced molecules. Additionally, the antifibrotic effect on HSFs was likely mediated by the inhibition of multiple intracellular signaling. The results of this study suggest the therapeutic potential of ASCs for HS treatment, which is worth of further investigation.

Low-intensity pulsed ultrasound upregulates pro-myelination indicators of Schwann cells enhanced by co-culture with adipose-derived stem cells.

Peripheral nerve injuries are a common occurrence, resulting in considerable patient suffering; it also represents a major economic burden on society. To improve treatment options following peripheral nerve injuries, scientists aim to find a way to promote Schwann cell (SC) myelination to help nerves to carry out their functions effectively. In this study, we investigated myelination ability of SCs, regulated by co-culture with adipose-derived stem cells (ASCs) or low-intensity pulsed ultrasound (LIPUS), and synergistic effects of combined treatments. Schwann cells were co-cultured with or without ASCs, and either left untreated or treated with LIPUS for 10min/d for 1, 4 or 7days. Effects of LIPUS and ASC co-culture on pro-myelination indicators of SCs were analysed by real-time PCR (RT-PCR), Western blotting and immunofluorescence staining (IF). Our results indicate that ASC-SC co-culture and LIPUS, together or individually, promoted mRNA levels of epidermal growth factor receptor 3 (EGFR3/ErbB3), neuregulin1 (NRG1), early growth response protein 2 (Egr2/Krox20) and myelin basic protein (MBP), with corresponding increases in protein levels of ErbB3, NRG1 and Krox20. Interestingly, combination of ASC-SC co-culture and LIPUS displayed the most remarkable effects. We demonstrated that ASCs upregulated pro-myelination indicators of SCs by indirect contact (through co-culture) and that effects could be potentiated by LIPUS. We conclude that LIPUS, as a mechanical stress, may have potential in nerve regeneration with potential clinical relevance.