Co-culture Of Adipose-derived Stem Cells Research Articles

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.

In Vitro Effects of Adipose-Derived Stem Cells on Breast Cancer Cells Harvested From the Same Patient.

Fat grafting for breast cancer (BrCa) reconstruction and breast augmentation has become increasingly more popular. A major area of debate and controversy is the effect of adipose-derived stem cells (ASCs) on remnant or undetected BrCa cells. We investigate the in vitro response of BrCa to ASCs in a coculture model with regards to cell migration. The study was approved by the institutional review board. BrCa and adipose tissue specimens either from subcutaneous breast tissue or abdominal lipoaspirate were obtained from the same patient. BrCa cells and ASCs were harvested with either explant culture and/or enzymatic digestion. Tissues were grown in cell culture flasks until adequate cell libraries were established. Adipose-derived stem cells from adipose specimens were characterized with flow cytometry. Immunofluorescence (IF) staining of the initial cell population harvested from the BrCa specimens confirmed the presence of CD24, an epithelial marker of BrCa. A homogenous CD 24+/CD 90- BrCa cell population was obtained with flowcytometric cell sorting. The in vitro migration of BrCa cells was examined in coculture with and without ASCs. Adipose-derived stem cells harvested from the adipose specimens were positive for mesenchymal stem cell markers CD 105, CD 90, CD 73, and CD 44 and negative for lymphocyte cell marker CD 34 and leukocyte marker CD 45. The percentage of the CD 24+/CD 90- BrCa cells in the initial cell population harvested from BrCa specimens was 0.61%. The BrCa cells morphologically had large nuclei and small cytoplasm in clusters under the light microscope, suggesting a cancer cell phenotype. CD 24 expression on the surface of BrCa cells was confirmed with IF staining. The number of BrCa cells migrated in ASCs coculture was approximately 10 times higher than the number of BrCa cells migrated in BrCa cell only cultures. Adipose-derived stem cells significantly increase the migration capacity of BrCa cells in vitro in cocultures. This should be taken into consideration when performing fat grafting to the breast especially in patients with a history of BrCa or strong family history of BrCa.

Adipose‐Derived Stem Cells Support Lymphangiogenic Parameters In Vitro

It is a common complication to develop a secondary lymphedema after surgery or radiation, for example, after axillary lymph node dissection due to breast cancer and current therapies are mainly symptomatic. Since these surgical procedures result in both, loss of adipose tissue and loss of lymphatic nodes and vessels, tissue engineering could be a new promising approach, to create an adipose tissue substitute comprised with a lymphatic network. We have conducted co-culture experiments to investigate the effects of human adipose-derived stem cells (ASCs) on human lymphatic endothelial cells (LECs) in terms of gene expression profile, proliferation, migration, and tube formation in vitro. In this respect, both cell types were co-cultured either indirectly or directly with or without the recombinant growth factor VEGF-C. Indirect co-cultures were performed with the aid of a transwell chamber. In case of direct co-culture, immunomagnetic separation by CD31 magnetic beads allowed examination of the LEC population. Direct and indirect co-culture of ASCs induced mRNA expression of lymphatic marker genes, proliferation, and migration by LECs without affecting tube formation. Thus, we have shown that co-culture of ASCs with LECs might be a feasible approach that could be used in cell-based tissue engineering therapies to heal or improve a secondary lymphedema. J. Cell. Biochem. 117: 2620-2629, 2016. © 2016 Wiley Periodicals, Inc.

Delivery of Adipose-Derived Stem Cells Attenuates Adipose Tissue Inflammation and Insulin Resistance in Obese Mice Through Remodeling Macrophage Phenotypes.

Adipose-derived stem cells (ADSCs) have been used to control several autoimmune or inflammatory diseases due to immunosuppressive properties, but their role in obesity-associated inflammation remains unestablished. This study aims to evaluate the effects of ADSCs on obesity-induced white adipose tissue (WAT) inflammation and insulin resistance. We found that diet-induced obesity caused a remarkable reduction of ADSC fraction in mouse WAT. Delivery of lean mouse-derived ADSCs, which could successfully locate into WAT of obese mice, substantially improved insulin action and metabolic homeostasis of obese mice. ADSC treatment not only reduced adipocyte hypertrophy but also attenuated WAT inflammation by reducing crown-like structures of macrophages and tumor necrosis factor (TNF)-α secretion. Importantly, ADSC treatment remodeled the phenotypes of adipose-resident macrophages from proinflammatory M1 toward anti-inflammatory M2-like subtypes, as characterized by decreased MHC class II-expressing but increased interleukin (IL)-10-producing macrophages together with low expression of TNF-α and IL-12. Coculture of ADSCs through the transwell or conditional medium with induced M1 macrophages also reproduced the phenotypic switch toward M2-like macrophages, which was substantiated by elevated arginase 1, declined inducible nitric oxide synthase, inhibition of NF-κB activity, and activation of STAT3/STAT6. Taken together, our data support that ADSC supplement in obese mice could sustain IL-10-producing M2-like macrophages in WAT through paracrine action, thereby suggesting the crucial role of ADSCs in resolving WAT inflammation, maintaining adipose homeostasis, and proposing a potential ADSC-based approach for the treatment of obesity-related diseases.

Fluid and cell behaviors along a 3D printed alginate/gelatin/fibrin channel.

Three-dimensional (3D) cell manipulation is available with the integration of microfluidic technology and rapid prototyping techniques. High-Fidelity (Hi-Fi) constructs hold enormous therapeutic potential for organ manufacturing and regenerative medicine. In the present paper we introduced a quasi-three-dimensional (Q3D) model with parallel biocompatible alginate/gelatin/fibrin hurdles. The behaviors of fluids and cells along the microfluidic channels with various widths were studied. Cells inside the newly designed microfluidic channels attached and grew well. Morphological changes of adipose-derived stem cells (ADSCs) in both two-dimensional (2D) and 3D milieu were found on the printed constructs. Endothelialization occurred with the co-cultures of ADSCs and hepatocytes. This study provides insights into the interactions among fluids, cells and biomaterials, the behaviors of fluids and cells along the microfluidic channels, and the applications of Q3D techniques.

Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) Based Electrospun 3D Scaffolds for Delivery of Autogeneic Chondrocytes and Adipose-Derived Stem Cells: Evaluation of Cartilage Defects in Rabbit.

The management of chondral defects has long been a challenge because of the poor self-healing capacity of articular cartilage. Many approaches ranging from symptomatic treatment to structural cartilage regeneration have obtained very limited satisfactory results. Cartilage tissue engineering, which involves an optimized combination of novel scaffolds, cell sources and growth factors, has emerged as a promising strategy for cartilage regeneration and repair. In this study, the cellular morphologies and the adhesion, migration and proliferation capabilities of adipose-derived stem cells (ASCs) and chondrocytes seeded on 3D scaffolds composed of electrospun poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P3HB4HB) were evaluated. Next, TGF-β1/scaffolds with 4:1 co-culture of ASCs and chondrocytes were implanted into the full thickness cartilage defects in rabbit knee for 16 weeks. ASCs and chondrocytes seeded on the scaffolds showed better adhesion, migration and proliferation than that on petri dishes in vitro. Importantly, implantation with TGF-β1/scaffolds with delivery of ASCs and chondrocytes revealed desirable in vivo healing outcomes. These results demonstrate that ASCs have great potential in the field of tissue engineering. It is possible that the improvement in ASC-seeded electrospun 3D P3HB4HB scaffolds may ultimately lead to improved repair of cartilage injuries.

Mechanisms of vasculogenesis in 3D fibrin matrices mediated by the interaction of adipose-derived stem cells and endothelial cells.

Vascularization of tissue-engineered constructs is essential to provide sufficient nutrient supply and hemostasis after implantation into target sites. Co-cultures of adipose-derived stem cells (ASC) with outgrowth endothelial cells (OEC) in fibrin gels were shown to provide an effective possibility to induce vasculogenesis in vitro. However, the mechanisms of the interaction between these two cell types remain unclear so far. The aim of this study was to evaluate differences of direct and indirect stimulation of ASC-induced vasculogenesis, the influence of ASC on network stabilization and molecular mechanisms involved in vascular structure formation. Endothelial cells (EC) were embedded in fibrin gels either containing non-coated or ASC-coated microcarrier beads as well as ASC alone. Moreover, EC-seeded constructs incubated with ASC-conditioned medium were used in addition to constructs with ASC seeded on top. Vascular network formation was visualized by green fluorescent protein expressing cells or immunostaining for CD31 and quantified. RT-qPCR of cells derived from co-cultures in fibrin was performed to evaluate changes in the expression of EC marker genes during the first week of culture. Moreover, angiogenesis-related protein levels were measured by performing angiogenesis proteome profiler arrays. The results demonstrate that proximity of endothelial cells and ASC is required for network formation and ASC stabilize EC networks by developing pericyte characteristics. We further showed that ASC induce controlled vessel growth by secreting pro-angiogenic and regulatory proteins. This study reveals angiogenic protein profiles involved in EC/ASC interactions in fibrin matrices and confirms the usability of OEC/ASC co-cultures for autologous vascular tissue engineering.

Co-culture of adipose-derived stem cells and endothelial cells in fibrin induces angiogenesis and vasculogenesis in a chorioallantoic membrane model

Neovascularization of adipose tissue equivalents is a crucial step in successful adipose tissue engineering, since insufficient vascularization results in graft resorption in an in vivo situation. A possible cellular approach to overcome this limitation is the co-implantation of adipose-derived stem cells (ASCs) with endothelial cells to stimulate the formation of a vascular network. We investigated the potential of ASCs derived from human abdominal fat tissue co-cultured with endothelial progenitor cells (EPCs) from human peripheral blood to stimulate neovascularization of fibrin constructs on the chorioallantoic membrane (CAM) of fertilized chicken eggs, in direct comparison to human umbilical vein endothelial cells (HUVECs). After 9 days of incubation, cell-fibrin constructs were explanted and histologically evaluated with respect to ingrowth of avian blood vessels into the construct and formation of human blood vessels by co-implanted endothelial cells. When administered on the CAM, ASCs successfully guided host vasculature into the construct (angiogenesis) and guided formation of capillary-like structures by co-implanted human endothelial cells (vasculogenesis), with HUVECs being superior to EPCs, leading to a perfused avian and human capillary network within the fibrin construct. However, the results also showed that perfused human blood vessels were only observed near the CAM compared to unperfused capillary-like structures near the top of the construct, indicating that perfusion of the cell-fibrin construct takes longer than 9 days. In conclusion, as blood vessel formation is an essential step during adipogenic differentiation, the data support our hypothesis that cellular communication between transplanted ASCs and endothelial cells is beneficial for vasculogenesis. Copyright © 2013 John Wiley & Sons, Ltd.

Regeneration of articular cartilage by adipose tissue derived mesenchymal stem cells: Perspectives from stem cell biology and molecular medicine

Adipose-derived stem cells (ASCs) have been discovered for more than a decade. Due to the large numbers of cells that can be harvested with relatively little donor morbidity, they are considered to be an attractive alternative to bone marrow derived mesenchymal stem cells. Consequently, isolation and differentiation of ASCs draw great attention in the research of tissue engineering and regenerative medicine. Cartilage defects cause big therapeutic problems because of their low self-repair capacity. Application of ASCs in cartilage regeneration gives hope to treat cartilage defects with autologous stem cells. In recent years, a lot of studies have been performed to test the possibility of using ASCs to re-construct damaged cartilage tissue. In this article, we have reviewed the most up-to-date articles utilizing ASCs for cartilage regeneration in basic and translational research. Our topic covers differentiation of adipose tissue derived mesenchymal stem cells into chondrocytes, increased cartilage formation by co-culture of ASCs with chondrocytes and enhancing chondrogenic differentiation of ASCs by gene manipulation.

Multilineage co-culture of adipose-derived stem cells for tissue engineering

Stem cell interactions through paracrine cell signalling can regulate a range of cell responses, including metabolic activity, proliferation and differentiation. Moving towards the development of optimized tissue-engineering strategies with adipose-derived stem cells (ASCs), the focus of this study was on developing indirect co-culture models to study the effects of mature adipocytes, chondrocytes and osteoblasts on bovine ASC multilineage differentiation. For each lineage, ASC differentiation was characterized by histology, gene expression and protein expression, in the absence of key inductive differentiation factors for the ASCs. Co-culture with each of the mature cell populations was shown to successfully induce or enhance lineage-specific differentiation of the ASCs. In general, a more homogeneous but lower-level differentiation response was observed in co-culture as compared to stimulating the bovine ASCs with inductive differentiation media. To explore the role of the Wnt canonical and non-canonical signalling pathways within the model systems, the effects of the Wnt inhibitors WIF-1 and DKK-1 on multilineage differentiation in co-culture were assessed. The data indicated that Wnt signalling may play a role in mediating ASC differentiation in co-culture with the mature cell populations.

Human Adipose-Derived Stem Cells Enhance the Angiogenic Potential of Endothelial Progenitor Cells, But Not of Human Umbilical Vein Endothelial Cells

One of the current challenges in the field of adipose tissue engineering is to promote sufficient vascularization to prevent cell death and to support adipose tissue formation. Thus, a novel strategy to enhance neovascularization of tissue-engineered adipose tissue might be the coimplantation of adipose-derived stem cells (ASCs) with endothelial progenitor cells (EPCs). However, no knowledge is given about the cellular interaction in vitro of human ASCs derived from subcutaneous fat tissue and EPCs derived from human peripheral blood. In this study, the first aim was to characterize ASCs and EPCs. Secondly, the two-dimensional Matrigel assay and the three-dimensional spheroid sprouting assay were applied for analyzing the ASC-EPC interaction in regard to formation of capillary-like structures by EPCs by ASC-conditioned medium (CM) or coculture of both cell types and compared to cocultures of ASCs and human umbilical vein endothelial cells (HUVECs). ASC-CM had no influence on the formation of capillary-like structures by EPCs. However, coculture with ASCs significantly enhanced the formation of capillary-like structures by EPCs; an effect that was not observed in cocultures of ASCs with HUVECs. Importantly, this increase in capillary-like structure formation by EPCs due to cell-cell contact was associated with significantly increased vascular endothelial growth factor (VEGF) secretion and VEGF-A mRNA expression, while inhibition of VEGF receptor tyrosine kinases completely abolished this effect. In conclusion, these data suggest that cellular communication occurs between ASCs and EPCs triggered by cell-cell contact or at least close proximity, which is partially mediated by secreted VEGF leading to the enhancement of angiogenic properties in EPCs, but not in HUVECs.

Differentiation of adipose stem cells into epidermal cells induced by two methods

Objective To find an optimal way to induce differentiation of adipose-derived stem cells (ASCs) into epidermal cells.Methods In co-culture group,HaCaT cells and ASCs were co-cultured by using Transwell apparatus.In epidermal growth factor (EGF) induction group,EGF was added into the culture medium of ASCs.After culture for 3 and 6 days,ASCs were detected by counting the number and dying the markers of epidermal cells such as cytokeratin 19 (CK-19),integrin β1 and pan-cytokeratin (Pan-CK).Results The positive rate of CK-19,integrin β1 and Pan-CK in ASCs was (25.8 ±4.1 )％,( 29.2 ± 3.9) ％ and ( 18.3 ± 2.7 ) ％ respectively at 3rd day after culture by co-culture method,which was significantly higher than in EGF induction group (P ＜ 0.05).The positive rate of CK-19,integrin β1 and Pan-CK in ASCs was ( 34.1 ± 5.7) ％,(42.8 ± 4.3 ) ％ and (29.4 ± 3.3 ) ％ respectively at 6th day after culturc by co culturc method,which was significantly higher than in EGF induction group ( P ＜0.01 ).The number of epidermal cells obtained by co-culture method was significantly more than that in EGF induction group.Conclusion Co-culture of ASCs with HaCaT cells is an optimal method to promote the differentiation of ASCs into epidermal cells. Key words: Adipose-derived stem cells; Epidermal cells; Induction; Differentiation

Coculture of Vascular Endothelial Cells and Adipose-Derived Stem Cells as a Source for Bone Engineering

The interaction between vascular endothelial cells (VECs) and osteoblasts (OBs) is the focus of this recent research. Vascular endothelial cells secrete bone morphogenetic protein, which promotes OB differentiation and stimulates OBs and their precursor cells to secrete vascular endothelial growth factor. Vascular endothelial growth factor is important in angiogenesis and angiopoiesis. Cloning studies have shown that adipose-derived stem cells (ADSCs) have the potential to differentiate into fat, bone, cartilage, and skeletal and smooth muscle cells, among others. Adipose-derived stem cells can express multiple growth factors, including vascular endothelial growth factor and hepatocyte growth factor. Our study examined the influence of coculturing VECs and ADSCs on osteogenic differentiation. Cord blood-derived VECs and ADSCs were isolated from rats and characterized with immunofluorescence staining and morphological observation. Coculture of third-generation ADSCs and VECs was induced for 6 weeks. Cell growth was analyzed using a modified MTT assay. Alkaline phosphatase (ALP) and osteocalcin (OC) was analyzed using immunofluorescence staining. When ADSCs and VECs were cocultured, the absorbance of cells gradually increased, reaching a peak on day 12. The highest absorbance was seen in a coculture system with a ratio of ADSCs and VECs of 1:1. The secretion of ALP and OC gradually increased in these cells and was significantly higher than controls (P < 0.01). Coculturing of ADSCs and VECs at a 1:1 ratio gave the highest secretion of ALP and OC at every time point, and was significantly higher than other groups (P < 0.01). Our results indicated that ADSCs can be induced to osteogenic differentiation by VECs in vitro, suggesting a coculture system of VECs and ADSC as a novel source of cells for bone engineering.

Human Adult Stem Cells Restore Endothelial Migratory Dysfunction in a Hypoxic Environment

Adipose-derived stem cells (ASCs) injected into the blood stream after an ischemic event promote therapeutic angiogenesis in affected tissues. It has been suggested that the stem cells exert their influence by way of a paracrine effect on native endothelial cells (ECs). Using an in vitro model, we evaluated the effect of ASC coculture on EC function in a hypoxic environment.

PS212. Human Adult Stem Cells Restore Endothelial Migratory Dysfunction in a Hypoxic Environment

Adipose-derived stem cells (ASC) injected into the blood stream following an ischemic event promote therapeutic angiogenesis in affected tissues. It has been suggested that the stem cells exert their influence via a paracrine effect on native endothelial cells (EC). Using an in vitro model, we evaluate the effect of ASC co-culture on EC function in a hypoxic environment. Confluent monolayers of human EC grown on the bottom of transwell plates were wounded to create an even 5mm defect and cultured in either normoxic (21%) or hypoxic (1%) conditions. Human ASC were co-cultured on the top of the transwell plates to evaluate a paracrine effect. Subsequently, EC migration was determined by measuring the wound size after 3d. Media samples were collected and VEGF concentration measured via ELISA. To confirm mechanism, ECs were treated with recombinant VEGF at various concentrations and migration measured. HIF-1α expression was evaluated in ASC by Western blot. Hypoxia inhibited EC migration (0.87 mm vs 0.78mm, p < 0.05) over 3d. Co-culture of ASC enhanced EC migration in both normoxic (0.87 mm to 0.93mm) and hypoxic (0.78 mm to 1.02mm; p < 0.05) environments. Media from co-cultures in hypoxia contained significantly more VEGF (708.3 pg/mL) than normoxic co-cultures (311.2 pg/mL) and EC alone (28.9 pg/mL). The addition of VEGF to wounded EC cultures improved migration, but not to the extent of ASC co-culture. Finally, hypoxia increased levels of HIF-1α in ASC. These results demonstrate that: 1) ASC restore and enhance endothelial cell migratory function in a hypoxic environment, and 2) the effect is primarily, but not totally, due to secretion of VEGF by ASC in response to hypoxia. These results suggest that hypoxic pre-conditioning of ASC might be of value in enhancing their role in therapeutic angiogenesis to treat ischemic heart or limb conditions.

Addition of Adipose-Derived Stem Cells in Cord Blood Cultures Stimulates Their Pluripotent Differentiation

Introduction Adipose tissue is recognized as an important source of postnatal mesenchymal stem cells for generative medicine applications. Moreover, cord blood stem cells have been shown to contain pluripotent stem cells called unrestricted somatic stem cells (USSCs). However, this population is rare and cannot be generated from every cord blood sample. In this study, we have presented a new method of co-culture of adipose-derived stem cells (ADPCs) and cord blood stem cells that results in pluripotent differentiation. Materials and Methods ADPCs were obtained from a piece of adipose tissue after treatment with 0.075% collagenase, which was subsequently inactivated with DMEM/10% FBS. The cellular pellet of centrifugation was plated at 5–7 × 10 6 cells/mL in T25 culture flasks in a low-glycose DMEM with 30% FCS. Cord blood stem cells were obtained by centrifugation following double-processing in the presence of 2% HES 200/0.5 and plated at 5–7 × 10 6 cells/mL in the same medium. To investigate the crucial role of ADPCs in pluripotent cord blood differentiation, we added a ADPCS as (1 × 10 4 cells/mL) to the cord blood cultures and analyzed the contribution of ADPCs using a microscope as well as with flow cytometry. Results After only 3 days, adherent cells (USSC colonies) of fibroblastic morphology were detected in all co-cultured samples, whereas this was observed later or not at all in the non–co-cultured samples. The greater density of colonies in the co-coltured samples was another point. Hematopoietic CD45 cells were no longer detected after the first passage. Pluripotent stem cells were obtained from all co-cultured samples that contained stem cells positive for CD29, CD44, CD49e, CD90, CD105, CD51 Stro, and C-kit antibodies but negative for CD34, CD45, CD133, and glycophorin A. Conclusion Addition of ADPCs was crucial to generate pluripotent-derived stem cells from cord blood samples. This double culture may be a useful tool for a universal allogeneic stem cell source for tissue repair or regeneration.