Human Adipose Tissue-derived Stem Cells Research Articles

Matrigel enhances differentiation of human adipose tissue-derived stem cells into dopaminergic neuron

BackgroundTherapy based stem cells have offered a novel therapeutic approach for the improvement of neurodegenerative diseases, specially Parkinson. Hence, developing a well-established culture model with appropriate stem cells is extremely crucial in regenerative engineering to provide efficient targeted cells. Human adult mesenchymal stem cells derived from adipose tissue (hADSCs) have emerged as a promising source of stem cells due to their unique potentials of self-renewal and differentiation into other stem cells. The purpose of this study was to investigate the differentiation capacity of hADSCs into dopaminergic and neuron-like cells in the 3D culture plate (Matrigel). Methods and materialshADSCs were obtained from adipose tissues of patients and then characterized morphologically with flowcytometry. Isolated cells were harvested to perform differentiation on Matrigel and tissue culture plate (TCP) supplemented with induction factors. The survival rate of cells during neural induction was monitored by MTT. The expression of specific cell markers was analyzed by QRT-PCR and immunocytochemistry on days 2, 8 and 14. The level of released dopamine was measured using HPLC technique. ResultsMatrigel had a positive effect on maintaining cell growth compared to those on TCP. Moreover, the number of TH and MAPII positive cells is substantially higher in Matrigel than in TCP. Sox2 and Nestin had a prominent expression in hADSCs within the first days of differentiation. The gene expression of neural markers such as TH, Nurr1, LMX1A and DAT was detected and increased after day 8. Moreover, the dopamine released in the cell harvested on Matrigel was greater than those seeded on TCP. ConclusionsOverall, hADSCs could generate dopaminergic cells, which suggest its strong capability to serve as a tool for Parkinson disease model in the regenerative medicine.

The Antioxidant Effects of Ginger Extract on Bioavailability and Oxidative Stress-induced Apoptosis in Mesenchymal Stem Cells of Human Adipose Tissue and Rat Bone Marrow

Background and Aim: Proliferate potential differentiate into different cell lineages and high self-renewal of Mesenchymal Stem Cells (MSCs); thus, they are ideal tools for regenerative medicine. However, a leading problem is an oxidative stress in the target tissue and the apoptosis of transplanted stem cells before tissue repair. The pretreatment of stem cells with antioxidants may make them resistant to oxidative stress. Ginger is the main medicinal plant with antioxidant properties. This study explored the antioxidant effects of ginger extract on bioavailability and oxidative stress-induced apoptosis in human adipose tissue-derived mesenchymal stem cells and rat bone marrow examined. Methods & Materials: In this study, human adipose tissue-derived mesenchymal stem cells and rat bone marrow were cultured in a DMEM medium with 20% FBS. The explored cells were incubated for 4 and 6 hours for pretreatment with different concentrations of ginger extract (50, 100, 200, & 400 mg/mL); then, they were treated with 200 μM H2O2 for 2 hours. Bioavailability was analyzed by ELISA reader using an MTS kit and apoptosis was analyzed by flow cytometry using an Annexin V-FITC/PI kit into the manufacturer’s protocol at both times. The obtained data were analyzed by Analysis of Variance (ANOVA) using SPSS. Ethical Considerations: This study was approved by the Ethics Research Committee of Shahrekord Branch, Islamic Azad University (Code: IR.IAU.SHK.REC.1397.028). Results: The MTS results indicated a dose- and time-dependent manner increase in the bioavailability of human adipose tissue-derived mesenchymal treated stem cells. Ginger extract treatment also dose- and time-dependently decreased the rate of apoptosis in rat bone marrow mesenchymal stem cells. Conclusion: Ginger extract, by reducing the oxidative stress in mesenchymal stem cells, elevates their lifespan in the target tissue, and increases the efficiency of these cells in tissue regeneration.

Human adipose tissue-derived MSCs improve psoriasis-like skin inflammation in mice by negatively regulating ROS

Background Psoriasis is chronic incurable skin inflammation. The anti-inflammatory properties of mesenchymal stem cells (MSCs) have been put forward to be involved in several inflammatory diseases. However, little was known about the role of human adipose tissue-derived stem cells (hAD-MSCs) in psoriasis. Objective We sought to explore the feasibility of using hAD-MSCs infusion as a therapeutic approach in psoriatic mice. Methods We constructed the psoriasis-like model by IMQ implication, treated with hAD-MSCs by subcutaneous injection. To evaluate the efficacy, we examined the histology, CD45 and ROS positive cells by HE and flow cytometry respectively. We also tested the key cytokines with PCR. Moreover, to achieve a better therapeutic effect, we treated the model by combing with vitamin E application. Results We found that the classic histological symptoms of psoriasis were relieved after treatment with hAD-MSCs, also, the splenic index, the infiltration of immune cells and several pro-inflammatory cytokines were decreased. Interestingly, we also found that hAD-MSCs could inhibit ROS generation. Moreover, the combination therapy of hAD-MSCs and vitamin E could promote the curative effect with greater ROS inhibition. Conclusion These results suggested that hAD-MSCs could be useful for treating psoriasis by negatively regulating ROS.

The Micro-RNA Cargo of Extracellular Vesicles Released by Human Adipose Tissue-Derived Mesenchymal Stem Cells Is Modified by Obesity.

Obesity is a chronic disease that interferes with normal repair processes, including adipose mesenchymal stem/stromal cells (ASCs) function. ASCs produce extracellular vesicles (EVs) that activate a repair program in recipient cells partly via their micro-RNA (miRNA) cargo. We hypothesized that obesity alters the miRNA expression profile of human ASC-derived EVs, limiting their capacity to repair injured cells. Human ASCs were harvested from obese and age- and gender-matched non-obese (lean) subjects during bariatric or cosmetic surgeries, respectively (n = 5 each), and their EVs isolated. Following high-throughput sequencing analysis, differentially expressed miRNAs were identified and their gene targets classified based on cellular component, molecular function, and biological process. The capacity of human lean- and obese-EVs to modulate inflammation, apoptosis, as well as mitogen-activated protein kinase (MAPK) and Wnt signaling in injured human proximal tubular epithelial (HK2) cells was evaluated in vitro. The number of EVs released from lean- and obese-ASCs was similar, but obese-EVs were smaller compared to lean-EVs. Differential expression analysis revealed 8 miRNAs upregulated (fold change > 1.4, p < 0.05) and 75 downregulated (fold change < 0.7, p < 0.05) in obese-EVs vs. lean-EVs. miRNAs upregulated in obese-EVs participate in regulation of NFk-B and MAPK signaling, cytoskeleton organization, and apoptosis, whereas those downregulated in obese-EVs are implicated in cell cycle, angiogenesis, and Wnt and MAPK signaling. Treatment of injured HK2 cells with obese-EVs failed to decrease inflammation, and they decreased apoptosis and MAPK signaling significantly less effectively than their lean counterparts. Obesity alters the size and miRNA cargo of human ASC-derived EVs, as well as their ability to modulate important injury pathways in recipient cells. These observations may guide development of novel strategies to improve healing and repair in obese individuals.

Interaction between Galectin-3 and Integrins Mediates Cell-Matrix Adhesion in Endothelial Cells and Mesenchymal Stem Cells.

Galectin-3 (Gal-3) is a β-galactoside-binding protein that influences various cell functions, including cell adhesion. We focused on the role of Gal-3 as an extracellular ligand mediating cell-matrix adhesion. We used human adipose tissue-derived stem cells and human umbilical vein endothelial cells that are promising for vascular tissue engineering. We found that these cells naturally contained Gal-3 on their surface and inside the cells. Moreover, they were able to associate with exogenous Gal-3 added to the culture medium. This association was reduced with a β-galactoside LacdiNAc (GalNAcβ1,4GlcNAc), a selective ligand of Gal-3, which binds to the carbohydrate recognition domain (CRD) in the Gal-3 molecule. This ligand was also able to detach Gal-3 newly associated with cells but not Gal-3 naturally present on cells. In addition, Gal-3 preadsorbed on plastic surfaces acted as an adhesion ligand for both cell types, and the cell adhesion was resistant to blocking with LacdiNAc. This result suggests that the adhesion was mediated by a binding site different from the CRD. The blocking of integrin adhesion receptors on cells with specific antibodies revealed that the cell adhesion to the preadsorbed Gal-3 was mediated, at least partially, by β1 and αV integrins—namely α5β1, αVβ3, and αVβ1 integrins.

Human adipose tissue-derived mesenchymal stem cells and their extracellular vesicles modulate lipopolysaccharide activated human microglia

Neurodegenerative diseases (NDs), such as Alzheimer’s disease (AD), are driven by neuroinflammation triggered by activated microglial cells; hence, the phenotypic regulation of these cells is an appealing target for intervention. Human adipose tissue-derived mesenchymal stem cells (hAD-MSCs) may be a potential therapeutic candidate to treat NDs given their immunomodulatory properties. Evidence suggests that the mechanism of action of hAD-MSCs is through their secretome, which includes secreted factors such as cytokines, chemokines, or growth factors as well as extracellular vesicles (EVs). Recently, EVs have emerged as important mediators in cell communication given, they can transfer proteins, lipids, and RNA species (i.e., miRNA, mRNA, and tRNAs) to modulate recipient cells. However, the therapeutic potential of hAD-MSCs and their secreted EVs has not been fully elucidated with respect to human microglia. In this study, we determined the therapeutic potential of different hAD-MSCs doses (200,000, 100,000, and 50,000 cells) or their secreted EVs (50, 20, or 10 µg/ml), on human microglial cells (HMC3) that were activated by lipopolysaccharides (LPS). Upregulation of inducible nitric oxide synthase (iNOS), an activation marker of HMC3 cells, was prevented when they were cocultured with hAD-MSCs and EVs. Moreover, hAD-MSCs inhibited the secretion of proinflammatory factors, such as IL-6, IL-8, and MCP-1, while their secreted EVs promoted the expression of anti-inflammatory mediators such as IL-10 or TIMP-1 in activated microglia. The present data therefore support a role for hAD-MSCs and their secreted EVs, as potential therapeutic candidates for the treatment of NDs.

An RNAi Screening of Clinically Relevant Transcription Factors Regulating Human Adipogenesis and Adipocyte Metabolism.

ContextHealthy hyperplasic (many but smaller fat cells) white adipose tissue (WAT) expansion is mediated by recruitment, proliferation and/or differentiation of new fat cells. This process (adipogenesis) is controlled by transcriptional programs that have been mostly identified in rodents.ObjectiveA systemic investigation of adipogenic human transcription factors (TFs) that are relevant for metabolic conditions has not been revealed previously.MethodsTFs regulated in WAT by obesity, adipose morphology, cancer cachexia, and insulin resistance were selected from microarrays. Their role in differentiation of human adipose tissue-derived stem cells (hASC) was investigated by RNA interference (RNAi) screen. Lipid accumulation, cell number, and lipolysis were measured for all screened factors (148 TFs). RNA (RNAseq), protein (Western blot) expression, insulin, and catecholamine responsiveness were examined in hASC following siRNA treatment of selected target TFs.ResultsAnalysis of TFs regulated by metabolic conditions in human WAT revealed that many of them belong to adipogenesis-regulating pathways. The RNAi screen identified 39 genes that affected fat cell differentiation in vitro, where 11 genes were novel. Of the latter JARID2 stood out as being necessary for formation of healthy fat cell metabolic phenotype by regulating expression of multiple fat cell phenotype-specific genes.ConclusionThis comprehensive RNAi screening in hASC suggests that a large proportion of WAT TFs that are impacted by metabolic conditions might be important for hyperplastic adipose tissue expansion. The screen also identified JARID2 as a novel TF essential for the development of functional adipocytes.

Identification of Angiogenic Cargo in Extracellular Vesicles Secreted from Human Adipose Tissue-Derived Stem Cells and Induction of Angiogenesis In Vitro and In Vivo.

Angiogenesis is defined as the generation of new blood vessels or the sprouting of endothelial cells from a pre-existing vascular network. Angiogenesis occurs during the growth and development of an organism, the response of organs or tissues to injury, and during cancer development and progression. The majority of studies on stem-cell-derived extracellular vesicles (EVs) have used cell lines, and have primarily focused on well-known solitary proteins. Here, we isolated stem cells from human adipose tissue (ADSCs), and we isolated EVs from them (ADSC-EVs). The ADSC-EVs were characterised and 20 angiogenic proteins were analysed using an angiogenic antibody array. Furthermore, we analysed the ability of ADSC-EVs to induce angiogenesis in vitro and in vivo. ADSC-EVs were positive for CD81 and negative for GM130, calnexin, and cytochrome-C. ADSC-EVs showed typical EV spherical morphology and were ~200 nm in size. ADSC-EVs were found to contain angiogenic proteins as cargo, among which interleukin 8 (IL-8) was the most abundant, followed by chemokine (C-C motif) ligand 2 (CCL2), a tissue inhibitor of metalloproteinases 1 (TIMP-1), TIMP-2, and vascular endothelial growth factor-D (VEGF-D). ADSC-EVs treatment increased the proliferation, migration, total vessel length, total number of junctions, and junction density of endothelial cells in vitro. The results of an in vivo Matrigel plug assay revealed that ADSC-EVs induced more blood vessels in the Matrigel compared with the control. These results demonstrate that ADSC-EVs contain angiogenic proteins as cargo and promote angiogenesis in vitro and in vivo. Therefore, ADSC-EVs have potential for therapeutic use in ischaemia.

Synergic effects of extremely low-frequency electromagnetic field and betaine on in vitro osteogenic differentiation of human adipose tissue-derived mesenchymal stem cells.

Human adipose tissue-derived mesenchymal stem cells (hADSCs) due to easy extraction, relative abundance, in vitro expansion and differentiation potential, frozen storage capability, and ability to secrete cytokines, compared to other stem cells, are appropriate candidate in regenerative medicine. Extremely low-frequency electromagnetic fields (ELF-EMF) and betaine are two safe factors in bone lesions repair. This study was designed to assess the osteogenic differentiation potential of these factors on hADSCs. The samples were collected from women undergoing liposuction after obtaining written consent. The hADSCs were extracted and treated with osteogenesis differentiation medium (OD) as the positive control, with OD and betaine (BET group), with OD and EMF (EMF group), and with OD and betaine and EMF (BET+EMF group) for 21 d; the negative control consisted of cells without treatment. Betaine 10 mM and EMF with 50-Hz frequency, 1-mT intensity (8 h daily), and in the form of sinus wave were used. Osteogenic differentiation was evaluated by Alizarin Red staining, alkaline phosphatase activity, calcium deposition, and real-time PCR. A significant increase in calcium deposition in the BET+EMF group was observed compared to the other groups. The activity of alkaline phosphatase in the positive control and BET groups was increased significantly compared to EMF and BET + EMF groups and a significant increase of this enzyme activity in the BET + EMF compared to EMF group was observed. The expression of RUNX2 and OCN genes in the EMF-treated groups were significantly reduced compared to the non-EMF-treated groups, and BET+EMF showed a significant increase of RUNX2 gene expression as compared the EMF group. The ELF-EMF leads to a decrease in the osteogenic differentiation and the expression RUNX2 and OCN genes in hADSCs. But osteogenic differentiation and RUNX2 gene expression were increased post-induction by betaine. The synergic effect of betaine and EMF on the osteogenic differentiation and related genes expression of hADSCs was higher than EMF.

Understanding of how the properties of medical grade lactide based copolymer scaffolds influence adipose tissue regeneration: Sterilization and a systematic in vitro assessment

Aliphatic polyesters are the synthetic polymers most commonly used in the development of resorbable medical implants/devices. Various three-dimensional (3D) scaffolds have been fabricated from these polymers and used in adipose tissue engineering. However, their systematic evaluation altogether lacks, which makes it difficult to select a suitable degradable polymer to design 3D resorbable implants and/or devices able to effectively mimic the properties of adipose tissue. Additionally, the impact of sterilization methods on the medical devices, if any, must be taken into account. We evaluate and compare five different medical-grade resorbable polyesters with l-lactide content ranging from 50 to 100 mol% and exhibiting different physiochemical properties depending on the comonomer (d-lactide, ε-caprolactone, glycolide, and trimethylene carbonate). The salt-leaching technique was used to prepare 3D microporous scaffolds. A comprehensive assessment of physical, chemical, and mechanical properties of the scaffolds was carried out in PBS at 37 °C. The cell-material interactions and the ability of the scaffolds to promote adipogenesis of human adipose tissue-derived stem cells were assessed in vitro. The diverse physical and mechanical properties of the scaffolds, due to the different composition of the copolymers, influenced human adipose tissue-derived stem cells proliferation and differentiation. Scaffolds made from polymers which were above their glass transition temperature and with low degree of crystallinity showed better proliferation and adipogenic differentiation of stem cells. The effect of sterilization techniques (electron beam and ethylene oxide) on the polymer properties was also evaluated. Results showed that scaffolds sterilized with the ethylene oxide method better retained their physical and chemical properties. Overall, the presented research provides (i) a detailed understanding to select a degradable polymer that has relevant properties to augment adipose tissue regeneration and can be further used to fabricate medical devices/implants; (ii) directions to prefer a sterilization method that does not change polymer properties.

Isolation of adipose tissue-derived stem cells by direct membrane migration and expansion for clinical application.

Mesenchymal stem cells (MSCs) have recently made significant progression in multiple clinical trials targeting several clinical disorders and in the modulation of immune responses. In the present study, we isolated human adipose tissue-derived stem cells (ADSCs) by direct membrane migration method without using enzymatic digestion via collagenase, and tried to extract adequate number of cells for clinical application. Hydroxyapatite-treated nonwoven fabric membrane made up of synthetic macromolecular fiber materials, polyethylene and polyester terephthalene was used. Expansion culture of ADSCs having plastic flask adherent characteristic in serum-free condition was successfully established, and adequate number of cells were obtained for clinical application. They were found to be positive for CD44, CD73, CD90 and CD105 and negative for CD11b, CD34, CD45, CD80 and HLA-DR. The resulting immunological marker profile satisfied the immunophenotype of previously reported MSCs. Also, microscopic findings demonstrated trilineage differentiation into adipogenic, osteogenic and chondrogenic cells as the characteristics of MSCs. The isolation by nonwoven fabric membrane and expanded cells under serum-free condition satisfied the criteria of MSCs, as proposed by the International Society for Cellular Therapy. Our direct membrane migration method without enzyme digestion is useful as ADSCs can be obtained from small pieces of adipose tissue and expanded under serum-free culture condition. This method was considered to be feasible for clinical application.

The angiogenic potential of CD271+ human adipose tissue-derived mesenchymal stem cells

BackgroundAutologous fat grafting is often a crucial aspect of reconstructive and aesthetic surgeries, yet poor graft retention is a major issue with this technique. Enriching fat grafts with adipose tissue-derived mesenchymal stem cells (AD-MSCs) improves graft survival—however, AD-MSCs represent a heterogeneous population. Selection of subpopulations of AD-MSCs would allow the targeting of specific AD-MSCs that may benefit fat graft survival more than the general AD-MSC population.MethodsHuman AD-MSCs were selected for the surface marker CD271 using magnetic-activated cell sorting and compared to the CD271 negative phenotype. These subpopulations were analysed for gene expression using Real-Time qPCR and RNA sequencing; surface marker characteristics using immunostaining; ability to form tubules when cultured with endothelial cells; and gene and protein expression of key angiogenic mediators when cultured with ex-vivo adipose tissue.ResultsHuman AD-MSCs with the surface marker CD271 express angiogenic genes at higher levels, and inflammatory genes at lower levels, than the CD271− AD-MSC population. A greater proportion of CD271+ AD-MSCs also possess the typical complement of stem cell surface markers and are more likely to promote effective neoangiogenesis, compared to CD271− AD-MSCs.ConclusionEnriching grafts with the CD271+ AD-MSC subpopulation holds potential for the improvement of reconstructive and aesthetic surgeries involving adipose tissue.

Co-transfection with BMP2 and FGF2 via chitosan nanoparticles potentiates osteogenesis in human adipose-derived stromal cells in vitro.

ObjectiveTo investigate if co-transfection of human bone morphogenetic protein 2 (BMP-2, BMP2) and human fibroblast growth factor 2 (FGF2, FGF2) via chitosan nanoparticles promotes osteogenesis in human adipose tissue-derived stem cells (ADSCs) in vitro.Materials and MethodsRecombinant BMP2 and/or FGF2 expression vectors were constructed and packaged into chitosan nanoparticles. The chitosan nanoparticles were characterized by atomic force microscopy. Gene and protein expression levels of BMP-2 and FGF2 in ADSCs in vitro were evaluated by real-time polymerase chain reaction (PCR), western blot, and enzyme-linked immunosorbent assay. Osteocalcin (OCN) and bone sialoprotein (BSP) gene expression were also evaluated by real-time PCR to assess osteogenesis.ResultsThe prepared chitosan nanoparticles were spherical with a relatively homogenous size distribution. The BMP2 and FGF2 vectors were successfully transfected into ADSCs. BMP-2 and FGF2 mRNA and protein levels were significantly up-regulated in the co-transfection group compared with the control group. OCN and BSP mRNA levels were also significantly increased in the co-transfection group compared with cells transfected with BMP2 or FGF2 alone, suggesting that co-transfection significantly enhanced osteogenesis.ConclusionsCo-transfection of human ADSCs with BMP2/FGF2 via chitosan nanoparticles efficiently promotes the osteogenic properties of ADSCs in vitro.

Comparative study of BMP-2 gene delivery to Human adipose tissue-derived mesenchymal stem cells with Turbofect and Polyethylenimine

Genetic modification of mesenchymal stem cells (MSCs) with plasmid encoding the bone morphogenetic protein 2 gene (BMP2) is a crucial task in the development of gene therapy and tissue engineering technologies for bone regeneration. The low transfection efficiency of MSC cultures is a limitation for practical applications and makes it necessary to search for optimal protocols that ensure efficient gene delivery while maintaining sufficient cell viability. Comparison of two transfecting reagents (TurboFect and Polyethylenimine) shows that TurboFect is the most effective for MSCs transfection. A higher level of target gene BMP2 expression and osteogenic differentiation can be achieved using the TaqRFP-N-BMP2 plasmid compared with pcDNA3-BMP2.

Neural-Induced Human Adipose Tissue-Derived Stem Cells Conditioned Medium Ameliorates Rotenone-Induced Toxicity in SH-SY5Y Cells.

Parkinson’s disease (PD) is an age-related neurodegenerative disease (NDD) characterized by the degenerative loss of dopaminergic neurons in the substantia nigra along with aggregation of α-synuclein (α-syn). Neurogenic differentiation of human adipose-derived stem cells (NI-hADSCs) by supplementary factors for 14 days activates different biological signaling pathways. In this study, we evaluated the therapeutic role of NI-hADSC-conditioned medium (NI-hADSC-CM) in rotenone (ROT)-induced toxicity in SH-SY5Y cells. Increasing concentrations of ROT led to decreased cell survival at 24 and 48 h in a dose- and time-dependent manner. Treatment of NI-hADSC-CM (50% dilution in DMEM) against ROT (0.5 μM) significantly increased the cell survival. ROT toxicity decreased the expression of tyrosine hydroxylase (TH). Western blot analysis of the Triton X-100-soluble fraction revealed that ROT significantly decreased the oligomeric, dimeric, and monomeric phosphorylated Serine129 (p-S129) α-syn, as well as the total monomeric α-syn expression levels. ROT toxicity increased the oligomeric, but decreased the dimeric and monomeric p-S129 α-syn expression levels. Total α-syn expression (in all forms) was increased in the Triton X-100-insoluble fraction, compared to the control. NI-hADSC-CM treatment enhanced the TH expression, stabilized α-syn monomers, reduced the levels of toxic insoluble p-S129 α-syn, improved the expression of neuronal functional proteins, regulated the Bax/Bcl-2 ratio, and upregulated the expression of pro-caspases, along with PARP-1 inactivation. Moreover, hADSC-CM treatment decreased the cell numbers and have no effect against ROT toxicity on SH-SY5Y cells. The therapeutic effects of NI-hADSC-CM was higher than the beneficial effects of hADSC-CM on cellular signaling. From these results, we conclude that NI-hADSC-CM exerts neuroregenerative effects on ROT-induced PD-like impairments in SH-SY5Y cells.

Accelerated in vitro recellularization of decellularized porcine pericardium for cardiovascular grafts

An ideal decellularized allogenic or xenogeneic cardiovascular graft should be capable of preventing thrombus formation after implantation. The antithrombogenicity of the graft is ensured by a confluent endothelial cell layer formed on its surface. Later repopulation and remodeling of the scaffold by the patient’s cells should result in the formation of living autologous tissue. In the work presented here, decellularized porcine pericardium scaffolds were modified by growing a fibrin mesh on the surface and inside the scaffolds, and by attaching heparin and human vascular endothelial growth factor (VEGF) to this mesh. Then the scaffolds were seeded with human adipose tissue-derived stem cells (ASCs). While the ASCs grew only on the surface of the decellularized pericardium, the fibrin-modified scaffolds were entirely repopulated in 28 d, and the scaffolds modified with fibrin, heparin and VEGF were already repopulated within 6 d. Label free mass spectrometry revealed fibronectin, collagens, and other extracellular matrix proteins produced by ASCs during recellularization. Thin layers of human umbilical endothelial cells were formed within 4 d after the cells were seeded on the surfaces of the scaffold, which had previously been seeded with ASCs. The results indicate that an artificial tissue prepared by in vitro recellularization and remodeling of decellularized non-autologous pericardium with autologous ASCs seems to be a promising candidate for cardiovascular grafts capable of accelerating in situ endothelialization. ASCs resemble the valve interstitial cells present in heart valves. An advantage of this approach is that ASCs can easily be collected from the patient by liposuction.

Human Chondrocytes from Human Adipose Tissue-Derived Mesenchymal Stem Cells Seeded on a Dermal-Derived Collagen Matrix Sheet: Our Preliminary Results for a Ready to Go Biotechnological Cartilage Graft in Clinical Practice.

Background The articular cartilage is unique in that it contains only a single type of cell and shows poor ability for spontaneous healing. Cartilage tissue engineering which uses mesenchymal stem cells (MSCs) and adipose tissue-derived mesenchymal stem cells (AT-MSCs) is considered an attractive treatment for cartilage lesions and osteoarthritis. The establishment of cartilage regenerative medicine is an important clinical issue, but the search for cell sources able to restore cartilage integrity proves to be challenging. The aim of this study was to create cartilage grafts from the combination of AT-MSCs and collagen substrates. Methods Mesenchymal stem cells were obtained from human donors' adipose tissue, and collagen scaffold, obtained from human skin and cleaned from blood vessels, adipose tissues, and debris, which only preserve dermis and epidermis, were seeded and cultured on collagen substrates and differentiated to chondrocytes. The obtained chondrocyte extracellular matrix of cartilage was then evaluated for the expression of chondrocyte-/cartilage-specific markers, the Cartilage Oligomeric Matrix Protein (COMP), collagen X, alpha-1 polypeptide (COL10A1), and the Collagen II, Human Tagged ORF Clone (COL2A1) by using the reverse transcription polymerase chain reaction (RT-PCR). Results Our findings have shown that the dermal collagen may exert important effects on the quality of in vitro expanded chondrocytes, leading in this way that the influence of collagen skin matrix helps to produce highly active and functional chondrocytes for long-term cartilage tissue regeneration. Conclusion This research opens up the possibility of generating cartilage grafts with the precise purpose of improving the existing limitation in current clinical procedures.

Growth Factors VEGF-A165 and FGF-2 as Multifunctional Biomolecules Governing Cell Adhesion and Proliferation.

Vascular endothelial growth factor-A165 (VEGF-A165) and fibroblast growth factor-2 (FGF-2) are currently used for the functionalization of biomaterials designed for tissue engineering. We have developed a new simple method for heterologous expression and purification of VEGF-A165 and FGF-2 in the yeast expression system of Pichia pastoris. The biological activity of the growth factors was assessed in cultures of human and porcine adipose tissue-derived stem cells (ADSCs) and human umbilical vein endothelial cells (HUVECs). When added into the culture medium, VEGF-A165 stimulated proliferation only in HUVECs, while FGF-2 stimulated the proliferation of both cell types. A similar effect was achieved when the growth factors were pre-adsorbed to polystyrene wells. The effect of our recombinant growth factors was slightly lower than that of commercially available factors, which was attributed to the presence of some impurities. The stimulatory effect of the VEGF-A165 on cell adhesion was rather weak, especially in ADSCs. FGF-2 was a potent stimulator of the adhesion of ADSCs but had no to negative effect on the adhesion of HUVECs. In sum, FGF-2 and VEGF-A165 have diverse effects on the behavior of different cell types, which maybe utilized in tissue engineering.

Cytochalasin B-Induced Membrane Vesicles from Human Mesenchymal Stem Cells Overexpressing IL2 Are Able to Stimulate CD8+ T-Killers to Kill Human Triple Negative Breast Cancer Cells.

Simple SummaryAlmost all human cells release extracellular vesicles participating in intercellular communication. Extracellular vesicles are rounded structures surrounded by the cytoplasmic membrane, which embody cytoplasmic contents of the parental cells, which makes extracellular vesicles a promising therapeutic tool for cell-free cancer therapy. In this study, human mesenchymal stem cells were genetically modified to overexpress human interleukin-2 (IL2), a cytokine which regulates the proliferation and activation of immune cells. Membrane vesicle release from native and genetically modified stem cells was induced by cytochalasin B treatment to increase the yield of membrane vesicles. To evaluate the immunomodulating properties of isolated membrane vesicles, immune cells were isolated from human peripheral blood and co-cultured with membrane vesicles from native or IL2 overexpressing stem cells. To analyze the anti-tumor activity of immune cells after interaction with IL2-enriched membrane vesicles, immune cells were co-cultured with triple negative breast cancer cells. As a result, IL2-enriched membrane vesicles were able to activate and stimulate the proliferation of immune cells, which in turn were able to induce apoptosis in breast cancer cells. Therefore, the production of IL2-enriched membrane vesicles represents a unique opportunity to meet the potential of extracellular vesicles to be used in clinical applications for cancer therapy.Interleukin 2 (IL2) was one of the first cytokines used for cancer treatment due to its ability to stimulate anti-cancer immunity. However, recombinant IL2-based therapy is associated with high systemic toxicity and activation of regulatory T-cells, which are associated with the pro-tumor immune response. One of the current trends for the delivery of anticancer agents is the use of extracellular vesicles (EVs), which can carry and transfer biologically active cargos into cells. The use of EVs can increase the efficacy of IL2-based anti-tumor therapy whilst reducing systemic toxicity. In this study, human adipose tissue-derived mesenchymal stem cells (hADSCs) were transduced with lentivirus encoding IL2 (hADSCs-IL2). Membrane vesicles were isolated from hADSCs-IL2 using cytochalasin B (CIMVs-IL2). The effect of hADSCs-IL2 and CIMVs-IL2 on the activation and proliferation of human peripheral blood mononuclear cells (PBMCs) as well as the cytotoxicity of activated PBMCs against human triple negative cancer MDA-MB-231 and MDA-MB-436 cells were evaluated. The effect of CIMVs-IL2 on murine PBMCs was also evaluated in vivo. CIMVs-IL2 failed to suppress the proliferation of human PBMCs as opposed to hADSCs-IL2. However, CIMVs-IL2 were able to activate human CD8+ T-killers, which in turn, killed MDA-MB-231 cells more effectively than hADSCs-IL2-activated CD8+ T-killers. This immunomodulating effect of CIMVs-IL2 appears specific to human CD8+ T-killer cells, as the same effect was not observed on murine CD8+ T-cells. In conclusion, the use of CIMVs-IL2 has the potential to provide a more effective anti-cancer therapy. This compelling evidence supports further studies to evaluate CIMVs-IL2 effectiveness, using cancer mouse models with a reconstituted human immune system.

Development of pH-Responsive Polymer Coating as an Alternative to Enzyme-Based Stem Cell Dissociation for Cell Therapy.

Cell therapy usually accompanies cell detachment as an essential process in cell culture and cell collection for transplantation. However, conventional methods based on enzymatic cell detachment can cause cellular damage including cell death and senescence during the routine cell detaching step due to an inappropriate handing. The aim of the current study is to apply the pH-responsive degradation property of poly (amino ester) to the surface of a cell culture dish to provide a simple and easy alternative method for cell detachment that can substitute the conventional enzyme treatment. In this study, poly (amino ester) was modified (cell detachable polymer, CDP) to show appropriate pH-responsive degradation under mild acidic conditions (0.05% (w/v) CDP, pH 6.0) to detach stem cells (human adipose tissue-derived stem cells (hADSCs)) perfectly within a short period (less than 10 min). Compared to conventional enzymatic cell detachment, hADSCs cultured on and detached from a CDP-coated cell culture dish showed similar cellular properties. We further performed in vivo experiments on a mouse hindlimb ischemia model (1.0 × 106 cells per limb). The in vivo results indicated that hADSCs retrieved from normal cell culture dishes and CDP-coated cell culture dishes showed analogous therapeutic angiogenesis. In conclusion, CDP could be applied to a pH-responsive cell detachment system as a simple and easy nonenzymatic method for stem cell culture and various cell therapies.