Enhanced cellular engraftment of adipose-derived mesenchymal stem cell spheroids by using nanosheets as scaffolds.
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ABSTRACT:
SUBMITTER: Nagano H
PROVIDER: S-EPMC8280158 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Adipose-derived mesenchymal stromal cells (Ad-MSCs) are a promising tool for articular cartilage repair and regeneration. However, the terminal hypertrophic differentiation of Ad-MSC-derived cartilage is a critical barrier during hyaline cartilage regeneration. In this study, we investigated the role of matrilin-3 in preventing Ad-MSC-derived chondrocyte hypertrophy in vitro and in an osteoarthritis (OA) destabilization of the medial meniscus (DMM) model. Methacrylated hyaluron (MAHA) (1%) was used to encapsulate and make scaffolds containing Ad-MSCs and matrilin-3. Subsequently, the encapsulated cells in the scaffolds were differentiated in chondrogenic medium (TGF-β, 1-14 days) and thyroid hormone hypertrophic medium (T3, 15-28 days). The presence of matrilin-3 with Ad-MSCs in the MAHA scaffold significantly increased the chondrogenic marker and decreased the hypertrophy marker mRNA and protein expression. Furthermore, matrilin-3 significantly modified the expression of TGF-β2, BMP-2, and BMP-4. Next, we prepared the OA model and transplanted Ad-MSCs primed with matrilin-3, either as a single-cell suspension or in spheroid form. Safranin-O staining and the OA score suggested that the regenerated cartilage morphology in the matrilin-3-primed Ad-MSC spheroids was similar to the positive control. Furthermore, matrilin-3-primed Ad-MSC spheroids prevented subchondral bone sclerosis in the mouse model. Here, we show that matrilin-3 plays a major role in modulating Ad-MSCs' therapeutic effect on cartilage regeneration and hypertrophy suppression.
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Project description:Emphysema, a pathologic component of the chronic obstructive pulmonary disease, causes irreversible destruction of lung. Many researchers have reported that mesenchymal stem cells can regenerate lung tissue after emphysema. We evaluated if spheroid human adipose-derived mesenchymal stem cells (ASCs) showed greater regenerative effects than dissociated ASCs in mice with elastase-induced emphysema. ASCs were administered via an intrapleural route. Mice injected with spheroid ASCs showed improved regeneration of lung tissues, increased expression of growth factors such as fibroblast growth factor-2 (FGF2) and hepatocyte growth factor (HGF), and a reduction in proteases with an induction of protease inhibitors when compared with mice injected with dissociated ASCs. Our findings indicate that spheroid ASCs show better regeneration of lung tissues than dissociated ACSs in mice with elastase-induced emphysema. [BMB Reports 2017; 50(2): 79-84].
| S-EPMC5342870 | biostudies-literature
Project description:The mechanical properties of adipose-derived stem cell (ASC) clones correlate with their ability to produce tissue-specific metabolites, a finding that has dramatic implications for cell-based regenerative therapies. Autologous ASCs are an attractive cell source due to their immunogenicity and multipotent characteristics. However, for practical applications ASCs must first be purified from other cell types, a critical step which has proven difficult using surface-marker approaches. Alternative enrichment strategies identifying broad categories of tissue-specific cells are necessary for translational applications. One possibility developed in our lab uses single-cell mechanical properties as predictive biomarkers of ASC clonal differentiation capability. Elastic and viscoelastic properties of undifferentiated ASCs were tested via atomic force microscopy and correlated with lineage-specific metabolite production. Cell sorting simulations based on these "mechanical biomarkers" indicated they were predictive of differentiation capability and could be used to enrich for tissue-specific cells, which if implemented could dramatically improve the quality of regenerated tissues.
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Project description:The paracrine function of mesenchymal stem cells (MSCs) during transplantation has been recently studied due to its poor differentiation ratio. Dimethyloxalylglycine (DMOG) has been used to promote angiogenesis in experimental animal models, however, comparable approaches for canine MSCs are not sufficient. In the present study, we assessed whether DMOG improves angiogenesis in canine adipose tissue-derived mesenchymal stem cells (cAT-MSCs). cAT-MSCs were treated with DMOG and their effect on angiogenesis was investigated by cell proliferation assay, western blotting, and tube formation assay. Dimethyloxalylglycine preconditioning enhanced the expression of vascular endothelial growth factor (VEGF) among pro-angiogenic factors in cAT-MSCs via hypoxia-inducible factor-1α stabilization. Dimethyloxalylglycine primed-cAT-MSC-conditioned media increased angiogenesis in human umbilical vein endothelial cells. These results suggest that DMOG conditioning of cAT-MSCs augmented the secretion of VEGF, which acted as a prominent pro-angiogenic factor during angiogenesis. DMOG-primed cAT-MSCs may have the potential to induce beneficial effects in ischemic diseases in clinical trials.
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Project description:IntroductionDifferentiation of hepatocytes and culture methods have been investigated in dogs as a tool to establish liver transplant and drug metabolism examination systems. However, mass culture techniques for canine hepatocytes (cHep) have not been investigated, and it is necessary to construct a suitable culture system. Recently, a protocol called Bud production has attracted attention, and a mixed culture of human and mouse hepatocytes, stem cells, and artificial blood vessels significantly improved the size and formation ratio of spheroids. The purpose of this study was to investigate and improve the in vitro culture of cHep by mixing canine adipose-derived mesenchymal stem cells (cASCs) and human umbilical vein endothelial cells (HUVECs).MethodsSpheroid formation ratio and histological examination were evaluated among four culture methods, including cHep alone, two-mix (cHep + cASCs and cHep + HUVEC), and three-mix (cHep + HUVEC + cASCs), on days 0, 4, and 7. Expression levels of liver-related genes (ALB, AFP, α1-AT, CDH1, CYP2E1, CYP3A12, and TAT) were evaluated by quantitative real-time polymerase chain reaction (RT-PCR). Protein expression of albumin, vimentin, and von Willebrand Factor (vWF) was investigated to confirm the location of the hepatocytes.ResultsThe ratio of spheroid formation was 60.2% in the three-mix culture and was significantly improved compared with cHep alone (5.9%) and two-mix; cHep + cASCs (36.2%) and cHep + HUVEC (26.4%) (P < 0.001). Histological evaluation revealed that the three-mix spheroids formed large canine hepatocyte spheroids (LcHS), and hepatocytes were distributed in the center of the spheroids. Quantitative gene expression analysis of LcHS showed that liver-related genes expression were maintained the same levels with that of a culture of cHep alone from days 4-7.ConclusionThese results revealed that the three-mix culture method using cHep, HUVECs, and cASCs was capable of promoting LcHS without impairing liver function in cHep, suggesting that LcHS could be used for the application of high-volume culture techniques in dogs.
| S-EPMC8728312 | biostudies-literature
Project description:BackgroundChronic low back pain is a prevalent disability, often caused by intervertebral disc (IVD) degeneration. Mesenchymal stem cell (MSC) therapy could be a safe and feasible option for repairing the degenerated disc. However, for successful translation to the clinic, various challenges need to be overcome including unwanted adverse effects due to acidic pH, hypoxia, and limited nutrition. Matrilin-3 is an essential extracellular matrix (ECM) component during cartilage development and ossification and exerts chondrocyte protective effects.MethodsThis study evaluated the effects of matrilin-3-primed adipose-derived MSCs (Ad-MSCs) on the repair of the degenerated disc in vitro and in vivo. We determined the optimal priming concentration and duration and developed an optimal protocol for Ad-MSC spheroid generation.ResultsPriming with 10 ng/ml matrilin-3 for 5 days resulted in the highest mRNA expression of type 2 collagen and aggrecan in vitro. Furthermore, Ad-MSC spheroids with a density of 250 cells/microwell showed the increased secretion of favorable growth factors such as transforming growth factor beta (TGF-β1), TGF-β2, interleukin-10 (IL-10), granulocyte colony-stimulating factor (G-CSF), and matrix metalloproteinase 1 (MMP1) and decreased secretion of hypertrophic ECM components. In addition, matrilin-3-primed Ad-MSC spheroid implantation was associated with optimal repair in a rabbit model.ConclusionOur results suggest that priming MSCs with matrilin-3 and spheroid formation could be an effective strategy to overcome the challenges associated with the use of MSCs for the treatment of IVD degeneration.
| S-EPMC7444036 | biostudies-literature
Project description:BackgroundAdipose-derived stem cell (ASC) has been considered as a desirable source for cell therapy. In contrast to combining scaffold materials with cells, ASCs can be fabricated into scaffold-free three-dimensional (3D) constructs to promote regeneration at tissue level. However, previous reports have found decreased expression of vascular endothelial growth factor (VEGF) in ASC sheets. In this study, we aimed to integrate ASC spheroids into ASC sheets to enhance the angiogenic capability of cell sheets.MethodsASCs were seeded in agarose microwells to generate uniform cell spheroids with adjustable size, while extracellular matrix deposition could be stimulated by ascorbic acid 2-phosphate to form ASC sheets. RNA sequencing was performed to identify the transcriptomic profiles of ASC spheroids and sheets relative to monolayer ASCs. By transferring ASC spheroids onto ASC sheets, the spheroid sheet composites could be successfully fabricated after a short-term co-culture, and their angiogenic potential was evaluated in vitro and in ovo.ResultsRNA sequencing analysis revealed that upregulation of angiogenesis-related genes was found only in ASC spheroids. The stimulating effect of spheroid formation on ASCs toward endothelial lineage was demonstrated by enhanced CD31 expression, which maintained after ASC spheroids were seeded on cell sheets. Relative to ASC sheets, enhanced expression of VEGF and hepatocyte growth factor was also noted in ASC spheroid sheets, and conditioned medium of ASC spheroid sheets significantly enhanced tube formation of endothelial cells in vitro. Moreover, chick embryo chorioallantoic membrane assay showed a significantly higher capillary density with more branch points after applying ASC spheroid sheets, and immunohistochemistry also revealed a significantly higher ratio of CD31-positive area.ConclusionIn the spheroid sheet construct, ASC spheroids can augment the pro-angiogenesis capability of ASC sheets without the use of exogenous biomaterial or genetic manipulation. The strategy of this composite system holds promise as an advance in 3D culture technique of ASCs for future application in angiogenesis and regeneration therapies.
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Project description:BackgroundAdipose-derived mesenchymal stem cells (ADSCs) have been extensively explored as a promising therapeutic agent due to their differentiation, proliferation and migration abilities. The epigenetic mechanisms that regulate the fate of mesenchymal stem cells (MSCs) have been described in detail. However, the epigenetic modulation of ADSCs proliferation and migration is poorly understood.MethodsThe present study examined histone demethylases roles and expression by RT-PCR, as well as through siRNA screening and ChIP-qPCR assay. Cellular proliferation and migration assays were employed in shRNA-mediated JMJD6 knockdown and control ADSCs. PDE1C inhibition studies were conducted to confirm its role in JMJD6-mediated epigenetic regulation of ADSCs.ResultsThe data demonstrate that the histone demethylase JMJD6 plays a critical role in regulating the proliferation and migration of ADSCs by removing H4R3me2a at the promoter regions of PDEC1 and suppressing PDEC1 expression. Importantly, the depletion of JMJD6 in ADSCs significantly increased cellular proliferation and motility, which was associated with increases in PDE1C expression and decreases in the levels of both cAMP and cGMP. The increase in proliferation and migration was reversed by treatment with a PDE1C inhibitor, suggesting that JMJD6 attenuates the proliferation and migration of ADSCs as an epigenetic regulator and PDE1C partially contributes to the JMJD6-mediated regulation.ConclusionsTaken together, our results indicate for the first time that JMJD6 plays an important role in the regulation of ADSCs proliferation and migration through the modulation of PDE1C expression.
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Project description:BackgroundPersistent epithelial defects (PED), associated with limbal stem cell deficiency (LSCD), require ocular surface reconstruction with a stable corneal epithelium (CE). This study investigated CE reformation using human adipose mesenchymal stem cells (ADSC), which derived epithelial progenitors via mesenchymal-epithelial transition (MET).MethodsSTEMPRO human ADSC were cultured with specific inhibitors antagonizing glycogen synthase kinase-3 and transforming growth factor-β signaling, followed by culture under a defined progenitor cell targeted-epithelial differentiation condition to generate epithelial-like cells (MET-Epi), which were characterized for cell viability, mesenchymal, and epithelial phenotypes using immunofluorescence and flow cytometry. Tissue-engineered (TE) MET-Epi cells on fibrin gel were transplanted to corneal surface of the rat LSCD model caused by alkali injury. Epithelial healing, corneal edema, and haze grading, CE formation were assessed by fluorescein staining, slit lamp bio-microscopy, anterior segment optical coherence tomography, and immunohistochemistry.ResultsCD73high/CD90high/CD105high/CD166high/CD14negative/CD31negative human ADSC underwent MET, giving viable epithelial-like progenitors expressing δNp63, CDH1 (E-cadherin), epidermal growth factor receptor, integrin-β4, and cytokeratin (CK)-5, 9. Under defined epithelial differentiation culture, these progenitors generated MET-Epi cells expressing cell junction proteins ZO1 and occludin. When transplanted onto rat corneal surface with LSCD-induced PED, TE-MET-Epi achieved more efficient epithelial healing, suppressed corneal edema, and opacities, when compared to corneas without treatment or transplanted with TE-ADSC. CE markers (CK3, 12, and CDH1) were expressed on TE-MET-Epi-transplanted corneas but not in other control groups.ConclusionHuman ADSC-derived epithelial-like cells, via MET, recovered the CE from PED associated with LSCD. ADSC can be a viable adult stem cell source for potential autologous epithelial cell-based therapy for corneal surface disorders.
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Project description:Hydrothermally carbonized sugarcane bagasse (SCB) has exceptional surface properties. Looking at the huge amount of SCB produced, its biocompatible nature, cheap-cost for carbonization, and its easy functionalization can give impeccable nano-biomaterials for tissue engineering applications. Herein, sugarcane bagasse was converted into hydrochar (SCB-H) by hydrothermal carbonation. The SCB-H produced was further modified with iron oxide (Fe3O4) nanoparticles (denoted as SCB-H@Fe3O4). Facile synthesized nano-bio-composites were characterized by SEM, HR-TEM, XRD, FT-IR, XPS, TGA, and VSM analysis. Bare Fe3O4 nanoparticles (NPs), SCB-H, and SCB-H@Fe3O4 were tested for cytocompatibility and osteoconduction enhancement of human adipose tissue-derived mesenchymal stem cells (hADMSCs). The results confirmed the cytocompatible and nontoxic nature of SCB-H@Fe3O4. SCB-H did not show enhancement in osteoconduction, whilst on the other hand, Fe3O4 NPs exhibited a 0.5-fold increase in the osteoconduction of hADMSCs. However, SCB-H@Fe3O4 demonstrated an excellent enhancement in osteoconduction of a 3-fold increase over the control, and a 2.5-fold increase over the bare Fe3O4 NPs. Correspondingly, the expression patterns assessment of osteoconduction marker genes (ALP, OCN, and RUNX2) confirmed the osteoconductive enhancement by SCB-H@Fe3O4. In the proposed mechanism, the surface of SCB-H@Fe3O4 might provide a unique topology, and anchoring to receptors of hADMSCs leads to accelerated osteogenesis. In conclusion, agriculture waste-derived sustainable materials like "SCB-H@Fe3O44" can be potentially applied in highly valued medicinal applications of stem cell differentiation.
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