Differentiation Of Adipose-derived Mesenchymal Stem Cells Research Articles

Framework nucleic Acid-MicroRNA mediated hepatic differentiation and functional hepatic spheroid development for treating acute liver failure

The specific induction of hepatic differentiation presents a significant challenge in developing alternative liver cell sources and viable strategies for clinical therapy of acute liver failure (ALF). The past decade has witnessed the blossom of microRNAs in regenerative medicine. Herein, microRNA 122-functionalized tetrahedral framework nucleic acid (FNA-miR-122) has emerged as an unprecedented and potential platform for directing the hepatic differentiation of adipose-derived mesenchymal stem cells (ADMSCs), which offers a straightforward and cost-effective method for generating functional hepatocyte-like cells (FNA-miR-122-iHep). Additionally, we have successfully established a liver organoid synthesis strategy by optimizing the co-culture of FNA-miR-122-iHep with endothelial cells (HUVECs), resulting in functional Hep:HUE-liver spheroids. Transcriptome analysis not only uncovered the potential molecular mechanisms through which miR-122 influences hepatic differentiation in ADMSCs, but also clarified that Hep:HUE-liver spheroids could further facilitate hepatocyte maturation and improved tissue-specific functions, which may provide new hints to be used to develop a hepatic organoid platform. Notably, compared to transplanted ADMSCs and Hep-liver spheroid, respectively, both FNA-miR-122-iHep-based single cell therapy and Hep:HUE-liver spheroid-based therapy showed high efficacy in treating ALF in vivo. Collectively, this research establishes a robust system using microRNA to induce ADMSCs into functional hepatocyte-like cells and to generate hepatic organoids in vitro, promising a highly efficient therapeutic approach for ALF.

Photobiomodulation Dose-Response on Adipose-Derived Stem Cell Osteogenesis in 3D Cultures.

Osteoporosis and other degenerative bone diseases pose significant challenges to global healthcare systems due to their prevalence and impact on quality of life. Current treatments often alleviate symptoms without fully restoring damaged bone tissue, highlighting the need for innovative approaches like stem cell therapy. Adipose-derived mesenchymal stem cells (ADMSCs) are particularly promising due to their accessibility, abundant supply, and strong differentiation potential. However, ADMSCs tend to favor adipogenic pathways, necessitating the use of differentiation inducers (DIs), three-dimensional (3D) hydrogel environments, and photobiomodulation (PBM) to achieve targeted osteogenic differentiation. This study investigated the combined effects of osteogenic DIs, a fast-dextran hydrogel matrix, and PBM at specific wavelengths and fluences on the proliferation and differentiation of immortalized ADMSCs into osteoblasts. Near-infrared (NIR) and green (G) light, as well as their combination, were used with fluences of 3 J/cm2, 5 J/cm2, and 7 J/cm2. The results showed statistically significant increases in alkaline phosphatase levels, a marker of osteogenic differentiation, with G light at 7 J/cm2 demonstrating the most substantial impact on ADMSC differentiation. Calcium deposits, visualized by Alizarin red S staining, appeared as early as 24 h post-treatment in PBM groups, suggesting accelerated osteogenic differentiation. ATP luminescence assays indicated increased proliferation in all experimental groups, particularly with NIR and NIR-G light at 3 J/cm2 and 5 J/cm2. MTT viability and LDH membrane permeability assays confirmed enhanced cell viability and stable cell health, respectively. In conclusion, PBM significantly influences the differentiation and proliferation of hydrogel-embedded immortalized ADMSCs into osteoblast-like cells, with G light at 7 J/cm2 being particularly effective. These findings support the combined use of 3D hydrogel matrices and PBM as a promising approach in regenerative medicine, potentially leading to innovative treatments for degenerative bone diseases.

Optimization of differentiation conditions for porcine adipose-derived mesenchymal stem cells and analysis of fatty acids in cultured fat

Cultured fat is an important part of cultured meat, and the ability of adipose-derived mesenchymal stem cells (ADSCs) to differentiate into mature adipose tissue affects the quality of cultured fat. Thus, the primary aim of this study was to screen for combinations of differentiation-inducing factors (DIF) using single-factor experiment and orthogonal experimental design under two-dimensional culture conditions for ADSCs. The results showed that a combination of DIF consisting of 1 μmol/L dexamethasone, 0.1 mmol/L 3-isobutyl-1-methylxanthine, 10 μg/mL insulin, 0.1 mmol/L indomethacin, and 2 μmol/L rosiglitazone was a good choice for the differentiation of ADSCs. An combination of DIF was applied to the preparation of cultured fat with collagen as scaffolds. Forty-eight fatty acids were detected in cultured fat by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Among them, the content of twenty-one fatty acids in cultured fat was significantly higher than that of conventional porcine subcutaneous adipose tissue (P < 0.05), and the content of 14 fatty acids was not significantly different (P > 0.05). The ratio of ω-6 polyunsaturated fatty acids content to ω-3 polyunsaturated fatty acids content was 1.23:1, which meant cultured fat was beneficial for human health. This study provides a method to improve the differentiation ability of ADSCs while also providing a reference for indicating the nutritional value of cultured fat.

Hmga2 knockdown enhances osteogenic differentiation of adipose-derived mesenchymal stem cells and accelerates bone defect healing in mice

To investigate the role of high-mobility group AT-hook 2 (HMGA2) in osteogenic differentiation of adipose-derived mesenchymal stem cells (ADSCs) and the effect of Hmga2 knockdown for promoting bone defect repair. Bioinformatics studies using the GEO database and Rstudio software identified HMGA2 as a key factor in adipogenic-osteogenic differentiation balance of ADSCs. The protein-protein interaction network of HMGA2 in osteogenic differentiation was mapped using String and visualized with Cytoscape to predict the downstream targets of HMGA2. Primary mouse ADSCs (mADSCs) were transfected with Hmga2 siRNA, and the changes in osteogenic differentiation of the cells were evaluated using alkaline phosphatase staining and Alizarin red S staining. The expressions of osteogenic markers Runt-related transcription factor 2 (RUNX2), osteopontin (OPN), and osteocalcein (OCN) in the transfected cells were detected using RT-qPCR and Western blotting. In a mouse model of critical-sized calvarial defects, mADSCs with Hmga2-knockdown were transplanted into the defect, and bone repair was evaluated 6 weeks later using micro-CT scanning and histological staining. GEO database analysis showed that HMGA2 expression was upregulated during adipogenic differentiation of ADSCs. Protein-protein interaction network analysis suggested that the potential HMGA2 targets in osteogenic differentiation of ADSCs included SMAD7, CDH1, CDH2, SNAI1, SMAD9, IGF2BP3, and ALDH1A1. In mADSCs, Hmga2 knockdown significantly upregulated the expressions of RUNX2, OPN, and OCN and increased cellular alkaline phosphatase activity and calcium deposition. In a critical-sized calvarial defect model, transplantation of mADSCs with Hmga2 knockdown significantly promoted new bone formation. HMGA2 is a crucial regulator of osteogenic differentiation in ADSCs, and Hmga2 knockdown significantly promotes osteogenic differentiation of ADSCs and accelerates ADSCs-mediated bone defect repair in mice.

Black phosphorus quantum dot-modified ADSCs as a novel therapeutic for periodontitis bone loss coupling of osteogenesis and osteoimmunomodulation

Alveolar bone defect repair remains a persistent clinical challenge for periodontitis treatment. The use of peripheral functional seed cells is a hot topic in periodontitis. Herein, we explored the cellular behaviors and osteogenic ability of adipose-derived mesenchymal stem cells (ADSCs) treated with black phosphorus quantum dots (BPQDs). Additionally, macrophage polarization, osteogenic effects and angiogenesis were investigated through the paracrine pathway regulated by BPQD-modified ADSCs. Our results demonstrated that BPQDs showed good biocompatibility with ADSCs and BPQD-modified ADSCs could improve the bone repair in vivo inflammatory microenvironment by regulating osteogenesis and osteoimmunomodulation. The BPQDs increased the osteogenic differentiation of ADSCs via the Wnt/β-catenin and BMP2/SMAD5/Runx2 signaling pathway. In addition, BPQD-modified ADSCs promoted the osteogenic effect of BMSCs and facilitated the polarization of macrophages from M1 towards M2 phenotype transformation through the paracrine pathway in the periodontitis microenvironment. This strategy provides a novel idea for treatment of alveolar bone defects for periodontitis in the foreseeable future.

P157 Differentiation of Tonsil-derived mesenchymal stem cells to intestinal stem cells-like cells for cell therapy of inflammatory bowel disease

Abstract Background Tonsil-derived mesenchymal stem cells (TMSCs) were found to exhibit a faster growth rate compared to mesenchymal stem cells derived from other tissues. This is attributed to the relatively younger age of the tonsil tissue donors. Moreover, it has been verified that TMSCs possess the capability to undergo differentiation into endoderm lineages, giving rise to cell types like parathyroid glands and insulin-producing cells. In this study, we explored the possibility of differentiation of TMSCs into intestinal stem cells-like cells (ISC-like cells), a cell type of endoderm lineages for utilizing these cells for therapeutic applications in inflammatory bowel disease (IBD). Methods TMSCs were cultivated in DMEM (10% FBS) and used in the experiment when 90-100% confluency was observed. Adipose-derived mesenchymal stem cells (AMSCs) were used as the control for assessing the differentiation efficacy of TMSCs. The morphology of cells was verified at each differentiation stage, and the expression of endoderm and intestinal stem cell markers was confirmed using real-time PCR (qPCR), western blotting, and immunofluorescence (IF) staining. Results Subjected to 100ng/ml of Activin A for endoderm differentiation and cultured for 2 to 5 days, TMSCs exhibited an upregulation in endoderm markers (SOX17, FOXA2), as confirmed by real-time PCR. Specifically, under the influence of Activin A at 100ng/ml and cultured for 5 days, there was a significant increase in the expression of SOX17 (pre-differentiation vs. endoderm-differentiation, 0.999 vs. 3.610, P=0.0394) and FOXA2 (1.047 vs. 3.912, P=0.0297). Following the endoderm differentiation phase, the medium was changed to include 500ng/ml of FGF4 and 500ng/ml of Wnt3a for a 7-day period. Post-culture, the expression level of Lgr5, an intestinal stem cell (ISC) marker, was significantly increased in TMSCs (1.061 vs. 134.0, P&amp;lt;0.001). Notably, AMSCs displayed a lower expression rate compared to TMSCs (TMSCs vs. AMSCs, 134.0 vs. 9.819, P&amp;lt;0.001). Moreover, the spheroids formation was observed 7 days after the initiation of FGF4 and Wnt3a treatment, a phenomenon absent in AMSCs differentiation. Immunofluorescence staining for differentiation markers further validated these findings: the expression of SOX17, an endoderm marker, and the fluorescence density of LGR5, an ISC marker, were notably higher in TMSCs compared to AMSCs. Protein expression levels also confirmed the upregulation of each marker in TMSCs. Conclusion This study validated the potential of TMSCs to differentiate into ISC-like cells, with a higher efficiency observed compared to AMSCs. The prospect of differentiating into ISC positions TMSCs as promising candidates for use as a tool in IBD.

ITGA7 loss drives the differentiation of adipose-derived mesenchymal stem cells to cancer-associated fibroblasts.

Cancer-associated fibroblasts (CAFs) represent a major cellular component of the tumor (pre-)metastatic niche and play an essential role in omental dissemination of ovarian cancer. The omentum is rich in adipose, and adipose-derived mesenchymal stem cells (ADSCs) have been identified as a source of CAFs. However, the molecular events driving the phenotype shift of ADSCs remain largely unexplored. In this research, we focus on integrins, transmembrane receptors that have been widely involved in cellular plasticity. We found that integrin α7(ITGA7) was the only member of the integrin family that positively correlated with both overall survival and progression-free survival in ovarian cancer through GEPIA2. The immunohistochemistry signal of ITGA7 was apparent in the tumor stroma, and a lower omental ITGA7 level was associated with metastasis. Primary ADSCs were isolated from the omentum of patients with ovarian cancer and identified by cellular morphology, biomarkers, and multilineage differentiation. The conditional medium of ovarian cancer cells induced ITGA7 expression decrease and phenotypic changes in ADSCs. Downregulation of ITGA7 in primary omental ADSCs led to decrease in stemness properties and emerge of characteristic morphology and biomarkers of CAFs. Moreover, the conditioned medium of ADSCs with ITGA7 depletion exhibited enhanced abilities to improve the migration and invasion of ovarian cancer cells in vitro. Overall, these findings indicate that loss of ITGA7 may induce the differentiation of ADSCs to CAFs that contribute to a tumor-supportive niche.

Combining Porous Se@SiO2 Nanocomposites and dECM Enhances the Myogenic Differentiation of Adipose-Derived Stem Cells.

Volumetric Muscle Loss (VML) denotes the traumatic loss of skeletal muscle, a condition that can result in chronic functional impairment and even disability. While the body can naturally repair injured skeletal muscle within a limited scope, patients experiencing local and severe muscle loss due to VML surpass the compensatory capacity of the muscle itself. Currently, clinical treatments for VML are constrained and demonstrate minimal efficacy. Selenium, a recognized antioxidant, plays a crucial role in regulating cell differentiation, anti-inflammatory responses, and various other physiological functions. We engineered a porous Se@SiO2 nanocomposite (SeNPs) with the purpose of releasing selenium continuously and gradually. This nanocomposite was subsequently combined with a decellularized extracellular matrix (dECM) to explore their collaborative protective and stimulatory effects on the myogenic differentiation of adipose-derived mesenchymal stem cells (ADSCs). The influence of dECM and NPs on the myogenic level, reactive oxygen species (ROS) production, and mitochondrial respiratory chain (MRC) activity of ADSCs was evaluated using Western Blot, ELISA, and Immunofluorescence assay. Our findings demonstrate that the concurrent application of SeNPs and dECM effectively mitigates the apoptosis and intracellular ROS levels in ADSCs. Furthermore, the combination of dECM with SeNPs significantly upregulated the expression of key myogenic markers, including MYOD, MYOG, Desmin, and myosin heavy chain in ADSCs. Notably, this combination also led to an increase in both the number of mitochondria and the respiratory chain activity in ADSCs. The concurrent application of SeNPs and dECM effectively diminishes ROS production, boosts mitochondrial function, and stimulates the myogenic differentiation of ADSCs. This study lays the groundwork for future treatments of VML utilizing the combination of SeNPs and dECM.

Enhancing osteogenic differentiation in adipose-derived mesenchymal stem cells with Near Infra-Red and Green Photobiomodulation

Worldwide, osteoporosis is the utmost predominant degenerative bone condition. Stem cell regenerative therapy using adipose-derived mesenchymal stem cells (ADMSCs) is a promising therapeutic route for osteoporosis. Photobiomodulation (PBM) has sparked considerable international appeal due to its’ ability to augment stem cell proliferation and differentiation properties. Furthermore, the differentiation of ADMSCs into osteoblast cells and cellular proliferation effects have been established using a combination of osteogenic differentiation inducers and PBM. This in vitro study applied dexamethasone, β-glycerophosphate disodium, and ascorbic acid as differentiation inducers for osteogenic induction differentiation media. In addition, PBM at a near-infrared (NIR) wavelength of 825 nm, a green (G) wavelength of 525 nm, and the novel combination of both these wavelengths using a single fluence of 5 J/cm2 had been applied to stimulate proliferation and differentiation effectivity of immortalised ADMSCs into early osteoblasts. Flow cytometry and ELISA were used to identify osteoblast antigens using early and late osteoblast protein markers. Alizarin red Stain was employed to identify calcium-rich deposits by cells within culture. The morphology of the cells was examined, and biochemical assays such as an EdU proliferation assay, MTT proliferation and viability assay, Mitochondrial Membrane Potential assay, and Reactive Oxygen Species assay were performed. The Central Scratch Test determined the cells' motility potential. The investigative outcomes revealed that a combination of PBM treatment and osteogenic differentiation inducers stimulated promising early osteogenic differentiation of immortalised ADMSCs. The NIR-Green PBM combination did appear to offer great potential for immortalised ADMSC differentiation into early osteoblasts amongst selected assays, however, further investigations will be required to establish the effectivity of this novel wavelength combination. This research contributes to the body of knowledge and assists in the establishment of a standard for osteogenic differentiation in vitro utilising PBM.

Substrate stiffness can affect the crosstalk between adipose derived mesenchymal stem cells and macrophages in bone tissue engineering.

Purpose: This study aimed to explore the effect of biomaterials with different stiffness on Adipose Derived Mesenchymal Stem Cells (ADSC)-macrophage crosstalk in bone tissue engineering and its role in bone repair. Methods: Biomaterials with Young's modulus of 64 and 0.2kPa were selected, and the crosstalk between ADSCs and macrophages was investigated by means of conditioned medium treatment and cell co-culture, respectively. Polymerase chain reaction (PCR) and flow cytometry were used to evaluate the polarization of macrophages. Alkaline phosphatase (ALP) and alizarin red staining (ARS) solutions were used to evaluate the osteogenic differentiation of ADSCs. Transwell assay was used to evaluate the chemotaxis of ADSCs and macrophages. Moreover, mass spectrometry proteomics was used to analyze the secreted protein profile of ADSCs of different substrates and macrophages in different polarization states. Results: On exploring the influence of biomaterials on macrophages from ADSCs on different substrates, we found that CD163 and CD206 expression levels in macrophages were significantly higher in the 64-kPa group than in the 0.2-kPa group in conditioned medium treatment and cell co-culture. Flow cytometry showed that more cells became CD163+ or CD206+ cells in the 64-kPa group under conditioned medium treatment or cell co-culture. The Transwell assay showed that more macrophages migrated to the lower chamber in the 64-kPa group. The proteomic analysis found that ADSCs in the 64-kPa group secreted more immunomodulatory proteins, such as LBP and RBP4, to improve the repair microenvironment. On exploring the influence of biomaterials on ADSCs from macrophages in different polarization states, we found that ALP and ARS levels in ADSCs were significantly higher in the M2 group than in the other three groups (NC, M0, and M1 groups) in both conditioned medium treatment and cell co-culture. The Transwell assay showed that more ADSCs migrated to the lower chamber in the M2 group. The proteomic analysis found that M2 macrophages secreted more extracellular remodeling proteins, such as LRP1, to promote bone repair. Conclusion: In bone tissue engineering, the stiffness of repair biomaterials can affect the crosstalk between ADSCs and macrophages, thereby regulating local repair immunity and affecting bone repair.

Mechanism of miR-26a-5p/cAMP response element binding protein 1 molecular axis regulating osteogenic differentiation of adipose-derived mesenchymal stem cells

To investigate the regulatory effects of miR-26a-5p on the osteogenic differentiation of adipose-derived mesenchymal stem cells (ADSCs) by regulating cAMP response element binding protein 1 (CREB1). The adipose tissues of four 3-4 weeks old female C57BL/6 mice were collected and the cells were isolated and cultured by digestion separation method. After morphological observation and identification by flow cytometry, the 3rd-generation cells were subjected to osteogenic differentiation induction. At 0, 3, 7, and 14 days after osteogenic differentiation induction, the calcium deposition was observed by alizarin red staining, ALP activity was detected, miR- 26a-5p and CREB1 mRNA expressions were examined by real-time fluorescence quantitative PCR, and CREB1 protein and its phosphorylation (phospho-CREB1, p-CREB1) level were measured by Western blot. After the binding sites between miR-26a-5p and CREB1 was predicted by the starBase database, HEK-293T cells were used to conduct a dual-luciferase reporter gene experiment to verify the targeting relationship (represented as luciferase activity after 48 hours of culture). Finally, miR-26a-p inhibitor (experimental group) and the corresponding negative control (control group) were transfected into ADSCs. Alizarin red staining, ALP activity, real-time fluorescent quantitative PCR (miR-26a-5p) and Western blot [CREB1, p-CREB1, Runt-related transcription factor 2 (RUNX2), and osteocalcin (OCN)] were performed at 7 and 14 days after osteogenic induction culture. The cultured cells were identified as ADSCs. With the prolongation of osteogenic induction culture, the number of calcified nodules and ALP activity significantly increased ( P<0.05). The relative expression of miR-26a-5p in the cells gradually decreased, while the relative expressions of CREB1 mRNA and protein, as well as the relative expression of p-CREB1 protein were increased. The differences were significant between 7, 14 days and 0 day ( P<0.05). There was no significant difference in p-CREB1/CREB1 between different time points ( P>0.05). The starBase database predicted that miR-26a-5p and CREB1 had targeted binding sequences, and the dual-luciferase reporter gene experiment revealed that overexpression of miR-26a-5p significantly suppressed CREB1 wild-type luciferase activity ( P<0.05). After 7 and 14 days of osteogenic induction, compared with the control group, the number of calcified nodules, ALP activity, and relative expressions of CREB1, p-CREB1, OCN, and RUNX2 proteins in the experimental group significantly increased ( P<0.05). There was no significant difference in p-CREB1/CREB1 between the two groups ( P>0.05). Knocking down miR-26a-5p promoted the osteogenic differentiation of ADSCs by up-regulating CREB1 and its phosphorylation.

Hypoxia regulates adipose mesenchymal stem cells proliferation, migration, and nucleus pulposus-like differentiation by regulating endoplasmic reticulum stress via the HIF-1α pathway

ObjectiveHypoxia can promote stem cell proliferation and migration through HIF-1α. Hypoxia can regulate cellular endoplasmic reticulum (ER) stress. Some studies have reported the relationship among hypoxia, HIF-α, and ER stress, however, while little is known about HIF-α and ER stress in ADSCs under hypoxic conditions. The purpose of the study was to investigate the role and relationship of hypoxic conditions, HIF-1α and ER stress in regulating adipose mesenchymal stem cells (ADSCs) proliferation, migration, and NPC-like differentiation.MethodADSCs were pretreated with hypoxia, HIF-1α gene transfection, and HIF-1α gene silence. The ADSCs proliferation, migration, and NPC-like differentiation were assessed. The expression of HIF-1α in ADSCs was regulated; then, the changes of ER stress level in ADSCs were observed to investigate the relationship between ER stress and HIF-1α in ADSCs under hypoxic conditions.ResultThe cell proliferation and migration assay results show that hypoxia and HIF-1α overexpression can significantly increase the ADSCs proliferation and migration, while HIF-1α inhibition can significantly decrease the ADSCs proliferation and migration. The HIF-1α and co-cultured with NPCs played an important role in the directional differentiation of ADSCs into NPCs. The hypoxia-regulated ER stress in ADSCs through the HIF-1α pathway, thereby regulating the cellular state of ADSCs, was also observed.ConclusionHypoxia and HIF-1α play important roles in proliferation, migration, and NPC-like differentiation of ADSCs. This study provides preliminary evidence that HIF-1α-regulated ER stress thus affects ADSCs proliferation, migration, and differentiation. Therefore, HIF-1α and ER may serve as key points to improve the efficacy of ADSCs in treating disc degeneration.

DNA methyltransferases inhibitor azacitidine improves the skeletal phenotype of mild osteogenesis imperfecta by reversing the impaired osteogenesis and excessive osteoclastogenesis.

Osteogenesis imperfecta (OI), as a disease of congenital bone dysplasia, is often accompanied by the abnormal alteration of bone absorption and bone formation. DNA methyltransferases (Dnmts) can regulate the gene expression involved in osteogenesis and osteoclastogenesis. Dnmts changes and their effects on bone cells under OI is poorly understood. The Dnmts expression in adipose derived mesenchymal stem cells (ADSCs), bone marrow derived pre-osteoclasts (pre-Ocs) and femurs of Col1a2oim/+ and Col1a1+/-365 mice, both modeling mild OI types, were determined. The effects of azacitidine (Aza) administration and Dnmt3a knockdown by ShRNA on the osteogenic differentiation of ADSCs together with osteoclasts (Ocs) production of pre-Ocs were studied in vitro. The synthesis and secretion of collagen fibers of OI derived ADSCs were examined. The therapeutic outcomes of intraperitoneal (i.p.) infused Aza (1mg/kg/2d) for 30days were evaluated in OI mice. Obviously elevated expression of Dnmts, especially Dnmt3a, existed in ADSCs, pre-Ocs, and femurs isolated from OI modeled mice. Much more collagen molecules of mutant ADSCs were secreted into the extracellular medium post Aza addition. Both Aza administration and Dnmt3a knockdown effectively enhanced the bone-forming capacity of affected ADSCs and reduced Ocs formation of OI mice in vitro. Aza treatment apparently improved the femora microstructure and biomechanical properties, increased bone formation and decreased the number of Ocs in mice with OI. Highly expressed Dnmt3a contributed to the impaired osteogenesis and enhanced osteoclastogenesis of collagen defect-related OI. Aza medication effectively improved the femora phenotype of the two types of OI modeled mice partly by Dnmts inhibition and modulating cell stress response. These findings facilitated understanding the role of Dnmts alteration in skeletal pathological development of mild OI and preliminary confirmed the therapeutic potential of Dnmts depressants in mild OI treatment. Still, further researches are needed to explore the specific function of Dnmts in OI bones and clarify the benefits of Aza administration in OI treatment.

Targeting G-quadruplex motifs interferes with differentiation of adipose-derived mesenchymal stem cells

BackgroundG-quadruplex (G4) motifs are nucleic acid secondary structures observed in mammalian genomes and transcriptomes able to regulate various cellular processes. Several small molecules have been developed so far to modulate G4 stability, frequently associated with anticancer activity. However, how G4 structures are regulated over homeostatic conditions is mostly unexplored. Here, we used human adipose-derived mesenchymal stem cells (ASCs) to address the role of G4 motifs during adipogenic differentiation.MethodsAdipocyte differentiation of ASCs was investigated in the presence or absence of a well-known G4 ligand, Braco-19. Cell viability was determined by sulforhodamine B assay. Cell dimension and granularity, DNA G4 motifs and cell cycle were detected by flow cytometry. Lipid droplet accumulation was assessed by Oil Red O staining. Cell senescence was evaluated by β-galactosidase staining. Gene expression was measured by qPCR. Protein release in the extracellular medium was quantified by ELISA.ResultsBraco-19 used at non-cytotoxic concentrations induced morphological changes in mature adipocytes partially restoring an undifferentiated-like status. Braco-19 reduced lipid vacuolization and PPARG, AP2, LEP and TNFA mRNA levels in terminally differentiated cells. No effect was observed in cell senescence, fibrotic markers, IL-6 and IL-8 production, while the secretion of VEGF was dose-dependently reduced. Interestingly, G4 structures were increased in differentiated adipocytes compared to their precursors. Braco-19 treatment reduced G4 content in mature adipocytes.ConclusionsOur data highlight a new role of G4 motifs as genomic structural elements related to human ASC differentiation into mature adipocytes, with potential implications in physio-pathological processes.

Selection and Validation of Reference Genes for Gene Expression Studies in an Equine Adipose-Derived Mesenchymal Stem Cell Differentiation Model by Proteome Analysis and Reverse-Transcriptase Quantitative Real-Time PCR.

Adipose-derived stem cells (ADSCs) are used in tissue regeneration therapies. The objective of this study is to identify stable reference genes (RGs) for use in gene expression studies in a characterized equine adipose-derived mesenchymal stem cell (EADMSC) differentiation model. ADSCs were differentiated into adipocytes (ADs) or osteoblasts (OBs), and the proteomes from these cells were analyzed by liquid chromatography tandem mass spectrometry. Proteins that were stably expressed in all three cells types were identified, and the mRNA expression stabilities for their corresponding genes were validated by RT-qPCR. PPP6R1, CCDC97, and then either ACTB or EPHA2 demonstrated the most stable mRNA levels. Normalizing target gene Cq data with at least three of these RGs simultaneously, as per MIQE guidelines (PPP6R1 and CCDC97 with either ACTB or EPHA2), resulted in congruent conclusions. FABP5 expression was increased in ADs (5.99 and 8.00 fold, p = 0.00002 and p = 0.0003) and in OBs (5.18 and 5.91 fold, p = 0.0011 and p = 0.0023) relative to ADSCs. RUNX2 expression was slightly higher in ADs relative to ADSCs (1.97 and 2.65 fold, p = 0.04 and p = 0.01), but not in OBs (0.9 and 1.03 fold, p = 0.58 and p = 0.91).

Engineered substrates incapable of induction of chondrogenic differentiation compared to the chondrocyte imprinted substrates

It is well established that surface topography can affect cell functions. However, finding a reproducible and reliable method for regulating stem cell behavior is still under investigation. It has been shown that cell imprinted substrates contain micro- and nanoscale structures of the cell membrane that serve as hierarchical substrates, can successfully alter stem cell fate. This study investigated the effect of the overall cell shape by fabricating silicon wafers containing pit structure in the average size of spherical-like chondrocytes using photolithography technique. We also used chondrocyte cell line (C28/I2) with spindle-like shape to produce cell imprinted substrates. The effect of all substrates on the differentiation of adipose-derived mesenchymal stem cells (ADSCs) has been studied. The AFM and scanning electron microscopy images of the prepared substrates demonstrated that the desired shapes were successfully transferred to the substrates. Differentiation of ADSCs was investigated by immunostaining for mature chondrocyte marker, collagen II, and gene expression of collagen II, Sox9, and aggrecan markers. C28/I2 imprinted substrate could effectively enhanced chondrogenic differentiation compared to regular pit patterns on the wafer. It can be concluded that cell imprinted substrates can induce differentiation signals better than engineered lithographic substrates. The nanostructures on the cell-imprinted patterns play a crucial role in harnessing cell fate. Therefore, the patterns must include the nano-topographies to have reliable and reproducible engineered substrates.

Zirconia substrate with periodic surface microstructures enhances osteogenic differentiation of rat adipose-derived stem cells

Zirconia substrates with periodic surface microstructures formed by irradiation with a femtosecond laser previously enhanced osteogenic differentiation of rat bone marrow mesenchymal stem cells (BMSCs), associated with a reduction in the cell attachment area. Adipose-derived mesenchymal stem cells (ADSCs) are clinically less difficult in harvesting the cells than BMSCs. In this study, we investigated the effects of zirconia substrates with the periodic surface microstructures on osteogenic differentiation of rat ADSCs. Zirconia substrates with the periodic surface microstructures enhanced not adipogenic differentiation but osteogenic differentiation of rat ADSCs, associated with a reduction in the cell attachment area. Therefore, zirconia substrates with surface periodic microstructures formed by irradiation with a femtosecond laser could be useful in bone tissue engineering using ADSCs when BMSCs are unavailable.

Swelling-Mediated Mechanical Stimulation Regulates Differentiation of Adipose-Derived Mesenchymal Stem Cells for Intervertebral Disc Repair Using Injectable UCST Microgels.

Mechanical stimulation is an effective approach for controlling stem cell differentiation in tissue engineering. However, its realization in in vivo tissue repair remains challenging since this type of stimulation can hardly be applied to injectable seeding systems. Here, it is presented that swelling of injectable microgels can be transformed to in situ mechanical stimulation via stretching the cells adhered on their surface. Poly(acrylamide-co-acrylic acid) microgels with the upper critical solution temperature property are fabricated using inverse emulsion polymerization and further coated with polydopamine to increase cell adhesion. Adipose-derived mesenchymal stem cells (ADSCs) adhered on the microgels can be omnidirectionally stretched along with the responsive swelling of the microgels, which upregulate TRPV4and Piezo1channel proteins and enhance nucleus pulposus (NP)-like differentiation of ADSCs. In vivo experiments reveal that the disc height and extracellular matrix content of NP are promoted after the implantation with the microgels. The findings indicate that swelling-induced mechanical stimulation has great potential for regulating stem cell differentiation during intervertebral disc repair.

Adipose-derived stem cells with miR-150-5p inhibition laden in hydroxyapatite/tricalcium phosphate ceramic powders promote osteogenesis via regulating Notch3 and activating FAK/ERK and RhoA

Adipose-derived mesenchymal stem cells (ADSCs) are multipotent stromal cells and play huge role in forming and repairing bone tissues. Emerging evidence shows that MicroRNAs (miRNAs) are involved in ADSCs differentiation. Here, we explored the role of miR-150-5p and its related mechanisms in ADSCs osteogenesis. Real-time PCR was used to determine miR-150-5p expression during ADSCs osteogenesis. miR-150-5p inhibitors, miR-150-5p ADV or short hairpin RNA (shRNA) of Notch3 were transfected to ADSCs for analyzing the effects on osteogenesis. The mixture of hydroxyapatite/tricalcium phosphate (HA/TCP) ceramic powders and transfected ADSCs was implanted into BALB/C nude mice. Micro-CT and histological methods were performed to evaluate the new bone formation. Compared with negative control (NC) and miR-150-5p overexpression, inhibition of miR-150-5p increased ADSCs osteogenesis by regulating Notch3. MiR-150-5p overexpression decreased the expression of pFAK, pERK1/2, and RhoA, while these were up-regulated when miR-150-5p was inhibited, or notch3 was silenced. Furthermore, miR-150-5p inhibition partially reversed the suppression effect of notch3 knockdown on osteogenesis in vitro and in vivo. This study demonstrated the critical function of miR-150-5p during osteogenesis. The combination of ADSCs with miR-150-5p inhibition and HA/TCP might be a promising strategy for bone damage repair. Statement of significanceOsteoporosis is a common chronic metabolic bone disease in humans. Bone tissue engineering based on mesenchymal stem cells, biomaterials, and growth factors, provides a promising way to treat osteoporosis and bone defects. ADSCs commonly differentiate into adipose cells, they can also differentiate into osteogenic cell lineages. Nucleic acids and protein have usually been considered as regulators of ADSCs osteogenic differentiation. In the current study, we demonstrated the combination of ADSCs with miR-150-5p inhibition and hydroxyapatite/tricalcium phosphate ceramic powders enhanced bone regeneration. Furthermore, miR-150-5p/Notch3 axis regulating osteogenesis via the FAK/ERK1/2 and RhoA pathway was assessed. The current study showed the application of ADSCs in bone regeneration might be a promising strategy for osteoporosis and bone damage repairing.

Adipose-Derived Mesenchymal Stem Cells Differentiation Toward Cardiomyocyte-Like Cells on the PCL/PANI Nanofibrous Scaffold: An Experimental Study.

Owing to the fact that the heart tissue is not able to repair itself. Biomaterial-based scaffolds are important cues in tissue engineering (TE) applications. Recent advances in TE have led to the development of suitable scaffold architecture for various tissue defects. Given the importance of cellular therapy, it was the aim of the present study to differentiate cardio myocyte cells from human adipose-derived mesenchymal stem cells (Ad-MSCs) using suitable induction reagents (namely, 5-azacytidine and transforming growth factor beta (TGF-β)) on poly-caprolactone (PCL)/Poly aniline (PANI) Nano fibrous scaffolds prepared by electrospinning. For this purpose, the adipose-derived mesenchymal stem cells (Ad-MSCs) were initially isolated and characterized before cultivation on the PCL/PANI Nano fibrous scaffold to be treated for 21 days with 5-azacytidine either singly or in combination with TGF-β in medium. The scaffold's morphological and cell attachment properties were investigated using electron microscopy (SEM). Finally, the cardio myocyte differentiation of Ad-MSCs on the scaffold was studied using both quantitative Real-time PCR (qPCR) and flow-cytometry while the expression rates of the cardio myocytes' specific genes (Gata4, NKX2.5, MYH-7, and Troponin I) were also determined. The results of Ad-MSCs culture, MTT assay, and SEM indicated that the cells had well proliferated on the PCL/PANI scaffolds, showing the biocompatibility of the nanofibers for cellular growth and adhesion. After 21 days of induced cardio myocyte differentiation by both agents, Real-time PCR revealed increases in the expressions of Gata4, Troponin I, MYH-7, and NKX2.5 genes in the cells cultured on the PCL/PANI scaffolds while the flow-cytometry test approved the expression of troponin I. The data obtained showed that the PCL/PANI Nano fibrous scaffolds were able to promote and support mesenchymal stem cell transformation to cardio myocyte cells. Generally speaking, the results of the study might be exploited in future in vitro and in vivo experimental model studies of cardio myocyte differentiation using co-polymer scaffolds.