Vascular organoids: unveiling advantages, applications, challenges, and disease modelling strategies.

Understanding mechanisms and manifestations of cardiovascular risk factors, including diabetes, on vascular cells such as endothelial cells, pericytes, and vascular smooth muscle cells, remains elusive partly due to the lack of appropriate disease models. Therefore, here we explore different aspects for the development of advanced 3D in vitro disease models that recapitulate human blood vessel complications using patient-derived induced pluripotent stem cells, which retain the epigenetic, transcriptomic, and metabolic memory of their patient-of-origin. In this review, we highlight the superiority of 3D blood vessel organoids over conventional 2D cell culture systems for vascular research. We outline the key benefits of vascular organoids in both health and disease contexts and discuss the current challenges associated with organoid technology, providing potential solutions. Furthermore, we discuss the diverse applications of vascular organoids and emphasize the importance of incorporating all relevant cellular components in a 3D model to accurately recapitulate vascular pathophysiology. As a specific example, we present a comprehensive overview of diabetic vasculopathy, demonstrating how the interplay of different vascular cell types is critical for the successful modelling of complex disease processes in vitro. Finally, we propose a strategy for creating an organ-specific diabetic vasculopathy model, serving as a valuable template for modelling other types of vascular complications in cardiovascular diseases by incorporating disease-specific stressors and organotypic modifications.

PLAC8-Mediated Activation of NOX4 Signalling Restores Angiogenic Function of Endothelial Colony-Forming Cells in Experimental Hypoxia.

Ischaemic cardiovascular disease is associated with tissue hypoxia as a significant determinant of angiogenic dysfunction and adverse remodelling. While cord blood-derived endothelial colony-forming cells (CB-ECFCs) hold clear therapeutic potential due to their enhanced angiogenic and proliferative capacity, their impaired functionality within the disease microenvironment represents a major barrier to clinical translation. The aim of this study was to define the specific contribution of NOX4 NADPH oxidase, which we previously reported as a key CB-ECFC regulator, to hypoxia-induced dysfunction and its potential as a therapeutic target. CB-ECFCs exposed to experimental hypoxia demonstrated downregulation of NOX4-mediated reactive oxygen species (ROS) signalling linked with a reduced tube formation, which was partially restored by NOX4 plasmid overexpression. siRNA knockdown of placenta-specific 8 (PLAC8), identified by microarray analysis as an upstream regulator of NOX4 in hypoxic versus normoxic CB-ECFCs, enhanced tube formation, NOX4 expression and hydrogen peroxide generation, and induced several key transcription factors associated with downstream Nrf2 signalling. Taken together, these findings indicated that activation of the PLAC8-NOX4 signalling axis improved CB-ECFC angiogenic functions in experimental hypoxia, highlighting this pathway as a potential target for protecting therapeutic cells against the ischaemic cardiovascular disease microenvironment.

Adipose-derived human mesenchymal stem cells seeded on denuded or stromal sides of the amniotic membrane improve angiogenesis and collagen remodeling and accelerate healing of the full-thickness wound.

Several strategies have been proposed to enhance wound healing results. Along with other forms of wound dressing, the human amniotic membrane (HAM) has long been regarded as a biological wound dressing that decreases infection and enhances healing. This study investigates the feasibility and effectiveness of wound healing using decellularized HAM (dAM) and stromal HAM (sAM) in combination with adipose-derived human mesenchymal stem cells (AdMSCs). The dAM and sAM sides of HAM were employed as wound dressing scaffolds, and AdMSCs were seeded on top of either dAM or sAM. Sixty healthy Wistar rats were randomly divided into three groups: untreated wound, dAM/AdMSCs group, and sAM/AdMSCs group. The gene expression of VEGF and COL-I was measured in vitro. Wound healing was examined after wounding on days 3, 7, 14, and 21. The expression level of VEGF was significantly higher in sAM/AdMSCs than dAM/AdMSCs (P ≤ 0.05), but there was no significant difference in COL-I expression (P ≥ 0.05). In vivo research revealed that on day 14, wounds treated with sAM/AdMSCs had more vascularization than wounds treated with dAM/AdMSCs (P ≤ 0.01) and untreated wound groups on days 7 (P ≤ 0.05) and 14 (P ≤ 0.0001), respectively. On days 14 (P < 0.05 for sAM/AdMSCs, P < 0.01 for dAM/AdMSCs), and 21 (P < 0.05 for sAM/AdMSCs, P < 0.01 for dAM/AdMSCs), the collagen deposition in the wound bed was significantly thicker in the sAM/AdMSCs and dAM/AdMSCs groups compared to untreated wounds. The study demonstrated that the combination of sAM and AdMSCs promotes wound healing by enhancing angiogenesis and collagen remodeling.

Scaphoid Bone Nonunions: Clinical and Functional Outcomes of Collagen/PGA Scaffolds and Cell-Based Therapy.

In this study, the procedure for treating the nonunion complication of scaphoid fractures using collagen/poly glycolic acid (CPGA) scaffolds with bone marrow mesenchymal stem cell (BM-MSC) therapy was adopted and compared with the commonly employed autologous bone tissue graft. With conducting a two-armed clinical trial, 10 patients with scaphoid nonunions were enrolled in this investigation. Patients were randomly assigned to two groups treated with (1) CPGA + cell therapy and (2) autologous iliac crest bone graft standard therapy. Treatment outcomes were evaluated three months after surgery, measuring the grip and pinch strengths and wrist range of motion, with two questionnaires: Patient-Rated Wrist Evaluation (PRWE) and Quick form of Disabilities of the Arm, Shoulder, and Hand (QDASH). We have also assessed the union rate using clinical and radiologic healing criteria one and three months post-operatively. Restorative effects of CPGA + cell therapy were similar to those of the autologous bone graft standard therapy, except for the grip strength (P = 0.048) and QDASH score (P = 0.044) changes, which were higher in the CPGA + cell therapy group. Three months following the surgery, radiographic images and computed tomography (CT) scans also demonstrated that the scaphoid union rate in the test group was comparable to that of scaphoids treated with the standard autograft method. Our findings demonstrate that the CPGA + cell therapy is a potential alternative for bone grafting in the treatment of bone nonunions.

Comparing the Effects of Two Cryoprotectant Protocols, Dimethyl-Sulfoxide (DMSO) and Glycerol, on the Recovery Rate of Cultured Keratinocytes on Amniotic Membrane.

Background: Off-the-shelf supply of viable engineered tissue is critical for effective and fast treatment of life-threatening injuries such as deep burns. An expanded keratinocyte sheet on the human amniotic membrane (KC sheet-HAM) is a beneficial tissue-engineering product for wound healing. To access an on-hand supply for the widespread application and overcome the time-consuming process, it is necessary to develop a cryopreservation protocol that guarantees the higher recovery of viable keratinocyte sheets after freeze-thawing. This research aimed to compare the recovery rate of KC sheet-HAM after cryopreservation by dimethyl-sulfoxide (DMSO) and glycerol. Methods: Amniotic membrane was decellularized with trypsin, and keratinocytes were cultured on it to form a multilayer, flexible, easy-to-handle KC sheet-HAM. The effects of 2 different cryoprotectants were investigated by histological analysis, live-dead staining, and proliferative capacity assessments before and after cryopreservation. Results: KCs well adhered and proliferated on the decellularized amniotic membrane and successfully represented 3 to 4 stratified layers of epithelialization after 2 to 3 weeks culture period; making it easy to cut, transfer, and cryopreserve. However, viability and proliferation assay indicated that both DMSO and glycerol cryosolutions have detrimental effects on KCs, and KCs-sheet HAM could not recover to the control level after 8 days of culture post-cryo. The KC sheet lost its stratified multilayer nature on AM, and sheet layers were reduced in both cryo-groups compared to the control. Conclusion: Expanding keratinocytes on the decellularized amniotic membrane as a multilayer sheet made a viable easy-to-handle sheet, nonetheless cryopreservation reduced viability and affected histological structure after thawing. Although some viable cells were detectable, our research highlighted the need for a better cryoprotectant protocol other than DMSO and glycerol, specific for the successful banking of viable tissue constructs.

MmCMS: mouse models' consensus molecular subtypes of colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) primary tumours are molecularly classified into four consensus molecular subtypes (CMS1-4). Genetically engineered mouse models aim to faithfully mimic the complexity of human cancers and, when appropriately aligned, represent ideal pre-clinical systems to test new drug treatments. Despite its importance, dual-species classification has been limited by the lack of a reliable approach. Here we utilise, develop and test a set of options for human-to-mouse CMS classifications of CRC tissue. METHODS: Using transcriptional data from established collections of CRC tumours, including human (TCGA cohort; n = 577) and mouse (n = 57 across n = 8 genotypes) tumours with combinations of random forest and nearest template prediction algorithms, alongside gene ontology collections, we comprehensively assess the performance of a suite of new dual-species classifiers. RESULTS: We developed three approaches: MmCMS-A; a gene-level classifier, MmCMS-B; an ontology-level approach and MmCMS-C; a combined pathway system encompassing multiple biological and histological signalling cascades. Although all options could identify tumours associated with stromal-rich CMS4-like biology, MmCMS-A was unable to accurately classify the biology underpinning epithelial-like subtypes (CMS2/3) in mouse tumours. CONCLUSIONS: When applying human-based transcriptional classifiers to mouse tumour data, a pathway-level classifier, rather than an individual gene-level system, is optimal. Our R package enables researchers to select suitable mouse models of human CRC subtype for their experimental testing.

Bioactive glass-collagen/poly (glycolic acid) scaffold nanoparticles exhibit improved biological properties and enhance osteogenic lineage differentiation of mesenchymal stem cells.

Today's using tissue engineering and suitable scaffolds have got attention to increase healing of non-union bone fractures. In this study, we aimed to prepare and characterize scaffolds with functional and mechanical properties suitable for bone regeneration. Porous scaffolds containing collagen-poly glycolic acid (PGA) blends and various quantities of bioactive glass (BG) 45S5 were fabricated. Scaffolds with different compositions (BG/collagen-PGA ratios (w/w): 0/100; 40/60; 70/30) were characterized for their morphological properties, bioactivity, and mechanical behavior. Then, biocompatibility and osteogenic differentiation potential of the scaffolds were analyzed by seeding mesenchymal stem cells (MSCs). Scaffolds made with collagen-PGA combined with the BG (45S5) were found to have interconnected pores (average pore diameter size 75-115 µm) depending on the percentage of the BG added. Simulated body fluid (SBF) soaking experiments indicated the stability of scaffolds in SBF regardless of their compositions, while the scaffolds retained their highly interconnected structure. The elastic moduli, cell viability, osteogenic differentiation of the BG/collagen-PGA 40/60 and 70/30 scaffolds were superior to the original BG/collagen-PGA (0/100). These results suggest that BG incorporation enhanced the physical stability of our collagen-PGA scaffold previously reported. This new scaffold composition provides a promising platform to be used as a non-toxic scaffold for bone regeneration and tissue engineering.

RNA-Binding Proteins: Emerging Therapeutics for Vascular Dysfunction.

Vascular diseases account for a significant number of deaths worldwide, with cardiovascular diseases remaining the leading cause of mortality. This ongoing, ever-increasing burden has made the need for an effective treatment strategy a global priority. Recent advances in regenerative medicine, largely the derivation and use of induced pluripotent stem cell (iPSC) technologies as disease models, have provided powerful tools to study the different cell types that comprise the vascular system, allowing for a greater understanding of the molecular mechanisms behind vascular health. iPSC disease models consequently offer an exciting strategy to deepen our understanding of disease as well as develop new therapeutic avenues with clinical translation. Both transcriptional and post-transcriptional mechanisms are widely accepted to have fundamental roles in orchestrating responses to vascular damage. Recently, iPSC technologies have increased our understanding of RNA-binding proteins (RBPs) in controlling gene expression and cellular functions, providing an insight into the onset and progression of vascular dysfunction. Revelations of such roles within vascular disease states have therefore allowed for a greater clarification of disease mechanisms, aiding the development of novel therapeutic interventions. Here, we discuss newly discovered roles of RBPs within the cardio-vasculature aided by iPSC technologies, as well as examine their therapeutic potential, with a particular focus on the Quaking family of isoforms.

Differentiation of human adipose-derived mesenchymal stem cells toward tenocyte by platelet-derived growth factor-BB and growth differentiation factor-6.

Mesenchymal Stem Cells (MSCs) are important in regenerative medicine and tissue engineering and will be a very sensible choice for repair and regeneration of tendon. New biological practices, such as cellular therapy using stem cells, are promising for facilitating or expediting tendon therapy. Before using these cells clinically, it is best to check and confirm the optimal conditions for differentiation of these cells in the laboratory. Hence, in the present study, the impacts of PDGF-BB and GDF-6 supplementation on adipose-derived MSCs (ASCs) culture were studied. The frozen ASC were recovered and expanded in basic culture medium (DMEM with 10%FBS). The cells after passage five (P5) were treated with basic medium containing L-Prolin, Ascorbic Acid and only PDGF-BB or GDF-6 (20 ng/ml) or both of them (mix) as 3 groups for 14 days to investigate efficiency of ASCs differentiation towards tenocytes. The cells culturing in basic medium were used as control group. To validate tenogenic differentiation, H&E and Sirius Red staining were used to assess cell morphology and collagen production, respectively. In addition, mRNA levels of collagen I and III, Scleraxis and Tenomodulin as tenogenic markers were analyzed using qPCR. In all test groups, cells appeared slenderer, elongated cytoplasmic attributes compared to the control cells. The intensity of Sirius Red staining was significantly higher in GDF-6, PDGF-BB alone, than in group without supplements. The optical density was higher in the GDF-6 than PDGF-BB and mix-group. QPCR results showed that Col I and III gene expression was increased in all groups compared to the control. SCX expression was significantly increased only in the PDGF-BB group. TNMD mRNA expression was not significant among groups. In this study, we have corroborated that human ASCs are reactionary to tenogenic induction by GDF-6 and PDGF-BB alone or in combination. These outcomes will help greater insight into GDF-6 and PDGF-BB driven tenogenesis of ASCs and new directions of discovery in the design of ASC-based treatments for tendon healing.

CRISPR/Cas9 mediated GFP-human dentin matrix protein 1 (DMP1) promoter knock-in at the ROSA26 locus in mesenchymal stem cell for monitoring osteoblast differentiation.

BACKGROUND: Dentin matrix protein 1 (DMP1) is highly expressed in mineralized tooth and bone, playing a critical role in mineralization and phosphate metabolism. One important role for the expression of DMP1 in the nucleus of preosteoblasts is the up-regulation of osteoblast-specific genes such as osteocalcin and alkaline phosphatase1 . The present study aimed to investigate the potential application of human DMP1 promoter as an indicator marker of osteoblastic differentiation. METHODS: In the present study, we developed DMP1 promoter-DsRed-GFP knock-in mesenchymal stem cell (MSCs) via the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system that enabled automatic detection of osteoblast differentiation. With the application of a homology-directed knock-in strategy, a 2-kb fragment of DMP1 promoter, which was inserted upstream of the GFP and DsRed reporter cassette, was integrated into the human ROSA locus to generate double fluorescent cells. We further differentiated MSCs under osteogenic media to monitor the fate of MSCs. First, cells were transfected using CRISPR/Cas9 plasmids, which culminated in MSCs with a green fluorescence intensity, then GFP-positive cells were selected using puromycin. Second, the GFP-positive MSCs were differentiated toward osteoblasts, which demonstrated an increased red fluorescence intensity. The osteoblast differentiation of MSCs was also verified by performing alkaline phosphatase and Alizarin Red assays. RESULTS: We have exploited the DMP1 promoter as a predictive marker of MSC differentiation toward osteoblasts. Using the CRISPR/Cas9 technology, we have identified a distinctive change in the fluorescence intensities of GFP knock-in (green) and osteoblast differentiated MSCs 2 . CONCLUSIONS: The data show that DMP1-DsRed-GFP knock-in MSCs through CRISPR/Cas9 technology provide a valuable indicator for osteoblast differentiation. Moreover, The DMP1 promoter might be used as a predictive marker of MSCs differentiated toward osteoblasts.

Induction of tenogenic differentiation of equine adipose-derived mesenchymal stem cells by platelet-derived growth factor-BB and growth differentiation factor-6.

Managing tendon healing process is complicated mainly due to the limited regeneration capacity of tendon tissue. Mesenchymal stem cells (MSCs) have potential applications in regenerative medicine and have been considered for tendon repair and regeneration. This study aimed to evaluate the capacity of equine adipose tissue-derived cells (eASCs) to differentiate into tenocytes in response to platelet-derived growth factor-BB (PDGF-BB) and growth differentiation factor-6 (GDF-6) in vitro. Frozen characterized eASCS of 3 mares were thawed and the cells were expanded in basic culture medium (DMEM supplemented with 10% FBS). The cells at passage 5 were treated for 14 days in different conditions including: (1) control group in basic culture medium (CM), (2) induction medium as IM (CM containing L-prolin, and ascorbic acid (AA)) supplemented with PDGF-BB (20 ng/ml), (3) IM supplemented with GDF-6 (20 ng/ml), and (4) IM supplemented with PDGF-BB and GDF-6. At the end of culture period (14th day), tenogenic differentiation was evaluated. Sirius Red staining was used to assess collagen production, and H&E was used for assessing cell morphology. mRNA levels of collagen type 1 (colI), scleraxis (SCX), and Mohawk (MKX), as tenogenic markers, were analyzed using real-time reverse-transcription polymerase chain reaction (qPCR). H&E staining showed a stretching and spindle shape (tenocyte-like) cells in all treated groups compared to unchanged from of cells in control groups. Also, Sirius red staining data showed a significant increase in collagen production in all treated groups compared with the control group. MKX expression was significantly increased in PDGF-BB and mixed groups and COLI expression was significantly increased only in PDGF-BB group. In conclusion, our results showed that PDGF-BB and GDF-6 combination could induce tenogenic differentiation in eASCs. These in vitro findings could be useful for cell therapy in equine regenerative medicine.

Endothelial Cells Derived From Patients With Diabetic Macular Edema Recapitulate Clinical Evaluations of Anti-VEGF Responsiveness Through the Neuronal Pentraxin 2 Pathway.

Diabetic macular edema (DME) remains a leading cause of vision loss worldwide. DME is commonly treated with intravitreal injections of vascular endothelial growth factor (VEGF)-neutralizing antibodies. VEGF inhibitors (anti-VEGFs) are effective, but not all patients fully respond to them. Given the potential side effects, inconvenience, and high cost of anti-VEGFs, identifying who may not respond appropriately to them and why is essential. Herein we determine first the response to anti-VEGFs, using spectral-domain optical coherence tomography scans obtained from a cohort of patients with DME throughout the 1st year of treatment. We found that fluid fully cleared at some time during the 1st year in 28% of eyes ("full responders"); fluid cleared only partly in 66% ("partial responders"); and fluid remained unchanged in 6% ("nonresponders"). To understand this differential response, we generated induced pluripotent stem cells (iPSCs) from full responders and nonresponders, from subjects with diabetes but no DME, and from age-matched volunteers without diabetes. We differentiated these iPSCs into endothelial cells (iPSC-ECs). Monolayers of iPSC-ECs derived from patients with diabetes showed a marked and prolonged increase in permeability upon exposure to VEGF; the response was significantly exaggerated in iPSC-ECs from nonresponders. Moreover, phosphorylation of key cellular proteins in response to VEGF, including VEGFR2, and gene expression profiles, such as that of neuronal pentraxin 2, differed between full responders and nonresponders. In this study, iPSCs were used in order to predict patients' responses to anti-VEGFs and to identify key mechanisms that underpin the differential outcomes observed in the clinic. This approach identified NPTX2 as playing a significant role in patient-linked responses and as having potential as a new therapeutic target for DME.

Comparison the effects of hypoxia-mimicking agents on migration-related signaling pathways in mesenchymal stem cells.

Adipose-derived mesenchymal stem cells (Ad-MSCs) have been designated as the promising agents for clinical applications for easy accessibility, multi-linage differentiation and immunomodulation capacity. Despite this, optimal cell delivery conditions have remained as a clinical challenge and improvement of stem cell homing to the target organs is being considered as a major strategy in cell therapy systemic injection. It has been shown that homing of mesenchymal stem cells are increased when treated with physical or chemical hypoxia-mimicking factors, however, efficiency of different agents remained to be determined. In this study, hypoxia-mimicking agents, including valproic acid (VPA), cobalt chloride (CoCl2) and deferoxamine (DFX) were examined to determine whether they are able to activate signaling molecules involved in migration of Ad-MSCs in vitro. We report that Ad-MSCs treated by DFX resulted in a significantly enhanced mRNA expression of MAPK4 (associated with MAPK signaling pathway), INPP4B (associated with Inositol polyphosphate pathway), VEGF-A and VEGF-C (associated with cytokine-cytokine receptor pathways), IL-8 and its receptor, CXCR2 (associated with IL-8 signaling pathway). While the cells treated with VPA did not show such effects and CoCl2 only upregulated VEGF-A and VEGF-C gene expression. Furthermore, results of wound-healing assays showed migration capacity of Ad-MSCs treated with DFX significantly increased 8 and 24 h of the treatment. This study provides credible evidence around DFX, which might be an effective drug for pharmacological preconditioning of Ad-MSCs to boost their homing capacity and regeneration of damaged tissues though, activation of the migration-related signaling pathways.

Enhanced biological properties of collagen/chitosan-coated poly(ε-caprolactone) scaffold by surface modification with GHK-Cu peptide and 58S bioglass.

Bioactive glasses and peptides have shown promising results in improving wound healing and skin repair. The present study explores the effectiveness of surface modification of collagen/chitosan-coated electrospun poly(ε-caprolactone) scaffold with 58S bioactive glass or GHK-Cu peptide. To coat scaffolds with the bioactive glass, we prepared suspensions of silanized bioactive glass powder with three different concentrations and the scaffolds were pipetted with suspensions. Similarly, GHK-Cu-coated scaffolds were prepared by pipetting adequate amount of 1-mM solution of peptide (in milli-Q) on the surface of scaffolds. ATR-FTIR spectroscopy indicated the successful modification of collagen/chitosan-coated electrospun poly(ε-caprolactone) scaffold with bioactive glass and GHK-Cu. Microstructural investigations and in vitro studies such as cell adhesion, cell viability and antibacterial assay were performed. All samples demonstrated desirable cell attachment. Compared to poly(ε-caprolactone)/collagen/chitosan, the cell proliferation of GHK-Cu and bioactive glass-coated (concentrations of 0.01 and 0.1) scaffolds increased significantly at days 3 and 7, respectively. Poly(ε-caprolactone)/collagen/chitosan-uncoated scaffold and scaffolds coated with GHK-Cu and bioactive glass revealed desirable antibacterial properties but the antibacterial activity of GHK-Cu-coated sample turned out to be superior. These findings indicated that biological properties of collagen/chitosan-coated synthetic polymer could be improved by GHK-Cu and bioactive glass.

The effect of adrenocorticotropic hormone on alpha-2-macroglobulin in osteoblasts derived from human mesenchymal stem cells.

Nowadays, alpha-2-macroglobulin (A2M) gene has allocated escalating interest among several genes involved in the pathogenesis of avascular necrosis of the femoral head (ANFH). This molecule could interact with several osteogenic-related proteins. It was reported that adrenocorticotropic hormone (ACTH) affects bones through its receptor located on osteoblasts, suggesting it as a potential target in ANFH treatment. In this study, the effect of ACTH on A2M expression was investigated in osteoblasts as well as during the differentiation of human mesenchymal stem cells (MSCs) into osteoblasts. In this study, MSCs derived from bone marrow were isolated and purified using Ficoll gradient and several passaging. MSCs were characterized by induction with osteogenic and adipogenic medium followed by Oil Red O, Alizarin Red and alkaline phosphatase staining. Besides, MSCs were exposed to various concentrations of ACTH to evaluate the cell variability by MTT assay. MSCs and differentiated osteoblasts were treated with 10-8 molar ACTH for 16 and 26 days, respectively. Then, the total RNA was extracted and A2M expression was quantified by real-time qPCR. The protein expression levels of osteoblast markers including alkaline phosphatase (ALPL) and bone gamma-carboxyglutamate protein (BGLAP) were also measured. The results showed that A2M expression in cells treated with ACTH was up-regulated significantly compared to the control group. Similarly, the expression of osteoblast gene markers including ALPL and BGLAP was significantly increased. ACTH, as an osteoblastic differentiation enhancer, up-regulates A2M, which promotes osteoblastic differentiation probably through TGF-ß induction.

T-Box20 inhibits osteogenic differentiation in adipose-derived human mesenchymal stem cells: the role of T-Box20 on osteogenesis.

BACKGROUND: Skeletal development and its cellular function are regulated by various transcription factors. The T-box (Tbx) family of transcription factors have critical roles in cellular differentiation as well as heart and limbs organogenesis. These factors possess activator and/or repressor domains to modify the expression of target genes. Despite the obvious effects of Tbx20 on heart development, its impact on bone development is still unknown. METHODS: To investigate the consequence by forced Tbx20 expression in the osteogenic differentiation of human mesenchymal stem cells derived from adipose tissue (Ad-MSCs), these cells were transduced with a bicistronic lentiviral vector encoding Tbx20 and an enhanced green fluorescent protein. RESULTS: Tbx20 gene delivery system suppressed the osteogenic differentiation of Ad-MSCs, as indicated by reduction in alkaline phosphatase activity and Alizarin Red S staining. Consistently, reverse transcription-polymerase chain reaction analyses showed that Tbx20 gain-of-function reduced the expression levels of osteoblast marker genes in osteo-inductive Ad-MSCs cultures. Accordingly, Tbx20 negatively affected osteogenesis through modulating expression of key factors involved in this process. CONCLUSION: The present study suggests that Tbx20 could inhibit osteogenic differentiation in adipose-derived human mesenchymal stem cells.

The Intricate Interplay between Epigenetic Events, Alternative Splicing and Noncoding RNA Deregulation in Colorectal Cancer.

Colorectal cancer (CRC) results from a transformation of colonic epithelial cells into adenocarcinoma cells due to genetic and epigenetic instabilities, alongside remodelling of the surrounding stromal tumour microenvironment. Epithelial-specific epigenetic variations escorting this process include chromatin remodelling, histone modifications and aberrant DNA methylation, which influence gene expression, alternative splicing and function of non-coding RNA. In this review, we first highlight epigenetic modulators, modifiers and mediators in CRC, then we elaborate on causes and consequences of epigenetic alterations in CRC pathogenesis alongside an appraisal of the complex feedback mechanisms realized through alternative splicing and non-coding RNA regulation. An emphasis in our review is put on how this intricate network of epigenetic and post-transcriptional gene regulation evolves during the initiation, progression and metastasis formation in CRC.

Overexpression of MicroRNA-148b-3p stimulates osteogenesis of human bone marrow-derived mesenchymal stem cells: the role of MicroRNA-148b-3p in osteogenesis.

BACKGROUND: Mesenchymal stem cells (MSCs) are attractive choices in regenerative medicine and can be genetically modified to obtain better results in therapeutics. Bone development and metabolism are controlled by various factors including microRNAs (miRs) interference, which are small non-coding endogenous RNAs. METHODS: In the current study, the effects of forced miR-148b expression was evaluated on osteogenic activity. Human bone marrow-derived mesenchymal stem cells (BM-MSCs) were transduced with bicistronic lentiviral vector encoding hsa-miR-148b-3p or -5p and the enhanced green fluorescent protein. Fourteen days post-transduction, immunostaining as well as Western blotting were used to analyze osteogenesis. RESULTS: Overexpression of miR-148b-3p increased the osteogenic differentiation of human BM-MSCs as demonstrated by anenhancement of mineralized nodular formation and an increase in the levels of osteoblastic differentiation biomarkers, alkaline phosphatase and collagen type I. CONCLUSIONS: Since lentivirally overexpressed miR-148b-3p increased osteogenic differentiation capability of BM-MSCs, this miR could be applied as a therapeutic modulator to optimize bone function.

Regeneration and Repair of Skin Wounds: Various Strategies for Treatment.

Skin as a mechanical barrier between the inner and outer environment of our body protects us against infection and electrolyte loss. This organ consists of 3 layers: the epidermis, dermis, and hypodermis. Any disruption in the integrity of skin leads to the formation of wounds, which are divided into 2 main categories: acute wounds and chronic wounds. Generally, acute wounds heal relatively faster. In contrast to acute wounds, closure of chronic wounds is delayed by 3 months after the initial insult. Treatment of chronic wounds has been one of the most challenging issues in the field of regenerative medicine, promoting scientists to develop various therapeutic strategies for a fast, qualified, and most cost-effective treatment modality. Here, we reviewed more recent approaches, including the development of stem cell therapy, tissue-engineered skin substitutes, and skin equivalents, for the healing of complex wounds.

Adipocyte lineage differentiation potential of MSCs isolated from reaming material.

Mesenchymal stem cells (MSCs) obtained from various sources have been used for different therapeutic applications including tissue regeneration. Reamer/irrigator/aspirator (RIA) has been increasingly used in recent years for the derivation of MSCs. Here in this investigation we have comparatively analyzed MSCs obtained from iliac crest bone marrow (ICBM) and RIA for their morphology, cluster determinant (CD) markers, and adipogenic differentiation capacity. MSCs were isolated, cultured, and purified from both sources and then flow cytometric studies were performed to study their characteristics. The differentiation potential of RIA and ICBM was examined by an Oil Red O staining protocol. Moreover, the tissue-specific markers related to adipogenesis were analyzed by real-time polymerase chain reaction (RT-PCR). The cells were cultured in the relevant induction medium and then adipogenic lineage differentiation was tested and confirmed for all MSC preparations. Additionally, analysis by flow cytometer was indicative of RIA derived MSCs (RIA-MSCs) having a more homogenous population than ICBM derived MSCs. The RIA-MSCs differentiation toward adipogenic lineage was more efficient compared with ICBM-MSCs. Direct comparative analysis of RIA to ICBM-MSCs indicated that the RIA-MSCs had a higher potential toward adipocyte lineage differentiation compared with ICBM-MSCs.