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Objective: Today, for the regenerative treatment of injured or early osteoarthritic (OA) cartilage, autologous chondrocyte implantation (ACI) or matrix assisted ACI (MACI) are clinically applied. Also mesenchymal stem cell (MSC) approaches have reached the clinic, combining microfracture with the implantation of a collagen membrane (AMIC) or PGA/hyaluronan scaffold (Chondrotissue). AMIC is a passive approach; endogenous MSC flow into the membrane. Chondrotissue, in whose development we were involved, is between passive and active; MSC flow into the scaffold is enhanced by the addition of MSC recruiting serum. Our aim is to develop an active in situ approach, wherein the implantation of scaffolds loaded with MSC recruiting chemokines (CK) and differentiation factors, combined with microfracture, allows the use of endogenous MSC for cartilage regeneration. In this lecture, we will present the international state of the art and our own results. Materials and Methods: Bone marrow MSC of normal donors (ND) and OA patients were characterized (growth kinetics, FACS CD marker profile, multilineage assays). Also their CK receptor profile (qPCR, antibodies), CK release profile (protein array) and CK dependent migration (chemotaxis assays) was elucidated. Moreover, expression profiles of CK stimulated ND and OA MSC were compared (microarrays). Selected CK were encapsulated in PLGA release particles, and in vitro and in vivo applied to attract superparamagnetic iron oxide nanoparticle (SPION) labeled MSC (rat model, MRI). Finally, comparative gene expression profiling was performed for ND and OA MSC fibrinogen/ PLGA implants after chondrogenic induction. Results: ND and OA MSC have a similar doubling time, differentiate to fat, bone and cartilage, are CD73, CD105 positive, and CD45 negative. Moreover, both express most CK receptors. Proteomics revealed similar CK release profiles. Based on 96-well chemotaxis assays, they also show a similar CK dependent migration potential. Promising CK like CCL25 (TECK) and CXCL12 (SDF1•) were analyzed in more detail. CCL25 recruited a high number of ND and OA MSC and represents a promising new candidate for in situ approaches. Expression profiling of CCL25 and CXCL12 induced ND and OA MSC gave us a deep insight into their mobilization. For example, 22 genes were differentially expressed in both ND and OA MSC. Most are involved in homing (PDE4B), movement (PTGS2) and cytoskeletal and membrane reorganization (IGFBP1). In an ongoing study, CCL25 loaded PLGA particles are tested in a rat model. Here, for in vivo MRI monitoring of MSC migration towards CK releasing particles, SPION labeled MSC are used. Finally, the regenerative potential of OA and ND MSC was studied in fibrinogen/PLGA transplants. Here, chondrogenesis resulted in fibro- and joint cartilage, and ND and OA expression profiles showed a similar expression of marker genes known in context of OA (COL10A1, MMP1 and -3). Conclusion: We have shown that end-stage OA MSC behave like ND MSC, and that we have established the key knowledge and tools for an active, MSC based in situ therapy of injured or OA joint cartilage. This will be presented in this keynote in context of the international state of the art. [ABSTRACT FROM AUTHOR]

Vascular organoids (VOs), derived from induced pluripotent stem cells (iPSCs), hold promise as in vitro disease models and drug screening platforms. However, their ability to faithfully recapitulate human vascular disease and cellular composition remains unclear. In this study, we demonstrate that VOs derived from iPSCs of donors with diabetes (DB-VOs) exhibit impaired vascular function compared to non-diabetic VOs (ND-VOs). DB-VOs display elevated levels of reactive oxygen species (ROS), heightened mitochondrial content and activity, increased proinflammatory cytokines, and reduced blood perfusion recovery in vivo. Through comprehensive single-cell RNA sequencing, we uncover molecular and functional differences, as well as signaling networks, between vascular cell types and clusters within DB-VOs. Our analysis identifies major vascular cell types (endothelial cells [ECs], pericytes, and vascular smooth muscle cells) within VOs, highlighting the dichotomy between ECs and mural cells. We also demonstrate the potential need for additional inductions using organ-specific differentiation factors to promote organ-specific identity in VOs. Furthermore, we observe basal heterogeneity within VOs and significant differences between DB-VOs and ND-VOs. Notably, we identify a subpopulation of ECs specific to DB-VOs, showing overrepresentation in the ROS pathway and underrepresentation in the angiogenesis hallmark, indicating signs of aberrant angiogenesis in diabetes. Our findings underscore the potential of VOs for modeling diabetic vasculopathy, emphasize the importance of investigating cellular heterogeneity within VOs for disease modeling and drug discovery, and provide evidence of GAP43 (neuromodulin) expression in ECs, particularly in DB-VOs, with implications for vascular development and disease., (© The Author(s) 2024. Published by Oxford University Press.)

Endothelial cell dysfunction is a complex process involving various causes, early and late events, and subsequent consequences. This review provides an overview of each aspect and outlines therapeutic interventions targeting these stages. Causes of endothelial dysfunction encompass a spectrum of risk factors including hypertension, diabetes, smoking, obesity, inflammation, oxidative stress, and genetic predispositions. Early events such as endothelial activation, inflammatory response, and dysregulated vasomotor tone precede late events like oxidative stress, endothelial apoptosis, and microvascular rarefaction. The consequences include endothelial remodelling, neovascularization, organ dysfunction, and clinical manifestations, highlighting the diverse impacts across multiple systems. While depicted linearly, the progression of endothelial dysfunction is dynamic, influenced by various factors such as the underlying cause and affected vascular bed. Understanding these dynamics is crucial for tailoring therapeutic interventions, ranging from lifestyle modifications to targeted therapies, to address the underlying causes and effects effectively. Here we provide comprehensive understanding of endothelial cell dysfunction that is essential for developing strategies to mitigate the impact of this dysregulation on health and cardiovascular diseases progression., Competing Interests: The authors declare no conflict of interest., (© 2024 The Author(s). Published by IMR Press.)

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., (© 2023. BioMed Central Ltd., part of Springer Nature.)

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.

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.

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., Competing Interests: Declaration of Competing Interest The authors declared no competing interest., (Copyright © 2023. Published by Elsevier GmbH.)

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., (© 2023. The Author(s).)

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.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Toosi, Naderi-Meshkin, Esmailzadeh, Behravan, Ramakrishna and Behravan.)

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.

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., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

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 (CoCl 2 ) 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 CoCl 2 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.

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 phosphatase 1 . 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., (© 2020 John Wiley & Sons, Ltd.)

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., (© 2020 by the American Diabetes Association.)

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.

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.

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-β induction., (© 2020 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Using cell-based engineered skin is an emerging strategy for treating difficult-to-heal wounds. To date, much endeavor has been devoted to the fabrication of appropriate scaffolds with suitable biomechanical properties to support cell viability and growth in the microenvironment of a wound. The aim of this research was to assess the impact of adipose tissue-derived mesenchymal stem cells (AD-MSCs) and keratinocytes on gelatin/chitosan/β-glycerol phosphate (GCGP) nanoscaffold in full-thickness excisional skin wound healing of rats. For this purpose, AD-MSCs and keratinocytes were isolated from rats and GCGP nanoscaffolds were electrospun. Through an in vivo study, the percentage of wound closure was assessed on days 7, 14, and 21 after wound induction. Samples were taken from the wound sites in order to evaluate the density of collagen fibers and vessels at 7 and 14 days. Moreover, sampling was done on days 7 and 14 from wound sites to assess the density of collagen fibers and vessels. The wound closure rate was significantly increased in the keratinocytes-AD-MSCs-scaffold (KMS) group compared with other groups. The expressions of vascular endothelial growth factor, collagen type 1, and CD34 were also significantly higher in the KMS group compared with the other groups. These results suggest that the combination of AD-MSCs and keratinocytes seeded onto GCGP nanoscaffold provides a promising treatment for wound healing., (© 2019 International Federation for Cell Biology.)

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., (© 2019 Wiley Periodicals, Inc.)

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., Competing Interests: Competing interestsThe authors declare that they have no competing interests.

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.

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.

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.

To develop osteogenic building blocks for modular bone tissue engineering applications, influence of gelatin as cell adhesive molecule and nano-hydroxyapatite (nHA) as osteoconductive component was examined on alginate-based hydrogel properties and microencapsulated osteoblast-like cell behavior by using factorial experimental design technique. nHA and alginate showed a statistically significant impact on swelling reduction, and improvement of stability and mechanical strength of hydrogels, respectively. Gelatin influence, however, was in a reverse manner. nHA played imperative roles in promoting microencapsulated osteoblastic cell proliferation and function due to its bioactivity and mechanical strength improvement of hydrogels to the modulus range of mineralized bone tissue in vivo. The results and their statistical analysis also revealed the importance of interaction effect of gelatin and nHA. Proliferation and osteogenic function of the cells fluctuated with increasing gelatin concentration of microcapsules in the presence of nHA, demonstrating that hydrogel properties should be balanced to provide an efficient 3D osteoconductive microcapsule. Alginate (1%)-gelatin (2.5%)-nHA (0.5%) microcapsule with compressive modulus of 0.19 MPa ± 0.02, swelling ratio of 52% ± 8 (24 h) and degradation rate of 12% ± 4 (96 h) revealed a maximum performance for the cell proliferation and function, indicating a potential microcapsule composition to prepare building blocks for modular bone tissue engineering., (Copyright © 2018 Elsevier B.V. All rights reserved.)

We have evaluated the capability of a collagen/poly glycolic acid (PGA) scaffold in regeneration of a calvarial bone defects in rabbits. 4 bone critical size defects (CSD) were created in the calvarial bone of each rabbit. The following 4 treatment modalities were tested (1) a collagen/PGA scaffold (0.52% w/w); (2) the collagen/PGA scaffold (0.52% w/w) seeded with adipose-derived mesenchymal stem cells (AD-MSCs, 1 × 10 6 cells per each defect); (3) AD-MSCs (1 × 10 6 cells) no scaffold material, and (4) blank control. The rabbits were then divided into 3 random groups (of 5) and the treatment outcomes were evaluated at 4, 8 and 12 weeks. New bone formation was histologically assessed. Experimental groups were analyzed by CT scan and real-time PCR. Histological analysis of bone defects treated with collagen/PGA alone exhibited significant fibrous connective tissue formation at the 12 weeks of treatments (P ≤ 0.05). There was no significant difference between collagen/PGA alone and collagen/PGA + AD-MSCs groups. The results were confirmed by CT scan data showing healing percentages of 34.20% for the collage/PGA group alone as compared to the control group and no difference with collagen/PGA containing AD-MSCs (1 × 10 6 cells). RT-PCR analysis also indicated no significant differences between collagen/PGA and collagen/PGA + AD-MSC groups, although both scaffold containing groups significantly express ALP and SIO rather than groups without scaffolds. Although there was no significant difference between the scaffolds containing cells with non-cellular scaffolds, our results indicated that the Collagen/PGA scaffold itself had a significant effect on wound healing as compared to the control group. Therefore, the collagen/PGA scaffold seems to be a promising candidate for research in bone regeneration.

ECM components include a number of osteoinductive and osteoconductive factors, which are involved in bone fracture healing. In this study, a combination of adipose derived mesenchymal stem cells (Ad-MSCs), cancellous bone graft (CBG), and chitosan hydrogel (CHI) was applied to the non-union bone fracture and healing effects were evaluated for the first time. After creation of animal models with non-union fracture in rats, they were randomly classified into seven groups. Radiography at 0, 2, 4, and 8 weeks after surgery, indicated the positive effects of Ad-MSCs + CBG + CHI and Ad-MSCs + CBG in treatment of bone fractures as early as 2 weeks after the surgery. These data were confirmed with both biomechanical and histological studies. Gene expression analyses of Vegf and Bmp2 showed a positive effect of Ad-MSCs on vascularization and osteogenic differentiation in all groups receiving Ad-MSCs, as shown by real-time PCR. Immunofluorescence analysis and RT-PCR results indicated existence of human Ad-MSCs in the fractured region 8 weeks post-surgery. In conclusion, we suggest that application of Ad-MSCs, CBG, and CHI, could be a suitable combination for osteoinduction and osteoconduction to improve non-union bone fracture healing. Further investigations are required to determine the exact mechanisms involved in this process before moving to clinical studies. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 301-311, 2019., (© 2018 Wiley Periodicals, Inc.)

Motivation: Extracellular vesicles (EVs), including exosomes and microvesicles, are potent and clinically valuable tools for early diagnosis, prognosis and potentially the targeted treatment of cancer. The content of EVs is closely related to the type and status of the EV-secreting cell. Circulating exosomes are a source of stable RNAs including mRNAs, microRNAs and long non-coding RNAs (lncRNAs)., Results: This review outlines the links between EVs, lncRNAs and cancer. We highlight communication networks involving the tumor microenvironment, the immune system and metastasis. We show examples supporting the value of exosomal lncRNAs as cancer biomarkers and therapeutic targets. We demonstrate how a system biology approach can be used to model cell-cell communication via exosomal lncRNAs and to simulate effects of therapeutic interventions. In addition, we introduce algorithms and bioinformatics resources for the discovery of tumor-specific lncRNAs and tools that are applied to determine exosome content and lncRNA function. Finally, this review provides a comprehensive collection and guide to databases for exosomal lncRNAs., Supplementary Information: Supplementary data are available at Bioinformatics online.

Recent advances in wound healing have made cell therapy a potential approach for the treatment of various types of skin defects such as trauma, burns, scars and diabetic leg ulcers. Cultured keratinocytes have been applied to burn patients since 1981. Patients with acute and chronic wounds can be treated with autologous/allograft cultured keratinocytes. There are various methods for cultivation of epidermal keratinocytes used in cell therapy. One of the important properties of an efficient cell therapy is the preservation of epidermal stem cells. Mesenchymal Stem Cells (MSCs) are major regulatory cells involved in the acceleration of wound healing via induction of cell proliferation, angiogenesis and stimulating the release of paracrine signaling molecules. Considering the beneficial effects of MSCs on wound healing, the main aim of the present study is investigating paracrine effects of Adipose-derived Mesenchymal Stem Cell (Ad-MSCs) on cultivation of keratinocytes with focusing on preservation of stem cells and their differentiation process. We further introduced a new approach for culturing isolated keratinocytes in vitro in order to generate epidermal keratinocyte sheets without using a feeder layer. To do so, Ad-MSC conditioned medium was applied as an alternative to commercial media for keratinocyte cultivation. In this study, the expression of several stem/progenitor cell (P63, K19 and K14) and differentition (K10, IVL and FLG) markers was examined using real time PCR on days 7, 14 and 21 of culture in keratinocytes in Ad-MSC conditioned medium. P63 and α6 integrin expression was also evaluated via flow cytometry. The results were compared with control group including keratinocytes cultured in EpiLife medium and our data indicated that this Ad-MSC conditioned medium is a good alternative for keratinocyte cultivation and producing epidermal sheets for therapeutic and clinical purposes. The reasons are the expression of stem cell and differentiation markers and overcoming the requirement for feeder layer which leads to a xenograft-free transplantation. Besides, this approach has low cost and is easier to perform. However, more in vitro and in vivo experiments as well as safety evaluation required before clinical applications.

One of the major obstacles in achieving a successful stem cell therapy is insufficient homing of transplanted cells. To overcome this obstacle, understanding the underlying mechanisms of stem cell homing is of obvious importance. Central to this review is the concept that cancer metastasis can be viewed as a role model to build up a comprehensive concept of stem cell homing. In this novel perspective, the prosurvival choices of the cancerous cells in the bloodstream, their arrest, extravasation, and proliferation at the secondary site can be exploited in favor of targeted stem cell homing. To date, tumor cells have been found to employ a wide variety of strategies to promote metastasis. One of these strategies is through their ability to activate platelets and subsequently activated platelets serve cancer cell survival and metastasis. Accordingly, in the first part of this review the roles of platelets in cancer metastasis as well as stem cell homing are discussed. Next, we provide some lessons learned from cancer metastasis in favor of developing strategies for improvement of stem cell homing with emphasis on the role of platelets. Based on direct or indirect evidence from metastasis, strategies such as manipulation of stem cells to enhance interaction with platelets, preconditioning-pretreatment of stem cells with platelets in vitro, and coinjection of both stem cells and platelets are proposed to improve stem cell homing., (© 2018 Wiley Periodicals, Inc.)

Stem cell therapy is considered as a promising treatment for cardiovascular diseases. Adipose-derived mesenchymal stem cells (ADMSCs) have the ability to undergo cardiomyogenesis. Medicinal plants are effective and safe candidates for cell differentiation. Therefore, the aim of our study was to investigate cardiogenic effects of characterized (HPLC-UV) extracts of Geum urbanum on ADMSCs of adipose tissue. The methanolic extracts of the root and aerial parts of G. urbanum were obtained and MTT assay was used for studying their cytotoxic effects. Then, cells were treated with 50 or 100 μg/mL of the extracts from root and aerial parts of G. urbanum. MTT assay showed that the extracts of G. urbanum did not have any toxic effects on ADMSCs. Immunostaining results showed increase in the expression of α-actinin and cardiac troponin I (cTnI), and quantitative real-time reverse-transcription PCR data confirmed the upregulation of ACTN, ACTC1, and TNNI3 genes in ADMSCs after treatment. According to HPLC fingerprinting, some cardiogenic effects of G. urbanum extracts are probably due to ellagic and gallic acid derivatives. Our findings indicated that G. urbanum extracts effectively upregulated some essential cardiogenic markers, which confirmed the therapeutic role of this plant as a traditional cardiac medicine.

Since the adult mammalian heart has limited regenerative capacity, cardiac trauma, disease, and aging cause permanent loss of contractile tissue. This has fueled the development of stem cell-based strategies to provide the damaged heart with new cardiomyocytes. Bone marrow-derived mesenchymal stem cells (BM-MSCs) are capable of self-renewal and differentiation into cardiomyocytes, albeit inefficiently. MicroRNAs (miRNAs, miRs) are non-coding RNAs that have the potential to control stem cell fate decisions and are employed in cardiac regeneration and repair. In this study, we tested the hypothesis that overexpression of miR-499a induces cardiomyogenic differentiation in BM-MSCs. Human BM-MSCs (hBM-MSCs) were transduced with lentiviral vectors encoding miR-499a-3p or miR-499a-5p and analyzed by immunostaining and western blotting methods 14 days post-transduction. MiR-499a-5p-transduced cells adopted a polygonal/rod-shaped (myocyte-like) phenotype and showed an increase in the expression of the cardiomyocyte markers α-actinin and cTnI, as cardiogenic differentiation markers. These results indicate that miR-499a-5p overexpression promotes the cardiomyogenic differentiation of hBM-MSCs and may thereby increase their therapeutic efficiency in cardiac regeneration.

Human amniotic membrane (HAM) has been widely used as a natural scaffold in tissue engineering due to many of its unique biological properties such as providing growth factors, cytokines and tissue inhibitors of metalloproteinases. This study aimed at finding the most suitable and supportive layer of HAM as a delivery system for autologous or allogeneic cell transplantation. Three different layers of HAM were examined including basement membrane, epithelial and stromal layers. In order to prepare the basement membrane, de-epithelialization was performed using 0.5 M NaOH and its efficiency was investigated by histological stainings, DNA quantification, biomechanical testing and electron microscopy. Adipose-derived stromal cells (ASCs) and a human immortalized keratinocyte cell line (HaCaT) were seeded on the three different layers of HAM and cultured for 3 weeks. The potential of the three different layers of HAM to support the attachment and viability of cells were then monitored by histology, electron microscopy and (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Moreover, mechanical strengths of the basement membrane were assessed before and after cell culture. The results indicated that the integrity of extra cellular matrix (ECM) components was preserved after de-epithelialization and resulted in producing an intact basement amniotic membrane (BAM). Moreover, all three layers of HAM could support the attachment and proliferation of cells with no visible cytotoxic effects. However, the growth and viability of both cell types on the BAM were significantly higher than the other two layers. We conclude that growth stimulating effectors of BAM and its increased mechanical strength after culturing of ASCs, besides lack of immunogenicity make it an ideal model for delivering allogeneic cells and tissue engineering applications.

Objectives: Renal ischemia-reperfusion injury (IRI) as a severe condition of acute kidney injury (AKI) is the most common clinical problem with high mortality rates of 35-60% deaths in hospital. Mesenchymal stem cells (MSC) due to unique regenerative characteristics are ideal candidates for the treatment of the ischemic injuries. This work is focused on the administration of MSC to IRI-induced AKI Wistar rats and evaluating their significance in AKI treatment., Material and Methods: Animals underwent surgical procedure and AKI was induced by 40 min bilateral renal pedicle clamping. Immediately after reperfusion, 2×106 rat bone marrow derived MSCs were injected via intra-parenchymal or intra-aortic route., Results: Animals subjected to AKI after days 1 and 3 showed significant increase in the serum creatinine and blood urea nitrogen (BUN) concentration along with a declined glomerular filtration rate (GFR) when compared with non-ischemic animals. On the other hand, treated animals showed a significant enhanced regeneration as compared to ischemic animals in both administration route groups., Conclusion: According to the results concluded from the renoprotective effects of MSC in IRI/AKI, MSCs could be considered as promising therapeutic approach for AKI in clinical applications.

Ischemic heart disease often results in myocardial infarction and is the leading cause of mortality and morbidity worldwide. Improvement in the function of infarcted myocardium is a main purpose of cardiac regenerative medicine. One possible way to reach this goal is via stem cell therapy. Mesenchymal stem cells (MSCs) are multipotent stromal cells that can differentiate into a variety of cell types but display limited cardiomyogenic differentiation potential. Members of the T-box family of transcription factors including Tbx20 play important roles in heart development and cardiomyocyte homeostasis. Therefore, in the current study, we investigated the potential of Tbx20 to enhance the cardiomyogenic differentiation of human adipose-derived MSCs (ADMSCs). Human ADMSCs were transduced with a bicistronic lentiviral vector encoding Tbx20 (murine) and the enhanced green fluorescent protein (eGFP) and analyzed 7 and 14 days post transduction. Transduction of human ADMSCs with this lentiviral vector increased the expression of the cardiomyogenic differentiation markers ACTN1, TNNI3, ACTC1, NKX2.5, TBX20 (human), and GATA4 as revealed by RT-qPCR. Consistently, immunocytological results showed elevated expression of α-actinin and cardiac troponin I in these cells in comparison to the cells transduced with control lentiviral particles coding for eGFP alone. Accordingly, forced expression of Tbx20 exerts cardiomyogenic effects on human ADMSCs by increasing the expression of cardiomyogenic differentiation markers at the RNA and protein level., (© 2018 Wiley Periodicals, Inc.)

Bioactive glasses have shown some interesting biological properties such as biocompatibility, biodegradation, and angiogenesis in skin tissue engineering. In the current research, the effects of MgO- or CoO-doped 64S bioactive glass with a composition of 64 SiO 2 -26 CaO-5 P 2 O 5 -5 MgO or CoO (mol%) were studied in relation with biological properties of electrospun [poly(lactic-co-glycolic acid) (PLGA)/collagen]. PLGA/collagen samples were rinsed in suspension of bioactive glass nanoparticles in distilled water with a concentration of 0.1 w/v and then freeze dried. Cell adhesion, viability, angiogenesis, and ionic release were performed and tested in culture medium containing fibroblast cells. Attachment and viability of fibroblast cells were increased significantly in bioglass-coated samples, while shrinkage in PLGA/collagen scaffold was reduced by the addition of bioactive glass. Vascular endothelial growth factor secretion in coated scaffold was dropped compared to the uncoated samples. This could be attributed to the fast degradation of glass nanoparticles, according to the inductively coupled plasma-atomic emission spectroscopy results.

Use of mesenchymal stem cells (MSCs) has been introduced as a promising tool, for structural and functional recovery of damaged tissues/organs. Studies have indicated that interactions between chemokine receptors and their ligands have a critical role in homing of MSCs to the site of injury. Although CXCR4 variants have been characterized, the exact role of each transcript in homing has remained unclear. In this study, cells were pretreated with various hypoxia-mimicking compounds (valproic acid, cobalt-chloride, and deferoxamine mesylate). Results indicated that both variants of CXCR4 were overexpressed after 24 hours of treatments and their expression could cooperatively induce and promote the cell migration. Moreover, deferoxamine mesylate was more effective in overexpression of variant A (lo), which resulted in higher level of CXCR4 protein and the highest rate of migration of the cells. In conclusion, our findings may have important potential implications in clinical applications, reinforcing the concept that manipulating the expression of specific CXCR4 variants may increase migration of MSCs.

Mesenchymal stem cells (MSCs) have been designated as the most reliable cells in clinics to treat osteo-diseases because of their versatile nature. MSCs, isolated from long bone (Lb-MSCs) are rarely reported and named as RIA-MSCs because of the reamer-irrigator-aspirator (RIA) device. The potential of these cells in the treatment of non-union bone fractures made them the ideal candidates to be studied for clinical practices. In this work, effect of cryopreservation on the proliferation and differentiation capabilities of long bone MSCs (Lb-MSCs) has been studied. For this purpose, Lb-MSCs were isolated via RIA device and characterized using flow cytometry and differentiation assays. Cells were cryopreserved for 3, 6 and 12 months and thereafter were characterized using differentiation assays and genetic markers specific for osteogenic, chondrogenic, and adipogenic potential quantitatively by qRT-PCR. Lb-MSCs were found expressing MSC characteristic markers defining their identity. The population doubling time (PDT) was about 2.5 ± 0.5 days and colonies appeared after 7-10 days. Differentiation potential and gene expression of 3, 6 and 12 months cryopreserved Lb-MSCs were unaltered. The results show that cryopreservation did not have an effect on the differentiation potential of human Lb-MSCs. Therefore, our work offers Lb-MSCs as clinically cells for treating osteo-diseases.

MSC-based therapy is providing a cure for degenerative diseases with unmet medical need and usually iliac crest bone marrow (ICBM) are being applied in clinics. Alternative sources, including adipose tissue and reamer/irrigator/aspirator hold great potential for isolating MCSs. Here, we compared original MSCs features of adipose tissue (Ad-MSCs) and bone marrow of long-bone (RIA-MSCs) or iliac crest, and the expression of chemokine receptors (including CXCR4 , CX3CR1 , CXCR6 , CXCR2 , CCR1 and CCR7 ) in these three sources, which are important in the context of homing. We further investigated the role of SDF-1/CXCR4 axis as a key player in motility of different population of MSCs using Transwell migration assay. All cells exhibited typical MSCs characteristics. However, different MSCs sources expressed different levels of chemokine receptors. Generally, the expression of these chemokine receptors was decreased with increasing passage (P) number from 2 to 3. Interestingly, it was observed that the CXCR4 expression and migration capacity in Ad-MSCs is significantly higher than ICBM and RIA-MSCs in P2. Although our data showed that CXCR4 had highest expression in P2 Ad-MSCs, but it dramatically declined following sub-culturing in the P3. Hence, to improve homing of MSCs by means of chemokine/their receptors axis, the source of isolation and passage number should be considered for clinical applications., Competing Interests: Authors declared no conflict of interest.This work has been approved by the Ethical Committee of ACECR-Khorasan Razavi Branch and all patients included in this study were informed regarding the usage of their samples for experimental analysis. (IRB No. 910750).

Bone defect is an important topic in public health. Novel therapies are based on osteogenic induction by natural antiosteoporotic compounds including plant-derived estrogens. In the current study, the osteogenic potential of Sophora pachycarpa root extract (SPRE) was explored on human adipose-derived mesenchymal stem cells. Herein, adipose-derived mesenchymal stem cells were osteoinducted in the presence of increased concentrations of the extract for 21 days. Then, cell viability was evaluated by MTT assay, and the differentiated cells were stained by Alizarin Red S for calcium deposition and subjected to alkaline phosphatase (ALP) assay for enzymatic activity. To assess the expression of bone-related genes, treated cells were evaluated by real-time polymerase chain reaction. The MTT test demonstrated that SPRE had no toxic effects on the cell viability. Treating the cells with SPRE noticeably promoted ALP activity, mineralization, and mRNA expression of runt-related transcription factor 2 (RUNX2), bone gamma-carboxyglutamate protein (BGLAP), secreted phosphoprotein 1 (SPP1), and collagen type I alpha 1 (COL1A1). Additionally, cells subjected to 0.1 μg/mL SPRE showed the highest osteogenic effects. According to high-performance liquid chromatography fingerprinting of SPRE, the osteoprotective effects of SPRE is probably due to presence of phytochemicals with estrogen-like activity in the extract. Thus, SPRE might be a suitable therapeutic agent for bone defects therapy in the future research. Copyright © 2017 John Wiley & Sons, Ltd., (Copyright © 2017 John Wiley & Sons, Ltd.)

Skin damage can occur for many reasons, including burns and injuries, which in extreme cases can even lead to death. Different methods such as electrospinning are used to produce scaffolds used in skin tissue engineering. Natural and synthetic polymers were used in this method. It was observed that the use of both natural and synthetic polymers gives better results for cell culturing rather than using of each material solely. In this study, scaffolds of poly(lactic-co-glycolic acid) and collagen were prepared using coating and common solvent methods. The characteristics of samples were evaluated through scanning electron microscopy, porosimetry, mechanical testing, degradation behavior, and in vitro assays. The mechanical and biocompatibility test results of the scaffold prepared by coating method were better than the other one. However, the degradation rate of the common solvent was nearly five times more than coating sample that leads to cytotoxicity in contact with the skin cells.