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Aim: To assess the safety and efficacy of a local skin substitute product in the treatment of chronic diabetic foot ulcers (DFUs). Materials and Methods: Five patients were evaluated over 6 months. Skin substitutes were applied twice at 2‐week intervals. Patients were monitored for any possible adverse effects and wound improvement. Results: The results indicated the overall safety of the skin substitute, with only few adverse effects unrelated to this product. Significant reduction in wound size was observed in four patients during the initial 12‐week treatment phase, with complete closure in two patients at 24 weeks. Conclusions: The application of a bi‐layered allogeneic keratinocyte and fibroblast skin substitute in patients with chronic DFU was safe and associated with favourable wound healing results. Adherence to standard treatment protocols, including optimal offloading, is essential to maximize the likelihood of successful wound healing. [ABSTRACT FROM AUTHOR]

A pH-sensitive shell-sheddable magnetic nanocarrier was prepared based on Fe3O4@SiO2 nanoparticles coated with polyethyleneimine (PEI) and polyethyleneglycol (PEG). The PEI was coated on Fe3O4@SiO2 nanoparticles through an electrostatic attraction and the PEG chains were connected to the PEI via pH-sensitive benzoicimine bonds. The synthesized products and final nanocarrier were characterized with FT-IR, 1H NMR, dynamic light scattering (DLS), zeta-potential, TEM, and vibrating sample magnetometer (VSM). The PEI, as a middle layer, was used to load the curcumin (an anticancer drug) via van der Waals interactions and the shedding of PEG as a hydrophilic corona at the tumor pH (acidic) could induce the release of the drug. The curcumin loading content was found to be ~15% and curcumin-loaded magnetic nanoparticles (C-LMNs) showed more triggered release at the tumor pH (5.6) than physiological pH (7.4). In comparison to pure magnetic nanoparticles (MNs), the C-LMNs showed more cytotoxicity to human prostate cancer cell (PC-3) and also resulted in an increased rate of late apoptotic/necrotic PC-3 cells, assessed by flow cytometry analysis. In conclusion, the C-LMN as pH- and magnetic responsive nano-carrier showed good potential for targeted curcumin delivery in therapy of prostate cancer. [ABSTRACT FROM AUTHOR]

"Organoid medicine" has rapidly progressed over the past decade as a new class of therapeutics with high functionality and complexity for addressing unmet medical needs such as effective treatment of patients suffering from chronic liver disease using liver organoids. Here, scalable and xeno‐free integrated differentiation platforms are established to generate hepatic progenitors, mesenchymal stromal cells, and endothelial cells using individual human pluripotent stem cell lines as starting cell types for vascularized liver organoids generation. A scalable microfluidic system is developed to continuously generate cells‐loaded microcapsules with self‐biodegradable 4‐arm‐PEG‐MMP1‐sensitive peptide hydrogel as shell material, to support cells proliferation, self‐condensation, and liver organoids generation through self‐organization. Self‐organized vascularized hepatobiliary organoids (VHOs) containing interconnected biliary networks and vascular structures are generated after optimizing the co‐culture conditions inside hydrogel microcapsules and transferring the organoids to 3D dynamic suspension culture for further maturation. The VHOs show key functional features similar to the fetal and adult liver tissue including the expression of liver‐specific marker genes, the ability to perform main liver metabolic functions, and inducing drug metabolism. The established platforms can be beneficial to the mass production of human liver organoids for liver organoid medicine and the development of safe, effective, and personalized drugs. [ABSTRACT FROM AUTHOR]

Cell culture‐conditioned medium (CCM) is a valuable source of extracellular vesicles (EVs) for basic scientific, therapeutic and diagnostic applications. Cell culturing parameters affect the biochemical composition, release and possibly the function of CCM‐derived EVs (CCM‐EV). The CCM‐EV task force of the Rigor and Standardization Subcommittee of the International Society for Extracellular Vesicles aims to identify relevant cell culturing parameters, describe their effects based on current knowledge, recommend reporting parameters and identify outstanding questions. While some recommendations are valid for all cell types, cell‐specific recommendations may need to be established for non‐mammalian sources, such as bacteria, yeast and plant cells. Current progress towards these goals is summarized in this perspective paper, along with a checklist to facilitate transparent reporting of cell culturing parameters to improve the reproducibility of CCM‐EV research. [ABSTRACT FROM AUTHOR]

The inner ear is a complex organ that encodes sound, motion, and orientation in space. Given the complexity of the inner ear, it is not surprising that treatments are relatively limited despite the fact that, in 2015, hearing loss was the fourth leading cause of years lived with disability worldwide. Inner ear organoid models are a promising tool to advance the study of multiple aspects of the inner ear to aid the development of new treatments and validate drug‐based therapies. The blood supply of the inner ear plays a pivotal role in growth, maturation, and survival of inner ear tissues and their physiological functions. This vasculature cannot be ignored in order to achieve a truly in vivo‐like model that mimics the microenvironment and niches of organ development. However, this aspect of organoid development has remained largely absent in the generation of inner ear organoids. The current review focuses on three‐dimensional inner ear organoid and how recent technical progress in generating in vitro vasculature can enhance the next generation of these models. [ABSTRACT FROM AUTHOR]

Diabetes mellitus may cause tau protein hyperphosphorylation and neurodegeneration, but the exact mechanism by which diabetic conditions induce tau pathology remains unclear. Tau protein hyperphosphorylation is considered a major pathological hallmark of neurodegeneration and can be triggered by diabetes. Various tau‐directed kinases, including P38, can be activated upon diabetic stress and induce tau hyperphosphorylation. Despite extensive research efforts, the exact tau specie(s) and kinases driving neurodegeneration in diabetes mellitus have not been clearly elucidated. We herein employed different techniques to determine the exact molecular mechanism of tau pathology triggered by diabetes in in vivo and in vitro models. We showed that diabetes‐related stresses and glucose metabolism deficiency could induce cis P‐tau (an early driver of the tau pathology) accumulation in the midbrain and corpus callosum of the diabetic mice models and cells treated with 2‐deoxy‐D‐glucose, respectively. We found that the active phosphorylated level of P38 was increased in the treated cells and diabetic mice models. We observed that oxidative stress activated P38, which directly and indirectly drove tau pathology in the GABAergic and glutamatergic neurons of the midbrain of the diabetic mice after 96 h, which accumulated in the other neighboring brain areas after 2 months. Notably, P38 inhibition suppressed tau pathogenicity and risk‐taking behaviors in the animal models after 96 h. The data establish P38 as a central mediator of diabetes mellitus‐induced tau pathology. Our findings provide mechanistic insight into the consequences of this metabolic disorder on the nervous system. [ABSTRACT FROM AUTHOR]

In this report, we look at the challenges posed by the outbreak of COVID‐19 and how the Executive Board of these two congresses succeeded in overcoming those challenges and holding two congresses. The approach for a large festival with different virtual setting components provided a suitable solution that led to exemplary achievements and created an appropriate model for future virtual or combined virtual and face‐to‐face events. These events proved that pandemic problems could not limit the organizers, pushing them to make better use of the facilities and turning this threat into an opportunity. [ABSTRACT FROM AUTHOR]

Developing technologies for scalable production of human organoids has gained increased attention for "organoid medicine" and drug discovery. We developed a scalable and integrated differentiation process for generation of hepatic organoid from human pluripotent stem cells (hPSCs) in a fully controlled stirred tank bioreactor with 150 ml working volume by application of physiological oxygen concentrations in different liver tissue zones. We found that the 20–40% dissolved oxygen concentration [DO] (corresponded to 30–60 mmHg pO2 within the liver tissue) significantly influences the process outcome via regulating the differentiation fate of hPSC aggregates by enhancing mesoderm induction. Regulation of the [DO] at 30% DO resulted in efficient generation of human fetal‐like hepatic organoids that had a uniform size distribution and were comprised of red blood cells and functional hepatocytes, which exhibited improved liver‐specific marker gene expressions, key liver metabolic functions, and, more important, higher inducible cytochrome P450 activity compared to the other trials. These hepatic organoids were successfully engrafted in an acute liver injury mouse model and produced albumin after implantation. These results demonstrated the significant impact of the dissolved oxygen concentration on hPSC hepatic differentiation fate and differentiation efficacy that should be considered ascritical translational aspect of established scalable liver organoid generation protocols for potential clinical and drug discovery applications. [ABSTRACT FROM AUTHOR]

Naïve pluripotency can be established in human pluripotent stem cells (hPSCs) by manipulation of transcription factors, signaling pathways, or a combination thereof. However, differences exist in the molecular and functional properties of naïve hPSCs generated by different protocols, which include varying similarities with pre‐implantation human embryos, differentiation potential, and maintenance of genomic integrity. We show here that short treatment with two chemical agonists (2a) of nuclear receptors, liver receptor homologue‐1 (LRH‐1) and retinoic acid receptor gamma (RAR‐γ), along with 2i/LIF (2a2iL) induces naïve‐like pluripotency in human cells during reprogramming of fibroblasts, conversion of pre‐established hPSCs, and generation of new cell lines from blastocysts. 2a2iL‐hPSCs match several defined criteria of naïve‐like pluripotency and contribute to human–mouse interspecies chimeras. Activation of TGF‐β signaling is instrumental for acquisition of naïve‐like pluripotency by the 2a2iL induction procedure, and transient activation of TGF‐β signaling substitutes for 2a to generate naïve‐like hPSCs. We reason that 2a2iL‐hPSCs are an easily attainable system to evaluate properties of naïve‐like hPSCs and for various applications. Synopsis: Short‐term exposure of nuclear receptors LRH‐1 and RARɣ with chemical agonists (2a) together with 2iL induces human naïve‐like pluripotency in fibroblasts, hPSC and blastocysts. 2a2iL‐hPSCs contribute to the three lineages of human‐mouse chimeras.2a2iL culture condition induces an intermediate state of naïve pluripotency.Short‐term exposure of 2a upregulates NODAL and LEFTY1/2 expression.TGFβ2iL increases expression of naïve specific genes more than 2a2iL. [ABSTRACT FROM AUTHOR]

Objectives: Genetic engineering of human‐induced pluripotent stem cell‐derived neural stem cells (hiPSC‐NSC) may increase the risk of genomic aberrations. Therefore, we asked whether genetic modification of hiPSC‐NSCs exacerbates chromosomal abnormalities that may occur during passaging and whether they may cause any functional perturbations in NSCs in vitro and in vivo. Materials and Methods: The transgenic cassette was inserted into the AAVS1 locus, and the genetic integrity of zinc‐finger nuclease (ZFN)‐modified hiPSC‐NSCs was assessed by the SNP‐based karyotyping. The hiPSC‐NSC proliferation was assessed in vitro by the EdU incorporation assay and in vivo by staining of brain slices with Ki‐67 antibody at 2 and 8 weeks after transplantation of ZFN‐NSCs with and without chromosomal aberration into the striatum of immunodeficient rats. Results: During early passages, no chromosomal abnormalities were detected in unmodified or ZFN‐modified hiPSC‐NSCs. However, at higher passages both cell populations acquired duplication of the entire long arm of chromosome 1, dup(1)q. ZNF‐NSCs carrying dup(1)q exhibited higher proliferation rate than karyotypically intact cells, which was partly mediated by increased expression of AKT3 located on Chr1q. Compared to karyotypically normal ZNF‐NSCs, cells with dup(1)q also exhibited increased proliferation in vivo 2 weeks, but not 2 months, after transplantation. Conclusions: These results demonstrate that, independently of ZFN‐editing, hiPSC‐NSCs have a propensity for acquiring dup(1)q and this aberration results in increased proliferation which might compromise downstream hiPSC‐NSC applications. [ABSTRACT FROM AUTHOR]

Cardiac tissue engineering, a fairly new concept in cardiovascular research, could substantially improve our success in both in vitro modeling of cardiac microtissue and in vivo cardiac regenerative medicine. To a large extent, this success was attributed to mechanical as well as electrical properties of cardiac‐designated biomaterials which inherit the fundamental characteristics of a native myocardial extracellular matrix. Large efforts have been made toward designation and construction of these scaffolds which paved the way for more natural‐like biomaterials. As an important characteristic, electrical conductivity has grabbed special attention, thus opening up a whole new area of research to achieve the best biomaterial. Electroconductive scaffolds have benefitted from both incorporation of conductive particles in polymeric matrix and fabrication of organic conductive polymers which supported cardiac tissue engineering. However, conductive scaffolds have not yet achieved full success and more work is required to obtain the optimal conductivity with highest similarity to the native heart for in vitro cardiac microtissue engineering. [ABSTRACT FROM AUTHOR]

Therapeutic contact lenses have attracted significant attention during the last decades. In this study, we used chitosan‐conjugated poly(2‐hydroxyethyl methacrylate) (PHEMA) for contact lens application. We aimed to increase affinity of anionic drugs, which are used in treatment of eye diseases. In this regard, we evaluated delivery of the small molecule anionic drug, ascorbic acid from the chitosan‐conjugated PHEMA. Chitosan immobilization improves drug loading efficiency and induces sustained release of ascorbic acid. The chitosan modified hydrogel also reduces the biofouling of tear fluid components. Our results showed that surface modification by chitosan inhibits protein and bacterial deposition on the contact lens. Protein absorption analysis revealed that neat PHEMA adsorbed tear proteins at a density of 28.4 ± 4.4 μg/cm2, whereas the chitosan‐conjugated hydrogel adsorbed tear proteins at a density of 18.5 ± 1.8 μg/cm2. Moreover, the neat PHEMA bacterial adhesion had a mean CFU value of 273 ± 27. However, a significant decrease in the number of bacterial colonies was observed in the chitosan group with a CFU value of 9 ± 6. [ABSTRACT FROM AUTHOR]

A large number of treatment approaches have been used for spinal cord injury improvement, a medically incurable disorder, and subsequently stem cell transplantation appears to be a promising strategy. The main objective of this study is to ascertain whether combinational therapy of human neural stem cells (hNSCs) together with lithium chloride improves cell survival, proliferation, and differentiation in a rat spinal contusion model, or not. Contusive spinal cord injury was implemented on Wistar male rats. Experimental groups comprised of: control, hNSCs transplanted, lithium chloride (Li), and hNSCs and lithium chloride (hNSCs + Li). In every experimental group, locomotor activity score and motor evoked potential (MEP) were performed to evaluate motor recovery as well as histological assessments to determine mechanisms of improvement. In accordance with our results, the hNSCs + Li and the Li groups showed significant improvement in locomotor scores and MEP. Also, Histological assessments revealed that transplanted hNSCs are capable of differentiation and migration along the spinal cord. Although NESTIN‐positive cells were proliferated significantly in the Lithium group in comparison with control and the hNSCs + Li groups, the quantity of ED1 cells in the hNSCs + Li was significantly larger than the other two groups. Our results demonstrate that combinational therapy of hNSCs with lithium chloride and lithium chloride individually are adequate for ameliorating more than partial functional recovery and endogenous repair in spinal cord‐injured rats. [ABSTRACT FROM AUTHOR]

Generating neural stem cells (NSCs) from astroglia as an abundant cell type in the mammalian brain has a promising outlook to be used in cell‐replacement therapy for treatment of neurodegenerative disorders and neuronal trauma. However, little is known about a single reprogramming factor that may lead to the generation of induced NSCs (iNSCs) from adult brain‐derived astrocytes in the absence of extrinsic inductive signals. Here, we show that zinc‐finger nuclear protein Zfp521 alone is sufficient for converting the adult mouse brain‐derived astrocytes into iNSCs. In vitro, Zfp521‐iNSCs demonstrated long‐term self‐renewal and multipotency and expressed related markers. Moreover, single‐seeded iNSCs were able to produce NSC colonies. These results suggest that application of Zfp521 to generate iNSCs could be regarded as a new approach for conversion of resident astrocytes into iNSCs in cell therapy for in vivo treatment of neural injuries. [ABSTRACT FROM AUTHOR]

Mesenchymal stromal cells (MSCs) can effectively contribute to tissue regeneration inside the inflammatory microenvironment mostly through modulating immune responses. MSC‐derived extracellular vesicles (MSC‐EVs) display immunoregulatory functions similar to parent cells. Interactions between MSC‐EVs and immune cells make them an ideal therapeutic candidate for infectious, inflammatory, and autoimmune diseases. These properties of MSC‐EVs have encouraged researchers to perform extensive studies on multiple factors that mediate MSC‐EVs immunomodulatory effects. Investigation of proteins involved in the complex interplay of MSC‐EVs and immune cells may help us to better understand their functions. Here, we performed a comprehensive proteomic analysis of MSC‐EVs that was previously reported by ExoCarta database. A total of 938 proteins were identified as MSC‐EV proteome using quantitative proteomics techniques. Kyoto Encyclopedia of Genes and Genomes analysis demonstrates that ECM–receptor interaction, focal adhesion, and disease‐specific pathways are enriched in MSC‐EVs. By detail analysis of proteins presence in immune system process, we found that expression of some cytokines, chemokines, and chemokine receptors such as IL10, HGF, LIF, CCL2, VEGFC, and CCL20, which leads to migration of MSC‐EVs to injured sites, suppression of inflammation and promotion of regeneration in inflammatory and autoimmune diseases. Also, some chemoattractant proteins such as CXCL2, CXCL8, CXCL16, DEFA1, HERC5, and IFITM2 were found in MSC‐EV proteome. They may actively recruit immune cells to the proximity of MSC or MSC‐EVs, may result in boosting immune response under specific circumstances, and may have protective role in infectious diseases. In this review, we summarize available information about immunomodulation of MSC‐EVs with particular emphasis on their proteomics analysis. Mesenchymal stromal cells‐derived EVs (MSC‐EVs) can regulate immune response post‐incorporating to innate and adoptive immune recipient cells similar to their corresponding parent cells with distinct protein cargoes. Herein, we summarize current in vitro and in vivo studies. In this way, to better understanding the mechanism underlying immune‐regulatory effects of MSC‐EVs, proteomic analysis was performed with emphasis on responsible proteins in regulation of immune response. [ABSTRACT FROM AUTHOR]

Organoids can be regarded as a beneficial tool for discovery of new therapeutics for diabetes and/or maturation of pancreatic progenitors (PP) towards β cells. Here, we devised a strategy to enhance maturation of PP by assembly of three‐dimensional (3D) pancreatic organoids (PO) containing human embryonic stem (ES) cell derivatives including ES‐derived pancreatic duodenal homeobox 1 (PDX1) + early PP, mesenchymal stem cells, and endothelial cells at an optimized cell ratio, on Matrigel. The PO was placed in a 3D‐printed tissue trapper and heterotopically implanted into the peritoneal cavity of immunodeficient mice where it remained for 90 days. Our results indicated that, in contrast to corresponding early PP transplants, 3D PO developed more vascularization as indicated by greater area and number of vessels, a higher number of insulin‐positive cells and improvement of human C‐peptide secretions. Based on our findings, PO‐derived β cells could be considered a novel strategy to study human β‐cell development, novel therapeutics, and regenerative medicine for diabetes. Organoids can be regarded as a beneficial tool for discovery of new therapeutics for diabetes and/or maturation of pancreatic progenitors (PP) towards β cells. Here, we devised a strategy to enhance maturation of PP by assembly of three‐dimensional (3D) pancreatic organoids (PO) containing human embryonic stem (ES) cell‐derivatives. The PO was placed in a 3D‐printed tissue trapper and heterotopically implanted into the peritoneal cavity of immunodeficient mice where it remained for 90 days. Our results indicate that 3D PO developed more vascularization, a higher number of insulin‐positive cells and improvement of human C‐peptide secretions. ES‐PP: ES cell‐derived pancreatic progenitors; ES‐MSC: ES cell‐derived mesenchymal stem cells; ES‐EC: ES cell‐derived endothelial cells; GF: growth factor; GFR: growth factor reduced; SM: small molecule; IP Tx: intraperitoneal transplantation [ABSTRACT FROM AUTHOR]

One of the major issues in cell therapy of myocardial infarction (MI) is early death of engrafted cells in a harsh oxidative stress environment, which limits the potential therapeutic utility of this strategy in the clinical setting. Increasing evidence implicates beneficial effects of omega‐3 fatty acids including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) and ascorbic acid (AA) in cardiovascular diseases, in particular their role in ameliorating fibrosis. In the current study, we aim to assess the cytoprotective role of EPA + DHA and AA in protecting embryonic stem cell (ESC)‐derived cardiac lineage cells and amelioration of fibrosis. Herein, we have shown that preincubation of the cells with EPA + DHA + AA prior to H2O2 treatment attenuated generation of reactive oxygen species (ROS) and enhanced cell viability. Gene expression analysis revealed that preincubation with EPA + DHA + AA followed by H2O2 treatment, upregulated heme oxygenase‐1 (HO‐1) along with cardiac markers (GATA4, myosin heavy chain, α isoform [MYH6]), connexin 43 [CX43]) and attenuated oxidative stress‐induced upregulation of fibroblast markers (vimentin and collagen type 1 [Col1]). Alterations in gene expression patterns were followed by marked elevation of cardiac troponin (TNNT2) positive cells and reduced numbers of vimentin positive cells. An injection of EPA + DHA + AA‐pretreated ESC‐derived cardiac lineage cells into the ischemic myocardium of a rat model of MI significantly reduced fibrosis compared to the vehicle group. This study provided evidence that EPA + DHA + AA may be an appropriate preincubation regimen for regenerative purposes. © 2019 BioFactors, 45(3):427–438, 2019 [ABSTRACT FROM AUTHOR]

Research that pertains to the molecular mechanisms involved in retinal pigment epithelial (RPE) development can significantly contribute to cell therapy studies. The effects of periocular mesenchymal cells on the expansion of RPE cells remain elusive. We have examined the possible proliferative role of hepatocyte growth factor (HGF) as a mesenchymal cell secretory factor against human embryonic stem cell derived RPE (hESC‐RPE). We found that the conditioned medium of human mesenchymal stem cells from apical papilla and/or exogenous HGF promoted proliferation of the hESC‐RPE cells as single cells and cell sheets, in addition to rabbit RPE sheets in vitro. Blockage of HGF signaling by HGF receptor inhibitor, PHA‐665752, inhibited proliferation of hESC‐RPE cells. However, differentiation of hESCs and human‐induced pluripotent stem cells to a rostral fate and eye‐field specification was unaffected by HGF. Our in vivo analysis showed HGF expression in periocular mesenchymal cells after optic cup formation in chicken embryos. Administration of HGF receptor inhibitor at this developmental stage in chicken embryos led to reduced eye size and disorganization of the RPE sheet. These findings suggested that HGF administration could be beneficial for obtaining higher numbers of hESC‐RPE cells in human preclinical and clinical trials. Dental mesenchymal stem cells originated from rostral neural crest secret hepatocyte growth factor (HGF), which promoted proliferation of hESC‐ derived RPE as a single cell and cell sheet in addition to rabbit RPE sheets in vitro. Inhibition of HGF secreted by periocular mesenchymal cells in chicken embryo led to reduced eye size and disorganization of the RPE sheets. [ABSTRACT FROM AUTHOR]

Cell death and differentiation appear to share similar cellular features. In this study, we aimed to investigate whether differentiation and mitochondrial cell death use a common pathway. We assessed the hallmarks of apoptosis during cardiomyocyte differentiation of human embryonic stem cells and found remarkable changes in P53, reactive oxygen species, apoptotic protease‐activating factor 1, poly[ADP‐ribose]polymerase 1, cellular adenosine triphosphate, and mitochondrial complex I activity. Furthermore, we observed reversible mitochondrial membrane permeabilization during cardiomyocyte differentiation accompanied by reversible loss of mitochondrial membrane potential, and these changes coincided with the fluctuating patterns of cytosolic cytochrome c accumulation and subsequent caspase‐9 and ‐3/7 activation. Moreover, the use of apoptosis inhibitors (BCL2‐associated X protein [BAX] inhibitor and caspase‐3/7 inhibitor) during differentiation impaired cardiomyocyte development, resulting in substantial downregulation of T, MESP1, NKX2.5, and α‐MHC. Additionally, although the expression of specific differentiation markers (T, MESP1, NKX2.5, MEF2C, GATA4, and SOX17) was enhanced in doxorubicin‐induced human embryonic stem cells, the stemness‐specific markers (OCT4 and NANOG) showed significant downregulation. With increasing doxorubicin concentration (0.03–0.6 µM; IC50 = 0.5 µM), we observed a marked increase in the expression of mesoderm and endoderm markers. In summary, we suggest that reversible mitochondrial outer membrane permeabilization promotes cardiomyocyte differentiation through an attenuated mitochondria‐mediated apoptosis‐like pathway. Mitochondria play a nonenergetic role during cardiomyocyte differentiation. An attenuated apoptosis‐like pathway promotes human embryonic stem cell–cardiomyocyte differentiation. Mitochondrial outer membrane permeabilization during cardiogenic differentiation does not lead to cell death. Doxorubicin increases the expression of some stage‐specific marker genes. [ABSTRACT FROM AUTHOR]

Critical‐sized bone defects constitute a major health issue in orthopedics and usually cause mal‐unions due to an inadequate number of migrated progenitor cells into the defect site or their incomplete differentiation into osteogenic precursor cells. The current study aimed to develop an optimized osteoinductive and angiogenic scaffold by incorporation of strontium (Sr) and bioglass (BG) into gelatin/nano‐hydroxyapatite (G/nHAp) seeded with bone marrow mesenchymal stem cells to enhance bone regeneration. The scaffolds were fabricated by a freeze‐drying technique and characterized in terms of morphology, structure, porosity and degradation rate. The effect of fabricated scaffolds on cell viability, attachment and differentiation into osteoblastic lineages was evaluated under in vitro condition. Micro computed tomography scan, histological and histomorphometric analysis were performed after implantation of scaffolds into the radial bone defects in rat. RT‐PCR analysis showed that G/nHAp/BG/Sr scaffold significantly increased the expression level of osteogenic and angiogenic markers in comparison to other groups (P < 0.05). Moreover, the defects treated with the BMSCs‐seeded scaffolds showed superior bone formation and mechanical properties compared to the cell‐free scaffolds 4 and 12 weeks post‐implantation. Finally, the BMSCs‐seeded G/nHAp/BG/Sr scaffold showed the greatest bone regenerative capacity which was more similar to autograft. It is concluded that combination of Sr, BG, and nHAp can synergistically enhance the bone regeneration process. In addition, our results demonstrated that the BMSCs have the potential to considerably increase the bone regeneration ability of osteoinductive scaffolds. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 107B: 50–64, 2019. [ABSTRACT FROM AUTHOR]

The present work reports the beneficial effects of using a microplatform on the development of mouse single blastomeres (SBs) to the blastocyst stage. Development of blastocysts from SBs separated from two‐ and four‐cell stage embryos (two‐ and four‐cell SBs) can provide a valuable supply both for couples who use fertility‐assisted techniques and farm animals. As a step forward, we introduce three chips that provide the possibility of culturing SBs separately, in groups, and in the vicinity of the intact embryo (co‐culture), while each well of the chips is assigned to an isolated SB. Two‐ and four‐cell SBs co‐cultured with intact embryos showed 97.1% and 76.6% developmental rates and up to 34.1% and 49.1% growth relative to the microdroplet method (control). We examined the quality of developed blastocysts by assessing the total cell number, the number of inner cell mass (ICM) according to the octamer‐binding transcription factor 4 marker (OCT4), and trophectoderm (TE). Co‐culture of SBs with an intact embryo in a chip with nanoscale culture medium volume also increased the cell population of the developed embryo. The ICM:TE ratio, which is the most important blastocyst quality parameter, also indicated that developed two‐cell SBs have a higher degree of similarity to intact embryos despite fewer numbers of total cells. The present work, for the first time, reports the beneficial effects of using a microplatform on development of mouse single blastomeres (SBs) to the blastocyst stage. Three chips were introduced that provide the possibility of culturing SBs separately, in groups, and in the vicinity of the intact embryo. The quality of developed blastocysts was also examined. [ABSTRACT FROM AUTHOR]

Various somatic tissue‐derived mesenchymal stromal cells (MSCs) have been considered as an attractive therapeutic tool for treatment of liver diseases in which the secretion of soluble factors or extracellular vesicles (EVs) is the most probable mechanism. The experimental application of human embryonic stem cell‐derived MSC (ES‐MSC) increased rapidly and showed promising results, in vitro and in vivo. However, possible therapeutic effects of human ES‐MSC and their EVs on Thioacetamide (TAA)‐induced chronic liver injury have not been evaluated yet. Our data indicated that human ES‐MSC can significantly suppress the proliferation of peripheral blood mononuclear cells compared to bone marrow (BM)‐MSC and adipose (AD)‐MSC. Moreover, ES‐MSC increased the secretion of anti‐inflammatory cytokines (i.e., TGF‐β and IL‐10) and decreased IFN‐γ, compared to other MSCs. ES‐MSC EVs demonstrated immunomodulatory activities comparable to parental cells and ameliorated cirrhosis in TAA‐induced chronic rat liver injury, that is, reduction in fibrosis and collagen density, necrosis, caspase density, portal vein diameter, and transaminitis. The gene expression analyses also showed upregulation in collagenases (MMP9 and MMP13), anti‐apoptotic gene (BCL‐2) and anti‐inflammatory cytokines (TGF‐β1 and IL‐10) and down‐regulation of major contributors to fibrosis (Col1α, αSMA, and TIMP1), pro‐apoptotic gene (BAX) and pro‐inflammatory cytokines (TNFα and IL‐2) following treatment with ES‐MSC and ES‐MSC‐EV. These results demonstrated that human ES‐MSC and ES‐MSC EV as an off‐the‐shelf product, that needs further assessment to be suggested as an allogeneic product for therapeutic applications for liver fibrosis. In present study, we found the superiority of ES‐MSC and ES‐MSC EVs regarding immunomodulatory potential in comparison to those from BM‐ and AD‐MSCs through the secretion of anti‐inflammatory cytokines in vitro. ES‐MSCs and ES‐MSC EVs also offer anti‐fibrotic, anti‐necrotic and immunosuppressive effects to modulate TAA‐induced cirrhotic livers after successfully engraftment in recipient damaged livers. To our knowledge, this is the preliminary evidence explaining hepatoprotective effects of ES‐MSCs and their EVs in chronic liver disease model. [ABSTRACT FROM AUTHOR]

Abstract: Background: Most two‐ dimensional in vitro models for studying host‐ H. pylori interactions rely on tumor‐derived cell lines, which harbor malignant alterations. The recent development of human gastric organoids has overcome this limitation and provides a highly sophisticated, yet costly, short‐term model for H. pylori infection, with restricted use in low‐budget centers. Method: Tissue specimens from upper, middle, and lower stomachs of H. pylori‐negative volunteers were collectively dispersed and cultured on mouse embryonic fibroblast (MEF) or collagen‐coated plates. Gastric primary cells (GPCs) were evaluated by light microscopy, immunostaining, qRT‐PCR and ELISA analysis of cellular secretions, before and after H. pylori infection. Results: The formation and long‐term (up to 1 year) maintenance of GPCs was highly dependent on adherent inactivated MEF cells, cultured in enriched media. These cells were multipassageable and able to undergo stable freezer storage and subsequent revival. The cellular composition of GPCs included the combination of cytokeratin 18 (CK18) and E‐cadherin (E‐cad)‐positive epithelial cells, MUC5AC‐positive gastric cells, and leucine‐rich repeat containing G protein‐coupled receptor 5 (LGR5)‐positive progenitor cells. These cells produced significant amounts of gastric pepsinogens I and II. GPCs also allowed for extended (up to 96 hours) H. pylori infection, during which they underwent morphological alterations (cellular vacuolation and elongation) and hyperproduction of gastric pepsinogens and inflammatory cytokines (IL‐8 and TNF‐α). Conclusion: We, hereby, present a simple, consistent, and cost‐efficient gastric cell culture system, which provides a suitable model for extended in vitro infection of H. pylori. This platform can be employed for a variety of gastric‐related research. [ABSTRACT FROM AUTHOR]

Human embryonic stem cell (hESC)-derived endodermal cells are of interest for the development of cellular therapies to treat disorders such as liver failure. The soluble form of activin A (Act) has been widely used as an in vitro inducer of definitive endoderm (DE). In this study, we have developed a nanofibrous poly (ɛ-caprolactone) substrate, biofunctionalized with Act, for directed differentiation of hESCs into DE. Bioconjugation of Act on nanofibrous meshes was confirmed by enzyme-linked immunosorbent assay (ELISA) and immunostaining. In order to investigate the bioactivity of immobilized Act (iAct), hESCs were cultivated on the Act-conjugated nanofibers for five days. The nanofibers with covalent iAct significantly increased expression levels of the endodermal markers SOX17, FOXA2, and CXCR4, compared with physically adsorbed Act (aAct) or without Act (noAct). In addition, iAct retained its bioactivity after storage for five days in the absence of cell seeding. The capability of cultivated cells to generate the DE-derived lineage was evaluated through further differentiation of seeded cells into hepatocyte-like cells (HLCs). Interestingly, the iAct sample showed a higher level of hepatic markers compared to the aAct sample. We also demonstrated that iAct in the presence of soluble Act (sAct) could improve the conventional protocol to generate HLCs from hESCs. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2861-2872, 2016. [ABSTRACT FROM AUTHOR]

Objectives To evaluate the effect of autologous muscle-derived cells injection in the treatment of complicated stress urinary incontinence in female patients. Methods Female patients presenting with severe and complicated stress urinary incontinence secondary to the bladder neck and/or urethral trauma or congenital epispadias (with or without exstrophy) were enrolled in this prospective study. They underwent transurethral injection of autologous muscle-derived cells. In selected cases, another injection was given after 6 months, as per the surgeon's assessment. All patients were monitored for 1 year, and the effect of autologous muscle-derived cells was evaluated by cough stress test, 1-h pad test and Incontinence Impact Questionnaire-short form score. A multichannel urodynamic study and maximum urethral closure pressure were carried out before and 12 months after the last treatment session. Cough stress test, 1-h pad test and uroflowmetry were repeated 36 months after the last injection. Severity and occurrence of complications were recorded at each visit. Results All 10 patients who completed the study were monitored for 36 months. Three patients were cured, four had improved and three did not respond to the treatment. There was no major adverse effect related to the treatment. Conclusions Muscle-derived cell therapy might represent a minimally-invasive and a safe procedure in the treatment of patients with severe and complicated stress urinary incontinence. [ABSTRACT FROM AUTHOR]

ABSTRACT Following myocardial infarction, heart muscle has a limited capacity of self-healing. Biological platforms providing the natural biochemical and biophysical cues of the native myocardium might be crucial to address current therapeutic shortcomings. The aim of this study was to assess the effect of decellularized human pericardium (DPc), as a bioactive platform, on viability, attachment, proliferation and differentiation of human cardiac progenitors (CPs), and evaluate the possibility of using DPc as a substitute of tissue culture polystyrene (TCPS) substrate for culturing CPs in vitro. The decellularization process removed more than 99% of the cellular components from Pc, yet well preserved its macro-/micro-structure and extracellular collagen and glycosaminoglycan content. DPc supported the viability, attachment, metabolism and proliferation of CPs, and enhanced their differentiation into mature cardiomyocytes compared to TCPS. Decellularized pericardium appeared thus to have a high potential for cardiac cell culture and could be applied as a superior alternative to common TCPS. DPc could be then utilized for further tissue engineering applications. © 2015 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2016, 133, 43255. [ABSTRACT FROM AUTHOR]

Derivation of definitive endoderm (DE) from human embryonic stem cells (hESCs) can address the needs of regenerative medicine for endoderm-derived organs such as the pancreas and liver. Fibrous substrates which topographically recapitulate native extracellular matrix have been known to promote the stem cell differentiation. However, the optimal fiber diameter remains to be determined for the desired differentiation. Here, we have developed a simple method to precisely fabricate electrospun poly(ε-caprolactone) fibers with four distinct average diameters at nano- and microscale levels (200, 500, 800, and 1300 nm). Human ESCs were cultured as clumps or single cells and induced into DE differentiation to determine the optimal topography leading to the promoted differentiation compared with planar culture plates. Gene expression analysis of the DE-induced cells showed significant upregulation of DE-specific genes exclusively on the 200-nm fibers. By Western blot analysis, significant expression of DE-specific proteins was found when hESCs were cultured on the 200 nm substrate as single cells rather than clumps, probably due to more efficient cell-matrix interaction realized by morphological observations of the cell colonies. The results indicated that nanofibrillar substrates, only at ultrathin fiber diameters, provided a better environment for DE differentiation of hESC, which holds great promise in prospective tissue engineering applications. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 103A: 3539-3553, 2015. [ABSTRACT FROM AUTHOR]

A novel biodegradable electroactive polyurethane containing aniline pentamer (AP) was blended with polycaprolactone (PCL). The prepared blend (PB) and PCL were further fabricated in to scaffolds using a mixture of poly(ethylene glycol) and salt particles in a double porogen particulate leaching and compression molding methodology. Scaffolds held open and interconnected pores having pore size ranging from several μm to 150 µm. PB scaffolds had compression modulus and strength of 4.1 and 1.3 MPa, respectively. The conductivity of the scaffold was measured as 10−5 ± 0.09 S .cm−1 and preserved for at least 100 h post fabrication. Scaffolds supported neonatal cardiomyocytes adhesion and growth with PB showing more extensive effect on the expression of the cardiac genes involved in muscle contraction and relaxation (troponin-T) and cytoskeleton alignment (actinin-4). Our results highlight the potential of incorporation of AP as an electroactive moiety for induction of cardiomyocyte proliferation and repair of damaged heart tissue. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 103A: 3179-3187, 2015. [ABSTRACT FROM AUTHOR]

Fndc5 has been recently recognized as a myokine which could be cleaved and secreted into blood stream. It is termed as irisin with an important role in thermogenesis and energy homeostasis. Increased expression of Fndc5 has been reported upon retinoic acid treatment during neural differentiation and its knockdown decreased neural differentiation and neurite outgrowth. This study tries to evaluate the effect of Fndc5 overexpression on rate of neural differentiation in mouse. (Thus, transduced cell line of mouse embryonic stem cell with ability to express Fndc5 under Doxycycline treatment was established. Subsequently, the effect of overexpression of Fndc5 on different stages of neural differentiation was studied). Our study showed an increase enhancement in neuronal precursor markers and mature neuron markers upon overexpression of Fndc5, concluding that Fndc5 facilitates neural differentiation. This effect might be related to increased expression of BDNF following overexpression of Fndc5. Our findings are consistent with recent studies reporting a similar role for Fndc5 in proliferation of neural cells and increase in the expression of neurotrophins like BDNF. [ABSTRACT FROM AUTHOR]

Artificial nanofiber nerve guides have gained huge interest in bridging nerve gaps and associated peripheral nerve regeneration due to its high surface area, flexibility and porous structure. In this study, electrospun poly (ε-caprolactone)/gelatin (PCL/Gel) nanofibrous mats were fabricated, rolled around a copper wire and fixed by medical grade adhesive to obtain a tubular shaped bio-graft, to bridge 10 mm sciatic nerve gap in in vivo rat models. Stem cells from human exfoliated deciduous tooth (SHED) were transplanted to the site of nerve injury through the nanofibrous nerve guides. In vivo experiments were performed in animal models after creating a sciatic nerve gap, such that the nerve gap was grafted using (i) nanofiber nerve guide (ii) nanofiber nerve guide seeded with SHED (iii) suturing, while an untreated nerve gap remained as the negative control. In vitro cell culture study was carried out for primary investigation of SHED-nanofiber interaction and its viability within the nerve guides after 2 and 16 weeks of implantation time. Walking track analysis, plantar test, electrophysiology and immunohistochemistry were performed to evaluate functional recovery during nerve regeneration. Vascularization was also investigated by hematoxilin/eosine (H&E) staining. Overall results showed that the SHED seeded on nanofibrous nerve guide could survive and promote axonal regeneration in rat sciatic nerves, whereby the biocompatible PCL/Gel nerve guide with cells can support axonal regeneration and could be a promising tissue engineered graft for peripheral nerve regeneration. © 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 102A: 4554-4567, 2014. [ABSTRACT FROM AUTHOR]

Transdifferentiation or direct reprogramming of somatic cells into neural lineage cells has provided an invaluable new tool to advance the regenerative neural medicine. Here, we provide an overview of the various strategies currently available for producing of induced neural lineage cells in vitro as well as the direct reprogramming of neural cells in vivo. We also discussing some of the challenges faced in harnessing the potential of induced neural lineage cells for biomedical applications. J. Cell. Physiol. 229: 728-742, 2014. © 2013 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

The hedgehog (Hh) signalling pathway is one of the key regulators in development with a dual role in cell fate specification, proliferation, and survival on different target cells. We have investigated the effect of recombinant sonic hedgehog (r-SHH) on extracted multipotent stem cells from human exfoliated deciduous teeth (SHED), which represent a potential stem cell population for therapeutic applications. Cell proliferation and cycle assays shown that r-SHH did not have a distinctive effect on cell cycle progression, nor did it increase cell number over a wide range of concentrations. Quantitative polymerase chain reaction (Q-PCR) also suggests that r-SHH treatment has no demonstrable influence on expression of proliferative genes ( CCNE1 and KI67); in contrast, the anti-proliferative gene ( CDKN1A) is overexpressed in response to SHH. Our findings have suggested the possibility that SHEDs demonstrate a different potential from human bone marrow mesenchymal stem cells (h-BMSCs) and dorsal neural progenitor in response to growth factors such as SHH. [ABSTRACT FROM AUTHOR]

Understanding the extracellular matrix (ECM) effect on pancreatic β cells is critical to optimise the derivation of functional β cells for transplantation and understand mechanisms that control islet neogenesis and glucose homeostasis. We assessed the effect of natural ECMs [collagen I, collagen IV, laminin and fibronectin (FN)] on rat islets of Langerhans' morphology, adhesion, viability, functionality and islet specific genes expression after 7 days in vitro culture. However, we could not detect a significant difference on the other parameters in these ECMs and islets interaction. To examine islets interactions, we used a synthetic three-dimensional surface composed of electrospun polyamide nanofibres. Laminin-coated nanofibrillar surfaces, but not laminin or nanosurface alone, induced comparable expression of the Ins1 and Ins2 genes in adult β cells. Using a glucose challenge test, a marked response of insulin secretion by islets occurred that were cultured on laminin-coated nanofibrillar surfaces. We contend that the reestablishment of cellular interactions by the combination of nanomaterials and natural ECMs can be useful in maintaining in vitro islet functions. [ABSTRACT FROM AUTHOR]

Despite its numerous limitations, liver transplants are the only definite cure for end-stage liver disease. Various stem cell populations may contribute to liver regeneration, of which there is accumulating evidence of the contribution of mesenchymal stem cells (MSCs). This study examines the hypothesis that repeated infusions of human bone marrow-derived MSCs (hBM-MSCs) can improve liver injury in an experimental model. MSCs were intravenously transplanted into immunosuppressed mice with carbon tetrachloride (CCl4)-induced liver fibrosis. Transplanting 3 × 106 MSCs in three divided doses improved survival, liver fibrosis and necrosis compared with injection of the same number of MSCs in a single dose. This was accompanied by increased influence on the expression of the fibrogenic/fibrolytic related genes Col1a1, Timp1 and Mmp13 in the repeated transplant group. Repeat administration of MSCs was three times more effective in homing of PKH-tagged transplanted cells 3 weeks post-transplant compared with the single transplant group. The benefits of repeated transplants may be of considerable significance in clinical trials on liver failure. [ABSTRACT FROM AUTHOR]

A critical element in tissue engineering involves the fabrication of a three-dimensional scaffold. The scaffold provides a space for new tissue formation, supports cellular ingrowth, and proliferation and mimics many roles of the extracellular matrix. Poly(3-hydroxybutyrate) (PHB) is the most thoroughly investigated member of the polyhydroxyalkanoates (PHAs) family that has various degrees of biocompatibility and biodegradability for tissue engineering applications. In this study, we fabricated PHB scaffolds by utilizing electrospinning and salt-leaching procedures. The behavior of monkey epithelial kidney cells (Vero) and mouse mesenchymal stem cells (mMSCs) on these scaffolds was compared by the MTS assay and scanning electron microscopy. Additionally, this study investigated the mechanical and physical properties of these scaffolds by measuring tensile strength and modulus, dynamic contact angle and porosity. According to our results, the salt-leached scaffolds showed more wettability and permeability, but inferior mechanical properties when compared with nanofibrous scaffolds. In terms of cell response, salt-leached scaffolds showed enhanced Vero cell proliferation, whereas both scaffolds responded similarly in the case of mMSCs proliferation. In brief, nanofibrous scaffolds can be a better substrate for cell attachment and morphology. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A 2012. [ABSTRACT FROM AUTHOR]

Abstract: The dynamics of chromatin structure are tightly regulated by multiple epigenetic mechanisms such as histone modifications and incorporation of histone variants. In the current work, differentiation of an embryonal carcinoma cell line, NT2, was induced by retinoic acid, and total histone proteins were compared throughout this process. The results showed a significant change in expression level of a variant of H1 histone named H1x. Chromatin immunoprecipitation coupled with real-time PCR analysis demonstrated a preferential incorporation of this protein in the regulatory region of Nanog, a marker gene of stemness that is significantly suppressed in differentiated cells. This finding reveals a dynamic role of H1x in differentiation, and implies a repressive role for this histone variant. [ABSTRACT FROM AUTHOR]