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Despite the numerous advantages of PDMS-based substrates in various biomedical applications, they are limited by their highly hydrophobic surface that does not optimally interact with cells for attachment and growth. Hence, the lack of lengthy and straightforward procedures for high-density cell production on the PDMS-based substrate is one of the significant challenges in cell production in the cell therapy field. In this study, we found that the PDMS substrate coated with a combination of polydopamine (PDA) and laminin-511 E8 fragments (PDA + LME8-coated PDMS) can support human-induced pluripotent stem cell (hiPSC) attachment and growth for the long term and satisfy their demands of differentiation into cardiomyocytes (iCMs). Compared with prior studies, the density of hiPSCs and their adhesion time on the PDMS surface were increased during iCM production. Although the differentiated iCMs beat and produce mechanical forces, which disturb cellular attachments, the iCMs on the PDA + LME8-coated PDMS substrate showed dramatically better attachment than the control condition. Further, the substrate required less manipulation by enabling one-step seeding throughout the process in iCM formation from hiPSCs under animal-free conditions. In light of the results achieved, the PDA + LME8-coated PDMS substrate will be an up-and-coming tool for cardiomyocyte production for cell therapy and tissue engineering, microfluidics, and organ-on-chip platforms.

Background: Chinese hamster ovary (CHO) cells are the widely used mammalian cell host for biopharmaceutical manufacturing. During cell cultures, CHO cells lose viability mainly from apoptosis. Inhibiting cell death is useful because prolonging cell lifespans can direct to more productive cell culture systems for biotechnology requests. Objectives: This study exploited a CRISPR/Cas9 technology to generate site-specific gene disruptions in the caspase-3 gene in the apoptosis pathway, which acts as an apoptotic regulator to extend cell viability in the CHO cell line. Methods: The STRING database was used to identify the key pro-apoptotic genes to be modified by CRISPR/Cas9 system. The guide RNAs targeting the caspase-3 gene were designed, and vectors containing sgRNA and Cas9 were transfected into CHO cells that expressed erythropoietin as a heterologous protein. Indel formation was investigated by DNA sequencing. Caspase-3 expression was quantified by real-time PCR and western blot. The effect of editing the caspase-3 gene on the inhibition of apoptosis was also investigated by induction of apoptosis in manipulated cell lines by oleuropein. Finally, the erythropoietin production in the edited cells was compared to the control cells. Results: The caspase-3 manipulation significantly prolongation of the cell viability and decreased the caspase-3 expression level of protein in manipulated CHO cells (more than 6-fold, P-value < 0.0001). Manipulated cells displayed higher threshold tolerance to apoptosis compared to the control cells when they were induced by oleuropein. They show a higher IC50 than the control ones (7271 µM/mL Vs. 5741 µM/mL). They also show a higher proliferation rate than the control cells in the presence of an apoptosis inducer (P-value < 0.0001). Furthermore, manipulated cell lines significantly produce more recombinant protein in the presence of 2,000 µM oleuropein compared to the control ones (P-value = 0.0021). Conclusions: We understood that CRISPR/Cas9 could be effectively applied to suppress the expression of the caspase-3 gene and rescue CHO cells from apoptosis induced by cell stress and metabolites. The CRISPR/Cas9 system-assisted caspase-3 gene ablation can potentially increase erythropoietin yield in CHO cells.

Direct neuronal reprogramming of somatic cells into induced neurons (iNs) has been recently established as a promising approach to generating neuron cells. Previous studies have reported that the biophysical cues of the

In the recent years, tissue engineering researchers have exploited a variety of biomaterials that can potentially mimic extracellular matrix (ECM) for tissue regeneration. Natural cellulose, mainly obtained from bacterial (BC) and plant-based (PC) sources, can serve as a high potential scaffold material for different regenerative purposes. Natural cellulose has drawn the attention of researchers due to its advantage over synthetic cellulose in terms of availability, cost-effectiveness, perfusablility, biocompatibility, negligible toxicity, mild immune response and due to imitating native tissues. In this article, we will review the recent in vivo and in vitro studies aimed to assess the potentials of natural cellulose for the purpose of soft (skin, heart, veins, nerve, among others) and hard (bone and tooth) tissue engineering.

Growth factors such as human epidermal growth factor (hEGF) have recently received high interest in regenerative medicine and pharmaceutical industries mainly due to their ability to restore tissues proliferation and improvement of their biological functions. In spite of various hEGF applications, its efficient expression in Escherichia coli could not yet reach an industrial reality mainly due to the lack of the ability of folding into the correct 3D structure because of three disulfide bonds in monomer hEGF. To address these challenges, here a fusion hEGF protein with a C terminus of collagen binding domain (CBD) along with intein protein with self-splicing property and ELP sequence was constructed by a three-step cloning procedure. This enabled us to purify recombinant hEGF without using chromatography columns. Following the confirmation of the construct by colony PCR, restriction enzymes analysis and sequencing, the 62[Formula: see text]kDa band of ELP-INTEIN-hEGF-CBD were observed on SDS-PAGE and confirmed by western blotting. Subsequently, the mitotic activity in Balb/c 3T3 cells proliferation in presence of recombinant hEGF-CBD compared with commercial hEGF using methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay which showed that our purified recombinant protein stimulates the cell proliferation similar to the commercial protein. Our strategy could be considered as a new feasible approach to produce hEGF in E. coli for pharmaceutical and clinical applications.

Various degenerative diseases, traumatic injuries and cancers are almost hard to treat with conventional therapies, causing death or at least permanent disability. The use of multipotent adipose-derived mesenchymal stem cells (ADMSCs) for cell-based therapeutic applications has lately drawn elevated attention primarily due to their potential for differentiation into mesodermal lineages, which can finally be converted into cardiomyocytes to replace present invasive methods used to treat cardiac diseases. However, some reports hindered their clinical implementation due to concerns about efficacy scalability and reproducibility. In this research, we tested the impacts of using multiple natural small molecules and plant extract on the effectiveness of differentiating human ADMSCs into the definitive mesoderm lineage and cardiac progenitors. First, mesenchymal stem cells separated from human adult fat tissue, are propagated and characterized using flow cytometry and appropriate markers for this purpose. Second, passage cells are transferred to 24 well plates and treated with various plant-derived small molecules — primarily one of the most preserved intracellular signals, WNT/[Formula: see text]-catenin stimulators including resveratrol, stilbene, and multiple plant extracts — and assessed their impacts on differentiation into the definitive mesoderm and cardiac lineages. Obtained results revealed that our suggested strategy to differentiation is more viable and safer than present approaches. Taken together, data presented here showed that in vitro differentiation using plant-derived small molecules could be a potential way to improve the effectiveness of their final differentiation into definitive cardiomyocytes for in vivo applications.

Background: The CRISPR-Cas9 system, a powerful tool, has revolutionized genome engineering in eukaryotic cells and living organisms. However, this approach poses unique concerns and limitations when used by conventional transfection methods, including limited packaging size and low delivery efficiency. Here, we aim at assessing the transfection efficiency of DNA encoding for the CRISPR-Cas9 system by PEI coated Magnetic NanoParticle (MNPs) to improve the delivery of CRISPR/Cas9 constructs into eukaryotic cells. Results: Superparamagnetic iron oxide nanoparticles (SPIONs) coated with polyethylenimine (PEI) and then complexed with pCXLE-dCas9VPH-T2A-GFP-shP53 plasmid DNA. We used HEK-293 (human embryonic kidney) and Human foreskin fibroblasts (HFF) cells to express GFP after transfection to evaluate delivery efficiency with MNPs and Lipofection methods. PEI-coated nanoparticles with magnetic iron oxide core were synthesized by co-precipitation technique resulting in an average size of ~ 20 nm in diameter. Characterization of Magnetic Nano Particle (MNPs) revealed that particles have narrow size distribution sufficient colloidal stability. The result showed that the magnetofection method with an efficiency around 85.7% for HEK-293 and 28.2% for HFF. Also, transfection efficiency by lipofection method was 83.2% and 7.89% for HEK-293 and HFF respectively. Conclusion: The magnetofection was revealed to be more efficient than classic Lipofectamine transfection as measured by GFP expression. We show that PEI-MNPs enable effective delivery and improved safety of plasmids encoding CRISPR/Cas9 into eukaryote cells.

Despite various efforts to produce strong recombinant bio-adhesive proteins for medical purposes, efficient production of a safe and feasible bio-glue is not yet a commercial reality due to the weak properties or low expression level. Here, a feasible expression system has been developed to produce strong recombinant fusion protein using Mfp3 and Mfp5 along with two curli proteins, GvpA and CsgA expressed under the control of alcohol oxidase (AOX1) promoter for high-level production in P. pastoris using pPICZα vector. Purified chimeric proteins were first evaluated using western blotting and its remaining dopa were measured in the modified proteins by NBT assay. We further elucidated mechanistic properties of obtained adhesive protein assembly in various pH based on its different subunits using atomic force microscopy (AFM) when adsorbed onto the mica surface. We found that combinational structural features of subunits and post-translational changes during expression in yeast host have led to potent adherence due to higher dopa residues majorly in acidic condition and tetrad complex that is higher than that of earlier reports in prokaryotic systems. We believe that our obtained chimeric protein resulted through fusion of GvpA and CsgA proteins with dopa-containing Mfp proteins expressed in the methylotrophic yeast, P. pastoris, not only presents a candidate for future biomedical applications, but also provides novel biological clues using for high-performance bioinspired biomaterials designation.

Despite various efforts to produce potent recombinant bio-adhesive proteins for medical purposes, efficient production of a safe and feasible bio-glue is not yet a commercial reality due to the weak properties or low expression levels. Here, a feasible expression system has been developed to produce strong recombinant fusion bioinspired protein using mussel foot protein 3 and 5 (Mfps) along with gas vesicle protein A (GvpA) of Anabaena flos-aquae, and a curli protein CsgA from E. coli, expressed under the control of alcohol oxidase (AOX1) promoter for high-level production in yeast P. pastoris using pPICZα vector. Purified chimeric proteins were first evaluated using western blotting, and their remaining dihydroxyphenylalanine (DOPA) was measured in the modified proteins by NBT assay. We further elucidated the mechanistic properties of obtained adhesive protein assembly in various pH levels based on its different subunits using atomic force microscopy (AFM) when adsorbed onto the mica surface. We found that both combinational structural features of subunits and post-translational changes during expression in yeast host have led to potent adherence due to higher DOPA residues specially in acidic condition and tetrad complex which is higher than that of earlier reports in prokaryotic systems. We believe that our obtained chimeric protein resulted from the fusion of GvpA and CsgA proteins with DOPA-containing Mfp proteins, expressed in the methylotrophic yeast, P. pastoris, not only presents a candidate for future biomedical applications but also provides novel biological clues used for high-performance bioinspired biomaterial designation. Graphical Abstract

Herbal-derived three-dimensional scaffolds have a unique structure that represents the natural cellular microenvironment and can be potentially used for tissue engineering applications. In the present study, cabbage (Cb) leaves were decellularized and then their characteristics, such as surface roughness, wettability, porosity, mechanical properties, and specific surface area, were investigated. After that, scaffold osteoinductivity was studied by bone-marrow-derived mesenchymal stem cells (BM-MSCs) osteogenic differentiation while growing on the decellularized Cb leaves. Cells mineralization, calcium secretion, alkaline phosphatase (ALP) activity, and expression levels of bone-related genes were determined during the differentiation process. Our results from the structural characterization of the scaffolds demonstrated that decellularized Cb leaves are good candidates for bone differentiation in terms of surface roughness, mechanical properties, and interconnected pores. Osteogenic differentiation evaluation of the BM-MSCs determined that the cell's ALP activity and mineralization were increased significantly while cultured on the decellularized Cb leaves compared to the cells cultured on the culture plate as a control. Besides, Runx2, ALP, collagen-1 (Col-I), and osteocalcin genes were expressed in cells cultured on decellularized Cb leaves significantly higher than cells cultured on the culture plate. Based on these results, it can be concluded that the decellularized Cb scaffold has great potential for promoting BM-MSCs proliferation and osteogenic differentiation.

In recent years, tissue engineering researchers have exploited a variety of biomaterials that can potentially mimic the extracellular matrix (ECM) for tissue regeneration. Natural cellulose, mainly obtained from bacterial (BC) and plant-based (PC) sources, can serve as a high-potential scaffold material for different regenerative purposes. Natural cellulose has drawn the attention of researchers due to its advantages over synthetic cellulose including its availability, cost effectiveness, perfusability, biocompatibility, negligible toxicity, mild immune response, and imitation of native tissues. In this article, we review recent in vivo and in vitro studies which aimed to assess the potential of natural cellulose for the purpose of soft (skin, heart, vein, nerve, etc.) and hard (bone and tooth) tissue engineering. Based on the current research progress report, it is sensible to conclude that this emerging field of study is yet to satisfy the clinical translation criteria, though reaching that level of application does not seem far-fetched.

Bio-adhesives play a pivotal role in a wide range of medical applications. However, there are some problems about their application in different pH values and low adhesion force under wet conditions. Here, we report new recombinant fusion protein achieved by mussel foot proteins (Mfps) of Mytilus Californianus and gas vesicle protein A (GvpA) of Anabaena flos-aquae by genetic engineering methods. Transmission electron microscopy verified that these chimeric proteins self-assembled into β-sheet rich fibres because of GvpA amyloid structure. Also, in comparison to other studies, their adhesion forces were significantly increased, especially in the alkaline environments based on Mfp-3 and Mfp-5, confirmed by the colloidal probe atomic force microscope. This study illustrates that copolymer of Mfp-5-GvpA:GvpA-Mfp-3 can be used as a sturdy underwater adhesive with tolerance to auto-oxidation, especially at basic conditions.

Background and objectives: Despite the advances made in the prevention and treatment of cardiovascular diseases (CVD) in the last decade, they are still the leading cause of death in males at the rate of 50% worldwide. Considering the protective role of estrogen to decrease CVD rates in young females, it was suggested that using hormone therapy can be considered to improve heart regeneration. Using in vitro induced pluripotent stem cells (iPSCs) has become one of the most significant tools in CVD treatment in both genders. We design a novel optimal protocol for the differentiation of iPSCs to cardiomyocytes which may be valuable for CVD treatment in men. Methods: Human iPSCs were initially cultivated on mouse embryonic fibroblasts and then, transferred to a specific culture medium for differentiation process. In vitro differentiation of iPSCs into cardiomyocytes was induced at three phases on RPMI-1640 medium including CHIR99021 (5 µM) on days 0–3, BMP4 (20 ng/mL), and bFGF (100 ng/mL) on days 3–5, 10 µM of XAV939 on 6–8, and phytoestrogen + ascorbic acid on days 8–13. Scanning electron microscopy and Real-time PCR using specific primers were applied to confirm produced cardiomyocytes.Results: We found that the simultaneous use of small chemical molecules such as CHIR99021 and XAV 939, growth factors, such as BMP4, bFGF, and herbal-derived phytoestrogen from red clover could efficiently differentiate hiPSCs from the mesoderm and cardiomyocytes after 13 days. Using phytoestrogen increased the induction of cardiac markers including cTnT and GATA-4 in a shorter time; consequently, the proposed formulation has the potential to be used in developing a novel approach for cardiac repair or regeneration.Conclusion: Presented data indicated that the serial use of XAV939 and phytoestrogen at different times and stages can successfully induce cardiogenesis from hiPSCs. Thus, the proposed approach can be used for improved translational strategies for cardiac regeneration with fewer side effects.

The challenge of tissue engineering remains for introducing the best substrate to support stem cell growth and proliferation, and also repair of damaged tissues. Scaffolds made of plant tissues can be considered as suitable substitutes for extracellular matrix. In this study, the outer skin of the onion was decellularized and after characterization, its supportive role on osteogenic differentiation was investigated by the culture of human mesenchymal stem cells (MSCs). Structural characterizations by Atomic Force and Scanning electron microscopes, Infrared spectroscopy and Brunauer-Emmett-Teller, Tensile, and Contact angle tests demonstrated that the decellularized scaffold has a proper 3D structure with interconnected pores, and moderate surface roughness. The Osteogenic differentiation evaluation revealed that alkaline phosphatase activity and calcium deposition in MSCs differentiated on the decellularized onion scaffold was significantly higher than those cells differentiated on tissue culture polystyrene as a 2D control. In addition, the expression level of common bone-related genes in MSCs differentiated on the decellularized onion scaffold was also increased significantly higher than those cells cultured on the 2D control group. These data demonstrated that 3D structure of the decellularized onion scaffold with supporting stem cell proliferation and differentiation has promising potential for use in tissue engineering as a new cost-effective and green scaffold technology.

Induced pluripotent stem cells (iPSCs) can self-renew indefinitely in culture and differentiate into all specialized cell types including gametes. iPSCs do not exist naturally and are instead generated (“induced” or “reprogrammed”) in culture from somatic cells through ectopic co-expression of defined pluripotency factors. Since they can be generated from any healthy person or patient, iPSCs are considered as a valuable resource for regenerative medicine to replace diseased or damaged tissues. In addition, reprogramming technology has provided a powerful tool to study mechanisms of cell fate decisions and to model human diseases, thereby substantially potentiating the possibility to (i) discover new drugs in screening formats and (ii) treat life-threatening diseases through cell therapy-based strategies. However, various legal and ethical barriers arise when aiming to exploit the full potential of iPSCs to minimize abuse or unauthorized utilization. In this review, we discuss bioethical, legal, and societal concerns associated with research and therapy using iPSCs. Furthermore, we present key questions and suggestions for stem cell scientists, legal authorities, and social activists investigating and working in this field.

Until now, various methods have been introduced to fabricate 3D scaffolds to provide a suitable substrate for cell growth and proliferation and subsequent use in tissue engineering to repair damaged tissues. The 3D scaffolds can simulate the natural cellular microenvironment well. Herein, the decellularized leaf spinach has been used which not only have no problems associated with artificial scaffolds, but they also do not cost significantly. Decellularized scaffolds surface properties were characterized by the investigation of scaffolds surface roughness, hydrophilicity, mechanical properties, size and shape of porosities and specific surface area. In the next step, osteogenic differentiation potential of bone marrow derived mesenchymal stem cells cultured on the scaffold and culture plate (as a control) was evaluated using alizarin staining and calcium content, alkaline phosphatase activity and bone related genes expression assays. The results indicated that the surface properties and shape of scaffold pores were effective in the stem cells binding, growth and proliferation. This higher biocompatibility due to the ideal surface hydrophilicity as well as high specific surface area due to the presence of a rough grid surface ultimately increased the efficiency of stem cell's bone differentiation. Taken together, it can be concluded that the decellularized spinach leaf scaffold, due to its easy availability, low prices and high efficiency, can be considered as a promising potential candidate for use as a proper substrate for stem cell growth and differentiation in bone tissue engineering.

Strategies based on growth factor (GF) delivery have attracted considerable attention in tissue engineering applications. Among different GFs, transforming growth factor beta 1 (TGF-β1) is considered to be a potent factor for inducing chondrogenesis. In the present study, an expression cassette encoding the TGF-β1 protein was prepared and transfected into the SP2/0-Ag14 cell line. The confocal microscopy of the transfected cells was performed to confirm the correct transfection process. The expression and in vitro release kinetics of the recombinant TGF-β1 were assessed by western blot analysis and ELISA, respectively. Moreover, the biological activity of the expressed protein was compared with that of a commercially available product. The chondrogenic effects of the sustained release of the recombinant TGF-β1 in an in vitro co-culture system were evaluated using a migration assay and real-time PCR. Results of confocal microscopy confirmed the successful transfection of the vector-encoding TGF-β1 protein into the SP2/0-Ag14 cells. The bioactivity of the produced protein was in the range of the commercial product. The sustained release of the TGF-β1 protein via SP2/0-Ag14 cells encapsulated in hydrogels encouraged the migration of adipose-derived MSCs. In addition, the expression analysis of chondrogenesis-related genes revealed that the pretreatment of encapsulated Ad-MSCs cells in alginate sulfate hydrogels through their exposure to the sustained release of TGF-β1 is an efficient approach before transplantation of cells into the body.

The capability of multi-stress-tolerant Saccharomyces cerevisiae diploid strain TJ14 for the production of cellulosic bio-ethanol by semi-simultaneous saccharification and fermentation (SSSF) technology was evaluated under high-temperature conditions. At 39°C, the TJ14 produced 45 g/l ethanol by SSSF of 100 g (w/v)/l cellulose - a significantly higher concentration than reported in prevailing literature.

In Saccharomyces cerevisiae, disruption of both protein phosphatase genes, PTP2 and MSG5, causes calcium sensitivity while additional disruption of protein kinase genes BCK1, MKK1, SLT2, MCK1, YAK1 and SSK2 confers calcium tolerance. Although the roles of BCK1, MKK1 and SLT2 have been characterized recently, the mechanism of suppression of the calcium sensitivity by SSK2 disruption is poorly understood. In this study, genetic analysis revealed a novel, high osmolarity glycerol (HOG)-independent suppressor function of Ssk2 in relation to the Ptp2 and Msg5-mediated calcium signaling. Through microarray analysis, we identified 19 genes with distinct pattern of expression that is likely involved in the calcium sensitive phenotype of the ptp2Δmsg5Δ double disruptant. Furthermore, we found msn2Δ and bcy1Δ as suppressors of the calcium sensitive phenotype. Our results suggest the interrelationship of a HOG-independent function of Ssk2, transcription factor Msn2, protein kinase A-related protein Bcy1 and 19 rise and fall genes as responsible for the suppression mechanism of the ptp2Δmsg5Δ double disruptant by ssk2Δ disruption.

The present study was undertaken to genetically evaluate Caspian horses for genetic diversity and to asses whether they have experienced recent population bottlenecks. A total of 100 individuals were characterized for within breed diversity using 16 microsatellite markers. The estimated mean number of alleles was 8.69 per locus, with a total of 139 alleles in the genotyped sample. The mean effective number of alleles in the Caspian horse population was 5.86, ranging from 3.49 to 8.49. The average observed heterozygosity in the present study (0.52) was lower than to the expected heterozygosity (0.82), which may reflect the narrow genetic base of the current population of this breed. All marker lociemployed in this study were very informative with an average of 0.80. The Chi-square and likelihood ratio tests performed to examine population for HWE showed some highly significant deviations from HWE. Estimated values of Wright’s fixation index, FIS (0.367) indicates a certain level of heterozygote deficiency. A significant heterozygote excess on the basis of different models, as revealed from Sign and Wilcoxon rank test suggested that Caspian horse population is not in mutation-drift equilibrium.But, the Mode-shift indicator test showed a normal ‘L’ shaped distribution for allelic class and proportion of alleles, thus indicating the absence of bottleneck events in the recent history of this breed. The present work is a contribution to the knowledge of population structure and to theassessment of genetic diversity that may be helpful to horse breeders in designing and managing breeding or conservation strategies for the Caspian horse breed.