An efficient method for cell sheet bioengineering from rBMSCs on thermo-responsive PCL-PEG-PCL copolymer.

Utilizing both medium enrichment and a thermos-responsive substrate to maintain the cell-to-cell junctions and extracellular matrix (ECM) intact, cell sheet technology has emerged as a ground-breaking approach. Investigating the possibility of using sodium selenite (as medium supplementation) and PCL-PEG-PCL (as vessel coating substrate) in the formation of the sheets from rat bone marrow-derived mesenchymal stem cells (rBMSCs) was the main goal of the present study. To this end, first, Polycaprolactone-co-Poly (ethylene glycol)-co-Polycaprolactone triblock copolymer (PCEC) was prepared by ring-opening copolymerization method and characterized by FTIR, 1 H NMR, and GPC. The sol-gel-sol phase transition temperature of the PCEC aqueous solutions with various concentrations was either measured. Next, rBMSCs were cultured on the PCEC, and let be expanded in five different media containing vitamin C (50 µg/ml), sodium selenite (0.1 µM), vitamin C and sodium selenite (50 µg/ml + 0.1 µM), Trolox, and routine medium. The proliferation of the cells exposed to each material was evaluated. Produced cell sheets were harvested from the polymer surface by temperature reduction and phenotypically analyzed via an inverted microscope, hematoxylin and eosin (H&E) staining, and field emission scanning electron microscopy (FESEM). Through the molecular level, the expression of the stemness-related genes (Sox2, Oct-4, Nanog), selenium-dependent enzymes (TRX, GPX-1), and aging regulator gene (Sirt1) were measured by q RT-PCR. Senescence in cell sheets was checked by beta-galactosidase assay. The results declared the improved ability of the rBMSCs for osteogenesis and adipogenesis in the presence of antioxidants vitamin C, sodium selenite, and Trolox in growth media. The data indicated that in the presence of vitamin C and sodium selenite, the quality of the cell sheet was risen by reducing the number of senescent cells and high transcription of the stemness genes. Monolayers produced by sodium selenite was in higher-quality than the ones produced by vitamin C.

Sodium selenite preserves rBM-MSCs' stemness, differentiation potential, and immunophenotype and protects them against oxidative stress via activation of the Nrf2 signaling pathway.

BACKGROUND: The physiological level of reactive oxygen species (ROS) is necessary for many cellular functions. However, during the in-vitro manipulations, cells face a high level of ROS, leading to reduced cell quality. Preventing this abnormal ROS level is a challenging task. Hence, here we evaluated the effect of sodium selenite supplementation on the antioxidant potential, stemness capacity, and differentiation of rat-derived Bone Marrow MSCs (rBM-MSCs) and planned to check our hypothesis on the molecular pathways and networks linked to sodium selenite's antioxidant properties. METHODS: MTT assay was used to assess the rBM-MSCs cells' viability following sodium selenite supplementation (concentrations of: 0.001, 0.01, 0.1, 1, 10 µM). The expression level of OCT-4, NANOG, and SIRT1 was explored using qPCR. The adipocyte differentiation capacity of MSCs was checked after Sodium Selenite treatment. The DCFH-DA assay was used to determine intracellular ROS levels. Sodium selenite-related expression of HIF-1α, GPX, SOD, TrxR, p-AKT, Nrf2, and p38 markers was determined using western blot. Significant findings were investigated by the String tool to picture the probable molecular network. RESULTS: Media supplemented with 0.1 µM sodium selenite helped to preserve rBM-MSCs multipotency and keep their surface markers presentation; this also reduced the ROS level and improved the rBM-MSCs' antioxidant and stemness capacity. We observed enhanced viability and reduced senescence for rBM-MSCs. Moreover, sodium selenite helped in rBM-MSCs cytoprotection by regulating the expression of HIF-1 of AKT, Nrf2, SOD, GPX, and TrxR markers. CONCLUSIONS: We showed that sodium selenite could help protect MSCs during in-vitro manipulations, probably via the Nrf2 pathway.

Immune evader cancer stem cells direct the perspective approaches to cancer immunotherapy.

Exploration of tumor immunity leads to the development of immune checkpoint inhibitors and cell-based immunotherapies which improve the clinical outcomes in several tumor types. However, the poor clinical efficacy of these treatments observed for other tumors could be attributed to the inherent complex tumor microenvironment (TME), cellular heterogeneity, and stemness driven by cancer stem cells (CSCs). CSC-specific characteristics provide the bulk tumor surveillance and resistance to entire eradication upon conventional therapies. CSCs-immune cells crosstalk creates an immunosuppressive TME that reshapes the stemness in tumor cells, resulting in tumor formation and progression. Thus, identifying the immunological features of CSCs could introduce the therapeutic targets with powerful antitumor responses. In this review, we summarized the role of immune cells providing CSCs to evade tumor immunity, and then discussed the intrinsic mechanisms represented by CSCs to promote tumors' resistance to immunotherapies. Then, we outlined potent immunotherapeutic interventions followed by a perspective outlook on the use of nanomedicine-based drug delivery systems for controlled modulation of the immune system.

Liver bioengineering: Recent trends/advances in decellularization and cell sheet technologies towards translation into the clinic.

Development of novel technologies provides the best tissue constructs engineering and maximizes their therapeutic effects in regenerative therapy, especially for liver dysfunctions. Among the currently investigated approaches of tissue engineering, scaffold-based and scaffold-free tissues are widely suggested for liver regeneration. Analogs of liver acellular extracellular matrix (ECM) are utilized in native scaffolds to increase the self-repair and healing ability of organs. Native ECM analog could improve liver repairing through providing the supportive framework for cells and signaling molecules, exerting normal biomechanical, biochemical, and physiological signal complexes. Recently, innovative cell sheet technology is introduced as an alternative for conventional tissue engineering with the advantage of fewer scaffold restrictions and cell culture on a Thermo-Responsive Polymer Surface. These sheets release the layered cells through a temperature-controlled procedure without enzymatic digestion, while preserving the cell-ECM contacts and adhesive molecules on cell-cell junctions. In addition, several novelties have been introduced into the cell sheet and decellularization technologies to aid cell growth, instruct differentiation/angiogenesis, and promote cell migration. In this review, recent trends, advancements, and issues linked to translation into clinical practice are dissected and compared regarding the decellularization and cell sheet technologies for liver tissue engineering.

Modulation of LXR signaling altered the dynamic activity of human colon adenocarcinoma cancer stem cells in vitro.

BACKGROUND: The expansion and metastasis of colorectal cancers are closely associated with the dynamic growth of cancer stem cells (CSCs). This study aimed to explore the possible effect of LXR (a regulator of glycolysis and lipid hemostasis) in the tumorgenicity of human colorectal CD133 cells. METHODS: Human HT-29 CD133+ cells were enriched by MACS and incubated with LXR agonist (T0901317) and antagonist (SR9243) for 72 h. Cell survival was evaluated using MTT assay and flow cytometric analysis of Annexin-V. The proliferation rate was measured by monitoring Ki-67 positive cells using IF imaging. The modulation of LXR was studied by monitoring the activity of all factors related to ABC transporters using real-time PCR assay and western blotting. Protein levels of metabolic enzymes such as PFKFB3, GSK3ß, FASN, and SCD were also investigated upon treatment of CSCs with LXR modulators. The migration of CSCs was monitored after being exposed to LXR agonist using scratch and Transwell insert assays. The efflux capacity was measured using hypo-osmotic conditions. The intracellular content of reactive oxygen species was studied by DCFH-DA staining. RESULTS: Data showed incubation of CSCs with T0901317 and SR9243 reduced the viability of CD133 cells in a dose-dependent manner compared to the control group. The activation of LXR up-regulated the expression and protein levels of ABC transporters (ABCA1, ABCG5, and ABCG8) compared to the non-treated cells (p < 0.05). Despite these effects, LXR activation suppressed the proliferation, clonogenicity, and migration of CD133 cells, and increased hypo-osmotic fragility (p < 0.05). We also showed that SR9243 inhibited the proliferation and clonogenicity of CD133 cells through down-regulating metabolic enzymes PFKFB3, GSK3ß, FASN, and SCD as compared with the control cells (p < 0.05). Intracellular ROS levels were increased after the inhibition of LXR by SR9243 (p < 0.05). Calling attention, both T0901317 and SR9243 compounds induced apoptotic changes in cancer stem cells (p < 0.05). CONCLUSIONS: The regulation of LXR activity can be considered as a selective targeting of survival, metabolism, and migration in CSCs to control the tumorigenesis and metastasis in patients with advanced colorectal cancers.

Corneal endothelium tissue engineering: An evolution of signaling molecules, cells, and scaffolds toward 3D bioprinting and cell sheets.

Cornea is an avascular and transparent tissue that focuses light on retina. Cornea is supported by the corneal-endothelial layer through regulation of hydration homeostasis. Restoring vision in patients afflicted with corneal endothelium dysfunction-mediated blindness most often requires corneal transplantation (CT), which faces considerable constrictions due to donor limitations. An emerging alternative to CT is corneal endothelium tissue engineering (CETE), which involves utilizing scaffold-based methods and scaffold-free strategies. The innovative scaffold-free method is cell sheet engineering, which typically generates cell layers surrounded by an intact extracellular matrix, exhibiting tunable release from the stimuli-responsive surface. In some studies, scaffold-based or scaffold-free technologies have been reported to achieve promising outcomes. However, yet some issues exist in translating CETE from bench to clinical practice. In this review, we compare different corneal endothelium regeneration methods and elaborate on the application of multiple cell types (stem cells, corneal endothelial cells, and endothelial precursors), signaling molecules (growth factors, cytokines, chemical compounds, and small RNAs), and natural and synthetic scaffolds for CETE. Furthermore, we discuss the importance of three-dimensional bioprinting strategies and simulation of Descemet's membrane by biomimetic topography. Finally, we dissected the recent advances, applications, and prospects of cell sheet engineering for CETE.

High-level Expression and Purification of Active Human FGF-2 in Escherichia coli by Codon and Culture Condition Optimization.

BACKGROUND: Basic fibroblast growth factor (bFGF) is a member of a highly conserved superfamily of proteins that are involved in cell proliferation, differentiation, and migration. OBJECTIVES: The objective of this study was to overexpress and purify the high-level active human bFGF in Escherichia coli (E. coli). MATERIALS AND METHODS: This experimental study was conducted in the Islamic Republic of Iran. After codon optimization and gene synthesis, the optimized FGF-2 gene was subcloned into plasmid pET-32a. pET32-FGF-2 was transformed into E. coli BL21 for expression. The cultivation parameters were optimized to produce a high yield of FGF-2. RESULTS: The optimal conditions were determined as follows: cultivation at 37°C in TB medium, with 1 mM isopropyl-ß-D-thiogalactopyranoside (IPTG), followed by post-induction expression for 6 h. Under the abovementioned conditions, the expression volumetric productivity of FGF-2 reached 1.48 g/L. CONCLUSIONS: A fusion tag from the pET32 expression plasmid permits the recovery of the recombinant fusion FGF-2 from E. coli, without affecting its biological activity.

Changes in Synaptic Transmission and Long-term Potentiation Induction as a Possible Mechanism for Learning Disability in an Animal Model of Multiple Sclerosis.

PURPOSE: Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system. It has been shown that memory deficits is common in patients with MS. Recent studies using experimental autoimmune encephalomyelitis (EAE) as an animal model of MS have shown that indicated that EAE causes hippocampal-dependent impairment in learning and memory. Thus far, there have been no in vivo electrophysiological reports describing synaptic transmission in EAE animals. The aim of the present work is to evaluate the synaptic changes in the CA1 region of the hippocampus of EAE rats. METHODS: To evaluate changes in synaptic transmission in the CA1 region of the hippocampus of EAE rats, field excitatory postsynaptic potentials (fEPSPs) from the stratum radiatum of CA1 neurons, were recorded following Schaffer collateral stimulation. RESULTS: The results showed that EAE causes deficits in synaptic transmission and long-term potentiation (LTP) in the hippocampus. In addition, paired-pulse index with a 120 msec interstimulus interval was decreased in the EAE group. These findings indicate that EAE might induce suppression in synaptic transmission and LTP by increasing the inhibitory effect of GABAB receptors on the glutamate-mediated EPSP. CONCLUSIONS: In conclusion, influence of inflammation-triggered mechanisms on synaptic transmission may explain the negative effect of EAE on learning abilities in rats.

High-level expression and purification of soluble bioactive recombinant human heparin-binding epidermal growth factor in Escherichia coli.

Heparin-binding epidermal growth factor (HB-EGF) is a member of highly conserved superfamily of proteins that has potential mitogenic activity and stimulates differentiation and migration of various cell types. Since HB-EGF has three intra-molecular disulfide bonds, a high expression pattern of active HB-EGF in an E. coli expression system was not successfully established. The aim of this study was to increase production of soluble bioactive recombinant human HB-EGF in E. coli by modifying growth conditions and codon optimization. The open reading frame codons of human HB-EGF were optimized to achieve high level expression in E. coli. The optimized codon was amplified, cloned into plasmid pET-32a, and transformed into E. coli BL21 for further expression. The cultivation parameters (temperature and inducer) were optimized to produce a high yield of soluble HB-EGF. The fusion protein was purified by Nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography. Amethylthiazole tetrazolium assay was used to evaluate the bioactivity of the produced recombinant protein. After codon optimization, the codon adaptation index (CAI) was increased from 0.255 in native gene to 0.829 using the optimized sequence. By lowering the temperature to 22°C and the inducer to 0.4 µM, we obtained 35% soluble expression of recombinant and biologically active human HB-EGF. Our data demonstrate that codon optimization increases the yield of HB-EGF in an E. coli expression system. Furthermore, the chosen modifications in cell culturing increase the solubility of recombinant human HB-EGF.