High yield expression and purification of Aspergillus flavus uricase cloned in Pichia pink™ expression system.

In this research, for the first time, A. flavus uricase gene was cloned in pPink-UOX plasmid under strong alcohol oxidase promoter of Pichia pink expression system after codon optimization. After selecting the best uricase producing clone with an activity of 0.7 U/ml at the Flask level, a 5-L fermenter was used to increase the expression of the enzyme. Within 60 h, the fermentation process produced 1500 g of biomass from 4 L of semi defined culture media and expressed 2.5 g/L of the enzyme. The purity of recombinant uricase production using three consecutive DEAE Sepharose, CM Sepharose and Phenyl Sepharose columns was above 99%, which was confirmed by SDS-PAGE and RP-HPLC analyses. Size exclusion chromatography analysis showed that the purified enzyme has comparable heterogeneity to the Rasburicase. The yield of recombinant uricase production in this study was 63% and its specific activity was 24 U/mg. The high expression of recombinant uricase in the Pichia pink strain and the increased enzyme activity compared to the standard sample indicate the potential of therapeutic and diagnostic applications of recombinant uricase in the present study.

Complete genome sequencing and molecular characterization of SARS-COV-2 from COVID-19 cases in Alborz province in Iran.

Iran was among countries which was hard hit at the early stage of the coronavirus disease 2019 (COVID-19) pandemic and dealt with the second wave of the pandemic in May and June 2020; however, there are a very limited number of complete genome sequences of acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from Iran. In this study, complete genome sequences of the virus in the samples obtained from three patients in Alborz province in May and June 2020 were generated and analyzed using bioinformatic methods. The sequenced genomes were positioned in a cluster with B.4 lineage along with the sequences from other countries namely, United Arab Emirates and Oman. There were seven single nucleotide variations (SNVs) in common in all samples and only one of the sequenced genomes showed the D614G amino acid substitution. Three SNVs, 1397 G > A, 28688T > C, 29742 G > T, which had already been reported in February, were found with high frequency in all the sequenced genomes in this study, implying that viral diversity reflected in the early stages of viral transmission in Iran were established in the second wave. Considering the importance of molecular epidemiology in response to ongoing pandemic, there is an urgent need for more complete genome sequencing and comprehensive analyses to gain insight into the transmission, adaptation and evolution of the virus in Iran.

The Role of MicroRNAs in the Induction of Pancreatic Differentiation.

Stem cell-based therapy is one of the therapeutic options with promising results in the treatment of diabetes. Stem cells from various sources are expanded and induced to generate the cells capable of secreting insulin. These insulin-producing cells [IPCs] could be used as an alternative to islets in the treatment of patients with diabetes. Soluble growth factors, small molecules, geneencoding transcription factors, and microRNAs [miRNAs] are commonly used for the induction of stem cell differentiation. MiRNAs are small non-coding RNAs with 21-23 nucleotides that are involved in the regulation of gene expression by targeting multiple mRNA targets. Studies have shown the dynamic expression of miRNAs during pancreatic development and stem cell differentiation. MiR- 7 and miR-375 are the most abundant miRNAs in pancreatic islet cells and play key roles in pancreatic development as well as islet cell functions. Some studies have tried to use these small RNAs for the induction of pancreatic differentiation. This review focuses on the miRNAs used in the induction of stem cells into IPCs and discusses their functions in pancreatic ß-cells.

PHBV nanofibers promotes insulin-producing cells differentiation of human induced pluripotent stem cells.

Tissue-engineering associated techniques have long been employed to improve the various elements of the therapeutic approaches toward the more efficient ones in diabetic states. The resultant constructs comprise of the polymeric scaffolds with proper degradation rates that produce bodily compatible components, and the pluripotent cells that are highly capable of generating islet-like cells. In this study, Poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) nanofibers were fabricated by the Electrospinning. After validation of its 3-D structure, fibers size and non-toxicity, insulin-producing cells (IPC) differentiation potential of the induced pluripotent stem cells (iPSCs) were evaluated during growing on the PHBV nanofibers in comparison with tissue culture polystyrene (TCPS). SEM analyses confirmed the 3-D and nanofibrous structure of the fabricated scaffold. The survival rate of the iPSCs cultured on the PHBV nanofibers was increased significantly compared to the cells cultured on the TCPS, which is an evidence for the non-toxicity of the nanofibers. Insulin and C-peptide secretion levels significantly increased in the differentiated iPSCs on PHBV nanofibers compared to those cells cultured on TCPS. Moreover, levels of the gene transcription and translation results revealed that insulin, Glut-2, and Pdx-1 genes and insulin protein, in IPC-differentiated iPSCs grown on PHBV nanofibers are significantly higher than those cells grown on TCPS. Taken together, these results go beyond previous reports, showing thatiPSCs-PHBV as a promising cell-copolymer construct, could potentially be applied in the pancreatic tissue engineering applications to diabetic patient treatment.

Aloe Vera-Derived Gel-Blended PHBV Nanofibrous Scaffold for Bone Tissue Engineering.

Today, composite scaffolds fabricated by natural and synthetic polymers have attracted a lot of attention among researchers in the field of tissue engineering, and given their combined properties that can play a very useful role in repairing damaged tissues. In the current study, aloe vera-derived gel-blended poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) nanofibrous scaffold was fabricated by electrospinning, and then, PHBV and PHBV gel fabricated scaffolds characterized by scanning electron microscope, protein adsorption, cell attachment, tensile and cell's viability tests. After that, osteogenic supportive property of the scaffolds was studied by culturing of human-induced pluripotent stem cells on the scaffolds under osteogenic medium and evaluating of the common bone-related markers. The results showed that biocompatibility of the PHBV nanofibrous scaffold significantly improved when combined with the aloe vera gel. In addition, higher amounts of alkaline phosphatase activity, mineralization, and bone-related gene and protein expression were detected in stem cells when grown on PHBV-gel scaffold in comparison with those stem cells grown on the PHBV and culture plate. Taken together, it can be concluded that aloe vera gel-blended PHBV scaffold has a great promising osteoinductive potential that can be used as a suitable bioimplant for bone tissue engineering applications to accelerate bone regeneration and also degraded completely along with tissue regeneration.

The Anti-Helicobacter pylori Effects of Lactobacillus acidophilus, L. plantarum, and L. rhamnosus in Stomach Tissue of C57BL/6 Mice.

BACKGROUND AND AIM: Helicobacter pylori is one of the most common pathogenic bacteria in the human gut, and is also one of the most important factors that cause digestive disorders such as chronic inflammation, gastric ulcers, and even gastric cancer. Since the use of various antibiotics to treat H. pylori infection is associated with the development of resistance in this bacterium, the aim of this study was to determine the anti-H. pylori effects of Lactobacillus acidophilus, L. plantarum, and L. rhamnosus in the stomach tissue of C57BL/6 mice. MATERIALS AND METHODS: In this experimental study, 70 mice in ten groups were evaluated from July to September 2017 in the microbiology laboratory of the School of Medicine, Alborz University of Medical Sciences, Karaj, Iran. After induction of H. pylori infection in mice with the standard strain of H. pylori (ATCC 43504), the infected mice were treated with drug and Lactobacillus species in different groups. Then, the anti-H. pylori effects of lactobacilli were evaluated by stool antigen test and tissue staining. RESULTS: Based on ELISA results and histological findings, a reduction of inflammation was observed. The group which was only exposed to L. rhamnosus and the one which was exposed to all three strains of Lactobacillus showed the highest antimicrobial effect on H. pylori. CONCLUSION: According to the results of this study, probiotic bacteria including L. acidophilus, L. plantarum, and L. rhamnosus could be useful in the reduction of H. pylori infection in the mouse model.

A Review of Evaluating Hematopoietic Stem Cells Derived from Umbilical Cord Blood's Expansion and Homing.

Transplantation of hematopoietic stem cells (HSCs) derived from umbilical cord blood (UCB) has been taken into account as a therapeutic approach in patients with hematologic malignancies. Unfortunately, there are limitations concerning HSC transplantation (HSCT), including (a) low contents of UCB-HSCs in a single unit of UCB and (b) defects in UCB-HSC homing to their niche. Therefore, delays are observed in hematopoietic and immunologic recovery and homing. Among numerous strategies proposed, ex vivo expansion of UCB-HSCs to enhance UCB-HSC dose without any differentiation into mature cells is known as an efficient procedure that is able to alter clinical treatments through adjusting transplantation-related results and making them available. Accordingly, culture type, cytokine combinations, O2 level, co-culture with mesenchymal stromal cells (MSCs), as well as gene manipulation of UCB-HSCs can have effects on their expansion and growth. Besides, defects in homing can be resolved by exposing UCB-HSCs to compounds aimed at improving homing. Fucosylation of HSCs before expansion, CXCR4-SDF-1 axis partnership and homing gene involvement are among strategies that all depend on efficiency, reasonable costs, and confirmation of clinical trials. In general, the present study reviewed factors improving the expansion and homing of UCB-HSCs aimed at advancing hematopoietic recovery and expansion in clinical applications and future directions.

Decellularized amniotic membrane Scaffolds improve differentiation of iPSCs to functional hepatocyte-like cells.

Human-induced pluripotent stem cells-derived hepatocyte-like cells (hiPSCs-HLCs) holds considerable promise for future clinical personalized therapy of liver disease. However, the low engraftment of these cells in the damaged liver microenvironment is still an obstacle for potential application. In this study, we explored the effectiveness of decellularized amniotic membrane (dAM) matrices for culturing of iPSCs and promoting their differentiation into HLCs. The DNA content assay and histological evaluation indicated that cellular and nuclear residues were efficiently eliminated and the AM extracellular matrix component was maintained during decelluarization. DAM matrices were developed as three-dimensional scaffolds and hiPSCs were seeded into these scaffolds in defined induction media. In dAM scaffolds, hiPSCs-HLCs gradually took a typical shape of hepatocytes (polygonal morphology). HiPSCs-HLCs that were cultured into dAM scaffolds showed a higher level of hepatic markers than those cultured in tissue culture plates (TCPs). Moreover, functional activities in term of albumin and urea synthesis and CYP3A activity were significantly higher in dAM scaffolds than TCPs over the same differentiation period. Thus, based on our results, dAM scaffold might have a considerable potential in liver tissue engineering, because it can improve hepatic differentiation of hiPSCs which exhibited higher level of the hepatic marker and more stable metabolic functions.

Improved osteogenic differentiation of human induced pluripotent stem cells cultured on polyvinylidene fluoride/collagen/platelet-rich plasma composite nanofibers.

Blood transfusion or blood products, such as plasma, have a long history in improving health, but today, platelet-rich plasma (PRP) is used in various medical areas such as surgery, orthopedics, and rheumatology in many ways. Considering the high efficiency of tissue engineering in repairing bone defects, in this study, we investigated the combined effect of nanofibrous scaffolds in combination with PRP on the osteogenic differentiation potential of human induced pluripotent stem cells (iPSCs). Electrospinning was used for fabricating nanofibrous scaffolds by polyvinylidene fluoride/collagen (PVDF/col) with and without PRP. After scaffold characterization, the osteoinductivity of the fabricated scaffolds was studied by culturing human iPSCs under osteogenic medium. The results showed that PRP has a considerable positive effect on the biocompatibility of the PVDF/col nanofibrous scaffold when examined by protein adsorption, cell attachment, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. In addition, the results obtained from alkaline phosphatase activity and calcium content assays demonstrated that nanofibers have higher osteoinductivity while grown on PRP-incorporated PVDF/col nanofibers. These results were also confirmed while the osteogenic differentiation of the iPSCs was more investigated by evaluating the most important bone-related genes expression level. According to the results, it can be concluded that PVDF/col/PRP has much more osteoinductivity while compared with the PVDF/col, and it can be introduced as a promising bone bio-implant for use in bone tissue engineering applications.

Micro-RNA-incorporated electrospun nanofibers improve osteogenic differentiation of human-induced pluripotent stem cells.

Smart scaffolds have a great role in the damaged tissue reconstruction. The aim of this study was developing a scaffold that in addition to its fiber's topography has also content of micro-RNAs (miRNAs), which play a regulatory role during osteogenesis. In this study, we inserted two important miRNAs, including miR-22 and miR-126 in the electrospun polycaprolactone (PCL) nanofibers and after scaffold characterization, osteoinductivity of the fabricated nanofibers was investigated by evaluating of the osteogenic differentiation potential of induced pluripotent stem cells (iPSCs) when grown on miRNAs-incorporated PCL nanofibers (PCL-miR) and empty PCL. MiRNAs incorporation had no effect on the fibers size and morphology, cell attachment, and protein adsorption, although viability and proliferation rate of the human iPSCs were increased after a week in PCL-miR compared to the empty PCL. The results obtained from alkaline phosphatase activity, calcium content, bone-related genes, and proteins expression assays demonstrated that the highest osteogenic markers were observed in iPSCs grown on the PCL-miR compared to the cells cultured on PCL and culture plate. According to the results, miR-incorporated PCL nanofibers could be considered as a promising potential tissue-engineered construct for the treatment of patients with bone lesions and defects.

Comparison of human-induced pluripotent stem cells and mesenchymal stem cell differentiation potential to insulin producing cells in 2D and 3D culture systems in vitro.

Diabetes is one of the most common diseases in the world that is chronic, progressive, and costly, and causes many complications. Common drug therapies are not able to cure it, and pancreas transplantation is not responsive to the high number of patients. The production of the insulin producing cells (IPCs) from the stem cells in the laboratory and their transplantation to the patient's body is one of the most promising new approaches. In this study, the differentiation potential of the induced pluripotent stem cells (iPSCs) and mesenchymal stem cells (MSCs) into IPCs was compared to each other while cultured on poly(lactic-co-glycolic) acid (PLGA)/polyethylene glycol (PEG) nanofibrous scaffold as a 3D substrate and tissue culture polystyrene (TCPS) as a 2D substrate. Although the expression level of the insulin, Glut2 and pdx-1 genes in stem cells cultured on 3D substrate was significantly higher than the stem cells cultured on 2D substrate, the highest expression level of these genes was detected in the iPSCs cultured on PLGA-PEG. Insulin and C-peptide secretions from differentiated cells were also investigated and the results showed that secretions in cultured iPSCs on the PLGA-PEG were significantly higher than cultured iPSCs on the TCPS and cultured MSCs on both PLGA-PEG and TCPS. In addition, insulin protein was also expressed in the cultured iPSCs on the PLGA-PEG significantly higher than cultured MSCs on the PLGA-PEG. It can be concluded that differentiation potential of iPSCs into IPCs is significantly higher than human MSCs at both 2D and 3D culture systems.

INVESTIGATION OF ENTEROCOCCUS FAECALIS POPULATION IN PATIENTS WITH POLYP AND COLORECTAL CANCER IN COMPARISON OF HEALTHY INDIVIDUALS.

BACKGROUND: Colorectal cancer is one of the most commonly diagnosed cancers around the world. One of the factors involved in the development of colorectal cancer is the changes in the normal flora of the intestine. OBJECTIVE: In this study, the mean copy number of Enterococcus faecalis in people with polyps and people with colorectal cancer has been evaluated in comparison with healthy controls. METHODS: In this study, 25 patients with colorectal cancer and 28 patients with intestinal polyps were selected and stool specimens were taken. In addition, 24 healthy individuals were selected as control group. Extraction of bacterial DNA from the stool sample were performed. The molecular methods of PCR for confirmation of standard strain and absolute Real Time PCR (qRT-PCR) method were used to evaluate the number of Enterococcus faecalis in the studied groups. RESULTS: The results of this study indicate that the mean copy number of Enterococcus faecalis in patients with colorectal cancer was 11.2x109 per gram of stool, and in patients with polyps was 9.4x108 per gram of stool. In healthy people, this number was 9x108 per gram of stool. There was a significant difference between the implicit copy numbers in the three groups. (P<0.05). CONCLUSION: Enterococcus faecalis in faecal flora of people with colorectal cancer was significantly higher than those with polyps and healthy people. This could potentially signify the ability of this bacterium to induce colorectal cancer. More studies are needed to prove this theory.

Synergistic effects of polyaniline and pulsed electromagnetic field to stem cells osteogenic differentiation on polyvinylidene fluoride scaffold.

Repairing the lost or damaged mandible is very difficult and time-consuming, so there is a great hope for tissue engineering to accelerate it. At the present study, electrospinning was applied to fabricate polyvinylidene fluoride (PVDF) and PVDF-polyaniline (PANI) composite scaffolds. In addition, extremely low frequency pulsed electromagnetic field (PEMF) was applied for treating the stem cells derived from dental pulp (DPSCs) when cultured on the nanofibrous scaffolds. Osteoinductive property of the fabricated PVDF, PVDF-PANI scaffold at the presence and absence of the PEMF was investigated by evaluating the common osteogenic differentiation markers in seeded-DPSCs on the scaffold. Results demonstrated that cell attachment, protein adsorption and cells viability were increased when PEMF was applied. In addition, ALP activity, calcium content, osteogenic genes and protein evaluations confirmed that PEMF could significantly increase osteoinductivity of the PVDF while composite with PANI. According to the results, the use of polymers with piezoelectricity and conductivity features plus PEMF exposure has a promising potential to improve the current treatment methods in bone and mandibular defects.

Investigação da população de Enterococcus faecalis em pacientes com pólipo e câncer colorretal em comparação a indivíduos saudáveis / Investigation of enterococcus faecalis population in patients with polyp and colorectal cancer in comparison of healthy individuals

ABSTRACT BACKGROUND: Colorectal cancer is one of the most commonly diagnosed cancers around the world. One of the factors involved in the development of colorectal cancer is the changes in the normal flora of the intestine. OBJECTIVE: In this study, the mean copy number of Enterococcus faecalis in people with polyps and people with colorectal cancer has been evaluated in comparison with healthy controls. METHODS: In this study, 25 patients with colorectal cancer and 28 patients with intestinal polyps were selected and stool specimens were taken. In addition, 24 healthy individuals were selected as control group. Extraction of bacterial DNA from the stool sample were performed. The molecular methods of PCR for confirmation of standard strain and absolute Real Time PCR (qRT-PCR) method were used to evaluate the number of Enterococcus faecalis in the studied groups. RESULTS: The results of this study indicate that the mean copy number of Enterococcus faecalis in patients with colorectal cancer was 11.2x109 per gram of stool, and in patients with polyps was 9.4x108 per gram of stool. In healthy people, this number was 9x108 per gram of stool. There was a significant difference between the implicit copy numbers in the three groups. (P<0.05). CONCLUSION: Enterococcus faecalis in faecal flora of people with colorectal cancer was significantly higher than those with polyps and healthy people. This could potentially signify the ability of this bacterium to induce colorectal cancer. More studies are needed to prove this theory.

RESUMO CONTEXTO: O câncer colorretal é um dos cânceres mais comumente diagnosticados em todo o mundo. Um dos fatores envolvidos no desenvolvimento do câncer colorretal é a mudança na flora normal do intestino. OBJETIVO: O número médio de cópias de Enterococcus faecalis em pessoas com pólipos e pessoas com câncer colorretal foram avaliados em comparação com controles saudáveis. MÉTODOS: Neste estudo, 25 pacientes com câncer colorretal e 28 pacientes com pólipos intestinais foram selecionados e amostras de fezes foram adquiridas. Além disso, 24 indivíduos saudáveis foram selecionados como grupo controle. A extração do DNA bacteriano da amostra coletada foi executada. Os métodos moleculares de PCR para confirmação da cepa padrão e o método absoluto de PCR em tempo real (qRT-PCR) foram utilizados para avaliar o número de Enterococcus faecalis nos grupos estudados. RESULTADOS: Os resultados deste estudo indicam que o número médio de cópias de Enterococcus faecalis em pacientes com câncer colorretal foi de 11,2x109 por grama de fezes, e em pacientes com pólipos foi de 9,4x108 por grama de fezes. Em pessoas saudáveis, este número foi de 9x108 por grama de fezes. Houve diferença significativa entre os números de cópia implícita nos três grupos. (P<0,05). CONCLUSÃO: Enterococcus faecalis na flora fecal de pessoas com câncer colorretal foi significativamente maior do que aqueles com pólipos e pessoas saudáveis. Isto poderia potencialmente significar a capacidade desta bactéria para induzir o câncer colorretal. Mais estudos são necessários para provar esta teoria.

Fucosylated umbilical cord blood hematopoietic stem cell expansion on selectin-coated scaffolds.

Despite the advantages of transplantation of umbilical cord blood's (UCB's) hematopoietic stem cells (uHSCs) for hematologic malignancy treatment, there are two major challenges in using them: (a) Insufficient amount of uHSCs in a UCB unit; (b) a defect in uHSCs homing to bone marrow (BM) due to loose binding of their surface glycan ligands to BM's endothelium selectin receptors. To overcome these limitations, after poly l-lactic acid (PLLA) scaffold establishment and incubation of uHSCs with fucosyltransferase-VI and GDP-fucose, ex vivo expansion of these cells on selectin-coated scaffold was done. The characteristics of the cultured fucosylated and nonfucosylated cells on a two-dimensional culture system, PLLA, and a selectin-coated scaffold were evaluated by flow cytometry, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, colony-forming unit (CFU) assay, and CXCR4 expression at the messenger RNA and protein levels. According to the findings of this study, optimized attachment to the scaffold in scanning electron microscopy micrograph, maximum count of CFU, and the highest 570 nm absorption were observed in fucosylated cells expanded on selectin-coated scaffolds. Furthermore, real-time polymerase chain reaction showed the highest expression of the CXCR4 gene, and immunocytochemistry data confirmed that the CXCR4 protein was functional in this group compared with the other groups. Considered together, the results showed that selectin-coated scaffold could be a supportive structure for fucosylated uHSC expansion and homing by nanotopography. Fucosylated cells placed on the selectin-coated scaffold serve as a basal surface for cell-cell interaction and more homing potential of uHSCs. Accordingly, this procedure can also be considered as a promising technique for the hematological disorder treatment and tissue engineering applications.

Incorporated-bFGF polycaprolactone/polyvinylidene fluoride nanocomposite scaffold promotes human induced pluripotent stem cells osteogenic differentiation.

Bioactive scaffolds that can increase transplanted cell survival time at the defect site have a great promising potential to use clinically since tissue regeneration or secretions crucially depend on the transplanted cell survival. In this study embedded basic fibroblast growth factor (bFGF)-polycaprolactone-polyvinylidene fluoride (PCL-PVDF) hybrid was designed and fabricated by electrospinning as a bio-functional nanofibrous scaffold for bone tissue engineering. After morphological characterization of the PCL-PVDF (bFGF) scaffold, nanofibers biocompatibility was investigated by culturing of the human induced pluripotent stem cells (iPSCs). Then, the bone differentiation capacity of the iPSCs was evaluated when grown on the PCL-PVDF and PCL-PVDF (bFGF) scaffolds in comparison with culture plate as a control using evaluating of the common osteogenic markers. The viability assay displayed a significant increase in iPSCs survival rate when grown on the bFGF content scaffold. The highest alkaline phosphatase activity and mineralization were detected in the iPSCs while grown on the PCL-PVDF (bFGF) scaffolds. Obtained results from gene and protein expression were also demonstrated the higher osteoinductive property of the bFGF content scaffold compared with the scaffold without it. According to the results, the release of bFGF from PCL-PVDF nanofibers increased survival and proliferation rate of the iPSCs, which followed by an increase in its osteogenic differentiation potential. Taking together, PCL-PVDF (bFGF) nanofibrous scaffold demonstrated that can be noted as a promising candidate for treating the bone lesions by tissue engineering products.

Comparison of osteogenic differentiation potential of induced pluripotent stem cells on 2D and 3D polyvinylidene fluoride scaffolds.

In recent decades, tissue engineering has been the most contributor for introducing 2D and 3D biocompatible osteoinductive scaffolds as bone implants. Polyvinylidene fluoride (PVDF), due to the unique mechanical strength and piezoelectric properties, can be a good choice for making a bone bioimplant. In the present study, PVDF nanofibers and film were fabricated as 3D and 2D scaffolds, and then, osteogenic differentiation potential of the human induced pluripotent stem cells (iPSCs) was investigated when grown on the scaffolds by evaluating the common osteogenic markers in comparison with tissue culture plate. Biocompatibility of the fabricated scaffolds was confirmed qualitatively and quantitatively by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and scanning electron microscopy assays. Human iPSCs cultured on PVDF nanofibers showed a significantly higher alkaline phosphate activity and calcium content compared with the iPSCs cultured on PVDF film. Osteogenic-related genes and proteins were also expressed in the iPSCs seeded on PVDF nanofibers significantly higher than iPSCs seeded on PVDF film, when investigated by real-time reverse transcription polymerase chain reaction and western blot analysis, respectively. According to the results, the PVDF nanofibrous scaffold showed a greater osteoinductive property compared with the PVDF film and due to the material similarity of the scaffolds, it could be concluded that the 3D structure could lead to better bone differentiation. Taken together, the obtained results demonstrated that human iPSC-seeded PVDF nanofibrous scaffold could be considered as a promising candidate for use in bone tissue engineering applications.

Improved chondrogenic response of mesenchymal stem cells to a polyethersulfone/polyaniline blended nanofibrous scaffold.

Owing to the fact that the cartilage tissue is not able to repair itself, the treatment of the joint damages is very difficult by current methods. Induction of tissue repair requires suitable cell and extracellular matrix. Providing these two parts can only be done using tissue engineering. In the present study, polyethersulfone (PES) and polyaniline (PANI) blend was electrospined for nanofibrous scaffold fabrication. Mesenchymal stem cells were isolated from human adipose tissue (AT-MSCs), and after characterization cultured on the PES-PANI scaffold and culture plate. Electron microscopic and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assays were used for biocompatibility evaluation of the scaffold and the chondrogenic differentiation potential of AT-MSCs were investigated by staining of proteoglycans and gene and protein expression evaluation. Alcian blue staining, real-time reverse-transcriptase polymerase chain reaction and Western blot results showed that chondrogenic differentiation potential of AT-MSCs was significantly increased when grown on PES-PANI nanofibers and was compared to the one grown on a culture plate. According to the results, PES-PANI has a promising potential to be used as a biomedical implant in patients with joints lesion, such as arthritis and osteoarthritis.

Umbilical cord blood mesenchymal stem cells application in hematopoietic stem cells expansion on nanofiber three-dimensional scaffold.

Umbilical cord blood (UCB) hematopoietic stem cells (HSCs) transplantation (HSCTs) is considered as a therapeutic strategy for malignant and nonmalignant hematologic disorders. Nevertheless, the low number of HSCs obtained from each unit of UCB can be a major challenge for using these cells in adults. In addition, UCB is a rich source of mesenchymal stem cells (MSCs) creating hopes for nonaggressive and painless treatment in tissue engineering compared with bone marrow MSCs. This study was designed to evaluate the effects of UCB-MSCs application in UCB-HSCs expansion on the nanoscaffold that mimics the cell's natural niche. To achieve this goal, after flow cytometry confirmation of isolated HSCs from UCB, they were expanded on three-dimensional (3D) poly-l-lactic acid (PLLA) scaffolds fabricated by electrospinning and two-dimensional (2D)-culture systems, such as (1) HSCs-MSCs culturing on the scaffold, (2) HSCs culturing on the scaffold, (3) HSCs-MSCs culturing on 2D, and (4) HSCs culturing on 2D. After 7 days, real-time polymerase chain reaction (PCR) was performed to evaluate the CXCR4 gene expression in the mentioned groups. Moreover, for the next validation, the number of total HSCs, 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide assay, scanning electron microscopy imaging, and colony-forming unit assay were evaluated as well. The results of the study indicated that UCB-MSCs interaction with HSCs in 3D-culture systems led to the highest expansion of UCB-HSCs on day 7. Flow cytometry results showed the highest purity of HSCs cocultured with MSCs. Real-time PCR showed a significant increase in gene expression of CXCR4 in the mentioned group. The highest viability and clonogenicity were detected in the mentioned group too. Considered together, our results suggest that UCB-HSCs and MSCs coculturing on PLLA scaffold could provide a proper microenvironment that efficiently promotes UCB-HSCs expansion and UCB-MSCs can also be considered as a promising candidate for UCB-HSCTs.

Efficient osteogenic differentiation of the dental pulp stem cells on ß-glycerophosphate loaded polycaprolactone/polyethylene oxide blend nanofibers.

Hard tissue lesion treatment in oral and maxillofacial has been challenging because of tissue complexities. This study aimed to investigate novel biopolymeric construct effects on the osteogenic differentiation potential of the dental pulp stem cells (DPSCs) for introducing a cell copolymer bioimplant. A blended polycaprolactone (PCL)-polyethylene oxide (PEO) was fabricated using electrospinning, simultaneously filled by ß-glycerophosphate (ß-GP). After that biocompatibility and release kinetics of the PCL-PEO+ß-GP was evaluated and compared with PCL-PEO and then the osteogenic differentiation potential of the DPSCs was examined while being cultured on the scaffolds and compared with those cultured on the culture plate. The results demonstrated that scaffolds have not any cytotoxicity and ß-GP can release in a long-term manner. Alkaline phosphatase activity and calcium content were significantly increased in DPSCs while being cultured on the PCL-PEO+ß-GP compared with the other groups. Runt-related transcription factor 2, collagen type-I, osteonectin, and osteocalcin (OSC) genes expression was upregulated in DPSCs cultured on the PCL-PEO+ß-GP and was significantly higher than those cultured on the PCL-PEO. Immunocytochemistry result also confirmed the positive effects of PCL-PEO+ß-GP on the osteogenic differentiation of the DPSCs by presenting a higher OSC protein expression. According to the results, incorporation of the ß-GP in PCL-PEO makes a better construct for osteogenic induction into the stem cells and it could be also considered as a great promising candidate for bone, oral, and maxillofacial tissue engineering applications.