Atezolizumab and granzyme B as immunotoxin against PD-L1 antigen; an insilico study.

CD274 gene encodes programmed death-ligand 1 (PD-L1) protein, also known as B7 homolog 1 (B7-H1), which is a crucial hallmark for highly proliferation cells including cancer cells. PD-1 and PD-L1 interaction is assumed as a negative regulator for immune response which can inhibit the T cell growth and cytokine secretion and supports tumor cells evasion from immune system. therefore, PD-L1 could be assumed as a candidate target for immune-therapy. The predicted structure of PD-L1 indicates (Gly4Ser) 3 linker-based chains links. In that line, different simulation softwares applied to explore the structure of granzyme B (GrB), a serine protease in cytotoxic lymphocytes granules as an apoptosis mediator, was attached to its specific antibody structure (atezolizumab) via an adaptor sequence. Evaluation of accuracy, energy minimization and characterization of biological properties of the final processed structure were performed and our computational outcomes indicated that the employed method for structure prediction has been successfully managed to design the immunotoxin structure. It is necessary to mention that, the precise and accurate design of the immune-therapeutic agents against cancer cells can be confirmed by employment of in-silico approaches. Consequently, based on this approach we could introduce a capable immunotoxin which specifically targeting PD-L1 in an accurate orientation and initiates cancer cell destruction by its toxin domain. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s40203-021-00076-z.

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.

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.

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.

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.

Immune therapy of melanoma: Overview of therapeutic vaccines.

Melanoma is the most serious type of skin cancer which develops from the occurrence of genetic mutations in the melanocytes. Based on the features of melanoma tumors such as location, genetic profile and stage, there are several therapeutic strategies including surgery, chemotherapy, and radiotherapy. However, because of the appearance resistance mechanisms, the efficiency of these treatments strategies may be reduced. It has been demonstrated that therapeutic monoclonal antibodies can improve the efficiency of melanoma therapies. Recently, several mAbs, such as nivolumab, pembrolizumab, and ipilimumab, were approved for the immunotherapy of melanoma. The antibodies inhibit immune checkpoint receptors such as CTL4 and pd-1. Another therapeutic strategy for the treatment of melanoma is cancer vaccines, which improve clinical outcomes in patients. The combination therapy using antibodies and gene vaccine give us a new perspective in the treatment of melanoma patients. Herein, we present the recent progressions in the melanoma immunotherapy, especially dendritic cells mRNA vaccines by reviewing recent literature.

Bladder smooth muscle cell differentiation of the human induced pluripotent stem cells on electrospun Poly(lactide-co-glycolide) nanofibrous structure.

Smooth muscle cell (SMC) regeneration plays an important role in retrieving the bladder-wall functionality and it can be achieved by a proper cell-co-polymer constructed by tissue engineering. Human induced pluripotent stem cells (iPSCs), which can be specifically prepared for the patient, was considered as cells in this study, and Poly(lactide-co-glycolide) (PLGA) as a most interesting polymer in biomedical applications was applied to the scaffold fabrication by electrospinning. After scaffold characterization, SMC differentiation potential of the human iPSCs was investigated while cultured on the PLGA nanofibrous scaffold by evaluation of the SMC related important gene and protein markers. Alpha-smooth muscle actin (ASMA), Smooth muscle 22 alpha (SM-22a) as two early SMC markers were significantly up regulated either two and three weeks after differentiation induction in human iPSCs cultured on PLGA compared to those cells cultured on the tissue culture polystyrene (TCPS). But Calponin-1, Caldesmon1 and myosin heavy chain (MHC) expression differences in human iPSCs cultured on PLGA and TCPS were significant only three weeks after differentiation induction based on its lately expression in the differentiation process. ASMA and MHC proteins were also considered for evaluation by immunocytochemistry on differentiated iPSCs whereas results showed higher expression of these proteins in stem cells grown on PLGA compared to the TCPS. According to the results, human iPSCs demonstrated a great SMC differentiation potential when grown on PLGA and it could be considered as a promising cell-co-polymer for use in bladder tissue engineering.

In vitro osteogenic differentiation potential of the human induced pluripotent stem cells augments when grown on Graphene oxide-modified nanofibers.

Due to the several limitations that surgeons are faced during bone tissue implantation there are daily increases in introducing new cell-co-polymer composites for use in bone tissue engineering approaches. In this study tried to develop a suitable nanostructured bio-composite for enhancing osteogenic differentiation of the human induced pluripotent stem cells (iPSCs). Polyvinylidene fluoride-Graphene oxide (PVDF-GO) nanofibers was fabricated by electrospinning and then characterized using scanning electron microscope, tensile and viability assays. After that osteogenic differentiation of the iPSCs was investigated in three groups, including PVDF, PVDF-GO and tissue culture plate as a control group. Alkaline phosphatase activity and calcium content of the iPSCs cultured on PVDF-GO were significantly higher than those cultured on other groups. In addition, Runx2, osteocalcin and osteonectin genes were up regulated in iPSCs cultured on PVDF-GO significantly higher than those cells cultured on PVDF and control. Finally, osteocalcin and osteopontin proteins expression evaluated and the results confirmed higher osteoinductivity of the PVDF-GO nanofibers in comparison with the PVDF nanofibers. According to the results, it was demonstrated that PVDF-GO nanofibers have a great osteoinductive potential and taking together iPSCs-PVDF-GO nanofibrous construct can be an appropriate bio-implant to use for bone tissue engineering applications.

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.

Coculture of conjunctiva derived mesenchymal stem cells (CJMSCs) and corneal epithelial cells to reconstruct the corneal epithelium.

Coculture systems are widely used in tissue engineering to mimic cell-cell interactions between different populations. This study aimed to find an improved and convenient system for the corneal epithelial differentiation of conjunctiva derived mesenchymal stem cells (CJMSCs). Thus, the cells were used to reconstruct corneal epithelial cells. Obtained by flow cytometry data, 51.9% of isolated CJMSCs were immune reactive for SSEA4+ antibody which are more potent to differentiate into corneal epithelial cells. A differential medium in a single culture plate was applied and compared to a Coculture with a SHEM medium and a Coculture with a commercial medium. It was found that CJMSCs can be induced to corneal epithelial cells through in vitro co-culturing in a SHEM medium; this was confirmed with immunostaining results. Moreover, relative gene expression results showed that a Coculture system with a SHEM medium can provide a more favorable microenvironment for cells to differentiate into epithelial cells than a single culture or a Coculture with a CnT-Prime commercial medium. Finally, as platforms for cell differentiation, CJMSCs can differentiate into epithelial lineages. This was proven using immunofluorescence staining.

Homing Genes Expression in Fucosyltransferase VI-Treated Umbilical Cord Blood CD133+ Cells which Expanded on Protein-Coated Nanoscaffolds.

Umbilical cord blood (UCB)-derived hematopoietic stem cells (HSCs) are considered because of their self-renewing, differentiating, proliferating, and readily available properties. Moreover, HSCs' homing to the hematopoietic microenvironment is an important step in their transplantation process. But low content of progenitor cells in one unit of UCB and defect in the bone marrow (BM) homing limit their applications. Hence, we decided to correct this deficiency with ex vivo incubation of CD133+ cells using fucosyltransferase VI and GDP-fucose. Then C-X-C chemokines receptor-4 (CXCR4), very late activation antigen-4 (VLA4), very late activation antigen-5 (VLA5), lymphocyte function-associated antigen-1 (LFA-1), and E-cadherin (E-cad) genes expressions were investigated with the goal of homing evaluation. The purity of MACS isolated CD133+ cells and confirmation of fucosylation were done by flow cytometry, and the viability of cells seeded on protein-coated poly L-lactic acid (PLLA) scaffold was proven via MTT assay. Scanning electron microscopy (SEM), CFU assays, and expression assays of CXCR4, VLA4, VLA5, LFA-1 and E-cad by real-time PCR were performed, too. Flow cytometry data showed that isolated cells were suitable for fucosyltransferase VI (FT-VI) incubation and expansion on nanoscaffolds. MTT, CFU assays, and SEM micrographs demonstrated fibronectin (FN)-collagen-selectin (FCS)-coated scaffold serve as best environment for viability, clonogenicity, and cell attachment. High levels of homing genes expression were also observed in cells seeded on FCS-coated scaffolds. Also, CXCR4 flow cytometry analysis confirmed real-time data. FCS-PLLA scaffolds provided optimal conditions for viability of FT-VI-treated CD133+ cells, and clonogenicity with the goal of improving homing following UCB-HSCs transplantation.

In vitro expansion of CD 133+ cells derived from umbilical cord blood in poly-L-lactic acid (PLLA) scaffold coated with fibronectin and collagen.

CONTEXT: Due to their renewal and potency, umbilical cord blood (UCB) stem cells have the ability to proliferate and serve as an attractive alternative source for bone marrow transplantation. However, insufficient number of haematopoietic stem cells (HSCs) in UCB is still a major constraint in clinical applications. OBJECTIVE: In vitro expansion of stem cells on fibronectin (Fn)-coated poly-L-lactic acid (PLLA) scaffold can be a proper way to overcome this limitation. MATERIALS AND METHOD: UCB CD133 + cells were isolated by magnetic cell sorting (MACS), and then the flowcytometry method was used for analysing CD133 + cells. Confirmed cells were seeded on the Fn-coated PLLA scaffold; also, collagen-coated PLLA scaffold, PLLA scaffold and two-dimensional (2D) culture system were expanded for 7 days. During this time, we used the flowcytometric method for analysing CD133 + cells and real-time PCR for the expression level of CXCR4 gene. The number of total cells and CD133 + cells, as well as MTT assay and colony-forming unit (CFU) assay were evaluated. RESULTS: Flowcytometry data indicated that the purity of CD133 + before expansion was 93%. After 7 days, there was higher number of CD133 + cells on the Fn-coated PLLA scaffold compared to other groups. Moreover, results of MTT and colony assays showed higher viability and proliferation of CD133 + on the Fn-coated PLLA scaffold. Also, the quantity of CXCR4 gene expression increased compared to other groups. DISCUSSION: The Fn-coated scaffold was the most effective scaffold for proliferation and improved the adhesiveness to the scaffold. CONCLUSION: The Fn-coated PLLA scaffold could be a suitable in vitro mimicry niche over a 2D system.