Improved biological behaviours and osteoinductive capacity of the gelatin nanofibers while composites with GO/MgO.

Among the many polymers introduced for bone tissue engineering, natural polymers have more advantages due to their high biocompatibility and biodegradability, despite their low mechanical properties. Herein, gelatin nanofibers with and without magnesium oxide (MgO) and graphene oxide (GO) nanoparticles were fabricated by electrospinning. The fabricated gelatin and gelatin/GO/MgO nanofibers were examined using scanning electron microscopy, protein adsorption, cell attachment and viability assays. The results revealed that biological behaviours of the gelatin nanofibers significantly improved while incorporated with MgO and GO nanoparticles. In the following, osteosupportive capacity of the fabricated scaffolds was investigated by Alizarin-red staining, alkaline phosphatase activity, and calcium content, and bone-related gene and protein assays. The results revealed that the highest osteogenic differentiation potential of human-induced pluripotent stem cells (hiPSCs) was detected while these cells were cultured on the gelatin/GO/MgO nanofibers. However, these makers in the hiPSCs cultured on the gelatin nanofibers were also significantly increased in comparison with the cells cultured on the tissue culture plates as a control. In conclusion, the results revealed that predictable disadvantages in gelatin nanofibers can be greatly improved by the addition of MgO and GO nanoparticles, and the resulting composite scaffold could be a potential candidate for use in bone tissue engineering.

In vivo bone regeneration using a bioactive nanocomposite scaffold and human mesenchymal stem cells.

Due to the osteoconductive role of bioceramics, use of these bioactive nanocomposite scaffolds that can maintain their structural integrity during bone tissue repair is one of the major goals of tissue engineering. Herein, a nanofibrous poly-L-lactic acid (PLLA) scaffold was fabricated by electrospinning and then gelatin and hydroxyapatite nanoparticles (nHA) were coated over the surface of the scaffold. Osteoconductivity of the fabricated nano-composite scaffolds was then studied while grafted on the rat calvarial defects. Our results indicated that the coating of PLLA scaffold with nHA and gelatin increased the adhesion and growth of the human bone marrow derived mesenchymal stem cells (BM-MSCs) and also significantly increased the level of mineralization over a week culture period. The results of radiographic and histological studies showed that the newly created bone tissue at the defect site was significantly higher in animals treated with nanocomposite scaffolds than the empty scaffolds and control groups. This increase in the defect reconstruction was significantly increased after culturing BM-MSCs on the scaffolds, especially nanocomposite scaffolds. It can be concluded that the combination of nanocomposite scaffolds and BM-MSCs could be a very good candidate for treatment of bone lesions and could be considered as a bony bioimplant.

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.

Incorporation of SPION-casein core-shells into silk-fibroin nanofibers for cardiac tissue engineering.

Mimicking the structure of extracellular matrix (ECM) of myocardium is necessary for fabrication of functional cardiac tissue. The superparamagnetic iron oxide nanoparticles (SPIONs, Fe3 O4 ), as new generation of magnetic nanoparticles (NPs), are highly intended in biomedical studies. Here, SPION NPs (1 wt%) were synthesized and incorporated into silk-fibroin (SF) electrospun nanofibers to enhance mechanical properties and topography of the scaffolds. Then, the mouse embryonic cardiac cells (ECCs) were seeded on the scaffolds for in vitro studies. The SPION NPs were studied by scanning electron microscope (SEM), X-ray diffraction (XRD), and transmission electron microscope (TEM). SF nanofibers were characterized after incorporation of SPIONs by SEM, TEM, water contact angle measurement, and tensile test. Furthermore, cytocompatibility of scaffolds was confirmed by 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. SEM images showed that ECCs attached to the scaffolds with elongated morphologies. Also, the real-time PCR and immunostaining studies approved upregulation of cardiac functional genes in ECCs seeded on the SF/SPION-casein scaffolds including GATA-4, cardiac troponin T, Nkx 2.5, and alpha-myosin heavy chain, compared with the ones in SF. In conclusion, incorporation of core-shells in SF supports cardiac differentiation, while has no negative impact on ECCs' proliferation and self-renewal capacity.

Electrical stimulation induces differentiation of human cardiosphere-derived cells (hCDCs) to committed cardiomyocyte.

Logistic complexities of heart transplantation embossed the necessity of utilizing novel methods, which enable heart regeneration. Human cardiosphere-derived cells (hCDCs) are taken into consideration as a promising cell resource in cell therapy in recent years. In this study, we designed an electrochemical stimulation system, which sends square pulses to the hCDCs and records their electrical response. Morphology, viability and differentiation of hCDCs are monitored at certain time courses of the treatment. Differentiating hCDCs aligned perpendicularly with respect to the direction of applied electric current, and obtained a spindle-like morphology, while they remained viable. At the same time, specific cardiac marker genes including GATA4, cTnT and α-MHC showed a considerable up-regulation. Our findings confirm that hCDCs differentiate to committed cardiomyocytes when hCDCs receive an electrical energy of 0.06 - 0.12 Wh. This amount of electrical energy could be applied to the stem cells using versatile electrical stimulation patterns via commercially available devices.

Vascular tissue engineering: Fabrication and characterization of acetylsalicylic acid-loaded electrospun scaffolds coated with amniotic membrane lysate.

As the incidence of small-diameter vascular graft (SDVG) occlusion is considerably high, a great amount of research is focused on constructing a more biocompatible graft. The absence of a biocompatible surface in the lumen of the engineered grafts that can support confluent lining with endothelial cells (ECs) can cause thrombosis and graft failure. Blood clot formation is mainly because of the lack of an integrated endothelium. The most effective approach to combat this problem would be using natural extracellular matrix constituents as a mimic of endothelial basement membrane along with applying anticoagulant agents to provide local antithrombotic effects. In this study, we fabricated aligned and random electrospun poly-L-lactic acid (PLLA) scaffolds containing acetylsalicylic acid (ASA) as the anticoagulation agent and surface coated them with amniotic membrane (AM) lysate. Vascular scaffolds were structurally and mechanically characterized and assessed for cyto- and hemocompatibility and their ability to support endothelial differentiation was examined. All the scaffolds showed appropriate tensile strength as expected for vascular grafts. Lack of cytotoxicity, cellular attachment, growth, and infiltration were proved using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and scanning electron microscopy. The blood compatibilities of different scaffolds examined by in vitro hemolysis and blood coagulation assays elucidated the excellent hemocompatibility of our novel AM-coated ASA-loaded nanofibers. Drug-loaded scaffolds showed a sustained release profile of ASA in 7 days. AM-coated electrospun PLLA fibers showed enhanced cytocompatibility for human umbilical vein ECs, making a confluent endothelial-like lining. In addition, AM lysate-coated ASA-PLLA-aligned scaffold proved to support endothelial differentiation of Wharton's jelly-derived mesenchymal stem cells. Our results together indicated that AM lysate-coated ASA releasing scaffolds have promising potentials for development of a biocompatible SDVG.

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.

Decellularized Wharton's jelly extracellular matrix as a promising scaffold for promoting hepatic differentiation of human induced pluripotent stem cells.

Liver tissue engineering as a therapeutic option for restoring of damaged liver function has a special focus on using native decellularized liver matrix, but there are limitations such as the shortage of liver donor. Therefore, an appropriate alternative scaffold is needed to circumvent the donor shortage. This study was designed to evaluate hepatic differentiation of human induced pluripotent stem cells (hiPSCs) in decellularized Wharton's jelly (WJ) matrix as an alternative for native liver matrix. WJ matrices were treated with a series of detergents for decellularization. Then hiPSCs were seeded into decellularized WJ scaffold (DWJS) for hepatic differentiation by a defined induction protocol. The DNA quantitative assay and histological evaluation showed that cellular and nuclear materials were efficiently removed and the composition of extracellular matrix was maintained. In DWJS, hiPSCs-derived hepatocyte-like cells (hiPSCs-Heps) efficiently entered into the differentiation phase (G1) and gradually took a polygonal shape, a typical shape of hepatocytes. The expression of hepatic-associated genes (albumin, TAT, Cytokeratin19, and Cyp7A1), albumin and urea secretion in hiPSCs-Heps cultured into DWJS was significantly higher than those cultured in the culture plates (2D). Altogether, our results suggest that DWJS could provide a proper microenvironment that efficiently promotes hepatic differentiation of hiPSCs.

Derivation of male germ cells from induced pluripotent stem cells by inducers: A review.

Induced pluripotent stem cells (iPSCs) refer to stem cells that are artificially produced using a new technology known as cellular reprogramming, which can use gene transduction in somatic cells. There are numerous potential applications for iPSCs in the field of stem cell biology becauase they are able to give rise to several different cell features of lineages such as three-germ layers. Primordial germ cells, generated via in vitro differentiation of iPSCs, have been demonstrated to produce functional gametes. Therefore, in this review we discussed past and recent advances in the in vitro differentiation of germ cells using pluripotent stem cells with an emphasis on iPSCs. Although this domain of research is still in its infancy, exploring development mechanisms of germ cells is promising, especially in humans, to promote future reproductive and developmental engineering technologies. While few studies have evaluated the ability and efficiency of iPSCs to differentiate toward male germ cells in vitro by different inducers, the given effect was investigated in this review.

The osmolyte type affects cartilage associated pathologic marker expression during in vitro mesenchymal stem cell chondrogenesis under hypertonic conditions.

Stem cells' fate during in vitro differentiation is influenced by biophysicochemical cues. Osmotic stress has proved to enhance chondrocyte marker expression, however its potent negative impacts had never been surveyed. We questioned whether specific osmotic conditions, regarding the osmolyte agent, could benefit chondrogenesis while dampening undesired concomitant hypertrophy and inflammatory responses. To examine the potential side effects of hypertonicity, we assessed cell proliferation as well as chondrogenic and hypertrophic marker expression of human Adipose Derived-MSC after a two week induction in chondrogenic media with either NaCl or Sorbitol, as the osmolyte agent to reach a +100 mOsm hypertonic condition. Calcium deposition and TNF-α secretion as markers associated with hypertrophy and inflammation were then assayed. While both hyperosmotic conditions upregulated chondrogenic markers, sorbitol had a nearly three times higher chondro-promotive effect and a lesser hypertrophic effect compared to NaCl. Also, a significantly lesser calcium deposition was observed in sorbitol hypertonic group. NaCl showed an anti-proinflammatory effect while sorbitol had no effect on inflammatory markers. The ossification potential and cartilage associated pathologic markers were affected differentially by the type of the osmolyte. Thus, a vigilant application of the osmotic agent is inevitable in order to avoid or reduce undesired hypertrophic and inflammatory phenotype acquisition by MSC during chondrogenic differentiation. Our findings are a step towards developing a more reliable chondrogenic regimen using external hypertonic cues for MSC chondrogenesis with potential applications in chondral lesions cell therapy.

Enhanced chondrogenesis differentiation of human induced pluripotent stem cells by MicroRNA-140 and transforming growth factor beta 3 (TGFß3).

Induced pluripotent stem cells (iPSCs) make an attractive source for regenerative medicine. The objective of our study was to establish a new method for differentiation of human iPSCs toward chondrocyte by overexpression of MicroRNA-140 (miR-140) and use of transforming growth factor beta 3 (TGFß3) in high-cell density culture systems. We prepared vectors and then was used for recombinant Lenti virus production in HEK-293 cell. Transducted cells were selected and cultured in pellet culture system and were harvested after days 7, 14 and 21. Real-time PCR was performed to evaluate the cartilage-specific genes in the mRNA levels. Also, in order to confirm our results, we have done immunological assay. iPSCs were transducted with recombinant Lenti virus and miR-140 was expressed. Immunological methods confirmed that differentiation of iPSC toward chondrocyte with handling cartilage matrix genes. Also real time PCR demonstrated that in transducted iPSCs significantly increased gene expression of collagen type II, SOX9 and aggrecan, and down-regulated expression of collagen type I when compared to the mRNA levels measured in non transducted iPSCs. In Conclusion, our data implies that miR-140 is a potent chondrogenic differentiation inducer for iPSCs and also, we have showed increasing chondrogenic differentiation by using overexpression of miR-140 and TGFß3.

Improved stem cell therapy of spinal cord injury using GDNF-overexpressed bone marrow stem cells in a rat model.

The use of stem cell base therapy as an effective strategy for the treatment of spinal cord injury (SCI) is very promising. Although some strategy has been made to generate neural-like cells using bone marrow mesenchymal stem cells (BMSCs), the differentiation strategies are still inefficiently. For this purpose, we improved the therapeutic outcome with utilize both of N-neurotrophic factor derived Gelial cells (GDNF) gene and differentiation medium that induce the BMSCs into the neural-like cells. The differentiated GDNF overexpressed BMSCs (BMSCs-GDNF) were injected on the third day of post-SCI. BBB score test was performed for four weeks. Two weeks before the end of BBB, biotin dextranamin was injected intracrebrally and at the end of the fourth week, the tissue was stained. BBB scores were significantly different in BMSCs-GDNF injected and control animals. Significant difference in axon counting was observed in BMSCs-GDNF treated animals compared to the control group. According to the results, differentiated BMSCs-GDNF showed better results in comparison to the BMSCs without genetic modification. This study provides a new strategy to investigate the role of simultaneous in stem cell and gene therapy for future neural-like cells transplantation base therapies for SCI.

The generation of islet-like insulin-producing cells from Wharton's jelly-derived mesenchymal stem cells on the PES/fish gelatin scaffold.

Diabetes Mellitus (DM) disrupts the body's capability to control blood glucose statuses. Type 1 diabetes mellitus (T1DM) arises from inadequate insulin production and is treated with insulin replacement therapy. Stem cell therapy is a hopeful treatment for T1DM that involves using adult stem cells to generate insulin-producing cells (IPCs). Mesenchymal stem cells (MSCs) are particularly advantageous for generating IPCs. The islet cells require interactions with the extracellular matrix for survival, which is lacking in conventional 2D culture systems. Natural or synthetic polymers create a supportive 3D microenvironment in tissue engineering. We aim to construct superior differentiation conditions employing polyethersulfone (PES)/Fish gelatin scaffolds to differentiate Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) to IPCs. In this study, the PES/fish gelatin scaffold (3D) was manufactured by electrospinning, and then its biocompatibility and non-toxicity were investigated by MTT assay. After that, scaffold-supportive effects on WJ-MSCs differentiation to IPCs were studied at the gene and protein levels. After exposure to the differentiation media, 2D and 3D (PES/Fish gelatin) cultured cells were slowly aggregated and developed spherical-shaped clusters. The viability of cells was found to be comparable in both 2D and 3D cultures. The gene expression analysis showed that efficiency of differentiation was more elevated in 3D culture. Additionally, ELISA results indicated that C-peptide and insulin release were more significant in 3D than in 2D culture. In conclusion, the PES/fish gelatin scaffold is highly promising for pancreatic tissue engineering because it supports the viability, growth, and differentiation of WJ-MSCs into IPCs.

Applications of extraembryonic tissue-derived cells in vascular tissue regeneration.

Vascular tissue engineering is a promising approach for regenerating damaged blood vessels and developing new therapeutic approaches for heart disease treatment. To date, different sources of cells have been recognized that offer assistance within the recovery of heart supply routes and veins with distinctive capacities and are compelling for heart regeneration. However, some challenges still remain that need to be overcome to establish the full potential application of these cells. In this paper, we review the different cell sources used for vascular tissue engineering, focusing on extraembryonic tissue-derived cells (ESCs), and elucidate their roles in cardiovascular disease. In addition, we highlight the intricate interplay between mechanical and biochemical factors in regulating mesenchymal stem cell (MSC) differentiation, offering insights into optimizing their application in vascular tissues.

Improved efficiency of genome editing by constitutive expression of Cas9 endonuclease in genetically-modified mice.

Despite its convenience and precision, CRISPR-based gene editing approaches still suffer from off-target effects and low efficiencies, which are partially rooted in Cas9, the nuclease component of the CRISPR/Cas9 system. In this study, we showed how mouse genome editing efficiency can be improved by constitutive and inheritable expression of Cas9 nuclease. For this goal, a transgenic mouse line expressing the Cas9 protein (Cas9-mouse) was generated. For in vitro assessment of gene editing efficiency, the Cas9-mice were crossed with the EGFP-mice to obtain mouse embryonic fibroblasts (MEF) expressing both EGFP and Cas9 (MEFCas9-EGFP). Transfection of these cells with in vitro transcribed (IVT) EGFP sgRNA or phU6-EGFPsgRNA plasmid led to robust decrease of Mean Fluorescent Intensity (MFI) to 8500 ± 1025 a.u. and 13,200 ± 1006 a.u. respectively. However, in the control group, in which the MEFEGFP cells were transfected with a pX330-EGFPsgRNA plasmid, the measured MFI was 16,800 ± 2254 a.u. For in vivo assessment, the Cas9-zygotes at two pronuclei stage (2PN) were microinjected with a phU6-HhexsgRNA vector and the gene mutation efficiency was compared with the wild-type (WT) zygotes microinjected with a pX330-HhexsgRNA plasmid. The analysis of born mice showed that while the injection of Cas9-zygotes resulted in 43.75% Hhex gene mutated mice, it was just 15.79% for the WT zygotes. In conclusion, the inheritable and constitutive expression of Cas9 in mice provides an efficient platform for gene editing, which can facilitate the production of genetically-modified cells and animals.

An in situ hydrogel-forming scaffold loaded by PLGA microspheres containing carbon nanotube as a suitable niche for neural differentiation.

The cell-extracellular matrix (ECM) interactions are known to have a strong impact on cell behaviors in neural tissues. Due to complex physiology system and limited regenerative capacity of nervous system, neural tissue engineering has attracted attention as a promising strategy. In this study, we designed a hydrogel loaded by poly (lactic-co-glycolic acid) (PLGA) microspheres containing carbon nanotubes (CNT) and the biochemical differentiation factors, as a scaffold, in order to replicate the neural niche for stem cell growth (and/or differentiation). Different formulations from Hyaluronic acid (H), Poloxamer (P), Ethoxy-silane-capped poloxamer (PE), and cross-linked Alginate (Alg) were utilized as an in situ gel structure matrix to mirror the mechanical properties of the ECM of CNS. Subsequently, conductivity, surface morphology, size of microspheres, and CNT dispersion in microsphere were measured using two probes electrical conductometer, scanning electron microscopy (SEM), dynamic light scattering (DLS), and Raman spectroscopy, respectively. According to SEM and fluorescent microscopy images, CNTs increased the porosity of polymeric structure, which, in turn, facilitated the adhesion of stem cells on the surface of microspheres compared with control. Microstructure and rheological behaviors of different gel compositions were investigated using SEM and parallel-plate oscillatory rheometer, respectively. The MTT assay showed the toxicity profile of hydrogels was appropriate for cell transplantation. The confocal images illustrated the 3D platform of P15%H10% and P20%H5% gel formulations containing the PLGA-CNT microspheres, which allows the proliferation of neural stem cells (NSCs) derived from MSC. The results of real-time PCR and immunocytochemistry showed neuronal differentiation capacity of cultured NSCs derived from MSC in the alginate gel that contained PLGA-CNT microspheres as well as other control groups. The dispersion of the CNT-PLGA microspheres, covered by NSCs, into alginate gel in the presence of induction factors was found to notably enhance the expression of Sox2-SYP and ß-Tubulin III neuronal markers.

Microfluidic-Based Droplets for Advanced Regenerative Medicine: Current Challenges and Future Trends.

Microfluidics is a promising approach for the facile and large-scale fabrication of monodispersed droplets for various applications in biomedicine. This technology has demonstrated great potential to address the limitations of regenerative medicine. Microfluidics provides safe, accurate, reliable, and cost-effective methods for encapsulating different stem cells, gametes, biomaterials, biomolecules, reagents, genes, and nanoparticles inside picoliter-sized droplets or droplet-derived microgels for different applications. Moreover, microenvironments made using such droplets can mimic niches of stem cells for cell therapy purposes, simulate native extracellular matrix (ECM) for tissue engineering applications, and remove challenges in cell encapsulation and three-dimensional (3D) culture methods. The fabrication of droplets using microfluidics also provides controllable microenvironments for manipulating gametes, fertilization, and embryo cultures for reproductive medicine. This review focuses on the relevant studies, and the latest progress in applying droplets in stem cell therapy, tissue engineering, reproductive biology, and gene therapy are separately evaluated. In the end, we discuss the challenges ahead in the field of microfluidics-based droplets for advanced regenerative medicine.

Development of Inactivated FAKHRAVAC® Vaccine against SARS-CoV-2 Virus: Preclinical Study in Animal Models.

The recent viral infection disease pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in a global public health crisis. Iran, as one of the countries that reported over five million infected cases by September 2021, has been concerned with the urgent development of effective vaccines against SARS-CoV-2. In this paper, we report the results of a study on potency and safety of an inactivated SARS-CoV-2 vaccine candidate (FAKHRAVAC) in a preclinical study so as to confirm its potential for further clinical evaluation. Here, we developed a pilot-scale production of FAKHRAVAC, a purified inactivated SARS-CoV-2 virus vaccine candidate that induces neutralizing antibodies in Balb/c mice, guinea pigs, rabbits, and non-human primates (Rhesus macaques-RM). After obtaining ethical code of IR.IUMS.REC.1399.566, immunizations of animals were conducted by using either of three different vaccine dilutions; High (H): 10 µg/dose, Medium (M): 5 µg/dose, and Low (L): 1 µg/dose, respectively. In the process of screening for viral seeds, viral strains that resulted in the most severe clinical manifestation in patients have been isolated for vaccine development. The viral seed produced the optimal immunity against SARS-CoV-2 virus, which suggests a possible broader neutralizing ability against SARS-CoV-2 strains. The seroconversion rate at the H-, M-, and L-dose groups of all tested animals reached 100% by 28 days after immunization. These data support the eligibility of FAKHRAVAC vaccine candidate for further evaluation in a clinical trial.

DKK1 expression is suppressed by miR-9 during induced dopaminergic differentiation of human trabecular meshwork mesenchymal stem cells.

Wnt/ß-catenin pathway has been recently identified as one of the key players in dopaminergic (DA) neuron differentiation. DKK1, the potent inhibitor of the Wnt/ß-catenin pathway, is expressed in a precisely controlled manner in ventral midbrain during brain development, however the molecular mechanism underlying this regulation is still unknown. Here we show that human trabecular meshwork mesenchymal stem cells (TM-MSCs) can be used as an efficient tool for in vitro differentiation of DA neurons. After differentiating TM-MSCs to DA neuron-like cells, ß-catenin protein accumulation was increased in the nucleus, indicating the increased activity of Wnt/ß-catenin pathway in the time-window of DA differentiation. Interestingly, DKK1 transcript level was reduced dramatically after DA induction in TM-MSCs which was accompanied by an increase in the in silico-predicted MIR9 and MIR101 levels. Measuring DKK1 expression level after overexpressing either MIR9 or MIR101 and performing luciferase assay alongside, revealed that both miR-9 and miR-101 suppress DKK1 expression and that miR-9 exerts a direct inhibitory effect on 3'UTR regulatory region. Therefore miR-9 and miR-101 might explain, at least in part, the underlying regulatory mechanism of DKK1 reduction and resulting Wnt/ß-catenin pathway activation during DA neuron differentiation process.

Zinc silicate mineral-coated scaffold improved in vitro osteogenic differentiation of equine adipose-derived mesenchymal stem cells.

In current study we aimed to coat the PLLA scaffold with zinc (Zn) silicate mineral nanoparticles. Then, using equine adipose-derived stem cells (ASCs) we intended to compare the osteogenic induction potency of Zn silicate mineral-coated PLLA scaffold with uncoated PLLA scaffold and tissue culture plastic (TCPS). Adipose tissues were collected from 3 horses, and isolation of ASCs was achieved by enzymatic digestion. PLLA scaffold was successfully prepared using a phase separation method and coated with Zn silicate mineral nanoparticles. The coating efficiency was then characterized by scanning electron microscopy and further evaluated with the application of fourier transform infrared microscopic imaging. Viability and growth characteristics of ASCs on TCPS, uncoated and coated PLAA scaffolds were investigated by MTT assay. Alizarin Red staining was performed for determination of calcium deposition following the osteogenic induction. Furthermore, other common osteogenic markers such as alkaline phosphatase (ALP) activity, calcium content, as well as osteogenic (Runx2, ALP, osteonectin, and collagen I) marker genes were also evaluated. Our data showed that Zn silicate mineral nanoparticles was coated successfully on PLLA scaffold and such scaffold had no detrimental effect on cell growth rate as indicated by MTT assay. Moreover, ASCs that differentiated on Zn silicate mineral-coated PLLA scaffold indicated higher ALP activity, more calcium content, and higher expression of bone-related genes than that on uncoated PLLA scaffold and TCPS. Adequate proliferation rate and higher expression of osteogenic markers of stem cells, provides this scaffold as a suitable substrate to support proliferation and differentiation of ASCs in equine.