Allogenic mesenchymal stromal cells and their extracellular vesicles in COVID-19 induced ARDS: a randomized controlled trial.

BACKGROUND AND AIMS: The main causes of death in patients with severe Coronavirus disease-2019 (COVID-19) are acute respiratory distress syndrome (ARDS) and multiorgan failure caused by a severe inflammatory cascade. Novel treatment strategies, such as stem-cell-based therapy and their derivatives can be used to relieve inflammation in these cases. In this study, we aimed to evaluate the safety and efficacy of therapy using mesenchymal stromal cells (MSCs) and their derived extracellular vesicles in COVID-19 patients. MATERIALS AND METHODS: COVID-19 patients with ARDS were included in this study and allocated into two study and control groups using block randomization. While all patients received recommended treatment based on guidelines from the national advisory committee for COVID-19 pandemic, the two intervention groups received two consecutive injections of MSCs (100 × 106 cells) or one dose of MSCs (100 × 106 cells) followed by one dose of MSC-derived extracellular vesicles (EVs). Patients were assessed for safety and efficacy by evaluating clinical symptoms, laboratory parameters, and inflammatory markers at baseline and 48 h after the second intervention. RESULTS: A total number of 43 patients (the MSC alone group = 11, MSC plus EV group = 8, and control group = 24) were included in the final analysis. Mortality was reported in three patients in the MSC alone group (RR: 0.49; 95% CI 0.14-1.11; P = 0.08); zero patient in the MSC plus EV group (RR: 0.08; 95% CI 0.005-1.26; P = 0.07) and eight patients in the control group. MSC infusion was associated with a decrease in inflammatory cytokines such as IL-6 (P = 0.015), TNF-α (P = 0.034), IFN-γ (P = 0.024), and CRP (P = 0.041). CONCLUSION: MSCs and their extracellular vesicles can significantly reduce the serum levels of inflammatory markers in COVID-19 patients, with no serious adverse events. Trial registration IRCT, IRCT registration number: IRCT20200217046526N2. Registered 13th April 2020, http://www.irct.ir/trial/47073 .

In Vitro Modeling as a Tool for Testing Therapeutics for Spinal Muscular Atrophy and IGHMBP2-Related Disorders.

Spinal Muscular Atrophy (SMA) is the leading genetic cause of infant mortality. The most common form of SMA is caused by mutations in the SMN1 gene, located on 5q (SMA). On the other hand, mutations in IGHMBP2 lead to a large disease spectrum with no clear genotype-phenotype correlation, which includes Spinal Muscular Atrophy with Muscular Distress type 1 (SMARD1), an extremely rare form of SMA, and Charcot-Marie-Tooth 2S (CMT2S). We optimized a patient-derived in vitro model system that allows us to expand research on disease pathogenesis and gene function, as well as test the response to the AAV gene therapies we have translated to the clinic. We generated and characterized induced neurons (iN) from SMA and SMARD1/CMT2S patient cell lines. After establishing the lines, we treated the generated neurons with AAV9-mediated gene therapy (AAV9.SMN (Zolgensma) for SMA and AAV9.IGHMBP2 for IGHMBP2 disorders (NCT05152823)) to evaluate the response to treatment. The iNs of both diseases show a characteristic short neurite length and defects in neuronal conversion, which have been reported in the literature before with iPSC modeling. SMA iNs respond to treatment with AAV9.SMN in vitro, showing a partial rescue of the morphology phenotype. For SMARD1/CMT2S iNs, we were able to observe an improvement in the neurite length of neurons after the restoration of IGHMBP2 in all disease cell lines, albeit to a variable extent, with some lines showing better responses to treatment than others. Moreover, this protocol allowed us to classify a variant of uncertain significance on IGHMBP2 on a suspected SMARD1/CMT2S patient. This study will further the understanding of SMA, and SMARD1/CMT2S disease in particular, in the context of variable patient mutations, and might further the development of new treatments, which are urgently needed.

A Roadmap for the Production of a GMP-Compatible Cell Bank of Allogeneic Bone Marrow-Derived Clonal Mesenchymal Stromal Cells for Cell Therapy Applications.

BACKGROUND: Allogeneic mesenchymal stromal cells (MSCs) have been used extensively in various clinical trials. Nevertheless, there are concerns about their efficacy, attributed mainly to the heterogeneity of the applied populations. Therefore, producing a consistent population of MSCs is crucial to improve their therapeutic efficacy. This study presents a good manufacturing practice (GMP)-compatible and cost-effective protocol for manufacturing, banking, and lot-release of a homogeneous population of human bone marrow-derived clonal MSCs (cMSCs). METHODS: Here, cMSCs were isolated based on the subfractionation culturing method. Afterward, isolated clones that could reproduce up to passage three were stored as the seed stock. To select proliferative clones, we used an innovative, cost-effective screening strategy based on lengthy serial passaging. Finally, the selected clones re-cultured from the seed stock to establish the following four-tired cell banking system: initial, master, working, and end of product cell banks (ICB, MCB, WCB, and EoPCB). RESULTS: Through a rigorous screening strategy, three clones were selected from a total of 21 clones that were stored during the clonal isolation process. The selected clones met the identity, quality, and safety assessments criteria. The validated clones were stored in the four-tiered cell bank system under GMP conditions, and certificates of analysis were provided for the three-individual ready-to-release batches. Finally, a stability study validated the EoPCB, release, and transport process of the frozen final products. CONCLUSION: Collectively, this study presents a technical and translational overview of a GMP-compatible cMSCs manufacturing technology that could lead to the development of similar products for potential therapeutic applications.

The therapeutic potential of adipose tissue-derived mesenchymal stromal cells in the treatment of busulfan-induced azoospermic mice.

PURPOSE: The generation of germ cells from mesenchymal stromal cells (MSCs) provides a valuable in vitro platform for infertility modeling. The establishment of these cells is a new approach for assisted reproductive technology (ART) to help infertile patients who lack functional gametes. METHODS: Human adipose-derived MSCs were isolated and then characterized for multipotency by flow cytometry, differentiation capacity, and cytogenetic assays. These cells were used in a male germ cell differentiation study. The expression of male germ cell markers was evaluated at day 21 of differentiation using an immunofluorescence assay, flow cytometry, and RT-qPCR. Undifferentiated MSCs were used for transplantation in busulfan-induced azoospermic mice. RESULTS: In this study, MSCs were successfully isolated from human adipose tissues which were positive for cell markers such as CD90, CD105, CD73, and CD29 but negative for CD34 and CD45. The results of flow cytometry, immunocytochemistry, and RT-qPCR analysis at day 21 of differentiation showed that the undifferentiated adipose-derived MSCs are able to differentiate into male germ cells. Additionally, transplantation of undifferentiated MSCs in busulfan-induced azoospermic mice caused spermatogenesis recovery in the majority of seminiferous tubules. CONCLUSION: In this study, we showed that differentiation of human adipose-derived MSCs into male germ cells is a useful tool for in vitro study of human germ cell development. Our results demonstrated that cell therapy with adipose-derived MSCs could help the repair of pathological changes in testicular seminiferous tubules. Therefore, it may have a clinical application for the treatment of azoospermia in infertile patients.

Cryopreservation of Iranian Markhoz goat fibroblast cells as an endangered national genetic resource.

BACKGROUND: The continuous accessibility of local animals for sustainable use is being eroded annually. Thus, a strategic vision for the conservation of biodiversity is of far-reaching emphasis to deal with unprecedented challenges in the local population extension facing in the future. This study aimed to establish and cryopreserve endangered Markhoz goat (Capra hircus) fibroblast cell lines in vitro. METHODS AND RESULTS: These primary fibroblast cells were isolated from 58 Iranian Markhoz goats and individually cultured by explant technique in DMEM medium supplemented with 10% FBS and 2 mM L-Glutamine, in the presence of Penicillin (200 U/ml)-Streptomycin (200 mg/ml) during the first passage number. The extracted cell lines were confirmed morphologically as fibroblast cells. The population doubling time for DMEM-cultured cells was 23 ± 0.5 h. Chromosomal analysis indicated a total chromosome number of 2n = 60 with > 95% frequency. The cultured cells were checked for bacteria, fungi, yeast, and mycoplasma contaminations and the results were reported negative. The efficiencies of the fluorescent protein encoded by VSV-G (pMDG) and lentiviral pCSGW vectors reported in a range of 65% value. According to the species identification analysis, the goat cell lines were banked and confirmed without any miss- and cross-contamination. CONCLUSIONS: The significant issue in this paper can be concluded about the first report of the establishment of endangered Markhoz goat cell banking inside the country. This study demonstrated the successful establishment of a genetically stable fibroblast bank as a valuable genetic resource for the endangered Iranian Markhoz goat breed.

The effect of aflibercept and arsenic trioxide on the proliferation, migration and apoptosis of oral squamous cell carcinoma in vitro.

Aflibercept and arsenic trioxide drugs apply a cytotoxic effect on some human cancer cell lines. However, no more study has followed the effects of both drugs, especially arsenic trioxide, on oral squamous cell carcinoma (OCC). We used three OCC lines as a model to show the effect of these drugs on the genetically complex disease and investigate its targeted therapy. In this study, three human OCC cell lines were used from different patients. We treated cell lines with both medications to detect the effect and relevant molecular basis. First, methyl thiazolyl tetrazolium (MTT) assay was performed to detect the cytotoxicity effect and cell growth. Second, flow cytometry, gene and protein expression were performed to evaluate the anti-angiogenic effect on OCC lines. Next apoptosis was analyzed by flow cytometry. Finally, clonogenesis capacity and cell migration were assessed by colony formation assay and wound healing, respectively. Aflibercept had no cytotoxic effect on the three OCC cell lines but decreased cell growth rate. Arsenic trioxide had a significant cytotoxic effect on three cell lines. Our results demonstrated that both drugs significantly decreased endoglin, VEGFA, and VEGFB expression. In addition, Migration and colony formation assays confirmed that these drugs have significant anti-proliferative and anti-migration effect on oral carcinoma cells. These results revealed that both medications might be a potential drug for the management of oral cancer patients.

Establishment and Preservation of Lymphoblastoid Cell Lines from Fresh and Frozen Whole Blood and Mononuclear Cells.

Although blood cells are interesting sources for genome investigations, one of the main problems in obtaining genomic DNA from blood is the restricted amount of DNA. This obstacle can be avoided by generating Epstein-Barr virus (EBV)-induced B cell lines. This study investigates the efficiency of four different methods to generate lymphoblastoid cell lines (LCLs). Blood samples (n = 120) were obtained from donors and categorized into four groups: fresh whole blood, frozen whole blood, fresh peripheral blood mononuclear cells (PBMCs), and frozen PBMCs. The samples were followed by EBV transformation to generate LCLs. Quality control and authentication of the cells were performed using multiplex PCR and short tandem repeat (STR) analyses. Finally, we assessed the success rate and amount of time to establish the cell lines in each group. The results showed that the cells were not contaminated nor were they misidentified or cross-contaminated with other cells. The success rate of LCLs generated from the whole blood groups was lower than the PBMC groups. The freezing procedures did not have any considerable effect on the establishment of lymphoblastoid cells. These established cells have been preserved in the human and animal cell bank of the Iranian Biological Resource Center (IBRC) and are available for researchers. Due to the management and transformation of a substantial number of blood samples, we recommend that researchers freeze PBMCs for further use with high efficiency and time-saving. We suggest that whole fresh blood should be directly transformed when the volume of the blood sample is less than 0.5 ml.

Three-dimensional decellularized amnion membrane scaffold promotes the efficiency of male germ cells generation from human induced pluripotent stem cells.

Cells grow differently in conventional 2D cell culture than when they grow in the physiological microenvironment. In this study, we developed a 3D cell culture model for generating male germ cells from human iPSCs using a human decellularized amnion membrane (DAM) scaffold. To this end, human iPSCs were generated using retroviral vectors and characterized for pluripotency properties by immunofluorescence assay, flow cytometry, ALP staining, cytogenetic assay, and differentiation capacity. The iPSCs were used for investigating male germ cells differentiation efficiency in both conventional 2D culture and 3D-DAM scaffold. The expression of male germ cell markers was evaluated at day 21 of differentiation using immunofluorescence assay, flow-cytometry, and RT-qPCR. The results indicated a successful reprogramming of human foreskin fibroblast cells into pluripotent iPSCs. The reprogrammed cells were positive for pluripotency markers and differentiated into the three germ layers. During male germ cell differentiation, the cells tend to aggregate and form colony-like structures in both 2D and 3D conditions. However, significant expression of VASA, DAZL, PLZF, STELLA, and NANOS3 markers and more efficient haploid male germ cell production were observed in the 3D condition when compared to the 2D model. Considering the effect of the 3D-DAM scaffold in prompting male germ cell-specific markers and increased efficiency of germ cell differentiation in 3D culture, it appears that DAM scaffold is a useful tool for in vitro studies of human germ cell development and ultimately future clinical application.

Simvastatin Induces Apoptosis in Medulloblastoma Brain Tumor Cells via Mevalonate Cascade Prenylation Substrates.

Medulloblastoma is a common pediatric brain tumor and one of the main types of solid cancers in children below the age of 10. Recently, cholesterol-lowering "statin" drugs have been highlighted for their possible anti-cancer effects. Clinically, statins are reported to have promising potential for consideration as an adjuvant therapy in different types of cancers. However, the anti-cancer effects of statins in medulloblastoma brain tumor cells are not currently well-defined. Here, we investigated the cell death mechanisms by which simvastatin mediates its effects on different human medulloblastoma cell lines. Simvastatin is a lipophilic drug that inhibits HMG-CoA reductase and has pleotropic effects. Inhibition of HMG-CoA reductase prevents the formation of essential downstream intermediates in the mevalonate cascade, such as farnesyl pyrophosphate (FPP) and gernaylgerany parophosphate (GGPP). These intermediates are involved in the activation pathway of small Rho GTPase proteins in different cell types. We observed that simvastatin significantly induces dose-dependent apoptosis in three different medulloblastoma brain tumor cell lines (Daoy, D283, and D341 cells). Our investigation shows that simvastatin-induced cell death is regulated via prenylation intermediates of the cholesterol metabolism pathway. Our results indicate that the induction of different caspases (caspase 3, 7, 8, and 9) depends on the nature of the medulloblastoma cell line. Western blot analysis shows that simvastatin leads to changes in the expression of regulator proteins involved in apoptosis, such as Bax, Bcl-2, and Bcl-xl. Taken together, our data suggests the potential application of a novel non-classical adjuvant therapy for medulloblastoma, through the regulation of protein prenylation intermediates that occurs via inhibition of the mevalonate pathway.

Three-dimensional decellularized amnion membrane scaffold as a novel tool for cancer research; cell behavior, drug resistance and cancer stem cell content.

BACKGROUND: Cancer is the second leading cause of human death. Therefore, comprehensive research and the appropriate tools are needed in this field. Animal models and cell culture studies are the most important preclinical tools in cancer research. In 2D cell culture models, cells are forced to grow in a 2D environment, which differs from their natural physiology. Recently, 3D cell culture models were developed to fill the gap between 2D cell culture and animal models. MATERIALS AND METHODS: Human amniotic membranes were obtained, decellularized, characterized and used as a natural 3D scaffold to investigate cancer cell behavior in 2D compared to 3D conditions. Time-lapse imaging of cells was used, and cell proliferation, velocity and migration were evaluated. Cisplatin was applied in 2D and 3D conditions, followed by evaluation of viability, apoptosis and cancer stem cell proteins by flow cytometry and western blot analysis. RESULTS: The results showed that in the decellularized amnion membrane (DAM) scaffold most cells did not spread and remain rounded and then penetrated into the scaffold with no cytotoxicity. Significant differences in migration, velocity, morphology and proliferation of cancer cells were observed between the 3D DAM scaffold and 2D model. Furthermore, the cells in the 3D DAM scaffold showed much more resistance to apoptosis and higher CSC content. CONCLUSION: In conclusion, considering the effect of the 3D DAM scaffold in cell behavior, apoptosis resistance and CSC content as well as the short processing time for decellularizing the AM, it appears that the 3D DAM scaffold offers an appropriate tool for in vitro cancer research.

Establishment and characterization of rough-tailed gecko original tail cells.

Some of lizard species have the ability to lose their tail in order to defend against predators and regenerate the new tail. Lizard's regenerated tail has attracted scientists' attention for unraveling the regeneration process, but less information is known about the cellular characterization and cell growth properties of original tail. This research aimed to report cell culture and banking process of rough-tailed gecko or Cyrtopodion scabrum's original tail cell sample from inner tissue without skin using tissue explant technique. For banking reports, it is essential to analyze this cells' potential to proliferate, to investigate biological aspects such as cell culture features, differentiation and chromosome number and to report its species identification and quality control. To achieve optimal growth conditions, three different temperatures for incubation including 18, 23 and 37 °C and two different media including DMEM and L-15 were applied. The expanded cells were studied for their potential to adipose and osteoblast differentiation. Results indicated that lizard's original tail cells could be successfully obtained by explant technique. The cells demonstrated fibroblast like morphology with population doubling times of approximately 24 ± 0.5 h. Karyotyping analysis showed a distribution of 2n = 40 chromosome number for this cell line. The comparison of different incubation media and temperatures showed that cell growth is equally optimal in all mentioned conditions according to growth curves. Adipose and osteoblast differentiation was obviously observed in these cells which confirms the hint of stem-ness in the produced mixed cells. According to cell banking policies, produced cells were also checked for bacterial, fungal, yeast and mycoplasma contaminations and no contamination was observed. Multiplex PCR for identification of species confirmed the species of lizard with no cross-contamination with other cells in the cell bank. Establishment of authenticated and well-characterized lizard's original tail cell line will provide a valuable source for subsequent in vitro regenerative research and molecular studies which are not feasible in in vivo methods. This finding will allow us to get an opportunity to create and preserve a new collection of lizard cell lines in the future.

Anti-angiogenic efficacy of aflibercept and bevacizumab in primary oral squamous cell carcinoma cells.

BACKGROUND: In recent decades, anti-angiogenic treatment strategy has been well described in cancer treatment. The anti-angiogenic activity of both bevacizumab and aflibercept has been researched on 10 previously established primary oral squamous cell carcinoma (OSCC) cells of an Iranian population with different levels of purity, in an attempt to find the most effective anti-angiogenic-targeted drug. METHODS: To investigate and compare the effect of bevacizumab and aflibercept on vascular endothelial growth factor (VEGF) secretion of 10 primary OSCC cells, cell proliferation and viability were assessed by ELISA and MTT assays. In addition, cell migration was studied using scratch assay. RESULTS: The results showed that VEGF impressively expressed in all primary cancer cells. Although both drugs significantly reduced the secretion of VEGF, the effect of aflibercept was more prominent. Also, bevacizumab-treated cells migration was lower than the control group and the cells treated with aflibercept showed the lowest migration rate compared to bevacizumab and control groups. CONCLUSION: The anti-angiogenic-targeted drugs, especially Af, might be effective in treatment of patients with OSCC in combination with conventional surgical treatments.

Establishment and Characterization of Primary Cultures from Iranian Oral Squamous Cell Carcinoma Patients by Enzymatic Method and Explant Culture.

OBJECTIVES: Oral Squamous Cell Carcinoma (OSCC) is the most frequent oral cancer worldwide. It is known as the eighth most common cancer in men and as the fifth most common cancer in women. Cytogenetic and biochemical studies in recent decades have emphasized the necessity of providing an appropriate tool for such researches. Cancer cell culture is a useful tool for investigations on biochemical, genetic, molecular and immunological characteristics of different cancers, including oral cancer. Here, we explain the establishment process of five primary oral cancer cells derived from an Iranian population. MATERIALS AND METHODS: The specimens were obtained from five oral cancer patients. Enzymatic, explant culture and magnetic-activated cell sorting (MACS) methods were used for cell isolation. After quality control tests, characterization and authentication of primary oral cancer cells were performed by short tandem repeats (STR) profiling, chromosome analysis, species identification, and monitoring the growth, morphology and the expression of CD326 and CD133 markers. RESULTS: Five primary oral cancer cells were established from an Iranian population. The flow cytometry results showed that the isolated cells were positive for CD326 and CD133 markers. Furthermore, the cells were free from mycoplasma, bacterial and fungal contamination. No misidentified or cross-contaminated cells were detected by STR analysis. CONCLUSIONS: Human primary oral cancer cells provide an extremely useful platform for studying carcinogenesis pathways of oral cancer in Iranian population. They may be helpful in explaining the ethnic differences in cancer biology and the individuality in anticancer drug response in future studies.

Establishment and characterization of Caspian horse fibroblast cell bank in Iran.

Caspian horse, a rare horse breed found in 1965 by Louise Firouz in northern Iran, is a small horse which is reported to be in danger of extinction in its original homeland. There seems to be a great need to prevent extinction of this valuable horse. In this study, 51 fibroblast cell lines from Caspian horse ear marginal tissue were successfully established by sampling 60 horses using primary explant technique. Cells were authenticated and growth curve was plotted. According to results obtained, population doubling time (PDT) was calculated 23 ± 0.5 h for all cell lines. Multiplex polymerase chain reaction (multiplex PCR) revealed that cell lines had no cross-contamination with other species. Bacteria, fungi, and mycoplasma contamination were checked using standard methods such as PCR, direct culture, and Hoechst staining. In addition to providing a valuable source for genomic, postgenomic, and somatic cloning researches, the established cell lines would preserve Caspian horse genetic resources. It will also create an accessible database for researchers.

Differentiation of adipocytes and osteocytes from human adipose and placental mesenchymal stem cells.

OBJECTIVES: Mesenchymal stem cells (MSC) can be isolated from adult tissues such as adipose tissue and other sources. Among these sources, adipose tissue (because of easy access) and placenta (due to its immunomodulatory properties, in addition to other useful properties), have attracted more attention in terms of research. The isolation and comparison of MSC from these two sources provides a proper source for clinical experimentation. The aim of this study was to compare the characteristics of MSC isolated from human adipose tissue and placenta. MATERIALS AND METHODS: Adipose and placental MSC were isolated from the subcutaneous adipose tissues of 10 healthy women (25 to 40 years) and from a fresh term placenta (n= 1), respectively. Stem cells were characterized and compared by flow cytometry using CD29, CD31, CD34, CD44, CD45, CD105, CD166 and HLA-DR markers. Osteocytes and adipocytes were differentiated from isolated human mesenchymal stem cells (HMSC). RESULTS: Adipose and placenta-derived MSC exhibited the same morphological features. ADSC differentiated faster than placenta; however, both were differentiated, taking up to 21 days for osteocyte and 14 days for adipocyte differentiation. About 90% of PLC-MSC and ADSC were positive for CD29, CD44, CD105, and CD166; and negative for CD31, CD34, CD45, and HLA-DR. CONCLUSION: The two sources of stem cells showed similar surface markers, morphology and differentiation potential and because of their multipotency for differentiating to adipocytes and osteocytes, they can be applied as attractive sources of MSC for regenerative medicine.

Mixture of fibroblast, epithelial and endothelial cells conditioned media induce monocyte-derived dendritic cell maturation.

Fully matured DCs with large amount cytoplasm and copious dendritic projections were visible at the end of culturing period in the presence of MCM, TNF-α and poly (I:C), with or without FEECM. Thus, DCs generated with these maturation factors are nonadherent and have typical satellite morphology. Flow cytometric analysis using anti-CD14, -CD80, -CD86, -HLA-DR and -CD83 revealed that expression of CD14 is decreased in particular in FEECM treated DCs, on day 5 and expression of CD80, CD86 and HLA-DR was the higher when FEECM are added to maturation factor. Functionally, when DCs matured in the presence of FEECM elicited stronger MLR, reduced phagocytic activity. These results support the use of the FEECM with MCM, TNF-α and poly (I-C) as maturation factor in DC generation that could result in functionally mature monocyte-derived DCs in comparison to either alone.