Establishment of A Three-Dimensional Culture Condition for The U266 Cell Line Based on Peripheral Blood Plasma-Derived Fibrin Gels.

OBJECTIVE: The study of pathophysiology as well as cellular and molecular aspects of diseases, especially cancer, requires appropriate disease models. In vitro three-dimensional (3D) structures attracted more attention to recapitulate diseases rather than in vitro two-dimensional (2D) cell culture conditions because they generated more similar physiological and structural properties. Accordingly, in the case of multiple myeloma (MM), the generation of 3D structures has attracted a lot of attention. However, the availability and cost of most of these structures can restrict their use. Therefore, in this study, we aimed to generate an affordable and suitable 3D culture condition for the U266 MM cell line. MATERIALS AND METHODS: In this experimental study, peripheral blood-derived plasma was used to generate fibrin gels for the culture of U266 cells. Moreover, different factors affecting the formation and stability of gels were evaluated. Furthermore, the proliferation rate and cell distribution of cultured U266 cells in fibrin gels were assessed. RESULTS: The optimal calcium chloride and tranexamic acid concentrations were 1 mg/ml and 5 mg/ml for gel formation and stability, respectively. Moreover, the usage of frozen plasma samples did not significantly affect gel formation and stability, which makes it possible to generate reproducible and available culture conditions. Furthermore, U266 cells could distribute and proliferate inside the gel. CONCLUSION: This available and simple fibrin gel-based 3D structure can be used for the culture of U266 MM cells in a condition similar to the disease microenvironment.

Investigating The Correction of IVS II-1 (G> A) Mutation in HBB Gene in TLS-12 Cell Line Using CRISPR/Cas9 System.

OBJECTIVE: Beta-thalassemia is a group of inherited hematologic. The most HBB gene variant among Iranian beta-thalassemia patients is related to two mutations of IVSII-1 (G>A) and IVSI-5 (G>C). Therefore, our aim of this study is to use the knock in capability of CRISPR Cas9 system to investigate the correction of IVSII-1 (G>A) variant in Iran. MATERIALS AND METHODS: In this experimental study, following bioinformatics studies, the vector containing Puromycin resistant gene (PX459) was cloned individually by designed RNA-guided nucleases (gRNAs), and cloning was confirmed by sequencing. Proliferation of TLS-12 was done. Then, the transfect was set up by the vector with GFP marker (PX458). The PX459 vectors carrying the designed gRNAs together with Single-stranded oligodeoxynucleotides (ssODNs) as healthy DNA pattern were transfected into TLS-12 cells. After taking the single cell clones, molecular evaluations were performed on single clones. Sanger sequencing was then performed to investigate homology directed repair (HDR). RESULTS: The sequencing results confirmed that all three gRNAs were successfully cloned into PX459 vector. In the transfection phase, The TLS-12 containing PX459-gRNA/ssODN was selected. Molecular evaluations showed that the HBB gene was cleaved by the CRISPR/Cas9 system, that indicates that the performance of non-homologous end joining (NHEJ) repair system. Sequencing in some clones cleaved by the T7E1 enzyme showed that HDR was not confirmed in these clones. CONCLUSION: IVS-II-1 (G> A) mutation, which is the most common thalassemia mutation especially in Iran, the CRISPR/ Cas9 system was able to specifically target the HBB gene sequence. This could even lead to a correction in the mutation and efficiency of the HDR repair system in future research.

Evaluation of Expansion and Maintenance of Umbilical Cord Blood CD34+ Cells in The Co-Culture with Umbilical Cord Blood-Derived Mesenchymal Stem Cells in The Presence of Microcarrier Beads.

OBJECTIVE: Umbilical cord blood (UCB) is an accessible and effective alternative source for hematopoietic stem cell (HSC) transplantation. Although the clinical application of UCB transplantation has been increased recently, quantitative limitation of HSCs within a single cord blood unit still remains a major hurdle for UCB transplantation. In this study we used microcarrier beads to evaluate the ex vivo expansion of UCB-derived HSCs in co-cultured with UCB-derived mesenchymal stem cells (MSC). MATERIALS AND METHODS: In this experimental study, we used microcarrier beads to expand UCB-derived MSCs. We investigated the simultaneous co-culture of UCB-derived CD34+ cells and MSCs with microcarrier beads to expand CD34+ cells. The colony forming capacity and stemness-related gene expression on the expanded CD34+ cells were assessed to determine the multipotency and self-renewal of expanded cells. RESULTS: Our results indicated that the microcarrier-based culture significantly increased the total number and viability of UCB-derived MSCs in comparison with the monolayer cultures during seven days. There was a significant increase in the UCB-derived CD34+ cells expanded in the presence of microcarrier beads in this co-culture system. The expanded UCB-derived CD34+ cells had improved clonogenic capacity, as evidenced by higher numbers of total colony counts, granulocyte, erythrocyte, monocyte, megakaryocyte colony forming units (CFU-GEMM), and granulocyte-monocyte colony forming units (CFU-GM). There were significantly increased expression levels of key regulatory genes (CXCR4, HOXB4, BMI1) during CD34+ cells self-renewal and quiescence in the microcarrier-based co-culture. CONCLUSION: Our results showed that the increase in the expansion and multipotency of CD34+ cells in the microcarrierbased co-culture can be attributed to the enhanced hematopoietic support of UCB-derived MSCs and improved cell-cell interactions. It seems that this co-culture system could have the potential to expand primitive CD34+ cells.

Circulating cell-free mitochondrial DNA as a diagnostic and prognostic biomarker in chronic and late acute graft-versus-host disease in children.

In an earlier study, we found that mitochondrial DNA (mtDNA) concentration is elevated in adults with chronic graft-versus-host disease (cGvHD), acting as an endogenous source of TLR9 agonists to augment B-cell responses. To validate this in children, we evaluated mtDNA plasma expression in a large pediatric cohort (ABLE/PBMTC 1202 study). Plasma cell-free mtDNA (cf-mtDNA) copy numbers were measured in 202 pediatric patients using quantitative Droplet Digital polymerase chain reaction (ddPCR). Two evaluations were performed: 1) before the onset of cGvHD or late acute graft-versus-host disease (aGvHD) at day 100 ± 14 days and 2) at the time of cGvHD onset compared with time-matched non-cGvHD controls. We found that cf-mtDNA copy numbers were not affected by immune reconstitution post-hematopoietic stem cell transplantation but were higher on day 100 before the onset of late aGvHD and at the onset of cGvHD. We found that cf-mtDNA was not impacted by previous aGvHD, but correlated with the early onset, NIH moderate/severe cGvHD, and did not correlate with other immune cell populations, cytokines, or chemokines but did with the metabolites spermine and taurine. Similar to adults, children have elevated plasma cf-mtDNA concentrations at the early onset of cGvHD, especially in NIH moderate/severe cGvHD, elevation with late aGvHD, and associated with metabolites involved in mitochondrial function.

Calprotectin as new potential clinical marker for multiple myeloma.

Increased levels of inflammatory cytokines in multiple myeloma (MM) patients and the role of inflammation in disease pathogenesis, have recently been considered. The aim of this study was to quantitatively evaluation of fecal calprotectin (CP) as a non-invasive biomarker for the evaluation of inflammation in patients with multiple myeloma. This study is a hospital-based case control study. MM patients referred to patients referred to medical centers of Tehran province, Iran, were identified and classified into two groups of new MM patients (n = 40) and patients undergoing treatment (n = 28). Healthy individuals were included in the study as healthy control (n = 25). Morning stool samples were collected and CP was extracted immediately. After collecting the samples, CP was measured according to ELISA method and was determined in µg/g of feces. Values ​​above 50 µg/g of feces are positive and indicate inflammation. The results revealed that there is a significant difference between groups in terms if CP mean (p = 0.001). The mean of CP among new cases, under treatment and control groups were 301.3 (SD: 141.0), 165.1 (SD: 153.9) and 36.9 (SD: 13.5), respectively. Then the groups were compared in pairs, the results showed that the new case group was significantly different from the under-treatment group (p = 0.001), and also the control group showed a significant difference with the new case group (p = 0.001) and the under-treatment group (p = 0.001) that the amount of CP in the control group was significantly lower than the other two groups. In addition, the results of the study showed a significant correlation between age and plasma cells with CP value, so that with increasing age and plasma cells, CP value also showed a significant increase. The results indicate that quantitative evaluation of CP as a non-invasive laboratory biomarker has a high potential as a clinical marker in patients with multiple myeloma and inflammation should considered as a hallmark of cancer. Further diagnostic studies are recommended.

NK cell therapy in relapsed refractory multiple myeloma.

Recent advances in adoptive cell therapy have considerably changed the paradigm of cancer immunotherapy. Although current immunotherapies could cure many patients with multiple myeloma (MM), relapsed/refractory MM (RR/MM) is still challenging in some cases. Natural killer (NK) cells are innate immune cells that exert effective cytotoxic activity against malignant cells like myeloma cells. In addition to their antitumor properties, NK cells do not induce graft versus host disease following transplantation. Therefore, they provide a promising approach to treating RR/MM patients. Currently, attempts have been made to produce large-scale and good manufacturing practices (GMP) of NK cells. Ex vivo expanded/activated NK cells derived from the own patient or allogenic donors are potential options for NK cell therapy in MM. Besides, novel cell-based products such as NK cell lines and chimeric antigen receptor (CAR)-NK cells may provide an off-the-shelf source for NK cell therapy. Here, we summarized NK cell activity in the MM microenvironment and focused on different NK cell therapy methods for MM patients.

Protective effect of human amniotic membrane extract against hydrogen peroxide-induced oxidative damage in human dermal fibroblasts.

OBJECTIVE: One of the main approaches to preventing skin ageing is to protect fibroblast cells from oxidative stress. The promoting effect of the human amniotic membrane extract (hAME) on re-epithelization, proliferation and migration of cells in wound healing has been already well studied. This experimental study aimed to investigate the antioxidant activity of hAME against hydrogen peroxide (H2 O2 )-induced dermal fibroblast damage. METHODS: Here, to establish the ageing model, human foreskin fibroblasts (HFFs) were exposed to 200 µM H2 O2 for 2 h. HFFs were treated with 0.1 mg/ml AME for 24 or 48 h before or/and after H2 O2 exposure. A total of 48 h following the H2 O2 treatment, we measured cell proliferation, viability, senescence-associated ß-galactosidase (SA-ß-Gal), antioxidants and preinflammatory cytokine (IL-6) levels, as well as the expression of senescence-associated genes (P53 and P21). RESULTS: The obtained results indicated that under oxidative stress, AME significantly increased cellular viability and not only promoted the cell proliferation rate but also attenuated apoptotic induction condition (p < 0.001). AME also significantly reversed the SA-ß-Gal levels induced by H2 O2 (p < 0.001). Additionally, both pre- and post-treatment regimen by AME down-regulated the expression of senescence marker genes (p < 0.001). Moreover, AME declined different oxidative stress biomarkers such as superoxide dismutase and catalase and increased the glutathione amount. CONCLUSION: Altogether, our results indicated that AME had a remarkable antioxidant and antiageing activity as pre- and post-treatment regimen, pointing to this compound as a potential natural-based cosmeceutical agent to prevent and treat skin ageing conditions.

The Effect of Exosomes Derived from Unrestricted Somatic Stem Cells on Murine Model of Sepsis.

Sepsis is a systemic infection mainly caused by bacterial infections. Despite all efforts and advances in the treatment of sepsis, it is still considered one of the leading causes of death in hospitalized patients. Today, we have to use novel therapies and one of the most important is cell-free therapy. Exosomes have been shown to contain the contents of their parent cells and that they do not generate an immune response between different individuals which makes them a good candidate for transplantation. Unrestricted somatic stem cells (USSC), also known as mesenchymal stem cell progenitors due to their high proliferative capacity and low immune response, may be a novel therapy for sepsis. In this study, the effect of USSC-derived exosomes on sepsis was investigated using a mouse model. USSCs were isolated from human cord blood and characterized by flow cytometry and multi-lineage differentiation. The exosomes were then harvested from USSCs and characterized by transmission electron microscopy, Western blotting, and dynamic light scattering. The harvested exosomes were injected into the mouse model of sepsis. Biochemical, histological, molecular, and survival studies were performed in different groups. Our observations showed that USSC-derived exosomes can reduce inflammation in septic mice. Histopathologic and biochemical findings in the sham group showed multiorgan involvement, but these changes disappeared after 7 days of exosome administration. Moreover, the expression of IRAK-1 and TRAF-6 (main adapter molecules in signaling pathways of inflammation) was decreased through negative regulation by miR-146a after 72 h of exosome administration. A 2-fold increase in the level of IL-10 and a 2-fold decrease in the levels of IL-6 and TNF-α was observed. In conclusion, we showed that direct injection of USSC-derived exosomes can be one of the important methods for the treatment of various aspects of sepsis due to their immunomodulatory properties.

Human Umbilical Cord Mesenchymal Stem Cells-Derived Small Extracellular Vesicles Can Be Considered as Cell-Free Therapeutics for Angiogenesis Promotion.

OBJECTIVE: Angiogenesis has critical roles in several physiological processes. Restoring angiogenesis in some pathological conditions such as a few vascular diseases can be a therapeutic approach to controlling this issue. Mesenchymal stem cells (MSCs) secrete specific intracellular products known as extracellular vesicles (EVs) with high therapeutic potential which compared to their source cells, do not have the limitations of cell therapy. The angiogenic effect of the human umbilical cord MSCs (hUCMSCs)-derived small EVs are evaluated in the present work. Aim of this research is to show that hUCMSCs-derived small EVs cause differentiation of genes involved in angiogenesis like FGFR-1, FGF, VEGF, and VEGFR-2. MATERIALS AND METHODS: In this experimental study, MSCs were isolated from the human umbilical cord, and after confirming their identities, their secreted EVs (including exosomes) were extracted by ultracentrifugation. The isolated small EVs were characterized by dynamic light scattering (DLS), transmission electron microscopy (TEM), bicinchoninic acid assay (BCA), and Western Blotting. Then, the human umbilical vein endothelial cells (HUVECs) were treated with derived small EVs for 72 hours, and the expression of the angiogenic factors including FGFR-1, FGF, VEGF, and VEGFR-2 was evaluated by quantitative real-time-polymerase chain reaction (qPCR). Angiogenesis was also evaluated via a tube formation assay. RESULTS: The results demonstrated that FGFR-1, FGF, VEGF, and VEGFR-2 could be elevated 2, 2, 3.5, and 2 times, respectively, in EVs treated HUVECs, and derivative EVs can encourage tube formation in HUVECs. CONCLUSION: These findings imply that hUCMSCs-derived small EVs are valuable resources in promoting angiogenesis and are very promising in cell-free therapy.

Analysis of the A549 cell line affected by anticancer bioactive compounds of Actinomycetes isolated from saline soils.

Actinomycetes are filamentous bacteria and the residents of the soil, prone to produce bioactive metabolites. This research aimed to isolate, classify, and investigate the anticancer properties of Actinomycetes secondary metabolites from various saline soils of Qom province. Actinomycetes isolates were molecularly recognized by 16SrRNA gene sequencing after the PCR procedure. The A549 cell line was then exposed to bacterial metabolites to find their cytotoxicity by MTT assay and their capacity to cause apoptosis by Flow cytometry. The expression levels of the bax and bcl-2 genes were determined using Real-time PCR. Bacterial metabolites were distinct by HPLC and GC-MS assays. Sequencing identified three novel Actinomycetes strains, Streptomyces griseoflavus, Streptomyces calvus, and Kitasatospora phosalacineus. The IC50 doses of bacterial metabolites were discovered equal to 1337, 2619, and 4874 µg/ml, respectively. Flow cytometric assay revealed that their secondary metabolites were capable of inducing apoptosis in A549 cells by 25%, 14.5%, and 7.58%, respectively. Real-time PCR findings displayed that the bax gene expression in A549 cells treated with S. griseoflavus and S. calvus, comparatively increased (P < 0.0008, P < 0.00056). The expression of the bcl-2 gene was significantly reduced in cells treated with S. griseoflavus and K. phosalacineus (P < 0.0006, P < 0.0004). The findings of this analysis showed the presence of new isolates in a soil sample from Qom province which can produce new anticancer agents and can be considered appropriate candidates for further research to employ as anticancer drugs.

Differentiation of Alginate-Encapsulated Wharton Jelly-Derived Mesenchymal Stem Cells into Insulin Producing Cells.

Objective: Insulin insufficiency due to the reduced pancreatic beta cell number is the hallmark of diabetes, resulting in
an intense focus on the development of beta-cell replacement options. One approach to overcome the problem is to
search for alternative sources to induce insulin-producing cells (IPCs), the advent of mesenchymal stem cells (MSCs)
holds great promise for producing ample IPCs. Encapsulate the MSCs with alginate improved anti-inflammatory effects
of MSC treatment. This study aimed to evaluate the differentiation of wharton jelly-derived MScs into insulin producing
cells using alginate encapsulation.
Materials and Methods: In this experimental study, we established an efficient IPCs differentiation strategy of human
MSCs derived from the umbilical cord's Wharton jelly with lentiviral transduction of Pancreas/duodenum homeobox
protein 1 (PDX1) in a 21-day period using alginate encapsulation by poly-L-lysine (PLL) and poly-L-ornithine (PLO)
outer layer. During differentiation, the expression level of PDX1 and secretion of insulin proteins were increased.
Results: Results showed that during time, the cell viability remained high at 87% at day 7. After 21 days, the differentiated beta-like cells in microcapsules were morphologically similar to primary beta cells. Evaluation of the expression of PDX1 and INS by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) on days 7, 14 and 21 of differentiation exhibited the highest expression on day 14. At the protein level, the expression of these two pancreatic markers was observed after PDX1 transduction. Results showed that the intracellular and extracellular insulin levels in the cells receiving PDX1 is higher than the control group. The current data showed that encapsulation with alginate by PLL and PLO outer layer permitted to increase the microcapsules' beta cell differentiation.
Conclusion: Encapsulate the transduced-MSCs with alginate can be applied in an in vivo model in order to do the further analysis.

Regenerative Effect of Low-Intensity Pulsed Ultrasound and Platelet-Rich Plasma on the Joint Friction and Biomechanical Properties of Cartilage: A Non-traumatic Osteoarthritis Model in the Guinea Pig.

This study was aimed at investigating the effects of platelet-rich plasma (PRP) and low-intensity pulsed ultrasound (LIPUS) on the joint friction parameters and biomechanical properties of articular cartilage in a non-traumatic knee osteoarthritis (OA) model. Fifty adult male Dunkin Hartley guinea pigs were randomly divided into five groups: control, OA60, OA + US, OA + PRP and OA + US + PRP). Non-traumatic knee OA was induced with a single dose of 3 mg of mono-iodoacetate (MIA) by intra-articular injection. Intra-articular PRP was injected twice in the OA + PRP and OA + US + PRP groups. LIPUS was delivered in 10 sessions in the OA + US and OA + US + PRP groups. By use of the pendulum free oscillation test, joint friction (coefficient of friction) was measured. In addition, the instantaneous elastic modulus and aggregate modulus were measured using the stress-relaxation test. MIA injection decreased cartilage thickness, instantaneous elastic modulus and aggregate modulus, and increased joint friction. The friction coefficients in the OA + US and OA + US + PRP groups reached near-normal values, and there was no significant difference compared with the control group (p = 0.232 and p = 0.459, respectively). The instantaneous elastic modulus and aggregate modulus in the OA + US group increased significantly compared with the OA + PRP group (p < 0.05). It seems that both LIPUS and PRP injection effectively improved joint lubrication, but LIPUS was superior to PRP in improving the mechanical properties of the articular cartilage.

Effects of bone marrow-derived mesenchymal stem cells exposed to endocrine-disrupting chemicals on the differentiation of umbilical cord blood hematopoietic stem cells.

Endocrine-disrupting chemicals (EDCs), a class of peripheral toxic substances, can cause many environmental and clinical side effects, particularly on the human body's endocrine system. Bisphenol A (BPA) and diethylhexyl phthalate (DEHP) are two well-known EDCs in the medicine industry. However, there are no comprehensive studies on their effects on hematopoiesis. Hence, this study aimed to investigate the effect of these two aforementioned substances on the clonogenic capacity of umbilical cord blood hematopoietic stem cells (CB-HSCs). The HSCs which express CD34 + were isolated from umbilical cord blood by the magnetic-activated cell sorting (MACS) system. To investigate the effects of different optimized concentrations of BPA and DEHP, CB-CD34+ HSCs were exposed to EDCs in semisolid medium. For evaluation of coexposures, CB-CD34+ HSCs were cocultured with bone marrow-derived mesenchymal stem cells (BM-MSCs) in the presence of BPA and DEHP. Finally, the number and types of colonies were evaluated after 14 days. Statistical analysis was performed by GraphPad Prism through ANOVA. CB-HSC treated by BPA and DEHP showed a lower absolute colony count than the control group (P < 0.05). Decrease in clonogenic potential of HSCs was more significant in coculture condition by MSCs. In particular, there was a significant decrease in the BFU-E colonies in comedicated-derived fractions (P < 0.0001). In the presence of EDCs such as BPA and DEHP, the patterns of differentiation in CD34+ CB-HSCs changed from suppressed erythroid differentiation toward stimulated myelogenesis pathways.

Investigation of diverse biosynthetic secondary metabolites gene clusters using genome mining of indigenous Streptomyces strains isolated from saline soils in Iran.

Background and Objectives: Bioactive secondary metabolites are the products of microbial communities adapting to environmental challenges, which have yet remained anonymous. As a result of demands in the pharmaceutical, agricultural, and food industries, microbial metabolites should be investigated. The most substantial sources of secondary metabolites are Streptomyces strains and are potential candidates for bioactive compound production. So, we used genome mining and bioinformatics to predict the isolates secondary metabolites, biosynthesis, and potential pharmaceuticals. Materials and Methods: This is a bioinformatics part of our previous experimental research. Here, we aimed to inspect the underlying secondary metabolite properties of 20 phylogenetically diverse Streptomyces species of saline soil by a rationalized computational workflow by several software tools. We examined the Metabolites' cytotoxicity and antibacterial effects using the MTT assay and plate count technique, respectively. Results: Among Streptomyces species, three were selected for genome mining and predicted novel secondary metabolites and potential drug abilities. All 11 metabolites were cytotoxic to A549, but ectoine (p≤0.5) and geosmin (p≤0.001) significantly operated as an anti-cancer drug. Metabolites of oxytetracycline and phosphinothricin (p≤0.001), 4Z-annimycin and geosmin (p≤0.01), and ectoine (p≤0.5) revealed significant antibacterial activity. Conclusion: Of all the 11 compounds investigated, annimycin, geosmin, phosphinothricin, and ectoine had antimicrobial properties, but geosmin also showed very significant anti-cancer properties.

AntagomiR-19a Induced Better Responsiveness to Bortezomib in Myeloma Cell Lines.

OBJECTIVE: Multiple myeloma (MM) is the clonal proliferation of neoplastic plasma cells in the bone marrow. Although bortezomib (BTZ) is a crucial drug for the treatment of MM, drug resistance is a major problem. OncomiR-19a plays an oncogenic role in many cancers, including MM; however, the function of miR-19a in the pathogenesis of MM and drug resistance has not been completely identified. The present research aims to investigate the inhibition of miR-19a by an antagomir to determine BTZ responsiveness, and determine if miR-19a can be a prognostic biomarker for MM. MATERIALS AND METHODS: In this experimental study, viability and apoptosis of myeloma cells were analysed by the colorimetric 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-Diphenyltetrazolium Bromide (MTT) and Annexin V/propidium iodide (PI) flow cytometry assays. Quantitative real-time polymerase chain reaction (qRT-PCR) was implemented to evaluate the expression levels of miR-19a, its targets SOCS3, STAT3, B-cell lymphoma 2 (BCL-2), PTEN and CDKN1A (antiapoptotic and cell cycle related genes) at the mRNA level. RESULTS: miR-19a was downregulated and exacerbated in transfected cells treated with BTZ. The rate of apoptosis in the myeloma cells after BTZ treatment considerably increased, which indicated an increase in the mRNA of SOCS3, PTEN, BCL-2, and CDKN1. A decrease in STAT3 was also observed. CONCLUSION: OncomiR-19a, as a biomarker, may induce better responsiveness to BTZ in myeloma cell lines through its targets SOCS3, STAT3 and PTEN. In the future, this biomarker may provide new therapeutic targets for MM.

Plasma Cell Proliferation Is Reduced in Myeloma-Induced Hypercalcemia and in Co-Culture with Normal Healthy BM-MSCs.

OBJECTIVE: In multiple myeloma (MM), stimulation of osteoclasts and bone marrow (BM) lesions lead to hypercalcemia, renal failure, and anemia. Co-culture of the myeloma cells in both hypocalcemia and hypercalcemia concentrations with bone marrow-mesenchymal stem cells were evaluated. MATERIALS AND METHODS: Viability and survival of myeloma cells were assessed by microculture tetrazolium test and flow cytometric assays. Mesenchymal stem cells (MSCs) were extracted from normal and myeloma patients and were co-cultured with myeloma cells. RESULTS: Myeloma cells showed less survival in both hypocalcaemia and hypercalcemia conditions (P <.01). The paracrine and juxtacrine conditions of demineralized bone matrix-induced hypercalcemia increased the proliferation and survival of the cells (P <.05). Unlike myeloma MSCs, normal MSCs reduced the survival of and induced apoptosis in myeloma cells (P <.1). CONCLUSION: Normal healthy-MSCs do not protect myeloma cells, but inhibit them. However, increasing the ratio of myeloma cells to MSCs reduces their inhibitory effects of MSCs and leads to their myelomatous transformation.

Mesenchymal stem cell-derived extracellular vesicles conditionally ameliorate bone marrow failure symptoms in an immune-mediated aplastic anemia mouse model.

Acquired forms of Aplastic anemia (AA) are characterized by T cell-mediated immune disease resulting in bone marrow (BM) failure and marrow hypoplasia. In these cases, it is a major challenge to modulate autoreactive T cell activity and thereby decrease the pro-inflammatory cytokine storm. Emerging evidence indicates that extracellular vesicles derived from mesenchymal stem cells (MSC-EVs) control and modulate immunity. The therapeutic potential of MSC-EVs has not been investigated in acquired AA. Hence, in this study, we constructed an AA mice model through irradiation and splenocyte infusion to test the benefits of hypoxic MSC-EVs (Hx-EVs) and normoxic MSC-EVs (Nx-EVs). We found that MSC-EVs treatment significantly prolonged the survival rate and increased the platelet (PLT) counts of the AA mice. Immunohistochemical staining and colony assay confirmed amelioration of hypoplasia in the BM and increased numbers of hematopoietic stem cells (HSCs). These effects of MSC-EVs were mediated by T cell suppression and inhibition of interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) production in the AA mouse model. In addition, an in vitro study revealed that MSC-EVs led to reduced IFN-γ and TNF-α levels and there was an association with decreased splenocyte viability. Previous studies examined the diagnostic and prognostic values of microRNAs (miRNAs) in AA and identified miR-199a, miR-146a, miR-223, and miR-126. We used quantitative real-time PCR to evaluate the expression of these miRNAs on isolated BM mononuclear cells (BM-MNCs) from treated and untreated AA mice. miR-223, miR-146a, and miR-199a expressions increased in the MSC-EVs treated AA mice. Treatment with MSC-EVs increased expression of miR-223 and miR-146a. Our findings showed that treatment with MSC-EVs significantly ameliorated immune destruction of HSCs in the AA mouse model and confirmed the importance of miRNAs in the clinical status of this model.

Stem Cell Therapy: A Promising Approach in Treatment of COVID-19.

COVID-19 pandemic is a global health crisis of the 21st Century. There are currently no approved vaccines and no particular anti-viral treatment for coronavirus disease. As COVID-19 has a broad range of illnesses, it is necessary to find a safe and effective therapeutic method for COVID-19. An attractive approach for treating COVID-19 is cell therapy. Cell therapy aims to inject new and healthy stem cells into a patient's body, to repair the damaged cells and tissues. Stem cell therapy is one of the most studied and important approaches in the treatment of COVID-19 these days. The significant clinical outcome was observed by the adoptive transfer of stem cells, specifically mesenchymal stem cells. This study reviews the characteristics of stem cells and clinical trials that have used stem cells in treating COVID-19.

Induced Pluripotent Stem Cell Meets Severe Combined Immunodeficiency.

Severe combined immunodeficiency (SCID) is classified as a primary immunodeficiency, which is characterized by impaired T-lymphocytes differentiation. IL2RG, IL7Ralpha, JAK3, ADA, RAG1/RAG2, and DCLE1C (Artemis) are the most defective genes in SCID. The most recent SCID therapies are based on gene therapy (GT) of hematopoietic stem cells (HSC), which are faced with many challenges. The new studies in the field of stem cells have made great progress in overcoming the challenges ahead. In 2006, Yamanaka et al. achieved "reprogramming" technology by introducing four transcription factors known as Yamanaka factors, which generate induced pluripotent stem cells (iPSC) from somatic cells. It is possible to apply iPSC-derived HSC for transplantation in patients with abnormality or loss of function in specific cells or damaged tissue, such as T-cells and NK-cells in the context of SCID. The iPSC-based HSC transplantation in SCID and other hereditary disorders needs gene correction before transplantation. Furthermore, iPSC technology has been introduced as a promising tool in cellular-molecular disease modeling and drug discovery. In this article, we review iPSC-based GT and modeling for SCID disease and novel approaches of iPSC application in SCID.

Factor V Leiden 1691G > A mutation and the risk of recurrent pregnancy loss (RPL): systematic review and meta-analysis.

BACKGROUND: Although numerous replication case-control studies have attempted to determine the association between Factor V Leiden (FVL) 1691G > A mutation and susceptibility to Recurrent pregnancy loss (RPL), there have been confliction among the results of various ethnic groups. To address this limitation, here we implemented first meta-analysis to provide with consistent conclusion of the association between FVL 1691G > A mutation and RPL risk. METHODS: After a systematic literature search, pooled odds ratio (OR) and their corresponding 95% confidence interval (CI) were used to evaluate the strength of the association. Additionally, meta-regression analyses were performed to find potential source of heterogeneity. RESULTS: In this meta-analysis, 62 studies, containing 10,410 cases and 9406 controls, were included in quantitative analysis. Overall population analysis revealed a significant positive association in the dominant (OR = 2.15, 95% CI = 1.84-2.50, P < 0.001), over-dominant (OR = 1.88, 95% CI = 1.61-2.19, P < 0.001), allelic (OR = 2.05, 95% CI = 1.79-2.35, P < 0.001), and heterozygote (OR = 1.97, 95% CI = 1.68-2.30, P < 0.001) models. Moreover, a significant association of dominant (OR = 3.04, 95% CI = 2.04-4.54, P < 0.001), over-dominant (OR = 2.65, 95% CI = 1.74-4.05, P < 0.001), and heterozygote (OR = 2.67, 95% CI = 1.81-4.22, P < 0.001) models was found in the Iranian population. The subgroup analysis indicated strong significant association in Asian, European, Africa population, and case-control studies but not in South Americans and cohort studies. CONCLUSION: The FVL 1691G > A mutation and the risk of RPL confers a genetic contributing factor in increasing the risk of RPL, particularly in Iranians, except for South Americans.