Preparation of poly(acrylic acid)/tricalcium phosphate nanoparticles scaffold: Characterization and releasing UC-MSCs derived exosomes for bone differentiation.

Introduction: This study focused on preparing a multiscale three-dimensional (3D) scaffold using tricalcium phosphate nanoparticles (triCaPNPs) in a substrate of poly(acrylic acid) (PAA) polymer for controlled release of exosomes in bone tissue engineering. Methods: A scaffold was fabricated with a material mixture containing acrylic acid (AA) monomer, N,N'-methylenebisacrylamide (MBAA), ammonium persulfate (APS), sodium bicarbonate (SBC), and triCaPNPs called composite scaffold (PAA/triCaPNPs) via cross-linking and freeze-drying methods. The synthesis process was easy and without complex multi-steps. Through mimicking the hybrid (organic-inorganic) structure of the bone matrix, we here chose triCaPNPs for incorporation into the PAA polymer. After assessing the physicochemical properties of the scaffold, the interaction of the scaffold with human umbilical cord mesenchymal stem cells (UC-MSCs) such as attachment, proliferation, and differentiation to osteoblast cells was evaluated. In addition, we used DiI-labeled exosomes to verify the exosome entrapment and release from the scaffold. Results: The polymerization reaction of 3D scaffold was successful. Based on results of physicochemical properties, the presence of nanoparticles in the composite scaffold enhanced the mechanical stiffness, boosted the porosity with a larger pore size range, and offered better hydrophilicity, all of which would contribute to greater cell penetration, proliferation, and then better bone differentiation. In addition, our results indicated that our scaffold could take up and release exosomes, where the exosomes released from it could significantly enhance the osteogenic commitment of UC-MSCs. Conclusion: The current research is the first study fabricating a multiscale scaffold using triCaPNPs in the substrate of PPA polymer using a cross-linker and freeze-drying process. This scaffold could mimic the nanoscale structure and chemical combination of native bone minerals. In addition, our results suggest that the PAA/triCaPNPs scaffold could be beneficial to achieve controlled exosome release for exosome-based therapy in bone tissue engineering.

Expression analysis of genes involved in the expansion of hematopoietic stem cells (SCF, Flt3-L, TPO, IL-3, and IL-6) in unrestricted somatic stem cells cultured on fibrin.

Umbilical cord blood (UCB) transplantation is a promising therapeutic approach for patients lacking HLA-matched donors. A main limitation to the use of UCB-derived HSCs (UCB-HSCs) is the low number of transplantable cells. Novel culture strategies are being developed to increase the number of HSCs. Unrestricted somatic stem cells (USSCs) have been identified as promising stromal cells for supporting HSC expansion. The current study aimed to explore the effect of fibrin on the expression of hematopoiesis-related genes (SCF, Flt3-L, TPO, IL-3, and IL-6) in USSCs. USSCs were isolated from UCB and characterized by flow cytometry and in vitro multilineage differentiation ability. DAPI staining and the MTT assay were used to assess the effect of fibrin on USSC viability. The cell attachment was evaluated using SEM. qRT-PCR was performed to evaluate the expression of SCF, Flt3-L, TPO, IL-3, and IL-6 in USSCs cultured on 3D fibrin scaffolds. USSCs were positive for CD73, CD105, and CD166 and negative for CD45. Alizarin red and Oil red O stains confirmed calcium deposition and lipid vacuoles in USSCs. Results obtained from DAPI and MTT assays revealed a positive effect of fibrin on USSC viability. Cells cultured on fibrin express significantly higher levels of SCF and TPO compared to those grown in a 2D environment. The positive effect of fibrin on IL-6 levels was reversed. Fibrin did not affect Flt3-L expression and IL-3 mRNA expression was not detected in either group. The results of this study provide the basis for developing further research on the ex vivo expansion of HSCs with USSCs.

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.

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.

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.

Therapeutic role of mesenchymal stem cell-derived exosomes in respiratory disease.

Exosomes are extracellular vesicles found in various tissues, blood circulation, and tissue fluids, secreted into the extracellular environment by fusing a multivesicular body with a plasma membrane. Various cell types release these vesicles to contribute to many cellular functions, including intercellular communication, cell proliferation, differentiation, angiogenesis, response to stress, and immune system signaling. These natural nanoparticles have therapeutic effects in various diseases and exhibit a behavior similar to the cell from which they originated. In the meantime, exosomes derived from mesenchymal stem cells have attracted the attention of many researchers and physicians due to their unique ability to modulate the immune system, repair tissue and reduce inflammation. Numerous clinical and preclinical studies have examined the effect of MSC-derived exosomes in various diseases, and their results have been published in prestigious journals. This review article discusses the biogenesis and sources of exosomes, MSC-derived exosomes, the use of these exosomes in regenerative medicine, and treatments based on exosomes derived from stem cells in respiratory diseases.

Reviewing the role of cardiac exosomes in myocardial repair at a glance.

Exosome-based therapy is an emerging novel approach for myocardial infarction (MI) treatment. Exosomes are identified as extracellular vesicles that are produced within multivesicular bodies in the cells' cytosols and then are secreted from the cells. Exosomes are 30-100 nm in diameter that are released from viable cells and are different from other secreted vesicles such as apoptotic bodies and microvesicles in their origin and contents such as RNAs, proteins, and nucleic acid. The recent advances in exosome research have demonstrated the role of these bionanovesicles in the physiological, pathological, and molecular aspects of the heart. The results of in vitro and preclinical models have shown that exosomes from different cardiac cells can improve cardiac function following MI. For example, mesenchymal stem cells (MSCs) and cardiac progenitor cells (CPCs) containing exosomes can affect the proliferation, survival, and differentiation of cardiac fibroblasts and cardiomyocytes. Moreover, MSCs- and CPCs-derived exosomes can enhance the migration of endothelial cells. Exosome-based therapy approaches augment the cardiac function by multiple means, such as reducing fibrosis, stimulation of vascular angiogenesis, and proliferation of cardiomyocytes that result in replacing damaged heart tissue with newly generated functional myocytes. This review article aims to briefly discuss the recent advancements in the role of secreted exosomes in myocardial repair by focusing on cardiac cells-derived exosomes.

Pre-transplant thrombocytopenia predicts engraftment time and blood products requirement in allogeneic hematopoietic stem cell transplantation patients.

INTRODUCTION: Thrombocytopenia is a common consequence of leukemia that affects the outcome of hematopoietic stem cell transplantation (HSCT). The stromal damage of bone marrow following pre-HSCT conditioning regimens can delay the hematopoietic engraftment and increased blood product transfusions. We tried to define threshold based on pre-transplant platelet count as a biomarker to predict engraftment time and blood product requirements in allogeneic HSCT patients. METHODS: This retrospective study was performed on 194 patients who received allogeneic HSCT. The median for platelet (PLT) count of patients at the admission day was considered as a cut off value. The association of platelet count with white blood cell (WBC) and PLT engraftment time and also the requirement of packed red blood cell or PLT transfusions as outcomes of interest were investigated. RESULTS: 164 patients (84.5 %) had successful WBC engraftment, and PLT engraftment was seen in 155 patients (79.9 %) in 30 and 50 days after HSCT, respectively. The patients with PLT count higher than 154,000/µL had better PLT engraftment (P = 0.060), and WBC engraftment (P = 0.014) than those with PLT count lower than this cut off. The pre-transplant PLT count had negative relations with SD platelet requests after HSCT (P = 0.008). CONCLUSION: The thrombocytopenia before HSCT might delay the platelet and WBC engraftment time, which should be taken into account before transplantation. Since the blood product transfusion is one of the factors associated with engraftment, the pre-transplant platelet count can be used as a predictive biomarker to manage the blood product requirement during the HSCT until engraftment occurs.

Combination Effect of Notch1 and PI3K/AKT/mTOR Signaling Pathways Inhibitors on T-ALL Cell Lines.

Background: Acute T lymphoblastic Leukemia (T-ALL) is a highly aggressive hematologic malignancy. Chemotherapy resistance is one of the most important challenges in T-ALL treatment. Alterations in cellular signaling pathways such as Notch1 and PI3K/AKT/mTOR play a role in cell proliferation, survival, and resistance to chemotherapy. Combination of Notch1 and PI3K/AKT/mTOR inhibitors is an interesting and rational strategy in treatment of T-ALL. Interaction of AZD5363 as an inhibitor of PI3k/AKT/mTOR and Compound E as an inhibitor of Notch1 signaling pathway was investigated in a T-ALL pre-clinical model. Materials and Methods: T-ALL cell lines included Jurkat, Molt-4, and HPB- ALL cells were treated with AZD5363 and Compound E alone and in combination. Cell viability was determined by MTT assay. Flow cytometry was used to measure apoptosis. Interaction between AZD5363 and Compound E was assessed by Chou-Talalay method. Results: Combination treatment with AZD5363 and Compound E decreased cell viability with synergistic effect in all cell lines at 72 hours. Drug combination increased apoptosis even in Jurkat and HPB-ALL cells resistant to Compound E and AZD5363, respectively. Conclusion: Combination of AZD5363 with Compound E in T-ALL cell lines exhibited a synergistic effect. Cytotoxicity of drug combination increased in all T-ALL cell lines compared to each as a single drug. Simultaneous inhibition of Notch1 and PI3K/AKT signaling pathways as a possible treatment of T-ALL, provides a basis for future investigations.

Generation of an in vitro model of ß-thalassemia using the CRISPR/Cas9 genome editing system.

ß-Thalassemia is a common monogenic disease characterized by defective ß-globin chains synthesis. In vitro ß-thalassemia-related research on increasing ß-like globin genes or identification of factors reducing the severity of the disease, has been performed on mouse erythroleukaemia or K562 cell lines. The aim of this study was the production of an in vitro model of ß-thalassemia using the highly efficient CRISPR-Cas9 system. Embryonic stem (ES) cells were nucleofected with guide RNA (gRNA)-Cas9 expression vectors. Molecular testing was done on extracted DNA to assess Hbb-b1 mutation. Analysis of transcription factors and hemoglobin genes were evaluated using quantitative reverse transcription-polymerase chain reaction following erythroid differentiation of ES cells. Sequencing data confirmed Hbb-b1 knockout alleles. Significant expression of erythroid transcription factors was observed in wild-type, Hbb-b1+/- and Hbb-b1-/- groups (P < .001). Compared with the wild-type group, the absolute number of Hbb-b1 mRNA in Hbb-b1+/- group significantly decreased from 6.44 × 106 to 3.23 × 106 copy number (P < .01), whereas in Hbb-b1-/- group had zero expression. The CRISPR/Cas9-mediated Hbb-b1 knockout in ES cells provides accessibility to an in vitro thalassemia model following erythroid differentiation. Considering the need for in vitro and mouse models to investigate the molecular basis of ß-thalassemia which also enables testing of therapeutic approaches, this method can be utilized to produce a mouse model of ß-thalassemia intermedia (Hbbth1/th1).

Parvovirus B19 Infection in Human Bone Marrow Mesenchymal Stem Cells Affects Gene Expression of IL-6 and TNF-α and also Affects Hematopoietic Stem Cells Differentiation.

Parvovirus B19 (B19V) has been known to induce transient erythroid aplasia, cytopenia and aplastic anemia. This virus persists in bone marrow mesenchymal stem cells (HBMSCs) of some immunocompetent individuals several years after primary infection. In B19V infected erythroid progenitor cells, the virus induces transactivation of Interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) gene expression. Due the critical role of HBMSCs in bone marrow niche and inhibitory effect of inflammatory cytokines on hematopoiesis, the aim of this study was to investigate the effect of B19V on IL-6 and TNF-α gene expression intransfected cells. In addition we assessed the clonogenicity potential of cord blood CD34+ stem cells that were co-cultured with infected cells. After 24 h of transfection, quantitative mRNA expression of IL-6 and TNF-α was evaluated and human cord blood CD34+ HSC were cultured with the transfected cells. At the end of 7 days of culture, HSCs colony forming units (CFUs) assay was performed. Our findings demonstrated statistically significant (18.1 and 21.9 fold) increase of TNF-α and IL-6 gene expression respectively and decrease in burst forming unit-erythrocyte (BFU-E) and colony forming unit-erythrocyte (CFU-E) enumeration(p < 0.05). We concluded that, inducing inflammatory cytokines gene expression in B19V-infected HBMSCs might influence on bone marrow microenvironment and hematopoiesis.

Generation of CCR5-ablated Human Induced Pluripotent Stem Cells as a Therapeutic Approach for Immune-mediated Diseases.

C-C chemokine receptor type 5 (CCR5) is a receptor for some pro-inflammatory chemokines which plays important roles in immunological disorder and host responses to infectious agents. Additionally, the prognosis of some immune-mediated diseases in the people who are naturally carrying the CCR5 32bp deletions is optimistic. However, the clinical application of CCR5 32bp mutant cells is very limited due to the rare availability of donors who are homozygous for CCR5 D32. The transfection efficiency of nucleofected placental mesenchymal stem cells derived - human induced pluripotent stem cells (PMSC-hiPSCs) was examined through the evaluation of green fluorescent protein (GFP) expression using flow cytometry. The nucleofected clonal populations were selected using colony picking. The CCR5 gene disrupted clonal populations were evaluated and confirmed by PCR and Sanger sequencing methods. Also, off-target sites were evaluated by the "Loss of a primer binding site" technique. The results of the flow cytometry revealed that among the six applied nucleofection programs for PMSC-iPSCs, the program of A-033 has achieved the best transfection efficiency (27.7%). PCR and then sequencing results confirmed the CCR5 gene was disrupted in two clonal populations of 16 (D6) and 62 (D20) by the Clustered regularly interspaced short palindromic repeats/CRISPR associated nuclease 9 (CRISPR/Cas9) system. The "Loss of a primer binding site" technique showed that no exonic off-target mutations were induced in both CCR5 gene disrupted clonal populations. We establish a CRISPR/Cas9 mediated CCR5 ablated PMSC-hiPSCs without detectable off-target damage. This approach can provide a stable supply of autologous/allogeneic CCR5-disrupted PMSC-hiPSCs that might be a feasible approach for the treatment of immune-mediated diseases.

Altered expression of MALAT1 lncRNA in chronic lymphocytic leukemia patients, correlation with cytogenetic findings.

BACKGROUND: Recent studies have devoted much attention to non-protein-coding transcripts in relation to a wide range of malignancies. MALAT1, a long non-coding RNA, has been reported to be associated with cancer progression and prognosis. Thus, we here determined MALAT1 gene expression in chronic lymphocytic leukemia (CLL), a genetically heterogeneous disease, and explored its possible relationships with cytogenetic abnormalities. METHODS: MALAT1 expression level was evaluated using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) on blood mononuclear cells from 30 non-treated CLL patients and 30 matched healthy controls. Cytogenetic abnormalities were determined in patients by fluorescence in situ hybridization (FISH). RESULTS: MALAT1 expression level was up-regulated in the CLL group compared to healthy controls (P=0.008). Del13q14, followed by Del11q22, were the most prevalent cytogenetic abnormalities. We found no association between the FISH results and MALAT1 expression in patients. CONCLUSION: Altered expression of MALAT1 is associated with CLL development. Further investigations are required to assess the relationship between this long non-coding RNA and CLL patient survival and prognosis.

Various Signaling Pathways in Multiple Myeloma Cells and Effects of Treatment on These Pathways.

Multiple myeloma (MM) results from malignancy in plasma cells and occurs at ages > 50 years. MM is the second most common hematologic malignancy after non-Hodgkin lymphoma, which constitutes 1% of all malignancies. Despite the great advances in the discovery of useful drugs for this disease such as dexamethasone and bortezomib, it is still an incurable malignancy owing to the development of drug resistance. The tumor cells develop resistance to apoptosis, resulting in greater cell survival, and, ultimately, develop drug resistance by changing the various signaling pathways involved in cell proliferation, survival, differentiation, and apoptosis. We have reviewed the different signaling pathways in MM cells. We reached the conclusion that the most important factor in the drug resistance in MM patients is caused by the bone marrow microenvironment with production of adhesion molecules and cytokines. Binding of tumor cells to stromal cells prompts cytokine production of stromal cells and launches various signaling pathways such as Janus-activated kinase/signal transduction and activator of transcription, Ras/Raf/MEK/mitogen-activated protein kinase, phosphatidyl inositol 3-kinase/AKT, and NF-KB, which ultimately lead to the high survival rate and drug resistance in tumor cells. Thus, combining various drugs such as bortezomib, dexamethasone, lenalidomide, and melphalan with compounds that are not common, including CTY387, LLL-12, OPB31121, CNTO328, OSI-906, FTY720, triptolide, and AV-65, could be one of the most effective treatments for these patients.

Comparison of anticancer effect of Pleurotus ostreatus extract with doxorubicin hydrochloride alone and plus thermotherapy on erythroleukemia cell line.

Background Recent studies have introduced Pleurotus ostreatus (Pleurotaceae) as a herbal medicine for treating different types of cancer. This survey utilizes P. ostreatus and doxorubicin hydrochloride (DOX) alone and then with hyperthermia to investigate the erythroleukemia cell line. This study evaluates and compares the apoptotic and necrotic effects of various treatments on the KG-1 cell line. Methods The proliferation of KG-1 cells was measured by using a tetrazolium salt (MTT)-based colorimetric assay during 96 h after treatment by gradient dilutions of 100 ng/mL to 100 mg/mL of P. ostreatus methanol extract and then the minimum inhibitory concentration (MIC) was determined and was applied in additional experiments. Afterward, the cells were treated using P. ostreatus extract, DOX (6.95 mg/L), and hyperthermia (42 and 44 °C), separately and then applying hyperthermia. Finally, the ratios of apoptosis and necrosis after 24 h incubation were evaluated by using flow cytometry. Results The MIC of the extract was determined (1 mg/mL), which significantly increased the ratio of apoptosis rather than necrosis, whereas the DOX treatment primarily induced necrosis on the KG-1 cells. The anticancer effects of the mushroom extract were significantly increased when it was combined with thermotherapy, which exhibited apoptotic effects at 42 °C but induced necrosis at 44 °C. Conclusions The results suggest that P. ostreatus extract induces apoptosis on KG-1 cells and its anticancer effects are significantly increased in combination with thermotherapy. Therefore, P. ostreatus could be considered as an alternative with anticancer effect for further studies in erythroleukemia patients.

Patterns of DNMT1 Promoter Methylation in Patients with Acute Lymphoblastic Leukemia.

Background: Acute lymphoblastic leukemia (ALL) is a clonal malignant disorder characterized by an uncontrolled proliferation of immature T or B lymphocytes. Extensive studies have shown that the epigenetic changes, especially modified DNA methylation patterns in the regulatory regions through the DNA methyltransferase (DNMTs), play an important role in the development of genetic disorders and abnormal growth and maturation capacity of leukemic stem cells (LSCs).The aim of this study was to evaluate the changes in DNMT1 promoter methylation and its expression pattern in patients with ALL. Materials and Methods: In this experimental study, methylation specific PCR (MSP) was used to assess the methylation status of DNMT1 promoter regions in samples collected from ALL patients (n=45) and healthy control subjects. According to this method, un-methylated cytosine nucleotides are converted to uracil by sodium bisulfite and the proliferation of methylated and un-methylated regions are performed using specific primers for target sequences. Results: None of the patients with B and T-ALL showed methylated promoter regions of the DNMT1 gene, while the methylation pattern of both pre-B ALL patients and the control group showed a relative promoter methylation. Conclusion: Analysis of promoter methylation patterns in various subgroups of ALL has revealed the importance of DNMT1 in the regulation of gene expression. Likewise, extensive data have also highlighted the methylation-based mechanisms exerted by DNAM1 as one of the main participants regulating gene expression in B-ALL and T-ALL patients. Investigation of the overall DNA methylation pattern offers significant improvements in the prediction of disease prognosis and treatment response.

Efficient Expansion of SALL4-Transduced Umbilical Cord Blood Derived CD133+Hematopoietic Stem Cells.

Hematopoietic stem cells (HSCs) were characterized by self-renewal and multilineage potential. Umbilical cord blood-derived (UCB) as an alternative source of HSCs is widely used especially in children for stem cells transplant (SCT). The main limitation in using UCB for transplantation especially in adults is low cell dose. To overcome this limitation besides using double dose UCB, ex vivo expansion is the most important way to increase cell number for transplantation. HSCs are mainly isolated using CD133 or CD34. CD133, as the most primitive marker, shows important physiological role in maintenance and expansion of HSCs. SALL4 plays crucial role in the development and maintaining the pluripotency and self-renewal ability of embryonic stem cells (ESCs) as well as HSCs. Moreover, SALL4 act as a regulator of HSCs expansion, normal hematopoiesis, and hematological malignancies. In the present study, CD133+ cells positively selected and ex vivo expanded in SALL-4 and GFP-transduced group. CD133 expression assessed using flow cytometry at day 0, 7 and 10. Moreover, multilineage differentiation and proliferation potential of expanded cells in both groups evaluated using colony forming unit (CFU) assay, and cells count assay. Karyotyping analysis was performed to assess any chromosomal instability after 7 days of expansion. Obtained results demonstrated that SALL-4 transduced cells showed significant increase in cell number compared to control group. Moreover, immunophenotyping results showed higher expression level of CD133 at day 7 and 10 following expansion in SALL-4 transduced (62 % and 42%) compared to control group (51% and 20.6%). Our results illustrated that SALL4 could act as a positive factor for the expansion of CD133+ derived UCB cells besides maintaining self-renewal and differentiation ability of expanded cell without any numerical and structural chromosomal aberrations .

MicroRNA Modulation during the In vitro Culture of Hematopoietic Stem Cells Prior to Transplantation.

BACKGROUND: Human umbilical cord blood (HUCB) is an acceptable and readily accessible source of stem cells. There is an ongoing interest in cord blood stem cell therapies; however, little is known about the possible unfavorable effects of laboratory modifications on the isolated HUCB cells. The involvement of miRNAs in several biological processes has been shown. The aim of this study was to evaluate the possible changes in miRNA expression profiles in CD133+ hematopoietic cells after in vitro culture. METHODS: HUCBCD133+ hematopoietic stem cells were isolated by magnetic-activated cell sorting, and then the cells were counted using flow cytometry. The cells were divided into 2 groups. In the first group, RNA was extracted and the cells of the second group were cultured in vitro for 12 days and then these cells were used to assay miRNAs expression using real-time qPCR. RESULTS: The results showed that the expression of 349 out of 1,151 screened miRNAs was upregulated following a 12-day in vitro culture of CD133+ cells, whereas the expression of 293 miRNAs was downregulated. In addition, the expression of 509 miRNAs was not significantly altered. Another in-silico analysis involving the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways related to the selected miRNAs was also conducted. CONCLUSION: Based on our results, the in vitro expansion of HUCB resulted in altered expression levels of miRNAs. This study provides information on the effects of 2-dimensional culture of hematopoietic cells prior to transplantation for more successful transplantation.

Effect of The Receptor Activator of Nuclear Factor кB and RANK Ligand on In Vitro Differentiation of Cord Blood CD133(+) Hematopoietic Stem Cells to Osteoclasts.

OBJECTIVE: Receptor activator of nuclear factor-kappa B ligand (RANKL) appears to be an osteoclast-activating factor, bearing an important role in the pathogenesis of multiple myeloma. Some studies demonstrated that U-266 myeloma cell line and primary myeloma cells expressed RANK and RANKL. It had been reported that the expression of myeloid and monocytoid markers was increased by co-culturing myeloma cells with hematopoietic stem cells (HSCs). This study also attempted to show the molecular mechanism of RANK and RANKL on differentiation capability of human cord blood HSC to osteoclast, as well as expression of calcitonin receptor (CTR) on cord blood HSC surface. MATERIALS AND METHODS: In this experimental study, CD133(+) hematopoietic stem cells were isolated from umbilical cord blood and cultured in the presence of macrophage colony-stimulating factor (M-CSF) and RANKL. Osteoclast differentiation was characterized by using tartrate-resistant acid phosphatase (TRAP) staining, giemsa staining, immunophenotyping, and reverse transcription-polymerase chain reaction (RT-PCR) assay for specific genes. RESULTS: Hematopoietic stem cells expressed RANK before and after differentiation into osteoclast. Compared to control group, flow cytometric results showed an increased expression of RANK after differentiation. Expression of CTR mRNA showed TRAP reaction was positive in some differentiated cells, including osteoclast cells. CONCLUSION: Presence of RANKL and M-CSF in bone marrow could induce HSCs differentiation into osteoclast.

Investigating Effects of Acidic pH on Proliferation, Invasion and Drug-Induced Apoptosis in Lymphoblastic Leukemia.

Some studies have shown that extracellular pH in tumors, which results in tumor progression, is less than that in normal tissues. The aim of this study was to investigate the effects of extracellular acidic pH on proliferation, invasion, and drug-induced apoptosis in acute lymphoblastic cells. The cells were cultured in different pH (pH 6.6 and pH 7.4) for 12 days. Cell proliferation was assessed by MTT assay and cell invasion was assayed by invasion assay and gene expression analysis of MMP-9. Drug-induced apoptosis was evaluated after exposure to doxorubicin for 24 hours by annexin V/PI staining and gene expression analysis of BAX pro-apoptotic protein. The results indicated the enhanced growth and invasion of leukemic cells at pH 6.6 (P ≤ 0.05). Furthermore, the cells at pH 6.6 were resistant to apoptosis by doxorubicin (P ≤ 0.05). It can be concluded that acidic pH increases the proliferation, invasion and reduces the drug-induced apoptosis in acute lymphoblastic leukemia. Extracellular acidity can influence the behavior of leukemic cells and therefore, the manipulation of extracellular liquid can be selected as a therapeutic strategy for leukemia, especially for acute lymphoblastic leukemia.