Effect of Replacement of Wharton Acellular Jelly With FBS on the Expression of Megakaryocyte Linear Markers in Hematopoietic Stem Cells CD34.

OBJECTIVE: Animal environments for the growth of stem cells cause the transmission of some diseases and immune problems for the recipient. Accordingly, replacing these environments with healthy environments, at least with human resources, is essential.  One of the media that can be used as an alternative to animal serums is Wharton acellular jelly (AWJ).  Therefore, in this study, we intend to replace FBS with Wharton jelly and investigate its effect on the expression of megakaryocyte-related genes and markers in stem cells. MATERIALS AND METHODS: In this study, cord blood-derived CD34 positive HSCs were cultured and expanded in the presence of cytokines including SCF, TPO, and FLT3-L. Then, the culture of expanded CD34 positive HSCs was performed in two groups: 1) IMDM culture medium containing 10% FBS and 100 ng / ml thrombopoietin cytokine 2) IMDM culture medium containing 10% AWJ, 100 ng / ml thrombopoietin cytokine.  Finally, CD41 expressing cells were analyzed with the flow cytometry method. The genes related to megakaryocyte lineage including FLI1 and GATA2 were also evaluated using the RT-PCR technique.  Results: The expression of CD41, a specific marker of megakaryocyte lineage in culture medium containing Wharton acellular jelly was increased compared to the FBS group. Additionally, the expression of GATA2 and FLI1 genes was significantly increased related to the control group. CONCLUSION: This study provided evidence of differentiation of CD34 positive hematopoietic stem cells from umbilical cord blood to megakaryocytes in a culture medium containing AWJ.
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Application of Emerging Plant-Derived Nanoparticles as a Novel Approach for Nano-Drug Delivery Systems.

Nanotechnology has enabled the delivery of small molecular drugs packaged in nanosized vesicles to the target tissues. Plant-Derived Nanoparticles (PDNPs) are vesicles with natural origin and unique properties. These nanoparticles have several advantages over synthetic exosomes and liposomes. They provide bioavailability and biodistribution of therapeutic agents when delivered into different tissues. These nanoparticles can be modified according to the specificity of their functions in target tissues. When PDNPs are internalized, they can induce stem cells proliferation, reduce colitis injury, activate intrinsic and extrinsic apoptosis pathways, and inhibit tumor growth and progression. These properties make them potential drug delivery systems in targeting diseased tissues, such as inflammatory regions and different cancers.

Down-regulation of intracellular anti-apoptotic proteins, particularly c-FLIP by therapeutic agents; the novel view to overcome resistance to TRAIL.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL or Apo2L) is a member of the tumor necrosis factor (TNF) superfamily that induces apoptosis in different types of cancer cells via activation of caspase cascade. TRAIL interacts with its cognate receptors that placed on cancer cells surface, including TRAIL-R1 (death receptor 4, DR4), TRAIL-R2 (death receptor 5, DR5), TRAIL-R3 (decoy receptor 1, DcR1), TRAIL-R4 (decoy receptor 2, DcR2), and osteoprotegerin (OPG). Despite high apoptosis-inducing ability of TRAIL, various cancerous cells gain resistance to TRAIL gradually, and consequently TRAIL potential for apoptosis stimulation in these cells diminishes intensely. According to diverse ranges of examinations, intracellular anti-apoptotic proteins, such as cellular-FLICE inhibitory protein (c-FLIP), apoptosis inhibitors (IAPs), myeloid cell leukemia sequence 1 (MCL-1), BCL-2, BCL-XL, and survivin play key role in cancer cells resistance to TRAIL. These proteins attenuate cancer cells sensitivity to TRAIL via various functions, importantly through caspase cascade suppression. The c-FLIP avoids from caspase 8 activation by FADD via binding to caspase 8 cleavage of FADD. Moreover, it activates signaling pathways that involved in cancer cells survival and proliferation. Intriguingly, it appears that the down-regulation of intracellular anti-apoptotic proteins, particularly c-FLIP is effectiveness goal for TRAIL-resistant cancers therapy, because their up-regulation in association with poor prognosis has been observed in various types of TRAIL-resistant cancers. In this review, we tried to collect and examine investigations that researchers have been able to sensitize cancer cells to TRAIL through targeting of c-FLIP alone or with other intracellular anti-apoptotic proteins directly or indirectly. It seems that co-treatment of resistant cells by TRAIL with other therapeutic agents with the aim of intracellular anti-apoptotic proteins inhibition is hopeful and attractive approach to overcome various TRAIL-resistant cancers.

Promoter Methylation Status of Survival-Related Genes in MOLT- 4 Cells Co-Cultured with Bone Marrow Mesenchymal Stem Cells under Hypoxic Conditions.

OBJECTIVES: DNA methylation is a well-studied epigenetic mechanism that is a potent arm of the gene expression controlling machinery. Since the hypoxic situation and the various cells of bone marrow microenvironment, e.g. mesenchymal stem cells, play a role in the in vivo and in vitro biology of leukemic cells, we decided to study the effects of hypoxia and mesenchymal stem cells (MSCs) on the promoter methylation pattern of BAX and BCL2 genes. MATERIALS AND METHODS: In this experimental study, the co-culture of MOLT-4 cells with MSCs and treatment with CoCl2 was done during 6, 12, and 24 hour periods. Total DNA was extracted using commercial DNA extraction kits, and sodium bisulfite (SBS) treatment was performed on the extracted DNA. Methylation specific polymerase chain reaction (MSP) was used to evaluate the methylation status of the selected genes' promoter regions. RESULTS: The BAX and BCL2 promoters of untreated MOLT-4 cells were in partial methylated and fully unmethylated states, respectively. After incubating the cancer cells with CoCl2 and MSCs, the MSP results after 6, 12, and 24 hours were the same as untreated MOLT-4 cells. In other words, the exposure of MOLT-4 cells to the hypoxia-mimicry agent and MSCs in various modes and different time frames showed that these factors have exerted no change on the methylation signature of the studied fragments from the promoter region of the mentioned genes. CONCLUSIONS: Hypoxia and MSCs actually have no notable effect on the methylation status of the promoters of BAX and BCL2 in the specifically studied regions. DNA methylation is probably not the main process by which MSCs and CoCl2 induced hypoxia regulate the expression of these genes. Finally, we are still far from discovering the exact functional mechanisms of gene expression directors, but these investigations can provide new insights into this field for upcoming studies.

Characterization of Common Chromosomal Translocations and Their Frequencies in Acute Myeloid Leukemia Patients of Northwest Iran.

OBJECTIVE: Detection of chromosomal translocations has an important role in diagnosis and treatment of hematological disorders. We aimed to evaluate the 46 new cases of de novo acute myeloid leukemia (AML) patients for common translocations and to assess the effect of geographic and ethnic differences on their frequencies. MATERIALS AND METHODS: In this descriptive study, reverse transcriptase-polymerase chain reaction (RT-PCR) was used on 46 fresh bone marrow or peripheral blood samples to detect translocations t (8; 21), t (15; 17), t (9; 11) and inv (16). Patients were classified using the French-American-British (FAB) criteria in to eight sub-groups (M0-M7). Immunophenotyping and biochemical test results of patients were compared with RT-PCR results. RESULTS: Our patients were relatively young with a mean age of 44 years. AML was relatively predominant in female patients (54.3%) and most of patients belonged to AML-M2. Translocation t (8; 21) had the highest frequency (13%) and t (15; 17) with 2.7% incidence was the second most frequent. CD19 as an immunophenotypic marker was at a relatively high frequency (50%) in cases with t (8; 21), and patients with this translocation had a specific immunophenotypic pattern of complete expression of CD45, CD38, CD34, CD33 and HLA-DR. CONCLUSION: Similarities and differences of results in Iran with different parts of the world can be explained with ethnic and geographic factors in characterizations of AML. Recognition of these factors especially in other comprehensive studies may aid better diagnosis and management of this disease.

MicroRNA-15b target Sall4 and diminish in vitro UCB-derived HSCs expansion.

Hematopoietic Stem Cells (HSCs) are cells that have the ability to self-renewal and differentiate into all of hematopoietic lineages. The lack of donors and unavailable efficient protocols for ex vivo expansion of HSCs, are obstacles in successful cell therapies. MicroRNAs (also refer as miRNAs or miRs) have significant roles in hematopoiesis; they can effect on HSCs expansion, maintaining undifferentiated state, self-renewal and differentiation. Recently attentions have been given to these small regulatory molecules to utilize them in order to expand HSCs. Using bioinformatics analysis we identified Sall4 as putative target of miR-15b and miR-219-5p. Relative expression levels of miRNAs and Sall4 were evaluated by qRT-PCR. Here we show 247-fold and 4.2-fold increasing Sall4 expression level compared to control group in CD34(+) cells nucleofected by anti-miR-15b and anti-miR-219-5p, respectively. These data showed that anti-miR-15b can promote clonogenic capacity of HSCs and also we found that miR-15b alone was able to increase the number of CD34(+)HSCs in vitro by more than 2 fold by targeting Sall4. Moreover, level of CD34 marker in HSCs nucleofected by anti-miR-15b increased more than 50 %. Our analysis showed no statistically difference in mRNA level of Sall4 after nucleofection of anti-miR-219-5p. Sall4 is a factor capable of enhancing HSC expansion significantly. We demonstrated that inhibition of miR-15b can enhance ex vivo expansion of UCB-derived HSCs and also expression of Sall4 allowed expansion and preserve self- renewal of CD34(+) HSCs.