ABSTRACT
Subject(s)
Humans , Anemia , Blood Platelets , Erythrocyte Indices , Erythrocytes , Ferritins , Hematology , Inflammation , Liver Diseases , Mean Platelet Volume , Non-alcoholic Fatty Liver Disease , Oxidative Stress , Pilot Projects , Public Health , QuercetinABSTRACT
Objective: MicroRNAs [miRNAs] are small endogenous non-coding regulatory RNAs that control mRNAs post-transcriptionally. Several mouse stem cells miRNAs are cloned differentially regulated in different hematopoietic lineages, suggesting their possible role in hematopoietic lineage differentiation. Recent studies have shown that specific miRNAs such as Mir-451 have key roles in erythropoiesis
Materials and Methods: In this experimental study, murine embryonic stem cells [mESCs] were infected with lentiviruses containing pCDH-Mir-451. Erythroid differentiation was assessed based on the expression level of transcriptional factors [Gata-1, Klf-1, Epor] and hemoglobin chains [alpha, beta, gamma, epsilon and zeta] genes using quantitative reverse transcriptase-polymerase chain reaction [qRT-PCR] and presence of erythroid surface antigens [TER-119 and CD235a] using flow cytometery. Colony-forming unit [CFU] assay was also on days 14thand 21thafter transduction
Results: Mature Mir-451 expression level increased by 3.434-fold relative to the untreated mESCs on day 4 after transduction [P<0.001]. Mir-451 up-regulation correlated with the induction of transcriptional factor [Gata-1, Klf-1, Epor] and hemoglobin chain [alpha, beta, gamma, epsilon and zeta] genes in mESCs [P<0.001] and also showed a strong correlation with presence of CD235a and Ter- 119 markers in these cells [13.084and 13.327-fold increse, respectively] [P<0.05]. Moreover, mESCs treated with pCDH-Mir-451 showed a significant raise in CFU-erythroid [CFU-E] colonies [5.2-fold] compared with untreated control group [P<0.05]
Conclusion: Our results showed that Mir-451 up-regulation strongly induces erythroid differentiation and maturation of mESCs. Overexpression of Mir-451 may have the potential to produce artificial red blood cells [RBCs] without the presence of any stimulatory cytokines
ABSTRACT
Hematopoietic stem cells [HSCs] transplantation using umbilical cord blood [UCB] has improved during the last decade. Because of cell limitations, several studies focused on the ex vivo expansion of HSCs. Numerous investigations were performed to introduce the best cytokine cocktails for HSC expansion The majority used the Fms-related tyrosine kinase 3 ligand [FLT3-L] as a critical component. According to FLT3-L biology, in this study we have investigated the hypothesis that FLT3-L only effectively induces HSCs expansion in the presence of a mesenchymal stem cell [MSC] feeder. In this experimental study, HSCs and MSCs were isolated from UCB and placenta, respectively. HSCs were cultured in different culture conditions in the presence and absence of MSC feeder and cytokines. After ten days of culture, total nucleated cell count [TNC], cluster of differentiation 34[+] [CD34[+]] cell count, colony forming unit assay [CFU], long-term culture initiating cell [LTC-IC], homeobox protein B4 [HoxB4] mRNA and surface CD49d expression were evaluated. The fold increase for some culture conditions was compared by the t test. HSCs expanded in the presence of cytokines and MSCs feeder. The rate of expansion in the co-culture condition was two-fold more than culture with cytokines [P<0.05]. FLT3-L could expand HSCs in the co-culture condition at a level of 20-fold equal to the presence of stem cell factor [SCF], thrombopoietin [TPO] and FLT3-L without feeder cells. The number of extracted colonies from LTC-IC and CD49d expression compared with a cytokine cocktail condition meaningfully increased [P<0.05]. FLT3-L co-culture with MSCs can induce high yield expansion of HSCs and be a substitute for the universal cocktail of SCF, TPO and FLT3-L in feeder-free culture
ABSTRACT
Background: Different processing methods are being used to improve the quality of hematopoietic stem cell transplantation. Using hydroxyethyl starch, simple centrifugation and Sepax automation, this study was aimed to compare these three conventional methods.
Material and Methods: 90 cord blood samples were taken and processed by hydroxyethyl starch, simple centrifugation and Sepax automation methods. Then they were subjected to total nucleated cell [TNC] counting and CD34 positive counting as well as colony assay. Finally, all data were analyzed using one-way analysis of variance [ANOVA] and ps less than 0.05 were considered statistically significant.
Results: The TNC recoveries in hydroxyethyl starch, simple centrifugation and Sepax automation methods were 76%, 71% and 80%, respectively [p> 0.05]. The CD34+ cell recoveries in the Sepax automation and in the other two methods were 91% and 85%, respectively [p> 0.05]. Also, the colony assay recoveries were not significantly different among the three methods [p> 0.05].
Conclusion: No significant difference was seen in TNC number, CD34 positive counting and colony formation among the three different methods.
ABSTRACT
Despite of many benefits, umbilical cord blood [UCB] hematopoietic stem cell [HSC] transplantation is associated with low number of stem cells and slow engraftment; in particular of platelets. So, expanded HSCs and co-transfusion of megakaryocyte [MK] progenitor cells can shorten this period. In this study, we evaluated the cytokine conditions for maximum expansion and MK differentiation of CD133[+] HSCs. In this experimental study, The CD133[+] cells were separated from three cord blood samples by magnetic activated cell sorting [MACS] method, expanded in different cytokine combinations for a week and differentiated in thrombopoietin [TPO] for the second week. Differentiation was followed by the flow cytometry detection of CD41 and CD61 surface markers. Colony forming unit [CFU] assay and DNA analysis were done for colonogenic capacity and ploidy assay. CD133[+] cells showed maximum expansion in the stem span medium with stem cell factor [SCF] + FMS-like tyrosine kinase 3-ligand [Flt3-L] + TPO but the maximum differentiation was seen when CD133[+] cells were expanded in stem span medium with SCF + Interleukin 3 [IL-3] + TPO for the first and in TPO for the second week. Colony Forming Unit-MK [CFU-MK] was formed in three sizes of colonies in the mega-cult medium. In the DNA analysis; 25.2 +/- 6.7% of the cells had more than 2n DNA mass. Distinct differences in the MK progenitor cell count were observed when the cells were cultured in stem span medium with TPO, SCF, IL-3 and then the TPO in the second week. Such strategy could be applied for optimization of CD133[+] cells expansion followed by MK differentiation