RESUMEN
Objective: Hemoglobin F [HbF] augmentation is considered a clinically beneficial phenomenon in beta-hemoglobinopathies. Prevention of gamma-globin gene silencing, inspired by the hereditary persistence of fetal hemoglobin, may be a suitable strategy to upregulate HbF expression in these patients. Therefore, our objective was to assess the potential feasibility of induced -117 G?A substitution in HBG promoter in prevention of transcriptional silencing of the gamma-globin
Materials and Methods: In this experimental study, human peripheral blood-derived hematopoietic stem cells [HSCs] and the K562 cell line were differentiated to erythroid cells. Erythroid maturation was examined using cell morphology parameters and flow cytometry analysis of CD235a expression. A synthesised chimeraplast was transfected to differentiating cells. The efficiency of chimeraplast delivery into target cells was assessed by flow cytometry. Restriction-fragment length polymorphism and DNA sequencing verified oligonucleotide-directed mutagenesis. Gene conversion frequency and globin genes expression was quantified through Allele specific-quantitaive polymerase chain reaction [AS-qPCR] and quantitative-PCR respectively
Results: Increase in CD235a-expressing cells along with observations made for different stages of erythroid maturation confirmed erythroid differentiation in HSCs and K562 cells. gamma to beta-globin gene switching was estimated to be on days 18-21 of HSC differentiation. Flow cytometry analysis showed that more than 70% of erythroid progenitor cells [EPCs] were transfected with the chimeraplast. The highest gene conversion efficiency was 7.2 and 11.1% in EPCs and K562 cells respectively. The induced mutation led to a 1.97-fold decrease in beta/gamma-globin gene expression in transfected EPCs at the experimental end point [day 28] whereas, due to the absence of beta-globin gene expression following K562 differentiation, this rate was not evaluable
Conclusion: Our results suggest the effectiveness of chimeraplasty in induction of the mutation of interest in both EPCs and K562 cells. We also demonstrate that the single nucleotide promoter variant was able to significantly inhibit gamma-globin gene silencing during erythroid differentiation
RESUMEN
Objective: In order to clarify the role of microRNAs [miRNA] in megakaryocyte differentiation, we ran a microRNA microarray experiment to measure the expression level of 961 human miRNA in megakaryocytes differentiated from human umbilical cord blood CD133+ cells
Materials and Methods: In this experimental study, human CD133+ hematopoietic stem cells were collected from three human umbilical cord blood [UCB] samples, and then differentiated to the megakaryocytic lineage and characterized by flow cytometry, CFU-assay and ploidy analysis. Subsequently, microarray analysis was undertaken followed by quantitative polymerase chain reaction [qPCR] to validate differentially expressed miRNA identified in the microarray analysis
Results: A total of 10 and 14 miRNAs were upregulated [e.g. miR-1246 and miR-148-a] and down-regulated [e.g. miR- 551b and miR-10a] respectively during megakaryocyte differentiation, all of which were confirmed by qPCR. Analysis of targets of these miRNA showed that the majority of targets are transcription factors involved in megakaryopoiesis
Conclusion: We conclude that miRNA play an important role in megakaryocyte differentiation and may be used as targets to change the rate of differentiation and further our understanding of the biology of megakaryocyte commitment