ABSTRACT
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
ABSTRACT
Objective: umbilical cord blood is used for transplantation purposes in regenerative medicine of hematological disorders. MicroRNAs are important regulators of gene expression that control both physiological and pathological processes such as cancer development and incidence. There is a new relation between p53 [tumor suppressor gene] and miR-145 [suppressor of cell growth] upregulation. In this study, we have assessed how adipose-derived stem cells [ADSCs] affect the expansion of hematopoietic stem cells [HSCs], as well as miR-145 and p53 expressions
Materials and Methods: in this experimental study, we cultured passage-3 isolated human ADSCs as a feeder layer. Flow cytometry analysis confirmed the presence of ADSC surface markers CD73, CD90, CD105. Ex vivo cultures of cordblood CD34+ cells were cultured under the following 4 culture conditions for 7 days: i. Medium only supplemented with cytokines, ii. Culture on an ADSCs feeder layer, iii. Indirect culture on an ADSCs feeder layer [Thin Cert[] plate with a 0.4 ?m pore size], and iv. Control group analyzed immediately after extraction. Real-time polymerase chain reaction [PCR] was used to determine the expressions of the p53 and miR-145 genes. Flow cytometry analysis of cells stained by annexin V and propidium iodide [PI] was performed to detect the rate of apoptosis in the expanded cells
Results: ADSCs tested positive for mesenchymal stem cell [MSC] markers CD105, CD90, and CD73, and negative for HSC markers CD34 and CD45. Our data demonstrated the differentiation potential of ASCs to osteoblasts by alizarin red and alkaline phosphatase staining. MTT assay results showed a higher proliferation rate of CD34+cells directly cultured on the ADSCs feeder layer group compared to the other groups. Direct contact between HSCs and the feeder layer was prevented by a microporous membrane p53 expression increased in the HSCs group with indirect contact of the feeder layer compared to direct contact of the feeder layer. p53 significantly downregulated in HSCs cultured on ADSCs, whereas miR-145 significantly upregulated in HSCs cultured on ADSCs
Conclusion: ADSCs might increase HSCs proliferation and self-renewal through miR-145, p53, and their relationship
ABSTRACT
Runt-related transcription factor 2 [RUNX2] and osterix [OSX] as two specific osteoblast transcription factors and distal-less homeobox 5 [DLX5] as a non-specific one are of paramount importance in regulating osteoblast related genes including osteocalcin, bone sialoprotein [BSP], osteopontin and collagen type I?1. The present study sets out to investigate whether epigenetic regulation of these genes is important in osteoblastic differentiation of mesenchymal stem cells [MSCs]. In this experimental study, MSCs were differentiated to osteoblasts under the influence of the osteogenic differentiation medium. DNA and RNA were extracted at days 0, 7, 14 and 21 from MSCs differentiating to osteoblasts. Promoter regions of RUNX2, OSX, DLX5 and BSP were analyzed by methylation-specific PCR [MSP]. Gene expression was analyzed during osteoblastic differentiation by quantitative real-time polymerase chain reaction [PCR]. MSP analysis revealed that promoter methylation status did not change in RUNX2, DLX5 and BSP during MSC osteoblastic differentiation. In contrast, OSX promoter showed a dynamic change in methylation pattern. Moreover, RUNX2, OSX, DLX5 and BSP promoter regions showed three different methylation patterns during MSC differentiation. Gene expression analyses confirmed these results. The results show that in differentiation of MSCs to osteoblasts, epigenetic regulation of OSX may play a leading role