[TGF-b downregulation by RNAi technique in ex vivo-expanded HSCs on 3D DBM scaffold]

Bone Marrow Transplantations [BMT] are limited by low CD34+ cell counts in umbilical cord blood [UCB] and these cells need to be expanded for success in such procedures. To achieve this goal, ex vivo expansion of hematopoietic stem cells [HSCs] by enhancing their self-renewal activity on demineralized bone matrix [DBM] scaffold coated with mesenchymal progenitor cells [MPCs] and unrestricted somatic stem cells [USSCs] was recommended. TGF-b pathway is a key inhibitory factor for HSCs self-renewal. In this study ex vivo expansion and downregulation of TGF-b pathway were simultaneously performed. USSC cells were isolated from UCB and then coated on DBM scaffold as a feeder layer. UCB CD34+ cells were isolated from UCB by magnetic activated cell sorting [MACS] method and were transfected by siRNA against TGFbR2 in two-dimensional [2D] and three-dimensional [3D] cultures by co-cultivation with USSC. TGFbR2 expression levels were evaluated by quantitative real-time PCR. Cell count and flow cytometry were performed and clonogenic activity was evaluated. Ex vivo expansion of CD34+ cells was significantly enhanced [41 +/- 0.7 folds] by TGFbR2 downregulation, especially in 2D than 3D cultures. Finally, 2D culture showed less TGFbR2 expression levels and higher increase in the percentage of CD34 markers by flow cytometry assay. The 3D siRNA delivery system would be of lower efficiency in contrast to 2D settings where the cells have less freedom and are in more contact with the feeder layer

[Differentiation of cord blood stem cells into erythroid progenitor cells in semisolid culture media containing SCF, IL-3, IL-6 and EPO]

The study of erythropoiesis needs to develop the methods for erythroid progenitor cells [EPCs] culture using stem cell potency to differentiate into variety of hematopoietic cells lineages. In this study, we induced differentiation of cord blood stem cells into erythroid progenitor cells in a semisolid culture media. Cord blood mononuclear cells were isolated and cultured in liquid culture media consist of erythroid differentiation factors [phase I]. Non-adherent cells were cultured in semisolid media containing SCF, IL-3, IL-6 and EPO [phase II]. After one week, the appeared erythroid colonies were picked up. Characterization of differentiated cells was performed by assessment of CD235a and CD36 using flowcytometry, giemsa cytological staining and gene expression analysis of GATA-1, EKLF, -globin genes by RT-PCR. Flowcytometry analysis to detect CD235a and CD36 positive cells showed that 94.3% and 95.5% of differentiated cells have erythroid specific cell markers, respectively. Morphology of the cells in giemsa stained slides demonstrated erythroid progenitor cells, ranged from proerythroblast to orthochromatic erythroblast. The RT-PCR results, confirmed the precursor state of erythroid differentiated cells by expression of GATA-1, EKLF, -globin genes. Cord blood stem cells have high potency to differentiate into erythroid progenitor cells using described method that can be utilized in the experimental studies