Abstract
Abstract 2534
Poster Board II-511
Induced pluripotent stem (iPS) cells, reprogrammed somatic cells with embryonic stem (ES) cell–like characteristics, are generated by the introduction of combinations of specific transcription factors. Despite the controversy surrounding the gene manipulation, it is expected that iPS cells should contribute to regenerative medicine, disease investigation, drug screening, toxicology, and drug development in future. In the fields of hematology, iPS cells could become used as a new feasible source for transplantation therapy without immunological barrier and for the investigation of various kinds of hematological defects. Previous studies on ES / iPS cells have already demonstrated that they can develop into various lineages of hematopoietic cells including erythrocytes following the similar processes occurred in embryo and fetus. However, it is important to establish the more effective system for developing functional blood cells. Here we present the methods for selectively inducing mature red blood cells from ES / iPS cells in vitro, and show the functional equality of them to natural blood cells. First, Flk1+ mesodermal progenitors were derived from ES / iPS cells on OP9 stromal cells at an efficacy of more than 50% and collected by fluorescence activated cell sorter. Then, those sorted cells were cultured in the presence of exogenous erythropoietin and stem cell factor. They highly selectively developed into erythroid lineages including enucleated red blood cells. Sequential FACS analysis using the antibodies against transferrin receptor CD71 and erythroid specific antigen Ter119 in combination with DNA staining dye Hoechst 33342 demonstrated that ES / iPS cell-derived erythropoiesis in our system follow the normal erythroid developmental pathway occurred in vivo. RT-PCR and Western blot analyses proved the expression of heme biosynthesis enzymes on the produced erythrocytes. Finally, the oxygen dissociation curve showed that ES / iPS cell-derived erythroid cells are functionally virtually equivalent to natural red blood cells as oxygen carriers. Taken together, our system can present the effective methods of investigating the mechanisms of normal erythropoiesis and the deficits in syndromes with disrupted red blood cell production.
Disclosures:
No relevant conflicts of interest to declare.
Cloning of cDNA Encoding NtEPc, a Marker Protein for the Embryogenic Dedifferentiation of Immature Tobacco Pollen Grains Cultured in Vitro
