RESUMO
The use of induced pluripotent stem cells (iPSCs) is an exciting frontier in the study and treatment of human diseases through the generation of specific cell types. Here we show the derivation of iPSCs from human nonmobilized peripheral blood (PB) and bone marrow (BM) mononuclear cells (MNCs) by retroviral transduction of OCT3/4, SOX2, KLF4, and c-MYC. The PB- and BM-derived iPSCs were quite similar to human embryonic stem cells with regard to morphology, expression of surface antigens and pluripotency-associated transcription factors, global gene expression profiles, and differentiation potential in vitro and in vivo. Infected PB and BM MNCs gave rise to iPSCs in the presence of several cytokines, although transduction efficiencies were not high. We found that 5 × 10(5) PB MNCs, which corresponds to less than 1 mL of PB, was enough for the generation of several iPSC colonies. Generation of iPSCs from MNCs of nonmobilized PB, with its relative efficiency and ease of harvesting, could enable the therapeutic use of patient-specific pluripotent stem cells.
Assuntos
Células Sanguíneas/citologia , Técnicas de Cultura de Células/métodos , Células-Tronco Pluripotentes Induzidas/citologia , Adulto , Biomarcadores/metabolismo , Células da Medula Óssea/citologia , Diferenciação Celular/genética , Células Clonais , Metilação de DNA/genética , Rearranjo Gênico/genética , Genoma Humano/genética , Células Germinativas/citologia , Células Germinativas/metabolismo , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Fator 4 Semelhante a Kruppel , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/metabolismo , Masculino , Regiões Promotoras Genéticas/genética , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Doadores de TecidosRESUMO
Reprogramming of somatic cells provides potential for the generation of specific cell types, which could be a key step in the study and treatment of human diseases. In vitro reprogramming of somatic cells into a pluripotent embryonic stem (ES) cell-like state has been reported by retroviral transduction of murine fibroblasts using four embryonic transcription factors or through cell fusion of somatic and pluripotent stem cells. Here we show that mouse adult bone marrow mononuclear cells (BM MNCs) are competent as donor cells and can be reprogrammed into pluripotent ES cell-like cells. We isolated BM MNCs and mouse embryonic fibroblasts (MEFs) from Oct4-GFP transgenic mice, fused them with ES cells, or infected them with retroviruses expressing Oct4, Sox2, Klf4, and c-Myc. Fused BM MNCs formed more ES-like colonies than did MEFs. Infected BM MNCs gave rise to induced pluripotent stem (iPS) cells, although transduction efficiencies were not high. It was more efficient to pick up iPS colonies as compared with MEFs. BM-derived iPS (BM iPS) cells expressed ES cell markers, formed teratomas, and contributed to chimera mice with germ line development. Clonal analysis revealed that BM iPS clones had diversity, although some clones were found to be genetically identical with different phenotypes. Our findings imply that BM MNCs have potential advantages to generate iPS cells for the clinical application.