In vitro generation of hematopoietic stem cells (HSCs) from induced pluripotent stem cells (iPSCs) has the potential to provide novel therapeutic approaches for replacing bone marrow (BM) transplantation without rejection or graft versus host disease. Hitherto, however, it has proved difficult to generate truly functional HSCs transplantable to adult host mice. Here, we demonstrate a unique in vivo differentiation system yielding engraftable HSCs from mouse and human iPSCs in teratoma-bearing animals in combination with a maneuver to facilitate hematopoiesis. In mice, we found that iPSC-derived HSCs migrate from teratomas into the BM and their intravenous injection into irradiated recipients resulted in multilineage and long-term reconstitution of the hematolymphopoietic system in serial transfers. Using this in vivo generation system, we could demonstrate that X-linked severe combined immunodeficiency (X-SCID) mice can be treated by HSCs derived from gene-corrected clonal iPSCs. It should also be noted that neither leukemia nor tumors were observed in recipients after transplantation of iPSC-derived HSCs. Taken our findings together, our system presented in this report should provide a useful tool not only for the study of HSCs, but also for practical application of iPSCs in the treatment of hematologic and immunologic diseases.

SummaryTo investigate the functional relationship between the expression of genes for ethylene-responsive transcription factors (ERFs) and the expression of ethylene-responsive genes, we examined the expression of genes for ERFs and the expression of a reporter gene in transgenic tobacco that carried a gene for β-glucuronidase (GUS) under the control of the ethylene-responsive element, which includes four copies of the 11-bp consensus sequence (designated the GCC-box, TAAGAGCCGCC). In strips of leaves of transgenic tobacco, the GCC-box-mediated expression of the reporter gene was induced in response to treatment with ethylene. We also observed the ethylene-independent immediate early induction of the synthesis of mRNAs for ERFs in wounded leaves and the enhancement of this induction by cycloheximide (CHX). Since CHX suppressed the induction of mRNAs for chitinase and GUS by ethylene, protein synthesis de novo was required for induction of the ethylenedependent GCC-box-mediated transcription of genes. In contrast, the enhancement by CHX of the wound-induced expression of ERFs suggested that no synthesis of new proteins was required for the wounding signal transduction leading to rapid expression of ERFs. Methyl jasmonate did not stimulate the wounding-responsive accumulation of ERF mRNAs, but it reduced such accumulation of mRNAs for ERF1, ERF2, ERF4 and the ethylene-dependent GCC-boxmediated transcription of the reporter gene. Thus, the immediate early induction of the expression of genes for ERFs in strips of tobacco leaves appears to be a novel type of wound-responsive activation of transcription. These results suggested that the expression of ERFs was not sufficient for activation of the GCC-box-mediated transcription but the expression of ERF1, ERF2 and ERF4, and that conversion of these ERFs by ethylene to their active form might be crucial for the GCC-box-mediated activation of the transcription of defense genes.

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