Development of an all-in-one inducible lentiviral vector for gene specific analysis of reprogramming.

Fair comparison of reprogramming efficiencies and in vitro differentiation capabilities among induced pluripotent stem cell (iPSC) lines has been hampered by the cellular and genetic heterogeneity of de novo infected somatic cells. In order to address this problem, we constructed a single cassette all-in-one inducible lentiviral vector (Ai-LV) for the expression of three reprogramming factors (Oct3/4, Klf4 and Sox2). To obtain multiple types of somatic cells having the same genetic background, we generated reprogrammable chimeric mice using iPSCs derived from Ai-LV infected somatic cells. Then, hepatic cells, hematopoietic cells and fibroblasts were isolated at different developmental stages from the chimeric mice, and reprogrammed again to generate 2nd iPSCs. The results revealed that somatic cells, especially fetal hepatoblasts were reprogrammed 1200 times more efficiently than adult hepatocytes with maximum reprogramming efficiency reaching 12.5%. However, we found that forced expression of c-Myc compensated for the reduced reprogramming efficiency in aged somatic cells without affecting cell proliferation. All these findings suggest that the Ai-LV system enables us to generate a panel of iPSC clones derived from various tissues with the same genetic background, and thus provides an invaluable tool for iPSC research.

Generation of germline-competent rat induced pluripotent stem cells.

BACKGROUND: Recent progress in rat pluripotent stem cell technology has been remarkable. Particularly salient is the demonstration that embryonic stem cells (ESCs) in the rat (rESCs) can contribute to germline transmission, permitting generation of gene-modified rats as is now done using mouse ESCs (mESCs) or mouse induced pluripotent stem cells (iPSCs; miPSCs). However, determinations of whether rat iPSCs (riPSCs) can contribute to germ cells are not published. Here we report the germline competency of riPSCs. METHODOLOGY/PRINCIPAL FINDINGS: We generated riPSCs by transducing three mouse reprogramming factors (Oct3/4, Klf4, and Sox2) into rat somatic cells, followed by culture in the presence of exogenous rat leukemia inhibitory factor (rLIF) and small molecules that specifically inhibit GSK3, MEK, and FGF receptor tyrosine kinases. We found that, like rESCs, our riPSCs can contribute to germline transmission. Furthermore we found, by immunostaining of testis from mouse-rat interspecific chimeras with antibody against mouse vasa homolog, that riPSCs can contribute to embryonic development with chimera formation in mice (rat-mouse interspecific chimeras) and to interspecific germlines. CONCLUSIONS/SIGNIFICANCE: Our data clearly demonstrate that using only three reprogramming factors (Oct3/4, Klf4, and Sox2) rat somatic cells can be reprogrammed into a ground state. Our generated riPSCs exhibited germline transmission in either rat-rat intraspecific or mouse-rat interspecific chimeras.

Definitive proof for direct reprogramming of hematopoietic cells to pluripotency.

Generation of induced pluripotent stem cells (iPSCs) generally uses fibroblastic cells, but other cell sources may prove useful in both research and clinical settings. Although proof of cellular origin requires genetic-marker identification in both target cells and established iPSCs, somatic cells other than mature lymphocytes mostly lack such markers. Here we show definitive proof of direct reprogramming of murine hematopoietic cells with no rearranged genes. Using iPSC factor transduction, we successfully derived iPSCs from bone marrow progenitor cells obtained from a mouse whose hematopoiesis was reconstituted from a single congenic hematopoietic stem cell. Established clones were demonstrated to be genetically identical to the transplanted single hematopoietic stem cell, thus proving their cellular origin. These hematopoietic cell-derived iPSCs showed typical characteristics of iPSCs, including the ability to contribute to chimerism in mice. These results will prompt further use of hematopoietic cells for iPSC generation while enabling definitive studies to test how cellular sources influence characteristics of descendant iPSCs.