Showing papers by "Keisuke Okita published in 2009"

Suppression of induced pluripotent stem cell generation by the p53–p21 pathway

Abstract: Induced pluripotent stem (iPS) cells can be generated from somatic cells by the introduction of Oct3/4 (also known as Pou5f1), Sox2, Klf4 and c-Myc, in mouse and in human. The efficiency of this process, however, is low. Pluripotency can be induced without c-Myc, but with even lower efficiency. A p53 (also known as TP53 in humans and Trp53 in mice) short-interfering RNA (siRNA) was recently shown to promote human iPS cell generation, but the specificity and mechanisms remain to be determined. Here we report that up to 10% of transduced mouse embryonic fibroblasts lacking p53 became iPS cells, even without the Myc retrovirus. The p53 deletion also promoted the induction of integration-free mouse iPS cells with plasmid transfection. Furthermore, in the p53-null background, iPS cells were generated from terminally differentiated T lymphocytes. The suppression of p53 also increased the efficiency of human iPS cell generation. DNA microarray analyses identified 34 p53-regulated genes that are common in mouse and human fibroblasts. Functional analyses of these genes demonstrate that the p53-p21 pathway serves as a barrier not only in tumorigenicity, but also in iPS cell generation.

Variation in the safety of induced pluripotent stem cell lines

Abstract: We evaluated the teratoma-forming propensity of secondary neurospheres (SNS) generated from 36 mouse induced pluripotent stem (iPS) cell lines derived in 11 different ways. Teratoma-formation of SNS from embryonic fibroblast-derived iPS cells was similar to that of SNS from embryonic stem (ES) cells. In contrast, SNS from iPS cells derived from different adult tissues varied substantially in their teratoma-forming propensity, which correlated with the persistence of undifferentiated cells.

Roles of Sall4 in the generation of pluripotent stem cells from blastocysts and fibroblasts

Abstract: Pluripotency of embryonic stem (ES) cells is maintained by a network consisting of multiple transcription factors, including Oct3/4, Sox2, Nanog, Klf4 and Sall4. Among these factors, the forced expressions of Oct3/4, Sox2 and Klf4 are sufficient to reprogram fibroblasts into induced pluripotent stem (iPS) cells. The current study analyzed the role of Sall4 during the generation of ES cells and iPS cells. The mouse Sall4 gene was deleted by homologous recombination. Sall4-null embryos died shortly after implantation, as has been reported. ES-like cell lines can be established from Sall4-null blastocysts, albeit with a lower efficiency and a slower time course. The knockdown of Sall4 significantly decreased the efficiency of iPS cell generation from mouse fibroblasts. Furthermore, retroviral transduction of Sall4 significantly increased the efficiency of iPS cell generation in mouse and some human fibroblast lines. These results demonstrated that Sall4 plays positive roles in the generation of pluripotent stem cells from blastocysts and fibroblasts.

Characterization of dendritic cells and macrophages generated by directed differentiation from mouse induced pluripotent stem cells.

Abstract: Methods have been established to generate dendritic cells (DCs) from mouse and human embryonic stem (ES) cells. We designated them as ES-DCs and mouse models have demonstrated the induction of anti-cancer immunity and prevention of autoimmune disease by in vivo administration of genetically engineered ES-DCs. For the future clinical application of ES-DCs, the histoincompatibility between patients to be treated and available human ES cells and the ethical concerns associated with human ES cells may be serious obstacles. However, recently developed induced pluripotent stem (iPS) cell technology is expected to resolve these issues. This report describes the generation and characterization of DCs derived from mouse iPS cells. The iPS cell-derived DCs (iPS-DCs) possessed the characteristics of DCs including the capacity of T-cell-stimulation, antigen-processing and presentation and cytokine production. DNA microarray analyses revealed the upregulation of genes related to antigen-presenting functions during differentiation into iPS-DCs and similarity in gene expression profile in iPS-DCs and bone marrow cell-derived DCs. Genetically modified iPS-DCs expressing antigenic protein primed T-cells specific to the antigen in vivo and elicited efficient antigen-specific anti-tumor immunity. In addition, macrophages were generated from iPS cells (iPS-MP). iPS-MP were comparable with bone marrow cell-derived macrophages in the cell surface phenotype, functions, and gene expression profiles.

Transplantation of mouse induced pluripotent stem cells into the cochlea.

Abstract: This study examined the potential of induced pluripotent stem (iPS) cells for use as a source of transplants for the restoration of auditory spiral ganglion neurons. We monitored neurite outgrowth from iPS cell-derived neural progenitors toward cochlear hair cells ex vivo, and followed their survival and fates after transplantation into mouse cochleae in vivo. Neurons derived from iPS cells projected neurites toward cochlear hair cells. The settlement of iPS cell-derived neurons was observed 1 week after transplantation into the cochlea. Some transplants expressed vesicular glutamate transporter 1, which is a marker for glutamatergic neurons. These findings indicate that iPS cells can be used as a source of transplants for the regeneration of spiral ganglion neurons.

Orderly hematopoietic development of induced pluripotent stem cells via Flk-1+ hemoangiogenic progenitors

Abstract: 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. Little is known about the differentiation of iPS cells in vitro. Here we demonstrate that murine iPS cells produce various hematopoietic cell lineages when incubated on a layer of OP9 stromal cells. During this differentiation, iPS cells went through an intermediate stage consisting of progenitor cells that were positive for the early mesodermal marker Flk-1 and for the sequential expression of other genes that are associated with hematopoietic and endothelial development. Flk-1(+) cells differentiated into primitive and definitive hematopoietic cells, as well as into endothelial cells. Furthermore, Flk-1(+) populations contained common bilineage progenitors that could generate both hematopoietic and endothelial lineages from single cells. Our results demonstrate that iPS cell-derived cells, like ES cells, can follow a similar hematopoietic route to that seen in normal embryogenesis. This finding highlights the potential use of iPS cells in clinical areas such as regenerative medicine, disease investigation, and drug screening.

Method of nuclear reprogramming

Abstract: This invention provides a method of producing an induced pluripotent stem cell comprising the step of introducing at least one kind of non-viral expression vector (more preferably a plasmid vector) incorporating at least one gene that encodes a reprogramming factor into a somatic cell. An induced pluripotent stem cell wherein no exogenous genes induced is integrated into the cellular genome is also provided.

Procédé consistant à établir efficacement des cellules souches pluripotentes induites

Abstract: La presente invention concerne un procede d'amelioration de l'efficacite d'etablissement de cellules souches pluripotentes induites (iPS), comprenant l'etape consistant a inhiber la fonction p53 dans l'etape de reprogrammation nucleaire des cellules somatiques. L'inhibition de la fonction p53 est obtenue en amenant une substance choisie dans le groupe comprenant (1) des inhibiteurs chimiques du p53, (2) des mutants negatifs dominants du p53 et des acides nucleiques qui codent pour ceux-ci, (3) des ARNsi et des ARNsh contre le p53 et des ADN qui codent pour ceux-ci, et (4) des inhibiteurs de la voie du p53, en contact avec une cellule somatique, et equivalents. La presente invention concerne egalement un agent pour ameliorer l'efficacite d'etablissement de cellules iPS, l'agent comprenant un inhibiteur de la fonction p53, en particulier (1) des inhibiteurs chimiques du p53, (2) des mutants negatifs dominants du p53 et des acides nucleiques qui codent pour ceux-ci, (3) des ARNsi et des ARNsh contre le p53 et des ADN qui codent pour ceux-ci, et (4) des inhibiteurs de la voie du p53. La presente invention concerne en outre un procede de production d'une cellule iPS, comprenant l'etape consistant a amener en contact une substance de reprogrammation nucleaire et un inhibiteur de la fonction p53 avec une cellule somatique.

Procédé de reprogrammation nucléaire

Abstract: La presente invention concerne un procede de production d’une cellule souche pluripotente induite comprenant l’etape consistant a introduire au moins un type de vecteur d’expression viral (plus preferablement un vecteur plasmidique) comprenant au moins un gene qui code pour un facteur de reprogrammation dans une cellule somatique. La presente invention concerne en outre une cellule souche pluripotente induite dans laquelle aucun gene exogene induit n’est integre dans le genome cellulaire.