Showing papers by "Keisuke Okita published in 2010"
TL;DR: An alternative method to generate iPS cells from mouse embryonic fibroblasts (MEFs) by continual transfection of plasmid vectors is described, which reduces the safety concern for iPS cell generation and application, and provides a source of cells for the investigation of the mechanisms underlying reprogramming and pluripotency.
Abstract: Reprogramming of somatic cells into pluripotent stem cells has been reported by introducing a combination of several transcription factors (Oct3/4, Sox2, Klf4 and c-Myc). The induced pluripotent stem (iPS) cells from patient's somatic cells could be a useful source for drug discovery and cell transplantation therapies. However, to date, most iPS cells were made using viral vectors, such as retroviruses and lentiviruses. Here we describe an alternative method to generate iPS cells from mouse embryonic fibroblasts (MEFs) by continual transfection of plasmid vectors. This protocol takes around 2 months to complete, from MEF isolation to iPS cell establishment. Although the reprogramming efficiency of this protocol is still low, the established iPS cells are most likely free from plasmid integration. This virus-free technique reduces the safety concern for iPS cell generation and application, and provides a source of cells for the investigation of the mechanisms underlying reprogramming and pluripotency.
243 citations
17 Jul 2010
TL;DR: The regulation of Sirt1 protein levels by miRNAs might provide new opportunities for therapeutic tissue-specific modulation of SIRT1 expression and for reprogramming of somatic cells into iPS cells.
Abstract: SIRT1 is increasingly recognized as a critical regulator of stress responses, replicative senescence, inflammation, metabolism, and aging. SIRT1 expression is regulated transcriptionally and post-transcriptionally, and its enzymatic activity is controlled by NAD+ levels and interacting proteins. We found that SIRT1 protein levels were much higher in mouse embryonic stem cells (mESCs) than in differentiated tissues. miRNAs post-transcriptionally downregulated SIRT1 during mESC differentiation and maintained low levels of SIRT1 expression in differentiated tissues. Specifically, miR-181a and b, miR-9, miR-204, miR-199b, and miR-135a suppressed SIRT1 protein expression. Inhibition of mir-9, the SIRT1-targeting miRNA induced earliest during mESC differentiation, prevented SIRT1 downregulation. Conversely, SIRT1 protein levels were upregulated post-transcriptionally during the reprogramming of mouse embryonic fibroblasts (MEFs) into induced pluripotent stem (iPS) cells. The regulation of SIRT1 protein levels by miRNAs might provide new opportunities for therapeutic tissue-specific modulation of SIRT1 expression and for reprogramming of somatic cells into iPS cells.
235 citations
TL;DR: In vivo analyses showed that iPS cells were intrinsically able to differentiate into fully mature hepatocytes that provided full liver function and were able to regenerate the liver after transplantation and two-thirds partial hepatectomy.
Abstract: The ability to generate induced pluripotent stem (iPS) cells from a patient's somatic cells has provided a foundation for organ regeneration without the need for immune suppression. However, it has not been established that the differentiated progeny of iPS cells can effectively reverse failure of a vital organ. Here, we examined whether iPS cell-derived hepatocytes have both the functional and proliferative capabilities needed for liver regeneration in mice with fumarylacetoacetate hydrolase deficiency. To avoid biases resulting from random genomic integration, we used iPS cells generated without viruses. To exclude compensation by hepatocytes not derived from iPS cells, we generated chimeric mice in which all hepatocytes were iPS cell derived. In vivo analyses showed that iPS cells were intrinsically able to differentiate into fully mature hepatocytes that provided full liver function. The iPS cell-derived hepatocytes also replicated the unique proliferative capabilities of normal hepatocytes and were able to regenerate the liver after transplantation and two-thirds partial hepatectomy. Thus, our results establish the feasibility of using iPS cells generated in a clinically acceptable fashion for rapid and stable liver regeneration.
189 citations
TL;DR: It is suggested that GFs from the easily obtainable gingival tissues can be readily reprogrammed into iPS cells, thus making them a promising cell source for investigating the basis of cellular reprogramming and pluripotency for future clinical applications.
Abstract: Background
Induced pluripotent stem (iPS) cells efficiently generated from accessible tissues have the potential for clinical applications Oral gingiva, which is often resected during general dental treatments and treated as biomedical waste, is an easily obtainable tissue, and cells can be isolated from patients with minimal discomfort
153 citations
TL;DR: Studies of the mechanisms underlying the reprogramming and establishment of non-integration methods contribute evidence to resolve the safety concerns associated with iPS cells.
95 citations
Patent•
18 Feb 2010TL;DR: In this article, a nuclear reprogramming factor has been used to reprogram a differentiated somatic cell to derive an induced pluripotent stem (iPS) cell, which can then be used for stem cell therapy.
Abstract: The present invention relates to a nuclear reprogramming factor having an action of reprogramming a differentiated somatic cell to derive an induced pluripotent stem (iPS) cell. The present invention also relates to the aforementioned iPS cells, methods of generating and maintaining iPS cells, and methods of using iPS cells, including screening and testing methods as well as methods of stem cell therapy. The present invention also relates to somatic cells derived by inducing differentiation of the aforementioned iPS cells.
41 citations
TL;DR: DNA methylation profile of T‐DMRs contributes to the mechanism of pluripotency, and can be a feasible solution for identification and evaluation of the pluripotent cells.
Abstract: DNA methylation profile, consisting of tissue-dependent and differentially methylated regions (T-DMRs), has elucidated tissue-specific gene function in mouse tissues. Here, we identified and profiled thousands of T-DMRs in embryonic stem cells (ESCs), embryonic germ cells (EGCs) and induced pluripotent stem cells (iPSCs). T-DMRs of ESCs compared with somatic tissues well illustrated gene function of ESCs, by hypomethylation at genes associated with CpG islands and nuclear events including transcriptional regulation network of ESCs, and by hypermethylation at genes for tissue-specific function. These T-DMRs in EGCs and iPSCs showed DNA methylation similar to ESCs. iPSCs, however, showed hypomethylation at a considerable number of T-DMRs that were hypermethylated in ESCs, suggesting existence of traceable progenitor epigenetic information. Thus, DNA methylation profile of T-DMRs contributes to the mechanism of pluripotency, and can be a feasible solution for identification and evaluation of the pluripotent cells.
28 citations
Patent•
18 May 2010
TL;DR: A method for inducing the differentiation of artificial pluripotent stem cells that have been induced from mammalian somatic cells or undifferentiated stem cells into epithelial progenitor cells, stem cells or corneal epithelial cells was proposed in this article.
Abstract: A method for inducing the differentiation of artificial pluripotent stem cells that have been induced from mammalian somatic cells or undifferentiated stem cells into epithelial progenitor cells, stem cells or corneal epithelial cells by culturing the artificial pluripotent stem cells under specified conditions; epithelial progenitor cells, stem cells or corneal epithelial cells produced by the method; and a cell preparation for treating epithelial diseases or a cell sheet, which is prepared using the cells.
9 citations
Patent•
06 Aug 2010TL;DR: In this article, the authors proposed a method of producing an iPS cell, comprising of Oct3/4, Klf4, Sox2, L-Myc, Lin28 and Lin28b.
Abstract: Provided is a method of producing an iPS cell, comprising bringing (a) Oct3/4 or a nucleic acid that encodes the same, (b) Klf4 or a nucleic acid that encodes the same, and (c) Sox2 or a nucleic acid that encodes the same, as well as (d1) L-Myc or a nucleic acid that encodes the same and/or (d2) a functional inhibitor of p53, into contact with a somatic cell. It is preferable that (a) a nucleic acid that encodes Oct3/4, (b) a nucleic acid that encodes Klf4, (c) a nucleic acid that encodes Sox2, (d1) a nucleic acid that encodes L-Myc and (e) a nucleic acid that encodes Lin28 or Lin28b be inserted into an episomal vector having loxP sequences placed in the same orientation on the 5′ and 3′ sides of a vector constituent essential for the replication of the vector, that (d2) a nucleic acid that encodes an shRNA against p53 be inserted into a vector ensuring transient expression (plasmid vector and the like), and that all these nucleic acids be transferred to a somatic cell.
9 citations
Patent•
28 May 2010TL;DR: In this paper, a method of preparing an induced pluripotent stem cell (iPS cell) deprived of a reprogramming gene, including providing an iPS cell having an expression vector wherein a loxP sequence is placed on each of the 5′ and 3′ sides of the reprogramging gene or a vector component necessary for the replication of the re-programming gene in the same orientation, was presented.
Abstract: Provided is a method of preparing an induced pluripotent stem cell (iPS cell) deprived of a reprogramming gene, including providing an iPS cell having an expression vector wherein a loxP sequence is placed on each of the 5′ and 3′ sides of the reprogramming gene or a vector component necessary for the replication of the reprogramming gene in the same orientation, and treating the IPS cell with Cre recombinase. Also provided are an iPS cell deprived of a reprogramming gene, as obtained by the method, and a use of the iPS cell as a cell source for producing somatic cells.
8 citations
Patent•
09 Dec 2010
TL;DR: In this paper, a method for preparing an induced pluripotent stem cell (iPS cell) wherein a reprogramming gene is eliminated, and comprising a step to provide an iPS cell having an expression vector containing a loxP sequence at 5' side and 3' side of a re-programming gene or a vector element necessary for replication of the re-registration gene.
Abstract: PROBLEM TO BE SOLVED: To provide a safe iPS cell applicable to cell transplantation therapy SOLUTION: There are provided a method for preparing an induced pluripotent stem cell (iPS cell) wherein a reprogramming gene is eliminated, and comprising a step to provide an iPS cell having an expression vector containing a loxP sequence at 5' side and 3' side of a reprogramming gene or a vector element necessary for replication of the reprogramming gene, and a step to treat the iPS cell with Cre recombinase; the iPS cell in which the reprogramming gene is eliminated and produced by the method; and a use of the iPS cell as a source for producing a somatic cell COPYRIGHT: (C)2011,JPO&INPIT
Patent•
18 May 2010
TL;DR: In this paper, a method for inducing differentiation into an epithelial progenitor cell/stem cell population or a corneal epithelial cell population by culturing, under particular conditions, induced pluripotent stem cells induced from mammalian somatic cells or undifferentiated stem cells was presented.
Abstract: The present invention relates to: a method for inducing differentiation into an epithelial progenitor cell/stem cell population or a corneal epithelial cell population by culturing, under particular conditions, induced pluripotent stem cells induced from mammalian somatic cells or undifferentiated stem cells; an epithelial progenitor cell/stem cell population or a corneal epithelial cell population obtained by the method; and a cell preparation for the treatment of epithelial disease and a cell sheet, which are prepared using these cell populations.
Patent•
06 Aug 2010
TL;DR: The authors concerne un procede de production d'une cellule iPS, comprenant une etape consistant a mettre en contact (a) Oct3/4 ou un acide nucleique l'encodant, (b) Klf4 or Klf 4 ou an acide noc encodant, and (c) Sox2 or Sox2 ou noc noc encodant.
Abstract: La presente invention concerne un procede de production d'une cellule iPS, comprenant une etape consistant a mettre en contact (a) Oct3/4 ou un acide nucleique l'encodant, (b) Klf4 ou un acide nucleique l'encodant et (c) Sox2 ou un acide nucleique l'encodant, ainsi que (d1) L-Myc ou un acide nucleique l'encodant et/ou (d2) un inhibiteur fonctionnel de p53, avec une cellule somatique. Il est preferable que (a) un acide nucleique codant pour Oct3/4, (b) un acide nucleique codant pour Klf4, (c) un acide nucleique codant pour Sox2, (d1) un acide nucleique codant pour L-Myc et (e) un acide nucleique codant pour Lin 28 ou Lin28b soient inseres dans un vecteur episomique comportant des sequence loxP disposees dans le meme sens sur les cotes 5' et 3' d'un constituant d'un vecteur essentiel a la replication dudit vecteur, et que (d2) un acide nucleique codant pour un ARNsh dirige contre p53 soit insere dans un vecteur assurant une expression transitoire (vecteur plasmidique et equivalent), et que tous ces acides nucleiques soient transferes vers une cellule somatique.