Generation of human induced pluripotent stem cells by direct delivery of reprogramming proteins.

doi:10.1016/J.STEM.2009.05.005

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Generation of human induced pluripotent stem cells by direct delivery of reprogramming proteins.

Journal Article•DOI•

Generation of human induced pluripotent stem cells by direct delivery of reprogramming proteins.

Dohoon Kim¹, Chun-Hyung Kim¹, Jung-Il Moon¹, Young-Gie Chung, Mi-Yoon Chang¹, Baek-Soo Han¹, Sanghyeok Ko¹, Eungi Yang¹, Kwang Yul Cha, Robert Lanza, Kwang Soo Kim¹ - Show less +7 more•Institutions (1)

Harvard University¹

05 Jun 2009-Cell Stem Cell (NIH Public Access)-Vol. 4, Iss: 6, pp 472-476

TL;DR: Document S1.

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About: This article is published in Cell Stem Cell.The article was published on 2009-06-05 and is currently open access. It has received 1890 citations till now.

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Journal Article•DOI•

Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA

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Luigi Warren¹, Philip D. Manos², Philip D. Manos¹, Tim Ahfeldt³, Tim Ahfeldt², Yuin-Han Loh², Hu Li⁴, Hu Li⁵, Frank H. Lau², Wataru Ebina¹, Pankaj Mandal¹, Zachary D. Smith⁶, Alexander Meissner⁶, Alexander Meissner², George Q. Daley, Andrew S. Brack², James J. Collins⁴, James J. Collins⁵, James J. Collins⁷, Chad A. Cowan, Thorsten M. Schlaeger², Thorsten M. Schlaeger¹, Derrick J. Rossi - Show less +19 more•Institutions (7)

Boston Children's Hospital¹, Harvard University², University of Hamburg³, Wyss Institute for Biologically Inspired Engineering⁴, Boston University⁵, Broad Institute⁶, Howard Hughes Medical Institute⁷

05 Nov 2010-Cell Stem Cell

TL;DR: It is shown that this approach can reprogram multiple human cell types to pluripotency with efficiencies that greatly surpass established protocols and represents a safe, efficient strategy for somatic cell reprogramming and directing cell fate that has broad applicability for basic research, disease modeling, and regenerative medicine.

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2,627 citations

Cites background or methods from "Generation of human induced pluripo..."

...Importantly, methods that rely on repeat administration of transient vectors, whether DNA or protein based, have so far shown very low iPSC derivation efficiencies (Jia et al., 2010; Kim et al., 2009; Okita et al., 2008; Stadtfeld et al., 2008; Yu et al., 2009; Zhou et al., 2009), presumably because of weak or inconstant expression of reprogramming factors....
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...iPSCs have also been derived with two DNA-free methods: serial protein transduction with recombinant proteins incorporating cell-penetrating peptide moieties (Kim et al., 2009; Zhou et al., 2009) and transgene...
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...…also been derived with two DNA-free methods: serial protein transduction with recombinant proteins incorporating cell-penetrating peptide moieties (Kim et al., 2009; Zhou et al., 2009) and transgene . delivery using the Sendai virus, which has a completely RNAbased reproductive cycle (Fusaki et…...
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...…repeat administration of transient vectors, whether DNA or protein based, have so far shown very low iPSC derivation efficiencies (Jia et al., 2010; Kim et al., 2009; Okita et al., 2008; Stadtfeld et al., 2008; Yu et al., 2009; Zhou et al., 2009), presumably because of weak or inconstant…...
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Journal Article•DOI•

A more efficient method to generate integration-free human iPS cells

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Keisuke Okita¹, Yasuko Matsumura¹, Yoshiko Sato¹, Aki Okada¹, Asuka Morizane¹, Satoshi Okamoto, Hyenjong Hong¹, Masato Nakagawa¹, Koji Tanabe¹, Ken-ichi Tezuka², Toshiyuki Shibata², Takahiro Kunisada², Masayo Takahashi¹, Jun Takahashi¹, Hiroh Saji, Shinya Yamanaka - Show less +12 more•Institutions (2)

Kyoto University¹, Gifu University²

01 May 2011-Nature Methods

TL;DR: A simple method is reported, using p53 suppression and nontransforming L-Myc, to generate human induced pluripotent stem cells (iPSCs) with episomal plasmid vectors, which may provide iPSCs suitable for autologous and allologous stem-cell therapy in the future.

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Abstract: Human induced pluripotent stem cells are generated with episomal plasmid vectors at increased efficiency using non-transforming L-Myc and knockdown of p53 Also in this issue, Chen et al report defined conditions for human cell reprogramming and culture

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1,712 citations

Journal Article•DOI•

Efficient induction of transgene-free human pluripotent stem cells using a vector based on Sendai virus, an RNA virus that does not integrate into the host genome

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Noemi Fusaki, Hiroshi Ban, Akiyo Nishiyama, Koichi Saeki, Mamoru Hasegawa - Show less +1 more

01 Jan 2009

TL;DR: It is shown that Sendai virus (SeV), an RNA virus and carries no risk of altering host genome, is an efficient solution for generating safe iPSC and will accelerate the clinical application.

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Abstract: Induced pluripotent stem cells (iPSC) have been generated from somatic cells by introducing reprogramming factors. Integration of foreign genes into the host genome is a technical hurdle for the clinical application. Here, we show that Sendai virus (SeV), an RNA virus and carries no risk of altering host genome, is an efficient solution for generating safe iPSC. Sendai-viral human iPSC expressed pluripotency genes, showed demethylation characteristic of reprogrammed cells. SeV-derived transgenes were decreased during cell division. Moreover, viruses were able to be easily removed by antibody-mediated negative selection utilizing cell surface marker HN that is expressed on SeV-infected cells. Viral-free iPSC differentiated to mature cells of the three embryonic germ layers in vivo and in vitro including beating cardiomyocytes, neurons, bone and pancreatic cells. Our data demonstrated that highly-efficient, non-integrating SeV-based vector system provides a critical solution for reprogramming somatic cells and will accelerate the clinical application.(Communicated by Kumao TOYOSHIMA, M.J.A.)

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1,370 citations

Journal Article•DOI•

Embryonic stem cell trials for macular degeneration: a preliminary report

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Steven D. Schwartz¹, Jean-Pierre Hubschman¹, Gad Heilwell¹, Valentina Franco-Cardenas¹, Carolyn K. Pan¹, Rosaleen Ostrick¹, Edmund Mickunas², Irina Klimanskaya², Robert Lanza² - Show less +5 more•Institutions (2)

University of California, Los Angeles¹, Advanced Cell Technology²

25 Feb 2012-The Lancet

TL;DR: The first description of hESC-derived cells transplanted into human patients with Stargardt's macular dystrophy and dry age-related macular degeneration is provided, with no signs of hyperproliferation, tumorigenicity, ectopic tissue formation, or apparent rejection after 4 months.

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1,319 citations

Journal Article•DOI•

Linking the p53 tumour suppressor pathway to somatic cell reprogramming

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Teruhisa Kawamura¹, Jotaro Suzuki¹, Jotaro Suzuki², Yunyuan V. Wang¹, Sergio Menendez, Laura Batlle Morera, Angel Raya³, Geoffrey M. Wahl¹, Juan Carlos Izpisua Belmonte¹ - Show less +5 more•Institutions (3)

Salk Institute for Biological Studies¹, Astellas Pharma², Catalan Institution for Research and Advanced Studies³

27 Aug 2009-Nature

TL;DR: It is shown that reprogramming factors can activate the p53 (also known as Trp53 in mice, TP53 in humans) pathway and silencing of p53 significantly increased the reprograming efficiency of human somatic cells.

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Abstract: Reprogramming somatic cells to induced pluripotent stem (iPS) cells has been accomplished by expressing pluripotency factors and oncogenes, but the low frequency and tendency to induce malignant transformation compromise the clinical utility of this powerful approach. We address both issues by investigating the mechanisms limiting reprogramming efficiency in somatic cells. Here we show that reprogramming factors can activate the p53 (also known as Trp53 in mice, TP53 in humans) pathway. Reducing signalling to p53 by expressing a mutated version of one of its negative regulators, by deleting or knocking down p53 or its target gene, p21 (also known as Cdkn1a), or by antagonizing reprogramming-induced apoptosis in mouse fibroblasts increases reprogramming efficiency. Notably, decreasing p53 protein levels enabled fibroblasts to give rise to iPS cells capable of generating germline-transmitting chimaeric mice using only Oct4 (also known as Pou5f1) and Sox2. Furthermore, silencing of p53 significantly increased the reprogramming efficiency of human somatic cells. These results provide insights into reprogramming mechanisms and suggest new routes to more efficient reprogramming while minimizing the use of oncogenes.

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1,111 citations

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References

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Journal Article•DOI•

Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.

[...]

Kazutoshi Takahashi¹, Shinya Yamanaka¹•Institutions (1)

Kyoto University¹

25 Aug 2006-Cell

TL;DR: Induction of pluripotent stem cells from mouse embryonic or adult fibroblasts by introducing four factors, Oct3/4, Sox2, c-Myc, and Klf4, under ES cell culture conditions is demonstrated and iPS cells, designated iPS, exhibit the morphology and growth properties of ES cells and express ES cell marker genes.

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23,959 citations

Additional excerpts

...In 2006, a new and less controversial method of reprogramming somatic cells to pluripotency was reported by viral expression of the transcription factors Oct4, Sox2, Klf4, and c-Myc (Takahashi and Yamanaka, 2006)....
[...]

Journal Article•DOI•

Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors

[...]

Kazutoshi Takahashi¹, Koji Tanabe¹, Mari Ohnuki¹, Megumi Narita¹, Tomoko Ichisaka¹, Kiichiro Tomoda, Shinya Yamanaka - Show less +3 more•Institutions (1)

Kyoto University¹

30 Nov 2007-Cell

TL;DR: It is demonstrated that iPS cells can be generated from adult human fibroblasts with the same four factors: Oct3/4, Sox2, Klf4, and c-Myc.

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18,175 citations

"Generation of human induced pluripo..." refers background or methods in this paper

...…cells can be reprogrammed to the pluripotent state via viral 472 Cell Stem Cell 4, June 5, 2009 ª2009 E transduction with the same or similar sets of reprogramming factors (Maherali et al., 2007; Okita et al., 2007; Park et al., 2008; Takahashi et al., 2007; Wernig et al., 2007; Yu et al., 2007)....
[...]
...…(p-hiPS01, first row; and p-hiPS02, second row): ectoderm, epidermal and neural tissue (rosette); mesoderm, bone and cartilage; and endoderm, respiratory epithelium and intestinal-like epithelium. protocols (about 0.01% of input cells) (Park et al., 2008; Takahashi et al., 2007; Yu et al., 2007)....
[...]
...…and the hESC H9 line, whereas the same regions were densely methylated in the parental HNF cells (Figure 2C). hiPSC lines from the starting HNFs were also generated using retroviral vectors expressing the same four reprogramming factors (Park et al., 2008; Takahashi et al., 2007; Yu et al., 2007)....
[...]
...Overall, the establishment of these hiPSC-like colonies took about 8 weeks, approximately double that seen with viral transduction (Park et al., 2008; Takahashi et al., 2007; Yu et al., 2007)....
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Journal Article•DOI•

Induced Pluripotent Stem Cell Lines Derived from Human Somatic Cells

[...]

Junying Yu¹, Maxim A. Vodyanik, Kim Smuga-Otto, Jessica Antosiewicz-Bourget, Jennifer L. Frane, Shulan Tian, Jeff Nie, Gudrun A. Jonsdottir, Victor Ruotti, Ron Stewart, Igor I. Slukvin, James A. Thomson - Show less +8 more•Institutions (1)

University of Wisconsin-Madison¹

21 Dec 2007-Science

TL;DR: This article showed that OCT4, SOX2, NANOG, and LIN28 factors are sufficient to reprogram human somatic cells to pluripotent stem cells that exhibit the essential characteristics of embryonic stem (ES) cells.

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Abstract: Somatic cell nuclear transfer allows trans-acting factors present in the mammalian oocyte to reprogram somatic cell nuclei to an undifferentiated state. We show that four factors (OCT4, SOX2, NANOG, and LIN28) are sufficient to reprogram human somatic cells to pluripotent stem cells that exhibit the essential characteristics of embryonic stem (ES) cells. These induced pluripotent human stem cells have normal karyotypes, express telomerase activity, express cell surface markers and genes that characterize human ES cells, and maintain the developmental potential to differentiate into advanced derivatives of all three primary germ layers. Such induced pluripotent human cell lines should be useful in the production of new disease models and in drug development, as well as for applications in transplantation medicine, once technical limitations (for example, mutation through viral integration) are eliminated.

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9,836 citations

"Generation of human induced pluripo..." refers background or methods in this paper

...…cells can be reprogrammed to the pluripotent state via viral 472 Cell Stem Cell 4, June 5, 2009 ª2009 E transduction with the same or similar sets of reprogramming factors (Maherali et al., 2007; Okita et al., 2007; Park et al., 2008; Takahashi et al., 2007; Wernig et al., 2007; Yu et al., 2007)....
[...]
...…(p-hiPS01, first row; and p-hiPS02, second row): ectoderm, epidermal and neural tissue (rosette); mesoderm, bone and cartilage; and endoderm, respiratory epithelium and intestinal-like epithelium. protocols (about 0.01% of input cells) (Park et al., 2008; Takahashi et al., 2007; Yu et al., 2007)....
[...]
...…and the hESC H9 line, whereas the same regions were densely methylated in the parental HNF cells (Figure 2C). hiPSC lines from the starting HNFs were also generated using retroviral vectors expressing the same four reprogramming factors (Park et al., 2008; Takahashi et al., 2007; Yu et al., 2007)....
[...]
...Overall, the establishment of these hiPSC-like colonies took about 8 weeks, approximately double that seen with viral transduction (Park et al., 2008; Takahashi et al., 2007; Yu et al., 2007)....
[...]

Journal Article•DOI•

Viable offspring derived from fetal and adult mammalian cells

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Ian Wilmut¹, Angelika Schnieke¹, Jim McWhir¹, Alex J. Kind¹, Keith H.S. Campbell¹ - Show less +1 more•Institutions (1)

The Roslin Institute¹

27 Feb 1997-Nature

TL;DR: The birth of lambs from differentiated fetal and adult cells confirms that differentiation of that cell did not involve the irreversible modification of genetic material required for development to term and reinforces previous speculation that by inducing donor cells to become quiescent it will be possible to obtain normal development from a wide variety of differentiated cells.

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Abstract: Fertilization of mammalian eggs is followed by successive cell divisions and progressive differentiation, first into the early embryo and subsequently into all of the cell types that make up the adult animal. Transfer of a single nucleus at a specific stage of development, to an enucleated unfertilized egg, provided an opportunity to investigate whether cellular differentiation to that stage involved irreversible genetic modification. The first offspring to develop from a differentiated cell were born after nuclear transfer from an embryo-derived cell line that had been induced to become quiescent. Using the same procedure, we now report the birth of live lambs from three new cell populations established from adult mammary gland, fetus and embryo. The fact that a lamb was derived from an adult cell confirms that differentiation of that cell did not involve the irreversible modification of genetic material required for development to term. The birth of lambs from differentiated fetal and adult cells also reinforces previous speculation that by inducing donor cells to become quiescent it will be possible to obtain normal development from a wide variety of differentiated cells.

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4,721 citations

"Generation of human induced pluripo..." refers background in this paper

...Over a decade ago, Wilmut and colleagues showed that adult somatic cells could be reprogrammed back to an undifferentiated embryonic state using somatic cell nuclear transfer (SCNT) (Wilmut et al., 1997)....
[...]
...However, since that time, attempts to generate patient-specific cells using SCNT have proven unsuccessful (Chung et al., 2009; French et al., 2008)....
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Journal Article•DOI•

Generation of germline-competent induced pluripotent stem cells

[...]

Keisuke Okita¹, Tomoko Ichisaka¹, Shinya Yamanaka¹•Institutions (1)

Kyoto University¹

19 Jul 2007-Nature

TL;DR: iPS cells competent for germline chimaeras can be obtained from fibroblasts, but retroviral introduction of c-Myc should be avoided for clinical application.

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Abstract: We have previously shown that pluripotent stem cells can be induced from mouse fibroblasts by retroviral introduction of Oct3/4 (also called Pou5f1), Sox2, c-Myc and Klf4, and subsequent selection for Fbx15 (also called Fbxo15) expression These induced pluripotent stem (iPS) cells (hereafter called Fbx15 iPS cells) are similar to embryonic stem (ES) cells in morphology, proliferation and teratoma formation; however, they are different with regards to gene expression and DNA methylation patterns, and fail to produce adult chimaeras Here we show that selection for Nanog expression results in germline-competent iPS cells with increased ES-cell-like gene expression and DNA methylation patterns compared with Fbx15 iPS cells The four transgenes (Oct3/4, Sox2, c-myc and Klf4) were strongly silenced in Nanog iPS cells We obtained adult chimaeras from seven Nanog iPS cell clones, with one clone being transmitted through the germ line to the next generation Approximately 20% of the offspring developed tumours attributable to reactivation of the c-myc transgene Thus, iPS cells competent for germline chimaeras can be obtained from fibroblasts, but retroviral introduction of c-Myc should be avoided for clinical application

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4,371 citations

"Generation of human induced pluripo..." refers background in this paper

...The use of genome-integrating viruses could cause insertional mutagenesis and unpredictable genetic dysfunction (Okita et al., 2007; Yamanaka, 2007)....
[...]
...…cells can be reprogrammed to the pluripotent state via viral 472 Cell Stem Cell 4, June 5, 2009 ª2009 E transduction with the same or similar sets of reprogramming factors (Maherali et al., 2007; Okita et al., 2007; Park et al., 2008; Takahashi et al., 2007; Wernig et al., 2007; Yu et al., 2007)....
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