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Bryce W Carey,
Styliani Markoulaki,
Jacob H Hanna,
Dina A Faddah,
Yosef Buganim,
Jongpil Kim,
Kibibi Ganz,
Eveline J Steine,
John P Cassady,
Menno P Creyghton,
G Grant Welstead,
Qing Gao,
Rudolf Jaenisch
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ABSTRACT: We compared two genetically highly defined transgenic systems to identify parameters affecting reprogramming of somatic cells to a pluripotent state. Our results demonstrate that the level and stoichiometry of reprogramming factors during the reprogramming process strongly influence the resulting pluripotency of iPS cells. High expression of Oct4 and Klf4 combined with lower expression of c-Myc and Sox2 produced iPS cells that efficiently generated "all-iPSC mice" by tetraploid (4n) complementation, maintained normal imprinting at the Dlk1-Dio3 locus, and did not create mice with tumors. Loss of imprinting (LOI) at the Dlk1-Dio3 locus did not strictly correlate with reduced pluripotency though the efficiency of generating "all-iPSC mice" was diminished. Our data indicate that stoichiometry of reprogramming factors can influence epigenetic and biological properties of iPS cells. This concept complicates efforts to define a "generic" epigenetic state of iPSCs and ESCs and should be considered when comparing different iPS and ES cell lines.
Cell stem cell 12/2011; 9(6):588-98. · 23.56 Impact Factor
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Mitchell Guttman,
Julie Donaghey, Bryce W Carey,
Manuel Garber,
Jennifer K Grenier,
Glen Munson,
Geneva Young,
Anne Bergstrom Lucas,
Robert Ach,
Laurakay Bruhn,
Xiaoping Yang,
Ido Amit,
Alexander Meissner,
Aviv Regev,
John L Rinn,
David E Root,
Eric S Lander
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ABSTRACT: Although thousands of large intergenic non-coding RNAs (lincRNAs) have been identified in mammals, few have been functionally characterized, leading to debate about their biological role. To address this, we performed loss-of-function studies on most lincRNAs expressed in mouse embryonic stem (ES) cells and characterized the effects on gene expression. Here we show that knockdown of lincRNAs has major consequences on gene expression patterns, comparable to knockdown of well-known ES cell regulators. Notably, lincRNAs primarily affect gene expression in trans. Knockdown of dozens of lincRNAs causes either exit from the pluripotent state or upregulation of lineage commitment programs. We integrate lincRNAs into the molecular circuitry of ES cells and show that lincRNA genes are regulated by key transcription factors and that lincRNA transcripts bind to multiple chromatin regulatory proteins to affect shared gene expression programs. Together, the results demonstrate that lincRNAs have key roles in the circuitry controlling ES cell state.
Nature 08/2011; 477(7364):295-300. · 36.28 Impact Factor
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Menno P Creyghton,
Albert W Cheng,
G Grant Welstead,
Tristan Kooistra, Bryce W Carey,
Eveline J Steine,
Jacob Hanna,
Michael A Lodato,
Garrett M Frampton,
Phillip A Sharp,
Laurie A Boyer,
Richard A Young,
Rudolf Jaenisch
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ABSTRACT: Developmental programs are controlled by transcription factors and chromatin regulators, which maintain specific gene expression programs through epigenetic modification of the genome. These regulatory events at enhancers contribute to the specific gene expression programs that determine cell state and the potential for differentiation into new cell types. Although enhancer elements are known to be associated with certain histone modifications and transcription factors, the relationship of these modifications to gene expression and developmental state has not been clearly defined. Here we interrogate the epigenetic landscape of enhancer elements in embryonic stem cells and several adult tissues in the mouse. We find that histone H3K27ac distinguishes active enhancers from inactive/poised enhancer elements containing H3K4me1 alone. This indicates that the amount of actively used enhancers is lower than previously anticipated. Furthermore, poised enhancer networks provide clues to unrealized developmental programs. Finally, we show that enhancers are reset during nuclear reprogramming.
Proceedings of the National Academy of Sciences 11/2010; 107(50):21931-6. · 9.68 Impact Factor
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ABSTRACT: Human and mouse embryonic stem cells (ESCs) are derived from blastocyst-stage embryos but have very different biological properties, and molecular analyses suggest that the pluripotent state of human ESCs isolated so far corresponds to that of mouse-derived epiblast stem cells (EpiSCs). Here we rewire the identity of conventional human ESCs into a more immature state that extensively shares defining features with pluripotent mouse ESCs. This was achieved by ectopic induction of Oct4, Klf4, and Klf2 factors combined with LIF and inhibitors of glycogen synthase kinase 3beta (GSK3beta) and mitogen-activated protein kinase (ERK1/2) pathway. Forskolin, a protein kinase A pathway agonist which can induce Klf4 and Klf2 expression, transiently substitutes for the requirement for ectopic transgene expression. In contrast to conventional human ESCs, these epigenetically converted cells have growth properties, an X-chromosome activation state (XaXa), a gene expression profile, and a signaling pathway dependence that are highly similar to those of mouse ESCs. Finally, the same growth conditions allow the derivation of human induced pluripotent stem (iPS) cells with similar properties as mouse iPS cells. The generation of validated "naïve" human ESCs will allow the molecular dissection of a previously undefined pluripotent state in humans and may open up new opportunities for patient-specific, disease-relevant research.
Proceedings of the National Academy of Sciences 05/2010; 107(20):9222-7. · 9.68 Impact Factor
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ABSTRACT: We report transgenic mouse models in which three or four reprogramming factors are expressed from a single genomic locus using a drug-inducible transgene. Multiple somatic cell types can be directly reprogrammed to generate induced pluripotent stem cells (iPSCs) by culture in doxycycline. Because reprogramming factors are carried on a single polycistronic construct, the mice can be easily maintained, and the transgene can be easily transferred into other genetic backgrounds.
Nature Methods 12/2009; 7(1):56-9. · 19.28 Impact Factor
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Jacob Hanna,
Styliani Markoulaki,
Maisam Mitalipova,
Albert W Cheng,
John P Cassady,
Judith Staerk, Bryce W Carey,
Christopher J Lengner,
Ruth Foreman,
Jennifer Love,
Qing Gao,
Jongpil Kim,
Rudolf Jaenisch
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ABSTRACT: Embryonic stem cells (ESCs) are isolated from the inner cell mass (ICM) of blastocysts, whereas epiblast stem cells (EpiSCs) are derived from the postimplantation epiblast and display a restricted developmental potential. Here we characterize pluripotent states in the nonobese diabetic (NOD) mouse strain, which prior to this study was considered "nonpermissive" for ESC derivation. We find that NOD stem cells can be stabilized by providing constitutive expression of Klf4 or c-Myc or small molecules that can replace these factors during in vitro reprogramming. The NOD ESCs and iPSCs appear to be "metastable," as they acquire an alternative EpiSC-like identity after removal of the exogenous factors, while their reintroduction converts the cells back to ICM-like pluripotency. Our findings suggest that stem cells from different genetic backgrounds can assume distinct states of pluripotency in vitro, the stability of which is regulated by endogenous genetic determinants and can be modified by exogenous factors.
Cell stem cell 06/2009; 4(6):513-24. · 23.56 Impact Factor
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Mitchell Guttman,
Ido Amit,
Manuel Garber,
Courtney French,
Michael F Lin,
David Feldser,
Maite Huarte,
Or Zuk, Bryce W Carey,
John P Cassady,
Moran N Cabili,
Rudolf Jaenisch,
Tarjei S Mikkelsen,
Tyler Jacks,
Nir Hacohen,
Bradley E Bernstein,
Manolis Kellis,
Aviv Regev,
John L Rinn,
Eric S Lander
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ABSTRACT: There is growing recognition that mammalian cells produce many thousands of large intergenic transcripts. However, the functional significance of these transcripts has been particularly controversial. Although there are some well-characterized examples, most (>95%) show little evidence of evolutionary conservation and have been suggested to represent transcriptional noise. Here we report a new approach to identifying large non-coding RNAs using chromatin-state maps to discover discrete transcriptional units intervening known protein-coding loci. Our approach identified approximately 1,600 large multi-exonic RNAs across four mouse cell types. In sharp contrast to previous collections, these large intervening non-coding RNAs (lincRNAs) show strong purifying selection in their genomic loci, exonic sequences and promoter regions, with greater than 95% showing clear evolutionary conservation. We also developed a functional genomics approach that assigns putative functions to each lincRNA, demonstrating a diverse range of roles for lincRNAs in processes from embryonic stem cell pluripotency to cell proliferation. We obtained independent functional validation for the predictions for over 100 lincRNAs, using cell-based assays. In particular, we demonstrate that specific lincRNAs are transcriptionally regulated by key transcription factors in these processes such as p53, NFkappaB, Sox2, Oct4 (also known as Pou5f1) and Nanog. Together, these results define a unique collection of functional lincRNAs that are highly conserved and implicated in diverse biological processes.
Nature 02/2009; 458(7235):223-7. · 36.28 Impact Factor
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Styliani Markoulaki,
Jacob Hanna,
Caroline Beard, Bryce W Carey,
Albert W Cheng,
Christopher J Lengner,
Jessica A Dausman,
Dongdong Fu,
Qing Gao,
Su Wu,
John P Cassady,
Rudolf Jaenisch
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ABSTRACT: Proviruses carrying drug-inducible Oct4, Sox2, Klf4 and c-Myc used to derive 'primary' induced pluripotent stem (iPS) cells were segregated through germline transmission, generating mice and cells carrying subsets of the reprogramming factors. Drug treatment produced 'secondary' iPS cells only when the missing factor was introduced. This approach creates a defined system for studying reprogramming mechanisms and allows screening of genetically homogeneous cells for compounds that can replace any transcription factor required for iPS cell derivation.
Nature Biotechnology 02/2009; 27(2):169-71. · 29.50 Impact Factor
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ABSTRACT: Directed reprogramming of somatic cells by defined factors provides a novel method for the generation of patient-specific stem cells with the potential to bypass both the practical and ethical concerns associated with somatic cell nuclear transfer (SCNT) and human embryonic stem (hES) cells. Although the generation of induced pluripotent stem (iPS) cells has proven a robust technology in mouse and human, a major impediment to the use of iPS cells for therapeutic purposes has been the viral-based delivery of the reprogramming factors because multiple proviral integrations pose the danger of insertional mutagenesis. Here we report a novel approach to reduce the number of viruses necessary to reprogram somatic cells by delivering reprogramming factors in a single virus using 2A "self-cleaving" peptides, which support efficient polycistronic expression from a single promoter. We find that up to four reprogramming factors (Oct4, Sox2, Klf4, and c-Myc) can be expressed from a single virus to generate iPS cells in both embryonic and adult somatic mouse cells and we show that a single proviral copy is sufficient to generate iPS cells from mouse embryonic fibroblasts. In addition we have generated human induced pluripotent stem (hiPS) cell lines from human keratinocytes, demonstrating that a single polycistronic virus can reprogram human somatic cells.
Proceedings of the National Academy of Sciences 01/2009; 106(1):157-62. · 9.68 Impact Factor
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Jacob Hanna,
Styliani Markoulaki,
Patrick Schorderet, Bryce W Carey,
Caroline Beard,
Marius Wernig,
Menno P Creyghton,
Eveline J Steine,
John P Cassady,
Ruth Foreman,
Christopher J Lengner,
Jessica A Dausman,
Rudolf Jaenisch
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ABSTRACT: Pluripotent cells can be derived from fibroblasts by ectopic expression of defined transcription factors. A fundamental unresolved question is whether terminally differentiated cells can be reprogrammed to pluripotency. We utilized transgenic and inducible expression of four transcription factors (Oct4, Sox2, Klf4, and c-Myc) to reprogram mouse B lymphocytes. These factors were sufficient to convert nonterminally differentiated B cells to a pluripotent state. However, reprogramming of mature B cells required additional interruption with the transcriptional state maintaining B cell identity by either ectopic expression of the myeloid transcription factor CCAAT/enhancer-binding-protein-alpha (C/EBPalpha) or specific knockdown of the B cell transcription factor Pax5. Multiple iPS lines were clonally derived from both nonfully and fully differentiated B lymphocytes, which gave rise to adult chimeras with germline contribution, and to late-term embryos when injected into tetraploid blastocysts. Our study provides definite proof for the direct nuclear reprogramming of terminally differentiated adult cells to pluripotency.
Cell 05/2008; 133(2):250-64. · 32.40 Impact Factor
