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

Clinical application of induced pluripotent stem cells (iPSCs) is limited by the low efficiency of iPSC derivation and the fact that most protocols modify the genome to effect cellular reprogramming. Moreover, safe and effective means of directing the fate of patient-specific iPSCs toward clinically useful cell types are lacking. Here we describe a simple, nonintegrating strategy for reprogramming cell fate based on administration of synthetic mRNA modified to overcome innate antiviral responses. We show that this approach can reprogram multiple human cell types to pluripotency with efficiencies that greatly surpass established protocols. We further show that the same technology can be used to efficiently direct the differentiation of RNA-induced pluripotent stem cells (RiPSCs) into terminally differentiated myogenic cells. This technology 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.

[1]  J. Utikal,et al.  Induced Pluripotent Stem Cells Generated Without Viral Integration , 2008, Science.

[2]  G. Vanham,et al.  Simultaneous Activation of Viral Antigen-specific Memory CD4+ and CD8+ T-cells Using mRNA-electroporated CD40-activated Autologous B-cells , 2006, Journal of immunotherapy.

[3]  P. Bevilacqua,et al.  A brilliant disguise for self RNA: 5’-end and internal modifications of primary transcripts suppress elements of innate immunity , 2008, RNA biology.

[4]  Zachary D. Smith,et al.  Dynamic single-cell imaging of direct reprogramming reveals an early specifying event , 2010, Nature Biotechnology.

[5]  Wei Wang,et al.  piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells , 2009, Nature.

[6]  Jeroen S. van Zon,et al.  Direct cell reprogramming is a stochastic process amenable to acceleration , 2009, Nature.

[7]  Lisheng Wang,et al.  Cytokines and BMP-4 promote hematopoietic differentiation of human embryonic stem cells. , 2003, Blood.

[8]  Robert Lanza,et al.  Generation of human induced pluripotent stem cells by direct delivery of reprogramming proteins. , 2009, Cell stem cell.

[9]  George Q. Daley,et al.  Reprogramming of human somatic cells to pluripotency with defined factors , 2008, Nature.

[10]  D. Campana,et al.  Synthetic messenger RNA as a tool for gene therapy. , 2006, Human gene therapy.

[11]  D. Weissman,et al.  Naturally occurring nucleoside modifications suppress the immunostimulatory activity of RNA: implication for therapeutic RNA development. , 2007, Current opinion in drug discovery & development.

[12]  P. Bevilacqua,et al.  Nucleoside modifications modulate activation of the protein kinase PKR in an RNA structure‐specific manner , 2008, RNA.

[13]  A. Pichlmair,et al.  RIG-I-Mediated Antiviral Responses to Single-Stranded RNA Bearing 5'-Phosphates , 2006, Science.

[14]  K. Plath,et al.  Generation of human induced pluripotent stem cells from dermal fibroblasts , 2008, Proceedings of the National Academy of Sciences.

[15]  R. Stewart,et al.  Human Induced Pluripotent Stem Cells Free of Vector and Transgene Sequences , 2009, Science.

[16]  J. Utikal,et al.  Immortalization eliminates a roadblock during cellular reprogramming into iPS cells , 2009, Nature.

[17]  J. Licht,et al.  Sprouty1 Regulates Reversible Quiescence of a Self-Renewing Adult Muscle Stem Cell Pool during Regeneration , 2010, Cell stem cell.

[18]  G. Wahl,et al.  Linking the p53 tumor suppressor pathway to somatic cell reprogramming , 2009, Nature.

[19]  S. Weissman,et al.  Chimeric receptor mRNA transfection as a tool to generate antineoplastic lymphocytes. , 2008, Human gene therapy.

[20]  Gunther Hartmann,et al.  5'-Triphosphate RNA Is the Ligand for RIG-I , 2006, Science.

[21]  Shizuo Akira,et al.  Antiviral signaling through pattern recognition receptors. , 2006, Journal of biochemistry.

[22]  N. Elango,et al.  Optimized transfection of mRNA transcribed from a d(A/T)100 tail-containing vector. , 2005, Biochemical and biophysical research communications.

[23]  D. Melton,et al.  Synthesis of long, capped transcripts in vitro by SP6 and T7 RNA polymerases. , 1989, Methods in enzymology.

[24]  S. Hoekstra.  Cationic lipid-mediated transfection in vitro and in vivo , 2001, Molecular membrane biology.

[25]  G. Daley,et al.  Live cell imaging distinguishes bona fide human iPS cells from partially reprogrammed cells , 2009, Nature Biotechnology.

[26]  Dong Wook Han,et al.  Generation of induced pluripotent stem cells using recombinant proteins. , 2009, Cell stem cell.

[27]  S. Nishikawa,et al.  A ROCK inhibitor permits survival of dissociated human embryonic stem cells , 2007, Nature Biotechnology.

[28]  K. Woltjen,et al.  Virus free induction of pluripotency and subsequent excision of reprogramming factors , 2009, Nature.

[29]  Michel Sadelain,et al.  Stoichiometric and temporal requirements of Oct4, Sox2, Klf4, and c-Myc expression for efficient human iPSC induction and differentiation , 2009, Proceedings of the National Academy of Sciences.

[30]  T. Graf.  Faculty Opinions recommendation of Induction of pluripotent stem cells from adult human fibroblasts by defined factors. , 2007 .

[31]  T. Ichisaka,et al.  Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors , 2007, Cell.

[32]  Hideyuki Okano,et al.  Variation in the safety of induced pluripotent stem cell lines , 2009, Nature Biotechnology.

[33]  G. Stein,et al.  Pluripotency: Toward a gold standard for human ES and iPS cells , 2009, Journal of cellular physiology.

[34]  A. Alcamí,et al.  Vaccinia virus encodes a soluble type I interferon receptor of novel structure and broad species soecificity , 1995, Cell.

[35]  H. Weintraub,et al.  Expression of a single transfected cDNA converts fibroblasts to myoblasts , 1987, Cell.

[36]  A. Mares,et al.  HIT AND RUN , 1999 .

[37]  S. Cole,et al.  Sequences Human Induced Pluripotent Stem Cells Free of Vector and Transgene , 2012 .

[38]  R. Stewart,et al.  Induced Pluripotent Stem Cell Lines Derived from Human Somatic Cells , 2007, Science.

[39]  S. Goodbourn,et al.  Interferons and viruses: an interplay between induction, signalling, antiviral responses and virus countermeasures. , 2008, The Journal of general virology.

[40]  U. Şahin,et al.  Modification of antigen-encoding RNA increases stability, translational efficacy, and T-cell stimulatory capacity of dendritic cells. , 2006, Blood.

[41]  Deepak M. Gupta,et al.  A nonviral minicircle vector for deriving human iPS cells , 2010, Nature Methods.

[42]  L. Gehrke,et al.  Nucleotide sequences and modifications that determine RIG-I/RNA binding and signaling activities. , 2009, Journal of virology.

[43]  Hiroki Kato,et al.  Incorporation of pseudouridine into mRNA yields superior nonimmunogenic vector with increased translational capacity and biological stability. , 2008, Molecular therapy : the journal of the American Society of Gene Therapy.

[44]  T. Ichisaka,et al.  Hypoxia enhances the generation of induced pluripotent stem cells. , 2009, Cell stem cell.

[45]  James A Thomson,et al.  Neural differentiation of human induced pluripotent stem cells follows developmental principles but with variable potency , 2010, Proceedings of the National Academy of Sciences.

[46]  Shizuo Akira,et al.  Innate Antiviral Responses by Means of TLR7-Mediated Recognition of Single-Stranded RNA , 2004, Science.

[47]  T. Schoeb,et al.  Polycistronic Lentiviral Vector for “Hit and Run” Reprogramming of Adult Skin Fibroblasts to Induced Pluripotent Stem Cells , 2009, Stem cells.

[48]  S. Akira,et al.  Toll-like Receptors and Type I Interferons* , 2007, Journal of Biological Chemistry.

[49]  Paul H Lerou,et al.  Generation of human-induced pluripotent stem cells , 2008, Nature Protocols.

[50]  Houping Ni,et al.  Suppression of RNA recognition by Toll-like receptors: the impact of nucleoside modification and the evolutionary origin of RNA. , 2005, Immunity.

[51]  S. Kain,et al.  Generation of Destabilized Green Fluorescent Protein as a Transcription Reporter* , 1998, The Journal of Biological Chemistry.

[52]  S. Yamanaka,et al.  Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors , 2006, Cell.

[53]  Shinya Yamanaka,et al.  Generation of Mouse Induced Pluripotent Stem Cells Without Viral Vectors , 2008, Science.

[54]  D. Weissman,et al.  HIV Gag mRNA Transfection of Dendritic Cells (DC) Delivers Encoded Antigen to MHC Class I and II Molecules, Causes DC Maturation, and Induces a Potent Human In Vitro Primary Immune Response1 , 2000, The Journal of Immunology.

[55]  Wenjun Guo,et al.  Induction of pluripotent stem cells by defined factors is greatly improved by small-molecule compounds , 2008, Nature Biotechnology.

[56]  M. Hasegawa,et al.  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 , 2009, Proceedings of the Japan Academy. Series B, Physical and biological sciences.

[57]  C. van Broeckhoven,et al.  Highly efficient gene delivery by mRNA electroporation in human hematopoietic cells: superiority to lipofection and passive pulsing of mRNA and to electroporation of plasmid cDNA for tumor antigen loading of dendritic cells. , 2001, Blood.