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Definitions

• the present invention relates to compositions and rapid, efficient methods for changing the state of differentiation of a eukaryotic cell.
• the present invention provides mRNA molecules and methods for their use to reprogram cells, such as to reprogram human somatic cells to pluripotent stem cells.
• pluripotent stem cells were also induced by transforming human somatic cells with genes encoding the similar human protein factors (OCT4, SOX2, KLF4, and c-MYC) (Takahashi et al. 2007), or by transforming human somatic cells with genes encoding human OCT4 and SOX2 factors plus genes encoding two other human factors, NANOG and LIN28 (Lin-28 homolog A) (Yu et al. 2007).
• All of these methods used retroviruses or lentiviruses to integrate genes encoding the reprogramming factors into the genomes of the transformed cells and the somatic cells were reprogrammed into iPS cells only over a long period of time (e.g., in excess of a week).
• the generation iPS cells from differentiated somatic cells offers great promise as a possible means for treating diseases through cell transplantation.
• the possibility to generate iPS cells from somatic cells from individual patients also may enable development of patient-specific therapies with less risk due to immune rejection.
• generation of iPS cells from disease-specific somatic cells offers promise as a means to study and develop drugs to treat specific disease states (Ebert et al. 2009, Lee et al. 2009, Maehr et al. 2009).
• iPSC factors protein reprogramming factors
• Induced pluripotent stem cells were also generated from human somatic cells by introduction of a plasmid that expressed genes encoding human OCT4, SOX2, c-MYC, KLF4, NANOG and LIN28 (Yu et al. 2009).
• Other successful approaches for generating iPS cells include treating somatic cells with: recombinant protein reprogramming factors (Zhou et al. 2009); non-integrating adenoviruses (Stadtfeld et al. 2008); or piggyBac transposons (Woltjen et al. 2009) to deliver reprogramming factors.
• recombinant protein reprogramming factors Zhou et al. 2009
• non-integrating adenoviruses Stadtfeld et al. 2008
• piggyBac transposons Wiltjen et al. 2009
• the present invention relates to compositions and rapid, efficient methods for changing the state of differentiation of a eukaryotic cell.
• the present invention provides mRNA molecules and methods for their use to reprogram cells, such as to reprogram human somatic cells to pluripotent stem cells.
• the present invention provides methods for changing the state of differentiation of a cell comprising: introducing an mRNA encoding an iPS cell induction factor into a somatic cell to generate a reprogrammed cell.
• the introducing comprises delivering the mRNA to the somatic cell with a transfection reagent.
• the reprogrammed cell is a dedifferentiated cell.
• the reprogrammed cell is a transdifferentiated cell.
• the mRNA is polyadenylated.
• the mRNA comprises a poly-A tail 100-200 nucleotides in length.
• the mRNA comprises capped mRNA.
• the mRNA is a population of mRNA molecules, the population having greater than 99% capped mRNA.
• the mRNA comprises pseudouridine in place of uridine.
• the iPS cell induction factor is selected from the group consisting of KLF4, LIN28, c-MYC, NANOG, OCT4, and SOX2.
• the introducing comprises introducing mRNA encoding a plurality of iPS cell induction factors into the somatic cell.
• the plurality of iPS cell induction factors comprises each of KLF4, LIN28, c-MYC, NANOG, OCT4, and SOX2.
• the cell is a fibroblast.
• the reprogrammed cell is a pluripotent stem cell.
• the dedifferentiated cell expresses NANOG and TRA-1-60.
• the cell is in vitro.
• the cell resides in culture.
• the cells reside in MEF- conditioned medium.
• the present invention provides compositions comprising an mRNA encoding an iPS cell induction factor, the mRNA having pseudouridine in place of uridine.
• the composition comprises mRNA encoding a plurality of iPS cell induction factors, selected from the group consisting of KLF4, LIN28, c-MYC, NANOG, OCT4, and SOX2.
• the plurality comprises three or more, or four or more, or five or more, or six.
• compositions described above are packaged in a kit.
• the compositions comprise a transfection reagent and an mRNA encoding an iPS cell induction factor.
• Figure 1 shows that mR As encoding each of the six human reprogramming factors, prepared as described in the EXAMPLES, are expressed when transfected into human newborn 1079 fibroblasts.
• Phase contrast images of the human 1079 fibroblasts which were not transfected with an mR A encoding a reprogramming factor (i.e., untreated) and which were not stained with a labeled antibody specific for a reprogramming factor are shown in A, E, I, M, Q, and U.
• Phase contrast images of untreated human 1079 fibroblasts which were stained using a labeled antibody specific for the indicated reprogramming factor show the endogenous expression of that respective reprogramming factor protein in B, F, J, N, R, and V.
• Phase contrast images of the human 1079 fibroblasts which were transfected with an mRNA encoding the indicated reprogramming factor (i.e., treated or transfected), but which were not stained with a labeled antibody specific for a reprogramming factor are shown in C, G, K, O, S, and W; and the corresponding images of the human 1079 fibroblasts that were transfected with mRNA encoding the indicated reprogramming factor and then stained with respective labeled antibody specific for that reprogramming factor 24 hours post-transfection are shown in D, H, L, P, T, and X.
• A-T are at 20x magnification.
• U-X are at lOx magnification.
• Figure 2 shows that mRNA encoding human reprogramming factors (KLF4, LIN28, c- MYC, NANOG, OCT4, and SOX2) produce iPS cells in human somatic cells.
• Figure 2 shows phase contrast images of an iPS cell colony at 12 days after the final transfection with mRNA encoding reprogramming factors (A, C). NANOG staining (green) is observed in colony #1 (B, D). Images A and B are at lOx magnification. C and D are at 20x magnification.
• Figure 3 shows that iPS colonies derived from human 1079 and IMR90 somatic cells are positive for NANOG and TRA-1-60.
• Figure 3 shows phase contrast images of iPS colonies derived from 1079 cells (A, D) and IMR90 cells (G). The same iPS colony shown in (A) is positive for both NANOG (B) and TRA-1-60 (C). The iPS colony shown in (D) is NANOG- positive (E) and TRA-l-60-positive (F). The iPS colony generated from IMR90 fibroblasts (G) is also positive for both NANOG (H) and TRA-1-60 (I). All images are at 20x magnification.
• Figure 4 shows that rapid, enhanced-efficiency iPSC colony formation is achieved by transfecting cells with mRNA encoding reprogramming factors in MEF-conditioned medium. Over 200 colonies were detected 3 days after the final transfection, in the 10-cm dish transfected three times with 36 ⁇ g of each reprogramming mRNA (i.e., encoding KLF4, LIN28, c-MYC, NANOG, OCT4, and SOX2). Representative iPSC colonies are shown at 4x (A, B), lOx (C-E) and 20x magnification (F).
• NANOG and TRA-1-60 expression Eight days after the final mRNA transfection with 36 ⁇ g of mRNA for each of the six reprogramming factors, the 1079-derived iPSC colonies (shown in A, D, and G) are positive for NANOG (B, E, and H) and TRA-1-60 (C, F, and I). Eight days after the final mRNA transfection with 18 ⁇ g (J-L) or 36 ⁇ g (M-O) of mRNA for each of the six reprogramming factors, IMR90-derived iPS colonies are also positive for NANOG (K, N) and TRA-1-60 (L, 0).
• Figure 6 provides the mRNA coding sequence for KLF4 (SEQ ID NO: l) and LIN28 (SEQ ID NO:2).
• Figure 7 provides the mRNA coding sequence for cMYC (SEQ ID NO:3) and NANOG (SEQ ID NO:4).
• Figure 8 provides the mRNA coding sequence for OCT4 (SEQ ID NO:5) and SOX2 (SEQ ID NO:6).
• derived such as for an R A (including m NA) or a polypeptide that is "derived” from a sample, biological sample, cell, tumor, or the like
• the RNA or polypeptide either was present in the sample, biological sample, cell, tumor, or the like, or was made using the RNA in the sample, biological sample, cell, tumor, or the like by a process such as an in vitro transcription reaction, or an RNA amplification reaction, wherein the RNA or polypeptide is either encoded by or a copy of all or a portion of the RNA or polypeptide molecules in the original sample, biological sample, cell, tumor, or the like.
• such RNA can be from an in vitro transcription or an RNA amplification reaction, with or without cloning of cDNA, rather than being obtained directly from the sample, biological sample, cell, tumor, or the like, so long as the original RNA used for the in vitro transcription or an RNA amplification reaction was from the sample, biological sample, cell, tumor, or the like.
• sample and “biological sample” are used in their broadest sense and encompass samples or specimens obtained from any source that contains or may contain eukaryotic cells, including biological and environmental sources.
• sample when used to refer to biological samples obtained from organisms, includes bodily fluids (e.g., blood or saliva), feces, biopsies, swabs (e.g., buccal swabs), isolated cells, exudates, and the like.
• the organisms include fungi, plants, animals, and humans. However, these examples are not to be construed as limiting the types of samples or organisms that find use with the present invention.
• a “sample” or “biological sample” comprises fixed cells, treated cells, cell lysates, and the like.
• the "sample” or “biological sample” also comprises bacteria or viruses.
• the term "incubating" and variants thereof mean contacting one or more components of a reaction with another component or components, under conditions and for sufficient time such that a desired reaction product is formed.
• nucleoside refers to a molecule consisting of a guanine (G), adenine (A), thymine (T), uridine (U), pseudouridine (abbreviated by the Greek letter psi- ⁇ ), or cytidine (C) base covalently linked to a pentose sugar
• nucleotide or “mononucleotide” refers to a nucleoside phosphorylated at one of the hydroxyl groups of the pentose sugar.
• Linear nucleic acid molecules are said to have a "5' terminus” (5' end) and a "3' terminus” (3' end) because, except with respect to adenylation (as described elsewhere herein), mononucleotides are joined in one direction via a phosphodiester linkage to make oligonucleotides, in a manner such that a phosphate on the 5' carbon of one mononucleotide sugar moiety is joined to an oxygen on the 3' carbon of the sugar moiety of its neighboring mononucleotide.
• an end of an oligonucleotide is referred to as the "5' end” if its 5' phosphate is not linked to the oxygen of the 3' carbon of a mononucleotide sugar moiety, and as the "3' end” if its 3' oxygen is not linked to a 5' phosphate of a subsequent mononucleotide sugar moiety.
• a terminal nucleotide is the nucleotide at the end position of the 3' or 5' terminus.
• a nucleic acid base, sugar moiety, or internucleoside linkage in one or more of the nucleotides of the mRNA that is introduced into a eukaryotic cell in any of the methods of the invention may comprise a modified base, sugar moiety, or internucleoside linkage.
• one or more of the nucleotides of the mRNA can have a modified nucleic acid base comprising or consisting of: xanthine; allyamino-uracil; allyamino-thymidine; hypoxanthine; 2-aminoadenine; 5-propynyl uracil; 5-propynyl cytosine; 4- thiouracil; 6-thioguanine; an aza or deaza uracil; an aza or deaza thymidine; an aza or deaza cytosines; an aza or deaza adenine; or an aza or deaza guanines; or a nucleic acid base that is derivatized with a biotin moiety, a digoxigenin moiety, a fluorescent or chemiluminescent moiety, a quenching moiety or some other moiety.
• a modified nucleic acid base comprising or consisting of: xanthine; allyamino-ura
• one or more of the nucleotides of the mRNA can have a sugar moiety, such as, but not limited to: 2'-fluoro-2'-deoxyribose or 2'-0- methyl-ribose, which provide resistance to some nucleases; or 2'-amino-2'-deoxyribose or 2'- azido-2'-deoxyribose, which can be labeled by reacting them with visible, fluorescent, infrared fluorescent or other detectable dyes or chemicals having an electrophilic, photoreactive, alkynyl, or other reactive chemical moiety.
• a sugar moiety such as, but not limited to: 2'-fluoro-2'-deoxyribose or 2'-0- methyl-ribose, which provide resistance to some nucleases; or 2'-amino-2'-deoxyribose or 2'- azido-2'-deoxyribose, which can be labeled by reacting them with visible, fluorescent,
• one or more of the nucleotides of the mRNA can have a modified internucleoside linkage, such as, but not limited to, a phosphorothioate, phosphorodithioate, phosphoroselenate, or phosphorodiselenate linkage, which are resistant to some nucleases.
• a modified internucleoside linkage such as, but not limited to, a phosphorothioate, phosphorodithioate, phosphoroselenate, or phosphorodiselenate linkage, which are resistant to some nucleases.
• the invention is not limited to the modified nucleic acid bases, sugar moieties, or internucleoside linkages listed, but the list is presented to show examples which may be used for a particular purpose in a method.
• nucleic acid or a “polynucleotide” is a covalently linked sequence of nucleotides in which the 3' position of the sugar moiety of one nucleotide is joined by a phosphodiester bond to the 5' position of the sugar moiety of the next nucleotide (i.e., a 3' to 5' phosphodiester bond), and in which the nucleotides are linked in specific sequence; i.e., a linear order of nucleotides.
• an "oligonucleotide” is a short polynucleotide or a portion of a polynucleotide.
• an oligonucleotide may be between 10- 60 nucleotides in length.
• the oligonucleotide consists of or comprises 2'- deoxyribonucleotides (DNA).
• the oligonucleotide consists of or comprises ribonucleotides (RNA).
• isolated or purified when used in relation to a polynucleotide or nucleic acid, as in “isolated RNA” or “purified RNA” refers to a nucleic acid that is identified and separated from at least one contaminant with which it is ordinarily associated in its source.
• an isolated or purified nucleic acid e.g., DNA and RNA
• a given DNA sequence e.g., a gene
• a specific RNA e.g., a specific mRNA encoding a specific protein
• the isolated or purified polynucleotide or nucleic acid may be present in single-stranded or double- stranded form.
• a “cap” or a “cap nucleotide” means a nucleoside-5 '-triphosphate that, under suitable reaction conditions, is used as a substrate by a capping enzyme system and that is thereby joined to the 5 '-end of an uncapped RNA comprising primary RNA transcripts or RNA having a 5'- diphosphate.
• the nucleotide that is so joined to the RNA is also referred to as a "cap nucleotide” herein.
• a “cap nucleotide” is a guanine nucleotide that is joined through its 5' end to the 5' end of a primary RNA transcript.
• RNA that has the cap nucleotide joined to its 5' end is referred to as "capped RNA” or “capped RNA transcript” or “capped transcript.”
• a common cap nucleoside is 7-methylguanosine or N -methylguanosine (sometimes referred to as “standard cap”), which has a structure designated as "m 7 G,” in which case the capped RNA or “m 7 G-capped RNA” has a structure designated as m 7 G(5')ppp(5')Ni(pN) x -OH(3'), or more simply, as m 7 GpppNi(pN) x or m 7 G[5']ppp[5']N, wherein m 7 G represents the 7-methylguanosine cap nucleoside, ppp represents the triphosphate bridge between the 5' carbons of the cap nucleoside and the first nucleotide of the primary RNA transcript, ⁇ ( ⁇ ) ⁇ - ⁇ (3') represents the primary RNA
• RNA that has any cap nucleotide is referred to as "capped RNA.”
• the capped RNA can be naturally occurring from a biological sample or it can be obtained by in vitro capping of RNA that has a 5' triphosphate group or RNA that has a 5' diphosphate group with a capping enzyme system (e.g., vaccinia capping enzyme system or Saccharomyces cerevisiae capping enzyme system).
• a capping enzyme system e.g., vaccinia capping enzyme system or Saccharomyces cerevisiae capping enzyme system.
• the capped RNA can be obtained by in vitro transcription (IVT) of a DNA template that contains an RNA polymerase promoter, wherein, in addition to the GTP, the IVT reaction also contains a dinucleotide cap analog (e.g., a m 7 GpppG cap analog or an N 7 -methyl, 2'-0-methyl-GpppG ARC A cap analog or an N 7 -methyl, 3'-0-methyl-GpppG ARC A cap analog) using methods known in the art (e.g., using an AMPLICAPTM T7 capping kit (EPICENTRE)).
• IVTT in vitro transcription
• Capping of a 5'-triphosphorylated primary mRNA transcript in vivo occurs via several enzymatic steps (Higman et al. 1992, Martin et al. 1975, Myette and Niles 1996).
• RNA triphosphatase cleaves the 5 '-triphosphate of mRNA to a diphosphate, ⁇ ( ⁇ ) ⁇ ⁇ - ⁇ (3') ⁇ ⁇ ( ⁇ ) ⁇ - ⁇ (3') + Pi; and then
• RNA guanyltransferase catalyzes joining of GTP to the 5 '-diphosphate of the most 5' nucleotide (Ni) of the mRNA, ppNi(pN) x -OH(3 * ) + GTP ⁇ G(5 * )ppp(5 * )Ni(pN) x -OH(3 * ) + PPi; and finally,
• guanine-7-methyltransferase using S-adenosyl-methionine (AdoMet) as a co-factor, catalyzes methylation of the 7-nitrogen of guanine in the cap nucleotide,
• RNA that results from the action of the RNA triphosphatase and the RNA guanyltransferase enzymatic activities, as well as RNA that is additionally methylated by the guanine-7-methyltransferase enzymatic activity is referred to herein as "5' capped RNA” or “capped RNA”, and a “capping enzyme system” or, more simply, a “capping enzyme” herein means any combination of one or more polypeptides having the enzymatic activities that result in "capped RNA.”
• Capping enzyme systems including cloned forms of such enzymes, have been identified and purified from many sources and are well known in the art (Banerjee 1980, Higman et al.
• capping enzyme system that can convert uncapped RNA that has a 5' polyphosphate to capped RNA can be used to provide a capped RNA for any of the embodiments of the present invention.
• the capping enzyme system is a poxvirus capping enzyme system.
• the capping enzyme system is vaccinia virus capping enzyme.
• the capping enzyme system is Saccharomyces cerevisiae capping enzyme.
• the capping enzyme system can originate from one source, or one or more of the RNA triphosphatase, RNA guanyltransferase, and/or guanine-7-methyltransferase activities can comprise a polypeptide from a different source.
• a “modified cap nucleotide” of the present invention means a cap nucleotide wherein the sugar, the nucleic acid base, or the internucleoside linkage is chemically modified compared to the corresponding canonical 7-methylguanosine cap nucleotide.
• a modified cap nucleotide examples include a cap nucleotide comprising: (i) a modified 2'- or 3'- deoxyguanosine-5'- triphosphate (or guanine 2'- or 3'- deoxyribonucleic acid-5 '-triphosphate) wherein the 2'- or 3'- deoxy position of the deoxyribose sugar moiety is substituted with a group comprising an amino group, an azido group, a fluorine group, a methoxy group, a thiol (or mercapto) group or a methylthio (or methylmercapto) group; or (ii) a modified guanosine-5 '-triphosphate, wherein the 06 oxygen of the guanine base is substituted with a methyl group; or (iii) 3'-deoxyguanosine.
• an "alkoxy-substituted deoxyguanosine- 5'-triphosphate” can also be referred to as an "O-alkyl-substituted guanosine-5'-triphosphate”; by way of example, but without limitation, 2'-methoxy-2'-deoxyguanosine-5 '-triphosphate (2 - methoxy-2'-dGTP) and 3 '-methoxy-3'-deoxy guanosine-5 '-triphosphate (3'-methoxy-3'-dGTP) can also be referred to herein as 2'-0-methylguanosine-5 '-triphosphate (2'-OMe-GTP) and 3'-0- methylguanosine-5 '-triphosphate (3'-OMe-GTP), respectively.
• the portion of said modified cap nucleotide that is joined to the uncapped RNA comprising primary RNA transcripts may be referred to herein as a "modified cap nucleoside" (i.e., without referring to the phosphate groups to which it is joined), but sometimes it is referred to as a "modified cap nucleotide".
• a “modified-nucleotide-capped RNA” is a capped RNA molecule that is synthesized using a capping enzyme system and a modified cap nucleotide, wherein the cap nucleotide on its 5' terminus comprises the modified cap nucleotide, or a capped RNA that is synthesize co- transcriptionally in an in vitro transcription reaction that contains a modified dinucleotide cap analog wherein the dinucleotide cap analog contains the chemical modification in the cap nucleotide.
• the modified dinucleotide cap analog is an anti-reverse cap analog or ARCA (Grudzien et al. 2004, Grudzien-Nogalska et al. 2007, Jemielity et al. 2003, Peng et al. 2002, Stepinski et al. 2001).
• a “primary RNA” or “primary RNA transcript” means an RNA molecule that is synthesized by an RNA polymerase in vivo or in vitro and which RNA molecule has a triphosphate on the 5'-carbon of its most 5' nucleotide.
• RNA amplification reaction or an “RNA amplification method” means a method for increasing the amount of RNA corresponding to one or multiple desired RNA sequences in a sample.
• the RNA amplification method comprises: (a) synthesizing first-strand cDNA complementary to the one or more desired RNA molecules by RNA-dependent DNA polymerase extension of one or more primers that anneal to the desired RNA molecules; (b) synthesizing double-stranded cDNA from the first-strand cDNA using a process wherein a functional RNA polymerase promoter is joined thereto; and (c) contacting the double-stranded cDNA with an RNA polymerase that binds to said promoter under transcription conditions whereby RNA corresponding to the one or more desired RNA molecules is obtained.
• an RNA amplification reaction means a sense RNA amplification reaction, meaning an RNA amplification reaction that synthesizes sense RNA (e.g., RNA having the same sequence as an mRNA or other primary RNA transcript, rather than the complement of that sequence).
• Sense RNA amplification reactions known in the art which are encompassed within this definition include, but are not limited to, the methods which synthesize sense RNA described in Ozawa et al. (Ozawa et al. 2006) and in U.S. Patent Application Nos. 20090053775; 20050153333; 20030186237; 20040197802; and 20040171041.
• RNA amplification method described in U.S. Patent Application No. 20090053775 is a preferred method for obtaining amplified RNA derived from one or more cells, which amplified RNA is then used to make mRNA for use in the methods of the present invention.
• PAP poly-A polymerase
• a "reprogramming factor” means a protein, peptide, or other biomolecule that, when used alone or in combination with other factors or conditions, causes a change in the state of differentiation of a cell in which the reprogramming factor is introduced or expressed.
• the reprogramming factor is a protein or peptide that is encoded by an mRNA that is introduced into a cell, thereby generating a cell that exhibits a changed state of differentiation compared to the cell in which the mRNA was introduced.
• the reprogramming factor is a transcription factor.
• One embodiment of a reprogramming factor used in a method of the present invention is an "iPS induction factor.”
• iPS cell induction factor is a protein, peptide, or other biomolecule that, when used alone or in combination with other dedifferentiation factors, causes the generation of iPS cells from somatic cells.
• iPS cell induction factors include OCT4, SOX2, c-MYC, KLF4, NANOG and LIN28.
• iPS cell induction factors include full length polypeptide sequences or biologically active fragments thereof.
• an mR A encoding an iPS cell induction factor may encode a full length polypeptide or biologically active fragments thereof.
• the present invention employs the sequences or similar sequences shown in these figures, including mRNA molecules that additionally comprise, joined to these mRNA sequences, oligoribonucleotides which exhibit any of the 5' and 3' UTR sequences, Kozak sequences, IRES sequences, cap nucleotides, and/or poly(A) sequences used in the experiments described herein, or which are generally known in the art and which can be used in place of those used herein by joining them to these protein-coding mRNA sequences for the purpose of optimizing translation of the respective mRNA molecules in the cells and improving their stability in the cell in order to accomplish the methods described herein.
• “Differentiation” or “cellular differentiation” means the process by which a cell that exhibits a less specialized state of differentiation or cell type becomes a cell that exhibits a more specialized state of differentiation or cell type.
• a cell is defined, described, or categorized with respect to its "cell type,” “differentiated state,” or “state of differentiation” based on one or more phenotypes exhibited by that cell, which phenotypes can include shape, a biochemical or metabolic activity or function, the presence of certain biomolecules in the cell (e.g., based on stains that react with specific biomolecules), or on the cell (e.g., based on binding of one or more antibodies that react with specific biomolecules on the cell surface).
• different cell types are identified and
• Dedifferentiation means the process by which a cell that exhibits a more specialized state of differentiation or cell type becomes a cell that exhibits a less specialized state of differentiation or cell type.
• a differentiated somatic cell e.g., a mammalian fibroblast
• iPS cell e.g., a mammalian fibroblast
• the present invention relates to compositions and methods for reprogramming somatic cells to pluripotent stem cells.
• the present invention provides mRNA molecules and their use to reprogram human somatic cells into pluripotent stem cells.
• mRNA molecules can be administered to cells and induce a dedifferentiation process to generate dedifferentiated cells—including pluripotent stem cells.
• the present invention provides compositions and methods for generating iPS cells.
• the administration of mRNA can provide highly efficient generation of iPS cells.
• the present invention provides methods for dedifferentiating a somatic cell comprising: introducing mRNA encoding one or more iPSC induction factors into a somatic cell to generate a dedifferentiated cell.
• the present invention provides methods for dedifferentiating a somatic cell comprising: introducing mRNA encoding one or more iPSC induction factors into a somatic cell and maintaining the cell under conditions wherein the cell is viable and the mRNA that is introduced into the cell is expressed in sufficient amount and for sufficient time to generate a dedifferentiated cell.
• the dedifferentiated cell is an induced pluripotent stem cell (iPSC).
• the present invention provides methods for changing the state of differentiation (or differentiated state) of a eukaryotic cell comprising: introducing mRNA encoding one or more reprogramming factors into a cell and maintaining the cell under conditions wherein the cell is viable and the mRNA that is introduced into the cell is expressed in sufficient amount and for sufficient time to generate a cell that exhibits a changed state of differentiation compared to the cell into which the mRNA was introduced.
• the present invention provides methods for changing the state of differentiation of a eukaryotic cell comprising: introducing mRNA encoding one or more reprogramming factors into a cell and maintaining the cell under conditions wherein the cell is viable and the mRNA that is introduced into the cell is expressed in sufficient amount and for sufficient time to generate a cell that exhibits a changed state of differentiation compared to the cell into which the mRNA was introduced.
• the changed state of differentiation is a dedifferentiated state of differentiation compared to the cell into which the mRNA was introduced.
• the cell that exhibits the changed state of differentiation is a pluripotent stem cell that is dedifferentiated compared to a somatic cell into which the mRNA was introduced (e.g., a somatic cell that is differentiated into a fibroblast, a cardomyocyte, or another differentiated cell type).
• a somatic cell into which the mRNA was introduced e.g., a somatic cell that is differentiated into a fibroblast, a cardomyocyte, or another differentiated cell type.
• the cell into which the mRNA is introduced is a somatic cell of one lineage, phenotype, or function
• the cell that exhibits the changed state of differentiation is a somatic cell that exhibits a lineage, phenotype, or function that is different than that of the cell into which the mRNA was introduced; thus, in these embodiments, the method results in transdifferentiation (Graf and Enver 2009).
• the methods of the invention are not limited with respect to a particular cell into which the mRNA is introduced.
• the cell into which the mRNA is introduced is derived from any multi-cellular eukaryote.
• the cell into which the mRNA is introduced is selected from among a human cell, an animal cell, a plant cell, and a fungal cell.
• the cell into which the mRNA is introduced is a normal cell that is from an organism that is free of a known disease.
• the cell into which the mRNA is introduced is a cell from an organism that has a known disease.
• the cell into which the mRNA is introduced is a cell that is free of a known pathology. In some embodiments of any of the above methods, the cell into which the mRNA is introduced is a cell that exhibits a disease state or a known pathology (e.g., a cancer cell, or a pancreatic beta cell that exhibits metabolic properties characteristic of a diabetic cell).
• a disease state or a known pathology e.g., a cancer cell, or a pancreatic beta cell that exhibits metabolic properties characteristic of a diabetic cell.
• the invention is not limited to the use of a specific cell type (e.g., to a specific somatic cell type) in embodiments of the methods comprising introducing mRNA encoding one or more iPSC cell induction factors in order to generate a dedifferentiated cell (e.g., an iPS cell).
• a dedifferentiated cell e.g., an iPS cell.
• Any cell that is subject to dedifferentiation using iPS cell induction factors is contemplated.
• Such cells include, but are not limited to, fibroblasts, keratinocytes, adipocytes, lymphocytes, T-cells, B-Cells, cells in mononuclear cord blood, buccal mucosa cells, hepatic cells, HeLa, MCF-7 or other cancer cells.
• the cells reside in vitro (e.g., in culture) or in vivo.
• a cell-free conditioned medium e.g., MEF- conditioned medium
• a feeder cell layer is used instead of conditioned medium for culturing the cells that are treated using the method.
• the step of introducing mRNA comprises delivering the mRNA into the cell (e.g., a human or other animal somatic cell) with a transfection reagent (e.g., TRANSITTM mRNA transfection reagent, MirusBio, Madison, WI).
• a transfection reagent e.g., TRANSITTM mRNA transfection reagent, MirusBio, Madison, WI.
• the invention is not limited by the nature of the transfection method utilized.
• the transfection reagent comprises a lipid (e.g., liposomes, micelles, etc.). In some embodiments, the transfection reagent comprises a nanoparticle or nanotube.
• the transfection reagent comprises a cationic compound (e.g., polyethylene imine or PEI).
• the transfection method uses an electric current to deliver the mRNA into the cell (e.g., by electroporation).
• a total of three doses with each dose comprising 18 micrograms of each of six different mRNAs, each encoding a different human reprogramming factor, was used to introduce the mRNA into approximately 3 x 10 5 human fibroblast cells in a 10-cm plate (e.g., delivered using a lipid-containing transfection reagent), although in other embodiments, higher or lower amounts of the mRNAs were used to introduce into the cells.
• the invention is not limited to a particular chemical form of the mRNA used, although certain forms of mRNA may produce more efficient results.
• the mRNA is polyadenylated.
• the mRNA comprises a poly-A tail (e.g., a poly-A tail having 50-200 nucleotides, e.g., preferably 100-200, 150-200 nucleotides, or greater than 150 nucleotides), although in some embodiments, a longer or a shorter poly-A tail is used.
• the mRNA used in the methods is capped. To maximize efficiency of expression in the cells, it is preferred that the majority of mRNA molecules contain a cap.
• the mRNA molecules used in the methods are synthesized in vitro by incubating uncapped primary RNA in the presence of with a capping enzyme system.
• the primary RNA used in the capping enzyme reaction is synthesized by in vitro transcription (IVT) of a DNA molecule that encodes the RNA to be synthesized.
• the DNA that encodes the RNA to be synthesized is joined to an RNA polymerase promoter, to which, an RNA polymerase binds and initiates transcription therefrom.
• the IVT can be performed using any RNA polymerase so long as synthesis of the template that encodes the RNA is specifically and sufficiently initiated from a respective cognate RNA polymerase promoter.
• the RNA polymerase is selected from among T7 RNA polymerase, SP6 RNA polymerase and T3 RNA polymerase.
• capped RNA is synthesized co-transcriptionally by using a dinucleotide cap analog in the IVT reaction (e.g., using an AMPLICAPTM T7 Kit; EPICENTRE Technologies Corporation, Madison, WI).
• a dinucleotide cap analog in the IVT reaction
• capping enzyme system which results in approximately 100% of the RNA being capped
• co-transcriptional capping typically results in only about 80% of the RNA being capped.
• a high percentage of the mRNA molecules used in a method of the present invention are capped (e.g., greater than 80%>, greater than 90%>, greater than 95%, greater than 98%, greater than 99%, greater than 99.5%, or greater than 99.9% of the population of mRNA molecules are capped).
• the mRNA used in the methods of the present invention has a cap with a capl structure, meaning that the penultimate nucleotide with respect to the cap nucleotide has a methyl group on the 2 '-position of the ribose.
• mRNA used in the methods has a cap with a capO structure, meaning that the penultimate nucleotide with respect to the cap nucleotide does not have a methyl group on the 2'-position of the ribose.
• transfection of eukaryotic cells with mRNA having a cap with a capl structure results in a higher level or longer duration of protein expression in the transfected cells compared to transfection of the same cells with the same mRNA but with a cap having a capO structure.
• the mRNA used in the methods of the present invention has a modified cap nucleotide.
• the present Applicants found that, when 1079 or IMR90 human fibroblast cells were transfected with OCT4 mRNA that contained either uridine or pseudouridine in place of uridine, the pseudouridine-containing mRNA was expressed at a higher level or for a longer duration than the mRNA that contained uridine. Therefore, in some preferred embodiments, one or more or all of the uridines contained in the mRNA(s) used in the methods of the present invention is/are replaced by pseudouridine (e.g., by substituting pseudouridine-5 ' -triphosphate in the IVT reaction to synthesize the RNA in place of uridine-5 '-triphosphate).
• pseudouridine e.g., by substituting pseudouridine-5 ' -triphosphate in the IVT reaction to synthesize the RNA in place of uridine-5 '-triphosphate.
• the mRNA used in the methods of the invention contains uridine and does not contain pseudouridine.
• a nucleic acid base, sugar moiety, or internucleoside linkage in one or more of the nucleotides of the mRNA that is introduced into a eukaryotic cell in any of the methods of the invention may comprise a modified nucleic acid base, sugar moiety, or internucleoside linkage.
• the invention is also not limited with respect to the source of the mRNA that is delivered into the eukaryotic cell in any of the methods of the invention.
• the mRNA is synthesized in vitro by transcription of a DNA template comprising a gene cloned in a linearized plasmid vector or a PCR or RT-PCR amplification product, capping using a capping enzyme system, and polyadenylation using a poly-A polymerase.
• the mRNA that is delivered into the eukaryotic cell in any of the methods of the invention is derived directly from a cell or a biological sample.
• the mRNA derived from a cell or biological sample is obtained by amplifying the mRNA from the cell or biological sample using an RNA amplification reaction.
• the invention is not limited by the nature of the iPS cell induction factors used. Any mRNA encoding one or more protein induction factors now known, or later discovered, that find use in dedifferentiation, are contemplated for use in the present invention. In some embodiments, one or more mRNAs encoding for KLF4, LIN28, c-MYC, NANOG, OCT4, or SOX2 are employed.
• Soxl, Sox2, Sox3, and Sox 15 have been identified as transcriptional regulators involved in the induction process. Additional genes, however, including certain members of the Klf family (Klfl, Klf2, Klf , and Klf5), the Myc family (C- myc, L-myc, and N-myc), Nanog, and LIN28, have been identified to increase the induction efficiency. Any one or more such factors may be used as desired.
• compositions and methods of the invention may be used to generated iPS cells, the invention is not limited to the generation of such cells.
• mRNA encoding one or more reprogramming factors is introduced into a cell in order to generate a cell with a changed state of differentiation compared to the cell into which the mRNA was introduced.
• mRNA encoding one or more iPS cell induction factors is used to generate a dedifferentiated cell that is not an iPS cells.
• Such cells find use in research, drug screening, and other applications.
• the present invention further provides methods employing the dedifferentiated cells generated by the above methods. For example, such cells find use in research, drug screening, and therapeutic applications in humans or other animals.
• the cells generated find use in the identification and characterization of iPS cell induction factors as well as other factors associated with differentiation or dedifferentiation.
• the generated dedifferentiated cells are transplanted into an organism or into a tissue residing in vitro or in vivo.
• an organism, tissue, or culture system housing the generated cells is exposed to a test compound and the effect of the test compound on the cells or on the organism, tissue, or culture system is observed or measured.
• a dedifferentiated cell generated using the above methods is further treated to generate a differentiated cell that has the same state of differentiation or cell type compared to the somatic cell from which the dedifferentiated cell was generated.
• the dedifferentiated cell generated using the above methods is further treated to generate a differentiated cell that has a different state of differentiation or cell type compared to the somatic cell from which the dedifferentiated cell was generated.
• the differentiated cell is generated from the generated dedifferentiated cell (e.g., the generated iPS cell) by introducing mRNA encoding one or more reprogramming factors into the generated iPS cell and maintaining the cell into which the mRNA is introduced under conditions wherein the cell is viable and is differentiated into a cell that has a changed state of differentiation or cell type compared to the generated dedifferentiated cell (e.g., the generated iPS cell) into which the mRNA encoding the one or more reprogramming factors is introduced.
• the generated differentiated cell that has the changed state of differentiation is used for research, drug screening, or therapeutic applications (e.g., in humans or other animals).
• the generated differentiated cells find use in the identification and characterization of reprogramming factors associated with differentiation.
• the generated differentiated cells are transplanted into an organism or into a tissue residing in vitro or in vivo.
• an organism, tissue, or culture system housing the generated differentiated cells is exposed to a test compound and the effect of the test compound on the cells or on the organism, tissue, or culture system is observed or measured.
• the method comprising introducing mRNA encoding one or more iPSC induction factors into a somatic cell and maintaining the cell under conditions wherein the cell is viable and the mRNA that is introduced into the cell is expressed in sufficient amount and for sufficient time to generate a dedifferentiated cell (e.g., wherein the dedifferentiated cell is an induced pluripotent stem cell), the sufficient time to generate a dedifferentiated cell is less than one week.
• the reprogramming efficiency for generating dedifferentiated cells is greater than or equal to 50 dedifferentiated cells (e.g., iPSCs) per 3 x 10 5 input cells into which the mRNA is introduced.
• the reprogramming efficiency for generating dedifferentiated cells is greater than or equal to 100 dedifferentiated cells (e.g., iPSCs) per 3 x 10 5 input cells into which the mRNA is introduced. In some preferred embodiments of this method, the reprogramming efficiency for generating dedifferentiated cells is greater than or equal to 150 dedifferentiated cells (e.g., iPSCs) per 3 x 10 5 input cells into which the mRNA is introduced. In some preferred embodiments of this method, the reprogramming efficiency for generating dedifferentiated cells is greater than or equal to 200 dedifferentiated cells (e.g., iPSCs) per 3 x 10 5 input cells into which the mRNA is introduced.
• 100 dedifferentiated cells e.g., iPSCs
• 150 dedifferentiated cells e.g., iPSCs
• the reprogramming efficiency for generating dedifferentiated cells is greater than or equal to 200 dedifferentiated cells (e.g.
• the reprogramming efficiency for generating dedifferentiated cells is greater than or equal to 300 dedifferentiated cells (e.g., iPSCs) per 3 x 10 5 input cells into which the mRNA is introduced. In some preferred embodiments of this method, the reprogramming efficiency for generating dedifferentiated cells is greater than or equal to 400 dedifferentiated cells (e.g., iPSCs) per 3 x 10 5 input cells into which the mRNA is introduced. In some preferred embodiments of this method, the reprogramming efficiency for generating dedifferentiated cells is greater than or equal to 500 dedifferentiated cells (e.g., iPSCs) per 3 x 10 5 input cells into which the mRNA is introduced.
• 300 dedifferentiated cells e.g., iPSCs
• 400 dedifferentiated cells e.g., iPSCs
• the reprogramming efficiency for generating dedifferentiated cells is greater than or equal to 500 dedifferentiated cells (e.g.
• the reprogramming efficiency for generating dedifferentiated cells is greater than or equal to 600 dedifferentiated cells per 3 x 10 5 input cells (e.g., iPSCs) into which the mRNA is introduced. In some preferred embodiments of this method, the reprogramming efficiency for generating dedifferentiated cells is greater than or equal to 700 dedifferentiated cells (e.g., iPSCs) per 3 x 10 5 input cells into which the mRNA is introduced.
• the reprogramming efficiency for generating dedifferentiated cells is greater than or equal to 800 dedifferentiated cells (e.g., iPSCs) per 3 x 10 5 input cells into which the mR A is introduced. In some preferred embodiments of this method, the reprogramming efficiency for generating dedifferentiated cells is greater than or equal to 900 dedifferentiated cells (e.g., iPSCs) per 3 x 10 5 input cells into which the mRNA is introduced.
• the reprogramming efficiency for generating dedifferentiated cells is greater than or equal to 1000 dedifferentiated cells (e.g., iPSCs) per 3 x 10 5 input cells into which the mRNA is introduced.
• this method was greater than 2-fold more efficient than the published protocol comprising delivery of reprogramming factors with a viral vector (e.g., a lentivirus vector).
• this method was greater than 5-fold more efficient than the published protocol comprising delivery of reprogramming factors with a viral vector (e.g., a lentivirus vector).
• this method was greater than 10-fold more efficient than the published protocol comprising delivery of reprogramming factors with a viral vector (e.g., a lentivirus vector). In some preferred embodiments, this method was greater than 20-fold more efficient than the published protocol comprising delivery of reprogramming factors with a viral vector (e.g., a lentivirus vector). In some preferred embodiments, this method was greater than 25 -fold more efficient than the published protocol comprising delivery of reprogramming factors with a viral vector (e.g., a lentivirus vector). In some preferred embodiments, this method was greater than 30-fold more efficient than the published protocol comprising delivery of reprogramming factors with a viral vector (e.g., a lentivirus vector).
• this method was greater than 35-fold more efficient than the published protocol comprising delivery of reprogramming factors with a viral vector (e.g., a lentivirus vector). In some preferred embodiments, this method was greater than 40-fold more efficient than the published protocol comprising delivery of reprogramming factors with a viral vector (e.g., a lentivirus vector).
• the present invention further provides compositions (systems, kits, reaction mixtures, cells, mRNA) used or useful in the methods and/or generated by the methods described herein.
• the present invention provides an mRNA encoding an iPS cell induction factor, the mRNA having pseudouridine in place of uridine.
• the present invention further provides compositions comprising a transfection reagent and an mR A encoding an iPS cell induction factor (e.g., a mixture of transfection reagent and mRNA).
• compositions comprise mRNA encoding a plurality (e.g., 2 or more, 3 or more, 4 or more, 5 or more, or 6) of iPS cell induction factors, including, but not limited to, KLF4, LIN28, c-MYC, NANOG, OCT4, and SOX2.
• a plurality e.g., 2 or more, 3 or more, 4 or more, 5 or more, or 6
• iPS cell induction factors including, but not limited to, KLF4, LIN28, c-MYC, NANOG, OCT4, and SOX2.
• compositions may further comprise any other reagent or component sufficient, necessary, or useful for practicing any of the methods described herein.
• reagents or components include, but are not limited to, transfection reagents, culture medium (e.g., MEF- condition medium), cells (e.g., somatic cells, iPS cells), containers, boxes, buffers, inhibitors (e.g., RNase inhibitors), labels (e.g., fluorescent, luminescent, radioactive, etc.), positive and/or negative control molecules, reagents for generating capped mRNA, dry ice or other refrigerants, instructions for use, cell culture equipment, detection/analysis equipment, and the like.
• the mRNAs are purified into purified RNA preparations that have most of the contaminating RNA molecules removed (e.g., molecules that cause an immunogenic response in the cells), such as described in U.S. Application Serial Number 12/962,468 filed December 7, 2010, which is herein incorporated by reference.
• Newborn human foreskin fibroblast 1079 cells (Cat# CRL-2097, ATCC, Manassas, VA) and human IMR90 cells (Cat# CCL-186, ATCC) were cultured in Advanced MEM Medium (Invitrogen, Carlsbad, CA) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Hyclone Laboratories, Logan, UT), 2mM Glutamax (Invitrogen), O. lmM ⁇ - mercaptoethanol (Sigma, St. Louis, MO), and Penicillin/Streptomycin (Invitrogen). All cells were grown at 37°C and 5% C0 2 .
• Advanced MEM Medium Invitrogen, Carlsbad, CA
• FBS heat-inactivated fetal bovine serum
• 2mM Glutamax Invitrogen
• O. lmM ⁇ - mercaptoethanol (Sigma, St. Louis, MO)
• Penicillin/Streptomycin Invitrogen
• human iPS cells that were induced using methods described herein were maintained on irradiated mouse embryonic fibroblasts (MEFs) (R&D Systems, Minneapolis, MN) on 10-cm plates pre-coated with 0.1% gelatin (Millipore, PhiUipsburg, NJ) in DMEM/F12 medium supplemented with 20% KnockOut serum replacer, O.lmM L-glutamine (all from Invitrogen), O. lmM ⁇ -mercaptoethanol (Sigma) and lOOng/ml basic fibroblast growth factor (Invitrogen).
• human iPS cells that were induced using methods described herein were maintained in MEF-conditioned medium that had been collected as previously described (Xu et al. 2001).
• the cDNAs for the open reading frames (ORFs) of KLF4, LIN28, NANOG, and OCT4 were PCR amplified from cDNA clones (Open Biosystems, Huntsville, AL), cloned into a plasmid vector downstream of a T7 RNA polymerase promoter (Mackie 1988, Studier and Moffatt 1986) (e.g., various pBluescriptTM, Agilent, La Jolla, CA or pGEMTM, Promega, Madison, WI, vectors) and sequenced.
• ORFs open reading frames
• the ORF of SOX2 was PCR amplified from a cDNA clone (Invitrogen) and the ORF of c-MYC was isolated by RT-PCR from HeLa cell total RNA. Both SOX2 and c-MYC ORF were also cloned into a plasmid vector downstream of a T7 RNA polymerase promoter and sequenced. Alternative plasmid vectors containing human open reading frames of (KLF4, LIN28, c-
• pBluescriptll MYC, NANOG, OCT4 and SOX2 were cloned into pBluescriptll. These pBluescriptll vectors where constructed by ligating the above open reading frames into the EcoRV (cMyc) or EcoRV/Spel (KLF4, LIN28, NANOG, OCT4, and SOX2) sites between the 5' and 3' Xenopus laevis B-globin untranslated regions described (Krieg and Melton 1984). mRNA Production.
• the mSCRIPTTM mRNA production system (EPICENTRE Technologies Corporation, Madison, WI) was used to produce mRNA with a 5' Capl structure and a 3' Poly (A) tail (e.g., with approximately 150 A residues), except that pseudouridine-5 ' -triphosphate (TRILINK, San Diego, CA) was used in place of uridine-5' -triphosphate in the T7 RNA polymerase in vitro transcription reactions.
• pseudouridine-5 ' -triphosphate (TRILINK, San Diego, CA) was used in place of uridine-5' -triphosphate in the T7 RNA polymerase in vitro transcription reactions.
• Reprogramming of Human Somatic Cells on MEFs 1079 fibroblasts were plated at 1 x 10 5 cells/well of a 6-well dish pre-coated with 0.1% gelatin (Millipore) and grown overnight.
• the 1079 fibroblasts were transfected with equal amounts of each reprogramming factor mRNA (KLF4, LIN28, c-MYC, NANOG, OCT4, and SOX2) using TransIT mRNA transfection reagent (MirusBio, Madison, WI). A total of three transfections were performed, with one transfection being performed every other day, with media changes the day after the first and second transfection. The day after the third transfection, the cells were trypsinized and 3.3 x 10 5 cells were plated in 1079 medium onto 0.1% gelatin pre-coated 10-cm plate seeded with 7.5 x 10 5 MEFs the day before.
• KLF4, LIN28, c-MYC, NANOG, OCT4, and SOX2 TransIT mRNA transfection reagent
• 1079 or IMR90 fibroblasts were plated at 3 x 104 cells/well of a 6 well dish seeded with MEFs the previous day and grown overnight at 37°C/5%C0 2 .
• the mScript Kit was then used to generate Capl/poly-adenylated mRNA from the following vectors (pT7-X g-KLF4, pT7-X g-LIN28, pT7-X g-c-MYC, pT7-Xpg-NANOG, pT7-X g-OCT4, and pT7-X g-SOX2) for use in these daily transfections. All six reprogramming mRNAs were diluted to 100 ng/ ⁇ of each mRNA.
• Equal molarity of each mRNA was added together using the following conversion factors (OCT4 is set at 1 and all of the other mRNAs are multiplied by these conversion factors to obtain equal molarity in each mRNA mix).
• OCT4 is set at 1 and all of the other mRNAs are multiplied by these conversion factors to obtain equal molarity in each mRNA mix.
• KLF 1.32
• SOX2 0.88.
• a 600 ⁇ g total dose for transfections would mean that lOOng (using molarity conversions above) of each of six reprogramming mRNAs was used.
• Trans-IT mRNA transfection reagent was used to transfect these mRNA doses.
• mRNA pools were added to 250 ⁇ 1 of either DMEM/F12 media without additives or Advanced MEM media without additives.
• IMR90 fibroblasts were plated at 3 x 10 5 cells per 10cm dishes pre-coated with 0.1% gelatin (Millipore) and grown overnight.
• the 1079 or IMR90 fibroblasts were transfected with equal amounts of reprogramming factor mRNA (KLF4, LIN28, c-MYC, NANOG, OCT4, and SOX2) using TransIT mRNA transfection reagent (MirusBio, Madison, WI).
• reprogramming factor mRNA KLF4, LIN28, c-MYC, NANOG, OCT4, and SOX2
• TransIT mRNA transfection reagent TransIT mRNA transfection reagent
• a total of three transfections were performed, with one transfection being performed every other day with the medium being changed the day after each of the first and second transfections. All transfections were performed in MEF-conditioned medium. The day after the third transfection, the cells were trypsinized and 3 x 10 5 cells were plated on new 10-cm dishes pre-coated with 0.1% gelatin (Millipore). The cells were grown in MEF-conditioned medium for the duration of the experiment.
• the iPS cells were then washed 3 times for 5 minutes each wash with PBS followed by three washes in PBS + 0.1% Triton X-100.
• the iPS cells were then blocked in blocking buffer (PBS + 0.1% Triton, 2% FBS, and 1% BSA) for 1 hour at room temperature.
• the cells were then incubated for 2 hours at room temperature with the primary antibody (mouse anti-human OCT4 Cat# sc- 5279, Santa Cruz Biotechnology, Santa Cruz, CA), (rabbit anti-human NANOG Cat #3580, rabbit anti-human KLF4 Cat # 4038, mouse anti-human LIN28 Cat# 5930, rabbit anti-human c- MYC Cat# 5605, rabbit anti-human SOX2 Cat# 3579, and mouse anti-TRA-1-60 all from Cell Signaling Technology, Beverly, MA) at a 1 :500 dilution in blocking buffer.
• the primary antibody mouse anti-human OCT4 Cat# sc- 5279, Santa Cruz Biotechnology, Santa Cruz, CA
• rabbit anti-human NANOG Cat #3580 rabbit anti-human KLF4 Cat # 4038
• mouse anti-human LIN28 Cat# 5930 mouse anti-human c- MYC Cat# 5605
• rabbit anti-human SOX2 Cat# 3579 rabbit anti-TRA-1-60 all from Cell Signaling Technology, Beverly
• the iPS cells were incubated for 2 hours with the anti-rabbit Alexa Fluor 488 antibody (Cat # 4412, Cell Signaling Technology), anti-mouse FITC secondary (Cat# F5262, Sigma), or an anti-mouse Alexa Fluor 555 (Cat# 4409, Cell Signaling Technology) at 1 : 1000 dilutions in blocking buffer. Images were taken on a Nikon TS100F inverted microscope (Nikon, Tokyo, Japan) with a 2-megapixel monochrome digital camera (Nikon) using NIS-elements software (Nikon).
• This example describes tests to determine if transfections with mRNA encoding KLF4, LIN28, c-MYC, NANOG, OCT4 and SOX2 resulted in expression and proper subcellular localization of each respective protein product in newborn fetal foreskin 1079 fibroblasts.
• the mRNAs used in the experiments were made with pseudouridine-5 ' -triphosphate substituting for uridine-5 '-triphosphate (Kariko et al. 2008).
• the 1079 fibroblasts were transfected with 4 ⁇ g of each mRNA per well of a 6-well dish and immunofluorescence analysis was performed 24 hours post-transfection.
• this example describes development of a protocol for iPS cell generation from somatic fibroblasts. Equal amounts (by weight) of KLF4, LIN28, c-MYC, NANOG, OCT4, and SOX2 mRNAs were transfected into 1079 fibroblasts three times (once every other day). The day after the third transfection, the cells were plated onto irradiated MEF feeder cells and grown in iPS cell medium.
• NANOG expression arising from the mRNAs that were transfected 12 days earlier would be negligible based on previous reports on the duration of mRNA stability and expression (Kariko et al. 2008). Staining for NANOG showed that both of the two iPS cell colonies were NANOG positive (Fig. 2 B, D, and not shown). The surrounding fibroblasts that were not part of the iPS cell colony were NANOG negative, suggesting that they were not reprogrammed into iPS cells. In a subsequent experiment using the same protocol, both 1079 fibroblasts and human
• IMR90 fibroblasts were transfected with the same reprogramming mRNAs. Multiple colonies were detected as early as 4 days after plating the transfected cells on irradiated MEFs. When 6 ⁇ g of each mRNA (KLF4, LIN28, c-MYC, NANOG, OCT4, and SOX2) were used in transfections in 6-well dishes, 3 putative iPS cell colonies were later detected in both cell lines after plating on MEFs in 10-cm plates ( Figure 3). In addition to analyzing these colonies for expression of NANOG, TRA-1-60, a more stringent marker of fully reprogrammed iPS cells (Chan et al. 2009), was also used for immunofluorescence analysis.
• iPS colonies generated from 1079 fibroblasts (Fig. 3 A-F) and from IMR90 fibroblasts (Fig. 3 G-I) were positive for both NANOG and TRA-1-60, indicating that these colonies are fully reprogrammed type III iPS cell colonies.
• This protocol comprising three transfections of mRNAs encoding all six reprogramming factors and then plating onto MEF feeder cells resulted in a similar reprogramming efficiency (3-6 iPS colonies per 1 x 10 6 input cells) as was previously reported by protocols comprising delivery of the same reprogramming factors by transfection of an expression plasmid (Aoi et al. 2008).
• Example 3 This example describes attempts to improve the efficiency of reprogramming differentiated cells using mR A.
• a protocol was used that comprised transfecting 1079 or IMR90 fibroblasts three times (once every other day) with the mR As encoding the six reprogramming factors in MEF-conditioned medium rather than in fibroblast medium and then growing the treated 1079 fibroblasts in MEF-conditioned medium rather than plating them on a MEF feeder layer after the treatments.
• the highest transfection dose utilized 36 ⁇ g of each reprogramming factor per 10-cm dish
• 208 iPS cell colonies were detected three days after the final transfection (Figure A-F).
• this protocol was over 7-40 times more efficient than the published protocol comprising delivery of reprogramming factors with lentiviruses, based on the published data that lentiviral delivery of reprogramming factors into 1079 newborn fibroblasts, which resulted in approximately 57 iPS cell colonies per 6 x 10 5 input cells (Aoi et al. 2008). This protocol is also much faster than the published methods.
• Takahashi K Yamanaka S. 2006. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126: 663-676. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S. 2007. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131 : 861-872.

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