WO2011046635A1 - Régions méthylées de manière différente de cellules souches pluripotentes induites reprogrammées, méthode et compositions correspondantes - Google Patents

Régions méthylées de manière différente de cellules souches pluripotentes induites reprogrammées, méthode et compositions correspondantes Download PDF

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WO2011046635A1
WO2011046635A1 PCT/US2010/033281 US2010033281W WO2011046635A1 WO 2011046635 A1 WO2011046635 A1 WO 2011046635A1 US 2010033281 W US2010033281 W US 2010033281W WO 2011046635 A1 WO2011046635 A1 WO 2011046635A1
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gene
nucleic acid
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acid sequences
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Andrew Paul Feinberg
George Q. Daley
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The Johns Hopkins University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6881—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention relates generally to differentially methylated regions (DMRs) in the genome outside CpG islands in induced pluripotent stem (iPS) cells, and more specifically to methods for detecting and analyzing alterations in the methylation status of DMRs in iPS cells, somatic cells and embryonic stem (ES) cells as well as methods for reprogramming somatic cells to generate an iPS cell.
  • DMRs differentially methylated regions
  • iPS induced pluripotent stem
  • Epigenetics is the study of non-sequence information of chromosome DNA during cell division and differentiation.
  • the molecular basis of epigenetics is complex and involves modifications of the activation or inactivation of certain genes. Additionally, the chromatin proteins associated with DNA may be activated or silenced.
  • Epigenetic changes are preserved when cells divide. Most epigenetic changes only occur within the course of one individual organism's lifetime, but some epigenetic changes are inherited from one generation to the next.
  • DNA methylation a covalent modification of the nucleotide cytosine.
  • DNAm DNA methylation
  • DNA methylation involves the addition of methyl groups to cytosine nucleotides in the DNA, to convert cytosine to 5-methylcytosine.
  • DNA methylation plays an important role in determining whether some genes are expressed or not.
  • Abnormal DNA methylation is one of the mechanisms known to underlie the changes observed with aging and development of many cancers.
  • DNAm DNA methylation
  • iPS cells are derived by epigenetic reprogramming.
  • iPS cells can be derived from somatic cells by introduction of a small number of genes: for example,
  • iPS cells offer considerable therapeutic promise, avoiding both immunologic and ethical barriers to their use. iPS cells differ from their somatic parental cells epigenetically, and thus a comprehensive comparison of the epigenome in iPS and somatic cells would provide insight into the mechanism of tissue reprogramming. Although two previous targeted studies examined a subset of the genome, 7,000 (Ball et al. (Nat. Biotechnol. (27)485 (2009)) and 66,000 (Deng et al. (Nat. Biotechnol. (27)353-360 (2009))) CpG sites in a small cohort of three iPS- fibroblast pairs, a global assessment of genome-wide methylation has not yet been performed.
  • the present invention is based on the discovery that alterations in DNA
  • methylation in iPS cells as compared to both ES cells and parental fibroblasts, occur not only in promoters or CpG islands, but in sequences up to 2 kb distant from such CpG islands (such sequences are termed "CpG island shores").
  • DMR of reprogrammed iPS cells R-DMRs
  • a method of generating an iPS cell includes contacting a somatic cell with an agent that alters the methylation status of one or more nucleic acid sequences of the somatic cell, the one or more nucleic acid sequences being outside of a promoter region of a gene and outside of a CpG island, and wherein the nucleic acid sequences are up to about 2 kb in distance from a CpG island, and wherein the nucleic acid sequences are differentially methylated in reprogrammed somatic cells as compared with parent somatic cells, thereby generating an iPS cell.
  • the one or more nucleic acid sequences are any combination of DMR sequences as set forth in Tables 2, 6, 9, Figures 1B-1C, Figures 4A-4G, the BMP7 gene, the GSC gene, the TBX3 gene, the HOXD3 gene, the PTPRT gene, the POU3F4 gene, the AZBPl gene, the ZNF184 gene, the IGFIR gene, and any combination thereof.
  • the method further comprises detecting the methylation status profile of the one or more nucleic acid sequences of the induced iPS.
  • the method further comprises comparing the methylation status profile to a methylation status profile of the one or more nucleic acid sequences of a parental somatic cell from which the iPS is induced.
  • the agent is a nuclear reprogramming factor.
  • the nuclear reprogramming factor is a nucleic acid encoding a SOX family gene, a KLF family gene, a MYC family gene, POU5F1, SALL4, OCT4, NANOG, LIN28, or the expression product thereof.
  • the nuclear reprogramming factor is one or more of POU5F1, OCT4, SOX2, KLF4, or C-MYC.
  • an iPS cell produced using the methods of the invention.
  • nucleic acid sequences are outside of a promoter region of a gene and outside of a CpG island, and wherein the nucleic acid sequence is up to about 2 kb in distance from a CpG island, and wherein the nucleic acid sequences are differentially methylated in the reprogramming of a somatic cell to generate an iPS cell.
  • the nucleic acid sequence are any DMR sequences as set forth in Tables 2, 6, 9, Figures 1B-1C, Figures 4A-4G, the BMP7 gene, the GSC gene, the TBX3 gene, the HOXD3 gene, the PTPRT gene, the POU3F4 gene, the AZBPl gene, the ZNF184 gene, and the IGFIR gene.
  • the plurality of nucleic acid sequences is a microarray.
  • nucleic acid sequences are outside of a promoter region of a gene and outside of a CpG island, and wherein the nucleic acid sequences are up to about 2 kb in distance from a CpG island, and wherein the methylation status of the nucleic acid sequences is altered in an iPS cell as compared to an ES cell.
  • the nucleic acid sequence are any DMR sequences as set forth in Table 7, Figures 4C-4G, the PTPRT gene, the POU3F4 gene, the AZBP1 gene, the ZNF184 gene, and the IGF1R gene.
  • the plurality of nucleic acid sequences is a microarray.
  • a method of identifying an iPS cell includes comparing the methylation status of one or more nucleic acid sequences of a putative iPS cell, with the proviso that the one or more nucleic acid sequences are outside of a promoter region of a gene and outside of a CpG island, and wherein the nucleic acid sequences are up to about 2 kb in distance from a CpG island, to a known methylation status of the one or more nucleic acid sequences of an iPS cell, wherein a similarity in methylation status is indicative of the putative cell being an iPS cell.
  • the one or more nucleic acid sequences are DMR sequences as set forth in Tables 2, 6, 7, 9, Figures 1B-1C, Figures 4C-4G, the BMP7 gene, the GSC gene, the TBX3 gene, the HOXD3 gene, the PTPRT gene, the POU3F4 gene, the AZBP1 gene, the ZNF184 gene, the IGF1R gene, and any combination thereof.
  • a method of identifying an iPS cell includes comparing the methylation status of one or more nucleic acid sequences of a putative iPS cell, with the proviso that the one or more nucleic acid sequences are outside of a promoter region of a gene and outside of a CpG island, and wherein the nucleic acid sequences are up to about 2 kb in distance from a CpG island, to a known methylation status of the one or more nucleic acid sequences of a corresponding somatic cell from which the iPS cell is induced or ES cell, wherein an alteration in methylation status is indicative of the putative cell being an iPS cell.
  • the method further includes comparing the methylation status of the one or more nucleic acid sequences of the putative iPS cell to a known methylation status of the one or more nucleic acid sequences of a corresponding somatic cell from which the iPS cell is induced.
  • the one or more nucleic acid sequences are DMR sequences as set forth in Tables 2, 6, 9, Figures 1B- 1C, Figures 4A-4G, the the BMP7 gene, the GSC gene, the TBX3 gene, the HOXD3 gene, the PTPRT gene, the POU3F4 gene, the AZBP1 gene, the ZNF184 gene, the IGF1R gene, and any combination thereof.
  • the method further includes comparing the methylation status of the one or more nucleic acid sequences of the putative iPS cell to the methylation status of the one or more nucleic acid sequences of a known ES cell.
  • the one or more nucleic acid sequences are DMR sequences as set forth in Table 6, Figures 4C-4G, the PTPRT gene, the POU3F4 gene, the AZBP1 gene, the ZNF184 gene, the IGF1R gene, and any combination thereof.
  • the one or more nucleic acid sequences are within a gene.
  • the one or more nucleic acid sequences are upstream or downstream of a gene.
  • determination of methylation status is performed by one or more techniques selected from the group consisting of a nucleic acid amplification, polymerase chain reaction (PCR), methylation specific PCR, bisulfite pyrosequencing, single-strand conformation polymorphism (SSCP) analysis, restriction analysis, microarray technology, and proteomics.
  • PCR polymerase chain reaction
  • SSCP single-strand conformation polymorphism
  • a method for providing a methylation map of a region of genomic DNA isolated from an iPS cell includes performing comprehensive high-through array-based relative methylation
  • the method further includes performing one or more techniques, such as a nucleic acid amplification, polymerase chain reaction (PCR), methylation specific PCR, bisulfite pyrosequencing, single-strand conformation polymorphism (SSCP) analysis, and restriction analysis.
  • PCR polymerase chain reaction
  • SSCP single-strand conformation polymorphism
  • a method of characterizing the methylation status of the nucleic acid of an iPS cell includes a) hybridizing labeled and digested nucleic acid of an iPS cell to a DNA microarray comprising at least 2000 nucleic acid sequences, with the proviso that the nucleic acid sequences are outside of a promoter region of a gene and outside of a CpG island, and wherein the nucleic acid sequences are up to about 2 kb in distance from a CpG island; and b) determining a pattern of methylation from the hybridizing of (a), thereby characterizing the methylation status for the iPS cell.
  • the one or more nucleic acid sequences are DMR sequences as set forth in Tables 2, 6, 7, 9, Figures 1B-1C, Figures 4A-4G, the BMP7 gene, the GSC gene, the TBX3 gene, the HOXD3 gene, the PTPRT gene, the POU3F4 gene, the AZBP1 gene, the ZNF184 gene, the IGF1R gene and any combination thereof.
  • the method further includes comparing the methylation status profile to a methylation profile from hybridization of the microarray with labeled and digested nucleic acid from a parental somatic cell from which the iPS is induced or from an ES cell.
  • a method of treating a subject includes a) obtaining a somatic cell from a subject; b) reprogramming the somatic cell into an iPS cell using the methods of the invention; c) culturing the pluripotent stem (iPS) cell to differentiate the cell into a desired cell type suitable for treating a condition; and d) introducing into the subject the differentiated cell, thereby treating the condition.
  • iPS pluripotent stem
  • Figure 1A shows plots depicting the distribution of distance of R-DMRs from CpG islands.
  • Figures IB and 1C, upper panels, show plots of M value versus genomic location for fibroblast and iPS cells and plots of CpG density versus genomic location, where the curve represents averaged smoothed M values; the location of CpG dinucleotides (black tick marks), CpG density, location of CpG islands (filled boxes along the x-axis (zero)), along with the gene annotation.
  • Figures IB and 1C lower panels show validation by bisulfite pyrosequencing of methylation percentage mapping to the unfilled box along the x-axis of the plots of CpG density versus genomic location in the upper panels of Figures IB and 1C for various iPS cells, fibroblasts, ES cells (BGOl, BG03 and H9) as well as the highly methylated HCT116 colon cancer cell line and a generally hypomethylated double DNA methyltransferase 1/3B double knockout line (DKO) derived from it.
  • DKO double DNA methyltransferase 1/3B double knockout line
  • Figure 2 shows plots depicting the distribution of distance of R-DMRs from CpG islands.
  • Figure 3A shows a clustering of M values of all tissues from the 4,401 regions (FDR ⁇ 0.05) corresponding to R-DMRs (iPS cells compared to parental fibroblasts) comparing normal brain, spleen and liver tissues (denoted as Br, Sp and Lv, respectively).
  • Figure 3B shows a clustering of M values of all tissues from the 4,401 regions (FDR ⁇ 0.05) corresponding to R-DMRs (iPS cells compared to parental fibroblasts) comparing colorectal cancer and matched normal colonic mucosa (denoted as T and N, respectively).
  • Figure 4 shows plots depicting differential DNA methylation (upper panels) and confirmation by bisulfite pyrosequencing (lower panels) for DMRs found by comparison between iPS cells and fibroblasts (A and B) as well as various genes (C-G).
  • Figures 4A-G, upper panels, show plots of M value versus genomic location, where the curve represents averaged smoothed M values.
  • CpG dinucleotide black tick marks on x-axis
  • CpG density smoothed black line
  • location of CpG islands filled boxes along the x-axis (zero)
  • gene annotation indicating the transcript thin outer gray line
  • coding region thin inner gray line
  • exons filled gray box
  • gene transcription directionality on the y-axis sense marked as +, antisense as -.
  • Figures 4A-G, lower panels depict plots showing the degree of DNA methylation as measured by bisulfite
  • the unfilled box indicated on the x-axis of the CpG density plot in the upper panel indicates the CpG sites that were measured. Reactions were performed in triplicate; bars represent the mean methylation ⁇ SD of iPS cells, fibroblasts, and ES cells (BGOl, BG03 and H9) as well as DKO (DNMT1 and DNMT3B Double KO cell line) and HCT116 (parental colon cancer cell line) for each individual CpG site measured.
  • DKO DNMT1 and DNMT3B Double KO cell line
  • HCT116 parental colon cancer cell line
  • Figure 5 shows plots of differential gene expression versus differential methylation for R-DMRs at CpG island shores.
  • the present invention is based on the discovery that alterations in DNA
  • CpG island shores cytosine-phosphate-guanine island shores
  • the DMRs in the reprogrammed cells were significantly enriched in tissue-specific (T-DMRs) and cancer-specific DMRs (C-DMRs).
  • T-DMRs tissue-specific
  • C-DMRs cancer-specific DMRs
  • iPS cells are derived from fibroblasts
  • their R-DMRs can distinguish between cells of normal tissue and between cancer and normal cells, e.g., colon cancer and normal colon cells.
  • DM s are broadly involved in tissue differentiation, epigenetic reprogramming and cancer.
  • hypomethylated C-DMRs and the absence of bivalent marks were observed, suggesting two mechanisms for epigenetic reprogramming in iPS cells and cancer.
  • a method of generating an iPS cell includes contacting a somatic cell with an agent that alters the methylation status of one or more nucleic acid sequences of the somatic cell, the one or more nucleic acid sequences being outside of a promoter region of a gene and outside of a CpG island, and wherein the nucleic acid sequences are up to about 2 kb in distance from a CpG island, and wherein the nucleic acid sequences are differentially methylated in reprogrammed somatic cells as compared with parent somatic cells, thereby generating an iPS cell.
  • reprogramming is intended to refer to a process that alters or reverses the differentiation status of a somatic cell that is either partially or terminally differentiated.
  • Reprogramming of a somatic cell may be a partial or complete reversion of the differentiation status of the somatic cell.
  • reprogramming is complete wherein a somatic cell is reprogrammed into an iPS cell.
  • reprogramming may be partial, such as reversion into any less differentiated state. For example, reverting a terminally differentiated cell into a cell of a less differentiated state, such as a multipotent cell.
  • pluripotent cells include cells that have the potential to divide in vitro for an extended period of time (greater than one year) and have the unique ability to differentiate into cells derived from all three embryonic germ layers, namely endoderm, mesoderm and ectoderm.
  • Somatic cells for use with the present invention may be primary cells or immortalized cells.
  • Such cells may be primary cells (non-immortalized cells), such as those freshly isolated from an animal, or may be derived from a cell line (immortalized cells).
  • the somatic cells are mammalian cells, such as, for example, human cells or mouse cells. They may be obtained by well-known methods, from different organs, such as, but not limited to skin, brain, lung, pancreas, liver, spleen, stomach, intestine, heart, reproductive organs, bladder, kidney, urethra and other urinary organs, or generally from any organ or tissue containing living somatic cells, or from blood cells.
  • Mammalian somatic cells useful in the present invention include, by way of example, adult stem cells, Sertoli cells, endothelial cells, granulosa epithelial cells, neurons, pancreatic islet cells, epidermal cells, epithelial cells, hepatocytes, hair follicle cells, keratinocytes, hematopoietic cells,
  • somatic cell is also intended to include adult stem cells.
  • An adult stem cell is a cell that is capable of giving rise to all cell types of a particular tissue.
  • Exemplary adult stem cells include hematopoietic stem cells, neural stem cells, and mesenchymal stem cells.
  • alterations in methylation patterns occur during differentiation or dedifferention of a cell which work to regulate gene expression of critical factors that are 'turned on' or 'turned off at various stages of differentiation.
  • agents are capable of altering the methylation status of one or more nucleic acid sequences of a somatic cell to induce pluripotency that may be suitable for use with the present invention.
  • an agent is intended to include any agent capable of altering the methylation status of one or more nucleic acid sequences of a somatic cell.
  • an agent useful in any of the method of the invention may be any type of molecule, for example, a polynucleotide, a peptide, a peptidomimetic, peptoids such as vinylogous peptoids, chemical compounds, such as organic molecules or small organic molecules, or the like.
  • the agent may be a polynucleotide, such as DNA molecule, an antisense oligonucleotide or RNA molecule, such as microRNA, dsRNA, siRNA, stRNA, and shRNA.
  • MicroRNA are single-stranded RNA molecules whose expression is known to be regulated by methylation to play a key role in regulation of gene expression during differentiation and dedifferentiation of cells.
  • an agent may be one that inhibits or induces expression of miRNA or may be a mimic miRNA.
  • miRNAs which are intended to mean a microRNA exogenously introduced into a cell that have the same or substantially the same function as their endogenous counterpart.
  • an agent that alters the methylation status of one or more nucleic acid sequences is a nuclear reprogramming factor.
  • Nuclear reprogramming factors may be genes that induce pluripotency and utilized to reprogram differentiated or semi-differentiated cells to a phenotype that is more primitive than that of the initial cell, such as the phenotype of a pluripotent stem cell.
  • genes and agents are capable of generating a pluripotent stem cell from a somatic cell upon expression of one or more such genes having been integrated into the genome of the somatic cell or upon contact of the somatic cell with the agent or expression product of the gene.
  • a gene that induces pluripotency is intended to refer to a gene that is associated with pluripotency and capable of generating a less differentiated cell, such as a pluripotent stem cell from a somatic cell upon integration and expression of the gene.
  • the expression of a pluripotency gene is typically restricted to pluripotent stem cells, and is crucial for the functional identity of pluripotent stem cells.
  • genes have been found to be associated with pluripotency and suitable for use with the present invention as reprogramming factors.
  • Such genes include, by way of example, SOX family genes (SOXl, SOX2, SOX3, SOX15, SOX18), KLF family genes (KLF1, KLF2, KLF4, KLF5), MYC family genes (C-MYC, L-MYC, N- MYC), SALL4, OCT4, NANOG, LIN28, STELLA, NOBOX, POU5F1 or a STAT family gene.
  • STAT family members may include for example STAT1 , STAT2, STAT3, STAT4, STAT5 (STAT5 A and STAT5B), and STAT6 .
  • pluripotency While in some instances, use of only one gene to induce pluripotency may be possible, in general, expression of more than one gene is required to induce pluripotency.
  • two, three, four or more genes may be simultaneously integrated into the somatic cell genome as a polycistronic construct to allow simultaneous expression of such genes.
  • four genes are utilized to induce pluripotency including OCT4, POU5F1, SOX2, KLF4 and C-MYC. Additional genes known as reprogramming factors suitable for use with the present invention are disclosed in U.S. Patent Application No. 10/997,146 and U.S. Patent Application No. 12/289,873, incorporated herein by reference.
  • genes commonly exist in mammals, including human, and thus homologues from any mammals may be used in the present invention, such as genes derived from mammals including, but not limited to mouse, rat, bovine, ovine, horse, and ape.
  • mutant gene products including substitution, insertion, and/or deletion of several (e.g., 1 to 10, 1 to 6, 1 to 4, 1 to 3, and 1 or 2) amino acids and having similar function to that of the wild-type gene products can also be used.
  • the combinations of factors are not limited to the use of wild-type genes or gene products.
  • Myc chimeras or other Myc variants can be used instead of wild-type Myc.
  • the present invention is not limited to any particular combination of nuclear reprogramming factors.
  • a nuclear reprogramming factor may comprise one or more gene products.
  • the nuclear reprogramming factor may also comprise a combination of gene products as discussed herein.
  • Each nuclear reprogramming factor may be used alone or in combination with other nuclear reprogramming factors as disclosed herein.
  • nuclear reprogramming factors of the present invention can be identified by screening methods, for example, as discussed in U.S. Patent Application No. 10/997,146, incorporated herein by reference.
  • the nuclear reprogramming factor of the present invention may contain one or more factors relating to differentiation, development, proliferation or the like and factors having other physiological activities, as well as other gene products which can function as a nuclear reprogramming factor.
  • the nuclear reprogramming factor may include a protein or peptide.
  • the protein may be produced from a gene as discussed herein, or alternatively, in the form of a fusion gene product of the protein with another protein, peptide or the like.
  • the protein or peptide may be a fluorescent protein and/or a fusion protein.
  • a fusion protein with green fluorescence protein (GFP) or a fusion gene product with a peptide such as a histidine tag can also be used.
  • fusion protein with the TAT peptide derived from the virus HIV, intracellular uptake of the nuclear reprogramming factor through cell membranes can be promoted, thereby enabling induction of reprogramming only by adding the fusion protein to a medium thus avoiding complicated operations such as gene transduction. Since preparation methods of such fusion gene products are well known to those skilled in the art, skilled artisans can easily design and prepare an appropriate fusion gene product depending on the purpose.
  • the agent alters the methylation status of one or more nucleic acid sequences, such as DMR sequences as set forth in Tables 2, 6, 9, Figures 1B-1C, Figures 4A-4G, the BMP7 gene, the GSC gene, the TBX3 gene, the HOXD3 gene, the PTPRT gene, the POU3F4 gene, the AZBP1 gene, the ZNF184 gene, the IGF1R gene, and any combination thereof.
  • DMR sequences as set forth in Tables 2, 6, 9, Figures 1B-1C, Figures 4A-4G
  • the BMP7 gene the GSC gene
  • the TBX3 gene the HOXD3 gene
  • the PTPRT gene the POU3F4 gene
  • the AZBP1 gene the ZNF184 gene
  • IGF1R gene any combination thereof.
  • Detecting the methylation status profile of the one or more nucleic acid sequences of the induced iPS and/or comparing the methylation status profile to a methylation status profile of the one or more nucleic acid sequences of a parental somatic cell from which the iPS is induced may also be performed to assess pluripotency characteristics.
  • expression profiling of reprogrammed somatic cells to assess their pluripotency characteristics may also be conducted. Expression of individual genes associated with pluripotency may also be examined. Additionally, expression of embryonic stem cell surface markers may be analyzed.
  • expression refers to the production of a material or substance as well as the level or amount of production of a material or substance. Thus, determining the expression of a specific marker refers to detecting either the relative or absolute amount of the marker that is expressed or simply detecting the presence or absence of the marker.
  • marker refers to any molecule that can be observed or detected.
  • a marker can include, but is not limited to, a nucleic acid, such as a transcript of a specific gene, a polypeptide product of a gene, a non-gene product polypeptide, a glycoprotein, a carbohydrate, a glycolipd, a lipid, a lipoprotein or a small molecule.
  • a nucleic acid such as a transcript of a specific gene, a polypeptide product of a gene, a non-gene product polypeptide, a glycoprotein, a carbohydrate, a glycolipd, a lipid, a lipoprotein or a small molecule.
  • Detection and analysis of a variety of genes known in the art to be associated with pluripotent stem cells may include analysis of genes such as, but not limited to OCT4, NANOG, SALL4, SSEA-1, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, or a combination thereof.
  • iPS cells may express any number of pluripotent cell markers, including: alkaline phosphatase (AP); ABCG2; stage specific embryonic antigen- 1 (SSEA-1); SSEA-3; SSEA-4; TRA-1-60; TRA-1-81; Tra-2-49/6E; ERas/ECAT5, E-cadherin; ⁇ - ⁇ -tubulin; ⁇ -smooth muscle actin ( ⁇ -SMA); fibroblast growth factor 4 (Fgf4), Cripto, Daxl; zinc finger protein 296 (Zfp296); N-acetyltransferase-1 (Natl); ES cell associated transcript 1 (ECAT1);
  • AP alkaline phosphatase
  • SSEA-1 stage specific embryonic antigen- 1
  • SSEA-3 stage specific embryonic antigen- 1
  • SSEA-4 SSEA-1-60
  • TRA-1-81 Tra-2-49/6E
  • ERas/ECAT5 E-cadherin
  • differentiation refers to a change that occurs in cells to cause those cells to assume certain specialized functions and to lose the ability to change into certain other specialized functional units.
  • Cells capable of differentiation may be any of totipotent, pluripotent or multipotent cells. Differentiation may be partial or complete with respect to mature adult cells.
  • Differentiated cell refers to a non-embryonic, non-parthenogenetic or non- pluripotent cell that possesses a particular differentiated, i.e., non-embryonic, state.
  • the three earliest differentiated cell types are endoderm, mesoderm, and ectoderm.
  • Pluripotency can also be confirmed by injecting the cells into a suitable animal, e.g., a SCID mouse, and observing the production of differentiated cells and tissues. Still another method of confirming pluripotency is using the subject pluripotent cells to generate chimeric animals and observing the contribution of the introduced cells to different cell types. Methods for producing chimeric animals are well known in the art and are described in U.S. Pat. No. 6,642,433, incorporated by reference herein.
  • Yet another method of confirming pluripotency is to observe cell differentiation into embryoid bodies and other differentiated cell types when cultured under conditions that favor differentiation (e.g., removal of fibroblast feeder layers).
  • the invention further provides iPS cells produced using the methods described herein, as well as populations of such cells.
  • the reprogrammed cells of the present invention capable of differentiation into a variety of cell types, have a variety of applications and therapeutic uses.
  • the present invention further provides a method of treatment or prevention of a disorder and/or condition in a subject using induced pluripotent stem cells generated using the methods described herein.
  • the method includes obtaining a somatic cell from a subject and reprogramming the somatic cell into an iPS cell using the methods described herein.
  • the cell is then cultured under suitable conditions to differentiate the cell into a desired cell type suitable for treating the condition.
  • the differentiated cell may then be introducing into the subject to treat or prevent the condition.
  • One advantage of the present invention is that it provides an essentially limitless supply of isogenic or synegenic human cells suitable for transplantation.
  • the iPS cells are tailored specifically to the patient, avoiding immune rejection. Therefore, it will obviate the significant problem associated with current transplantation methods, such as, rejection of the transplanted tissue which may occur because of host versus graft or graft versus host rejection.
  • iPS cells or fully differentiated somatic cells prepared from iPS cells from somatic cells derived from healthy humans can be stored in an iPS cell bank as a library of cells, and one kind or more kinds of the iPS cells in the library can be used for preparation of somatic cells, tissues, or organs that are free of rejection by a patient to be subjected to stem cell therapy.
  • the iPS cells of the present invention may be differentiated into a number of different cell types to treat a variety of disorders by methods known in the art.
  • iPS cells may be induced to differentiate into hematopoetic stem cells, muscle cells, cardiac muscle cells, liver cells, cartilage cells, epithelial cells, urinary tract cells, neuronal cells, and the like.
  • the differentiated cells may then be transplanted back into the patient's body to prevent or treat a condition.
  • the methods of the present invention may be used to treat a subject having a myocardial infarction, congestive heart failure, stroke, ischemia, peripheral vascular disease, alcoholic liver disease, cirrhosis, Parkinson's disease, Alzheimer's disease, diabetes, cancer, arthritis, wound healing, immunodeficiency, aplastic anemia, anemia, Huntington's disease, amyotrophic lateral sclerosis (ALS), lysosomal storage diseases, multiple sclerosis, spinal cord injuries, genetic disorders, and similar diseases, where an increase or replacement of a particular cell type/ tissue or cellular de-differentiation is desirable.
  • ALS amyotrophic lateral sclerosis
  • the method increases the number of cells of the tissue or organ by at least about 5%, 10%, 25%, 50%, 75% or more compared to a corresponding untreated control tissue or organ.
  • the method increases the biological activity of the tissue or organ by at least about 5%, 10%, 25%, 50%, 75% or more compared to a corresponding untreated control tissue or organ.
  • the method increases blood vessel formation in the tissue or organ by at least about 5%, 10%, 25%, 50%, 75% or more compared to a corresponding untreated control tissue or organ.
  • the cell is administered directly to a subject at a site where an increase in cell number is desired either before or after differentiation of the cell to a desired cell type.
  • the present invention provides a method of identifying an iPS cell.
  • the method includes comparing the methylation status of one or more nucleic acid sequences of a putative iPS cell, with the proviso that the one or more nucleic acid sequences are outside of a promoter region of a gene and outside of a CpG island, and wherein the nucleic acid sequences are up to about 2 kb in distance from a CpG island, to a known methylation status of the one or more nucleic acid sequences of an iPS cell, wherein a similarity in methylation status is indicative of the putative cell being an iPS cell.
  • the known methylation status of the one or more nucleic acid sequences of an iPS cell may include the R-DMRs set forth in Tables 2, 6, 7, 9, Figures 1B-1C, Figures 4C-4G, the BMP7 gene, the GSC gene, the TBX3 gene, the HOXD3 gene, the PTPRT gene, the POU3F4 gene, the AZBP1 gene, the ZNF184 gene, the IGF1R gene, and any combination thereof.
  • the method of identifying an iPS cell includes comparing the methylation status of one or more nucleic acid sequences of a putative iPS cell, with the proviso that the one or more nucleic acid sequences are outside of a promoter region of a gene and outside of a CpG island, and wherein the nucleic acid sequences are up to about 2 kb in distance from a CpG island, to a known methylation status of the one or more nucleic acid sequences of a corresponding somatic cell from which the iPS cell is induced and/or an ES cell, wherein an alteration in methylation status is indicative of the putative cell being an iPS cell.
  • the one or more nucleic acid sequences may be DMR sequences as set forth in Tables 2, 6, 7, 9, Figures 1B-1C, Figures 4A-4G, the BMP7 gene, the GSC gene, the TBX3 gene, the HOXD3 gene, the POU3F4 gene, the AZBP1 gene, the ZNF184 gene, the IGF1R gene, and any combination thereof.
  • the invention further provides a plurality of nucleic acid sequences, wherein the nucleic acid sequences are outside of a promoter region of a gene and outside of a CpG island, and wherein the nucleic acid sequence is up to about 2 kb in distance from a CpG island, and wherein the nucleic acid sequences are differentially methylated in the reprogramming of a somatic cell to generate an iPS cell.
  • the nucleic acid sequences are the DMR sequences as set forth in Tables 2, 6, 9, Figures 1B-1C, Figures 4A- 4G, the BMP7 gene, the GSC gene, the TBX3 gene, the HOXD3 gene, the POU3F4 gene, the AZBP1 gene, the ZNF184 gene, and the IGF1R gene.
  • the invention further provides a plurality of nucleic acid sequences, wherein the nucleic acid sequences are outside of a promoter region of a gene and outside of a CpG island, and wherein the nucleic acid sequences are up to about 2 kb in distance from a CpG island, and wherein the methylation status of the nucleic acid sequences is altered in an iPS cell as compared to an ES cell.
  • the nucleic acid sequences are the DMR sequences as set forth in Table 7, Figures 4C-4G, the PTPRT gene, the POU3F4 gene, the AZBP1 gene, the ZNF184 gene, and the IGF1R gene.
  • the plurality of nucleic acid sequences may be utilized to provide a microarray for performing the methods described herein.
  • One skilled in the art would appreciate the many techniques that are well known for attaching nucleic acids on a substrate that may be utilized along with the various types of substrates and configurations.
  • the invention further provides a method of characterizing the methylation status of the nucleic acid of an iPS cell.
  • the method includes a) hybridizing labeled and digested nucleic acid of an iPS cell to a DNA microarray comprising at least 2000 nucleic acid sequences, with the proviso that the nucleic acid sequences are outside of a promoter region of a gene and outside of a CpG island, and wherein the nucleic acid sequences are up to about 2 kb in distance from a CpG island; and b) determining a pattern of methylation from the hybridizing of (a), thereby characterizing the methylation status for the iPS cell.
  • the one or more nucleic acid sequences are DMR sequences as set forth in Tables 2, 6, 7, 9, Figures 1B-1C, Figures 4A-4G, the BMP7 gene, the GSC gene, the TBX3 gene, the HOXD3 gene, the POU3F4 gene, the AZBP1 gene, the ZNF184 gene, the IGF1R gene, and any combination thereof.
  • Characterizing the methylation status of the nucleic acid of an iPS cell may further include comparing the methylation status profile to a methylation profile from hybridization of the microarray with labeled and digested nucleic acid from a parental somatic cell from which the iPS is induced or from an ES cell.
  • the one or more nucleic acid sequences are DMR sequences as set forth in Tables 2, 6, 7, 9, Figures 1B-1C, Figures 4A-4G, the BMP7 gene, the GSC gene, the TBX3 gene, the HOXD3 gene, the POU3F4 gene, the AZBP1 gene, the ZNF184 gene, the IGF1R gene, and any combination thereof.
  • methylation status is converted to an M value.
  • an M value can be a log ratio of intensities from total (Cy3) and McrBC- fractionated DNA (Cy5): positive and negative M values are quantitatively associated with methylated and unmethylated sites, respectively.
  • DMR may be hypermethylated or
  • hypomethylated Hypomethylation of a DMR is present when there is a measurable decrease in methylation of the DMR. In some embodiments, a DMR can be determined to be hypomethylated when less than 50% of the methylation sites analyzed are not methylated. Hypermethylation of a DMR is present when there is a measurable increase in methylation of the DMR. In some embodiments, a DMR can be determined to be hypermethylated when more than 50% of the methylation sites analyzed are methylated. Methods for determining methylation states are provided herein and are known in the art. In some embodiments methylation status is converted to an M value.
  • an M value can be a log ratio of intensities from total (Cy3) and McrBC-fractionated DNA (Cy5): positive and negative M values are quantitatively associated with methylated and unmethylated sites, respectively. M values are calculated as described in the Examples. In some embodiments, M values which range from -0.5 to 0.5 represent unmethylated sites as defined by the control probes, and values from 0.5 to 1.5 represent baseline levels of methylation.
  • determining of methylation status in the methods of the invention is performed by one or more techniques selected from the group consisting of a nucleic acid amplification, polymerase chain reaction (PCR), methylation specific PCR, bisulfite pyrosequenceing, single-strand conformation polymorphism (SSCP) analysis, restriction analysis, microarray technology, and proteomics.
  • analysis of methylation can be performed by bisulfite genomic sequencing.
  • Bisulfite treatment modifies DNA converting unmethylated, but not methylated, cytosines to uracil.
  • Bisulfite treatment can be carried out using the
  • bisulfite pyrosequencing which is a sequencing-based analysis of DNA methylation that quantitatively measures multiple, consecutive CpG sites individually with high accuracy and reproducibility
  • Exemplary primers for such analysis are set forth in Table 11.
  • Table 11 It will be recognized that depending on the site bound by the primer and the direction of extension from a primer, that the primers listed above can be used in different pairs.
  • additional primers can be identified within the DMRs, especially primers that allow analysis of the same methylation sites as those analyzed with primers that correspond to the primers disclosed herein.
  • Altered methylation can be identified by identifying a detectable difference in methylation. For example, hypomethylation can be determined by identifying whether after bisulfite treatment a uracil or a cytosine is present a particular location. If uracil is present after bisulfite treatment, then the residue is unmethylated. Hypomethylation is present when there is a measurable decrease in methylation.
  • the method for analyzing methylation of the DMR can include amplification using a primer pair specific for methylated residues within a DMR.
  • selective hybridization or binding of at least one of the primers is dependent on the methylation state of the target DNA sequence (Herman et al., Proc. Natl. Acad. Sci. USA, 93:9821 (1996)).
  • the amplification reaction can be preceded by bisulfite treatment, and the primers can selectively hybridize to target sequences in a manner that is dependent on bisulfite treatment.
  • one primer can selectively bind to a target sequence only when one or more base of the target sequence is altered by bisulfite treatment, thereby being specific for a methylated target sequence.
  • Methods using an amplification reaction can utilize a real-time detection amplification procedure.
  • the method can utilize molecular beacon technology (Tyagi et al., Nature Biotechnology, 14: 303 (1996)) or TaqmanTM technology (Holland et al., Proc. Natl. Acad. Sci. USA, 88:7276 (1991)).
  • methyl light Trinh et al., Methods 25(4):456-62 (2001), incorporated herein in its entirety by reference
  • Methyl Heavy Methyl Heavy
  • SNuPE single nucleotide primer extension
  • selective hybridization or “selectively hybridize” refers to hybridization under moderately stringent or highly stringent physiological conditions, which can distinguish related nucleotide sequences from unrelated nucleotide sequences.
  • the conditions used to achieve a particular level of stringency will vary, depending on the nature of the nucleic acids being hybridized. For example, the length, degree of complementarity, nucleotide sequence composition (for example, relative GC:AT content), and nucleic acid type, for example, whether the oligonucleotide or the target nucleic acid sequence is DNA or RNA, can be considered in selecting hybridization conditions. An additional consideration is whether one of the nucleic acids is immobilized, for example, on a filter. Methods for selecting appropriate stringency conditions can be determined empirically or estimated using various formulas, and are well known in the art ⁇ see, e.g., Sambrook et al., supra, 1989).
  • An example of progressively higher stringency conditions is as follows: 2X SSC/0.1% SDS at about room temperature (hybridization conditions); 0.2X SSC/0.1% SDS at about room temperature (low stringency conditions); 0.2X SSC/0.1% SDS at about 42°C (moderate stringency conditions); and 0.1X SSC at about 68°C (high stringency conditions). Washing can be carried out using only one of these conditions, for example, high stringency conditions, or each of the conditions can be used, for example, for 10 to 15 minutes each, in the order listed above, repeating any or all of the steps listed.
  • the degree of methylation in the DNA associated with the DMRs being assessed may be measured by fluorescent in situ hybridization (FISH) by means of probes which identify and differentiate between genomic DNAs, associated with the DMRs being assessed, which exhibit different degrees of DNA methylation.
  • FISH fluorescent in situ hybridization
  • the biological sample will typically be any which contains sufficient whole cells or nuclei to perform short term culture.
  • the sample will be a sample that contains 10 to 10,000, or, for example, 100 to 10,000, whole cells.
  • methyl light, methyl heavy, and array-based methylation analysis can be performed, by using bisulfite treated DNA that is then PCR- amplified, against microarrays of oligonucleotide target sequences with the various forms corresponding to unmethylated and methylated DNA.
  • nucleic acid molecule is used broadly herein to mean a sequence of deoxyribonucleotides or ribonucleotides that are linked together by a phosphodiester bond.
  • nucleic acid molecule is meant to include DNA and RNA, which can be single stranded or double stranded, as well as DNA/RNA hybrids.
  • nucleic acid molecule includes naturally occurring nucleic acid molecules, which can be isolated from a cell, as well as synthetic molecules, which can be prepared, for example, by methods of chemical synthesis or by enzymatic methods such as by the polymerase chain reaction (PCR), and, in various embodiments, can contain nucleotide analogs or a backbone bond other than a phosphodiester bond.
  • PCR polymerase chain reaction
  • polynucleotide and oligonucleotide also are used herein to refer to nucleic acid molecules. Although no specific distinction from each other or from “nucleic acid molecule” is intended by the use of these terms, the term “polynucleotide” is used generally in reference to a nucleic acid molecule that encodes a polypeptide, or a peptide portion thereof, whereas the term “oligonucleotide” is used generally in reference to a nucleotide sequence useful as a probe, a PCR primer, an antisense molecule, or the like. Of course, it will be recognized that an "oligonucleotide” also can encode a peptide. As such, the different terms are used primarily for convenience of discussion.
  • a polynucleotide or oligonucleotide comprising naturally occurring nucleotides and phosphodiester bonds can be chemically synthesized or can be produced using recombinant DNA methods, using an appropriate polynucleotide as a template.
  • a polynucleotide comprising nucleotide analogs or covalent bonds other than phosphodiester bonds generally will be chemically synthesized, although an enzyme such as T7 polymerase can incorporate certain types of nucleotide analogs into a polynucleotide and, therefore, can be used to produce such a polynucleotide recombinantly from an appropriate template.
  • kits that are useful for carrying out the methods of the present invention.
  • the components contained in the kit depend on a number of factors, including: the particular analytical technique used to detect methylation or measure the degree of methylation or a change in methylation, and the one or more DMRs is being assayed for methylation status.
  • the present invention provides a kit for determining a methylation status of one or more DMRs of the invention.
  • the one or more DMRs are selected from one or more of the sequences as set forth in Tables 2, 6, 7, 9, Figures 1B- 1C, Figures 4A-4G, the BMP7 gene, the GSC gene, the TBX3 gene, the HOXD3 gene, the PTPRT gene, the POU3F4 gene, the AZBP1 gene, the ZNF184 gene, the IGF1R gene, and any combination thereof.
  • the kit includes an oligonucleotide probe, primer, or primer pair, or combination thereof for carrying out a method for detecting hypomethylation, as discussed above.
  • the probe, primer, or primer pair can be capable of selectively hybridizing to the DMR either with or without prior bisulfite treatment of the DMR.
  • the kit can further include one or more detectable labels.
  • the kit can also include a plurality of oligonucleotide probes, primers, or primer pairs, or combinations thereof, capable of selectively hybridizing to the DMR with or without prior bisulfite treatment of the DMR.
  • the kit can include an oligonucleotide primer pair that hybridizes under stringent conditions to all or a portion of the DMR only after bisulfite treatment.
  • the kit can provide reagents for bisulfite pyrosequencing including one or more primer pairs set forth in Table 11.
  • the kit can include instructions on using kit components to identify, for example, the presence of cancer or an increased risk of developing cancer.
  • CHARM is 100% specific at 90% sensitivity for known methylation marks identified by other methods (for example, in promoters) and includes the approximately half of the genome not identified by conventional region preselection .
  • the CHARM results were also extensively corroborated by quantitative bisulfite pyrosequencing analysis.
  • IPS cells are derived by epigenetic reprogramming, but their DNA methylation patterns have not peviously been analyzed on a genome-wide scale. Substantial
  • cytosine-phosphate-guanine (CpG) island shores in nine human iPS cell lines as compared to their parental fibroblasts was determined.
  • the R- DMRs in the reprogrammed cells were significantly enriched in tissue-specific (T-DMRs; 2.6-fold, P ⁇ I Q- 4 ) and cancer-specific DMRs (C-DMRs; 3.6-fold, P ⁇ 10 "4 ).
  • T-DMRs tissue-specific
  • C-DMRs cancer-specific DMRs
  • P ⁇ 10 "4 cancer-specific DMRs
  • many DMRs are broadly involved in tissue differentiation, epigenetic reprogramming and cancer. Colocalization of hypomethylated R-DMRs with hypermethylated C-DMRs and bivalent chromatin marks, and colocalization of hypermethylated R-DMRs with
  • hypomethylated C-DMRs and the absence of bivalent marks were observed, suggesting two mechanisms for epigenetic reprogramming in iPS cells and cancer.
  • methylation density is determined for a region of nucleic acid. Density may be used as an indication of production of an iPS cell, for example. A density of about 0.2 to 0.7, about 0.3 to 0.7 , 0.3 to 0.6 or 0.3 to 0.4, or 0.3, may be indicative of generation of an iPS cell (the calculated DNA methylation density is the number of methylated CpGs divided by the total number of CpGs sequenced for each sample).
  • Methods for determining methylation density are well known in the art. For example, a method for determining methylation density of target CpG islands has been established by Luo et al. Analytical Biochemistry, Vol. 387:2 2009, pp. 143-149. In the method, DNA microarray was prepared by spotting a set of PCR products amplified from bisulfite- converted sample DNAs. This method not only allows the quantitative analysis of regional methylation density of a set of given genes but also could provide information of methylation density for a large amount of clinical samples as well as use in the methods of the invention regarding iPS cell generation and detection. Other methods are well known in the art (e.g., Holemon et al., BioTechniques, 43:5, 2007, pp. 683-693).
  • RNA and genomic DNA from fibroblast, hES cells and iPS cells.
  • iPS cell lines and their parental fibroblasts used were described in Park et al. (Nature (451)141-146 (2008)) and Park et al. Cell (134)877-886 (2008)).
  • MRC5 14-week-old fetal lung fibroblast from the ATCC cell biology collection
  • Detroit 551 551, fetal skin fibroblast from ATCC
  • hFib2 adult dermal fibroblast
  • SBDS SBDS
  • DMD GM04981 from Coriell
  • GD GM00852A from Coriell
  • PD AG20446 from Coriell
  • JDM GM02416 from Coriell
  • ADA GM01390 from Coriell.
  • Human ES cells BGOl, BG03 and H9 were used. Fibroblasts were grown in a-MEM containing 10% W 201
  • RNA and genomic DNA were isolated using RNeasyTM kit (Qiagen) with in-column DNase treatment and DNeasyTM kit (Qiagen), respectively, according to manufacturer's protocol.
  • CHARM DNA methylation analysis For each sample, 5 ⁇ g of genomic DNA was digested, fractionated, labeled and hybridized to a CHARM microarray as described in Irizarry et al. (Nat. Genet. (41)178-186 (2009)) and Irizarry et al. ⁇ Genome Res. (18)780-790 (2008)).
  • FDR false discovery rates
  • the parental fibroblast lines for this experiment were MRC5, SBDS, DMD, GD, Detroit551 and PD, and they were compared to the iPS cell lines derived from them.
  • ⁇ n 3 for each cell type
  • an absolute FDR could not be calculated, so we used an absolute area cutoff of 10.0, which corresponded in magnitude to the 5% FDR cutoff of the first set of experiments.
  • the parental fibroblast lines for the second experiment were JMD, ADA and hFib2 and were compared to the iPS cell lines derived from them, as well as to three ES cell lines, BGOl, BG03 and H9.
  • Unsupervised cluster analysis Using the R-DMRs, we performed unsupervised cluster analysis to determine to what degree the methylation at these locations distinguished normal brain, liver and spleen as well as colon cancer from its matched counterpart. As a test of significance, we randomly generated 1,000 CHARM array regions of length and number equal to those of the R-DMRs and then assessed the median euclidean distance among samples of a given tissue type and the median euclidean distance among samples of different tissue types. This test was also applied to the cancer and normal samples.
  • Table 1 1 provides the primer sequences used for the bisulfite pyrosequencing reactions, as well as the chromosomal coordinates in the University of California at Santa Cruz March 2006 human genome assembly for each CpG site interrogated.
  • the annealing temperature used for all PCR reactions was 50 °C.
  • Affymetrix microarray expression analysis Genome-wide gene expression analysis was done using Affymetrix U133 Plus 2.0TM microarrays. For each sample, 1 ⁇ g of high-quality total RNA was amplified, labeled and hybridized onto the microarray according to Affymetrix's specifications, and data were normalized as previously described in Irizarry et al. (Biostatistics (4)249-264 (2003)).
  • NCBI GEO Gene expression microarray data and CHARM microarray data have been submitted under accession number GSE18111.
  • IPS cells can be derived from somatic cells by introduction of a small number of genes: for example, POU5F1, MYC, KLF4 and SOX2.
  • iPS cells offer considerable therapeutic promise, avoiding both immunologic and ethical barriers to their use.
  • iPS cells differ from their somatic parental cells epigenetically, and thus a comprehensive comparison of the epigenome in iPS and somatic cells would provide insight into the mechanism of tissue reprogramming.
  • two previous targeted studies examined a subset of the genome, 7,000 (Ball et al. (Nat. Biotechnol. (27)485 (2009)) and 66,000 (Deng et al. (Nat. Biotechnol. (27)353-360 (2009))) CpG sites in a small cohort of three iPS-fibroblast pairs, a global assessment of genome- wide methylation has not yet been performed.
  • T-DMRs normal tissue types
  • C-DMRs colorectal cancer tissue from matched normal tissues
  • Genomic DNA from iPS cells, their parental fibroblasts and human embryonic stem (hES) cells was digested with the enzyme McrBC, fractionated, labeled and hybridized to a
  • Figure 1 details reprogramming differentially methylated regions (R-DMRs).
  • Figure 1 A depicts enrichment of R-DMRs at CpG island shores.
  • the CHARM array (left, labeled CpG regions) is enriched in CpG islands, and the R-DMRs (right, labeled R-DMR) show marked enrichment at CpG island shores.
  • Islands are denoted as regions that include >50% of a CpG island or are wholly contained in an island, and overlap regions are denoted as regions that include 0.1-50% of a CpG island. Specific base intervals of regions not overlapping islands are indicated; (0-500) means from 1 to 500 bases.
  • Percentage of the distribution is given for the CpG regions (CHARM array, null hypothesis) and reprogramming differentially methylated regions (R-DMRs).
  • Figures IB and C show examples of DMRs.
  • the gene encoding bone morphogenetic protein 7 (BMP7) is indicated in B, and the gene encoding goosecoid (GSC) is indicated in C.
  • BMP7 bone morphogenetic protein 7
  • GSC gene encoding goosecoid
  • the upper panels show a plot of methylation ( value; see Methods) versus genomic location, where the curve represents averaged smoothed M values; the location of CpG dinucleotides (black tick marks), CpG density, location of CpG islands (filled boxes along the x-axis (zero)), as well as the gene annotation are shown.
  • the bottom panels show validation by bisulfite
  • Figure 2 depicts the distribution of distance of reprogramming differentially methylated regions (R-DMRs) from CpG islands.
  • Islands are regions that are inside, cover, or overlap more than 50% of a CpG island. Overlap are regions that overlap 0.1-50% of a CpG island.
  • Regions denoted by (0, 500] are regions located ⁇ 500 bp but do not overlap an island.
  • Regions denoted by (500, 1000] are regions located >500 bp and ⁇ 1000 bp from an island.
  • Regions denoted by (1000, 2000] are regions located >1000 bp and ⁇ 2000 bp from an island.
  • Regions denoted by (2000, 3000] are regions located >2000 bp and ⁇ 3000 bp from an island. Regions denoted by >3000 are >3000 bp from an island. Percentage are given for the CpG regions (CHARM array, null hypothesis) and reprogramming differentially methylated regions (R-DMRs) as well as the R-DMRs subdivided into hypermethylation and
  • the methylation in iPS cells was intermediate between differentiated fibroblasts and ES cells; this was true, for example, of TBX5, which encodes a transcription factor that is involved in cardiac and limb development.
  • methylation in iPS cells differed from both fibroblasts and ES cells, suggesting that the iPS cells occupy a distinct and possibly aberrant epigenetic state.
  • An example was PTPRT, encoding a protein tyrosine phosphatase involved in many cellular processes including differentiation.
  • methylation levels changed in the same direction as for ES cells compared to fibroblasts, but to a greater degree; for example, methylation of the homeobox gene HOXA9 was greater in iPS compared to ES, whose methylation at this gene was greater than in fibroblasts.
  • Figure 4 includes examples of differential DNA methylation (upper panels) and confirmation by bisulfite pyrosequencing (lower panels).
  • Upper panels are a plot of M value versus genomic location, where the curve represents averaged smoothed M values. Also shown in the upper panels are the locations of CpG dinucleotide (black tick marks on x axis), CpG density (smoothed black line) calculated across the region using a standard density estimator, location of CpG islands (filled boxes along the x-axis (zero)), as well as gene annotation indicating the transcript (thin outer gray line), coding region (thin inner gray line), exons (filled gray box) and gene transcription directionality on the y axis (sense marked as +, antisense as -).
  • the lower panels represent the degree of DNA methylation as measured by bisulfite pyrosequencing.
  • the unfilled box indicated on the x axis of the CpG density plot in the upper panel indicates the CpG sites that were measured. Reactions were done in triplicate; bars represent the mean methylation ⁇ SD of iPS cells, fibroblasts, and ES cells (BGOl, BG03 and H9) as well as DKO (DNMT1 and DNMT3B Double KO cell line) and HCT116 (parental colon cancer cell line) for each individual CpG site measured.
  • A TBX3 (T-box 3 protein),
  • B HOXD3 (Homeobox D3),
  • C POU3F4 (POU domain, class 3, transcription factor 4),
  • D A2BP1 (ataxin 2-binding protein 1),
  • E ZNF184 (zinc finger protein 184),
  • W W
  • IGF1R insulin-like growth factor 1 receptor
  • G PTPRT (protein tyrosine phosphatase, receptor type, T).
  • HGU133 PlusTM 2.0 microarray There was a strong inverse correlation between differential gene expression and differential DNA methylation at R-DMRs that are within 500 bp of the transcriptional start site (TSS) of a gene: P ⁇ 10-3 for both hypermethylation and
  • FIG. 5 illustrates that gene expression strongly correlates with reprogramming differentially methylated regions (R-DMRs) at CpG island shores.
  • Red circles represent R- DMRs that are within 2kb from a CpG island
  • blue circles represent those that are more than 2kb away from a CpG island
  • black circles represent log ratios for all genes not within (A) 500bp or (B) lkb from the transcriptional start site (TSS) of an annotated gene.
  • TSS transcriptional start site
  • the log2 ratios of fibroblast to iPS expression were plotted against ⁇ values (fibroblast minus iPS) for R-DMRs in which one of the two points had approximately no methylation.
  • A DMRs that are within 500bp from a TSS of a gene.
  • B DMRs that are within lkb from a TSS of a gene.
  • FIG. 3 shows that DNA methylation at R-DMRs distinguishes normal tissues from each other and colon cancer from normal colon.
  • a and B The M values of all tissues from the 4,401 regions (FDR ⁇ 0.05) corresponding to R-DMRs (iPS cells compared to parental fibroblasts) were used for unsupervised hierarchical clustering comparing (A) normal brain, spleen and liver (denoted as Br, Sp and Lv, respectively) and (B) colorectal cancer and matched normal colonic mucosa (denoted as T and N, respectively).
  • R-DMRs colorectal cancer and matched normal colonic mucosa
  • the R-DMRs were compared to those obtained in a genome-scale comparison of DNA methylation in colorectal cancer and matched normal colonic mucosa from the same individuals (C-DMRs) as discussed in Irizarry et al. (Nat. Genet. (41)178-186 (2009)).
  • a secondary finding is that certain loci in iPS cells remain incompletely reprogrammed, whereas others are aberrantly reprogrammed, thus establishing that the methylation pattern of iPS cells differs both from those of the parent somatic cells and from those of human ES cells.
  • R-DMRs are regions that differ in DNA methylation between 6 iPS lines and the fibroblasts from which they were derived.
  • FDR false discovery rate.
  • R-DMRs were defined by using a FDR cutoff of 5%.
  • chrl5 94686085 94687818 1.33256 -0.0655 66.1928 NR2F2 11136 downstream -548 disjoint 0.00966 chrl4 64239302 64240573 1.82985 -0.0939 65.9892 PLE HG3 372 promoter 56 disjoint 0.00966 chrl6 71654610 71657245 1.01951 -0.0039 65.925 ATBF1 14836 upstream 0 overlap 0.00966 chrl2 128900598 128903997 0.11442 1.39687 65.8968 TMEM132D 50168 inside intron 1639 disjoint 0.00966 chrl2 6531060 6534095 0.50762 1.47267 65.1372 HOM-TES- 1394 inside intron 591 disjoint 0.00967
  • chr4 174664434 174666750 1.5136 0.35561 52.1861 HAND2 21202 downstream 0 cover 0.00994 chrl 112853554 112854782 1.61085 0.09865 51.8868 NT2B 166 overlaps exon 0 overlap 0.00995 downstream chr9 102276338 102277667 1.71233 0.25285 51.8343 TMEFF1 801 inside intron 0 overlap 0.00995 chr22 36S28744 36530199 1.21846 -0.0042 51.8264 H1F0 860 promoter 419 disjoint 0.00995 chrl9 16049883 16051512 -0.1787 1.11299 51.5279 TPM4 1559 inside intron -608 disjoint 0.00996 chrl 154961985 154963849 0.75971 1.95945 51.4944 ISG20L2 479 overlaps exon 886 disjoint 0.00997 downstream
  • chrl8 52963741 52965315 -0.0673 2.05326 45.079 WDR7 494128 downstream -23549 disjoint 0.01031 chr2 63132869 63134574 1.39985 0.39203 44.9938 OTX1 1401 covers exon(s) 0 overlap 0.01032 chr6 101020166 101021773 1.17303 0.13573 44.8811 SMI 1895 promoter 0 overlap 0.01033 chrlO 102485639 102487566 0.86061 0.0674 44.8699 PAX2 7755 upstream 0 overlap 0.01033 chr8 68139975 68141466 0.2847 1.56861 44.8163 CSPP1 819 inside intron 0 overlap 0.01033 chr2 100088245 100089913 1.14828 0.28393 44.6781 AFF3 35555 covers exon(s) -1072 disjoint 0.01034 chrl2 121
  • BHLHB2 1171 covers exon(s) -155 disjoint 0.01117 chr4 174667570 174669252 1.48981 0.49152 37.1108 HAND2 18700 downstream -134 disjoint 0.01118 chr6 31777467 31778888 -0.1092 0.71282 37.0848
  • BAT5 178 covers exon(s) 0 overlap 0.01 118 chrl4 103935048 103937957 0.18051 1.60718 37.048 LOC400258 179143 upstream 28992 disjoint 0.01119 chrl7 7491 5 087 74916508 0.47516 1.48744 37.0215 LOC146713 15057 inside intron 55332 disjoint 0.01119 chrl3 9267 5 166 92676682 1.04767 0.1 36.941
  • CLK1 713 covers exon(s) 184 disjoint 0.01232 chrlO 104669314 104670494 0.9997 0.01097 31.8062
  • C NM2 1211 overlaps exon -12 disjoint 0.01232 downstream
  • chrlO 26264471 26266406 0.27575 0.97024 23.8259 MY03A 1270 covers exon(s) -443 disjoint 0.01595 chrl4 92460706 92462209 0.28844 1.34755 23.8137 CHGA 1462 inside intron -1054 disjoint 0.01595 chr3 171240759 171241541 0.75819 1.81464 23.7874 GPR160 1363 inside intron -1221 disjoint 0.01597 chr6 150501895 150502887 0.30216 1.19675 23.7868 PPP1R14C 3038 upstream 2577 disjoint 0.01597 chrl 5 65601462 65602211 -0.1765 0.92526 23.7578 MAP2K5 19863 upstream -227 disjoint 0.01599 chr21 37000964 37001823 0.96679 -0.096 23.7463 SIM2 7104 inside intron 0
  • chrX 9939268 9940550 1.35799 0.2174 22.6607 WWC3 3061 upstream 1963 disjoint 0.01677 chrl8 54683040 54684032 0.40773 1.24646 22.6458 ZNF532 2000 covers exon(s) -772 disjoint 0.01678 chr9 126044511 126045440 0.785 1.84458 22.638 NEK6 14629 upstream 14630 disjoint 0.01679 chr2 115135470 115136151 0.29254 1.65873 22.6326 DPP10 219102 inside intron 47 disjoint 0.01679 chr7 43763958 43764428 1.78764 0.10568 22.6326 BLVRA 368 promoter 0 overlap 0.01679 chrl3 45322884 45324348 0.14484 0.75737 22.6322 LOC283514 0 overlaps 5' -136669 disjoint 0.01679 ch
  • GSTA4 846 covers exon(s) 272 disjoint 0.0177
  • chr6 31739482 31740684 0.34771 1.01018 21.6425
  • BAT4 457 covers exon(s) 110 disjoint 0.01771 chrl l 2659433 2660734 0.32085 0.93189 21.6423
  • MGC39900 375 inside in
  • chr2 238863009 238863692 1.89525 0.84663 19.2023
  • PER2 1179 promoter -370 disjoint 0.02031 chr7 130890067 130891273 0.75923 0.19851 19.2 PODXL 634 inside intron 0 overlap 0.02031 chr 5 180168460 180169309 0.96098 1.8665 19.1971 MGAT1 5807 upstream 289 disjoint 0.02032 chrl 8 66850594 66851589 0.51106 1.34133 19.1943 SOCS6 743352 downstream -601518 disjoint 0.02032 chr 5 72562287 7256303030 1.64336 0.50733 19.1905 TMEM174 62458 downstream -34 disjoint 0.02032 chr6 27837986 27838651 1.4716 0.4615 19.1862 HIST1H2BL 45036 downstream 44657 disjoint 0.02033 chr5
  • EIF4A1 1394 covers exon(s) -120 disjoint 0.02134 chrl6 67779903 67780862 0.17074 0.94074 18.4204 SNTB2 1353 inside intron -295 disjoint 0.02135 chr9 95755626 95756444 1.01266 0.26401 18.4154 BARX1 978 inside intron 0 inside 0.02136 chrl 149212099 149212953 0.17984 0.93111 18.4124 LASS2 1110 inside intron 167 disjoint 0.02136 chrl8 491107 491721 1.51967 0.34696 18.4021 COLEC12 423 promoter -385 disjoint 0.02138 chr9 136676317 136677114 0.39819 1.1694 18.4019 COL5A1 2845 inside intron -2099 disjoint 0.02138 chr 5 150059
  • chrl2 56294113 56294691 2.79263 1.74026 16.353 GEFT 2151 covers exon(s) -2495 disjoint 0.02472 chrl 6 19993114 19994020 0.04981 0.92352 16.3503 GPR139 514 promoter -308 disjoint 0.02473 chrl 33667959 33668537 1.082 -0.0701 16.3492 ZNF31 42308 upstream 52 disjoint 0.02473 chrl 8 42169270 42170295 0.52437 -0.0768 16.3444 RNF165 1086 inside intron -795 disjoint 0.02474 chr22 44850960 44851727 0.4 5 29 1.23622 16.3394 FLJ27365 8819 upstream -3768 disjoint 0.02474 chrlO 22655295 22655951 1.13875 0.15556 16.3371 BMI1 5150 covers exon(s) -355 disjoint 0.0
  • chrl 9 60838061 60839128 0.07448 0.56366 16.1214 ZNF580 5075 upstream 4200 disjoint 0.02517 chr20 21320460 21321039 0.97801 -0.0483 16.1132 XRN2 88519 downstream 0 overlap 0.02518 chr 5 50295795 50296517 1.27811 0.29459 16.1102 PARP8 297226 downstream 4325 disjoint 0.02518 chr2 71358949 71359598 1.18824 0.2614 16.1043 ZNF638 52798 upstream -1208 disjoint 0.0252 chr4 81104306 81104941 1.51951 0.50483 16.1038 ANTXR2 108335 inside intron 0 overlap 0.0252 chr6 41483828 41484646 1.07438 0.34236 16.0982 NCR2 72324 downstream 0 overlap 0.02521 chrl 212219253 212219828 1.51148
  • chr2 176723900 176724580 1.17517 0.3365 15.585 HOXD4 0 overlaps 5' 82 disjoint 0.02624 chr9 138810115 138812923 0.52237 1.23234 15.5828 KIAA1984 0 overlaps 5' 881 disjoint 0.02625 chr3 197107717 197108427 0.28981 1.13194 15.582 TN 2 11849 inside intron -290 disjoint 0.02625 chrX 136479176 136479820 0.64895 -0.2463 15.58 ZIC3 3165 overlaps exon 0 overlap 0.02625 upstream
  • chrl7 25468769 25469534 -0.1972 0.73541 15.5395 CCDC55 810 covers exon(s) -632 disjoint 0.02633 chrl l 2233400 2234180 0.8216 1.63515 15.5367 ASCL2 14577 downstream 12500 disjoint 0.02634 chr9 132701631 132702763 0.14232 0.83722 15.5352 ABL1 980 inside intron -525 disjoint 0.02635 chr4 171205682 171206224 0.15767 1.21789 15.5327 AADAT 41533 downstream -20199 disjoint 0.02635 chrI9 47121534 47122274 0.08171 0.84473 15.5182 ARHGEF1 41249 downstream 1482 disjoint 0.02639 chrl4 52326302 52327205 1.20365 0.30153 15.5168 GNPNAT1 927 inside intron 209 disjoint 0.02639 chrl
  • chrl 7 43403472 43404023 0.19458 1.02332 11.5793 CDK5RAP3 45 covers exon(s) 0 overlap 0.03783 chr5 140190451 140191134 -0.0502 0.59791 11.5783 PCDHA6 2618 inside intron -310 disjoint 0.03784 chr9 131409275 131409911 0.661 1.45848 11.5771 C9orf50 12964 downstream 2253 disjoint 0.03784 chrl l 69597308 69598097 0.77316 1.29387 11.5754 TMEM16A 4196 upstream 3889 disjoint 0.03785 chr7 29814368 29815312 1.04575 1.69584 11.5744 SC N1 180589 downstream -1092 disjoint 0.03785 chr7 2639523 2640101 0.467 1.19223 11.5735 TTYH3 1395 inside intron -1060 disjoint 0.03785
  • chrl 9 49023637 49023969 0.84112 1.92747 9.99264 LYPD5 7012 upstream -6788 disjoint 0.04525 chrl l 59138466 59139005 0.21259 0.88183 9.99106 OSBP 1187 inside intron 258 disjoint 0.04525 chr7 100398484 100398984 0.31237 1.02067 9.99061 MUC17 51099 upstream -1971 disjoint 0.04526 chr2 176698813 176699280 1.04925 0.17915 9.98569 HOXD9 3480 downstream -980 disjoint 0.04528 chrl 7 4404264 4404980 0.33318 0.79S26 9.98192 MYBBP1A 412 overlaps two exons 0 overlap 0.0453 chrl 148126311 148127372 -0.0647 0.27136 9.98162 HIST2H2AB 222 promoter -217 dis
  • chr4 40954889 40955503 1.32665 0.78182 9.39132 UCHL1 1204 inside intron -265 disjoint 0.04856 chrlO 42571857 42572513 0.89274 1.46374 9.38879 BMS1L 25751 upstream -939 disjoint 0.04858 chrl 7 33920924 33921412 1.20868 0.42304 9.38675 SNIP 94296 downstream -164 disjoint 0.04859 chrl 7 69708441 69708917 1.07719 1.78064 9.38381 RPL38 2472 promoter 2610 disjoint 0.04861 chrl4 70179420 70179959 0.82681 0.2202 9.38342 MED6 42284 upstream -335 disjoint 0.04861 chrl 8 32018645 32019115 1.29571 0.54563 9.38226 MOCOS 2364 promoter 1274 disjoint 0.0
  • chrX 118708883 118709458 0.72868 1.34083 9.19249 1902 inside intron 1022 disjoint 0.04972 chr4 90446622 90447152 2.55265 1.8928 9.1883 GPRJN3 1031 inside intron 585 disjoint 0.04975 chr5 88021299 88021841 0.61157 -0.001 9.18808 MEF2C 192938 downstream 0 overlap 0.04975 chr20 61477279 61477695 0.82053 1.66728 9.188 CHRNA4 14088 upstream -3802 disjoint 0.04975 chrl 65387779 65388180 0.80959 -0.0079 9.18249 AK3L2 1101 inside intron -293 disjoint 0.04979 chrl 7 45560093 45560766 0.93987 0.41026 9.18193 SAMD14 1399 inside intron 896 disjoint 0.04979 chr8 143400176 14
  • chr6 34966181 34966594 0.56238 1.52146 9.15576 AN S1A 1163 inside intron -539 disjoint 0.04995 chrl 7 34116469 34117469 0.8161 0.44096 9.15554 MLLT6 1069 covers exon(s) -1246 disjoint 0.04996 chr2 16708633 16709362 0.89128 1.57406 9.15118 FAM49A 1214 inside intron -155330 disjoint 0.04998
  • R-DMRs differentially methylated regions
  • chrl l 31784606 31787983 1.41933931 -0.31644601 159.697521
  • PAX6 1450 covers exon(s) -109 disjoint chr4 111780296 111782892 1.63173739 -0.17319743 132.277796
  • PITX2 2340 promoter -39 disjoint chr 6 101009118 101012166 1.56292862 0.07999288 121.511837
  • PAX6 9303 covers exon(s) -260 disjoint chr 5 174086712 174088442 1.75529701 0.00109963 85.6376081 MSX2 2532 inside intron -1742 disjoint chr2 66523155 66525137 1.53956949 -0.09867546 85.0156191
  • MEIS1 7120 covers exon(s) 798 disjoint chr 5 122456101 122458523 1.38936079 -0.02772582 82.6647917 PPIC 55778 upstream 52 disjoint chrl4 69416842 69419462 -0.19824968 1.20834882 82.6223597 SMOCl 947 inside intron -562 disjoint chrlO 22661208 22663161 1.4779070707 -0.07457995
  • chrl 2 113615569 113617118 1.45655962 -0.0416652 63.996903 TBX3 9218 upstream -944 disjoint chr2 144408857 144410536 -0.10667617 1.22862162 63.4659312 GTDC1 395962 downstream 600 disjoint chr20 49847751 49849925 1.04888608 -0.18236327 62.6992872 SALL4 2495 inside intron 794 disjoint chrl3 49600378 49601736 1.55239767 -0.106977 62.687492 KCNRG 112988 downstream 0 overlap chrlO 21827602 21828807 1.40520939 -0.48129466 62.5304957 C10orfl l4 1406 promoter 0 overlap chrl2 79626961 79628584 1.3730142 0.04232383 62.2483215 MYF6 1385 overlaps 3'
  • chr2 176736313 176737800 1.63327343 0.19509096 58.7718208 H0XD3 0 overlaps 5' 0 overlap chr7 27106893 27108134 1.61198644 0.01775409 58.6008133 H0XA2 784 overlaps exon 1572 disjoint upstream
  • chr3 148620314 148621273 1.90219036 -0.15922394 53.257094 ZICl 10444 downstream -299 disjoint chr4 151722401 151723404 1.86044619 -0.29765235 53.0965912 AB21L2 0 overlaps 5' 57 disjoint chr3 148614542 148615993 1.33962917 -0.03015777 52.9759123 ZICl 4672 inside exon -522 disjoint chrl l 31780431 31782210 1.31971017 -0.02747236 52.8408389 PAX6 7223 covers exon(s) 109 disjoint chr3 148587638 148589395 1.2363237 0.19347292 52.7520873 ZIC4 17701 overlaps exon 1803 disjoint upstream
  • chrlO 22652675 22653622 1.54940518 -0.48360594 52.6684368 BMI1 2530 inside intron 659 disjoint chr4 81403269 81404477 1.71294929 0.14782841 52.6540894 FGF5 2288 promoter 2041 disjoint chr 6 10513143 10514243 1.64823552 -0.16251431 52.4938953 TFAP2A 6349 inside intron 0 overlap chrl 212223887 212225199 1.41267876 -0.1036052 52.2142965 PROX1 3283 upstream 150 disjoint chr 5 72631433 72632752 1.55907375 0.03752966 51.5934543 TME 174 131604 downstream 0 overlap chr4 54663407 54664534 1.52539942 -0.1795399 51.3548036 GSH2 2453 close to 3' -587 disjoint chrl 7
  • chr 5 154219106 154220032 -0.13990552 1.31206448 35.9618646 CNOT8 716 inside intron -551 disjoint chrl9 36538829 36539809 1.08996256 -0.3296489 35.9512967 TSHZ3 76815 upstream 0 overlap chrl 162811165 162812119 1.58327994 0.21300667 35.9431306 PBX1 15605 inside intron 45 disjoint chr8 976495 978327 3.5304042 2.196131 35.9429244 ERICH1 305270 upstream 0 cover chr 6 134540510 134541574 -0.02928008 1.30769243 35.9333102 SG 2816 upstream -1061 disjoint chrl 219128497 219129717 1.40212372 0.15148187 35.792526 HLX1 9116 downstream -803 disjoint chr5
  • chrlO 124218089 124218979 0.06066256 1.27337721 29.5720366 HTRA1 7043 inside intron -5859 disjoint chr8 22355564 22356594 -0.1443314 0.83979129 29.5297431 PPP3CC 1024 inside intron -477 disjoint chr4 174659289 174660227 1.43921915 0.30342524 29.5293354 HAND2 27725 downstream -271 disjoint chrl4 51849417 51850354 -0.22788891 1.02669463 29.4617456 PTGER2 508 promoter 83 disjoint chr 5 11955022 11955804 0.24408633 1.6879937 29.3342179 CTNND2 1305 inside intron 746 disjoint chr8 75396686 75397561 -0.35371547 0.94986888 29.331202 JPH1 570 promoter -30
  • chr 5 87476895 87477930 1.2118908 0.33832881 25.812755 TMEM161B 122490 downstream -3634 disjoint chr3 198959742 198960636 -0.00700548 1.14414767 25.8028177 FYTTDl 443 promoter 20 disjoint chr5 87990657 87991382 1.40850902 0.17300754 25.7994468 MEF2C 223397 downstream 12 disjoint chrl 6 78193900 78194718 -0.25593462 0.86733562 25.7980389 MAF 1789 promoter -862 disjoint chr 5 45732504 45733256 0.11040816 1.40923399 25.7931255 HCN1 528 promoter -237 disjoint chr4 108964323 108964958 0.98319248 -0.4743061 25.7718278 MGC26963 68920 upstream
  • chrl 6 83877307 83877974 -0.09566556 1.16751429 24.6951009 FAM92B 173693 upstream 101 disjoint chr6 26152486 26153633 -0.08242374 1.23088597 24.6457558 HIST1H3C 0 overlaps 5' 0 overlap chrl7 35473832 35474651 -0.24125587 1.04278392 24.6187759 THRA 1244 inside intron -426 disjoint chrl7 56884781 56885461 1.13423668 -0.09680232 24.5924833 TBX4 3127 upstream 0 overlap chr3 24847250 24848266 -0.1072094 0.8417926 24.5730156 THRB 335934 upstream -877 disjoint chrl 119349858 119350709 1.63505916 0.44995063 24.5494468 TBX15 16157 upstream
  • chrl 7 78634924 78635571 -0.03917123 1.3151442 23.3594199 ETRNL 4069 inside intron 344 disjoint chr7 27169657 27170448 -0.00150461 0.96758684 23.3384608 HOXA9 1225 overlaps exon 0 overlap upstream
  • chrl 4 102744495 102745371 -0.08638888 0.97119929 23.3303348
  • TNFAIP2 82079 downstream -399 disjoint chrl 7 39992624 39993398 1.09543418 -0.02947563 23.3280657
  • FZD2 2174 close to 3' -247 disjoint chr2 60637613 60638356 1.04020067 -0.15752936 23.2394687 BCL11A 3477 upstream -1330 disjoint chr6 107544512 107545588 0.5903851 -0.22704599 23.2119459 PDSS2 341859 downstream -509 disjoint chrl 6 85168280 85168919 1.39358483 -0.03315958 23.1997553 FOXL1 696 promoter -314 disjoint chr2 96563766 96564515 0.02491383 1.04824304 23
  • chr6 1337367 1338080 0.90474756 -0.13948429 20.9499525 FOXF2 2300 inside intron 0 overlap chrlO 80872778 80873673 0.22766148 1.08428375 20.929173 C10orf56 1712 inside intron 785 disjoint chrl2 28015483 28016172 1.10397108 -0.15753488 20.9143598 PTHLH 10 overlaps exon -443 disjoint downstream
  • chr5 43080648 43081358 1.32666084 0.21708381 20.8618919 LOC389289 4551 upstream -1414 disjoint chr 6 34468801 34469546 -0.0602856 1.25743283 20.838668 NUDT3 383 promoter -21 disjoint chrl 168896553 168897161 1.41376343 0.18887288 20.8231393 PRRX1 2775 upstream 0 overlap chr7 25866943 25867761 1.58011652 0.37347007 20.8204436 NFE2L3 290623 upstream 885 disjoint chr4 141437002 141437853 0.42826366 1.36598175 20.8158184 SCOC 39113 inside intron -42985 disjoint chr2 28471161 28471700 -0.12084794 1.29905966 20.7885446 FOSL2 1879 inside intron -909 disjoint chrl
  • chrl 6 23756839 23758051 0.24801637 1.11289981 19.9848659 PRKCB1 2017 inside intron -1236 disjoint chr2 139252241 139253103 0.81921501 -0.0704156 19.9665534 NXPH2 1177 inside intron 1059 disjoint chr9 20614264 20614909 0.86475669 -0.16525658 19.9608637 MLLT3 1815 promoter -2402 disjoint chr3 170867640 170868332 0.83735758 -0.12887168 19.9211925 MDS1 3473 upstream 627 disjoint chr22 29696207 29696866 1.81017429 0.45033463 19.9047729 M0RC2 2021 promoter -632 disjoint chr8 1494643 1495250 0.41947829 1.58733939 19.8995937 DLGAP2 57668 inside intron 2007 disjoint ch
  • chr2 120019890 120020363 -0.23899867 1.25658172 19.442545 MGC33657 1374 covers exon(s) -965 disjoint chrl3 44782546 44783223 0.11652972 1.07695178 19.4397457 TPT1 30073 downstream 531 disjoint chr6 44147359 44147862 -0.11434842 1.28576208 19.4385456 MRPL14 55306 downstream 1130 disjoint chr5 92930972 92931733 1.16465651 0.25631026 19.4249583 NR2F1 13065 upstream 262 disjoint chrl6 52882066 52882578 1.1163736 -0.38002026 19.4201993 IRX3 4188 upstream 0 overlap chr6 1560799 1561626 1.27314557 0.3309289 19.4179463 FOXC1 5120 downstream 0 overlap chr7 27171652
  • chrl 2 45510421 45511068 1.29054192 0.22500936 19.2418273
  • NPEPL1 1650 promoter 1316 disjoint chrl 6221722 6222315 1.25712975 0.0500916 19.2328187
  • PCDHB6 442 promoter 1761 disjoint chr6 101024531 101025091 1.63725372 0.35335814 19.209085
  • chrl 6 48746440 48747336 -0.1732433 0.5566583 19.0881656
  • PAPD5 872 inside intron -88405 disjoint chr3 62330365 62331099 0.91118533 -0.10977465 19.080657
  • FEZF2 2961 overlaps 3' 0 overlap chrlO 69318244 69318888 -0.19935737 0.97219612 19.0792984
  • SIRT1 3812 covers exon(s) -3060 disjoint chr7 153213687 153214225 -0.08637869 1.31414203 19.0737415 DPP6 1126 promoter 25 disjoint chrl4 60191248 60192143 0.95850211 0.19798588 19.0612964 SKI 5316 upstream 1223 disjoint chr7 115639913 115640451 -0.31251243 1.01967809 19.0491695 TES 2097 inside intron
  • chrl 47466118 47466876 0.95632283 0.01352992 15.6017797 TALI 1153 inside intron -1804 disjoint chrl 7 5614070 5614799 -0.47487606 0.9144511 15.5936072 NALP1 185518 upstream -268744 disjoint chrl 7 70660138 70660729 1.07432545 0.07260763 15.5919191 HN1 1639 inside intron 696 disjoint chr3 165201691 165202224 0.24859832 1.4654294 15.5877523 SI 1076751 downstream 2517 disjoint chr2 205119425 205120105 -0.02652207 0.93653152 15.5801167 PARD3B 665 inside intron -270 disjoint chr2 218423333 218423914 0.09888264 1.28928094 15.5751583 TNS1 93091 inside intron -93590 disjoint ch
  • chrl 3 49598457 49599030 1.15950904 0.09131337 15.2857251 KCNRG 111067 downstream 0 inside chr7 27173819 27174511 0.10011545 0.88385961 15.2832056 HOXA9 2146 promoter -832 disjoint chr22 24129361 24130027 -0.15022157 0.94820338 15.2813576 LRP5L 40838 upstream 527 disjoint chr 6 7488826 7489329 1.16963066 0.10705188 15.259984 DSP 1958 inside intron -355 disjoint chrl 4 89917469 89918260 0.02019087 0.78768982 15.2481162 CALM1 14865 upstream 487 disjoint chrlO 127573396 127574007 -0.2562742 0.59364582 15.2413475 FANK1 1093 promoter 232 disjoint chrl 8 27991860 2799
  • chrl4 55115260 55115838 0.06026981 1.09635989 15.0477114 KTN1 954 promoter 549 disjoint chr3 188943023 188943515 1.6105657 0.50409136 15.0425538 BCL6 2653 inside intron 1150 disjoint chrl 184914668 184915177 0.17700789 1.23620617 15.0401466
  • PTGS2 1001 covers exon(s) 756 disjoint chrl2 52061585 52062272 -0.21970938 0.74744373 15.030235
  • SP1 1340 covers exon(s) -715 disjoint chrl 6 51136372 51136986 0.58749123 -0.30327507 15.0205933
  • chrl 7 6859295 6859972 0.06620085 0.8593832 14.5886562 C17orf49 448 covers exon(s) -32 disjoint chrll 125733920 125734803 0.12385162 1.24439347 14.5763201 ST3GAL4 2615 inside intron -2637 disjoint chrl 3 23050160 23050851 0.61891944 1.58584558 14.5760977 TNFRSF19 7438 inside intron 48 disjoint chr4 75936247 75936936 0.45554409 1.24062466 14.5747766 BTC 1916 inside intron 1189 disjoint chrlO 9488815 9489495 2.93836283 1.96294354 14.5698227 LOC389936 0 overlaps 5' 0 overlap chrlO 6663566 6664277 1.80616017 0.96291777 14.5633303 PRKCQ 1323 promoter -9
  • chrl l 31782580 31782945 1.10190013 -0.23662348 13.8188127 PAX6 6488 inside intron 0 inside chr6 27755547 27756240 1.13397798 0.16548022 13.8140645 HIST1H2BL 127447 downstream 0 cover chrl2 56523029 56523635 0.1909465 1.0978757 13.8108104 CTDSP2 3378 inside intron 1472 disjoint chrlO 118881250 118881870 0.92836145 0.04348301 13.8095704 VAX1 5931 overlaps exon 281 disjoint upstream
  • chr20 5 0156049 50156749 1.22096739 0.293302 13.1724017 ZFP64 85181 inside intron -730 disjoint chr5 66336576 66337220 0.81359734 0.08392192 13.1722767 LOC375449 408613 downstream -737 disjoint chrl 36625214 36626043 0.11955891 1.10377077 13.1690871 STK40 1143 promoter -754 disjoint chr2 120233007 120233357 0.9856377 -0.4603832 13.1562836 PTPN4 319 promoter 179 disjoint chr5 50296152 50296517 1.2761158 0.02949335 13.1485789 PARP8 297583 downstream 4325 disjoint chr2 211048605 211049051 -0.1551726 1.04103474 13.1461749 LANCL1 624 inside intron 375 disjoint chrl2 52655707
  • chr7 31341062 31341670 0.30081097 1.140024 13.0989209 NEUROD6 5392 downstream 700 disjoint chr2 176748865 176749335 0.81165516 -0.20298357 13.0976195 HOXD3 11815 downstream -668 disjoint chrlO 116382204 116382707 -0.21133972 0.71330692 13.0927624
  • ABLIM1 25340 inside intron -387 disjoint chrl 3 21140853 21141359 0.97782451 -0.005568 13.0810497 FGF9 2515 upstream 0 overlap chr3 158358839 158359426 -0.07770354 0.72699375 13.0517254 CCNL1 1749 overlaps exon 440 disjoint downstream
  • chrl 6 30596705 30597566 1.06026734 0.08908104 13.0511148 FBS1 12578 downstream -18049 disjoint chr8 102574006 102574620 1.11597252 0.04037331 13.0463219 GRHL2 0 overlaps 5' 0 overlap chrl 182621283 182621981 1.09931176 0.12661556 13.0417553 Clorf21 791 promoter 57 disjoint chrl 5 46722744 46723391 -0.53647504 0.19198763 13.0403565 FBN1 1818 inside intron 711 disjoint chr7 157064591 157064986 0.08689137 1.2722443 13.0388822 PTPRN2 1008192 inside intron -2436 disjoint chr2 200033986 200034541 0.8934441 -0.12674405 13.0319511 SATB2 541 promoter 0 inside ch
  • GDAP1 737 covers exon(s) -345 disjoint chr6 1557371 1557966 0.88765319 0.06395249 12.871152 FOXC1 1692 close to 3' 0 inside chr6 3693931 3694510 0.24966374 1.05134019 12.8677269 C6orfl45 2424 inside intron 1277 disjoint chr20 31748839 31749277 0.50588464 1.57785806 12.8636811 PXMP4 22519 downstream -10673 dis
  • chrll 7230822 7231223 1.01295717 -0.07891836 11.7446629 SYT9 1066 inside intron -11 disjoint chrl 6 71654712 71655274 0.9935094 0.21870991 11.744265 ATBF1 14938 upstream -96 disjoint chr6 24512676 24513041 1.52213034 0.26582346 11.7414747 MRS2L 1545 inside intron -1011 disjoint chr 5 122449676 122450107 1.18345786 0.14911913 11.7159406 PPIC 49353 upstream 2129 disjoint chr5 77181413 77181882 1.4502124 0.38568228 11.7098314 TBCA 73473 upstream 872 disjoint chrl 7 44062558 44063061 0.4059112 1.2010684 11.7085174 H0XB9 3725 upstream 2750 disjoint chr3
  • chr9 76305586 76306126 0.72579163 -0.10460337 11.0305174 RO B 3515 inside intron -1839 disjoint chrl 7 67623025 67623616 1.02502585 0.20738829 11.0251212 SOX9 5139 upstream 0 overlap chrX 136474847 136475400 1.17127285 0.07339791 11.0198597 ZIC3 611 promoter 0 overlap chrX 102489173 102489608 0.97582405 -0.01364143 11.0152644 WBP5 8427 upstream 28369 disjoint chr2 171385173 171385930 1.03045756 0.35936051 11.0138522 GADl 3728 inside intron 0 overlap chr3 185805542 185806029 1.43642482 0.50212969 11.0095501 EPHB3 43262 downstream -574 disjoint chr2 175058791 1750
  • chr20 55271171 55271722 1.03787897 0.37135108 10.9080068 BMP7 2985 inside intron 972 disjoint chr7 56141094 56141420 -0.34870077 0.92816033 10.906647 CHCHD2 260 inside intron 0 overlap chrl 7 41214998 41215399 0.09873197 1.0752817 10.8924431 CRHR1 2049 promoter 1048 disjoint chrl2 26157097 26157579 0.98283759 -0.20596333 10.8905497 BHLHB3 11533 downstream 746 disjoint chrX 119014627 119015199 -0.47682618 0.24357145 10.8805575 NKAP 52865 upstream 2680 disjoint chr4 36924025 36924570 0.27188648 1.08052563 10.8630867 C4orfl9 337679 upstream -651 disjoint chrl
  • chrl 2 70121556 70122098 0.10724426 0.79213639 10.2135731 LGR5 1477 inside intron -900 disjoint chr2 144807731 144808096 1.47855782 0.46355056 10.2126472 GTDC1 1233 promoter -864 disjoint chrlO 134388042 134388491 2.67713318 1.67824327 10.2016004 INPP5A 186700 inside intron 1316 disjoint chr 5 151765213 151765608 0.52900023 1.41472367 10.1997065 NMUR2 197 promoter -480196 disjoint chrl3 32060799 32061194 -0.11708394 0.80996368 10.1975238 APRIN 2176 inside intron -964 disjoint chr2 174904668 174905162 0.85683204 0.09466623 10.1974261 FLJ46347 5377 downstream 2547 disjo
  • chrl 6 2902926 2903396 0.52022455 1.35335458 10.1461996 FLYWCH1 946 inside intron -393 disjoint chr5 170765553 170766023 0.16191373 1.01722263 10.1418714 PM1 18151 overlaps exon 12342 disjoint upstream
  • Table 7 Regions of differential methylation between iPS cells and ES cells.
  • DMRs were defined by using a absolute area cutoff of 10.0 which corresponds in magnitude to the 5% FDR cutoff of the R-DMRs.
  • chrl 5 97225652 97227801 0.227471611 1.080152523 51.5937901 IGF1R 215365 inside intron -12153 disjoint chr6 27633621 27634805 0.108399472 1.289575618 38.81697032 ZNF184 84764 upstream -38573 disjoint chr5 43072850 43074823 1.466157509 0.5817001 37.26230862 LOC389289 1274 close to 3' 0 cover chr20 9437038 9438216 0.051275945 0.987698218 31.22453135 C20orfl03 5054 upstream -1149 disjoint chrl 4 28316507 28317651 0.760956759 -0.17642005 28.07
  • Table 8 Gene ontology functional categories enriched in differentially methylated regions between iPS cells and ES cells.

Abstract

La présente invention concerne des régions méthylées de manière différente (DMR) de cellules souches pluripotentes induites (iPS) reprogrammées (R-DMR) ainsi que des méthodes d'utilisation de ces dernières. La présente invention porte sur des procédés de détection et d'analyse d'altérations de l'état de méthylation de DMR dans des cellules iPS, des cellules somatiques et des cellules souches embryonnaires (ES) ainsi que sur des méthodes de reprogrammation de cellules somatiques pour générer une cellule iPS.
PCT/US2010/033281 2009-10-14 2010-04-30 Régions méthylées de manière différente de cellules souches pluripotentes induites reprogrammées, méthode et compositions correspondantes WO2011046635A1 (fr)

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012037456A1 (fr) * 2010-09-17 2012-03-22 President And Fellows Of Harvard College Analyse de génomique fonctionnelle pour caractérisation de l'utilité et de l'innocuité de cellules souches pluripotentes
WO2014194884A3 (fr) * 2013-06-07 2015-04-09 Klaus Olek Procédé de détection d'acides nucléiques, acides nucléiques et oligonucléotides complémentaires destinés à l'identification de matériel biologique, procédé et kit d'identification de matériel biologique
US10626445B2 (en) 2013-06-10 2020-04-21 President And Fellows Of Harvard College Early developmental genomic assay for characterizing pluripotent stem cell utility and safety
US11085067B2 (en) 2013-06-10 2021-08-10 President And Fellows Of Harvard College Early developmental genomic assay for characterizing pluripotent stem cell utility and safety
CN105233290A (zh) * 2015-07-31 2016-01-13 北京泱深生物信息技术有限公司 C22orf26基因及其表达产物在制备帕金森诊疗试剂中的应用
CN105233290B (zh) * 2015-07-31 2018-02-09 北京泱深生物信息技术有限公司 C22orf26基因及其表达产物在制备帕金森诊疗试剂中的应用
CN105238790A (zh) * 2015-11-26 2016-01-13 河南省农业科学院畜牧兽医研究所 一种调控猪aebp1的启动子及构建方法、转染载体及构建方法、应用
CN105238790B (zh) * 2015-11-26 2018-01-30 河南省农业科学院畜牧兽医研究所 一种调控猪aebp1的启动子及构建方法、转染载体及构建方法、应用

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