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  • C12N2510/00—Genetically modified cells

Definitions

• DNA damage depending on the nature of the damage, they activate different DNA damage repair mechanisms (Sinclair et al. (2004) Am. Nat., 164: 396-414). In parallel, cells also activate checkpoint pathways, which delay cell cycle progression until genome integrity has been restored (Shiloh (2001) Curr. Opin. Genet. Dev., 11 : 71-77).
• One aspect of the stem cell hypothesis of aging postulates that the gradual and coordinated age-related loss of DNA damage repair capacity results in DNA damage accumulation over time. This damage would pose a significant threat to adult stem cell survival by altering proliferation and differentiation patterns, ultimately triggering cellular senescence. Therefore, the ability of adult stem cells to monitor and faithfully repair DNA damage is key to the prevention of aging and neoplastic transformations.
• Retrotransposon transcriptional activities trigger and guide the processes of (i) assembly of centromeric chromatin, (ii) gene transcription, (iii) compartmentalization of chromatin and, (iv) nuclear organization of chromatin insulation during X-chromosome inactivation.
• Retrotransposons also serve a distinct function in non-random chromosomal translocations in tumors (Allen et al. (2004) Nat. Struct. Mol. Biol., 11 : 816-821; Chueh et al. (2005) Hum. Mol.
• SINE/Alus, and other interspersed retrotransposons have variable degrees of H3K9, H3K27, and H4K20 histone methylation, raising the possibility that posttranscriptional
• PTM retrotransposal chromatin
• Efficient repair of DNA double-strand breaks and authentic genome maintenance at the chromatin level are fundamental to faithful human adult stem cell self- renewal. Stem cell aging can be linked to deficiencies in these two processes. In one example, we report that -65% of naturally occurring repairable damage in self-renewing adult stem cells occurs in transposable elements. Upregulation of transcriptional activity from SINE/Alu retrotransposons interferes with the recruitment of condensin I and cohesin complexes in pericentric chromatin, resulting in the loss of efficient DNA repair and, in turn, senescence.
• Stable knockdown of generic SINE/Alu transcripts in senescent human adult stem cells reinstates the cells self-renewing properties and unexpectedly increases their plasticity as manifested by upregulation of Nanog and Oct4.
• the results provided herein demonstrate the functional significance of SINE/Alu retrotransposons and provide mechanistic insight into their novel role in mediating crosstalk between chromatin, DNA repair and aging of human adult stem cells.
• methods are provided for restoring a non-senescent phenotype, or aspects of a non-senescent phenotype to a senescent cell ⁇ e.g., a senescent adult stem cell).
• methods are provided for maintaining a non- senescent phenotype, or aspects of a non-senescent phenotype in a cell ⁇ e.g., a senescent adult stem cell).
• methods are provided for inducing and/or restoring and/or maintaining a non-senescent phenotype, or aspects thereof ⁇ e.g., proliferative capacity and/or pluripotency) in a mammalian cell.
• methods of transdifferentiating a mammalian cell from a first cell type or lineage into a second cell type or lineage typically involve transforming a differentiated cell into a pluripotent cell (or restoring pluripotency to a stem cell or a progenitor cell) by one of the methods described herein (e.g.
• the first cell type is a mesodermal cell type. In certain embodiments the first cell type is an ectodermal cell type. In certain embodiments the first cell type is an endodermal cell type. In certain embodiments the first cell type or lineage is a mesodermal cell type and the second cell type or lineage is a neuroectodermal cell type.
• the first cell type is an ectodermal cell type and the second lineage is a mesodermal or an endodermal cell type. In certain embodiments, the first cell type is an endodermal cell type and the second lineage is an ectodermal or mesodermal cell type. In certain embodiments the first cell type is an adipocyte or a bone marrow cell. In certain embodiments the second cell type is a cell type selected from the group consisting of a blood cell, a fetal cell, an epithelial cell, an adipocyte, a smooth muscle cell, a nerve cell, a pancreatic beta cell, and a cardiomyocyte.
• the culturing the cell under conditions that induce or permit differentiation of said cell comprises culturing said cells in a medium lacking or having a reduced quantity of leukemia inhibitory factor (LIF) and/or contacting (or culturing) the cell with one or more reagents (e.g., retinoic acid, PDGF, insulin, Arctigenin, ATRA (vitamin A), boswellic acid, bromelain and other proteolytic enzymes, CAPE , flavonoids (including apigenin, luteolin, quercetin, genistein,, and daidzein), emodin, EPA and DHA,
• LIF leukemia inhibitory factor
• Micro-R As are single-stranded R As of typically 22-nucleotides that are processed from ⁇ 70-nucleotide hairpin R A precursors by the Rnase III nuclease, Dicer. Similar to siRNAs, miRNAs can silence gene activity through destruction of homologous mRNA in plants or blocking its translation in plants and animals.
• shRNA or short hairpin RNA is an RNA molecule that contains a sense strand, antisense strand, and a short loop sequence between the sense and antisense fragments. Due to the complementarity of the sense and antisense fragments in their sequence, such RNA molecules tend to form hairpin-shaped double-stranded R A
• shRNA is cloned into a vector, allowing for expression by a pol III type promoter. The expressed shRNA is then exported into the cytoplasm where it is processed by dicer into siRNA which then get incorporated into the siRNA induced silencing complex (RISC).
• RISC siRNA induced silencing complex
• Small Interfering RNA are typically 21 -23 nucleotide double- stranded RNA molecules. Once incorporated into the RNA-induced silencing complex (RISC) they facilitate the cleavage and degradation of its recognized mRNA.
• RISC RNA-induced silencing complex
• Piwi-interacting RNA is class of small non-coding RNA molecules that is expressed in, or can be introduced into animal cells (see, e.g., Seto et al. (2007)
• piRNAs form RNA-protein complexes through interactions with piwi proteins. These piRNA complexes have been linked to both epigenetic and post-transcriptional gene silencing of retrotransposons and other genetic elements.
• pluripotency and “pluripotent stem cells” it is meant that such cells have the ability to differentiate into all types of cells in an organism. Pluripotent cells are characterized by the expression of one or more pluripotency markers known by one of ordinary skill in the art.
• pluripontent cells are capable of forming or contributing to ectoderm, mesoderm, or endoderm tissues in a living organism.
• primary cells are used interchangeably herein to refer to cells and cell cultures that have been derived from a subject and allowed to grow in vitro for a limited number of passages, i.e. splittings, of the culture.
• primary cultures are cultures that may have been passaged 0 times, 1 time, 2 times, 4 times, 5 times, 10 times, or 15 times, but not enough times go through the crisis stage.
• the primary adipose cells of the present invention are maintained for fewer than 10 passages in vitro prior to use.
• adipose is meant any fat tissue.
• the adipose tissue may be brown or white adipose tissue, derived from subcutaneous, omental/visceral, mammary, gonadal, or other adipose tissue site.
• the adipose is subcutaneous white adipose tissue or visceral adipose tissue or a lipoaspirate sample.
• the adipose tissue may be from any organism having fat tissue.
• the adipose tissue is mammalian, most preferably the adipose tissue is human.
• a convenient source of adipose tissue is from liposuction surgery, however, the source of adipose tissue need not be so limited.
• Figures 1A-1C illustrate features of the ex-vivo aging properties of hADSCs.
• Figure 1A Representative cumulative long term growth curve. Three distinct states are shown: SR- self-renewing (population doubling ⁇ 17); preSEN- presenescent (population doubling 29-38); SEN- senescent (population doubling >39).
• Figure IB illustrates features of the ex-vivo aging properties of hADSCs.
• Figure 1A Representative cumulative long term growth curve. Three distinct states are shown: SR- self-renewing (population doubling ⁇ 17); preSEN- presenescent (population doubling 29-38); SEN- senescent (population doubling >39).
• Figure IB illustrate features of the ex-vivo aging properties of hADSCs.
• Figure 1A Representative cumulative long term growth curve. Three distinct states are shown: SR- self-renewing (population doubling ⁇ 17); preSEN- presenescent (population doubling 29-38); SEN-
• FIG. 1C DNA damage response (DDR) in senescent hADSCs. Representative immunostaining for persistent ⁇ 2 ⁇ (green)/53BPl (red) foci formation upon senescence of hADSCs. Quantification of ⁇ 2 ⁇ /53 ⁇ 1 foci formation upon ex-vivo expansion of hADSCs is given in Figure 8.
• Figures 2A-2G illustrate genome-wide location analysis of ⁇ 2 ⁇ .
• FIG. 2A Schematic flow-chart for nucleosomal ChlP-seq analysis using SOLiD (ABI) platform.
• Figure 2B Relative chromosomal distributions of ⁇ 2 ⁇ tags in self-renewing and senescent cells illustrated for chromosomes 10 and 21.
• ⁇ 2 ⁇ tag enrichment levels calculated as the log2 ratio of position-specific tag counts normalized by the genomic background, are shown for self-renewing and senescent cells.
• Relative differences in ⁇ 2 ⁇ tag enrichment levels between cells calculated as the absolute values of the differences in cell stage specific enrichment levels, are shown below the individual cell tracks. Below the difference tracks, the chromosomal locations of large clusters of ⁇ 2 ⁇ modified sites are shown for the self-renewing and senescent cell types.
• Figure 2C Relative chromosomal distributions of ⁇ 2 ⁇ tags in self-renewing and senescent cells illustrated for chromosomes 10 and 21.
• Figure 2E Scatter plots showing the relationship between GC content and ⁇ 2 ⁇ tag density for human chromosomes in self-renewing (SR) and senescent cells (SEN). The slopes and intercepts of the linear trends are shown (y- value) along with Spearman's rank correlation coefficients (R-value) and statistical significance levels (P-value).
• Figure 2F The enrichment levels of ⁇ 2 ⁇ in promoter regions, surrounding transcriptional start sites (TSS), are shown for self-renewing (blue) and senescent (red) cells. Enrichment levels are calculated as the log2 ratio of the position- specific tag counts normalized to the genomic background.
• Figure 2G The numbers of genes with ⁇ 2 ⁇ modified sites in promoter regions, defined as ⁇ 2Kb from transcription start sites, are shown for self-renewing (SR) and senescent (SEN) cells. The number of genes with ⁇ 2 ⁇ modified promoters found in both cell phenotypes is indicated in the intersection.
• SR self-renewing
• SEN senescent
• Figures 3A-3E show peri-telomeric and pericentric accumulation of ⁇ 2 ⁇ .
• Figure 3A ⁇ 2 ⁇ enrichment levels in peritelomeric regions for self-renewing (SR - blue) and senescent (SEN - red) cell lines. Chromosome ends (telomeres) are shown at the origin of the x-axis, which then extends into the chromosome arms. Average ⁇ 2 ⁇ enrichment levels are calculated as the log2 ratio of the position-specific tag counts normalized to the genomic background averaged over all chromosome arms.
• Figure 3B ⁇ 2 ⁇ enrichment levels in pericentric regions for self-renewing (SR - blue) and senescent (SEN - red) cell lines.
• Centromeres are shown as gap centered on the x-axis, which extends into the chromosome arms in either direction. Average ⁇ 2 ⁇ enrichment levels are calculated as the log2 ratio of the position-specific tag counts normalized to the genomic background averaged over all chromosomes.
• Figure 3E Differences in the numbers of large ⁇ 2 ⁇ clusters in pericentric regions between senescent versus self- renewing cells. The absolute values of the normalized differences for ⁇ 2 ⁇ clusters between cell phenotypes are shown on the y-axis.
• FIG. 4 Panel A: Immunofluorescent labeling of self-renewing hADSCs. Cells were seeded on coverslips and co-stained with anti-CENP-A (green) and anti-53BPl (red) antibodies. DAPI staining is shown in blue. Confocal image of representative interphase nucleus is shown as separate channels and as a merged image.
• Panel C Association of persistent DNA damage sites upon senescence with regions of high transcriptional activity. Co-immunostaining of DNA damage foci depicted by 53BP1 antibodies (green) with PML bodies (blue) and nascent RNA (red) studied by confocal microscopy. Senescent hADSCs were incubated with halogenated precursor FUr for 10 min in vivo, fixed and stained with antibodies. BrdU antibodies were used to detect FUr labeled RNA. Representative image of a single nucleus is shown. Arrow points at the site of DNA damage focus co-localized with RNA. Spatial relationship between FUr incorporation sites, 53BP1 and PML bodies, is shown.
• C) Loss of cohesin and condensin I in the peri-centric location of persistent DNA damage in senescent hADSC. ChIP analysis of the peri-centric repeats on chromosome 10 in self- renewing (blue bars) and senescent (red bars) hADSCs. Repeats were assessed as locations for recruitment of TFIIIC, Ecol as well as components of cohesin (Rad21) and condensin I (CAP-H) complexes (n 3, ⁇ SEM). Schematic representation of the subunits of the cohesin and condensin I complexes, as well as a cartoon of previously reported function of Ecol, are shown. *p ⁇ 0.02 , **p ⁇ 0.2.
• Figures 6A-6G illustrate that stable knockdown of generic SINE/Alu transcript in senescent human adult stem cells restores cell's proliferative properties and produces iPS-like phenotype.
• Figure 6A Model of SINE/Alu retrotransposon. Secondary structure of generic SINE/Alu RNA (SEQ ID NO: 1). Regions for shRNA design are shaded.
• Figure 6B Representative example of the efficiency of lentiviral transduction of hADSCs depicted by GFP.
• Figure 6C Northern blot hybridization of the RNA recovered from hADSCs cells stably expressing sh-RNA against SINE/Alu.
• Senescent hADSCs were infected with lentiGFP sh-193Alu, lentiGFP sh-132Alu or control no shRNA insert lentiGFP. RNA was isolated after 24hrs post transduction and Northern hybridization was performed with a SINE/Alu specific oligonucleotide. Senescent hADSCs stably expressing sh-132Alu show near complete knockdown of the SINE/Alu transcripts.
• Figure 6D
• Figure 6F Morphological changes in the reversed-senescence hADSCs with stable knockdown of SINE/Alu transcripts.
• Figure 6G Model of SINE/Alu transcriptional interference in triggering persistent DDR causing senescence of human adult mesenchymal stem cells.
• Figures 7A-7B show FACS analysis and proliferative properties of hADSCs.
• FIG. 7A FACS analysis of hADSCs. Early PD hADSCs were stained with FITC (CD 31, CD44 and CD 45) or AlexaFlour-488 (CD 105) conjugated antibodies against cell surface markers and subjected to flow cytometric analysis. The cells were positive for CD 105 and CD 45, and negative for CD 34 and CD 44. The cell populations are shown as fluorescence to side scatter graphs (left), and the histograms (right) of stained cells (blue line) compared to un-stained cells (red line); with percentage of positive cells indicated.
• Figure 7B Replication capacity of hADSCs declines with ex-vivo aging.
• SR self-renewing
• preSEN pre-senescent
• SEN senescent cells
• Figure 8 shows quantification of accumulation of persistent DNA damage foci with ex-vivo passaging of hADSCs.
• ⁇ 2 ⁇ was stained with affinity-purified rabbit polyclonal antibody. Histogram indicates the percentage of the cells with 1, 2, 3 or more than 3 foci. Representative examples are shown below.
• Foci formation was scored in self- renewing, SR (population doubling less than 17), pre-senescent, preSEN (population doubling more than 29, but less than 38) and senescent, SEN (population doubling more than 39) hADSCs cultures.
• n total number of nuclei counted in all 3 independent experiments.
• Figure 9 shows DNA damage response activation in senescent hADSC.
• Senescent hADSC cultures were immunostained with antibodies against phosphorylated forms of Chkl (S345), Chk2 (T68) and cdc2 (Tyrl5) (brighter) and DAPI (dimmer). 50 mm confocal sections are shown. Chkl and Chk2 are transducer kinases that act downsteam of ATM/ATR kinase to provide for DNA damage checkpoint control. Contrary to genotoxic stress or irradiation induced DNA damage, senescent hADSCs do not show robust nuclear localization of phosphorylated forms of Chkl (S345) and Chk2 (T68). [0027] Figure 10.
• FIGS. 1 lA-11C show a comparison of mono-nucleosomal sized ⁇ 2 ⁇ modified positions to genomic clusters of ⁇ 2 ⁇ modified nucleosomes. ⁇ 2 ⁇ modified mono-nucleosomes and ⁇ 2 ⁇ modified clusters were identified as described in Supplemental Methods.
• Figure 11 A Frequency distributions for ⁇ 2 ⁇ modified genomic positions in self-renewing (SR) and senescent (SEN) hADSCs. Mono- nucleosomal sized positions dominate the distributions. Accordingly, the frequencies of mid-size and large ⁇ 2 ⁇ modified nucleosome clusters are enlarged and shown as insets for clarity.
• Figure 11B Percentages of genomic features occupied by ⁇ 2 AX modified mono-nucleosomes and large clusters of ⁇ 2 ⁇ modified nucleosomes.
• Figure 11C Percentages of genomic features occupied by ⁇ 2 AX modified mono-nucleosomes and large clusters of ⁇ 2 ⁇ modified nucleosomes.
• Relative entropy was calculated as a measure of the difference between the SR versus SEN ⁇ 2 ⁇ cluster size frequency distributions (see panel Fig. 11A). The relationship between relative entropy and cluster lengths was used to calculate a threshold (7,400 nt) between mid-size and large clusters as described in Supplemental Methods.
• Figure 12 shows relative enrichment of ⁇ 2 ⁇ modified nucleosomes in peri-telomeric versus pericentric genomic regions. Enrichment values were calculated as log2 normalized ratios of the ⁇ 2 ⁇ ChlP-seq tag counts per position in each region divided the genomic background tag counts per position. Values for self-renewing (SR) cells are shown in blue and for senescent (SEN) cells in red. Peri-telomeric regions are depleted for ⁇ 2 ⁇ , whereas pericentric regions are enriched
• FIG. 13 panels A-D, show persistent ⁇ 2 ⁇ /53 ⁇ 1 foci in senescent hADSCs are associated with centromeric regions.
• Panel A Quantification of CENP-A and 53BP1 co-localization in senescence. Senescent hADSCs were stained with antibodies against CENP-A (green) and 53BP1 (red) and DAPI (blue). Total of 200 cells were scored from three independent experiments. Error bars represent +/- SAM. Example of higher magnification of the image is shown Panel B. Scale bar ltm. Images were analyzed by IMARIS software with optical sections representation as depicted in Figures 4C and 4D.
• FIG. 14 illustrates SINE/Alu expression in SR and SEN hADSCs.
• RNA of 2 ⁇ g per lane was loaded as described in Experimental Procedures. Ribosomal small RNAs can be seen in the ethidium bromide stained gel for loading comparison. The ssRNA ladder sizes are indicated on the right.
• Figure 15A and 15B illustrates lentiviral shRNA-mediated knockdown of generic Alu transcripts in ADSCs and the effects of such knock down on cell senescence and proliferation.
• Figure 15A schematically illustrates a protocol for stably knocking-down Alu transcripts using an shRNA delivered by a lentiviral vector.
• Figure 15B illustrates the delivery vector, transfected cells and a Northern blot showing the results of transfection.
• Figure 16 illustrates the effect of lentiviral shRNA-mediated knockdown of generic Alu transcript in ADSCs. Cell proliferation is show as a function of time.
• Figure 17 illustrates the morphology of Alu shRNA mutant ADSCs in culture and their initial characterization.
• Figure 18 shows a comparison between "standard” protocols for the generation of induced pluripotent stem cells (iPSCs) and one of the protocols described herein.
• iPSCs induced pluripotent stem cells
• Figures 19A and 19B illustrate the subsequent differentiation of Alu shRNA mutant cells.
• Figure 19A illustrates the differentiation of ADSCs into neuroblasts.
• Figure 19B illustrates the use of the methods described herein to transdifferentate cells into numerous other pathways/lineages.
• Figures 20A-20E illustrates the transdifferentiation of kidney fibroblasts into neuroblasts.
• Figure 20A illustrates HEK 293T cells two days after infection with sh- 132Alu.
• Figure 20B illustrates colony formation 7 days after single colony isolation and desegregation into individual cells (ES medium supplemented with LIF).
• Figure 20C shows the expression of pluripotency markers (nanog, oct4, and alkaline phosphatase).
• Figure 20 shows the expression of mesodermal markers two days after the formation of embryoid bodies (EB).
• Figure 20F shows the expression of osterogenic (osteopontin), adipogenic (lipoprotein lipase), and glial (GAFP) genes 2 days after formation Embryoid bodies (EB).
• FIG 21 panels A (left) and B (right), illustrate alternative models of Alu shRNA action.
• Panel A shRNA against Alu forms a hairpin shRNA, which directs either nuclear PIWI or Dicer machinery to the genomic SINE/ Alu repeat location, initiating transcriptional silencing via heterochromatinization involving both DNA methylation and histone modification.
• Panel B shRNA against Alu activates the PTGS Dicer-dependent Ago2 pathway, leading to the cytoplasmic degradation of unprocessed Alu RNA transcripts.
• Figure 22A top shows a representation of 7SL-conserved region within Alu full-length sequence.
• Figure 22A (bottom) shows secondary structure of generic full-length Alu RNAv (SEQ ID NO:2).
• FIG. 22B shows sequence conservation of the Alu shRNA sequence compared to the rest of the element. Average percent identity levels among dispersed repeated element copies are compared for the shRNA Alu sequence regions versus other Alu sequence regions across four Alu subfamilies. Significance values for the differences are shown for each comparison.
• Figure 22C shows a step-by-step schematic representation of unbiased RNA affinity assay.
• Figure 22D shows silver-stained denaturing 4-12% NuP AGE No vex 4-12% Bis-Tris gel loaded with RNA affinity assay precipitants, with excised bands labeled A-E.
• Figure 23 shows a brightness-coded representation of abundance of members in major functional categories. Most abundant proteins within each group are depicted with the brightest color, fading to black for those with an abundance of only 1.
• Figure 24 A shows confidence levels for the involvement of isolated complex proteins in various cellular processes, represented Gene Ontology (GO) terms as a -logi 0 (p- value), using p-values calculated by DAVID software.
• 24B shows confidence levels for the presence of isolated complex proteins in various cellular components, represented as a - logio(p-value), as calculated using DAVID software.
• Figure 24C shows an interaction web of isolated proteins produced in STRING 9.0 software. Thicker lines represent higher confidence (experimentally derived) interactions while dotted lines represent inferred (lower confidence) interactions.
• Figure 25 shows a representation of protein complex using Gene Ontology
• retrotransposons interferes with the recruitment of condensin I and cohesin complexes in pericentric chromatin, resulting in the loss of efficient DNA repair and, in turn, senescence.
• retrotransposon transcripts in senescent human adult stem cells reinstates the cells' self- renewing properties and unexpectedly increases their plasticity (e.g., as manifested by upregulation of the pluripotency markers Nanog and Oct4).
• the results presented herein demonstrate the functional significance of SINE/Alu retrotransposons and provide mechanistic insight into their novel role in mediating crosstalk between chromatin, DNA repair and aging of human adult stem cells.
• methods are provided for inducing and/or restoring and/or maintaining proliferative capacity and/or pluripotency in a mammalian cell. More generally, the methods reduce the rate of onset of senescence or prevent senescence, or rescuing a cell from senescence. In various embodiments the methods induce or restore a pluripotent phenotype to a differentiated and/or senescent cell. In various embodiments the methods typically involve downregulating or inhibiting the level or activity of SINE/Alu retrotransposon transcripts in the cell (see, e.g., Figure 18). [0046] As demonstrated herein, such down regulation/inhibition rescues cells (e.g.
• stem cell populations e.g., stem cell lines
• SINE/Alu retrotransposon transcripts in those cells.
• the stem cells can be induced to differentiate into embryoid bodies, precursors or particular lineages, or terminally differentiated cells according to standard methods well known to those of skill in the art.
• cells including previously terminally differentiated mammalian cells, progenitor cells, stem cells, stem cell lines, and induced pluripotent stem cells (iPSCs) are provided where the cells have SINE/Alu retrotransposon transcripts
• the cells include normal (non-senescent) stem cells, stem cells that have been "rescued” from senescence by downregulating SINE/Alu retrotransposon transcript production or activity, induced pluripotent stem cells (IPSCs) that contain a construct that downregulates SINE/Alu retrotransposon transcript level or activity, embryonic stem cells, and the like.
• ISCs induced pluripotent stem cells
• cells are excluded that have been treated in a manner to form IPSCs (e.g., cells are excluded in which one or more of Nanog, and/or LIN-28, and/or Oct3/4, and/or Sox2, and/or Klf4, and/or c-myc are directly upregulated and/or that contain heterologous constructs that express Oct3/4, and/or Sox2, and/or Klf4, and/or c-myc).
• differentiated cells comprising cells that have been induced to differentiate from pluripotent cells generated according to the methods described herein are
• transdifferentiating cells typically comprise inducing or restoring a cell to a pluripotent phenotype according to the methods described herein (e.g., by inhibiting the Alu retrotransposon or other components of the pathway), and then culturing the resulting pluripotent cells under conditions that allow or induce differentiation.
• Cells that differentiate into a desired cell type e.g., pancreatic beta cells, motoneurons, hematopoietic progenitor cells, neural cells, dopaminergic neurons, adipocytes,
• cardiomyocytes, and the like are then selected and can be subsequently cultured (e.g., expanded in culture) or directly utilized.
• the methods described herein can be used to restore pluripotency and/or proliferative capacity to a cell (e.g., to a cell that has committed to a differentiation pathway).
• the methods described herein can be used to restore to a lesser senescent state or to a non-senescent state a cell that shows one or more indications of senescence.
• stem cell comprises a cell selected from the group consisting of an embryonic stem cell, a cord blood stem cell, an adult stem cell, and an IPSC.
• the mammalian stem cell is a stem cell derived from a tissue selected from the group consisting of human adipose tissue, human bone marrow, human neurological tissue, human smooth muscle, human adipose tissue, human cardiomyocytes, human endothelial tissue, human epithelial tissue, human pancreatic tissue, human bone or cartilage and the like.
• the cell is one that has committed to differentiation to a cell type selected from the group consisting of ectoderm, mesoderm, and endoderm. In certain embodiments the cell is one that has committed to differentiation to a cell type selected from the group consisting of human adipose cells, human blood cells, human nerve cells, human smooth muscle cells, human adipocytes, human chondrocytes, human osteoclasts and hosteoblasts, human cardiomyocytes, human endothelial cells, and human epithelial cells. In certain embodiments the cell comprises a non-renewing progenitor cell. In certain embodiments the mammalian cell comprises a terminally differentiated cell.
• SINE/Alu retrotransposon transcripts can restore proliferative capacity and/or pluripotency to a senescent stem cell or can maintain proliferative capacity and/or pluripotency in a non-senescent stem cell. Any of a variety of methods to inhibit SINE/Alu retrotransposon transcripts can be used.
• SINE/Alu retrotransposon transcripts can be reduced/inhibited using inhibitory RNAs.
• Suitable inhibitory RNAs include, but are not limited to siRNAs, shRNAs, miRNAs, dicer-substrate 27-mer duplexes, single-stranded interfering RNA, and the like.
• siRNAs typically refer to a double-stranded interfering RNA unless otherwise noted.
• suitable siRNA molecules to inhibit SINE/Alu retrotransposon transcripts include double-stranded ribonucleic acid molecules comprising two nucleotide strands, each strand having about 19 to about 28 nucleotides (i.e.
• interfering RNA having a length of 19 to 49 nucleotides when referring to a double-stranded interfering RNA means that the antisense and sense strands independently have a length of about 19 to about 49 nucleotides, including interfering RNA molecules where the sense and antisense strands are connected by a linker molecule.
• interfering RNA molecules and RNA- like molecules can to inhibit SINE/Alu retrotransposon transcripts.
• interfering RNA molecules that can to inhibit SINE/Alu retrotransposon transcripts include, but are not limited to short hairpin RNAs (shRNAs), single-stranded siRNAs, microRNAs (miRNAs), and dicer-substrate 27-mer duplexes.
• interfering RNAs RNA or RNA-like molecules that can interact with SINE/Alu retrotransposon transcripts RISC and participate in RISC-related changes in gene expression
• SiRNAs single-stranded siRNAs, shRNAs, miRNAs, and dicer-substrate 27-mer duplexes are, therefore, subsets of "interfering RNAs” or “interfering RNA molecules.”
• siRNA that inhibit SINE/Alu retrotransposon transcripts can comprise partially purified RNA, substantially pure RNA, synthetic RNA, recombinantly produced RNA, as well as altered RNA that differs from naturally-occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides.
• Such alterations can include, for example, addition of non-nucleotide material, such as to the end(s) of the siRNA or to one or more internal nucleotides of the siRNA, including modifications that make the siRNA resistant to nuclease digestion.
• one or both strands of the siRNA can comprise a 3' overhang.
• a "3' overhang” refers to at least one unpaired nucleotide extending from the 3 '-end of an RNA strand.
• the siRNA comprises at least one 3' overhang of from 1 to about 6 nucleotides (which includes ribonucleotides or deoxynucleotides) in length, from 1 to about 5 nucleotides in length, from 1 to about 4 nucleotides in length, or about 2 to about 4 nucleotides in length.
• the length of the overhangs can be the same or different for each strand.
• the 3' overhang is present on both strands of the siRNA, and is one, two, or three nucleotides in length.
• each strand of the siRNA can comprise 3' overhangs of dithymidylic acid ("TT") or diuridylic acid ("uu").
• the 3' overhangs can be also stabilized against degradation.
• the overhangs are stabilized by including purine nucleotides, such as adenosine or guanosine nucleotides.
• substitution of pyrimidine nucleotides by modified analogues e.g., substitution of uridine nucleotides in the 3' overhangs with 2'-deoxythymidine, is tolerated and does not affect the efficiency of RNAi degradation.
• the siRNA comprises the sequence AA(N19)TT
• siRNAs comprise approximately 30%-70% GC, and preferably comprise approximately 50% G/C.
• the sequence of the sense siRNA strand corresponds to (N19)TT (SEQ ID NO:6) or N21 (SEQ ID NO:7) (i.e., positions 3 to 23), respectively. In the latter case, the 3' end of the sense siRNA is converted to TT.
• the rationale for this sequence conversion is to generate a symmetric duplex with respect to the sequence composition of the sense and antisense strand 3' overhangs.
• the antisense RNA strand is then synthesized as the complement to positions 1 to 21 of the sense strand.
• the 3 '-most nucleotide residue of the antisense strand can be chosen deliberately.
• the penultimate nucleotide of the antisense strand (complementary to position 2 of the 23 -nt sense strand in either embodiment) is generally complementary to the targeted sequence.
• the siRNA comprises the sequence
• NAR(N17)YNN SEQ ID NO: 8
• R is a purine (e.g., A or G) and Y is a pyrimidine ⁇ e.g., C or U/T).
• Y is a pyrimidine ⁇ e.g., C or U/T.
• the respective 21-nt sense and antisense RNA strands of this embodiment therefore generally begin with a purine nucleotide.
• Such siRNA can be expressed from pol III expression vectors without a change in targeting site, as expression of RNAs from pol III promoters is only believed to be efficient when the first transcribed nucleotide is a purine.
• the siRNA of the invention can be targeted to any stretch of approximately 10-30, or 15-25, or 19-25 contiguous nucleotides in any of the target mRNA sequences (the "target sequence”).
• Target sequence any of the target mRNA sequences.
• Techniques for selecting target sequences for siRNA are given, for example, in Tuschl et ⁇ , "The siRNA User Guide,” revised May 6, 2004.
• the "siRNA User Guide” is available on the world wide web at a website maintained by Dr. Thomas Tuschl, and can be found by accessing the website of
• siRNA User Guide can be located by performing a google search for "siRNA User Guide” and can also be found at "www.rockefeller.edu/labheads/tuschl/sirna.html. Techniques for selecting target sequences for siRNA and miRNA can also be found in Sioud (2008) siRNA and miRNA Gene Silencing: From Bench to Bedside (Methods in Molecular Biology) , Humana Press.
• the sense strand of the present siRNA comprises a nucleotide sequence identical to any contiguous stretch of about 19 to about 25 nucleotides in the target SINE/Alu retrotransposon transcript(s).
• the SINE/Alu retrotransposon transcript silencing siRNAs can be obtained using a number of techniques known to those of skill in the art.
• the siRNA can be chemically synthesized or recombinantly produced using methods known in the art, such as the Drosophila in vitro system described in U.S. published application US
• the siRNAs are chemically synthesized using appropriately protected ribonucleoside phosphoramidites and a conventional DNA/RNA synthesizer.
• the siRNAs can be synthesized as two separate, complementary RNA molecules, or as a single RNA molecule with two complementary regions.
• Commercial suppliers of synthetic RNA molecules or synthesis reagents include Proligo (Hamburg, Germany), Dharmacon Research (Lafayette, Colo., USA), Pierce Chemical (part of Perbio Science, Rockford, III, USA), Glen Research (Sterling, Va., USA), ChemGenes (Ashland, Mass., USA) and Cruachem (Glasgow, UK).
• siR A can also be expressed from recombinant circular or linear DNA plasmids using any suitable promoter.
• suitable promoters for expressing siRNA from a plasmid include, for example, the U6 or HI RNA pol III promoter sequences and the cytomegalovirus promoter. Selection of other suitable promoters is within the skill in the art.
• the recombinant plasmids can also comprise inducible or regulatable promoters for expression of the siRNA in a particular tissue or in a particular intracellular environment.
• siRNA expressed from recombinant plasmids can either be isolated from cultured cell expression systems by standard techniques, or can be expressed intracellularly at or near the target area(s) in vivo.
• the use of recombinant plasmids to deliver siRNA to cells in vivo is discussed in more detail below.
• siRNA can be expressed from a recombinant plasmid either as two separate, complementary RNA molecules, or as a single RNA molecule with two complementary regions. Selection of plasmids suitable for expressing siRNAs, methods for inserting nucleic acid sequences for expressing the siRNA into the plasmid, and methods of delivering the recombinant plasmid to the cells of interest are within the skill in the art ⁇ see, e.g., Tuschl (2002) Nat. Biotechnol, 20: 446-448; Brummelkamp et al. (2002) Science 296: 550 553; Miyagishi et al. (2002) Nat. Biotechnol.
• a plasmid comprising nucleic acid sequences for expressing an siRNA for inhibiting SINE/Alu retrotransposon transcripts comprises a sense RNA strand coding sequence in operable connection with a polyT termination sequence under the control of a human U6 RNA promoter, and an antisense RNA strand coding sequence in operable connection with a polyT termination sequence under the control of a human U6 RNA promoter.
• the plasmid is ultimately intended for use in producing a recombinant adeno-associated viral vector comprising the same nucleic acid sequences for expressing the siRNA.
• operable connection with a polyT termination sequence means that the nucleic acid sequences encoding the sense or antisense strands are adjacent to the polyT termination signal in the 5' direction or sufficiently close so that during transcription of the sense or antisense sequences from the plasmid, the polyT termination signals act to terminate transcription after the desired product is transcribed.
• under the control of a promoter means that the nucleic acid sequences encoding the sense or antisense strands are located 3' of the promoter, so that the promoter can initiate transcription of the sense or antisense coding sequences.
• the siRNA can be delivered as a small hairpin RNA or short hairpin RNA (shRNA).
• shRNA is a sequence of RNA that makes a tight hairpin turn that can be used to silence gene expression via RNA interference.
• shRNA uses a vector introduced into cells and utilizes the U6 promoter to ensure that the shRNA is always expressed. This vector is usually passed on to daughter cells, allowing the gene silencing to be inherited.
• the shRNA hairpin structure is cleaved by the cellular machinery into siRNA, which is then bound to the RNA-induced silencing complex (RISC). This complex binds to and cleaves mRNAs that match the siRNA that is bound to it.
• RISC RNA-induced silencing complex
• the sense sequence of the shRNA will be from about
• the antisense sequence will be from about 19 to about 30, more nucleotides (e.g. about 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides) in length, more typically from about 19 to about 22 nucleotides in length
• the antisense sequence will be from about 19 to about 30, more typically from 19 to about 22 nucleotides (e.g. about 19, 20, 21 or 22 nucleotides), in length
• the loop region will be from about 3 to about 19 nucleotides (e.g., about 3, 4, 5, etc., . . . up to about 19) nucleotides in length.
• the sense and antisense sequences are the same length, i.e. the shRNA will form a symmetrical hairpin, but this is not necessarily the case.
• the sense or antisense strand may be shorter than its complementary strand, and an asymmetric hairpin is formed.
• the base pairing between the sense and antisense sequences is exact, this also need not be the case. In other words, some mismatch between the sequences may be tolerated, or even desired, e.g. to decrease the strength of the hydrogen bonding between the two strands.
• the sense and antisense sequences are the same length, and the base pairing between the two is exact and does not contain any mismatches.
• the shRNA molecule can also comprise a 5 '-terminal phosphate group that can be chemically modified.
• the loop portion of the shRNA molecule can comprise, for example, nucleotides, non- nucleotides, linker molecules, conjugate molecules, etc.
• the shRNA/siRNA /piRNA described herein targets and causes the RNAi- mediated degradation of SINE/Alu retrotransposon transcripts, or alternative splice forms, or participates in genomic silencing via (PIWI RNA pathways).
• methods for inhibiting SINE/Alu retrotransposon transcripts in a cell comprising administering an effective amount of an SINE/Alu retrotransposon transcript siRNA/shRNA/piRNA to the cell, such that the target mRNA is degraded.
• the siRNA/shRNA/piRNA can be expressed from recombinant viral vectors introduced into the subject cells.
• the recombinant viral vectors comprise sequences encoding the siRNA/shRNA and any suitable promoter for expressing the siRNA/shRNA/piRNA sequences.
• suitable promoters include, but are not limited to, the U6 or HI RNA pol III promoter sequences and the cytomegalovirus promoter.
• the recombinant viral vectors can also comprise inducible or regulatable promoters for expression of the siRNA/shRNA in a particular tissue or in a particular intracellular environment.
• the siRNA/shRNA/piRNA can be expressed from a recombinant viral vector either as two separate, complementary RNA molecules, or as a single RNA molecule with two complementary regions.
• Any viral vector capable of accepting the coding sequences for the siRNA/shRNA/piRNA molecule(s) to be expressed can be used, for example vectors derived from adenovirus (AV); adeno-associated virus (AAV); retroviruses (e.g. lentiviruses (LV), Rhabdoviruses, murine leukemia virus); herpes virus, and the like.
• the tropism of the viral vectors can also be modified by pseudotyping the vectors with envelope proteins or other surface antigens from other viruses.
• an AAV vector can be pseudotyped with surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, and the like.
• suitable viral vectors include, but are not limited to lentiviral vectors.
• lentiviral shRNA constructs to knockdown genetic SINE/Alu transcript are designed.
• the shRNA sense and anti-sense strands are chemically synthesized and the strands are annealed with equal amounts of each other creating restriction site specific overhangs for cloning, and ligated into a vector (e.g., a Hindlll and Bglll digested, gel purified pENTR/pTER+ vector).
• Equal amounts of each construct is mixed with pLenti-CMV-GFP DEST vector in LR Clonase reaction to recombine cloned shRNA production elements into a destination vector according to the manufacturer's instructions (Invitrogen).
• the produced lentiviral plasmid is transformed into E. coli Stbl3 cells (Invitrogen) for amplification.
• suitable viral vectors include those derived from AV and AAV.
• the siRNA/shRNA/piRNA is expressed as two separate, complementary single-stranded RNA molecules from a recombinant AAV vector comprising, for example, either the U6 or HI RNA promoters, or the cytomegalovirus (CMV) promoter.
• a suitable AV vector for expressing the siRNA, a method for constructing the recombinant AV vector, and a method for delivering the vector into target cells are described in Xia et al. (2002) Nat. Biotech. 20: 1006 1010.
• Suitable AAV vectors for expressing the siRNA/shRNA, methods for constructing the recombinant AV vector, and methods for delivering the vectors into target cells are also described in Samulski et al. (1987) J. Virol. 61 : 3096-3101; Fisher et al.
• Stem cells ⁇ e.g., adult stem cells, embryonic stem cells, cord stem cells,
• IPSCs IPSCs, etc.
• stem cells are also commercially available.
• stem cells in which level or activity of SINE/Alu retrotransposon transcripts is reduced/inhibited. It is believed these stem cells can be differentiated into embryoid bodies or terminally differentiated cells using standard differentiation methods well known to those of skill in the art.
• LIF leukemia inhibitory factor
• BM- MSCs bone marrow-mesenchymal stem cells
• BMP4, BMP7, and BMP8b can increase differentiation of human germ cells from human ES cells (see e.g., Gonsalves et al. (2006) Stem Cells Dev., 15(6): 831-837).
• neurotrophin family is one of the most important inducible signals for the differentiation. Among them, nerve growth factor is well known to induce neurogenesis, and neurotrophin 3 is involved in
• CNTF ciliary neurotrophic factor
• LIF leukemia inhibitory factor
• AICAR 5-aminoimidazole-4-carboxamide-l-P-D-ribofuranoside
• a number of natural compounds are also known to induce differentiation of stem cells in vitro.
• Such compounds include, but are not limited to retinoic acid, PDGF, insulin, Arctigenin, ATRA (vitamin A), boswellic acid, bromelain and other proteolytic enzymes, CAPE , flavonoids (including apigenin, luteolin, quercetin, genistein,, and daidzein), emodin, EPA and DHA, monoterpenes, resveratrol, 1,25-D3 (vitamin D3), and the like.
• the pluripotent cells produced using the methods described herein can be subsequently induced to differentiate using methods well known to induce differentiation if iPSCs.
• methods of differentiating induced pluripotent cells into CD34 + CD43 + hematopoietic progenitors and CD31 CD43 " endothelial cells are described by Choi et al. (2009) Stem Cells 27: 559. These cells can be further separated into phenotypically defined subsets of primitive hematopoietic cells in a pattern of differentiation resembling that of ES cells.
• Methods of differentiating induced pluripotent cells into pancreatic insulin-producing cells are described by Zhang et al. (2009) Cell Res.
• human iPS cells were further differentiated into pancreatic cells expressing MafA, Glut2, insulin, and in some cases, amylase and C-peptide.
• Functional cardiomyocytes demonstrating sarcomeric organization and expressing cardiac markers including Nkx2.5, cardiac Troponin T, atrial natriuretic factor, and myosin heavy and light chains, have also been derived from human iPS cells and are indistinguishable from those generated from ES cells ⁇ see, e.g., Zhang et al. (2009) Circ. Res. 104:e30).
• cells and compositions comprising cells that have been differentiated into embryoid bodies or further differentiated ⁇ e.g., terminally differentiated) are also contemplated.
• Such differentiated cells include but are not limited to embryoid bodies and/or progenitor cells and/or terminally differentiated cells that are differentiated into lineages for cardiomyocytes, blood cells, epithelial cells, osteoblasts, osteoclasts, chondrocytes, adipocytes, smooth muscle cells, nerve cells (neurons), glial cells, pancreatic ⁇ -cells, motoneurons, and the like.
• hADSCs Human adult adipose derived mesenchymal stem cells
• Fig. 7A cell surface antigens
• hADSC can be induced to differentiate along several mesenchymal tissue lineages, including adipocytes, osteoblasts, myocytes, and chondrocytes (Erickson et al. (2002) Biochem. Biophys. Res. Commun. 290: 763-769; Zuk et al. (2001) Tissue Eng., 7: 211-228).
• hADSCs in these experiments bore strong resemblance to mesenchymal stem cells (MSCs).
• SR self-renewing
• hADSCs remained consistent until population doubling (PD17), after which they displayed characteristic phenotypes of "old age” with prolonged ex vivo passages shown in Fig. 1 A (and also described by (Bonab et al. (2006) BMC Cell Biol, 7: 14; Fehrer et al. (2007) Aging Cell, 6: 745-757; Kern et al. (2006) Stem Cells, 24: 1294-1301).
• the culture's morphological abnormalities are typical of the Hayflick model of cellular aging (Juckett (1987) Mech. Ageing Dev., 38: 49-71).
• hADSC cultures accumulated non-dividing giant cells expressing the enzyme lysosomal pH6 senescence-associated J3- galactosidase (SA-J3-Gal) (Dimri et al. (1995) Proc. Natl. Acad. Sci. USA, 92: 9363-9367), as shown in Fig. IB.
• SA-J3-Gal lysosomal pH6 senescence-associated J3- galactosidase
• Fig. IB Cells self-renewed poorly due to a decrease in the number of dividing cells as determined by incorporation of bromodeoxyuridine (BrdU) and 3 [H] -thymidine into DNA (Fig. IB and Fig. 7B).
• Persistent ⁇ 2 ⁇ /53 ⁇ 1 foci formation upon cellular senescence has been associated with the presence of unresolved DSB, as determined by colocalization with several DNA repair factors (d'Adda di Fagagna et al. (2003) Nature, 426: 194-198). Occurrences of ⁇ 2 ⁇ /53 ⁇ 1 foci formations were very rare in self-renewing ADSCs, and their formation increased as cultured hADSCs approached senescence (SEN hADSCs) (Fig. 8).
• the phosphorylation of these sites by ATM/ATR is required for full execution of DNA-damage-induced cell-cycle arrest in human somatic cells (d'Adda di Fagagna et al. (2003) Nature, 426: 194-198; Sedelnikova et al. (2008) Aging Cell 7: 89- 100; Tanaka et al. (2006) Cell Prolif., 39: 313-323).
• differential SR vs.
• SEN hADSCs transcriptional analysis of the 96 genes involved in multiple aspects of the cell cycle (Human Cell Cycle qPCR array), described in detail in Experimental Procedures. Genes that were statistically significantly downregulated with a p value ⁇ 0.05 in SEN hADSCs are shown in Table. 1, and include genes involved in cell cycle regulation, DNA replication, and mitosis, suggesting that senescent hADSC cultures follow a DDR program directly or indirectly related to the formation of senescence-associated DNA-damage foci. The causal factors that might mediate this process in human adult stem cells are as yet unknown.
• ChlP-seq was performed on four replicates each of SR and SEN hADSC cultures. Genomic mapping of the resulting sequence tags (Table 2) was followed by outlier removal, noise reduction, and merging of the data from replicate experiments. Details of the algorithms developed and approaches used for ChlP-seq data analyses can be found in Supplemental Methods. The ChlP-seq experimental protocol and data merging we employed were supported by the highly consistent mapping results seen for the replicate experiments.
• this genomic element also showed the greatest difference in relative frequencies of ⁇ 2 ⁇ marks for SR versus SEN hADSCs (Fig. 2C).
• SINE/Alu elements were the most enriched for ⁇ 2 ⁇ modified nucleosomes in SR cells compared to SEN cells, whereas Lis had the greatest relative increase of ⁇ 2 ⁇ modified nucleosomes in SEN cells (Fig. 2C).
• Relative entropy values calculated as a measurement of the difference between the two distributions (Supplemental Methods), were calculated over a range of cluster length threshold values.
• Transcriptional activity may represent an obstacle for progressing replication forks, resulting in their collapse and the accumulation of DSB (reviewed in Zegerman and Diffley (2009) DNA Repair (Amst) 8: 1077-1088). Therefore, functional genie regions such as exons and promoters are possibly relatively enriched with ⁇ 2 ⁇ modified nucleosomes in SR hADSCs, in contrast to SEN cells that have ceased their replication activity. Also, one may expect that gene-dense human chromosomes would be prone to ⁇ 2 ⁇ accumulation, and if this damage were not resolved during checkpoint activation, perhaps due to wear-and-tear of the DNA repair machinery, this could trigger the senescent phenotype. If this were the case, one would expect to observe ⁇ 2 ⁇ enrichment at similar locations within the chromatin of SR and SEN cells, and the amounts of ⁇ 2 ⁇ chromatin would be positively correlated with gene density in both phenotypes.
• GC-rich genomic areas are also prone to the accumulation of DNA damage (Cha and Kleckner (2002) Science, 297: 602-606), but there is no a priori reason to expect a difference in GC-related damage accumulation between SR and SEN cells.
• chromosomal GC content and ⁇ 2 ⁇ tag density were compared, as was done for gene density, we observed significantly positive correlations for both SR and SEN hADSCs (Fig. 2E and Table 4).
• the positive correlation of GC content and ⁇ 2 ⁇ tag density in SEN cells stood in contrast to the negative correlation between gene density and ⁇ 2 ⁇ tag density in the same cells. This result is unexpected, since it is known that GC content is positively correlated with gene density.
• ⁇ 2 ⁇ showed periodic enrichment at specific positions from -2kb to +2kb relative to transcription start sites (TSS) (Fig. 2F). These enrichment patterns are consistent with nucleosome phasing around TSS in both SR and SEN cells; however, the distributions were shifted, suggesting a repositioning of ⁇ 2 ⁇ modified nucleosomes relative to TSS in SEN samples.
• telomeric erosion might provide the reservoir of persistent DNA damage signal resulting in sustained p53 activation and manifestation of the SEN phenotype.
• telomeres are directly engaged in DDR in SEN hADSCs.
• Telomeric regions are highly repetitive and have not been extensively characterized at the sequence level; therefore, we could not unambiguously map telomeric ⁇ 2 ⁇ ChlP-seq tags to the human genome.
• ChlP-seq tags that bore a specific telomeric repeat sequence motif (TTAGGG/AATCCC, (SEQ ID NO:9)) among all sequencing tags (mapped uniquely and unmapped due to their repetitive nature).
• SR cell samples had an average of 1.66% of such telomeric tags while SEN cells had 1.63%. These percentages did not reveal an over-representation of telomeric tags, and the difference between the two cell types was not significant.
• peri-telomeres as lOOkb regions from the end of the sequenced human chromosomes, and evaluated 15Mb of genomic DNA extending into the
• SR samples showed an enrichment of ⁇ 2 ⁇ over the entire region, while SEN samples were depleted for ⁇ 2 ⁇ .
• SEN samples were depleted for ⁇ 2 ⁇ .
• chromosomes 10, 12, 17, and 18 had ⁇ 2 ⁇ enriched telomeres, as did chromosomes 4, 7, and 18 in SEN hADSCs. Despite the differences in peri-telomeric ⁇ 2 ⁇ accumulation between SR and SEN cells, the chromosome arm asymmetry was always preserved. The relative levels of chromosome arm-specific ⁇ 2 ⁇ peri-telomeric accumulation were identical for SR and SEN cells (Fig. 3C).
• peri-centromeres exhibited 2.0- and 2.1 -fold enrichments for SR and SEN cells respectively (Fig. 12), thus indicating intrinsic susceptibility ("fragility") of these chromosomal regions.
• Peri-centromeres were not only enriched for ⁇ 2 ⁇ , but they were also one of the few genomic features that showed relatively greater ⁇ 2 ⁇ presence in SEN cells (Fig. 2B, Fig. 3D, 3E).
• Chromosomes 6, 14, 15, 16 and 21 demonstrated the most significant ⁇ 2 ⁇ enrichment in SEN cells (Fig. 3E).
• CENP-A is required to recruit many other centromere and kinetochore proteins (McClelland et al. (2007) EMBO J. 26: 5033- 5047), with the exception of proteins located in adjacent heterochromatin domains, such as heterochromatic protein 1 (HP1) (Blower and Karpen (2001) Nat. Cell Biol, 3: 730-739).
• HP1 heterochromatic protein 1
• Senescence- Associated ⁇ 2 ⁇ Foci are Sites of Active Pol-III Transcription
• Mammalian chromatin modifying activities associated with cellular aging have been previously reported to contribute not only to the formation of facultative heterochromatin (Vaquero et al. (2007) Nature 450, 440-444), but to also be involved in the mediation of repression at constitutive heterochromatic regions such as pericentromeric chromatin.
• the generation of DSB by oxidative stress leads to increased transcription of pericentric satellite repeat DNA in a model of mammalian embryonic stem cells (Oberdoerffer et al. (2008) Cell, 135: 907-918).
• Senescent hADSCs were either cultured in the presence of 10 tM inhibitor of
• Pol III transcriptional activity tagetin, for 2 hrs at 37°C (+tagetin) or in the absence of the inhibitor treatment (-tagetin).
• Nuclear RNA was labeled by addition of 2mM FUr to the culture for 10 min at 37°C. After fixation, cells were immunolabelled with anti-BrdU antibody (red) to detect FUr incorporation sites in combination with anti-53BPl .
• Double labeling experiment revealed FUr incorporation sites exclusively localized with persistent DNA damage sites throughout entire depth of z-stack images. Tagetin inhibition of Pol III dependent transcription resulted in complete disappearance of FUr incorporation, and loss of compaction of the DNA damage sites as detected by more defuse 53BP1 staining.
• AluSx and AluJb revealed that upregulation of Alu transcriptional activity correlates with the presence of persistent DNA damage in SEN samples, as shown for the Alu repeat in the vicinity of the 7.6kb ⁇ 2 ⁇ cluster.
• Our data demonstrate that, despite the fact that the transcriptional activity of the chosen repeats (AluJb, AluSx, and Alu) is recorded regardless of the state of the hADSC. Alu transcription is upregulated on chromosome 10 where damage is observed only upon cellular senescence. No
• the Sccl/Rad21/Mcdl subunit of this complex is central to cohesin function and has been shown to be necessary for sister chromatid cohesion and kinetochore function in vertebrate cells (Sonoda et al. (2001) Dev. Cell, 1 : 759-770), as well as Gl and G2-M DNA damage checkpoints (Jessberger (2009) EMBOJ., 28: 2491-2493). Reportedly, the cohesin complex becomes enriched at DSB sites and facilitates DNA repair by homologous recombination (HR) (Bekker- Jensen et al. (2006) J.
• Cap-H depletion does not affect CENP-A incorporation into the centric chromatin or kinetochore assembly, but does result in severe depletion of cohesin Sccl/Rad21/Mcdl, triggering cell cycle defects.
• SINE/Alu elements occupy 6% of the human genome, significantly outnumbering other families of pseudogenes generated by retrotransposition (Weiner et al, 1986).
• the human SINE/Alu retrotransposon is a tandem repeat of two Bl elements connected by an A-rich linker (Fig. 6A), and the secondary structure of its RNA was previously reported (Sinnett et al. (1991) J. Biol. Chem., 266: 8675-8678).
• SEN hADSCs were genetically manipulated to stably express shRNA, targeting the generic SINE/Alu transcript.
• Fig. 6C Northern Blot hybridization
• Fig. 6B Knockdown of the generic SINE/Alu transcript in transduced hADSCs stably expressing shRNA against different portions of SINE/Alu was demonstrated by Northern Blot hybridization (Fig. 6C) with a transduction efficiency of nearly 99% (Fig. 6B).
• hADSCs transduced with lentiGFP expressing sh-132Alu RNA demonstrated a near complete knockdown of the generic SINE/Alu transcript.
• hADSCs transduced with lentiGFP (control) or lentiGFP sh- 193 Alu exhibited little or no change at the SINE/Alu transcription level.
• SEN hADSCs lines stably expressing sh-132Alu RNA had a dramatically altered morphology and exhibited an increase in proliferation as detected in 3 [H] thymidine uptake DNA synthesis experiments (Fig. 6D).
• SINE/Alu retrotransposons Increased transcriptional activity of SINE/Alu retrotransposons in the aging human adult stem cells significantly correlates with the formation of persistent DNA damage foci, indicating a role in interfering with DNA repair.
• SINE/Alu transposons act by altering chromatin structure in the vicinity of DNA lesions, thus blocking their repair (Fig. 6G). This event is integral to establishing/mediating persistent DDR upon ex-vivo aging of human adult stem cells.
• Human adipose derived stem cells were isolated from human subcutaneous white adipose tissue collected during liposuction procedures. The lipoaspirate was suspended in Hank"s Buffered Salt Solution (HBSS), 3.5% Bovine Serum Albumin (BSA), 1% Collagenase, type II (Sigma) in 1 :3 w/v ratio and shaken at 37°C for 50 min. The cells were filtered through a 70 ⁇ mesh cell strainer (BD Falcon #352350), treated with Red Blood Cell Lysis buffer (150 mM NH 4 C1, 10 mM KHC0 3 , 0.1 mM EDTA, pH 7.3), and expanded ex-vivo in DMEM/F12 complete medium (DMEM/F12, 10% FBS, 100 U/ml penicillin, 100 ⁇ g/ml streptomycin, 2.5 ⁇ g/ml amphotericin B; Invitrogen) in 10% C02 at 37°C and passaged at 80%> confluency, changing
• N 0 is the number of cells plated in the flask and N is the number of cells harvested at this passage
• Senescence-associated J3-galactosidase activity assay was done as described in manufacturer's kit (Bio Vision). Cellular phenotype towards induced pluripotent cells was assayed with alkaline phosphatase staining kit (Stemgent). Immunofluorescence
• Epifluorescence images were acquired on an Olympus BX60 fluorescence microscope with Spotfire 3.2.4 software (Diagnostics Instruments). Confocal images (z- series slice thickness 0.39 ⁇ ) were acquired on Zeiss LSM 510 NLO with 488nm Argon, 543 nm HeNe, and Coherent Chameleon 2-photon lasers using a 63x planapo objective and 0.08x0.08x0.39 ⁇ voxel dimensions. Image stacks were deconvolved using Huygens Professional 3.4.0 (Scientific Volume Imaging, Netherlands) and visualized in Bitplane Imaris 6.3.1 (Bitplane Inc., Saint Paul, MN).
• RNAs from -1.5 x 10 6 SR and SEN ADSCs were prepared by
• ChlP-Seq Nucleosomal ChIP and SOLiD Library Preparation
• Immuno-complexes bound to beads were re- suspended in TE buffer, de-crosslinked, and purified by Proteinase K treatment, phenol: chloroform extraction, and isopropanol precipitation.
• DNA fragments were prepared for adapter ligation by filling-in ends by DNA polymerase I (Klenow fragment) and phosphorylating 5' ends of PCR primers by Polynucleotide kinase (NEB), and ligated to 30- fold molar excess of SOLIDTM System 2.0 (Applied Biosystems) library adapters according to manufacturer's protocol.
• DNA libraries were amplified by PCR using cloned Pfu DNA Polymerase (Stratagene, Agilent Technologies. Mononucleosomal size DNA fragments were size-selected in a 2% agarose gel, cut around 200 bp size, and purified with
• Emulsion PCR for sequencing bead preparations were done with 0.05 and 0.1 pg/ ⁇ of library DNA for each sample. Samples were sequenced on SOLiDTM System 2.0 (Applied Biosystems) according to
• Genomic coordinates of the SINE/ Alu elements tested in RT-PCR were from the March 2006 Assembly (NCBI36/hgl 8) of the Human Genome Browser at UCSC (genome.ucsc.edu); MIR: chrl0:41922815- 41922906, Alu: chrl0:41928992-41929118, AluJb: chr21 : 10141132-10141429 and AluSx: chr21 : 10145344- 10145644. 100 ng of total RNA was used with the RT2 First Strand Kit (SABiosciences) per reaction.
• the primers for first strand synthesis are at locations outside of the SINE/Alu element sequences (external or reverse primers, Table 9) and forward primers within the SINE/Alu element sequence (internal forward primers, Table 9).
• RPL13A, GAPDH was used as a positive control.
• RNA was used for first strand cDNA synthesis with SUPERSCRIPT® III First-Strand Synthesis System for RT- PCR (Invitrogen) with random priming.
• qPCR was performed using RT2 SYBR Green qPCR MasterMix (SABiosciences) and run in LIGHTCYCLER® 480 II (Roche). qPCR primers are listed in Table 9. Data analysis of relative gene expression was done by 2-DCt method.
• Lentiviral shRNA constructs to knockdown genetic SINE/Alu transcript were designed as follows: oligonucleotides Lenti sh-132 Alu RNA: 5'-GAT CCC CCC
• A-3' (SEQ ID NO: l 1), were annealed with equal amounts of their complementary strands, creating restriction site specific overhangs for cloning, and ligated into Hindlll and Bglll digested, gel purified pENTR/pTER+ vector (Campeau et al, 2009). The constructs were confirmed by sequencing (sense strand sequence is shown above). Equal amounts of each constructs was mixed with pLenti-CMV-GFP DEST vector (Campeau et al., 2009) in LR Clonase reaction to recombine cloned shRNA production elements into a destination vector according to manufacturer's instructions (Invitrogen). The produced lentiviral plasmid was transformed into E. coli Stbl3 cells (Invitrogen) for amplification. Lentiviral production and transduction
• Virus was precipitated with PEG and frozen in aliquots (-80oC). Lentiviral transductions were done in complete medium with 5 ⁇ g/ml Polybrene (Santa Cruz Biotechnology) for 12 hours. Viral titers were determined by comparing GFP positive cells counts to total population.
• Hybridizations were performed in 6xSSC, 4xDenhardts', 0.1% SDS at 37°C.
• Oligonucleotide probes were labeled with Biotin-16-dUTP (Roche) by terminal transferase (NEB). Northern was visualized with streptavidin-HRP (Invitrogen) using ECL Plus Western Blotting Detection Reagent (Amersham, GE Healthcare) and Amersham Hyperfilm (GE Healthcare).
• Oligonucleotide used as probes were: SINE/Alu 132:5 * - CCA CCA CGC CCG GCT AAT TT-3 * (SEQ ID NO: 12) and SINE/Alu 90: 5 * -CGC GCG CCA CCA CGC CCG GCT AAT TTT TGT ATT TTT AGT AGA GAC GGG GTT TCA CCA TGT TGG CC-3 * (SEQ ID NO: 13).
• ChlP-seq tags were mapped to the March 2006 human genome reference sequence (NCBI build 36.1 , UCSC hg 18) using the SOLiDTM System Analysis Pipeline Tool (Corona Lite). After tag-to-genome mapping of the ChlP-Seq data for each biological replicate experiment, tag counts were compared between replicates for identical genomic positions across all chromosomes. To do this, tag counts for each individual nucleotide position in each replicate experiment were determined. Then nucleotide positions that have a tag count of 0 in either replicate were eliminated from consideration. This is because if a position has a tag count of 0 in both data sets, it will not be considered in the subsequent analysis since there is no signal there.
• a standard deviation - s(y) - is computed based on the data distribution around E(y) for the corresponding value of x.
• the summation of position-specific tag counts across the genome can also be modeled by a Poissson distribution with a parameter equal to the sum of the individual 200-values from each replicate. This procedure resulted in a classification of all nucleosome size genomic positions as modified or unmodified based on data that has been merged between replicates and purged of outliers.
• Clusters were operationally defined as contiguous genomic regions where the number of modified mono-nucleosome size fragments is significantly greater than the average genomic background level of modified positions.
• Maximal Segment algorithm we first devised a binary scoring scheme that characterizes mono-nucleosome size fragments (200bp) as either modified or unmodified. This procedure is used to define a binary genome-wide map of nucleosome scores. Then the Maximal Segment algorithm was applied to the genomic map of binary nucleosome scores to define clusters.
• a binary scoring scheme is implemented in such a way as to assign the log likelihood that an individual mono-nucleosome size fragment either modified or
• This scoring scheme results in an assignment of a single positive score to all modified nucleosome positions and a single negative score to all unmodified positions. This approach is taken based on the proof that log likelihood ratios of this kind are optimal scores for the identification of contiguous genomic segments (Karlin and Altschul (1990) Proc. Natl. Acad. Sci. U SA, 87: 2264-2268).
• the value of q chosen for the scoring scheme allows for control over cluster selection in the sense that the density of modified nucleosomes per cluster will be greater than or equal to the value of q.
• Table 6 List of genes with promoter ⁇ 2 ⁇ accumulation in SR or SEN cells.
• NM 00108959 UQCRHL NM 017645 FAM29A NM_024323 C19orf57 NM 024699 ZFAND1
• NM 182828 GDF7 NM 00102524 IRAKI NM 007128 VPREB1 NM OO 100446 OR10A6 NM_181713 UBXN2A NR 000011 SNORA70 NM 002073 GNAZ NM 00102538 AMPD3
• NM OO 108042 GRIP2 NM_005838 GLYAT NMJ 3418 Clorfl56 NM 014212 H0XC 11
• NM_205853 MUSTN1 NM_033388 ATG16L2 NM 014812 CEP 170 NR 002979 SNORA49
• NM 00103311 PAIP2 NM 033426 KIAA1737 NM_005787 ALG3 NM l 52766 C17orf61
• NM 00109820 GPER NM 030579 CYB5B NR 003038 SNHG5 NM_080748 R0M01
• NM OOl 10554 PLXNA4 NM_175882 IMP5 NM O 16489 NT5C3 NM 001106 ACVR2B
• ECS ElonginB/C-CUL2/5-SOCS-box protein
• E3 ubiquitin-protein ligase complexes which mediate the ubiquitination of target proteins. May serve as a rigid scaffold in the complex and may contribute to catalysis through positioning of the substrate and the ubiquitin-conjugating enzyme.
• GAS7 plays a putative role in neuronal development.
• HERC5 hect domain GO:0007049 HERC5 hect domain and This gene is a member of the HERC family
• RLD 5 of ubiquitin ligases encodes a protein with a HECT domain and five RCC1 repeats.
• Pro-inflammatory cytokines upregulate expression of this gene in endothelial cells.
• the protein localizes to the cytoplasm and perinuclear region and functions as an interferon-induced E3 protein ligase that mediates ISGylation of protein targets.
• GLI2 GLI-Kruppel This gene encodes a protein which belongs family member to the C2H2-type zinc finger protein
• GLI2 subclass of the Gli family zinc finger proteins are mediators of Sonic hedgehog (Shh) signaling and they are implicated as potent oncogenes in the embryonal carcinoma cell.
• the protein encoded by this gene localizes to the cytoplasm and activates patched Drosophila homolog (PTCH) gene expression. It is also thought to play a role during embryogenesis.
• the encoded protein is associated with several phenotypes- Greig
• cephalopolysyndactyly syndrome Pallister- Hall syndrome, preaxial Polydactyly type IV, postaxial Polydactyly types Al and B.
• G superfamily expressed on the surface of T cells. It
• immunoglobulin class switch and is associated with hyper-IgM syndrome.
• CALCA calcitonin/ calcitoni This gene encodes the peptide hormones n-related calcitonin, calcitonin gene-related peptide polypeptide, alpha and katacalcin by tissue-specific alternative
• Calcitonin is involved in calcium regulation and acts to regulate phosphorus metabolism.
• cytokine 5 factor (ligand) cytokine that belongs to the tumor necrosis superfamily, factor (TNF) ligand family.
• This cytokine member 15 is a ligand for receptor TNFRSF25 and decoy receptor TNFRSF21/DR6. It can activate NF-kappaB and MAP kinases, and acts as an autocrine factor to induce apoptosis in endothelial cells. This cytokine is also found to inhibit endothelial cell proliferation, and thus may function as an angiogenesis inhibitor.
• ADRA2 adrenergic, alpha- Alpha-2-adrenergic receptors are members
• This protein is a receptor for interleukin 8 (IL8). It binds to IL8 with high affinity, and transduces the signal through a G-protein activated second messenger system. This receptor also binds to chemokine (C-X-C motif) ligand 1
• CXCL1/MGSA melanoma growth stimulating activity
• factor (ligand) cytokine that belongs to the tumor necrosis superfamily, factor (TNF) ligand family.
• This cytokine is member 8 a ligand for TNFRSF8/CD30, which is a cell surface antigen and a marker for Hodgkin lymphoma and related hematologic malignancies. This cytokine was shown to enhance cell proliferation of some lymphoma cell lines, while to induce cell death and reduce cell proliferation of other lymphoma cell lines.
• G tyrosine kinase 3 hematopoietic cells Synergizes well with a ligand number of other colony stimulating factors and interleukins.
• CD 164 is a type I integral transmembrane sialomucin sialomucin that functions as an adhesion receptor.
• the protein encoded by this gene is a brain protein 7, brain fatty acid binding protein.
• alpha member of the interleukin 1 cytokine family alpha member of the interleukin 1 cytokine family.
• This cytokine is a pleiotropic cytokine involved in various immune responses, inflammatory processes, and hematopoiesis.
• RERG RAS-like, RERG a member of the RAS superfamily of estrogen-regulated, GTPases, inhibits cell proliferation and growth inhibitor tumor formation.
• the kinase initiation factor 2- becomes autophosphorylated and can alpha kinase 2 catalyze the phosphorylation of the
• protein 1 belongs to the LIM/double zinc finger (intestinal) protein family.
• CRIP may be involved in intestinal zinc transport.
• REG IB regenerating islet- This gene encodes a protein secreted by the derived 1 beta exocrine pancreas that is highly similar to (pancreatic stone the REG 1 A protein.
• Protein complex assembled on piRNA derived from Alu retrotransposal transcript indicates putative participation of retroRNA in the cell cycle, DNA repair and
• RNA interference (RNAi) machinery is involved in these events.
• transcripts generated from telomere-repeat-encoded RNA interact with heterochromatin protein 1 (HP1), trimethlyated histone H3 Lysine 9 (H3K9me3), core components of the Shelterin complex as well as members of the DNA-damage-sensing pathway (Deng et al. (2009) Mol. Cell, 35: 403-413).
• RNA participates in the cytoplasmic assembly of the signal recognition particle (SRP) in mammalian cells (He et al. (1994) J. Cell Sci., 107(Pt 4): 903-912).
• SRP signal recognition particle
• the mammalian SRP is composed of a single RNA, the 7SL RNA, and six proteins (Walter and Blobel (1981) J. Cell Biol, 91 : 557-561).
• SRP9 and SRP 14 bind to the 5' end of the RNA (Alu-domain), which functions in translational arrest (Siegel and Walter (1988) Trends Biochem. Sci., 13: 314-316; Siegel and Walter (1986) Nature 320:81-84).

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