# Patent

## Title
Long noncoding RNAS and cell reprogramming and differentiation

## Abstract

Long noncoding RNAs (lncRNAs) are identified that enhance pluripotency reprogramming of somatic cells as well as differentiation of pluripotent cells. Induced pluripotent stem (iPS) cell generation from somatic cells leads to the upregulation and downregulation of identified lncRNAs. The modulation of these lncRNAs are capable of enhancing pluripotency of somatic cells as well as enhancing differentiation of a pluripotent cell.

## Background
CROSS-REFERENCE TO RELATED APPLICATION(S)
The present application claims priority to and the benefit of U.S. Provisional Application Ser. No. 61/840,306 filed on Jun. 27, 2013, the entire contents of which are incorporated herein by reference.


STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
This invention was made with government support under HG006998 awarded by the National Institutes of Health. The government has certain rights in the invention.”


SEQUENCE LISTING
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Nov. 17, 2014, is named 75207-C766_SL.txt and is 19,013,879 bytes in size.
BACKGROUND
The most well-known type of pluripotent stem cell is the embryonic stem (ES) cell. However, the generation of embryonic stem cells can only be derived from embryos, and it has so far not been feasible to create patient-matched embryonic stem cell lines. Induced pluripotent stem (iPS) cells are a type of pluripotent stem cell that can be generated directly from somatic (differentiated) cells. Since iPS cells can be derived directly from adult tissues, they not only bypass the need for embryos, but can be made in a patient-matched manner, which means that each individual could have their own pluripotent stem cell line. iPS cells may be generated through the ectopic expression of Oct 4, Sox2, Lkf4, and c-Myc (OSKM) transcription factors in somatic cells (Takahashi and Yamanaka, 2006), leading to global epigenetic changes during reprogramming (Papp and Plath, 2013). Chromatin regulatory proteins mediate epigenetic remodeling during iPS cell formation, and loss-of-function studies have shown that Polycomb proteins are potent regulators of cell fate reprogramming (Onder et al., 2012). However, iPS cells may retain epigenetic signatures of their somatic cell of origin (Kim et al., 2010; Polo et al., 2010) that can persist through extended passaging (Kim et al., 2011), and the molecular mechanisms responsible for epigenetic memory are unclear.
In ES cells, long noncoding RNAs (lncRNAs) associate with chromatin regulators such as Polycomb proteins (Guttman et al., 2011; Zhao et al., 2010; Zhao et al., 2008) and are required to repress lineage-specific genes in the pluripotent state (Guttman et al., 2011). LncRNAs have been shown to target chromatin regulatory complexes throughout the genome in various developmental settings (Lee and Bartolomei, 2013; Rinn and Chang, 2012), but relatively little is known about lncRNAs in the context of cellular reprogramming.
SUMMARY
Some embodiments of the present invention are directed to a method of enhancing reprogramming of a somatic cell to a pluripotent cell in a human or mouse, the method including upregulation of at least one long noncoding RNA (lncRNA) selected from SEQ ID NOs. 1-347 and 368-408 and isoforms, fragments, and homologs thereof in the somatic cell.
In some embodiments, the method of enhancing reprogramming of a somatic cell also includes downregulating at least one lncRNA selected from SEQ ID NOs. 348-367 and 415-424 and SEQ ID NOs. 408-414 and isoforms, fragments, and homologs thereof.
Some embodiments of the present invention are directed to a method of enhancing differentiation of a pluripotent cell in a human or mouse, the method including downregulation of at least one long noncoding RNA (lncRNA) selected from SEQ ID NOs. 1-347 and 368-407 and isoforms, fragments and homologs thereof in the pluripotent cell.
In some embodiments, the method of enhancing differentiation of a pluripotent cell also includes upregulating at least one lncRNA selected from SEQ ID NOs. 348-367 and 415-424 and SEQ ID NOs. 408-414 and isoforms, fragments, and homologs thereof.
Some embodiments of the present invention include a composition for enhancing pluripotency reprogramming in a human or mouse somatic cell, the composition includes an inhibiting nucleic acid directed against a long noncoding RNA (lncRNA) selected from SEQ ID NOs. 358-367 and SEQ ID NOs. 408-414 and isoforms, fragments, and homologs thereof.
Some embodiments of the present invention include a composition for enhancing pluripotency reprogramming in a human or mouse somatic cell, the composition includes a vector, synthetic nucleic acid or in vitro transcribed nucleic acid encoding a long noncoding RNA (lncRNA) selected from SEQ ID NOs. 1-347 and 368-407 and isoforms, fragments, and homologs thereof.



BRIEF DESCRIPTION OF THE DRAWINGS
These and other features and advantages of the present invention will be better understood by reference to the following detailed description when considered in conjunction with the accompanying drawings.
FIG. 1 is a schematic showing induction of pluripotency in somatic cells, according to embodiments of the invention.
FIGS. 2A, 2B, and 2C show induced pluripotent stem cells as described herein, according to embodiments of the invention.
FIG. 3 is a schematic showing induction of pluripotency in tail-tip fibroblasts, according to embodiments of the invention.
FIG. 4A shows activated genes in tail tip fibroblasts as described herein, according to embodiments of the invention. FIG. 4B lists 100 induced proteins as described herein. FIGS. 4C-4D show a timeline of induced pluripotency as described herein, according to embodiments of the invention.
FIGS. 5A, 5B, and 5C each show a forward scatter plot of iPS cells at week 3 (FIG. 5A), week 6 (FIG. 5B), and week 9 (FIG. 5C), according to embodiments of the invention.
FIG. 6 is a self-organizing map to identify dynamic changes in the single-cell transcriptomes of reprogramming cells to facilitate visualization of gene sets that are coordinately expressed during the reprogramming timecourse, according to embodiments of the invention.
FIG. 7A is an analysis of tail tip fibroblasts (TTFs) that strongly express genes involved in body axis anatomy and development of muscle, as described herein. Intermediate reprogramming cells at week 2 (Wk2) expressed numerous genes that are required for proper germ cell development and fertility, including Blimp1. By week 3 (Wk3) of reprogramming, individual cells began to express genes that are involved in RNA metabolism and noncoding RNA processing Late-stage iPS cells at week 6 (Wk6) strongly expressed hundreds of pseudogenes which can be processed into small regulatory RNAs in oocytes. Late-stage iPS cells at week 8 (Wk8) expressed known pluripotency factors at high levels, including Oct4, Nanog, Esrrb, and Sall4, but did not exhibit full activation of germ cell regulatory genes such as Stella and Prdm14, which are highly expressed in ES cells, according to embodiments of the invention.
FIG. 7B is a table summarizing the tail tip fibroblast (TTFs) analysis of FIG. 7A, as described herein, in which GO (gene ontology) the annotated genes as well as the Cluster frequency, Genome frequency, Corrected P-value, FDR, and False positive values are shown as indicated.
FIG. 8 shows the expression pattern of germ cell-related genes determined by log 2(RPKM+1) (RPKM=reads per kilobase per million) using small molecule fluorescent in situ hybridization (smFISH) showing that Stella and Prdm14 appear to be heterogeneously expressed during the reprogramming timecourse, as described herein.
FIG. 9 is a fluorescence image of a single cell at week 6 (wk6) of iPS cell reprogramming using 4-color smFISH to characterize the full distribution of Stella, Prdm14, Blimp1, Rex1, Oct4, and Sox2 RNAs in hundreds of late-stage iPS cells (n=303), as described herein.
FIGS. 10A-10D show a histogram (FIG. 10A) and box plots (FIGS. 10B, 10C, 10D) of RNA molecules per cell for each indicated gene, as determined by 4-color smFISH showing that cells expressing higher levels of Rex1, also expressed Stella, Prdm14, and Blimp1, while cells with low Rex1 expression generally did not express these genes, as described herein.
FIG. 11A shows the expression pattern of germ cell-related genes using small molecule fluorescent in situ hybridization (smFISH) showing that late-stage iPS cells began to express Piwi12 which associates with Piwi-interacting RNAs (piRNAs), as described herein.
FIG. 11B is a graph showing that at the population level, iPS cells expressed genes involved in piRNA function and biogenesis, as described herein.
FIG. 11C is a graph showing that the 2′-O-methylated small RNAs that were cloned and sequenced from iPS cells exhibited a characteristic size distribution peak of 26 nucleotides (nt), as described herein.
FIG. 11D is a graph showing that of the 2′-O-methylated small RNAs sequenced in FIG. 11C, 40% had a 5′U, indicative of primary piRNAs in the germline, as described herein.
FIG. 11E is a graph showing that 40% of the 2′-O-methylated small RNAs of FIG. 11C mapped to retrotransposons, as described herein.
FIG. 12 shows long noncoding RNAs (lncRNAs) that are activated during reprogramming (Ladr), according to embodiments of the invention.
FIG. 13A is a fluorescent image of a single cell at week 6 (Wk6) of iPS cell reprogramming using smFISH, with Ladr37 probe, as described herein.
FIGS. 13B and 13C are graphs of cumulative frequency of Ladr37 lncRNAs at week 6 (Wk6) (FIG. 13B) and week 9 (Wk9) (FIG. 13C) compared with ESC (embryonic stem cells), as described herein.
FIG. 14A is a fluorescent image of a single cell at week 6 (Wk6) of iPS cell reprogramming using smFISH, with Ladr80 probe, as described herein.
FIGS. 14B and 14C are graphs of cumulative frequency of Ladr80 lncRNAs at week 6 (Wk6) (FIG. 14B) and week 9 (Wk9) (FIG. 14C) compared with ESC (embryonic stem cells), as described herein.
FIG. 15A shows graphs measuring relative expression of Ladr80, Ladr37, and Ladr246 compared to controls, in late stage iPS cells in the presence or absence (control) of Ladr80 siRNA, Ladr37 siRNA, or Ladr246 siRNA, as indicated, as described herein.
FIG. 15B shows graphs measuring fold change of reprogramming efficiencies in the presence or absence (control) of Ladr37 siRNA or Ladr80 siRNA, as indicated, as described herein.
FIG. 16A shows differential expression analysis of significantly upregulated or downregulated genes in iPS cells in the presence (siLadr80) or absence (control) of Ladr80 siRNA, as determined by population level RNA-seq, and gene ontology (GO) analysis for significantly enriched GO terms in upregulated genes, as described herein.
FIG. 16B shows the expression patterns of the indicated genes in the indicated cell types, as described herein.
FIG. 16C is a graph showing gene expression of the indicated muscle genes in week 9 (Wk9) iPS cells in the presence of Ladr37 siRNA, Ladr80 siRNA, or control, as indicated, as described herein.
FIG. 16D is a graph showing gene expression of the indicated reprogramming/pluripotency-related genes Oct4, Sox2, Klf4, C-Myc, Lin28, and Nanog in Wk9 iPS cells in the presence of Ladr37 siRNA, Ladr80 siRNA, or control, as described herein.
FIG. 16E shows a graph of differential expression analysis of significantly upregulated or downregulated lncRNA genes in ES cells versus TFF cells, as described herein.
FIG. 16F shows two graphs indicating the fraction of upregulated or downregulated lncRNAs that associated with Polycomb proteins in ES cells, as described herein.
FIG. 17A shows long noncoding RNAs (lncRNAs) that are activated during reprogramming (Ladr) in IPS cells, ES cells, and primordial germ cells (PGC), as determined by single-cell RNA-seq, as described herein.
FIG. 17B is a graph showing single-cell average expression levels of Stella in the indicated cell types, as determined by single-cell RNA-seq, as described herein.
FIG. 18A is graph comparing Ladr lncRNAs with lncRNAs in ES cells, as described herein.
FIG. 18B shows the expression patterns of the indicated lncRNAs in the indicated cell types, as described herein.
FIG. 18C shows the expression patterns of the indicated lncRNAs in the indicated cell types including relative Oct4 correlation as indicated, as described herein.
FIG. 19A shows a graph indicating the fraction of the Ladr lncRNAs (115) that associate with Polycomb proteins (Polycomb-bound) in ES cells, as described herein.
FIG. 19B is a graph showing the enrichment of elements (e.g., ERVK) in Ladr lncRNAs versus, as described herein.
FIG. 19C is a schematic showing that Ladr37 is flanked by two different ERVK elements whose sequences overlap with its 5′ and 3′ exons, as described herein.
FIG. 20A is a graph showing expression of AP2γ (in RPKM) in the indicated cell types, as described herein.
FIG. 20B shows graphs indicating the fraction of Oct4, Prdm14, or Ap2γ binding sites within lncRNA promoters out of the total number of Ladr lncRNAs, as described herein.
FIG. 20C shows ChIP sequence data for Ladr80 lncRNA, Ladr169 lncRNA, Ladr37 lncRNA, or Ladr43 lncRNA, as indicated, analyzed with respect to Oct 4, Sox2, Nanog, Prdm14 and AP2γ, as described herein.
FIG. 21A is a graph showing the amount of upregulated lncRNAs or downregulated lncRNAs, as indicated, in iPS cells derived from hematopoietic progenitor cells (HPC-iPS cells) compared to HPC cells, as described herein.
FIG. 21B is a graph showing the fraction of lncRNAs activated during HPC reprogramming, as described herein.
FIG. 21C shows long noncoding RNAs (lncRNAs) that are activated in HPC-iPS cells during reprogramming, according to embodiments of the invention.
FIG. 21D is a fluorescent image of a single cell at week 6 (Wk6) of iPS cell reprogramming using smFISH, with Ladr246 probe, as described herein.
FIG. 21E shows two graphs of cumulative frequency of Ladr246 lncRNAs at week 6 (Wk6) and week 9 (Wk9) compared with ESC (embryonic stem cells), as described herein.
FIG. 21F is a graph showing differential expression analysis of upregulated or downregulated genes, as indicated, in week 9 (Wk9) iPS cells in the presence (siLadr246) or absence (control) of Ladr246 siRNA, as described herein.
FIG. 22A is a graph showing differential expression analysis of upregulated or downregulated genes, as indicated, in week 9 (Wk 9) iPS cells in the presence (siLadr272) or absence (control) of Ladr272 siRNA, as described herein.
FIG. 22B is a graph showing differential expression analysis of upregulated or downregulated genes, as indicated, in week 9 (Wk 9) iPS cells in the presence (siLadr43) or absence (control) of Ladr43 siRNA, as described herein.
FIG. 23 is a graph showing differential expression analysis of upregulated or downregulated genes in human iPS cells compared to human fibroblasts, as described herein.



DETAILED DESCRIPTION
Some embodiments of the present invention include at least one long noncoding RNA (lncRNA) that regulates the induction of pluripotency in somatic cells. As disclosed herein, the lncRNAs in Tables 1 and 2 increase effective pluripotency of iPS cells. Some embodiments of the present invention include methods for improved induction of pluripotency in somatic cells. These methods for inducing pluripotency in somatic cells include the presence of (activation of) and/or absence (repression of) at least one of the long lncRNAs disclosed in Tables 1 and/or 2. Induction of pluripotency in tail-tip fibroblasts from an OSKM mouse to produce iPS cells for comparative expression analysis with embryonic stem (ES) cells is shown schematically in FIG. 1.
As used herein, “reprogramming” in the context of a somatic cell refers to the erasure and remodeling of the differentiated somatic cell to a pluripotent embryonic state, Conversely, “reprogramming” in the context of a pluripotent cell is also referred to as differentiation, and refers to the remodeling of the pluripotent cell to a more specialized differentiated cell.
As used herein, “induction of pluripotency” and “pluripotency reprogramming” refers to the induction of pluripotency by induction of Oct4, Sox2, Klf4, c-Myc (OSKM) transcription factors and/or the “dual inhibition” of Mek and Gsk3 also known as “2i.”
In some embodiments, methods for improved induction of pluripotency in somatic cells includes the presence of the lncRNAs disclosed herein to be activated during reprogramming of the somatic cells. These lncRNAs activated during reprogramming are also referred to herein as Ladrs. Induction of pluripotency activated mouse Ladrs 1-314 (SEQ ID NOs. 1-314) are listed in Table 1 and the orthologous human lncRNA (SEQ ID NOs. 315-347) identified by Liftover analysis, are listed in Table 2.














TABLE 1





SEQ ID









NO:
lncRNA
Gene name
chr
left
right
Str*
ID






















1
Ladr 1
Gm17586
chr11
120495082
120496439
+
ENSMUSG00000090998


2
Ladr 2
4930444M15Rik
chr14
76914365
76920662
−
ENSMUSG00000085974


3
Ladr 3
1810019D21Rik
chr8
108659300
108662425
+
ENSMUSG00000086390


4
Ladr 4
B230208H11Rik
chr10
12636446
12642898
−
ENSMUSG00000085233


5
Ladr 5
Gm15728
chr5
117839148
117843507
+
ENSMUSG00000086075


6
Ladr 6
Gm2529
chr8
87189027
87193305
+
ENSMUSG00000073822


7
Ladr 7
2700086A05Rik
chr6
52125216
52165075
+
ENSMUSG00000085696


8
Ladr 8
C430039J16Rik
chr13
98053137
98055889
+
ENSMUSG00000091451


9
Ladr 9
Gm17291
chr10
62755034
62758541
−
ENSMUSG00000050249


10
Ladr 10
A330048O09Rik
chr13
48367785
48369253
−
ENSMUSG00000087671


11
Ladr 11
Gm17698
chr1
165231921
165233751
−
ENSMUSG00000090335


12
Ladr 12
4930481B07Rik
chr3
94819547
94824060
+
ENSMUSG00000085956


13
Ladr 13
A730011C13Rik
chr3
94798332
94801272
−
ENSMUSG00000084846


14
Ladr 14
4930526L06Rik
chr19
11271307
11374376
+
ENSMUSG00000085490


15
Ladr 15
Gm15787
chr5
110583419
110605523
−
ENSMUSG00000086247


16
Ladr 16
2310043M15Rik
chr16
93792395
93795133
−
ENSMUSG00000091302


17
Ladr 17
4930566F21Rik
chr5
31789002
31797552
−
ENSMUSG00000086967


18
Ladr 18
Meg3
chr12
110779211
110809936
+
ENSMUSG00000021268


19
Ladr 19
3110056K07Rik
chr12
72092602
72116819
−
ENSMUSG00000085622


20
Ladr 20
Gm15441
chr3
96359688
95370724
−
ENSMUSG00000074398


21
Ladr 21
Rian
chr12
110842155
110899919
+
ENSMUSG00000091793


22
Ladr 22
9830144P21Rik
chr2
129031694
129056315
−
ENSMUSG00000087528


23
Ladr 23
4930513N10Rik
chr8
98330730
98345628
+
ENSMUSG00000074136


24
Ladr 24
4930404I05Rik
chr16
91011494
91016988
+
ENSMUSG00000087354


25
Ladr 25
GM17250
chr2
71586132
71588219
−
ENSMUSG00000090953


26
Ladr 26
Gm16641
chr11
50050511
50102341
+
ENSMUSG00000085364


27
Ladr 27
2310010G23Rik
chrX
34354766
34357180
−
ENSMUSG00000090647


28
Ladr 28
2810442I21Rik
chr11
16835157
16851285
−
ENSMUSG00000087060


29
Ladr 29
9230114K14Rik
chr5
52581920
52589223
+
ENSMUSG00000087676


30
Ladr 30
Vax2os1
chr6
83642800
83662195
−
ENSMUSG00000085794


31
Ladr 31
1700007L15Rik
chr16
33379941
33380813
−
ENSMUSG00000090918


32
Ladr 32
4632427E13Rik
chr7
99886168
99889978
−
ENSMUSG00000074024


33
Ladr 33
Gm17491
chr8
23590763
23593802
+
ENSMUSG00000078859


34
Ladr 34
4930461G14Rik
chr9
58302980
58317430
+
ENSMUSG00000086516


35
Ladr 35
Gm16957
chr17
17526907
17532267
−
ENSMUSG00000087696


36
Ladr 36
2410003L11Rik
chr11
97459825
97484207
+
ENSMUSG00000085860


37
Ladr 37
4930500J02Rik
chr2
104399320
104411586
+
ENSMUSG00000086454


38
Ladr 38
Gm17335
chr11
22500338
22510882
−
ENSMUSG00000090797


39
Ladr 39
Gm13110
chr4
154019863
154029427
−
ENSMUSG00000086810


40
Ladr 40
9530027109Rik
chrX
45629969
45638603
−
ENSMUSG00000085283


41
Ladr 41
2500002B13Rik
chr8
59966851
59987674
+
ENSMUSG00000086276


42
Ladr 42
4933404O12Rik
chr5
137395016
137412982
+
ENSMUSG00000085062


43
Ladr 43
Gm2694
chr8
89996711
90049453
+
ENSMUSG00000087391


44
Ladr 44
A230072C01Rik
chrX
20538810
20563863
+
ENSMUSG00000086877


45
Ladr 45
C330046G13Rik
chr10
84010162
84016083
−
ENSMUSG00000085671


46
Ladr 46
Gm17300
chr4
131907687
131909297
+
ENSMUSG00000091021


47
Ladr 47
Xist
chrX
100655714
100678556
−
ENSMUSG00000086503


48
Ladr 48
4930558J18Rik
chr1
57416066
57434348
−
ENSMUSG00000084958


49
Ladr 49
Gm10143
chr19
10272858
10276874
+
ENSMUSG00000064032


50
Ladr 50
1110002J07Rik
chr10
66375237
66383006
−
ENSMUSG00000087275


51
Ladr 51
1700086O06Rik
chr18
38398059
38410219
−
ENSMUSG00000086988


52
Ladr 52
6720401G13Rik
chrX
47908921
47988498
−
ENSMUSG00000085396


53
Ladr 53
Gm17418
chr1
93885628
93889205
+
ENSMUSG00000079420


54
Ladr 54
Gm16986
chr13
66604096
66620459
−
ENSMUSG00000086120


55
Ladr 55
Gm17656
chr13
66450332
66466708
+
ENSMUSG00000090969


56
Ladr 56
B230206L02Rik
chr11
93994799
94017090
+
ENSMUSG00000086003


57
Ladr 57
Abhd1
chr5
31252439
31257464
+
ENSMUSG00000006638


58
Ladr 58
5930412G12Rik
chr5
129084981
129106568
−
ENSMUSG00000072591


59
Ladr 59
E130018N17Rik
chr2
167978103
167980013
−
ENSMUSG00000087648


60
Ladr 60
Gm14022
chr2
128886682
128891422
−
ENSMUSG00000087151


61
Ladr 61
Gm2788
chr7
56133328
56141978
+
ENSMUSG00000085995


62
Ladr 62
Gm16880
chr1
138592788
138622305
+
ENSMUSG00000085011


63
Ladr 63
Gm4419
chr12
21423772
21425664
+
ENSMUSG00000090621


64
Ladr 64
9330185C12Rik
chr1
115787962
115860711
−
ENSMUSG00000086520


65
Ladr 65
Gm17659
chr9
89989016
89990740
+
ENSMUSG00000091035


66
Ladr 66
Gm17710
chr7
95427297
95432039
−
ENSMUSG00000091953


67
Ladr 67
Gm10785
chr16
91689156
91715887
+
ENSMUSG00000085169


68
Ladr 68
Gm17561
chr17
32506250
32515390
+
ENSMUSG00000091872


69
Ladr 69
4930509G22Rik
chr16
11178269
11192385
+
ENSMUSG00000085780


70
Ladr 70
C430002E04Rik
chr3
41292726
41297114
−
ENSMUSG00000091878


71
Ladr 71
Gm17605
chr8
4088149
4089166
−
ENSMUSG00000091309


72
Ladr 72
Gm17362
chr8
23755333
23756758
+
ENSMUSG00000074911


73
Ladr 73
Gm17525
chr4
128986325
128995687
+
ENSMUSG00000091673


74
Ladr 74
Mirg
chr12
110973191
110987665
+
ENSMUSG00000091158


75
Ladr 75
1010001B22Rik
chr5
110424530
110425417
−
ENSMUSG00000091434


76
Ladr 76
Gm17597
chr15
81631221
81633043
−
ENSMUSG00000091934


77
Ladr 77
Gm16283
chr18
49915448
49916843
+
ENSMUSG00000087020


78
Ladr 78
4933407K13Rik
chrX
72952271
73010038
−
ENSMUSG00000087396


79
Ladr 79
Gm17279
chr8
19888055
20016359
+
ENSMUSG00000091588


80
Ladr 80
Gm16096
chr9
40588825
40592264
−
ENSMUSG00000087135


81
Ladr 81
Gm17440
chr7
30983649
30985908
+
ENSMUSG00000092079


82
Ladr 82
Gm17594
chrX
78316033
78318441
−
ENSMUSG00000078930


83
Ladr 83
Gm13657
chr2
75615245
75620363
+
ENSMUSG00000086813


84
Ladr 84
C330018A13Rik
chr5
116573623
116579119
+
ENSMUSG00000086655


85
Ladr 85
Gm17637
chrX
20937991
20941748
−
ENSMUSG00000091801


86
Ladr 86
Gm16827
chr18
31940880
31948699
−
ENSMUSG00000086768


87
Ladr 87
Atp10d
chr5
72594568
72690014
+
ENSMUSG00000046808


88
Ladr 88
5730457N03Rik
chr6
52258383
52264826
−
ENSMUSG00000086126


89
Ladr 89
Gm11714
chr11
107289590
107297000
+
ENSMUSG00000087113


90
Ladr 90
Gm16159
chr8
26659133
26666555
−
ENSMUSG00000086134


91
Ladr 91
Gm12898
chr4
118895779
118897420
+
ENSMUSG00000085626


92
Ladr 92
Gm16973
chr14
57315117
57320105
−
ENSMUSG00000086985


93
Ladr 93
6030442K20Rik
chr1
134363010
134373622
−
ENSMUSG00000090678


94
Ladr 94
Gm13830
chr5
115751129
115769217
+
ENSMUSG00000086368


95
Ladr 95
C330013F16Rik
chrX
135774765
135892277
−
ENSMUSG00000086807


96
Ladr 96
4930467K11Rik
chr10
57198187
57206159
−
ENSMUSG00000085621


97
Ladr 97
2010300F17Rik
chr11
96574296
96608750

ENSMUSG00000091444


98
Ladr 98
Gm2464
chr3
13471906
13474596
−
ENSMUSG00000078587


99
Ladr 99
9530080011Rik
chr4
95626406
95634346
−
ENSMUSG00000044125


100
Ladr 100
5C046401
chr2
165683184
165684534
+
ENSMUSG00000085274


101
Ladr 101
Gm17481
chr15
78693386
78697319
−
ENSMUSG00000086640


102
Ladr 102
Gm14817
chrX
71040554
71042955
−
ENSMUSG00000085669


103
Ladr 103
Gn717501
chr3
145313276
145315057
+
ENSMUSG00000090771


104
Ladr 104
Gm17502
chr7
6296993
6300606
+
ENSMUSG00000091822


105
Ladr 105
1700023H05Rik
chr13
81023485
81024785
−
ENSMUSG00000089827


106
Ladr 106
4930480K23Rik
chr14
70132591
70166920
+
ENSMUSG00000085243


107
Ladr 107
1110020A21Rik
chr17
85354307
85357050
−
ENSMUSG00000087023


108
Ladr 108
Gm16723
chr6
87858837
87864453
−
ENSMUSG000000857O3


109
Ladr 109
Gm16972
chr4
43058906
43066125
+
ENSMUSG00000086983


110
Ladr 110
GM807
chr13
99870661
99877535
+
ENSMUSG00000074744


111
Ladr 111
D030068K23Rik
chr8
111599122
111797711
+
ENSMUSG00000085859


112
Ladr 112
Gm8378
chr12
92913252
92924553
−
ENSMUSG00000090722


113
Ladr 113
Gm12059
chr11
22967998
22975574
+
ENSMUSG00000085665


114
Ladr 114
Gm17716
chr14
26175287
26198121
−
ENSMUSG00000091164


115
Ladr 115
Gm16244
chr19
42853522
42855944
−
ENSMUSG00000090235


116
Ladr 116
3110045C21Rik
chr1
171899540
171902526
+
ENSMUSG00000085494


117
Ladr 117
Gm16845
chr9
21875476
21890566
+
ENSMUSG00000087161


118
Ladr 118
Gm10658
chr9
56904437
56919773
−
ENSMUSG00000074284


119
Ladr 119
A930011O12Rik
chr14
65206952
65212796
+
ENSMUSG00000091456


120
Ladr 120
C030010L15Rik 
chr16
98215766
98218584
−
ENSMUSG00000091751


121
Ladr 121
4833407H14Rik
chr19
53535121
53537213
+
ENSMUSG00000090800


122
Ladr 122
Nespas
chr2
174106738
174120937
−
ENSMUSG00000086537


123
Ladr 123
Gm10492
chr17
95234246
95235982
+
ENSMUSG00000073366


124
Ladr 124
Gm17713
chr1
135506988
135510999
+
ENSMUSG00000092104


125
Ladr 125
A930029G22Rik
chr17
69766000
69788644
−
ENSMUSG00000085144


126
Ladr 126
Gm16046
chr17
13812667
13820523
−
ENSMUSG00000085705


127
Ladr 127
GM15522
chr5
34993367
34995126
+
ENSMUSG00000085166


128
Ladr 128
Gm11818
chr4
12833984
12908632
+
ENSMUSG00000055963


129
Ladr 129
1700016P03Rik
chr11
74986062
74991135
+
ENSMUSG00000085609


130
Ladr 130
Hoxa11as
chr6
52195051
52199794
+
ENSMUSG00000086427


131
Ladr 131
Gm17477
chr9
79966702
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−
ENSMUSG00000084917


132
Ladr 132
C030037DO9Rik
chr11
88579959
88590207
+
ENSMUSG00000087574


133
Ladr 133
A330040F15Rik
chr19 
12660358
12671056
−
ENSMUSG00000086213


134
Ladr 134
Mir155
chr16
84713268
84715487
+
ENSMUSG00000091875


135
Ladr 135
Gm4890
chr8
81819022
81831584
+
ENSMUSG00000085259


136
Ladr 136
1700028E1ORik
chr5
152171284
152216658
+
ENSMUSG00000087548


137
Ladr 137
Gm10575
chr7
148647380
148653670
−
ENSMUSG00000073787


138
Ladr 138
Gm15545
chr7
52242270
52249967
+
ENSMUSG00000087138


139
Ladr 139
A030009H04Rik
chr11 
69153963
69156143
+
ENSMUSG00000043419


140
Ladr 140
0610005C13Rik
chr7
52823165
52830697
−
ENSMUSG00000085214


141
Ladr 141
Gm17344
chr8
112385625
112392355
−
ENSMUSG00000078143


142
Ladr 142
Gm16761
chr16 
29907605
29946603
−
ENSMUSG00000084810


143
Ladr 143
Gm11538
chr11
96064768
96066699
+
ENSMUSG00000085983


144
Ladr 144
A230056P14Rik
chr7
63217901
63236238
+
ENSMUSG00000087178


145
Ladr 145
Gm16685
chr3
7612705
7690001
+
ENSMUSG00000086143


146
Ladr 146
Gm17690
chr3
95971387
95976337
−
ENSMUSG00000091244


147
Ladr 147
4933431E20Rik
chr3
107691768
107699131
−
ENSMUSG00000086968


148
Ladr 148
Gm17565
chr4
145686275
145688945
+
ENSMUSG00000090554


149
Ladr 149
1700030C12Rik
chr11
23397753
23399661
+
ENSMUSG00000086459


150
Ladr 150
2410133F24Rik
chr9
56947757
56979097
−
ENSMUSG00000086728


151
Ladr 151
C530005A16Rik
chr4
116262338
116270235
−
ENSMUSG00000085408


152
Ladr 152
Gm17254
chr9
96968602
96973848
+
ENSMUSG00000091219


153
Ladr 153
Gm17701
chr6
127058407
127060485
+
ENSMUSG00000091856


154
Ladr 154
Gm15489
chr7
129282522
129302915
−
ENSMUSG00000086942


155
Ladr 155
9530059O14Rik
chr9
122481615
122588714
+
ENSMUSG00000086476


156
Ladr 156
Gm17102
chr7
48649068
48652817
+
ENSMUSG00000091864


157
Ladr 157
Dio3os
chr12
111513594
111516278
−
ENSMUSG00000090962


158
Ladr 158
Snord123
chr15
32170324
32174417
−
ENSMUSG00000090401


159
Ladr 159
1700023L04Rik
chr6
29935329
29943776
+
ENSMUSG00000045709


160
Ladr 160
Gdap10
chr12
33506737
33511769
+
ENSMUSG00000059937


161
Ladr 161
Gm17500
chr11
51938032
51941281
−
ENSMUSG00000092028


162
Ladr 162
Gm17692
chr17
6314199
6492328
+
ENSMUSG00000091795


163
Ladr 163
Gm12784
chr7
35332225
35341223
+
ENSMUSG00000086631


164
Ladr 164
2810011L19Rik
chr12
106574804
106622141
+
ENSMUSG00000086023


165
Ladr 165
Gm16618
chr16
20511206
20517839
−
ENSMUSG00000086837


166
Ladr 165
Gm16912
chr17
29598841
29628870
−
ENSMUSG00000087551


167
Ladr 167
Gm12592
chr11
3205429
3231181
−
ENSMUSG00000053263


168
Ladr 168
Gm13261
chr2
10260910
10295668
−
ENSMUSG00000086748


169
Ladr 169
C330002G04Rik
chr19
23111880
23150343
−
ENSMUSG00000087169


170
Ladr 170
Gm16869
chr9
3000282
3038313
−
ENSMUSG00000087580


171
Ladr 171
Gn16938
chr7
105325704
105334107
+
ENSMUSG00000086325


172
Ladr 172
F630040L22Rik
chr9
108007296
108020674
+
ENSMUSG00000087645


173
Ladr 173
Gm17445
chr7
26601673
26605673
+
ENSMUSG00000084812


174
Ladr 174
AY512931
chr8
46146056
46150772
−
ENSMUSG00000066158


175
Ladr 175
Gm6410
chr8
4678462
4688494
+
ENSMUSG00000090435


176
Ladr 176
A930007I19Rik
chr19
29560845
29597649
−
ENSMUSG00000086309


177
Ladr 177
3010001F23Rik
chrX
148803116
148851241
+
ENSMUSG00000084885


178
Ladr 178
A330076H08Rik
chr7
69075799
69127242
−
ENSMUSG00000087490


179
Ladr 179
Gm15850
chr1
138022755
138027760
−
ENSMUSG00000086264


180
Ladr 180
Gm10425
chr12
112665085
112669157
−
ENSMUSG00000072830


181
Ladr 181
Gm16629
chr15
92174790
92208519
+
ENSMUSG00000085294


182
Ladr 182
2900052L18Rik
chr11
120091116
120092899
−
ENSMUSG00000043993


183
Ladr 183
2310031A07Rik
chr11
46253864
46260750
−
ENSMUSG00000091190


184
Ladr 184
4930506C21Rik
chr17
8486231
8503945
−
ENSMUSG00000087478


185
Ladr 185
Gm16733
chr7
20355960
20360752
+
ENSMUSG00000085994


186
Ladr 186
Gm11574
chr11
96910005
96912746
−
ENSMUSG00000085262


187
Ladr 187
2810429I04Rik
chr13
3477489
3493644
+
ENSMUSG00000086566


188
Ladr 188
A230004M16Rik
chr11
41523844
41786619
+
ENSMUSG00000087306


189
Ladr 189
Gm17392
chr17
55696464
55702805
+
ENSMUSG00000090426


190
Ladr 190
Gm17699
chr1
130667176
130670998
−
ENSMUSG00000091877


191
Ladr 191
A730099G02Rik
chr10
48939228
48945459
−
ENSMUSG00000091943


192
Ladr 192
Gm17644
chr1
12657644
12663171
+
ENSMUSG00000066918


193
Ladr 193
Gm17255
chr9
80849043
81097197
+
ENSMUSG00000090679


194
Ladr 194
Gm17638
chr15
77792104
77797556
+
ENSMUSG00000091802


195
Ladr 195
Gm16755
chr11
120347653
120349896
−
ENSMUSG00000086218


196
Ladr 196
Gm16702
chr17
8571910
8582333
−
ENSMUSG00000086627


197
Ladr 197
Gm11934
chr4
33390544
33397112
−
ENSMUSG00000086127


198
Ladr 198
Gm15396
chr7
51835338
51845994
−
ENSMUSG00000087560


199
Ladr 199
Gm3906
chr9
43921006
43930030
+
ENSMUSG00000092003


200
Ladr 200
Gm12976
chr4
128939209
128942254
+
ENSMUSG00000087575


201
Ladr 201
Gm16862
chr17
45871455
45925543
+
ENSMUSG00000086615


202
Ladr 202
Gm17496
chr16
95237977
95288624
+
ENSMUSG00000092023


203
Ladr 203
BC051077
chr5
117762529
117765548
−
ENSMUSG00000091333


204
Ladr 204
Gm17632
chr3
116191884
116196276
+
ENSMUSG00000090721


205
Ladr 205
Gm17275
chr1
182732699
182739614
+
ENSMUSG00000090986


206
Ladr 206
Gm17596
chr9
57379094
57385436
−
ENSMUSG00000091932


207
Ladr 207
Gm17327
chr7
35009774
35015255
+
ENSMUSG00000090311


208
Ladr 208
Gm17599
chr4
43043871
43045354
+
ENSMUSG00000091065


209
Ladr 209
Gm17517
chr8
60650232
60777663
+
ENSMUSG00000091944


210
Ladr 210
Gm17336
chr10
128082940
128086867
+
ENSMUSG00000091431


211
Ladr 211
A330032B11Rik
chr19
37248333
37271031
+
ENSMUSG00000085432


212
Ladr 212
1700007J10Rik
chr11
59539419
59553656
−
ENSMUSG00000086330


213
Ladr 213
Gm11542
chr11
94538879
94548978
+
ENSMUSG00000085051


214
Ladr 214
4833417C18Rik
chr11
95720179
95722358
+
ENSMUSG00000086015


215
Ladr 215
Gm17282
chr13
69948792
69954972
−
ENSMUSG00000091909


216
Ladr 216
Mir706
chr6
119983540
119986489
−
ENSMUSG00000090388


217
Ladr 217
6430562O15Rik
chr13
100166879
100182819
−
ENSMUSG00000085195


218
Ladr 218
Gm7782
chr13
65480701
65553664
−
ENSMUSG00000090514


219
Ladr 219
2610027K06Rik
chr11
85605163
85645725
−
ENSMUSG00000087013


220
Ladr 220
1110046J04Rik
chr13
34027689
34028499
+
ENSMUSG00000085457


221
Ladr 221
Gm17675
chr2
120358640
120365582
+
ENSMUSG00000091714


222
Ladr 222
A730081D07Rik
chr13 
41051783
41096520
−
ENSMUSG00000086693


223
Ladr 223
2610206C17Rik
chr7
91838150
91925059
+
ENSMUSG00000085236


224
Ladr 224
1110019D14Rik
chr6
13821526
13994373
+
ENSMUSG00000084931


225
Ladr 225
Gm16706
chr11
79830856
79843370
−
ENSMUSG00000086787


226
Ladr 226
Gm17317
chr4
146510387
146831128
+
ENSMUSG00000091833


227
Ladr 227
Gm16889
chr4
146392344
146866310
−
ENSMUSG00000084976


228
Ladr 228
Gm17452
chr4
146120857
146399068
−
ENSMUSG00000092143


229
Ladr 229
Gm16739
chr5
110351668
110352500
−
ENSMUSG00000085131


230
Ladr 230
Gm11602
chr1
63161045
63204864
+
ENSMUSG00000084799


231
Ladr 231
Gm16684
chr17 
32947089
32958642
+
ENSMUSG00000085948


232
Ladr 232
Gm17548
chr3
63767555
63772581
+
ENSMUSG00000091540


233
Ladr 233
Gm2366
chr8
46102570
46107739
−
ENSMUSG00000091546


234
Ladr 234
Gm17529
chr12 
60054082
60068998
+
ENSMUSG00000092098


235
Ladr 235 
Gm17451
chr6
38501531
38504007
−
ENSMUSG00000092140


236
Ladr 236
3100003L05Rik
chr7
131769184
131852449
−
ENSMUSG00000086254


237
Ladr 237
2810425M01Rik
chr10 
76949409
76979175
+
ENSMUSG00000086832


238
Ladr 238
Gm17238
chr18 
67933366
67934511
−
ENSMUSG00000091127


239
Ladr 239
2010110K18Rik
chr18 
34911463
34918339
+
ENSMUSG00000085410


240
Ladr 240
Gm17499
chr10
90612260
90613339
+
ENSMUSG00000092024


241
Ladr 241
Gm9917
chr9
107461141
107470746 
−
ENSMUSG00000053666


242
Ladr 242
Gm16568
chr9
15135017
15139789
+
ENSMUSG00000089702


243
Ladr 243
Gm16624
chr5
24202720
24210508
−
ENSMUSG00000084903


244
Ladr 244
1500026H17Rik
chr10
89149116
89163611
+
ENSMUSG00000087686


245
Ladr 245
1700109K24Rik
chr15
76914873
76926744
+
ENSMUSG00000087126


246
Ladr 246
2010204K13Rik
chrX
6988943
7022269
−
ENSMUSG00000063018


247
Ladr 247
2010320O07Rik
chr18
38369426
38388799
+
ENSMUSG00000089983


248
Ladr 248
2610035F20Rik
chr14
122869506
122872801
−
ENSMUSG00000085555


249
Ladr 249
2810430I11Rik
chr2
27738445
27742572
−
ENSMUSG00000085766


250
Ladr 250
2900041M22Rik
chr11
117472561
117475171
+
ENSMUSG00000054418


251
Ladr 251
4732463B04Rik
chr12
85380419
85394757
−
ENSMUSG00000086299


252
Ladr 252
4833412C05Rik
chr7
74929611
74948382
−
ENSMUSG00000085850


253
Ladr 253
4930544I03Rik
chr12
91991648
92208208
−
ENSMUSG00000092100


254
Ladr 254
4932430I15Rik
chr5
93233246
93238514
−
ENSMUSG00000072828


255
Ladr 255
4933427G23Rik
chr5
23321436
23337715
−
ENSMUSG00000086697


256
Ladr 256
5330411J11Rik
chr2
59220559
59225528
−
ENSMUSG00000087455


257
Ladr 257
5430416N02Rik
chr5
100849861
100858535
−
ENSMUSG00000084877


258
Ladr 258
7530420F21Rik
chr1
151922003
151947004
−
ENSMUSG00000084952


259
Ladr 259
8030451A03Rik
chr4
63640888
63810958
+
ENSMUSG00000073821


260
Ladr 260
A230028O05Rik
chr16
25059725
25069144
+
ENSMUSG00000085040


261
Ladr 261
A330094K24Rik
chr18
77968420
77972209
−
ENSMUSG00000090400


262
Ladr 262
A930001C03Rik
chr19
4439003
4448332
+
ENSMUSG00000087132


263
Ladr 263
AI854517
chr7
86645003
86679283
+
ENSMUSG00000085554


264
Ladr 264
Airn
chr17
12934177
13052988
+
ENSMUSG00000078247


265
Ladr 265
B230354K17Rik
chr17
45570801
45579493
+
ENSMUSG00000073393


266
Ladr 266
C130021I20Rik
chr2
33496713
33501869
+
ENSMUSG00000052951


267
Ladr 267
C130071C03Rik
chr13
83861160
83880913
+
ENSMUSG00000050334


268
Ladr 268
Emx2os
chr19
59499594
59533125
−
ENSMUSG00000087095


269
Ladr 269
Fam150a
chr1
6349537
6384812
+
ENSMUSG00000087247


270
Ladr 270
G730013B05Rik
chr16
50526417
50559548
+
ENSMUSG00000085617


271
Ladr 271
Gm10561
chr1
55283095
55292466
+
ENSMUSG00000073675


272
Ladr 272
Gm11019
chr13
98204925
98266960
+
ENSMUSG00000078952


273
Ladr 273
Gm12100
chr11
30650832
30656147
+
ENSMUSG00000087474


274
Ladr 274
Gm13939
chr2
109742553
109753476
−
ENSMUSG00000063751


275
Ladr 275
Gm14261
chr2
168591643
168593608
+
ENSMUSG00000085322


276
Ladr 276
Gm15270
chr10
24269247
24316914
−
ENSMUSG00000087400


277
Ladr 277
Gm15283
chr12
75027468
75050772
−
ENSMUSG00000087700


278
Ladr 278
Gm16070
chr1
17666108
17717727
−
ENSMUSG00000085125


279
Ladr 279
Gm16091
chr8
74671500
74684754
−
ENSMUSG00000087502


280
Ladr 280
Gm16233
chr3
144610525
144617842
+
ENSMUSG00000085773


281
Ladr 281
Gm16704
chr19
34549298
34556036
+
ENSMUSG00000085164


282
Ladr 282
Gm16707
chr9
119349396
119353948
−
ENSMUSG00000086780


283
Ladr 283
Gm16789
chr16
35806033
35809032
−
ENSMUSG00000086400


284
Ladr 284
Gm16882
chr18
37906213
37927896
−
ENSMUSG00000085906


285
Ladr 285
Gm16896
chr6
89254624
89277615
+
ENSMUSG00000085988


286
Ladr 286
Gm16952
chr17
71094669
71104922
−
ENSMUSG00000087105


287
Ladr 287
Gm17256
chr12
88673438
88676605
+
ENSMUSG00000090672


288
Ladr 288
Gm17278
chr13
65691229
65692497
−
ENSMUSG00000091589


289
Ladr 289
Gm17311
chr1
152006173
152012078
−
ENSMUSG00000091113


290
Ladr 290
Gm17322
chr9
57845848
57855258
+
ENSMUSG00000091267


291
Ladr 291
Gm17396
chr9
117161654
117163814
+
ENSMUSG00000079669


292
Ladr 292
Gm17431
chr5
5781530
5783636
+
ENSMUSG00000091282


293
Ladr 293
Gm17443
chr8
33118976
33126231
+
ENSMUSG00000091819


294
Ladr 294
Gm17513
chr2
177758513
177763441
−
ENSMUSG00000090944


295
Ladr 295
Gm17514
chr13
66284015
66304572
−
ENSMUSG00000090945


296
Ladr 296
Gm17520
chr1
173353316
173367449
−
ENSMUSG00000092117


297
Ladr 297
Gm17560
chr1
93193780
93200982
−
ENSMUSG00000091871


298
Ladr 298
Gm17575
chr3
96179569
96192787
−
ENSMUSG00000091380


299
Ladr 299
Gm17609
chr4
145191866
145301871
−
ENSMUSG00000090398


300
Ladr 300
Gm17685
chr7
152080465
152083366
−
ENSMUSG00000090767


301
Ladr 301
Gm17718
chr1
138518675
138521227
−
ENSMUSG00000091667


302
Ladr 302
Gm17724
chr5
110814854
110817092
+
ENSMUS000000090661


303
Ladr 303
Gm17735
chr13
66345152
66365458
+
ENSMUSG00000090502


304
Ladr 304
Gm2115
chr7
91677485
91726847
+
ENSMUSG00000085128


305
Ladr 305
Gm4349
chr3
95231593
95235009
+
ENSMUSG00000091761


306
Ladr 306
Gm6634
chr3
70576301
70611213
−
ENSMUSG00000086538


307
Ladr 307
Gm6846
chr10
21550305
21564657
−
ENSMUSG00000085422


308
Ladr 308
Igf2as
chr7
149845598
149856261
+
ENSMUSG00000086266


309
Ladr 309
Miat
chr5
112642248
112657968
−
ENSMUSG00000086878


310
Ladr 310
Rgs22
chr15
36067976
36070140
−
ENSMUSG00000091092


311
Ladr 311
Sox2ot
chr3
34459303
34579773
+
ENSMUSG00000090828


312
Ladr 312
Tdrd5
chr1
158185426
158233795
−
ENSMUSG00000060985


313
Ladr 313
Tsix
chrX
100626856
100680296
+
ENSMUSG00000085715


314
Ladr 314
Ttc28
chr5
111308822
111718800
+
ENSMUSG00000033209





*DNA strand: + = positive; − = negative













TABLE 2





Upregulated Mouse and Human lncRNA


orthologs upon induction of pluripotency



















SEQ ID






NO:
lncRNA
chr-mm9
left-mm9
right-mm9





50
Ladr50
chr10
66375237
66383006


129
Ladr129
chr11
74986062
74991135


136
Ladr136
chr5
152171284
152216658


150
Ladr150
chr9
56947757
56979097


164
Ladr164
chr12
106574804
106622141


236
Ladr236
chr7
131769184
131852449


32
Ladr32
chr7
99886168
99889978


48
Ladr48
chr1
57416066
57434348


88
Ladr88
chr6
52258383
52264826


58
Ladr58
chr5
129084981
129106568


259
Ladr259
chr4
63640888
63810958


176
Ladr176
chr19
29560845
29597649


263
Ladr263
chr7
86645003
86679283


264
Ladr264
chr17
12934177
13052988


56
Ladr56
chr11
93994799
94017090


267
Ladr267
chr13
83861160
83880913


84
Ladr84
chr5
116573623
116579119


111
Ladr111
chr8
111599122
111797711


157
Ladr157
chr12
111513594
111516278


272
Ladr272
chr13
98204925
98266960


230
Ladr230
chr1
63161045
63204864


277
Ladr277
chr12
75027468
75050772


154
Ladr154
chr7
129282522
129302915


279
Ladr279
chr8
74671500
74684754


145
Ladr145
chr3
7612705
7690001


282
Ladr282
chr9
119349396
119353948 


142
Ladr142
chr16
29907605
29946603


201
Ladr201
chr17
45871455
45925543


141
Ladr141
chr8
112385625
112392355 


192
Ladr192
chr1
12657644
12663171


43
Ladr43
chr8
89996711
90049453


135
Ladr135
chr8
81819022
81831584


110
Ladr110
chr13
99870661
99877535


130
Ladr130
chr6
52195051
52199794


18
Ladr18
chr12
110779211
110809936


134
Ladr134
chr16
84713268
84715487


74
Ladr74
chr12
110973191
110987665 


21
Ladr21
chr12
110842155
110899919 


158
Ladr158
chr15
32170324
32174417


311
Ladr311
chr3
34459303
34579773


313
Ladr313
chrX
100626856
100680296


30
Ladr30
chr6
83642800
83662195


47
Ladr47
chrX
100655714
100678556













SEQ ID NO:
chr-hc19
left-hg19
right-hg19





315
chr10
65473608
65477165


316
chr17
1946762
1954455


317
chr13
34116876
34220938


318
chr15
75660404
75699561


319
chr14
96342568
96391908


320
chr16
26306223
26376651


321
chr11
82783096
82790139


322
chr2
200761331 
200775882


323
chr7
27277134
27283643


324
chr12
130624758 
130646888


325
chr9
117806328 
117983873


326
chr9
5582670
5629781


327
chr15
89905832
89941709


328
chr6
160303155 
160413129


329
chr17
49002131
49028278


330
chr5
87960259
87980578


331
chr12
119969729 
119982930


332
chr16
72438983
72698902


333
chr14
102023636 
102026673


334
chr5
73669250
73730264


335
chr2
206950705 
207007651


336
chr14
62187023
62217777


337
chr16
23673394
23690117


338
chr19
16205669
16222211


339
chr8
79716708
79814703


340
chr3
38532681 
38537742


341
chr3
193692107
193721538


342
chr6
43991381 
44043849


343
chr16
71757936
71765413


344
chr8
70356940 
70361515


345
chr16
49315971 
49372596


346
chr4
146521236
146540150


347
chr5
71895453 
71900248


348
chr7
27224096
27228917


349
chr14
101292457
101327362


350
chr21
26945089
26947227


351
chr14
101521835
101539271


352
chr14
101361216
101417326


353
chr5
9546382
9550405


354
chr3
181328327
181461409


355
chrX
73014340
73073529


356
chr2
71099447
71128657


357
chrX
73040495
73072548









Additional human Lairs were identified in human skin fibroblasts upon induction of pluripotency reprogramming. These human lncRNAs include SEQ ID NOs. 368-408 as listed in Table 3.







TABLE 3







Human lncRNAs upregulated during pluripotency


reprogramming of primary skin fibroblasts.












SEQ ID







NO:
lncRNA
chr-hg19
str
left-hg19
right-hg19















368
RP11-426L16.8
chr1
−
113362792
113393265


369
AC008069.3
chr2
−
16973247
16978722


370
RP11-5N23.2
chr10
+
6622381
6627641


371
AL513497.1
chr1
−
28835514
28837109


372
AC021224.1
chr18
+
29992145
29993199


373
RP11-308B16.1
chr5
+
12574969
12804475


374
AC022409.1
chr19
−
23582041
23598873


375
AP002856.5
chr11
+
131123317
131170666


376
AC074289.1
chr2
+
64370373
64479993


377
RP11-342C23.4
chr9
+
97320996
97330312


378
AL133167.1
chr14
+
96342729
96391899


379
RP11-277P12.10
chr12
−
10485460
10490891


380
AP003486.1
chr11
−
130434325
130628495


381
RMST
chr12
+
97858799
97958793


382
KIAA0040
chr1
−
175126123
175162079


383
AC010627.1
chr5
+
14651755
14653492


384
RP11-69I8.2
chr6
+
132223103
132241705


385
AC009163.1
chr16
+
75507023
75529305


386
AL136362.1
chrX
−
91354536
91360178


387
MIR17HG
chr13
+
92000074
92006833


388
RP11-771K4.1
chr12
−
31516415
31522235


389
AC078819.1
chr12
−
104424522
104426026


390
AP000689.1
chr21
−
37502670
37648524


391
RP11-562F9.2
chr4
−
93189918
93198226


392
RP11-168P6.1
chr13
−
54689924
54707001


393
RP11-168O16.1
chr1
+
200993077
200997920


394
RP11-713B9.1
chr11
+
115045697
115046044


395
SOX2OT
chr3
+
180721562
181508734


396
RP11-403C10.2
chr8
−
9757574
9762876


397
RP11-697K23.1
chr3
−
45720535
45730626


398
AC020928.1
chr19
+
37264055
37267978


399
RP11-366M4.3
chr4
+
165798156
165820117


400
RP11-20D14.6
chr12
+
8940853
8948385


401
AC126775.1
chr5
+
146939557
147041572


402
AL691420.1
chr9
+
118235807
118353358


403
AC005753.1
chr5
+
141227143
141231803


404
RP11-799O21.1
chr10
+
6821560
6884868


405
RP11-129K20.2
chr3
+
62936105
63110738


406
AC112484.1
chr3
−
128679210
128684200


407
AC005394.1
chr19
−
28926300
29218587





DNA strand: + = positive strand; − = negative strand






In other embodiments of the present invention, improved somatic cell reprogramming includes the absence or repression of the lncRNAs disclosed herein to be downregulated during reprogramming of the somatic cells. The lncRNAs which are disclosed herein to be downregulated during induced pluripotency include the mouse lncRNAs (SEQ ID NOs. 415-424) as listed in Table 4A and the human lncRNAs (SEQ ID NOs. 358-367) as listed in Table 4B.
In some embodiments, decreasing the expression or cellular activity in mouse cells includes using one or more of the mouse lncRNAs encoded by SEQ ID NOs: 415-424, or decreasing the expression or cellular activity in human cells using one or more of the human lncRNAs enclosed by SEQ ID NOs. 358-367 as listed in Table 4B.







TABLE 4A







Mouse (mm9) downregulated lncRNAs during


iPS cell reprogramming













SEQ ID
chr-





1ncRNA
NO:
mm9
Str*
left-mm9
right-mm9















2210408F21Rik 
415
chr6
+
31170351
31287404


D030054H15Rik
416
chr17
−
8075373
8139217


Gm13986
417
chr2
−
117683027
117936938


Gm14005
418
chr2
−
128021729
128255085


Gm14488
419
chr2
−
30568667
30575577


Gm16625
420
chr8
+
25532876
25550480


1700020I14Rik
421
chr2
+
119420033
119433238


2410006H16Rik
422
chr11
+
62416379
62418309


9430037G07Rik
423
chr9
−
88490163
88494354


Gm17480
424
chr17
+
25931839
26101729





*DNA strand: + = positive strand; − = negative strand













TABLE 4B







Human (hg19) liftOver coordinates of mm9 in Table 4A.












SEQ ID





lncRNA
NO:
chr-hg19
left-hg19
right-hg19














2210408F21Rik
358
chr7
130794788
130934427


D030054H15Rik
359
chr6
159420370
159509319


Gm13986
360
chr15
39537299
39872516


Gm14005
361
chr2
111966340
112252695


Gm14488
362
chr9
132251908
132259983


Gm16625
363
chr8
40009983
40028634


1700020I14Rik
364
chr15
41576169
41598737


2410006H16Rik
365
chr17
16342356
16345222


9430037G07Rik
366
chr6
86386845
86388510


Gm17480
367
chr16
597538
767429









Additional human downregulated lncRNAs were identified in human skin fibroblasts upon induction of pluripotency reprogramming. These human lncRNAs include SEQ ID NOs. 408-414 as listed in Table 5.
Table 5. Downregulated human lncRNAs during induced pluripotency in human skin fibroblasts















SEQ ID NO:
lncRNA
chr-hg19
str*
left-hg19
right-hg19




















408
C17orf91
chr17
−
1614805
1619504


409
AC005323.1
chr17
+
10286461
10527704


410
RP11-834C11.4
chr12
+
54519882
54526627


411
RP11-792D21.2
chr4
+
79567057
79603853


412
AP000769.4
chr11
+
65211929
65212028


413
RP11-90J7.3
chr10
+
80008497
80434724


414
NEAT1
chr11
+
65190245
65213011





*DNA strand: + = positive strand; − = negative strand






Embodiments of the present invention include lncRNAs which are upregulated or downregulated upon induction of pluripotency and/or inhibition of Mek and Gsk3, referred to herein as “2i inhibition” or “2i conditions.” In some embodiments of the present invention, improved pluripotency by reprogramming of a somatic cell, includes the presence of the activated lncRNAs (i.e., Ladrs) in a pluripotency cell reaction. Methods for reprogramming of human induced pluripotent stem cells is described herein and has been previously described in Loewer et al., 2010, Nature Genetics, 42: 1113-1117, the entire contents of which are herein incorporated by reference. For example, reprogramming of induced pluripotent stem cells may include induction of the Oct4, Sox2, Klf4, c-Myc (OSKM) transcription factors and/or the “dual inhibition” of Mek and Gsk3 also known as “2i.”
In some embodiments, the lncRNA and lncRNA fragments of the present invention include fragments of the sequence that are at least 20 nucleotides (nt) in length. In one embodiment, an lncRNA molecule includes a nucleotide sequence that is at least about 85% or more homologous or identical to the entire length of a lncRNA sequence shown herein, e.g., in Tables 1, 2, 3, 4, or 5, or a fragment comprising at least 20 nt thereof (e.g., at least 25, 30, 35, 40, 50, 60, 70, 80, 90, or 100 nt thereof, e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50% or more of the full length lncRNA). In some embodiments, the nucleotide sequence is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homologous or identical to a lncRNA sequence shown herein.
In Tables 1, 2, 3, 4, and 5 disclosed herein the genomic coordinates are provided for each lncRNA. As understood by a person having ordinary skill in the art, any lncRNA transcripts that overlap by at least 1 base pair with the genomic coordinates of the lncRNAs disclosed herein, should be considered isoforms of these lncRNAs with analogous functional roles during somatic cell reprogramming or pluripotent stem cell differentiation.
In order to determine the percent identity of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in an nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). The length of a reference sequence aligned for comparison purposes is at least 80% of the length of the reference sequence, and in some embodiments is at least 90% or 100%. The nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein nucleic acid “identity” is equivalent to nucleic acid “homology”). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
For purposes of the present invention, the comparison of sequences and determination of percent identity between two sequences may be accomplished using a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
Methods of Pluripotency Reprogramming or Pluripotent Cell Differentiation.
Methods of enhancing pluripotency reprogramming or pluripotent cell differentiation are disclosed herein. The lncRNAs described herein, including fragments thereof that are at least 20 nt in length, and inhibitory nucleic acids and small molecules targeting (e.g., complementary to) them, can be used to modulate pluripotency reprogramming. Methods for enchanced pluripotency reprogramming include the addition or activation of Ladr molecules disclosed in Tables 1, 2, and/or 3 and/or the inhibition of the lncRNAs disclosed in Tables 4A, 4B and/or 5. Conversely, enchancing pluripotent cell differentiation includes the absence or repression of the Ladr sequences in Tables 1, 2, and/or 3 and/or the addition or activation of the lncRNA sequences in Tables 4A, 4B and/or 5.
For enhancing pluripotency reprogramming of a somatic cell or differentiation of a pluripotent cell, addition or activation of at least one lncRNA may include one of many known suitable methods. For example, the somatic cell or pluripotent cell may be contacted with (e.g., cultured with) synthetic lncRNAs or in vitro transcribed lncRNA encoding the lncRNA or a fragment thereof. Additional non-limiting examples for increasing the presence of lncRNA in the presence of somatic cell for induced pluripotency reprogramming includes delivery vectors, viral viruses, and chemical synthesis.
Enhancing pluripotency reprogramming of a somatic cell or differentiation of a pluripotent cell may include inhibition or repression of at least one lncRNA may include one of many known suitable methods. For example, the somatic cell or pluripotent cell may be contacted with (e.g., cultured with) an inhibiting nucleic acid of at least one lncRNA. Inhibiting nucleic acid molecules include antisense oligonucleotides, interfering RNA (RNAi) including small interfering RNA (siRNA) and short hairpin RNA (shRNA). Inhibiting nucleic acid molecules used to practice the methods described herein, whether RNA, cDNA, genomic DNA, vectors, viruses or hybrids thereof, can be isolated from a variety of sources, genetically engineered, amplified, and/or expressed/generated recombinantly. If desired, nucleic acid sequences of the invention can be inserted into delivery vectors and expressed from transcription units within the vectors. The recombinant vectors can be DNA plasmids or viral vectors. Generation of the vector construct can be accomplished using any suitable genetic engineering techniques well known in the art, including, without limitation, the standard techniques of PCR, oligonucleotide synthesis, restriction endonuclease digestion, ligation, transformation, plasmid purification, and DNA sequencing, for example as described in Sambrook et al. Molecular Cloning: A Laboratory Manual. (1989)), Coffin et al. (Retroviruses. (1997)) and “RNA Viruses: A Practical Approach” (Alan J. Cann, Ed., Oxford University Press, (2000), the entire contents of all of which are herein incorporated by reference).
In some embodiments, inhibitory nucleic acids of the invention are synthesized chemically. Nucleic acid sequences used to practice this invention can be synthesized in vitro by well-known chemical synthesis techniques, as described in, e.g., Adams (1983) J. Am. Chem. Soc. 105:661; Belousov (1997) Nucleic Acids Res. 25:3440-3444; Frenkel (1995) Free Radic. Biol. Med. 19:373-380; Blommers (1994) Biochemistry 33:7886-7896; Narang (1979) Meth. Enzymol. 68:90; Brown (1979) Meth. Enzymol. 68:109; Beaucage (1981) Tetra. Lett. 22:1859; U.S. Pat. No. 4,458,066; WO/2008/043753 and WO/2008/049085, and the references cited therein, the entire contents of all of which are herein incorporated by reference.
In some embodiments, a method of enhancing pluripotency reprogramming of a somatic human cell includes the addition or activation of at least one human lncRNA selected from Tables 2 and/or 3. In other embodiments, a method of enhancing human pluripotent cell differentiation includes the addition or activation of at least one human lncRNA selected from Tables 4B and/or 5.
In some embodiments, a method for enhancing pluripotency reprogramming of a somatic cell or differentiation of a pluripotent cell, includes inhibition or repression of at least one human lncRNA selected from Tables 4B or 5. In other embodiments, a method of enhancing human pluripotent cell differentiation includes the addition or activation of at least one human lncRNA selected from Tables 2 and/or 3.
It is understood by a person having ordinary skill in the art, that any of the modified chemistries or formats of inhibitory nucleic acids described herein can be combined with each other, and that one, two, three, four, five, or more different types of modifications can be included within the same molecule.
The following Examples are presented for illustrative purposes only, and do not limit the scope or contents of the present application.
Example 1
Single-Cell Transcriptome Analysis of Activated Genes During Reprogramming
In order to characterize the transcriptomes of individual reprogramming cells at defined timepoints, single-cell RNA-sequencing was performed as described (Ramskold et al., 2012, Nat Biotechnol 30, 777-782, the entire contents of which are herein incorporated by reference) using cells from the “reprogrammable” mouse (Carey, B. W. et al., 2010, Nat Methods 7, 56-59, the entire contents of which are herein incorporated by reference). Tail-tip fibroblasts (TTF) from these mice, which harbor doxycycline (dox)-inducible OSKM transgenes (FIG. 2A) were generated, and these cells were cultured in the presence of dox for 3 weeks. ES cell-like colonies that expressed SSEA1 began to appear after 3 weeks, and these early-stage iPS cells were cultured for an additional 3-6 weeks in the absence of dox. Late-stage iPS cells at week 6 (Wk6), which had single-cell cloning efficiencies of  ̃50% when compared to ES cells (FIG. 2B), were also cultured in 2i conditions to facilitate establishment of the pluripotent ground state (Silva, J. et al., 2008, PLoS Biol 6, e253, the entire contents of which are herein incorporated by reference). Additionally, two different ES cell lines (E14 and EL16.7) were cultured to compare similarities between ES and iPS cell transcriptomes at the single-cell level (FIG. 3).
Protein-coding genes that were activated during reprogramming and the acquisition of pluripotency were first examined. That is, approximately 3,000 activated genes were expressed at 10 RPKM (reads per kilobase per million mapped reads) (Mortazavi, A. et al., 2008, Nat Methods 5, 621-628, the entire contents of which are herein incorporated by reference) or higher in non-TTF cells, while being off in TTF cells (expressed at less than 1 RPKM) (FIG. 4A). When hierarchical clustering was performed, the cells from both ES lines clustered together, along with the late-stage iPS cells that were cultured in 2i conditions. Both early- and late-stage iPS cells cultured in non-2i conditions clustered together, suggesting different latencies in their reprogramming/remodeling kinetics (Hanna, J. et al., 2009, Nature 462, 595-601, the entire contents of which are herein incorporated by reference). In late-stage iPS cells, imposing 2i conditions pushed their protein-coding transcriptomes (FIG. 4B) to more closely resemble the pluripotent ground state of ES cells (FIG. 4C).
The expression patterns of known pluripotency-related genes that are widely used as faithful predictors of proper iPS cell reprogramming were next examined (Buganim, Y. et al., 2012, Cell 150, 1209-1222, the entire contents of which are herein incorporated by reference). It was found that the genes Esrrb, Utf1, Lin28, and Dppa2 were all expressed starting at week 3 (Wk3) in this reprogramming timecourse, as well as Nanog and Rex1. However, Polycomb histone methyltransferase Ezh2, which physically binds to numerous lncRNAs according to (Guttman, M. et al., 2011, Nature 477, 295-300; Zhao, J. et al., 2010, Mol Cell 40, 939-953, the entire contents of which are herein incorporated by reference), was heterogeneously expressed in both early- and late-stage iPS cells (FIG. 4D and FIGS. 5A, 5B, 5C). This heterogeneity in Ezh2 expression was attenuated under 2i conditions (FIG. 4D).
Example 2
Visualizing Single-Cell Transcriptome Dynamics Using the Self Organizing Map (SOM)
In order identify dynamic changes in the single-cell transcriptomes of reprogramming cells, a self-organizing map (SOM) (Kohonen, T., 2013, Neural Netw 37, 52-65, the entire contents of which are herein incorporated by reference) was generated, which facilitates visualization of gene sets that are coordinately expressed during the reprogramming timecourse (FIGS. 6 and 7A). The 5,000 protein-coding and lncRNA genes were used with the greatest variance among the single-cell RNA-seq data to train the SOM. The results of FIG. 7A show that TTFs strongly express genes involved in body axis anatomy and development of muscle. Notably, intermediate reprogramming cells at week 2 (Wk2) expressed numerous genes that are required for proper germ cell development and fertility, including Blimp1 (Ohinata, Y. et al., 2005, Nature 436, 207-213, the entire contents of which are herein incorporated by reference), Spin1, Mfge8, Usp2, Inpp5b, Jam3, and Zfp37 (FIGS. 7A-7B) (Eppig, J. T. et al., 2012, Nucleic Acids Res 40, D881-886, the entire contents of which are herein incorporated by reference). These results suggest that somatic cell reprogramming takes advantage of existing developmental programs in germ cells, where global reprogramming of the epigenome occurs as part of normal embryonic development (Hayashi, K. et al., 2009, Cell Stem Cell 4, 493-498, the entire contents of which are herein incorporated by reference).
By week 3 (Wk3) of reprogramming, individual cells began to express genes that are involved in RNA metabolism and noncoding RNA processing (FIG. 7A). Surprisingly, late-stage iPS cells at Wk6 strongly expressed hundreds of pseudogenes (FIG. 7A), which can be processed into small regulatory RNAs in oocytes (Tam, O. H. et al., 2008, Nature 453, 534-538, the entire contents of which are herein incorporated by reference). Late-stage iPS cells at week 8 (Wk8) expressed known pluripotency factors at high levels, including Oct4, Nanog, Esrrb, and Sall4, but they failed to exhibit full activation of germ cell regulatory genes such as Stella and Prdm14, which are highly expressed in ES cells (FIG. 7A). These results indicate that while the transcriptional landscape of late-stage iPS cells are quite similar to ES cells, there are also notable differences in expression of both protein-coding and noncoding genes at the single-cell level.
Example 3
Germ Cell-Related Genes are Expressed in Late-Stage iPS Cells
In order to validate the expression patterns of germ cell-related genes in the single-cell RNA-seq data of FIG. 8, smFISH as described in (Raj, A. et al., 2008, Nat Methods 5, 877-879, the entire contents of which are herein incorporated by reference) was used as an orthogonal, amplification-independent method to characterize the full distribution of Stella, Prdm14, Blimp1, Rex1, Oct4, and Sox2 RNAs in hundreds of late-stage iPS cells (n=303) at Wk6 (FIG. 9). The results of this amplification methods, Stella and Prdm14 appeared to be heterogeneously expressed during the reprogramming timecourse and were first detected at Wk6, whereas ES cells expressed these genes more uniformly (FIG. 8). Using 4-color smFISH, it was observed that Stella, Prdm14, and Blimp1 levels were all higher in individual iPS cells that also expressed greater levels of Rex1, while cells with low Rex1 expression generally did not express these genes (FIGS. 10A, 10B, 10C, 10D). These results suggest that late-stage iPS cells with high levels of Rex1 may exhibit more germ cell character.
Additionally, late-stage iPS cells began to express Piwi12 (FIG. 11A), which associates with Piwi-interacting RNAs (piRNAs) in the germline to epigenetically silence retrotransposons (Aravin, A. A. et al., 2007, Science 316, 744-747, the entire contents of which are herein incorporated by reference). At the population level, iPS cells expressed genes involved in piRNA function and biogenesis (FIG. 11B). The 2′-O-methylated small RNAs were cloned and sequenced (Li, X. Z. et al., 2013, Mol Cell 50, 67-81, the contents of which are herein incorporated by reference) from iPS cells, which exhibited a characteristic size distribution peak of 26 nt (FIG. 11C), analogous to Piwil2-associated piRNAs (Aravin, A. A. et al., 2008, Mol Cell 31, 785-799, the entire contents of which are herein incorporated by reference). The sequencing showed that approximately 40% of 2′-O-methylated small RNAs had a 5′U (FIG. 11D), reminiscent of primary piRNAs in the germline (Luteijn, M. J. et al., 2013, Nat Rev Genet 14, 523-534, the entire contents of which are herein incorporated by reference). Moreover, approximately 40% of small RNAs mapped to retrotransposons (FIG. 11E). Taken together with a recent study in human iPS cells (Marchetto, M. C. et al., 2013, Nature 503, 525-529, the entire contents of which are herein incorporated by reference), the results suggest that piRNAs are activated during epigenetic reprogramming.
Example 4
Long Noncoding RNAs Activated During Reprogramming (Ladr)
The changes in the lncRNA transcriptome during reprogramming were examined. Similar to the analysis of protein-coding genes, the focus was on activated lncRNAs (FIG. 1; Tables 1 and 3) that were expressed at 10 RPKM or higher in non-TTF cells, while being repressed in TTFs (below 1 RPKM). The results showed that approximately 150 lncRNAs were activated during the acquisition of pluripotency, and some lncRNAs showed apparent heterogeneity in their expression patterns, even in 2i conditions (FIG. 12). To validate these observations for specific lncRNAs, smFISH was used to characterize the expression patterns of 3 Polycomb-associated IneRNAs (FIGS. 13A, 13B, 13C, 14A, 14B, 14C) in hundreds of cells (n=351). Ladr37 was strongly expressed in both iPS and ES cells, and Ladr80 was weakly expressed in both cell types (FIG. 12 and FIGS. 13B, 13C, 14B, 14C). The distributions of Ladr80 expression in late-stage iPS cells at Wk6 and Wk9 were indistinguishable from ES cells, but Ladr37 expression was aberrantly high in a subset of Wk6 iPS cells relative to ES cells (FIGS. 13B, 13C, 14B, 14C). Also, a subset of Wk9 iPS cells expressed even higher levels of Lad37 (61-113 lncRNA molecules/cell) relative to ES cells (median: 14 lncRNA molecules/cell) (FIGS. 13B, 13C, 14B, 14C), revealing a broad range of heterogeneity in Ladr37 expression, even in late-stage iPS cells.
Example 5
Polycomb-Associated Ladr lncRNAs Repress Developmental Genes in iPS Cells
In order to examine the functional roles of Ladr80 and Ladr37, a pool of 2-4 small interfering RNAs (siRNAs) per lncRNA were used to knock down their expression levels in late-stage iPS cells at Wk9 (FIG. 15A). While knockdown of Ladr80 or Ladr37 had modest effects on reprogramming efficiencies (FIG. 15B), Ladr80 knockdown resulted in the significant upregulation of muscle-related genes in iPS cells. Gene ontology (GO) analysis showed significant enrichment for the GO terms “contractile fiber,” “sarcomere,” and “striated muscle thin filament” (FIG. 16A). These muscle-related genes were also expressed in TTFs and intermediate cells at Wk2, but their repression at subsequent timepoints coincided with the activation of the Polycomb genes Ezh2 and Suz12 (FIG. 16B), along with the Polycomb-associated Ladr80 lncRNA (FIG. 12). These results indicate that Ladr80 activation is required to repress a subset of the myogenic program during the course of iPS cell reprogramming.
Furthermore, Ladr37 knockdown resulted in the upregulation of a subset of muscle genes that were also upregulated upon Ladr80 loss-of-function (FIG. 16C), suggesting that lncRNAs may act redundantly during cell fate reprogramming. In addition, Ladr37 knockdown also led to the upregulation of two homeodomain transcription factors involved in limb development: Alx4 and Six2. However, both Ladr80 and Ladr37 knockdowns did not detectably perturb the expression levels of the reprogramming/pluripotency-related genes Oct4, Sox2, Klf4, c-Myc, Lin28, and Nanog (FIG. 16D), consistent with a specific role for these lncRNAs in repressing developmental genes during reprogramming.
When the differential expression of all Ensembl-annotated lncRNAs was analyzed in populations of ES cells and TTFs, Ladr37 and Ladr80 were among the 48 most significantly upregulated lncRNAs in ES cells versus TTFs (FIG. 16E). Of these 48 upregulated lncRNAs, 22 lncRNAs (46%) associate with Polycomb in ES cells (Guttman et al., 2011; Zhao et al., 2010, supra), whereas of the 37 lncRNAs that are significantly upregulated in TTFs (downregulated in ES cells), only 3 lncRNAs (8%) associate with Polycomb in the pluripotent state (FIG. 16F). These results suggest that additional Polycomb-associated Ladr lncRNAs have functional roles in silencing lineage-specific genes.
Example 6
2i Induces a PGC-Like State in the lncRNA Transcriptome of Late-Stage iPS Cells
It was next examined whether 2i conditions altered the lncRNA landscape in late-stage iPS cells at Wk6. For example, 2i conditions produced a coherent activation of 92 additional lncRNAs (FIG. 17, Tables 1 and 3). Given the delayed activation kinetics of germ cell-related genes in late-stage iPS cells, it was examined whether 2i conditions might enhance their germ cell-like character. Single-cell RNA-seq data from E6.5-E8.5 PGCs was analyzed (Magnusdottir, E. et al., 2013, Nat Cell Biol 15, 905-915, the entire contents of which are herein incorporated by reference) and it was found that a coherent cluster of 2i-activated lncRNAs in iPS cells was also coherently expressed at high levels in PGCs (FIG. 17A). The 2i conditions also resulted in a significant upregulation of Stella (FIG. 17A, FIG. 17B), which is the definitive marker of PGC specification (Leitch, H. G. et al., 2013, Development 140, 2495-2501; and Saitou, M. et al., 2002, Nature 418, 293-300, the entire contents of which are herein incorporated by reference). These results show that iPS cells treated with 2i adopt a more germ cell-like character, similar to ES cells (Hayashi, K. et al., 2008, Cell Stem Cell 3, 391-401, the entire contents of which are herein incorporated by reference), that is especially prominent in their lncRNA transcriptome.







TABLE 6A







Early activation: Wk 2/Wk 3









SEQ ID NO:
Ladr No
lncRNA












34
Ladr34
4930461G14Rik


142
Ladr142
Gm16761


130
Ladr130
Hoxa11as


49
Ladr49
Gm10143


158
Ladr158
Snord123


125
Ladr125
A930029G22Rik


131
Ladr131
Gm17477


79
Ladr79
Gm17279


94
Ladr94
Gm13830


91
Ladr91
Gm12898


141
Ladr141
Gm17344


107
Ladr107
1110020A21Rik


136
Ladr136
1700028E10Rik


147
Ladr147
4933431E20Rik


144
Ladr144
A230056P14Rik


133
Ladr133
A330040F15Rik


132
Ladr132
C030037D09Rik


137
Ladr137
Gm10575


143
Ladr143
Gm11538


128
Ladr128
Gm11818


113
Ladr113
Gm12059


138
Ladr138
Gm15545


115
Ladr115
Gm16244


145
Ladr145
Gm16685


117
Ladr117
Gm16845


148
Ladr148
Gm17565


146
Ladr146
Gm17690


114
Ladr114
Gm17716


135
Ladr135
Gm4890


134
Ladr134
Mir155


37
Ladr37
4930500J02Rik


17
Ladr17
4930566F21Rik


46
Ladr46
Gm17300


36
Ladr36
2410003L11Rik


41
Ladr41
2500002B13Rik


30
Ladr30
Vax2os1


16
Ladr16
2310043M15Rik


29
Ladr29
9230114K14Rik


122
Ladr122
Nespas


80
Ladr80
Gm16096


70
Ladr70
C430002E04Rik


127
Ladr127
Gm15522


35
Ladr35
Gm16957


54
Ladr54
Gm16986


129
Ladr129
1700016P03Rik


116
Ladr116
3110045C21Rik


69
Ladr69
4930509G22Rik


40
Ladr40
9530027J09Rik


22
Ladr22
9830144P21Rik


111
Ladr111
D030068K23Rik


68
Ladr68
Gm17561


71
Ladr71
Gm17605


55
Ladr55
Gm17656


66
Ladr66
Gm17710


110
Ladr110
Gm807
















TABLE 6B







Late activation: Wk 6+









SEQ ID NO:
Ladr No
lncRNA












18
Ladr18
Meg3


21
Ladr21
Rian


3
Ladr3
1810019D21Rik


23
Ladr23
4930513N10Rik


95
Ladr95
C330013F16Rik


150
Ladr150
2410133F24Rik


57
Ladr57
Abhd1


39
Ladr39
Gm13110


26
Ladr26
Gm16641


108
Ladr108
Gm16723


25
Ladr25
Gm17250


38
Ladr38
Gm17335


97
Ladr97
2010300F17Rik


96
Ladr96
4930467K11Rik


99
Ladr99
9530080O11Rik


119
Ladr119
A930011O12Rik


4
Ladr4
B230208H11Rik


100
Ladr100
BC046401


83
Ladr83
Gm13657


102
Ladr102
Gm14817


86
Ladr86
Gm16827


81
Ladr81
Gm17440


103
Ladr103
Gm17501


82
Ladr82
Gm17594


85
Ladr85
Gm17637


65
Ladr65
Gm17659


98
Ladr98
Gm2464


14
Ladr14
4930526L06Rik


2
Ladr2
4930444M15Rik


19
Ladr19
3110056K07Rik


27
Ladr27
2310010G23Rik


28
Ladr28
2810442I21Rik


42
Ladr42
4933404O12Rik


139
Ladr139
A030009H04Rik


90
Ladr90
Gm16159


77
Ladr77
Gm16283


62
Ladr62
Gm16880


109
Ladr109
Gm16972


72
Ladr72
Gm17362


53
Ladr53
Gm17418


33
Ladr33
Gm17491


104
Ladr104
Gm17502


76
Ladr76
Gm17597


124
Ladr124
Gm17713


43
Ladr43
Gm2694


61
Ladr61
Gm2788


63
Ladr63
Gm4419


74
Ladr74
Mirg


47
Ladr47
Xist


87
Ladr87
Atp10d


84
Ladr84
C330018A13Rik


149
Ladr149
1700030C12Rik


24
Ladr24
4930404I05Rik


10
Ladr10
A330048O09Rik


5
Ladr5
Gm15728


48
Ladr48
4930558J18Rik


64
Ladr64
9330185C12Rik


123
Ladr123
Gm10492


20
Ladr20
Gm15441


1
Ladr1
Gm17586


7
Ladr7
2700086A05Rik


121
Ladr121
4833407H14Rik


12
Ladr12
4930481B07Rik


93
Ladr93
6030442K20Rik


13
Ladr13
A730011C13Rik


126
Ladr126
Gm16046


92
Ladr92
Gm16973


9
Ladr9
Gm17291


101
Ladr101
Gm17481


73
Ladr73
Gm17525


11
Ladr11
Gm17698


6
Ladr6
Gm2529


8
Ladr8
C430039J16Rik


112
Ladr112
Gm8378


140
Ladr140
0610005C13Rik


75
Ladr75
1010001B22Rik


50
Ladr50
1110002J07Rik


31
Ladr31
1700007L15Rik


105
Ladr105
1700023H06Rik


51
Ladr51
1700086O06Rik


32
Ladr32
4632427E13Rik


106
Ladr106
4930480K23Rik


78
Ladr78
4933407K13Rik


88
Ladr88
5730457N03Rik


58
Ladr58
5930412G12Rik


52
Ladr52
6720401G13Rik


44
Ladr44
A230072C01Rik


56
Ladr56
B230206L02Rik


120
Ladr120
C030010L15Rik


45
Ladr45
C330046G13Rik


151
Ladr151
C530005A16Rik


59
Ladr59
E130018N17Rik


118
Ladr118
Gm10658


67
Ladr67
Gm10785


89
Ladr89
Gm11714


60
Ladr60
Gm14022


15
Ladr15
Gm15787
















TABLE 6C







2i-induced activation: Wk 6









SEQ ID NO:
Ladr No
lncRNA





220
Ladr220
1110046J04Rik


224
Ladr224
1110019D14Rik


212
Ladr212
1700007J10Rik


159
Ladr159
1700023L04Rik


239
Ladr239
2010110K18Rik


183
Ladr183
2310031A07Rik


219
Ladr219
2610027K06Rik


223
Ladr223
2610206C17Rik


164
Ladr164
2810011L19Rik


237
Ladr237
2810425M01Rik


187
Ladr187
2810429I04Rik


182
Ladr182
2900052L18Rik


177
Ladr177
3010001F23Rik


236
Ladr236
3100003L05Rik


214
Ladr214
4833417C18Rik


184
Ladr184
4930506C21Rik


217
Ladr217
6430562O15Rik


155
Ladr155
9530059O14Rik


188
Ladr188
A230004M16Rik


211
Ladr211
A330032B11Rik


178
Ladr178
A330076H08Rik


222
Ladr222
A730081D07R1k


191
Ladr191
A730099G02Rik


176
Ladr176
A930007I19Rik


174
Ladr174
AY512931


203
Ladr203
BC051077


169
Ladr169
C330002G04Rik


157
Ladr157
Dio3os


172
Ladr172
F630040L22Rik


160
Ladr160
Gdap10


180
Ladr180
Gm10425


194
Ladr194
Gm17638


192
Ladr192
Gm17644


221
Ladr221
Gm17675


162
Ladr162
Gm17692


190
Ladr190
Gm17699


153
Ladr153
Gm17701


233
Ladr233
Gm2366


199
Ladr199
Gm3906


175
Ladr175
Gm6410


218
Ladr218
Gm7782


241
Ladr241
Gm9917


216
Ladr216
Mir706


213
Ladr213
Gm11542


186
Ladr186
Gm11574


230
Ladr230
Gm11602


197
Ladr197
Gm11934


167
Ladr167
Gm12592


163
Ladr163
Gm12784


200
Ladr200
Gm12976


168
Ladr168
Gm13261


198
Ladr198
Gm15396


154
Ladr154
Gm15489


179
Ladr179
Gm15850


242
Ladr242
Gm16568


165
Ladr165
Gm16618


243
Ladr243
Gm16624


181
Ladr181
Gm16629


231
Ladr231
Gm16684


196
Ladr196
Gm16702


225
Ladr225
Gm16706


185
Ladr185
Gm16733


229
Ladr229
Gm16739


195
Ladr195
Gm16755


201
Ladr201
Gm16862


170
Ladr170
Gm16869


227
Ladr227
Gm16889


166
Ladr166
Gm16912


171
Ladr171
Gm16938


156
Ladr156
Gm17102


238
Ladr238
Gm17238


152
Ladr152
Gm17254


193
Ladr193
Gm17255


205
Ladr205
Gm17275


215
Ladr215
Gm17282


226
Ladr226
Gm17317


207
Ladr207
Gm17327


210
Ladr210
Gm17336


189
Ladr189
Gm17392


173
Ladr173
Gm17445


235
Ladr235
Gm17451


228
Ladr228
Gm17452


202
Ladr202
Gm17496


240
Ladr240
Gm17499


161
Ladr161
Gm17500


209
Ladr209
Gm17517


234
Ladr234
Gm17529


232
Ladr232
Gm17548


206
Ladr206
Gm17596


208
Ladr208
Gm17599


204
Ladr204
Gm17632









Ladr lncRNAs have both common and distinctive characteristics when compared with lncRNAs expressed in ES cells (FIG. 18A, 18B, 18C). Of the 243 Ladr lncRNAs, 115 (47%) associate with Polycomb proteins in ES cells (FIG. 19A) (Guttman et al., 2011; Zhao et al., 2010, supra). Ladr lncRNAs are also enriched for the LTR transposable element ERVK, an endogenous retrovirus (FIG. 19B). It was previously shown that lncRNAs containing endogenous retroviral elements are expressed at higher levels in ES cells relative to somatic cells (Kelley, D. et al., 2012, Genome Biol 13, R107, the entire contents of which are herein incorporated by reference). Although Ladr80 was not comprised of any ERVK sequence, Ladr37 was flanked by two different ERVK elements whose sequences overlapped with its 5′ and 3′ exons (FIG. 19C), which may contribute to its high levels of expression in both ES and iPS cells (FIG. 12) (Kelley and Rinn, 2012, supra). Of the 92 Ladr lncRNAs that were strongly upregulated in response to 2i, more than half (n=47) were associated with ERVK elements. Taken together with results from a previous study (Kelley and Rinn, 2012, supra), the results suggest that endogenous retroviral elements are involved in Ladr lncRNA regulation during reprogramming.
Example 7
Ladr lncRNA Regulation by ES Cell and PGC Transcriptional Networks
In order to examine whether Ladr lncRNAs were regulated by ES cell or PGC transcription factor networks, chromatin immunoprecipitation high-throughput sequencing (ChIP-seq) data was analyzed for the core pluripotency factors Oct4/Sox2/Nanog (Marson, A. et al., 2008, Cell 134, 521-533, the entire contents of which are herein incorporated by reference) and for Prdm14/AP2γ, which initiate the specification of PGCs (Magnusdottir et al., 2013, supra). Late-stage iPS cells, both in the absence and presence of 2i, expressed 5- to 20-fold higher levels of AP2γ relative to ES cells (FIG. 20A). Moreover, of the 243 Ladr lncRNAs, AP2γ had the highest number of binding sites within lncRNA promoters (n=86) (FIG. 20B). Prdm14 and Oct4 exhibited the second (n=71) and third (n=31) highest number of binding sites, respectively (FIG. 20B). Ladr lncRNAs whose expression patterns correlated most strongly with Oct4 were generally expressed in ES cells, while lncRNAs that were anti-correlated with Oct4 tended to be expressed in TTFs (FIG. 18C). It was also observed that many of the Ladr lncRNA genes were bound by multiple transcription factors. For example, the Ladr80 promoter region was bound by Sox2, Nanog, and Prdm14, while a 2i-induced lncRNA, Ladr169, was bound by Oct4/Sox2/Nanog and Prdm14/AP2γ (FIG. 20C). However, the Ladr37 promoter was not bound by any of these factors, while Ladr43 was bound by AP2γ (FIG. 20C). These findings suggest that both ES cell and PGC transcriptional networks regulate Ladr activation during iPS cell reprogramming.
Example 8
Ladr lncRNA Suppression of Lineage-Specific Genes
To search for additional lineage-specific Ladr lncRNAs, upregulated genes were analyzed in iPS cells derived from hematopoietic progenitor cells (HPC) (Chang, G. et al., 2014, Cell Res 24, 293-306, the entire contents of which are herein incorporated by reference). This analysis revealed that 130 lncRNAs were activated during HPC reprogramming into iPS cells, and more than half (n=71) were upregulated specifically in HPC-iPS cells, while the remaining lncRNAs (n=59) were activated during both HPC and TTF reprogramming (FIGS. 21B and 12). Of the 71 HPC-iPS lncRNAs, most were not expressed in ES or TTF-iPS cells (FIG. 21C). However, a small number of HPC-iPS lncRNAs were prominently expressed in ES cells, while exhibiting apparent heterogeneity in our iPS cells and TTFs (FIG. 21C). One of these lncRNAs (Ladr246) was examined using smFISH, which showed that Wk6 iPS cells expressed Ladr246 at aberrantly low levels relative to ES cells (FIG. 21D). In Wk9 iPS cells, however, the distribution of Ladr246 expression was indistinguishable from that of ES cells, indicating relatively late activation kinetics for Ladr246 (FIG. 21D, 21E). Additionally, when Ladr246 loss-of-function studies were performed in Wk9 iPS cells (FIGS. 15A, 15B), it was observed that Ladr246 was required to repress three genes involved in interferon signaling: Irgm1, Usp18, and Ifit3 (FIG. 21F). These results are consistent with a lineage-specific role for Ladr246 in repressing hematopoietic genes during HPC reprogramming.
Example 9
Knockdown Analysis of Ladr272 and Ladr43
Mouse Ladr 272 and Mouse Ladr 43 having orthologous sequences in the human genome as determined by liftOver analysis were analyzed in knockdown experiments. Specifically, differential expression analysis of significantly upregulated or downregulated genes in week 9 (Wk9) iPS cells were made deficient for the Ladr272 or Ladr43 using siLadr272 and siLadr43, as indicated in FIGS. 22A and 22B, respectively. Gene expression was determined by population level RNA-seq as described herein, and gene ontology (GO) analysis for significantly enriched GO terms in downregulated genes. Bonferroni-corrected P-values are shown in FIGS. 22A and 22B. Individually labeled points are a common set of genes downregulated in both Ladr 272 and Ladr43 loss-of-function experiments.
Example 10
FIG. 23 shows a graph of human lncRNAs that are significantly upregulated or downregulated during reprogramming of human primary skin fibroblasts into induced pluripotent stem (IPS) cells. The upregulation or downregulation of genes in the human fibroblasts and the human IPS cells was determined by population RNA-seq (GEO accession #:GSE41716).
Materials and Methods
Example 11
iPS Cell Reprogramming
Tail-tip fibroblast (TTF) cultures were established from 3-8 day old reprogrammable mice homozygous for both the tet-inducible OSKM polycistronic cassette and the ROSA26-M2rtTA allele (Carey, B. W. et al., 2010, supra). Maintenance of animals and tail tip excision were performed according to a mouse protocol approved by the Caltech Institutional Animal Care and Use Committee (IACUC). TTFs (+doxycycline), iPS cells, and ES cells were cultured in ES medium (DMEM, 15% FBS, sodium bicarbonate, HEPES, nonessential amino acids, penicillin-streptomycin, L-glutamine, b-mercaptoethanol, 1000 U/ml LIF) and grown on 6-well plates coated with 0.1% gelatin and irradiated MEF feeder cells (GlobalStem). For “2i” conditions, iPS cells were grown in ESGRO-2i medium (Millipore). For lncRNA loss-of-function, iPS cells were transfected with siRNAs (IDT) using Lipofectamine RNAiMAX (Life). For SSEA-1 detection, StainAlive SSEA-1 DyLight 488 antibody (Stemgent) was used to detect SSEA-1 positive cells at specified time-points during reprogramming, which were isolated using flow cytometry on an iCyt Mission Technology Reflection Cell Sorter inside a Baker Bioguard III biosafety cabinet.
Example 12
Single-Cell and Bulk Sample cDNA Synthesis and Amplification
cDNA synthesis was performed using the Smart-Seq protocol as previously described (Ramskold et al., 2012, supra). Specifically, the SMARTer Ultra Low RNA kit for Illumina sequencing (Clontech) was used to generate and amplify cDNA from single cells isolated using a micromanipulator or from bulk samples. Intact single cells were deposited directly into hypotonic lysis buffer. Poly(A)+RNA was reverse transcribed through oligo dT priming to generate full-length cDNA, which was then amplified using 20-22 cycles. cDNA length distribution was assessed using High Sensitivity DNA kits on a Bioanalyzer (Agilent), and only samples showing a broad length distribution peak centered at 2 kb were subsequently used for library generation.
Example 13
Single-Cell and Bulk Sample RNA-Seq Library Generation and Sequencing
Single-cell and bulk sample RNA-seq libraries were constructed using the Nextera DNA Sample Prep kit (Illumina). Briefly, cDNA was ‘tagmentated’ at 55° C. with Nextera transposase, and tagmented DNA was purified using Agencourt AMPure XP beads (Beckman Coulter). Purified DNA was amplified using 5 cycles of Nextera PCR, and library quality was assessed using High Sensitivity DNA kits on a Bioanalyzer (Agilent). Libraries were sequenced on the Illumina HiSeq2000. Single-end reads of 50 bp or 100 bp length were obtained.
Example 14
Read Mapping and Analysis
All reads were trimmed down to 50 bp (if necessary) and mapped to the mouse genome (version mm9) with TopHat (Trapnell, C. et al., 2009, Bioinformatics 25, 1105-1111, the entire contents of which are herein incorporated by reference) (version 1.2.1) while supplying splice junctions annotated in the ENSEMBL63 set of transcript models. RPKMs for the ENSEMBL63 annotation were obtained using Cufflinks (Trapnell, C. et al., 2010, Nat Biotechnol 28, 511-515, the entire contents of which are herein incorporated by reference) (version 1.0.3) with otherwise default settings. For downstream analysis, the biotype classification of genes and transcripts in the ENSEMBL annotation was used to identify noncoding genes. Hierarchical clustering was carried out using Cluster 3.0 (de Hoon, M. J. et al., 2004, Bioinformatics 20, 1453-1454, the entire contents of which are herein incorporated by reference) and visualized using Java Treeview (Saldanha, A. J., 2004, Bioinformatics 20, 3246-3248, the entire contents of which are herein incorporated by reference). For differential expression analysis, reads were aligned against the refSeq mouse transcriptome using Bowtie version 0.12.7 (Langmead, B. et al., 2009, Genome Biol 10, R25, the entire contents of which are herein incorporated by reference). Expression levels were then estimated using eXpress (Roberts, A. et al., 2013, Nat Methods 10, 71-73, the entire contents of which are herein incorporated by reference) (version 1.3.0), with gene-level effective counts and RPKM values derived from the sum of the corresponding values for all isoforms of a gene. The effective count values were then used as input to DESeq (Anders, S. et al., 2010, Genome Biol 11, R106, the entire contents of which are herein incorporated by reference) to assess differential expression. LncRNA transposon enrichment/depletion analysis was performed as previously described (Kelley, 2012, supra). For ChIP-seq analysis, sequencing data were downloaded from accession numbers GSM307140, GSM623989, GSM307137, GSM307138, E-MTAB-1600, GSM307155, and GSM623991. Reads were extracted using the fastq-dump program in the SRA ToolKit and mapped to the mm9 version of the mouse genome using Bowtie 0.12.7 with the following settings: “-v 2 -k 2 -m 1 -t --best --strata”, i.e. retaining only unique reads and allowing for up to 2 mismatches in a read. Enriched regions were called using ERANGE 3.2 (Johnson, D. S. et al., 2007, Science 316, 1497-1502, the entire contents of which are herein incorporated by reference) with the following settings: “-minimum 2 -ratio 3 -shift learn -revbackground”.
Example 15
Small RNA Sequencing and Analysis
Oxidation and beta-elimination of small RNAs were performed as previously described (Ameres, S. L. et al., 2010, Science 328, 1534-1539, the entire contents of which are herein incorporated by reference). The Illumina-compatible NEBNext Small RNA Sample Prep Set 1 (New England Biolabs) was used to prepare small RNA libraries for sequencing on the Illumina platform. Sequencing adapters were removed from reads by finding the 3′-most complete match to the adapter sequence and trimming the read after that position. The resulting were first mapped to the collection of ribosomal repeats (annotated using the RepeatMasker file downloaded from the UCSC genome browser), snoRNAs and snRNAs in the mouse genome (version mm9) using Bowtie version 0.12.7 in order to remove common contaminant reads. The unmapped reads from this filtering step were then aligned against the mm9 genome to determine the number of mappable reads. Both bowtie mapping steps were carried out with the following settings: ‘-v 0 -a -t --best --strata’, i.e. no mismatches and an unlimited number of locations to which a read could map to. Enrichment of repeat classes in sequencing was estimated by calculating RPM (Reads Per Million mapped reads) scores for each individual repeat annotated in the UCSC RepeatMasker file, then summing over all the instances of each repeat class in order to derive a total repeat class RPM score.
Example 16
Single-molecule fluorescence in situ hybridization (smFISH)
smFISH was performed as previously described ((Raj et al., 2008, supra). Up to 48 DNA probes per target mRNA or lncRNA were synthesized and conjugated to Alexa fluorophore 488, 555, 594, or 647 (Life Technologies) and then purified by HPLC. Cells were trypsinized, fixed in 4% Formaldehyde, and permeabilized in 70% ethanol overnight. Cells were then hybridized with probe overnight at 30° C., in 20% Formamide, 2×SSC, 0.1 g/ml Dextran Sulfate, 1 mg/ml E. coli tRNA, 2 mM Vanadyl ribonucleoside complex, 0.1% Tween 20 in nuclease free water. Samples were washed twice in 20% Formamide, 2×SSC, and Tween 20 at 30° C., and then twice in 2×SSC+0.1% Tween at RT. 1 μl of hybridized cells was placed between #1 coverslips and flattened. Automated grid-based acquisition was performed on a Nikon Ti-E with Perfect Focus System, Semrock FISH filtersets, Lambda LS Xenona Arc Lamp, 60×1.4 NA oil objective, and Coolsnap HQ2 camera. Semi-automated dot detection and segmentation was performed using custom-built MATLAB software with a Laplacian-of-Gaussian Kernel, using Otsu's method to determine “dotness” threshold across all cells in the dataset.
Example 17
Self-Organizing Maps (SOM)
The 5000 genes with the greatest variance among the libraries were used for training a self-organizing map. Prior to SOM training, the data vectors were normalized on a gene-by-gene basis by subtracting each vector mean and dividing by its standard deviation. The SOM was constructed using the R package ‘kohonen.’ The total number of map units was set to the heuristic value 5*sqrt(N), where N is the number of data vectors. The map grid was initialized with the first two principal components of the data multiplied by a sinusoidal function to yield smooth toroidal boundary conditions. Training lasted 200 epochs (presentations of the data) during which the radius within which units were adapted toward the winning unit decreased linearly from h/8 to 2 units, where h is the map height (always chosen as the direction of largest length). Further analysis, including clustering and visualization, was performed with custom python code. Clusters were seeded by the local minima of the u-matrix, with a value for each unit defined as the average of the vector difference between that unit's prototype and its six neighbors on the hexagonal grid. All other unit prototypes were then assigned to clusters according to the minimum vector distance to a seed unit. The lists of clustered genes were submitted to the Princeton GO TermFinder (Boyle, E. I. et al., 2004, Bioinformatics 20, 3710-3715, the entire contents of which are herein incorporated by reference) server (http://go.princeton.edu) in order to determine enriched terms.
Example 18
lncRNA Analysis of Induced Pluripotent Stem (iPS) Cells
Raw RNA sequencing data were downloaded from the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) and analyzed for differential expression of lncRNAs during mouse hematopoietic progenitor cell (FIG. 21A) and human skin fibroblast (FIG. 23) reprogramming into induced pluripotent stem (iPS) cells. The GEO accession number for FIG. 21A is GSE36290. The GEO accession number for FIG. 23 is GSE41716.
As disclosed throughout and as evidenced by, for example, FIGS. 15A, 15B, 16A-16F, 20A-20C, 21A-21F and 22A, 22B, and Tables 1-4, the disclosed lncRNAs participate in modulating pluripotency reprogramming and pluripotent cell differentiation, and may be utilized to enhance these cell fate methodologies.
While the present invention has been illustrated and described with reference to certain exemplary embodiments, those of ordinary skill in the art will understand that various modifications and changes may be made to the described embodiments without departing from the spirit and scope of the present invention, as defined in the following claims.

## Claims

1. A method of enhancing reprogramming of a somatic cell to a pluripotent cell in a human or mouse, comprising:
upregulating in the somatic cell at least one long noncoding RNA (lncRNA) selected from SEQ ID NOs. 43, 272, 304, 334, 345, 395, and homologs thereof having at least 85% homology to the at least one lncRNA, the upregulating comprising adding or activating the at least one lncRNA.

2. The method of claim 1, wherein the adding or activating comprises contacting the somatic cell with the at least one synthetic lncRNA and/or a vector encoding the at least one lncRNA.

3. The method of claim 1, further comprising downregulating in the somatic cell at least one lncRNA selected from SEQ ID NOs. 361, 408, 414, 415, 418, 419, and homologs thereof having at least 85% homology to the at least one lncRNA, the downregulating comprising contacting the somatic cell with antisense oligonucleotides and/or interfering RNA (RNAi) targeting the at least one lncRNA.

4. The method of claim 3, wherein the somatic cell is human and the upregulating of the at least one lncRNA comprises upregulating lncRNAs selected from SEQ ID NOs. 334, 345, 395, and the homologs thereof having at least 85% homology to the at least one lncRNA, and the downregulating of the at least one lncRNA comprises contacting the somatic cell with antisense oligonucleotides and/or interfering RNAi targeting the at least one lncRNA selected from SEQ ID NOs. 361, 408, 414, and the homologs thereof having at least 85% homology to the at least one lncRNA.

5. The method of claim 1, further comprising expressing Oct 4, Sox2, Klf4, and c-Myc transcription factors in the somatic cell.

6. The method of claim 1, wherein the homologs have at least 90% homology.

7. A method of enhancing differentiation of a cell in a pluripotent state in a human or mouse, comprising:
downregulating in the cell at least one long noncoding RNA (lncRNA) selected from SEQ ID NOs. 272, 304, 334, 395, and homologs thereof having at least 85% homology to the at least one lncRNA, the downregulating comprising contacting the cell with antisense oligonucleotides and/or interfering RNA (RNAi) targeting the at least one lncRNA.

8. The method of claim 7, further comprising upregulating at least one lncRNA selected from SEQ ID NOs. 361, 408, 414, 415, 418, 419, and homologs thereof having at least 85% homology to the at least one lncRNA, the upregulating comprising adding or activating the at least one lncRNA.