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Epigenetic memory in induced pluripotent stem cells

Epigenetic memory in induced pluripotent stem cells

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12/17/2013

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ARTICLES
Epigenetic memory in induced pluripotentstem cells
K. Kim
1
, A. Doi
2
, B. Wen
2
, K. Ng
1
, R. Zhao
1
, P. Cahan
1
, J. Kim
3
, M. J. Aryee
4
, H. Ji
2
, L. I. R. Ehrlich
5
{
, A. Yabuuchi
1
,A. Takeuchi
1
, K. C. Cunniff
1
, H. Hongguang
1
, S. Mckinney-Freeman
1
, O. Naveiras
1
, T. J. Yoon
6
{
, R. A. Irizarry
2
,N.Jung
2
,J.Seita
5
,J.Hanna
7
,P.Murakami
2
,R.Jaenisch
7
,R.Weissleder
6
,S.H.Orkin
3
,I.L.Weissman
5
,A.P.Feinberg
2
& G. Q. Daley
1
Somatic cell nuclear transfer and transcription-factor-based reprogramming revert adult cells to an embryonic state, andyield pluripotent stem cells that can generate all tissues. Through different mechanisms and kinetics, these tworeprogramming methods reset genomic methylation, an epigenetic modification of DNA that influences gene expression,leading us to hypothesize that the resulting pluripotent stem cells might have different properties. Here we observe thatlow-passageinducedpluripotentstemcells(iPSCs)derivedbyfactor-basedreprogrammingofadultmurinetissuesharbourresidualDNAmethylationsignaturescharacteristicoftheirsomatictissueoforigin,whichfavourstheirdifferentiationalonglineages related to the donor cell, while restricting alternative cell fates. Such an ‘epigenetic memory’ of the donor tissuecould be reset by differentiation and serial reprogramming, or by treatment of iPSCs with chromatin-modifying drugs. Incontrast,thedifferentiationandmethylationofnuclear-transfer-derivedpluripotentstemcellsweremoresimilartoclassicalembryonic stem cells than were iPSCs. Our data indicate that nuclear transfer is more effective at establishing the groundstate of pluripotency than factor-based reprogramming, which can leave an epigenetic memory of the tissue of origin thatmay influence efforts at directed differentiation for applications in disease modelling or treatment.
Direct reprogramming of somatic cells with the transcription factorsOct4 (also calledPou5f1), Sox2, Klf4 and Myc
1
 yields iPSCs withmarked similarity to embryonic stem cells from fertilized embryos(fESCs). Like fESCs, iPSCs form teratomas, differentiated tumourswith tissues from all three embryonic germ layers, and when injectedinto murine blastocysts contribute to all tissues, including the germline. iPSCs from mouse embryo fibroblasts generate ‘all-iPSC mice’after injection into tetraploid blastocysts
2
, thereby satisfying the moststringent criterion of pluripotency 
3
. Embryonic tissues are the mostefficientlyreprogrammed,producingiPSCsthatarenearlyidenticaltofESCs.Incontrast,reprogrammingfromaccessibleadulttissues,mostapplicable for modelling diseases and generating therapeutic cells, isinefficient and limited by barriers related to the differentiation stateandageofthedonor’scells
4–6
.AgedcellshavehigherlevelsofInk4/Arf,whichlimitstheefficiencyandfidelityofreprogramming
5
.Moreover,terminally differentiated blood cells reprogram less efficiently thanblood progenitors
6
. As with cloning by nuclear transfer in frogs andmice,theefficiencyandyieldofreprogrammedgenomesdeclineswithincreasing age and differentiation status of the donor cell
7
, and varieswith the methylation state of the donor nucleus
8
.Different tissues show variable susceptibility to reprogramming.Keratinocytesreprogrammorereadilythanfibroblasts
9
,andiPSCsfromstomach or liver cells harbour fewer integrated proviruses than fibro-blasts, indicating that they require lower levels of the reprogrammingfactors to achieve pluripotency 
10
. When differentiated into neuro-spheres, iPSCs from adult tail-tip fibroblasts retain more teratoma-formingcellsthaniPSCsfromembryonicfibroblasts,againindicatingheterogeneitybased onthetissueof origin
11
. Moreover,cellscanexistin intermediate states of reprogramming that interconvert with con-tinuous passage or treatment with chromatin-modifying agents
12,13
.Although generic iPSCs are highly similar to fESCs, in practice iPSCsgenerated from various tissues may harbour significant differences,both functional and molecular.Transcription factor reprogramming differs markedly from nuc-lear transfer, particularly with regard to DNA demethylation, whichcommences immediately upon transfer of a somatic nucleus intoooplasm
14
, but occurs over days to weeks during the derivation of iPSCs
13
. Because demethylation is a slow and inefficient process infactor-based reprogramming, we postulated that residual methyla-tionmightleaveiPSCswithan‘epigeneticmemory’,andthatmethy-lation might be more effectively erased by nuclear transfer. Here wecompare the differentiation potential and genomic methylation of multiple classes of pluripotent stem cells—iPSCs, nuclear transferESCs (ntESCs) and fESCs—and find evidence that iPSCs indeedretain a methylation signature of their tissue of origin.Initially wesought to compare the
in vivo 
engraftment potential of haematopoietic stemcellsderivedfromfESCs,ntESCsandiPSCsinatherapeutic mouse model of thalassaemia. However, even
in vitro 
we
1
StemCellTransplantationProgram,DivisionofPediatricHematology/Oncology,MantonCenterforOrphanDiseaseResearch,HowardHughesMedicalInstitute,Children’sHospitalBoston and Dana Farber Cancer Institute; Division of Hematology, Brigham and Women’s Hospital; Department of Biological Chemistry and Molecular Pharmacology, HarvardMedical School; Harvard Stem Cell Institute; Boston,Massachusetts 02115, USA.
2
Center for Epigenetics and Department of Medicine, Johns Hopkins UniversitySchool of Medicine,Baltimore, Maryland 21205, USA.
3
Department of Pediatric Oncology, Howard Hughes Medical Institute, Children’s Hospital Boston and Dana Farber Cancer Institute; Boston,Massachusetts02115,USA.
4
DepartmentofBiostatistics,JohnsHopkinsBloombergSchoolofPublicHealth,SidneyKimmelComprehensiveCancerCenter,JohnsHopkinsUniversity,Baltimore, Maryland 21205, USA.
5
Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, California 94305, USA.
6
Center forSystems Biology, Massachusetts General Hospital/Harvard Medical School, 185 Cambridge Street, CPZN 5206, Boston, Massachusetts 02114, USA.
7
Whitehead Institute forBiomedical Research, Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02142, USA.
{
Present addresses: University of Texas at Austin,Molecular Genetics and Microbiology, 2506 Speedway, NMS 2.314, Austin, Texas 78712, USA (L.I.R.E.); Department of Applied Bioscience, CHA University, Seoul 135-081, Korea(T.J.Y.).
1
Macmillan Publishers Limited. All rights reserved
 ©2010
 
observed markedly different blood-forming potential; thus, wefocusedhereinstead onunderstanding thisphenomenon.Ourinitialset of pluripotent stem cells was derived from the hybrid C57BL/6
3
CBA (B6/CBAF1) strain carrying a deletion in the
b
-globin locus
15
,which for this study is otherwise immaterial (Fig. 1a). We isolatedfESCs from naturally fertilized embryos and derived ntESCs fromnuclei of dermal fibroblasts
8
. We infected early bone marrow cells(Kit
1
, Lin
2
, CD45
1
) or dermal fibroblasts from aged mice withretroviral vectors carrying
Oct4 
,
Sox2 
,
Klf4 
and
Myc 
, and selectedblood-derived and fibroblast-derived iPSC colonies (B-iPSCs,F-iPSCs). Haematopoietic progenitors and fibroblasts from agedmice yielded a comparable frequency of reprogrammed colonies(0.02%), which consistent with previous reports
5
, was lower thantheyieldfromfibroblastsofajuvenilemouse(0.1%).Wecharacterizedthe fESC, ntESC and iPSC lines for expression of Oct4 and Nanog by immunohistochemistry, and demonstrated multilineage differenti-ation potential in teratomas (Fig. 1b, c and Supplementary Fig. 1).By criteria typically applied to human samples and appropriate for atherapeutic model
3
, all stem cell lines manifest pluripotency.
Differentiation of pluripotent stem cells
To test blood potential, we differentiated multiple pluripotent stemcell clones into embryoid bodies, dissociated cells, and assayed forhaematopoieticcolonyformingcells
16
.Allpluripotentcellsgeneratedcomparable embryoid bodies but markedly different numbers of haematopoietic colonies.Consistently,blood-derived B-iPSCs yieldedmorehaematopoieticcoloniesthanF-iPSCs(Fig.2a).HaematopoieticcolonyformationfromntESCandfESClineswashigherthantheiPSClines.We then tested differentiation into osteoblasts, a mesenchymallineage that can be derived from fibroblasts
17,18
. By Alizarin red stain-ing, a marker of osteogenic cells
19
, F-iPSCs produced more sharply defined osteogenic colonies (Fig. 2b), deposited more elemental cal-cium (Fig. 2c), and showed higher expression of three osteoblast-associated genes (Fig. 2d) than B-iPSCs. By these criteria, F-iPSCs
ab
ESC ntESC B-iPSC F-iPSC
   N  a  n  o  g   D   A   P   I   D   A   P   I   O  c   t   4
B6/CBA
β
-thal
FibroblastBloodBlastocystReprogrammingESCntESCF-iPSCB-iPSCBoneBloodDierentiation
c
ESC ntESCB-iPSCF-iPSC
Figure 1
|
Pluripotentstemcellsandtheircharacterization. a
,Experimentalschema. fESCs, ntESCs, F-iPSCs and B-iPSCs were derived from B6/CBAF1mice by reprogramming and/or cell culture, characterized for pluripotency by criteria applied to human cells, followed by differentiation analysis forosteogenic or haematopoietic lineages.
b
, Expression of pluripotency markers Nanog and Oct4 by immunohistochemistry. 4,6-Diamidino-2-phenylindole (DAPI) staining for total cell content. Feeder fibroblasts serveas internal negative controls.
c
, Teratoma analysis: tumour histology fromindicatedcelllinesshowshighlycysticstructuresconsistingofdifferentiatedelements of all three embryonic germ layers. Scale bar, 200
m
m.
bcd
B-iPSC F-iPSC
   C  a   l  c   i  u  m    d  e  p  o  s   i   t   i  o  n   (
     μ
  g   )
B-iPSCs
 n
=11)F-iPSCs
 n
=10)
2468100
ntESCs
 n
=3)fESCs
 n
=3)
BglapSp7 Runx2
B-iPSCs
 n
=11)F-iPSCs
 n
=10)ntESCs
 n
=3)fESCs
 n
=3)
   R  e   l  a   t   i  v  e  e  x  p  r  e  s  s   i  o  n
248610
a
   C  o   l  o  n  y  n  u  m   b  e  r  p  e  r   1   0   0 ,   0   0   0  c  e   l   l  s
ESCs
 n
=3)ntESCs
 n
=3)F-iPSCs
 n
=7)B-iPSCs
 n
=8)20406080100120d(E)MGMGEMM00
Figure 2
|
Differentiationofcelllines. a
,Haematopoieticcolonynumberper100,000embryoidbodycellsdifferentiatedfromtheindicatedcelllines.d(E),erythrocyte; GEMM, granulocyte-erythrocyte-macrophage-megakaryocyte;GM, granulocyte-macrophage; M, macrophage.
b
, Alizarin red staining of osteogenic cultures of B-iPSCs (left) and F-iPSCs (right). Top: 3-cm culturedish; bottom: stained colonies. Scale bar, 500
m
m.
c
, Quantification of elemental calcium by inductively coupled plasma–atomic emissionspectroscopy 
48
in5
3
10
5
cellsafterosteogenicdifferentiationofindicatedcelllines.
d
, Q-PCR of osteogenic genes
Bglap
,
Sp7 
and
Runx2
in indicated celllines after osteogenic differentiation. Gene expression was normalized toactin.
n
5
number of independent clones tested. All error bars indicate s.d.
ARTICLES
NATURE
2
Macmillan Publishers Limited. All rights reserved
 ©2010
 
show enhanced osteogenic potential, reflecting a propensity to differ-entiate towards a mesenchymal lineage. In contrast, ntESCs behavedcomparably to fESCs in haematopoietic and osteogenic assays.
DNA methylation of pluripotent stem cells
We hypothesized that the different pluripotent cells might harbourdifferent patterns of genomic DNA methylation; thus, we performedcomprehensive high-throughput array-based relative methylation(CHARM) analysis, which interrogates
,
4.6million CpG sites,including almost all CpG islands and nearby sequences, termedshores
20,21
, but does not assess non-CpG methylation. We determinedthenumberofdifferentiallymethylatedregions(DMRs)betweenpair-wisecomparisons,usingathresholdareacutoffof2,correspondingtoa 5% false discovery rate (FDR 
22
; Supplementary Table 1a). By thisanalysis,ntESCsweremostsimilartofESCs(only229DMRs),whereasF-iPSCs differed most extensively (5,304 DMRs). Relative to fESCs,hypermethylated DMRs predominated for F-iPSCs (3,349
5
63%)and B-iPSCs (516
5
74%). Highlighting their functional differences,5,202 DMRs were identified between B-iPSCs and F-iPSCs. We con-firmedtheresultsofCHARManalysisbybisulphitepyrosequencingof multiple loci (Supplementary Fig. 2).Unsupervised hierarchical clustering of DMRs between B-iPSCsand F-iPSCs easily distinguished iPSCs from ntESCs and fESCs,which cluster together (Fig. 3a). B-iPSCs cluster nearer to ntESCsand fESCs than do F-iPSCs, which represent a markedly separatecluster. These data indicate that the methylation patterns of ntESCsare more like fESCs than are either B-iPSCs or F-iPSCs.Several lines of evidence support a mechanistic link between differ-ential methylation and haematopoietic propensity of iPSC lines. First,literaturesurveyofgenesforthetop24DMRsthatdistinguishB-iPSCsand F-iPSCs links 11 to haematopoiesis and 3 to osteogenesis (Sup-plementaryTable2).Ofthe11haematopoieticloci,10arehypermethy-lated in F-iPSCs relative to B-iPSCs. Second, of 74 haematopoietictranscription factors, 20are in or near DMRs that are hypermethylatedin F-iPSCs versus B-iPSCs, twicethat predicted by chance (
5
0.0034;Fig. 3b left panel, Supplementary Fig. 3a and Supplementary Table 3).Similarly, of 764 fibroblast-specific genes, 115 are hypermethylatedin B-iPSCs, twice that predicted by chance (
5
10
2
5
; Fig. 3b rightpanel). Given the correlation between methylation and transcriptionalsilencing
23
, our data suggest that iPSCs harbour epigenetic marksantagonistic to cell lineages distinct from the donor cell type.We asked whether DMRs that distinguish B-iPSCs from fESCsmight allow us to identify their haematopoietic lineage of origin. Ina separate CHARM experiment, we examined genome-wide methyla-tion in highly purified multipotent and lineage-specific haematopoie-tic progenitors (H. Ji, I. L. Weissman and A. P. Feinberg, unpublisheddata).ComparingDMRsinB-iPSCstothosethatdefinehaematopoie-tic progenitors, we observed that B-iPSCs cluster alongside commonmyeloidprogenitors(CMPs)andaredistantfromcommonlymphoidprogenitors (CLPs; Supplementary Fig. 4a and Supplementary Table4), which is notable given that B-iPSCs were derived from Kit
1
, lin-eage-negativemyeloidmarrowprecursors.Next,weaskedwhetherthetissueof origin (bone marrow versusfibroblast)could beidentified by themethylationstateoftissue-specificDMRsinF-iPSCs,B-iPSCsandBl-iPSCs (a B-lymphocyte-derived iPSC line described below). UsingDMRs that distinguish fibroblast and bone marrow, and examiningmethylation in iPSCs and somatic cells from two different geneticbackgrounds (B6CBA and B6129), we found that F-iPSCs clusteralongside fibroblasts, and are distant from bone marrow (Sup-plementary Fig. 4b). Similarly, the haematopoietic-derived B-iPSCsand Bl-iPSCs grouped with somatic cells from bone marrow. Thus,residual methylation indicates the tissue of origin of iPSCs, and forblood derivatives even their precise lineage, further supporting thephenomenon of epigenetic memory in iPSCs.
Reprogrammed state of iPSCs and ntESCs
We postulated that the differing methylation signatures of B-iPSCs,F-iPSCsandntESCsreflectdisparatereprogramming,andconfirmedthis by two independent computational analyses. First, we over-lapped DMRs that distinguish B-iPSCs, F-iPSCs and ntESCs fromfESCs with genes specifically expressed in undifferentiated murinefESCs
24
. By this analysis, ntESCs showed the fewest DMRs at locicorresponding to the most highly expressed fESC-specific genes,and B-iPSCs showed fewer DMRs at these loci than F-iPSCs (Sup-plementary Fig. 5a). Second, we overlapped DMRs with the DNAbinding locations for seven transcription factors that compose a coreprotein network of pluripotency 
25
, and found the fewest DMRs atcore transcription factor binding sites in ntESCs, and less overlap inB-iPSCs than in F-iPSCs (Supplementary Table 5). These analysesindicate that F-iPSCs harbour more residual methylation thanB-iPSCs at loci directly linked to the gene expression and pluripo-tency networks of fESCs, whereas ntESCs show the least differentialmethylation and seem closest to fESCs at these critical loci.Further analysis of Oct4 and Nanog indicates that although bothare detected by immunohistochemistry in B-iPSCs and F-iPSCs(Fig. 1b),
Oct4 
mRNA is fully expressed from a demethylated pro-moter in both types of iPSC, whereas
Nanog 
mRNA is sub-optimally expressed from a promoter that retains considerable methylation inF-iPSCs (Supplementary Fig. 6). When assessed by blastocyst chi-maerism, B-iPSCs contribute to all tissues, including the germ line,whereas F-iPSCs contribute only poorly (Supplementary Fig. 7a),although they can be found in SSEA1
1
germ cells of the gonadalridge (Supplementary Fig. 7b). Thus, whereas both B-iPSCs andF-iPSCs generate robust multilineage teratomas, satisfying criteriaforpluripotencytypicallyappliedtohumancells
3
,broaderfunctionalassessments available in the mouse system confirm their differentialdegree of reprogramming. In this comparison of iPSCs derived fromaccessible tissues of aged adult mice, bone marrow yields stem cellswith superiorfeatures of pluripotency, butneither iPSC isequivalentto ntESCs or fESCs.
   B  -   i   P   S   C  -   2   B  -   i   P   S   C  -   1   8   B  -   i   P   S   C  -   8   B  -   i   P   S   C  -   1   0   B  -   i   P   S   C  -   1   2  n   t   E   S   C  -   B   3  n   t   E   S   C  -   V   2  n   t   E   S   C  -   V   3  n   t   E   S   C  -   F   1  n   t   E   S   C  -   V   1  n   t   E   S   C  -   O   f   E   S   C  -   E   f   E   S   C  -   B   f   E   S   C  -   C   f   E   S   C  -   A   f   E   S   C  -   D   f   E   S   C  -   G   f   E   S   C  -   F   f   E   S   C  -   IF  -   i   P   S   C  -   1   6   F  -   i   P   S   C  -   6   3   F  -   i   P   S   C  -   1   F  -   i   P   S   C  -   1   3   F  -   i   P   S   C  -   5   9   F  -   i   P   S   C  -   5   7   F  -   i   P   S   C  -   5   4   F  -   i   P   S   C  -   5   6
ESC:
 n
=8 clonesntESC:
 n
=6 clonesB-iPSC:
 n
=7 clonesF-iPSC:
 n
=8 clones
   B  -   i   P   S   C  -   1   5   B  -   i   P   S   C  -   1   1
120
   H  e   i  g   h   t
a
   F  r  e  q  u  e  n  c  y   F  r  e  q  u  e  n  c  y
b
Observedoverlap = 20
P
-value = 0.00337Observedoverlap = 115
P
-value = 10
5
Haematopoietic transcription actors:Hypermethylated in F-iPSCsNumber o overlapping genes Number o overlapping genesFibroblast-specifc genes:Hypermethylated in B-iPSCs20401008060
15,00010,000
5,00005,0007,0003,0001,000
0 10 20 30 40 0 20 40 60 80 100 120
0
Figure 3
|
Analysisofmethylationinstemcelllines. a
, Cluster dendrogramusing probes from DMRs that distinguish B-iPSCs and F-iPSCs. Cell clonesaredescribedinSupplementaryTable5.Height(
 y 
axis)isEuclideandistance.
b
, Enrichment of DMRs for haematopoiesis and fibroblast-relatedtranscription factors in B-iPSCs and F-iPSCs, relative to chance (bluehistogram; 100,000 random permutations). Left panel: 20 of 74haematopoiesis-relatedtranscriptionfactorsoverlapDMRshypermethylatedin F-IPSCs (
5
0.0034). Right panel: 115 of 764 fibroblast-specific genesoverlap DMRs hypermethylated in B-iPSCs (
5
10
2
5
).
NATURE
ARTICLES
3
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 ©2010

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