1、Deficiency of microRNA miR-34a expands cell fate potential in pluripotent stem cellsYong Jin Choi1,Chao-Po Lin1,*,Davide Risso2,Sean Chen1,Meng How Tan3,Jin B Li3,Yalei Wu4,Caifu Chen5,Zhenyu Xuan6,Todd Macfarlan7,Weiqun Peng8,Sang Yong Kim9,Terence P Speed10,and Lin He1,*1Division of Cellular and D
2、evelopmental Biology,MCB department,University of California at Berkeley,Berkeley,CA 94705,USA2Division of Biostatistics,School of Public Health,University of California at Berkeley,Berkeley,CA 94720,USA3Department of Genetics,Stanford University,Stanford,CA 94305,USA4Thermo Fisher Scientific,180 Oy
3、ster Point Blvd.,South San Francisco,CA 940805Integrated DNA Technologies,200 Chesapeake Drive,Redwood City,CA 94063,USA6Department of Molecular and Cell Biology,University of Texas at Dallas,800 West Campbell Road,Richardson,Texas 750807The Eunice Kennedy Shriver National Institute of Child Health
4、and Human Development,the National Institutes of Health,Bethesda,MD,USA8Department of Physics,the George Washington University,Washington,DC 20052,USA9Department of Pathology,NYU School of Medicine,540 First Ave,New York,NY 10016,USA10Department of Statistics,University of California,Berkeley,CA 947
5、20 USA;Department of Mathematics and Statistics,The University of Melbourne,Parkville,VIC 3010 Australia and Bioinformatics Division.The Walter and Eliza Hall Institute of Medical Research,Parkville,VIC 3052 AustraliaAbstractEmbryonic stem cells and induced pluripotent stem cells have pluripotent de
6、velopmental potential,efficiently giving rise to all embryonic cell types,but rarely extraembryonic lineages(1).Here,we identify a microRNA miR-34a,whose deficiency in mouse pluripotent stem cells expands their developmental potential to generate both embryonic and extra-embryonic lineages in vitro
7、and in vivo.miR-34a/pluripotent stem cells with this bidirectional cell fate potential resemble*Correspondence to:lheberkeley.edu(LH)and (CPL).These authors contributed equally.Supplementary Materials:Materials and MethodsSupplementary TextFig.S1S6Tables S1S6Supplementary InformationHHS Public Acces
8、sAuthor manuscriptScience.Author manuscript;available in PMC 2018 September 14.Published in final edited form as:Science.2017 February 10;355(6325):.doi:10.1126/science.aag1927.Author ManuscriptAuthor ManuscriptAuthor ManuscriptAuthor Manuscripttotipotent 2-cell(2C)blastomeres not only in their cell
9、 fate potential,but also in the key molecular signature,namely a strong induction of the MuERV-L(MERVL)family of murine endogenous retroviruses(ERVs).miR-34a represses MERVL expression through transcriptional regulation,at least in part,by repressing the transcription factor GATA-binding protein 2(G
10、ata2).Consistently,the miR-34a/Gata2 pathway restricts the acquisition of bidirectional cell fate potential in pluripotent stem cells.Altogether,our findings provide vital insights into the complex molecular network that defines and restrict the developmental potential of pluripotent stem cells.Mous
11、e embryonic stem cells(ESCs)derived from the inner cell mass(ICM)of blastocysts,as well as induced pluripotent stem cells(iPSCs)generated by somatic reprogramming,are classically defined as pluripotent stem cells(24).As a population,ESCs and iPSCs efficiently contribute to all embryonic cell types i
12、n vitro and in vivo,but rarely to extra-embryonic cell lineages in placenta and yolk sac(1).This restricted pluripotent potential contrasts with that of early blastomeres,which give rise to both embryonic and extra-embryonic cell lineages during normal development(5,6).Interestingly,rare pluripotent
13、 stem cell populations with expanded cell fate potential have been identified in culture as a result of genetic alterations,specific culture and derivation conditions,or enrichment with specific molecular markers(710).Such pluripotent stem cells exhibit bidirectional cell fate potential,contributing
14、 to both embryonic and extra-embryonic lineages in a variety of in vitro and in vivo functional assays(710).In addition,these rare ESCs/iPSCs with bidirectional potential often share a key molecular signature,namely a strong induction of the MuERV-L(MERVL)family of murine endogenous retroviruses(ERV
15、s),which only occurs in totipotent 2C blastomeres during normal mouse development(7,9).While these studies suggest that a subset of cultured ESCs/iPSCs retain the cell fate plasticity to acquire features of early blastomeres,there clearly exists a strong molecular barrier restricting the ESC/iPSC de
16、velopmental potential to a pluripotent cell state.In this study,we identified the miR-34a miRNA as the first non-coding regulator that restricts the pluripotent cell fate potential in cultured ESCs/iPSCs,the deficiency of which yields bidirectional cell fate potential and MERVL induction in pluripot
17、ent stem cells.miR-34a/pluripotent stem cells exhibit expanded cell fate potentialmicroRNAs(miRNAs)are a class of small,regulatory non-coding RNAs that regulate gene expression post-transcriptionally through a combined mechanism of mRNA degradation and translational repression(1113).These small non-
18、coding RNAs are increasingly recognized as key regulators of cell fate specification in normal development and in pluripotent stem cells(14,15).Initially identified as bona fide p53 transcriptional targets in tumor suppression,the miR-34 miRNAs(miR-34a,miR-34b and miR-34c),particularly miR-34a,have
19、been previously characterized as a key barrier for somatic reprogramming(16).miR-34a deficiency significantly enhances the efficiency of iPSC generation(16),producing iPSCs with normal self-renewal and pluripotency(Fig.S1A,S1B and S1C;ref.16).Surprisingly however,teratomas generated from miR-34a/iPS
20、Cs,but not wild-type iPSCs,contained cellular features reminiscent of trophoblast giant cells in the placenta,characterized by PL-1 Choi et al.Page 2Science.Author manuscript;available in PMC 2018 September 14.Author ManuscriptAuthor ManuscriptAuthor ManuscriptAuthor Manuscript(placental lactogen 1)
21、expression,large cell volume,enlarged nuclei,and close proximity to internal hemorrhages(Fig.1A).In ESCs,miR-34a constitutes the majority of expressed miR-34 miRNAs(Fig.S1D).Similarly,miR-34a/ESC derived teratomas,but not the wild-type controls,also contained areas reminiscent of extraembryonic plac
22、ental cell lineages(Fig.1A)and exhibited an induction of trophectoderm(TE)markers(Fig.S1E),including cdx2,elf5,psx1,fgfr2,egfr and mdfi(17,18).While we did not identify any areas morphologically resembling the visceral endoderm of the yolk sac,we detected a strong induction of primitive endoderm(PE)
23、markers(gata4,gata6 and sox17)in miR-34a/teratomas,but not in wild-type controls(Fig.S1E).These findings suggest that miR-34a/pluripotent stem cells likely differentiate towards both embryonic and extra-embryonic cell lineages during teratoma formation.The expanded potential of miR-34a/ESCs/iPSCs is
24、 also evident upon embryoid body(EB)differentiation(Fig.1B and 1C).While markers from all three germ layers were similarly induced in wild-type and miR-34a/EBs,significant upregulation of TE markers(cdx2,elf5,esx1,tfap2c and gata3)(17,19,20)was observed only in miR-34a/EBs(Fig.1B,1C and S1F).Immunof
25、luorescence(IF)staining confirmed that a significant percentage of miR-34a/EBs was Cdx2 positive(Fig.1B),and these Cdx2-positive cells preferably localized to the periphery(Fig.1B,S1G and S1H).Additionally,the extra-embryonic endoderm marker gata4 and pdgfra,as well as the trophoblast lineage marker
26、 mash2(ascl2)and pl1(prl3d1),were also induced in miR-34a/EBs(Fig.S1F).Thus,upon EB differentiation,miR-34a/ESCs exhibited expanded cell fate potential,generating cells with molecular features characteristic of both embryonic and extra-embryonic lineages.To define the cell fate potential of miR-34a/
27、pluripotent stem cells in normal development,we traced their lineage in chimeric blastocysts following microinjection or aggregation with recipient morulae.Initially,four GFP-labeled wild-type or miR-34a/ESCs were injected into each C57BL/6N recipient morula to generate chimeric blastocysts(Fig.1D).
28、While wild-type ESCs exclusively gave rise to cells localized to the ICM(Fig.1D;Table S1),miR-34a/ESC progenies localized to both ICM and TE in 60%of chimeric blastocysts(Fig.1D;Table S1).This expanded cell fate potential is unlikely due to extra-embryonic contamination during miR-34a/ESC derivation
29、,as miR-34a/iPSCs derived from mouse embryonic fibroblasts(MEFs)phenocopied miR-34a/ESCs in their developmental potential.When aggregated with recipient C57BL/6J morulae,miR-34a/ESCs and miR-34a/iPSCs colonize both ICM and TE of chimeric blastocysts,while passage-and littermate-controlled wild-type
30、ESCs and iPSCs exclusively colonized the ICM(Fig.S1I).The expanded cell fate potential of miR-34a/ESCs in chimeric blastocysts could be due to the presence of cells with bidirectional potential;alternatively,miR-34a/ESCs could contain a heterogeneous population of cells that preferentially different
31、iate into embryonic or extra-embryonic cell lineages.To distinguish between these two possibilities,we injected single,GFP-labeled miR-34a/ESCs into each recipient morula to generate chimeric blastocysts(Fig.1E).In two independent miR-34a/ESC lines tested,single miR-34a/ESCs colonized both ICM and T
32、E in 33%and 38%of chimeric blastocysts(n=13/40 and 8/21)(Fig.1E;Table S1)respectively,suggesting that a significant portion of miR-34a/ESCs exhibit a bidirectional developmental potential at the single-cell level.Choi et al.Page 3Science.Author manuscript;available in PMC 2018 September 14.Author Ma
33、nuscriptAuthor ManuscriptAuthor ManuscriptAuthor ManuscriptWe then generated chimeric embryos by microinjecting 1015 GFP-labeled wild-type or miR-34a/ESCs into C57BL/6N recipient blastocysts.While wild-type ESCs contributed exclusively to lineages of the three embryonic germ layers,miR-34a/ESCs cont
34、ributed to both embryonic and extra-embryonic cell lineages in E9.5,E12.5 and E14.5 chimeric embryos(Fig.1F,1G and S1J;Table S1).In particular,we observed clusters of GFP-positive,miR-34a/ESC progenies in the visceral endoderm of the yolk sac,as well as in multiple extra-embryonic trophoblast lineag
35、es of the placenta(trophoblast giant cells,spongiotrophoblasts,syncytiotrophoblasts(STBs)and sinusoidal trophoblast giant cells(s-TGCs),Fig.1F,1G;Table S1).In these chimeric embryos,the number of GFP-positive cells in extra-embryonic cell lineages greatly surpasses the number of miR-34a/ESCs injecte
36、d(Fig.1F and 1G),suggesting that injected miR-34a/ESCs had undergone substantial proliferation before committing to multiple terminally differentiated extra-embryonic lineages.miR-34a/pluripotent stem cells exhibit an induction of MERVL ERVsTo investigate the molecular basis for the bidirectional po
37、tential of miR-34a/pluripotent stem cells,we compared the transcriptomes of wild-type and miR-34a/iPSCs using RNA-sequencing(RNA-seq).We compared the abundance of all annotated transcripts between wild-type and miR-34a/iPSCs,including protein-coding genes,long non-coding RNAs(ncRNAs),pseudogenes,ant
38、isense transcripts,and retrotransposons using 100 bp paired end RNA-seq data(Fig.2A).Given the repetitive nature of retrotransposons,we quantified retrotransposon expression at the family level using both uniquely and non-uniquely mapped reads(Supplemental Information S1).Surprisingly,the most highl
39、y expressed and differentially regulated transcript in miR-34a/iPSCs was transcribed from the MERVL family of ERVs(Fig.2A and S2A),which were also highly induced in totipotent 2C blastomeres and reported ESCs with expanded potential(7,9,21,22).ERV induction in miR-34a/ESCs/iPSCs was largely specific
40、 to the MERVL family(Fig.2A,2B and S2A;Table S2).The majority of differentially expressed retrotransposons in miR-34a/iPSCs belonged to the canonical MERVL family of ERVs(a class-III ERV)(Fig.S2A;Table S2);a small fraction of differentially expressed loci belonged to the MT2A,MT2B,MT2B1,and MT2B2 ER
41、V families that are highly related to the canonical MERVL solo LTR,MT2_Mm(Fig.S2A;Table S3).Consistent with our RNA-seq results,we invariably detected a significant increase of MERVL expression in miR-34a/iPSCs and ESCs,using real-time PCR primer pairs designed from multiple highly conserved MERVL r
42、egions(Fig.2B and 2C;data not shown).Interestingly,while MERVL induction in miR-34a/iPSCs persisted for more than 27 passages(Fig.S2B),MERVL was only induced in early passages of miR-34a/ESCs and became completely silenced around passage 12(Fig.S2B).It is conceivable that MERVL expression in miR-34a
43、/ESCs triggers additional mechanisms to re-establish their silencing.The expanded cell fate potential of miR-34a/ESCs was highly correlated with the strong MERVL induction,as the late passage(passage 17)miR-34a/ESCs lost both MERVL induction and the bidirectional potential(Fig.S2B and S2C).Choi et a
44、l.Page 4Science.Author manuscript;available in PMC 2018 September 14.Author ManuscriptAuthor ManuscriptAuthor ManuscriptAuthor ManuscriptThe MERVL ERVs have been retained throughout mammalian evolution,with independent expansion in the murine and primate genomes(23).There are 2502 loci in the C57B6/
45、J mouse genome,26%of which encode elements with an intact retroviral structure,comprising 5-and 3-LTRs flanking the coding sequences for gag,pol,and dUTPase,but lacking env-like open reading frames(ORFs)(23)(Fig.2C).Another 32%of MERVL loci exhibit truncated retroviral structure,missing one or both
46、LTRs(Fig.2C).The remaining 41%of MERVL loci have undergone homologous recombination,yielding solo LTRs(MT2_Mm)with varying degrees of sequence degeneration(Fig.2C).We obtained bioinformatic estimates of locus-specific MERVL expression in wild-type and miR-34a/iPSCs using our RNA-seq data(Table S3;Su
47、pplemental Information).Notably,definitive evidence for MERVL reactivation in miR-34a/iPSCs was observed predominantly for loci harboring MERVLs with a complete retroviral structure,but not for those with truncated structure(Fig.2D and S2D;Table S3).A fraction of MT2_Mm solo LTRs,along with a few el
48、ements from the highly related MERVL solo LTRs(MT2B,MT2B1,MT2B2 and MT2A),also exhibited a similar induction(Fig.S2A;Table S3).Approximately 300 MERVL loci still encode intact Gag viral protein(7).We observed a significant increase in MERVL-Gag expression and in the percentage of MERVL-Gag-positive
49、cells in miR-34a/pluripotent stem cells(Fig.2E and 2F).Interestingly,miR-34a/ESCs and iPSCs were heterogeneous populations,containing 12%and 20%MERVL-Gag-positive cells,respectively,in otherwise Oct4-positive colonies(Fig.2E,2F,S2E and S2G).Consistent with this observation,a fraction of individual m
50、iR-34a/iPSC colonies exhibited a significantly greater MERVL induction than the bulk population(Fig.S2F),suggesting that the extent of MERVL induction in individual cells was largely underestimated using the bulk population.In miR-34a/ESCs and iPSCs,the expression of MERVL-Gag and Oct4 were mutually