mESC do not require gp130 mediated signaling to affect self-renewal .... Cell name followed by total number of passages in culture (compound named first.
Cell Stem Cell, Volume 4
Supplemental Data Histone Deacetylase Inhibition Elicits an Evolutionarily Conserved Self-Renewal Program in Embryonic Stem Cells Carol B. Ware, Linlin Wang, Brigham H. Mecham, Lanlan Shen, Angelique M. Nelson, Merav Bar, Deepak A. Lamba, Derek S. Dauphin, Brian Buckingham, Bardia Askari, Raymond Lim, Muneesh Tewari, Stanley M. Gartler, Jean-Pierre Issa, Paul Pavlidis, Zhijun Duan, and C. Anthony Blau
Figure S1. Butyrate treated H1 cells grown in hESM on Matrigel maintain an undifferentiated state relative to cells in CM. A. Flow cytometry profile of H1 cells cultured in butyrate (top) or CM (below). Profiles shaded in gray indicate control staining with secondary antibodies alone. B. Pou5f1 (Oct 4) and Nanog mRNA levels in H1 cells cultured in butyrate [orange bars, H1p48(CM3;B6)] or CM [blue bars, H1p48(CM9)]. Error bars denote standard error of the mean. C. Telomerase activity assay of H1 cells cultured in CM or butyrate in the presence (+) or absence (-) of heat inactivation. HFF: human foreskin fibroblasts. Cells were exposed to > 3 passages in butyrate in all assays to compare to cells grown in CM.
Figure S2. Schematic depiction of hESC karyotypes during culture in HDACi versus standard conditions. A. H1 cells cultured on Matrigel starting on passage 39. B. H7 cells cultured continuously on feeders. C. H13 cells cultured continuously on feeders. D. BG02 cells cultured on Matrigel beginning at passage 31. E. BG02 cells cultured on Matrigel plus butyrate starting at passage 41. F. BG02 cells cultured on Matrigel beginning at passage 39. Open circles indicate normal karyotype. ? indicates unknown karyotype. Closed circles indicate abnormal karyotypes. Hatched circles indicate inferred abnormal karyotype. Numbers of circles represent numbers of karyotypes examined.
Figure S3. mESC do not require gp130 mediated signaling to affect self-renewal in response to HDACi. Phase contrast appearance (top panels) and alkaline phosphatase staining (lower panels) of a. R1 cells, b. Lifr-/- mESC, or c. gp130 null mESC. In panel c, the lower panels are alkaline phosphatase labeled (left) and Pou5f1 labeled (right) under the respective unstained image. Control cultures for R1 and Lifr-/- cells used IL6/sIL6 receptor (to bypass LIFR and signal through gp130) whereas gp130 null mESC used parietal endoderm conditioned medium (that can sustain mESC without functional gp130 signaling) to maintain self-renewal. All cultures were performed in the absence of feeders. Control cultures [using IL6 + sIL6R (A, B) or parietal endoderm conditioned medium (C)] were compared to cells cultured for 4 passages in butyrate (without IL6/sIL6R or parietal endoderm conditioned medium). Note that differentiating cells could be detected in all culture conditions. Image areas were selected to reflect the inherent heterogeneity irrespective of culture conditions. The size bar indicates 38 μM.
Figure S4. hESC cultured in butyrate retain full differentiation capacity. A. Teratomas excised at day 49 – left panel generated from BG02p66(CM35) and the right panel generated from BG02p80(CM29;B20). The arrows placed in the butyrate tumor indicate the presence of melanin. B. Top panels: Trimmed paraffin blocks of teratomas shown in panel A. Bottom panels: Trimmed paraffin blocks of teratomas generated 10 weeks after injection of BG02 cells cultured in CM (left) or 8 weeks for cells cultured in butyrate without CM and FGF2 (right). Arrows indicate avascular areas. Triangles point toward melanin containing spots. C. Histological sections of teratomas from H1 cells cultured in CM (left) or butyrate (right). The top 3 images in each column are the same view at increasing magnification. The number of days between implantation and harvest is indicated at the top of the columns. Representatives of the 3 germ lineages are indicated EN: Endoderm. EC: ectoderm. M: mesoderm. The size bar indicates 250 μm in the upper panels, 100 μm in the second and fourth panels,
and 50 μm in the third panels. Note the pigment in the ectoderm of the butyrate tumors. D. A section from BG02p80(CM29;B20) 7 week tumor shown in panel B highlighting melanin expression (upper arrow). Lower arrow indicates cells labeled with an antibody directed against synaptophysin, a glycoprotein present in the membrane of neuronal presynaptic vesicles and an early neuroectodermal marker. The size bar indicates 12.5 mm in panel D.
Figure S5. A relatively small number of genes are regulated by butyrate. Unsupervised analysis (panels A & C) showing all genes and supervised analysis (panels B & D) showing only significantly altered expression from Agilent mRNA arrays probed with mRNA from H1 cells cultured in CM [hES-CM; H1p48(CM9)], H1 cells from the same pool cultured in butyrate for 6 passages [hES-But; H1p48(CM3;B6)] or H1 cells from the same pool converted to butyrate for 4 passages followed by CM for 3 passages [hES-rCM; H1p49(CM3;B4;CM3)]. Note that genome-wide expression patterns are only modestly affected by butyrate (C).
Figure S6. Characterizing the transcriptional response to butyrate. A. qRTPCR confirmation of 14 transcripts identified by microarray as being differentially regulated by butyrate [H1p48(CM;B6) relative to H1p48(CM9)] B. Addition of butyrate to H13 cells cultured on feeders (without added bFGF when butyrate was present and with 2 ng/ml added bFGF when grown in CM) induces Dppa5, Piwil2 and Ecat1. Butyrate was added for the last 3 passages. C. qRTPCR demonstrates the persistence of Ddx43/HAGE, Dppa5 and Ecat1 expression 3 passages after returning H1 cells cultured in butyrate for 4 passages back to CM for 3 passages [H1p49(CM3;B4;CM3)]. Error bars denote standard error of the mean.
Figure S7. Unsupervised (left) and supervised (right) profiles from whole mouse genome Agilent arrays using mRNA from R1 mESC cultured in LIF versus butyrate for four passages. Arrays were run independently, in quadruplicate.
Figure S8. Morphology of EpiSCs cultured in standard hESC-like conditions for 12 passages (left), or cultured under the same conditions except for the addition of butyrate for 3 passages (right). Size bar indicates 38 μm.
Figure S9. hESC respond to a variety of HDACi. A. Phase contrast appearance of hESC cultured in CM (BG02), sodium butyrate (BG02), butyryl CoA (BG03), trichostatin A (BG02), valproic acid (H1) and vorinostat/SAHA (BG02) at the indicated concentrations and passage numbers. Size bar indicates 38 μm. B. Transcriptional response of hESC (BG02) cultured in 0.2 mM butyrate (without feeders) for 38 passages or 10 nM trichostatin A for 34 passages relative to CM.
Table S1. Summary of karyotype data.
Cell line
H1p47(CM8) H1p46(B5) H1p49(B7) H1p55(B13) H7p43 H7p43(B8) H13p41 H13p40(B12) BG02p65(CM28) BG02p51(CM15,B5) BG02p58(CM6,SAHA13) BG02p59(CM21,BCoA5) BG02p64(CM24,GABA3)
BG02p62(CM10,TSA17) BG02p69(B28) BG02p74(CM35) BG02p71(CM10,B22)
Karyotype
Panel in Supplemental Figure 2
48XY+12;+17[5] 46XY[5] 47XY+12[1] 46XY[4] 46XY [6] 47XY+12 [9] 46XX[5] 46XX[5] 46XY[5] 46XY[5] 46XY[5] 46XYdup(1)(q21q42)[2] 46XY[23] 46XY[5] 46XYdup(1)(q21q42)[5] 44-46,XY dup(1)(q21q42)[2] 46XY[4] 92XXYY[1] 47XY+20 46XY
A A A
46XY[14] 46XY,t(4:13)(q25;q14)[1] 46XY[15]
A B B C C D D D D D
D E F F
Bold – grown in conditioned medium (H1, BG02) or on feeders (H7, H13) Italics – related to nearest line above in non-italicized print Cell name followed by total number of passages in culture (compound named first indicates culture presence for the indicated number of passages, followed by the second compound for the indicated number of passages - CM – conditioned medium; B – butyrate; SAHA – vorinostat; TSA – trichostatin A; BCoA - butyryl CoA; GABA – γaminobutyric acid.
Supplemental Table 2. Effect of butyrate on chimera formation in mice. Group Difficult clones R1 on feeders C57BL/6 line started on butyrate
Cultured in LIF # embryos # pups transferred born 112 25
# chimeras (%) 0
39
0
-
Cultured in Butyrate # embryos # pups # chimeras transferred born (%) 72 5 4 (70, 85, 2 x 100) 28 8 2 (100)
-
-
-
20
6
1 (85)
“Difficult clones” refer to transgenic mESC lines that had been unable to generate chimeric animals in the absence of butyrate.
Supplemental Table 3. 15 top butyrate induced and repressed genes in hESC Induced Symbol Dppa5 Ddx43 Piwil2 Tnni3 Cbr1 Rcn3 Slc16a3 Cxcl5 Bhmt2 Rasip1 Txnip Plek2 Crlf1 Kirrel2 Aif1 Repressed Sp5 Frzb Sfrp2 Dusp6 Ier5 Kcnj16 Cxcr4 Grb10 Prex1 C21orf129 Igfbp5 C9orf86 Id1 Tcf3 Stox2
Gene Name Developmental pluripotency associated 5 DEAD (Asp-Glu-Ala-As[) box polypeptide 43 PIWI-like 2 Troponin I, cardiac Carbonyl reductase Reticulocalbin 3, EF-hand calcium binding domain Solute carrier family 16 (monocarboxylic acid transporters), member 3 Chemokine (C-X-C motif) ligand 5 Betaine-homocysteine methyltransferase 2 Ras interacting protein 1 Thioredoxin interacting protein Pleckstrin 2 Cytokine receptor-like factor 1 Kin of IRRE like 2 (Drosophila) Allograft inflammatory factor 1
Chrm. 6 6 8 19 21 19 17
Sp5 transcription factor Frizzled-related protein Secreted frizzled-related protein 2 Dual specificity phosphatase 6 Immediate early response 5 Potassium inwardly-rectifying channel, subfamily J, member 16 Chemokine (C-X-C motif) receptor 4 Growth factor receptor bound protein 10 Phosphatidylinositol 3,4,5-triphosphate-dependent RAC exchanger 1 Chromosome 21 open reading frame 129 Insuling-like growth factor binding protein 5 Chromosome 9 open reading frame 86 Inhibitor of DNA binding 1, dominant negative helix-loop-helix protein Transcription factor 3 (E2A immunoglobulin enhancer binding factors E12/E47 Storkhead box 2
2 2 4 12 1 17 2 7 20
4 5 19 1 14 19 19 6
21 2 9 20 19 4
Table S4. Expression changes caused by culture of H1 in sodium butyrate as organized by function. Slowly returns to CM levels upon butyrate removal [chromosome location] Down Up EMBRYO/GERM ASSOC. Expressed in early embryos & germ cells Dppa5 [6q13] Tcf3 [19p13.3] Piwil2 [8p21.3] Gata6 [18q11.1] Bnc1 [15q25.2] Lrrc8e [19p13.2] Ecat8 [19q13.11] Mbd3 [19p13.3] DNA Methylation/nucleosome Mbd3 [19p13.3] H2afj [12p12] Recombination Rnf212 [4p16.3] Transcription factors Tbx1 [22q11.21] C3orf41 [3p22.1] Bnc1 [15q25.2] Heyl [1p34.3] Tcf15 [20p13] Npas1 [19q13.2-13.3] Flj10324 [7p22.1] Tceal5 [Xq22.1] Loc401233 [6p25.2] Transcriptional repressor Txnip [1q21.1] C1orf103 [1p13.3] DNA helicase
Sox17 [8q11.23]
Id1 [20q11] Id2 [2p25] Chd7 [8q12.2]
DNA-binding/Zinc finger Znf717 [3p12.3] Bnc1 [15q25.2] Zdhhc11 [5p15.33] Gtf2e1 [3q21-q24] Znf502 [3p21-31]
Znf229 [19q13.31] Phf10 [6q27] Pdlim5 [4q22] Lmo7 [13q22.2] Zic2 [13q32] Gata4 [8p23.1-p22] Zc3h6 [2q13] Znf219 [14q11]
Nucleosome structure Bhmt2 [5q13] Bhmt [5q13.1-q15] Centromere Aurkc [19q13.43] C19orf63 [19q13.33] RNA RNA-binding (KH domain) Dppa5 [6q13] RNA helicase Ddx43 [6q12-q13] Ecat8 [19q13.11] Ddx25 [11q24] RNAi/heterochromatin Piwil2 [8p21.3] Interacts with RNA polymerase Rbp7 [1p36.22] Bnc1 [15q25.2]
PROTEIN ASSOCIATED Ribonucleoprotein core (ribosome biogenesis) Pus7l [12q12] Protein chaperone Hspa2 [14q24.1] Hspb2 [11q22-q23] METABOLISM Cholesterol/lipid/adipose Ch25h [10q23] C7orf16 [7p15] Fabp7 [6q22-q23] Cpt1c [19q13.33] C10orf10 [10q11.21] Df [19p13.3] Energy Bhmt2 [5q13] Bhmt [5q13.1-q15] Nmu [4q12] Atp5e [20q13.32] C10orf96 [10q25.3] Atpbd4 [15q14] NADPH activity Cbr1 [21q22.13] Cbr3 [21q22.2] Undefined
Apoa2 [1q21-q23] Dagla [11q12.2] Dennd5b [12p11.21]
Gad1 [2q31]
Isoc1 [5q22.1-q33.3] CELLULAR LOCALIZATION Cytoskeleton Plek2 [14q23.3] Mark1 [1q41] Nefh [22q12.2] Pdlim5 [4q22] Magl2 [7q21] Tnnt1 [19q13.4] Protein scaffolds/matrix/adhesion Mxra8 [1p36.33] Frem1 [9p22.3] Adamts4 [1q21-q23] Pitx2 [4q25-q27] Silv [12q13-q14] Lamb1 [7q22] Lgals1 [22q13.1] Fam20c [7p22.3] Cthrc1 [8q22.3] Has3 [16q21.2] Semg1 [20q12-q13.2] Madcam1 [19p13.3] lysosome Ctsf [11q13] Golgi associated Ap1s2 [Xp22.2] Mitochondrial Aksl1 [1p31.3] Calcium channel Rcn3 [19q13.33] Tthy1 [19q13.4] Chloride channel Cilic6 [21q22.12] Tthy1 [19q13.4] Potasssium channel Kchh2 [7q35-q36] Kchj16 [17q23.1-q24.2] Kctd1 [18q11.2] CELL PROCESS Signal Transduction Matk [19p13.3]
Ptpn21 [14q31.3]
Sh2d5 [1p36.12] Gpc6 [13q32] Cblc [19q13.2] GTP/Ras/Rho Rasip1 [19q13.33] Prex1 [20q13.13] Vav1 [19p13.2] C9orf86 [9q34.3] Flj21438 [19p13.12] Flj10357 [14q11.2] Rasgrp2 [ 11q13] Solo [14q11.2] Rab37 [17q25.1] Rhobtb3 [5q15] Protease inhibitor Wfdc2 [20q12-q13.2] SerpinB9 [6p25] Ubiquitin Fbxo2 [1p36.22] Mark1 [1q41] Tle2 [19p13.3] Cblc [19q13.2] Tumor associated Aurkc [19q13.43] Sfrp2 [4q31.3] Gstt2 [22q11.2; 22q11.23] Cxcr4 [2q21] Hspa2 [14q24.1] Klf8 [Xp11.21] Hspb2 [11q22.3-q23] Sox11 [2p25] Matk [19p13.3] Cyyr1 [21q21.2] Magea1 [Xq28] Phlda1 [12q15] Magea2b [Xq28] Ctdspl [3p21.3] Cthrc1 [8q22.3] Mtcbp-1 [2p25.3] Nmu [4q12] Hpn [19q11-q13.2] Klk8 [19q13.3-q13.4] Anti-apoptotic Piwil2 [8p21.3] Tie1 [1p34-p33] Klk8 [19q13.3-q13.4] Inhba [7p15-p13] Stat6 [12q13] Klk10 [19q13.3-q13.4 Rp3-452h17.2 [Xq23] Cxorf61 [Xq23] Intercellular recognition/cell membrane transport/membrane associated Slc16a3 [17q25] Tspan5 [4q23] Ervwe1 [7q21-q22] Flrt3 [20p11] Atp8b3 [19p13.3] Gpc6 [13q32] Sectm1 [17q25] Hpn [19q11-q13.2] Slco4a1 [20q13.33] Gp1bb [22q11.21] Abca1 [9q31.1] Pprt2 [16p11.2] Lrrc8e [19p13.2] Prrt2 [16p11.2] Lypd5 [19q13.31] Stress induced/response Txnip [1q21.1] Ppm1b [2p21] Oxidation Protection Selm [22q12.2] Tubulin/centomere/growth inhibition – cell cycle control Aurkc [19q13.43] Sertad4 [1q32.1-q41] Bicd1 [12p11.2-11.1] Pftk1[7q21-q22] Cgref1 [2p23.3] Ppm1b [2p21] CHEMOKINE/CYTOKINE Cxcl5 [4q12-q13]
Crlf1 [19p12] Pf4 [4q12-q21] Stat6 [12q13] Cxcl12 [10q11.1] Cd70 [19p13] GROWTH FACTORS Mitogen Ier5 [1q25.3] TGF Inhba [7p15-p13]
Lefty1 [1q42.1] Foxq1 [6p25] Fst [5q11.2]
FGF Fgf12 [3q28] Fgfbp3 [10q23.32] Notch Heyl [1p34.3] Dner [2q36.3] MagI2 [7q21] Wnt Kremen2 [16p13.3] Tle2 [19p13.3] Apc2 [19p13.3]
Hormone Inhba [7p15-p13] Csh2 [17q24.2] Csh1 [17q24.2] Cshl1 [17q24.2] Gh1 [17q24.2] Rln2 [9p24.1] Insulin Igfbp6 [12q13]
Sfrp2 [4q31.3] Frzb [2qter] Sp5 [2q31.1] Tcf3 [19p13.3] Fzd2 [17q21.1] Wnt3 [17q21] C21orf129 [21q22.3] Fst [5q11.2] Dio3 [14q32] Oxtr [3p25]
Grb10 [7p12-p11.2] Igfbp5 [2q33-q36] Htra1 [10q26.3] Grb14 [2q22-q24] Phlda1 [12q15]
Stat3-P Responsive Dppa5 [6q13] expression Piwil2 [8p21.3] MAPK-ERK inactivation Dusp6 [12q22-23] DIFFERENTIATION Mesoderm Tcf15 [20p13]
Mesdc1 [15q13] Gata6 [18q11.1]
Homeobox Gsc [14q32.1] Pitx2 [4q25-q27] Differentiated tissues: Retinoic Acid Rbp7 [1p36.22] C1orf103 [1p13.3] Cardiac Tnni3 [19q13.4]
Nppb [1p36.2]
Muscle Eno3 [17pter-p11] Tnnt1 [19q13.4] Cdh15 [16q24.3] Pancreas Kirrel2 [19q13.12] Vascular Aif1 [6p21.3] Tie1 [1p34-p33] Tac3 [12q13-q21] Ednrb [13q22] Neural Tbx1 [22q11.21] Atcay [19p13.3] Gap43 [3q13.1-q13.2] Nrxn2 [11q13] Gdap1l1 [20q12] Pnmt [ 17q21-q22] Nptx2 [7q21.3-q22.1] Npas1 [19q13.2-13.3] Bone Bst2 [19p13.2] Immune associated Hla-f [6p21.3] Hla-a [6p21.3] Lag3 [12p13.32] Ifi30 [19p13.1] Nlrp7 [19q13.42] Calcb [11p15.2-p15.1] Lck [1p34.3] Indo [8p12-p11] Psme1 [14q11.2] Blood Cox6b2 [19q13.42] Matk [19p13] Testes/Ovary Piwil2 [8p21.3] Bnc1 [15q25.2] Nyd-Sp18 [7q32.1] Liver/Lung Arg1 [6q23] Intestine
Myl4 [17q21-qter] Myl7 [7p21-p11.2]
Sox11 [2p25] Cyyr1 [21q21.2] Ypel2 (7) [17q22] Coch [14q12-q13]
Fzd2 [17q21.1] Tff1 [21q22.3]
ALTERNATIVELY SPLICED/TRANSCRIPTS Tbx1 [22q11.21] Ap1s2 [Xp22.2] Aif1 [6p21.3] Pdlim5 [4q22] Dppa5 [6q13] Cyyr1 [21q21.2] Bnc1 [15q25.2] Flj10357 [14q11.2] Wdr21a [14q24.3] Solo [14q11.2] Klk10 [19q13.3-q13.4] Wfdc2 [20q12-q13.2] Bicd1 [12p11.2-p11.1] Csh2 [17q24.2] Cshl1 [17q24.2] Matk [19p13.3] Clic6 [21q22.12] [ Ednrb [13q22] Eno3 [17pter-p11] Atp5e [20q13.32] Gh1 [17q24.2] Arg1 [6q23]
Kcnh2 [7q35-q36] Nrxn2 [11q13] Vcx [Xp22] UNKNOWN FUNCTION C17orf51 [17p11.2] Rilpl2 [12q24.31] Arrdc4 [15q26.3] Wdr21a [14q24.3] Lrrc3b [3p24] Ttc29 [4q31.23] Vcx [Xp22] Fam12b [12q13.2] Snhg12 [1p35.3] Cxorf61 [Xq23] Prrt2 [16p11.2] Gtsf1 [12q13.2] C17orf51 [17p11.2]
Flj30428 [2q21.1] Cap2 [6p22.3] Rp11-38l15.1 [10q11.22] Stox2 [4q35.1] Flj45187 [10p12.31] Rnf150 [4q31.21] Fam35b2 [10q11.22] C2orf12 [2q24.3]
Supplemental Experimental Procedures Primate (hESC and rhESC) culture ES cells were cultured on primary mouse embryonic fibroblasts (MEF’s) from E12.5 CD-1 mice established following the protocol of Abbondanzo et
al.1.
Passage 4-6 MEF’s were γ-irradiated with 3000 Rads and plated at 104 cells per cm2. To make CM, a confluent 10 cm plate of γ-irradiated MEF’s was plated with hESC medium containing 2ng/ml human FGF2 and allowed to condition for 3 days at 37oC, at which time the medium was harvested and filtered through a 0.22 μm filter. For passage, cells were exposed to 1.2 U/ml dispase (Invitrogen) dissolved in PBS supplemented with either 10% fetal bovine serum (FBS;ESqualified FBS, Invitrogen) or containing SR in place of FBS.
Once colonies
began to lift from the plate, the cell layer was washed off using a Pasteur pipette, collected in a centrifuge tube containing culture medium, washed once by centrifugation, mechanically dispersed to ~100 μm diameter clusters using a 5 ml pipette, and washed again before plating. Cells cultured off of feeders did not require mechanical disruption. Cells exposed to butyrate were cultured in hESM but with no MEF conditioning and without added FGF2 and contained 0.2-0.3 mM sodium butyrate (Sigma). absorbed by polypropylene.
Sodium butyrate is a small molecule easily It was held as a stable additive at 1000x in
boroscillicate glass at 4oC and added directly to the culture plates as needed. The other iHDACs were either placed in medium and held at 4oC before use or treated like butyrate.
mESC culture and derivation mESC were passaged by washing once with PBS without calcium and magnesium (Invitrogen), followed by exposure to trypsin-EDTA (Invitrogen) until the cells were digested to single cells.
Cells were pelleted in DMEM
supplemented with 10% FBS and penicillin and streptomycin, the pellet resuspended and plated. Cells were routinely cultured on an irradiated MEF feeder layer and when cultured off of feeders, they were plated directly onto gelatinized plates in the media specified in the text. In addition, LIF receptor null
cells (a gift of Austin Smith) were cultured in 500 ng/ml IL-6 + 5000 ng/ml soluble IL-6 receptor (R&D Systems) to allow signaling through gp130 and gp130 null cells (a gift of Ian Chambers) were cultured in parietal endoderm conditioned medium, as an alternative to using LIFR/gp130 to sustain self-renewal, made as previously described2. Briefly, parietal endoderm cells were established from mESC using retinoic acid. Medium was conditioned by fluid changing a confluent 10 cm plate of parietal endoderm cells with 10 ml of mESC culture medium and culturing overnight. Medium was collected the next day and filtered through a 0.22 μm filter.
mESC lines were derived by culture of C57BL/6 embryonic day 3.5 (E3.5) embryos in mESC medium containing LIF on feeders with or without added 0.2 mM sodium butyrate. Embryos were allowed to hatch and plate and the inner cell mass was plucked and dispersed into 2-5 fragments for selective subculture. Using this method, approximately 10% of the embryos generated ESC lines irrespective of whether butyrate was present or absent.
Mouse Epiblast Cell (EpiSC) Culture Mouse EpiSC#5 (a gift of Paul Tesar and Ron McKay) was cultured as described3.
In addition, new mEpiSC lines were derived as previously
described3. Essentially, all mEpiSC were passaged using the same method as described above for hESC, on feeders, in medium containing serum replacer and passaged in clusters using dispase.
Once the mEpiSC were exposed to
butyrate, the medium was changed to mESC medium containing serum and mLIF, but the cells were passaged in clusters using dispase rather than as a single-cell suspension and were always cultured on a MEF feeder.
Generation of Chimeric Mice Chimeric mice were created through blastocyst microinjection of 8-12 mESC or mEpiSC as previously described4. Cells cultured in butyrate were passaged at least three times in butyrate before introduction into the blastocyst and injected
embryos were soaked in 0.2 mM sodium butyrate added to the M16 culture medium for 1 hour or overnight prior to transfer, depending on the availability of embryo transfer recipients. Assessment of germline transmission utilized a standard coat color assay with evidence of contribution of the ESC or EpiSC to the germline through a difference in the coat color between the host embryo versus the contributing stem cell.
Differentiation of hESC Cells for undirected differentiation were seeded at ~2 x 105 cells per uncoated, gelatinized or Matrigel treated 10 cm plate in DMEM (Invitrogen) containing 20% FBS (ES cell tested, Invitrogen) with penicillin and streptomycin (Invitrogen). Cells were allowed to attach and were fluid changed every other day. Directed differentiation toward retinal neurons has been previously described5.
Teratoma formation Teratomas were induced in either Balb/c Scid mice (Jackson Labs in the case of H1) or Scid-Beige (Charles River Labs) in the case of BG02. Two million cells suspended in 50μl were injected intramuscularly into the rear hindleg. The mice were monitored for tumor growth by palpation.
When the tumors became
evident, mice were euthanized using CO2 asphyxiation and the tumors harvested (usually around 6 weeks following injection), fixed in 4% paraformaldehyde in PBS, paraffin embedded and sectioned at 5-7 μm and stained for hematoxylin and eosin.
Karyotype G-banded karyotypes were performed by the Cytogenetics Laboratory at the University of Washington. At times, cells were sent to Cell Line Genetics (Madison, WI) for karyotype analysis.
Cell Growth Assay by BrdU Activity
Cells were analyzed for BrdU uptake using an ELISA-based kit (Roche, 5-Bromo2’-deoxyuridine Labeling and Detection Kit III) following manufacturer’s instructions. The cells were hand picked as clusters – 10 clusters per well in a 96-well plate, allowed to plate and grown for 2-3 days, when they were labeled with BrdU for 1.5 to 2 hours, then fixed and held for analysis by ELISA. Internal control wells with no cells were present on each plate and results were compared only within runs within a plate.
Immunohistochemistry Maintenance
of
an
undifferentiated
phenotype
was
established
by
immunohistochemistry using antibodies for OCT4 (R&D Systems; 1:200 dilution) and SSEA-4 (Chemicon International; 1:50 dilution) and by staining for alkaline phosphatase (AP) activity using a Black Alkaline Phosphatase Substrate Kit II (Vector Laboratories, Burlingame, CA, USA). For immunolabeling, the clusters were fixed in 4% paraformaldehyde and held in 70% ethanol at 4oC until processing. Antibody binding to the cells was visualized using a biotinylated secondary antibody included in the Universal Quick Kit using Nova Red (both from Vector Laboratories). All immunolabeling was compared to the Universal Quick Kit secondary antibody only control.
Animals All procedures with the mice followed the recommendations and approval of the Institutional Animal Care and Use Committee of the University of Washington and were performed within an American Association for the Advancement of Laboratory Animal Care accredited specific pathogen free facility at the University of Washington
Flow Cytometry Cytometric analyses were performed using a FACScan Cytometer and Cell Quest software (BD Biosciences). Antibodies used to detect cell surface antigens were: anti-SSEA4, anti-TRA-1-60, anti-TRA-1-81 (Chemicon) and anti-SSEA-3
(R&D Systems). Appropriate PE-conjugated secondary antibodies (Southern Biotech or Jackson Immuno Research) were used to detect unlabeled primary antibodies and secondary-only antibodies were used as negative controls. For cell cycle studies, cells were harvested, washed with phosphate-buffered saline (PBS), and fixed in 70% ethanol. Fixed cells were stained with propidium iodide (PI) and DNA content was measured by the intensity of the fluorescence produced by PI. Data were analyzed with the Modfit 3.0 software (Verity House Software).
Telomerase Activity Assay Telomerase activity in H1 cells cultured in CM or butyrate was analyzed with a TRAPEZE telomerase detection kit (Chemicon), according to the manufacturer’s instructions. About 0.5 µg cell lysate was used per reaction. The samples were separated by 10% non-denaturing acrylamide gel electrophoresis in 0.5xTBE buffer. The gel was stained with SYBR Green (1:10,000, Invitrogen).
Quantitative RT-PCR Total RNA was purified using the RNeasy Micro Kit (Qiagen, Valencia, CA) following the manufacturer-specified protocol. Reverse transcription of total RNA was performed using random hexamers with the SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen, Carlsbad, CA). qPCR was performed in triplicate using TaqMan Universal PCR Master Mix, No AmpErase UNG (Applied Biosystems, Foster City, CA) or SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA) in 25 µl reactions in an Applied Biosystems 7900HT Fast Real-Time PCR System set to the following: 50°C for 2 min, 95°C for 10 min, and 40 cycles of 95°C for 15 sec and 60°C for 1 min. The following genespecific primer pairs were used in this study.
Primers Target Aif1
Ddx43
Dusp6
Grb10
Kcnj16
Kirrel2
Rasip1
Rcn3
Sfrp2
Slc4a1ap
Tie1
Tnni3
Txnip
Primer Sequence forward: 5’-ATGAGCCAAACCAGGGATTTAC-3’ reverse:5’-GGGATCGTCTAGGAATTGCTTGT-3’ forward:5’-TACGTGGGTCGTTGCTAGTC-3’ reverse:5’-GCCGACACTATATCCCTCAGG-3’ forward:5’-TTCCTCGGACATCGAGTCTGA-3’ reverse:5’-GCAAATTGGGGGTGACGTTC-3’ forward:5’-TTGCACCATCCGTACTACCAG-3’ reverse:5’-CCTGCCGGGTCTTGTTGAC-3’ forward:5’-TATCAATGCGGACGCAAAATACC-3’ reverse:5’-ACCACATAGCTTCCCCATTCT-3’ forward:5’-GGCTCCAGTTCACGAGGTC-3’ reverse:5’-CCAGGTGTGGGTTGAAGTCATA-3’ forward:5’-TCTGGTGAACGGAAGGAGG-3’ reverse:5’-CGAAGAAGACTTGACAGAGGC-3’ forward:5’-GCGCCCGGTGAAGAATTTC-3’ reverse:5’-CCGCATGTGAGGGAACTCC-3’ forward:5’-CACGGCATCGAATACCAGAAC-3’ reverse:5’-CTCGTCTAGGTCATCGAGGCA-3’ forward: 5’-TTCGGCAGGAAGCAGTATCT-3’ reverse: 5’-AACTTGGCTTGAGGCTTTCA-3’ forward:5’-CACGACCATGACGGCGAAT-3’ reverse:5’-CGGCAGCCTGATATGCCTG-3’ forward:5’-TTTGACCTTCGAGGCAAGTTT-3’ reverse:5’-TCCAGGGACTCCTTAGCCC-3’ forward:5’-GGTCTTTAACGACCCTGAAAAGG-3’ reverse:5’-CGAAGTCTGTTTGCACTGCT-3’
TaqMan Gene Expression Assays (Applied Biosystems) used in this study include the following: Dppa5 Hs00988349_g1, Nanog Hs02387400_g1, Nubp1 Hs01895061_u1,
Oct4
Hs01895061_u1,
Piwil2
Hs00216263_m1,
Sox2
Hs01053049_s1. Slc4a1ap and Nubp1 were used as endogenous controls for normalization of target mRNA expression. SDS Relative Quantification Software (Applied Biosystems) was used for data analysis. TaqMan Assays Target Dppa5
Nanog
Oct4
Piwil2
Sox2
Assay ID Hs00988349_g1
Hs02387400_g1
Hs01895061_u1
Hs00216263_m1
Hs01053049_s1
miRNA qPCR 10 ng of template total RNA was reverse transcribed using the TaqMan® MicroRNA Reverse Transcription Kit and miRNA-specific stem-loop primers following the manufacturer’s protocol (Applied Biosystems, Inc.).
1.33 µl RT
product was introduced into a 20 µl PCR reaction, using TaqMan® Universal PCR master mix, No AmpErase UNG. Real-time PCR was carried out on an Applied Biosystems 7900HT thermocycler (Applied Biosystems, Inc.) at 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min. Target miRNA expression was normalized between different samples based on the
values of RNU 24 TaqMan® snoRNA endogenous control assay. Data was analyzed with SDS Relative Quantification Software (Applied Biosystems, Inc).
Microarray Analysis Human ES cells: Agilent HGUG4112a microarrays containing 43,931 spots were hybridized with total RNA from H1 cells cultured in each of the following conditions in independent triplicate: A) Cultured on Matrigel on CM; B) cultured for 6 passages in butyrate; C) cultured for 4 passages in butyrate, followed by 3 passages in CM. Mouse ES cells: Agilent Whole Mouse Genome Oligo Microarrays containing 41,000+ spots were hybridized with total RNA from R1 cells cultured in each of the following conditions in independent quadruplicate: A) R1 cells grown without feeders with LIF supplementation; B) R1 cells grown without feeders or LIF with 0.2 mM butyrate supplementation for 4 passages. Defining differentially expressed genes All analysis was done using the BioConductor software. Annotation information was obtained from the Hgug4112a and Mgug4122a libraries. Control spots were removed from all analyses. Using MA plots we were unable to detect any effects related to the Cy5 and Cy3 dyes, thus we avoided using any traditional dye-bias normalization.
To account for differences between the arrays, Cy5 and Cy3
single channel values from the same study were jointly quantile normalized. For all two-class comparisons (e.g. butyrate vs conditioned medium), we used a twostep approach to identify differentially expressed genes. First, we calculated the probability that a gene is expressed at equal amounts in the two groups using a paired t-test. The corresponding p-values were adjusted for multiple hypothesis tests using the q-value methodology. Diagnostic plots available in the q-value library within the BioConductor software were used to identify unique cutoffs for each comparison resulting in lists of putatively differentially expressed genes. Next, we filtered these lists to only include genes with large effects.
To
accomplish this we used the 95% confidence interval around the expected
difference between the two groups. per-comparison basis.
Cutoffs for this criterion were defined on a
A panel of 14 differentially regulated genes identified
using this approach was confirmed using qRTPCR (Supplemental Figure 6).
Analysis of Differentially Expressed Genes Genes were matched across the mouse and human data sets using Homologene (release 58)7. We developed a hypothesis test to assess the relationship between gene expression changes in response to butyrate exposure with gene expression changes between mouse ES cells and mouse EpiSCs. Specifically, we calculated the difference in the average expression level between the mouse ES cells and the mouse EpiSCs.
Next we compared the means of these
differences for groups defined by the analysis of the butyrate data using a twosample t-test; for example, one group consists of genes shown to be up regulated in response to butyrate, the other consists of genes shown to be down regulated. See the supplementary web site for access to the R-code used to analyze the data, the raw data itself, and more details regarding this analysis (https://depts.washington.edu/iscrm/GS_data/gsdata.html).
Supervised Analysis of ES cell related genes Supervised analysis was performed starting with a list of 125 genes previously tested in reports by Tesar et al.3 and Yu et al.,8 Sequences were obtained for the microarray platforms used in this study and by Tesar et al.,3 (GEO accessions GPL1708 and GPL4134, respectively)9. Annotation was performed essentially as described in Barnes et al10. Briefly, the sequences were aligned to the genome (hg18 and mm8 assemblies, respectively) using BLAT11 with default settings except the step size was 7. The UCSC GoldenPath genome annotation database12 was used to identify transcripts overlapping the aligned regions. In a few cases, probe annotations were corrected by hand for probes that aligned to predicted transcripts for otherwise well-annotated genes. Genes were matched across the mouse and human data sets using Homologene (release 58)7,
allowing comparison of results of 87 genes that had human homologues and probes available in both data sets. The fold changes for these genes in the two data sets were plotted (Figure 4) and compared statistically using Spearman’s rank correlation.
Xist RNA Cells grown on Matrigel coated cover slips were were placed in CSK buffer on ice for 30 seconds, exposed to 0.5% Triton X in CSK buffer for three minutes at room temperature, fixed in 4% paraformaldehyde in 1X PBS for 10 minutes at room temperature, washed with 70% ethanol and stored in 70% ethanol at 4oC until assayed. Cells were dehydrated in ethanol washes and hybridized with an Xist probe, pXIST GIA (a gift from C. Brown, U. of British Colombia). The probe was labeled with SpectrumGreen direct label kit from Vysis (Downers Grove, IL). Hybridization and detection were carried out according to the manufacturer's instructions.
Bisulfite-sequencing analysis of promoter methylation Bisulfite-sequencing analysis of Dppa5 promoter methylation was performed as reported previously13. Briefly, using bisulfite treated DNA, the primers (forward: 5’-GGTTAATGTAGGTGGAAATTTTA-3’,
reverse:
5’-
ACCAAAACCCTAAATCCATAC-3’) were used to amplify a 140bp fragment of the Dppa5 promoter.
These primers amplify a fragment from –264 to –124
relative to the putative transcription start site, and were based on GenBank NM_001025290. PCR conditions were as follows: the reaction volume was 50 μl; initial denaturation was 5 min at 95°C, followed by 35 cycles of 30 s at 95°C, 45 s at 55°C and 45 s at 72°C, with a final extension at 72°C for 8 min. The PCR products were then cloned into the TA vector pCR2.1 (Invitrogen).
Plasmid
DNAs were extracted from the resulting clones with the use of a QIAprep Spin
Miniprep kit (Qiagen, Valencia, CA) and sequenced at the M. D. Anderson Core Sequencing Facility) References 1. Abbondanzo SJ, Gadi I & Stewart CL. Derivation of embryonic stem cell lines. Meth Enzymol 225,803-823 (1993). 2. Dani, C., Chambers, I., Johnstone, S., Robertson, M., Ebrahimi, B., Saito, M., Taga, T., Li, M., Burdon, T., Nichols, J. & Smith, A. Paracrine induction of stem cell renewal by LIF-deficient cells: a new ES cell regulatory pathway. Dev. Biol. 203:149-162 (1998). 3. Tesar, P.J., Chenoweth, J.G., Brook, F.A., Davies, T.J., Evans, E.P., Mack, D.L.,. Gardner, R.L. & McKay, R.D. New cell lines from mouse epiblast share defining features with human embryonic stem cells. Nature 448, 196-199 (2007). 4. Nagy, A., Gertsenstein, M., Vintersten, K. & Behringer, R. Manipulating the Mouse embryo: A Laboratory Manual (Cold Spring Harbor Press, New York, 2003). 5. Lamba, D.A., Karl, M.O., Ware, C.B. & Reh, T.A. Efficient generation of retinal progenitor cells from human embryonic stem cells. Proc. Natl. Acad. Sci. USA 103, 12769-12774 (2006). 6. Smyth, G. K. Linear models and empirical Bayes for assessing differential expression in microarray experiments. Stat. Appl. Genet. Mol. Biol. 3, Article 1 (2004). 7. Wheeler, D.L., Barrett, T., Benson, D.A., Bryant, S.H., Canese, K., Chetvernin, V., Church, D.M., Dicuccio, M., Edgar, R., Federhen, S., Feolo, M., Geer, L.Y., Helmberg, W., Kapustin, Y., Khovavko, O., Landsman, D., Lipman, D.J., Madden, T.L., Maglott, D.R., Miller, V., Ostell, J., Pruitt, K.D., Schuler, C.D., Shumway, M., Sequeria, E., Sherry, S.T., Sirotkin, K., Souvaorov, A., Starchenko, G., Tatusov, R.L., Tatusova, T.A., Wagner, L. & Yaschenko, E. Database resources of the National Center for Biotechnology Information. Nucleic Acids Res. 36, D13-D21 (2008). 8. Yu, J., Vodyanik, M.A., Smuga-Otto, K., Antosiewicz-Bourget, J., Frane, J.L., Tian, S., Nie, J., Jonsdottir, G.A., Ruotti, V., Stewart, R., Slukvin, I.I. & Thomson, J.A. Induced Pluripotent Stem Cell Lines Derived from Human Somatic Cells. Science 318, 1917-1920 (2007)
9. Barrett, T., Troup, D.B., Wilhite, S.E., Ledoux, P., Rudnev, D., Evangelista, C., Kim, I.F., Soboleva, A., Tomashevsky, M. & Edgar, R. NCBI GEO: mining tens of millions of expression profiles – database and tools update. 10. Barnes, M., Freudenberg, J., Thompson, S., Aronow, B. & Pavlidis, P. Experimental comparison and cross-validation of the Affymetrix and Illumina gene expression analysis platforms. Nucleic Acids Res. 33, 5914-5923 (2005). 11. Kent, W.J. BLAT—the BLASST-like alignment tool. Genome Res.12, 656664 (2002). 12. Karolchik, D., Kuhn R.M., Baertsch, R., Barber, G.P., Clawson, H., Diekhans, M., Giardine, B., Harte, R.A., Hinrichs, A.S., Hsu, F., Kober, K.M., Miller, W., Pedersen, J.S., Pohl, A., Raney, B.J., Rhead, B., Rosenbloom, K.R., Smith, K.E., Stanke, M., Thakkapallayil, S., Trumbower, H., Wang, T., Zweig, A.S., Haussler, D. & Kent, W.J. The UCSC Genome Browser Database: 2008 update. Nucleic Acids Res.36, D773-D779 (2008). 13. Shen, L., Kondo, Y., Guo, Y., Zhang, J., Zhang, L., Ahmend, S., Shu, J., Chen, X., Waterland, R.A. & Issa, J.P. Genome-wide profiling of DNA methylation reveals a class of normally methylated CpG island promoters. PLoS Genet. 3, 2023-2036 (2007).
