Derivation of UCSFB lines from biopsied blastomeres of cleavage-stage human. Spare embryos were obtained through the UCSF IVF Tissue Bank from donors

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1 Supplementary Materials and Methods Derivation of UCSFB lines from biopsied blastomeres of cleavage-stage human embryos Spare embryos were obtained through the UCSF IVF Tissue Bank from donors undergoing IVF who gave informed consent for hesc derivation. The cells were derived and maintained in 6% CO2/8% O2 in air. Embryos frozen on day 3 post-fertilization were thawed using an Embryo Thaw Medium Kit (Irvine Scientific) according to the manufacturer's instructions. Immediately thereafter they were transferred to Quinn s Blastocyst Medium (1 embryo/20 l drop; Cooper Surgical) and cultured in a humidified atmosphere at 37 o C for at least 3 h prior to blastomere biopsy, performed as described (Chung et al., 2008). Biopsied blastomeres were cultured in a drop of Quinn s Cleavage Medium for 24 h. Then they were transferred to γ-irradiated human foreskin fibroblast (HFF) feeders (p12) in drops of Quinn s Blastocyst Medium supplemented with 10 µg/ml human laminin (day 0; Sigma). Three days later cultures were assessed for blastomere attachment to feeders. Thereafter, the medium of cultures with adherent blastomeres was refreshed daily by replacing one-third of the volume with Quinn s Blastocyst Medium supplemented with 10 µg/ml laminin, 10 ng/ml leukemia inhibitory factor (LIF; Chemicon International), and 25 ng/ml basic fibroblast growth factor (bfgf; Invitrogen), all human proteins. On day 5, Quinn s Blastocyst Medium was replaced with standard hesc medium [80% KnockOut-DMEM, 20% Knockout Serum Replacement, 25 ng/ml bfgf, 1x 2-ME, 10 mm nonessential amino acids (NEAA), 20 mm L-glutamine; all from Invitrogen] containing 10% fetal calf serum (FCS, Invitrogen) and 10 ng/ml LIF. The medium was changed on a daily basis. By day 7, initial hesc

2 colonies were well established. Two days later, they were dissected and plated in the same drop. The procedure was repeated on day 14. After the second dissection, small hesc clumps were transferred to 4-well dishes (Nunc, Sigma) and plated on freshly seeded γ-irradiated HFF p14 feeders. The next day, medium without FCS and LIF was added. Thereafter, the cells were grown until p7 in standard serum-free hesc medium. Then they were transitioned to tissue culture wells coated with CellStart (Invitrogen) and cultured in mtesr (Stem Cell Technologies) without FCS.

3 Figure S1. Derivation and characterization of the UCSFB lines. (A) hescs were derived from single blastomeres of five embryos that were donated by one couple. Four

4 lines were derived from one embryo (UCSFB1-4) and three from another (UCSFB5-7); the remaining embryos yielded one line each (UCSFB8, 9 and 10). (B) In an undifferentiated state, they immunostained for markers of pluripotency: OCT4, NANOG, Tra-1-60 and SSEA-4. Nuclei were visualized by DAPI staining. (C) Embryoid bodies stained for markers of ectodermal ( -III-tubulin), mesodermal (smooth muscle actin, SMA) and endodermal ( -fetoprotein, FP) derivatives. Data from UCSFB6 are shown. (D) The UCSFB lines formed teratomas in SCID-beige mice with cell types representative of the three germ layers. Data for UCSFB6 and -B8 are shown. NE, neuroectoderm; IE, intestinal epithelium; CAR, cartilage; MC, melanocytes; CH, chondrocytes. Scale bars: A, B, D, 100 µm; C, 50 µm.

5 Figure S2. Transcriptomes of the UCSFB hescs vs. the 15 conventional lines that were separated by one branch of the dendrogram (Figure 2B). (A) Heatmap showing gene expression data for the 953 differentially expressed transcripts (p<0.01) between the USCFB and most similar conventional hescs. (B) Functional enrichments for the differentially expressed genes as determined by GREAT. Bars showing the fold enrichments for each gene set are annotated with the log10 p-values. (C) Heatmap showing data for the 50 genes with the highest expression in UCSFB vs. the most similar conventional hescs. (D) Heatmap showing data for the 50 genes with the lowest expression in UCSFB vs. the most similar conventional hescs.

6 Figure S3. Comparison of the UCSFB5-7 methylation patterns to the methylation patterns of the most similar conventional lines (Figure 3B). (A) The conventional hesc lines with the most similar methylation patterns were derived in physiologic hypoxia as were the UCSFB lines. Nevertheless, 1164 cytosines were differentially methylated between the two groups of cells. (B) Heatmap showing a subset of the

7 differentially methylated cytosines that were robustly hypomethylated in UCSFB lines in regions near genes that control many key aspects of early developmental processes. (C) Functional enrichments suggested that these regions were involved in trophoblast/placental development and BMP signaling. (D) Heatmap showing DNA methylation data for imprinted genes. Blue labels, paternally imprinted/maternally expressed; pink labels, maternally imprinted/paternally expressed; green, isoformdependent imprinting. (E) mrna expression data for imprinted genes that were differentially expressed between UCSFB and conventional hescs. (F) Immunolocalization of PEG3 showed higher expression in UCSFB7 as compared to WA09. P29, passage 29. Scale bars = 100 µm.

8 Figure S4. Functional enrichments for gene expression modules that were differentially expressed among the UCSFB hescs. (A-D) Bar graphs depicting the fold enrichment for each gene module shown in Figure 5 annotated with log10 p-values. The size of each term in the word clouds reflected the frequency of the term among all the enrichments.

9 Figure S5. qrt-pcr validation of the microarray results. Concordance between microarray and qrt-pcr data showed differences in mrna levels among the lines (UCSFB1-7). Data points (top) show relative gene expression levels as determined by the microarray analyses. Bars (bottom) show mrna levels as determined by qrt-pcr.

10 Figure S6. Mapping of immunostained cells within the embryos. The centroid position of each blastomere nucleus was mapped according to its X,Y, Z coordinates. Each cell was depicted as a circle with the shade denoting signal intensity and the size proportional to the volume.

11 Figure S7. EBs formed from UCSFB5-7 contained trophoblast. (A) Fifteen EBs were plated per well. Each EB was comprised of ~700 cells. At days 3 and 5, conditioned medium (CM) from the three UCSFB lines contained higher levels of hcg than CM of WA09 cells. The error bars were calculated based on 5 wells. The entire experiment was repeated 3 times. (B) Markers of hesc pluripotency (Oct 3/4) or trophoblasts (Cdx2, CK7 or hcgb) were quantified at the mrna level by using a qrt-pcr approach (n = 3, error bars = SEM). (C-F) Immunoanalyses of UCSFB6 EBs at day 6 showed that the cultures contained cells with the antigenic profile of human TBs (expression of GCM1, HMGA2, GATA3 and GDF15). Scale bars = 50 µm.

12 Table S1. Table S1. Antibodies, dilutions and sources. Antigen/Antibody Host Dilution Source OCT3/4 rabbit 1:100 Santa Cruz Biotechnologies NANOG goat 1:50 R&D Systems TRA-1-60 mouse 1:100 Millipore SSEA-4 mouse 1:100 Millipore III TUBULIN mouse 1:500 Sigma SMOOTH MUSCLE ACTIN mouse 1:500 Sigma -FETOPROTEIN mouse 1:50 DAKO SOX1 rabbit 1:100 Millipore NESTIN rat 1:500 BD Biosciences SOX17 goat 1:100 R&D Systems PDX1 goat 1:100 R&D Systems INSULIN guniea pig 1:100 Linco T goat 1:100 Santa Cruz Biotechnologies CDX2 mouse 1:100 BioGenex CK7 rat 1:75 Produced in lab ELF5 mouse 1:100 Santa Cruz Biotechnologies GATA3 goat 1:30 R&D Systems GCM1 rabbit 1:100 Abcam GDF15 rabbit 1:100 Abcam HCG rabbit 1:100 Abcam HMGA2 goat 1:100 R&D Systems HLA-G mouse 1:100 Produced in lab HPL mouse 1:100 Serotec INTEGRIN 6 rat 1:100 Chemicon VLA (INTEGRIN 1) mouse 1:100 Thermo scientific EOMES rabbit 1:100 Orbigen TYROSINE HYDROXYLASE rabbit 1:500 Pel-Freeze

13 Table S2. Primer sequences. Target gene ISL1 SLN MLC2a HCN4 MLC2v ctnt ACTA1 CTSK GDF15 ACTC1 EOMES PDE4B ACTG2 FOXA1 T KRT7 (CK7) CDX2 POU5F1 CGB Taqman Assay Hs _m1;Hs _m1 Hs _m1;Hs _s1 Hs _m1 Hs _m1;Hs _m1 Hs _m1;Hs _m1 Hs _m1 Hs _m1 Hs _m1 Hs _m1 Hs _m1 Hs _m1 Hs _m1 Hs _m1 Hs _m1 Hs _m1 Hs _m1 Hs _m1 Hs _gh Hs _gh

14 Table S3. Differential mrna expression between UCSFB and conventional hescs mapped to the functional enrichments. (Provided as Excel file). (A) p-values and fold changes for 1651 probes/1544 unique transcripts that were differentially expressed. (B) Functional enrichments for the differentially expressed genes in Table S3A as determined by GREAT. (C) p-values and fold changes for 1004 probes/953 unique transcripts that were differentially expressed between UCSFB and the 15 conventional hesc lines with the most similar transcriptomes. (D) Functional enrichments as determined by GREAT for the differentially expressed genes in Table S3C. Click here to Download Table S3

15 Table S4. Differential methylation between UCSFB and conventional hescs and functional enrichments of these regions. (Provided as Excel file). (A) p-values, Beta value differences, and associated transcripts for 3506 genomic cytosines that were differentially methylated (p<0.01, Beta-value difference 0.1) between UCSFB5-7 and a set of 46 samples from 21 conventional hesc lines. (B) Functional enrichments for the differentially methylated cytosines as determined by GREAT. (C) p-values, Beta value differences, and associated transcripts for 1164/3506 genomic cytosines that were differentially methylated (p<0.01, Beta-value difference 0.1) between UCSFB5-7 and a set of 5 samples from 5 conventional hesc lines, derived in physiologic hypoxia, that were most similar to UCSFB5-7. (D) Functional enrichments for the differentially methylated cytosines from Table 4C, as determined by GREAT. Click here to Download Table S4

16 Table S5. Differential mrna expression among UCSFB lines. (Provided as Excel file). (A) p-values and fold changes for 4088 probes/3620 differentially expressed transcripts (p<0.01, fold change 1.5). (B) Results from Weighted Gene Correlation Network Analysis. For genes with multiple probes, only the most significant probe from the ANOVA test was used as input. (Columns D, G) Eigengenes were calculated for each module and correlated with each input probe. (Columns H, K) The significance of each probe in each module. (Column L) Hierarchies within each module. (C) Functional enrichments for gene modules that were differentially expressed among the UCSFB lines as determined by GREAT. Click here to Download Table S5