Single-cell RNA-Seq profiling of human pre-implantation embryos and embryonic stem cells

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1 Single-cell RNA-Seq profiling of human pre-implantation embryos and embryonic stem cells Liying Yan,2,5, Mingyu Yang,5, Hongshan Guo, Lu Yang, Jun Wu, Rong Li,2, Ping Liu, Ying Lian, Xiaoying Zheng, Jie Yan, Jin Huang, Ming Li, Xinglong Wu, Lu Wen, Kaiqin Lao 4, Ruiqiang Li,3*, Jie Qiao,2*, Fuchou Tang * Biodynamic Optical Imaging Center & Center for Reproductive Medicine, College of Life Sciences, Third Hospital, Peking University, Beijing 0087, P. R. China 2 Key Laboratory of Assisted Reproduction, Ministry of Education, Beijing 009, P. R. China 3 Genetic Systems, Applied Biosystems, Life Technologies, Foster City, California, USA 4 Peking-Tsinghua Center for Life Sciences, College of Life Sciences, Peking University, Beijing 0087, P. R. China 5 These authors contributed equally to this work. * Correspondence should be addressed to: R. L. (lirq@pku.edu.cn), J. Q. (jie.qiao@263.net), or F. T. (tangfuchou@pku.edu.cn) Nature Structural & Molecular Biology: doi:0.038/nsmb.2660

2 zygote oocyte zygote 2-cell 4-cell 8-cell Morluae c cell 4-cell 8-cell cell #2-C 4-cell #2-C3 4-cell #2-C2 4-cell #2-C4 4-cell #-C3 4-cell #-C4 4-cell #-C 4-cell #-C2 4-cell #3-C2 4-cell #3-C4 4-cell #3-C3 4-cell #3-C 8-cell #-C2 8-cell #-C3 8-cell #-C4 8-cell #-C 8-cell #3-C4 8-cell #3-C7 8-cell #3-C6 8-cell #3-C8 8-cell #3-C2 8-cell #3-C 8-cell #3-C3 8-cell #3-C5 8-cell #2-C3 8-cell #2-C8 8-cell #2-C7 8-cell #2-C 8-cell #2-C2 8-cell #2-C5 8-cell #2-C4 8-cell #2-C6 Supplemental Materials a b Morluae Blastocyst d Nature Structural & Molecular Biology: doi:0.038/nsmb cell 8-cell

3 Supplementary Fig.. Expression dynamics of known RefSeq genes during human pre-implantation development. (a) Number of RefSeq genes showing up- or downregulation during pre-implantation development (fold change, FC>2 or <0.5, p- value <0.0). (b) Heatmap of the genes showing differential expression between 4- and 8-cell stage embryos. (FC>4 or <0.25, p<0.0) (c) Correlation plots for the expression of RefSeq genes for individual blastomeres from three 2-cell-stage embryos. The correlation coefficient between blastomeres within the same 2-cell embryo is higher than 0.99, indicating that the technical variation is reasonably low. (d) Correlation plots for the expression of RefSeq genes of single mature oocytes and single blastomeres of pre-implantation embryos. Nature Structural & Molecular Biology: doi:0.038/nsmb.2660

4 a b c e d oocyte zygote 2-cell 4-cell 8-cell Morluae zygote 2- cell 4-cell 8-cell Morluae Blastocyst Nature Structural & Molecular Biology: doi:0.038/nsmb.2660

5 Supplementary Fig. 2. Global expression profile of known and novel lncrnas during human pre-implantation development. (a) Correlation plots of the expression of known lncrna genes for individual blastomeres from three 2-cell-stage embryos. (b) Correlation plots of the expression of known lncrna genes for mature oocytes and blastomeres of pre-implantation embryos. (c) Correlation plots of the expression of novel lncrna transcripts for individual blastomeres from three 2-cell-tage embryos. (d) Number of novel lncrna transcripts showing up- or downregulation during pre-implantation development (Fold Change, FC>2 or <0.5, p<0.0). (e) Correlation plots of the expression of novel lncrna transcripts for mature oocytes and blastomeres from pre-implantation embryos. Nature Structural & Molecular Biology: doi:0.038/nsmb.2660

6 XK F2 a d Color Key RPS6 Ro POU5F 00 EPI #5 EPI #2 EPI #4 EPI #3 EPI # TE #7 TE #4 TE # TE #3 TE #8 TE #5 TE #6 TE #3 TE #2 TE #6 TE #5 TE #8 TE #2 TE #4 TE # TE #7 TE #0 TE #9 0 SOX2 EPI TE NANOG 000 b KLF4 Color Key 0 CDX2 0 2 Ro CLDN0 000 PE #2 PE #3 PE #6 PE #5 PE #7 PE # PE #4 EPI #2 EPI #4 EPI #3 EPI #5 EPI # 00 0 GDF3 PE EPI 00 0 FOXD3 c 00 0 ESRRB Color Key Ro PRDM CLDN3 PE #4 PE #3 PE #2 PE #6 PE #7 PE #5 PE # TE #3 TE #4 TE #2 TE #5 TE #6 TE #7 TE #4 TE #3 TE #8 TE #6 TE #9 TE #7 TE #8 TE #2 TE # TE #5 TE #0 TE # 00 PE TE 0 FGFR4 TE PE EPI Nature Structural & Molecular Biology: doi:0.038/nsmb.2660

7 Supplementary Fig. 3. Heatmap and expression pattern of known RefSeq genes during pre-implantation development. (a) Heatmap of the genes showing differential expression between the epiblast and trophectoderm lineages of cells in late blastocysts. (b) Heatmap of the genes showing differential expression between the epiblast and primitive endoderm lineages of cells in late blastocysts. (c) Heatmap of the genes showing differential expression between the primitive endoderm and trophectoderm lineages of cells in late blastocysts. (d) Expression pattern of lineage specific marker genes in individual cells of blastocysts. EPI: epiblast; PE: primitive endoderm; TE: trophectoderm. Nature Structural & Molecular Biology: doi:0.038/nsmb.2660

8 a Expression Level (r.p.k.m) Expression Level (r.p.k.m) Expression Level (r.p.k.m) Expression Level (r.p.k.m) Expression Level Expression Level Expression Level Expression Level (r.p.k.m) (r.p.k.m) (r.p.k.m) (r.p.k.m) Oocyte Zygote 2-cell 4-cell 8-cell Morluae TE PE EPI ESC(P0) ESC(P0) RPS6 STELLA SALL2 KLF5 LIN28A LIN28B FGFR4 CLDN0 b Expression Level Expression Level Expression Level Expression Level Expression Level Expression Level Expression Level Expression Level (r.p.k.m) (r.p.k.m) (r.p.k.m) (r.p.k.m) (r.p.k.m) (r.p.k.m) (r.p.k.m) (r.p.k.m) Oocyte Zygote 2-cell 4-cell 8-cell Morluae TE PE EPI ESC(P0) ESC(P0) RPS6 TET TET2 TET3 DNMT3A DNMT3B DNMT3L HDAC7 Nature Structural & Molecular Biology: doi:0.038/nsmb.2660

9 Supplementary Fig. 4. Expression of marker genes in individual cells during human pre-implantation development and derivation of hescs. (a) Expression of marker genes (including transcription factors) in individual cells during human preimplantation development and derivation of hescs. (b) Expression of epigenetic regulator genes in individual cells during human pre-implantation development and derivation of hescs. The housekeeping gene RPS6 was used as a control. Each vertical bar represents the expression (RPKM) of the gene in an individual cell. Each column represents the expression of different genes within the same cell. TE: trophectoderm; PE: primitive endoderm; EPI: epiblast; P0: hescs of passage #0; P0: hescs of passage #0. Nature Structural & Molecular Biology: doi:0.038/nsmb.2660

10 0 a TE PE Relative expression EPI ESC P0 SOX2 KIT TDGF NODAL LEFTY LEFTY2 OCT4 LIN28A FOXD3 IFITM ZFP42 REST TFCP2L GBX2 GDF3 NANOG b EPI # EPI #3 EPI #4 EPI #2 EPI #5 hesc P0 #5 hesc P0 #7 hesc P0 #2 hesc P0 #6 hesc P0 #7 hesc P0 #3 hesc P0 #8 hesc P0 #4 hesc P0 #4 hesc P0 # hesc P0 #8 hesc P0 #5 hesc P0 #2 hesc P0 # hesc P0 #5 hesc P0 #3 hesc P0 #6 hesc P0 #0 hesc P0 #8 hesc P0 #6 hesc P0 #7 hesc P0 #4 hesc P0 #25 hesc P0 #22 hesc P0 #23 hesc P0 #3 hesc P0 #9 hesc P0 #20 hesc P0 #2 hesc P0 # hesc P0 #2 hesc P0 #9 hesc P0 #26 hesc P0 #24 c Nature Structural & Molecular Biology: doi:0.038/nsmb.2660

11 Supplementary Fig. 5. Gene expression dynamics of blastocysts and hescs. (a) Expression pattern of pluripotency-related genes in human embryonic stem cells (hescs) and human late blastocysts. The RPKM values of the genes are labeled at the top of the highest bar for every gene. (b) Unsupervised hierarchical clustering analysis for single cells of EPI and hescs. (c) Expression pattern of pluripotency-related genes in human embryonic stem cells (hescs) and mouse embryonic stem cells (mescs). The RPKM values of the genes are labeled at the top of the highest bar for every gene. Nature Structural & Molecular Biology: doi:0.038/nsmb.2660

12 a b T T over r over r 5 end T r 5 end T r Nature Structural & Molecular Biology: doi:0.038/nsmb.2660

13 Supplementary Fig. 6. Compare between Smart-Seq and our single cell RNA-Seq methods. (a) Number of genes detected in individual hescs. (b) Mean read coverage over transcripts for single cell RNA-Seq data of our technique (n=8) or Smart-Seq technique (n=8) for human embryonic stem cells. For comparison, we included data from standard RNA-Seq (n=2) on bulk amount of human embryonic stem cells (nonamplified). Error bar: s.d. Nature Structural & Molecular Biology: doi:0.038/nsmb.2660

14 Nature Structural & Molecular Biology: doi:0.038/nsmb.2660

15 Supplementary Fig. 7 Characterization of the hesc cell line and the lineage differentiation of the blastocysts. (a) Unsupervised hierarchical clustering analysis for single cells at morula and blastocyst stages. (b) Principal component analysis (PCA) of the transcriptomes of single cells at morula and blastocyst stages. (c) The karyotype (46, XY) of the hesc line used in the present study and Western blot of the pluripotency markers OCT4, NANOG, and SOX2 in the analyzed hescs. HeLa cells were used as a control. (d) Immunostaining for TRA--60 (green) and corresponding nuclear staining (PI staining; red) in hescs. (e) Teratomas formed by the hesc line analyzed in SCID mice. Ectoderm tissues (chromatophores, neuroepithelial) in teratoma sections. Mesoderm tissues (bone) in teratoma sections. Endoderm tissues (pseudostratified columnar epithelium) in teratoma sections. Scale bar: 20 µm. Nature Structural & Molecular Biology: doi:0.038/nsmb.2660

16 Supplementary Note As one example, the seven transcript isoforms of RING-H2 finger protein ANAPC, produce two different proteins with 84 and 96 amino acids in the same hesc, respectively. The expression level of the mrnas for these two proteins is RPKM 8 and 6, respectively. It is known that ANAPC is a core component of anaphasepromoting complex and together with E2 enzyme UBC4, it is sufficient to ubiquitinate a wide variety of substrates during cell cycle regulation 2. We found that the DNA methyltransferases DNMT3A and DNMT3B and the histone deacetylase HDAC7 are strongly upregulated from epiblast cells to passage #0 hescs (Supplementary Fig. 4b. Conversely, DNMT3L is highly expressed in epiblast cells but is downregulated by over a hundred fold in hescs, which implies that DNA methylation-related factors is likely involved in the derivation of hesc cells from epiblast cells (Supplementary Fig. 4b). In fact, of the 3 known epigenetic regulators and chromatin remodeling factors, 2 show clear changes from epiblast cells to passage #0 hescs (Supplementary Table 8). Therefore, our results show that during the derivation process of hescs, the most dramatic changes in the expression of epigenetic regulators occur at the earliest time point during the first ten days in culture, which can probably be attributed to the accommodation of the cells to the culture conditions, such as the addition of serum and cytokines. Nature Structural & Molecular Biology: doi:0.038/nsmb.2660

17 References. Gifford, Casey A. et al. Transcriptional and Epigenetic Dynamics during Specification of Human Embryonic Stem Cells. Cell 53, (203). 2. Gmachl, M., Gieffers, C., Podtelejnikov, A.V., Mann, M. & Peters, J.-M. The RING-H2 finger protein APC and the E2 enzyme UBC4 are sufficient to ubiquitinate substrates of the anaphase-promoting complex. Proceedings of the National Academy of Sciences 97, (2000). Nature Structural & Molecular Biology: doi:0.038/nsmb.2660