Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

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

Abstract

Measuring gene expression in individual cells is crucial for understanding the gene regulatory network controlling human embryonic development. Here we apply single-cell RNA sequencing (RNA-Seq) analysis to 124 individual cells from human preimplantation embryos and human embryonic stem cells (hESCs) at different passages. The number of maternally expressed genes detected in our data set is 22,687, including 8,701 long noncoding RNAs (lncRNAs), which represents a significant increase from 9,735 maternal genes detected previously by cDNA microarray. We discovered 2,733 novel lncRNAs, many of which are expressed in specific developmental stages. To address the long-standing question whether gene expression signatures of human epiblast (EPI) and in vitro hESCs are the same, we found that EPI cells and primary hESC outgrowth have dramatically different transcriptomes, with 1,498 genes showing differential expression between them. This work provides a comprehensive framework of the transcriptome landscapes of human early embryos and hESCs.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Morphology and marker gene expression of human early embryos and hESCs.
Figure 2: Global expression patterns of known RefSeq genes during the seven consecutive stages of human preimplantation development.
Figure 3: Dynamic patterns of alternative splicing during the seven consecutive stages of human preimplantation development and derivation of hESCs.
Figure 4: Expression patterns of known long noncoding RNA (lncRNA) genes during human preimplantation development and derivation of hESCs.
Figure 5: Expression patterns of novel lncRNAs during human preimplantation development.
Figure 6: The EPI, PE and TE lineage segregation in the blastocysts.

Accession codes

Primary accessions

Gene Expression Omnibus

References

  1. Martinez Arias, A. & Brickman, J.M. Gene expression heterogeneities in embryonic stem cell populations: origin and function. Curr. Opin. Cell Biol. 23, 650–656 (2011).

    Article  CAS  PubMed  Google Scholar 

  2. Hardy, K. et al. Future developments in assisted reproduction in humans. Reproduction 123, 171–183 (2002).

    Article  CAS  PubMed  Google Scholar 

  3. Niakan, K.K., Han, J., Pedersen, R.A., Simon, C. & Pera, R.A.R. Human pre-implantation embryo development. Development 139, 829–841 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Reijo Pera, R.A. Non-invasive imaging of human embryos to predict developmental competence. Placenta 32 (Suppl. 3), S264–S267 (2011).

    Article  PubMed  Google Scholar 

  5. Aghajanova, L. et al. Comparative transcriptome analysis of human trophectoderm and embryonic stem cell-derived trophoblasts reveal key participants in early implantation. Biol. Reprod. 86, 1–21 (2012).

    Article  CAS  PubMed  Google Scholar 

  6. Assou, S. et al. Dynamic changes in gene expression during human early embryo development: from fundamental aspects to clinical applications. Hum. Reprod. Update 17, 272–290 (2011).

    Article  CAS  PubMed  Google Scholar 

  7. Assou, S. et al. Transcriptome analysis during human trophectoderm specification suggests new roles of metabolic and epigenetic genes. PLoS ONE 7, e39306 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Assou, S. et al. A gene expression signature shared by human mature oocytes and embryonic stem cells. BMC Genomics 10, 10 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Bai, Q. et al. Dissecting the first transcriptional divergence during human embryonic development. Stem Cell Rev. Rep. 8, 150–162 (2012).

    Article  CAS  PubMed  Google Scholar 

  10. Dobson, A.T. et al. The unique transcriptome through day 3 of human preimplantation development. Hum. Mol. Genet. 13, 1461–1470 (2004).

    Article  CAS  PubMed  Google Scholar 

  11. Galán, A. et al. Functional genomics of 5- to 8-cell stage human embryos by blastomere single-cell cDNA analysis. PLoS ONE 5, e13615 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Haouzi, D. et al. Transcriptome analysis reveals dialogues between human trophectoderm and endometrial cells during the implantation period. Hum. Reprod. 26, 1440–1449 (2011).

    Article  CAS  PubMed  Google Scholar 

  13. Vassena, R. et al. Waves of early transcriptional activation and pluripotency program initiation during human preimplantation development. Development 138, 3699–3709 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Wong, C.C. et al. Non-invasive imaging of human embryos before embryonic genome activation predicts development to the blastocyst stage. Nat. Biotechnol. 28, 1115–1121 (2010).

    Article  CAS  PubMed  Google Scholar 

  15. Xie, D. et al. Rewirable gene regulatory networks in the preimplantation embryonic development of three mammalian species. Genome Res. 20, 804–815 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Kiessling, A.A. et al. Evidence that human blastomere cleavage is under unique cell cycle control. J. Assist. Reprod. Genet. 26, 187–195 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  17. Kiessling, A.A. et al. Genome-wide microarray evidence that 8-cell human blastomeres over-express cell cycle drivers and under-express checkpoints. J. Assist. Reprod. Genet. 27, 265–276 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  18. Guttman, M. & Rinn, J.L. Modular regulatory principles of large non-coding RNAs. Nature 482, 339–346 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Thomson, J.A. et al. Embryonic stem cell lines derived from human blastocysts. Science 282, 1145–1147 (1998).

    Article  CAS  PubMed  Google Scholar 

  20. Reijo Pera, R.A. et al. Gene expression profiles of human inner cell mass cells and embryonic stem cells. Differentiation 78, 18–23 (2009).

    Article  CAS  PubMed  Google Scholar 

  21. O'Leary, T. et al. Tracking the progression of the human inner cell mass during embryonic stem cell derivation. Nat. Biotechnol. 30, 278–282 (2012).

    Article  CAS  PubMed  Google Scholar 

  22. Tang, F. et al. mRNA-Seq whole-transcriptome analysis of a single cell. Nat. Methods 6, 377–382 (2009).

    Article  CAS  PubMed  Google Scholar 

  23. Tang, F., Lao, K. & Surani, M.A. Development and applications of single-cell transcriptome analysis. Nat. Methods 8, S6–S11 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Ramsköld, D. et al. Full-length mRNA-Seq from single-cell levels of RNA and individual circulating tumor cells. Nat. Biotechnol. 30, 777–782 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Hashimshony, T., Wagner, F., Sher, N. & Yanai, I. CEL-Seq: single-cell RNA-Seq by multiplexed linear amplification. Cell Reports 2, 666–673 (2012).

    Article  CAS  PubMed  Google Scholar 

  26. Tang, F. et al. Tracing the derivation of embryonic stem cells from the inner cell mass by single-cell RNA-Seq analysis. Cell Stem Cell 6, 468–478 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Braude, P., Bolton, V. & Moore, S. Human gene expression first occurs between the four- and eight-cell stages of preimplantation development. Nature 332, 459–461 (1988).

    Article  CAS  PubMed  Google Scholar 

  28. Cockburn, K. & Rossant, J. Making the blastocyst: lessons from the mouse. J. Clin. Invest. 120, 995–1003 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Rossant, J. & Tam, P.P.L. Blastocyst lineage formation, early embryonic asymmetries and axis patterning in the mouse. Development 136, 701–713 (2009).

    Article  CAS  PubMed  Google Scholar 

  30. Gabut, M. et al. An alternative splicing switch regulates embryonic stem cell pluripotency and reprogramming. Cell 147, 132–146 (2011).

    Article  CAS  PubMed  Google Scholar 

  31. Khalil, A.M. et al. Many human large intergenic noncoding RNAs associate with chromatin-modifying complexes and affect gene expression. Proc. Natl. Acad. Sci. USA 106, 11667–11672 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  32. Ørom, U.A. et al. Long noncoding RNAs with enhancer-like function in human cells. Cell 143, 46–58 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Wang, K.C. & Chang, H.Y. Molecular mechanisms of long noncoding RNAs. Mol. Cell 43, 904–914 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Guttman, M. et al. lincRNAs act in the circuitry controlling pluripotency and differentiation. Nature 477, 295–300 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Derrien, T., Guigó, R. & Johnson, R. The long non-coding RNAs (lncRNAs): a new (p)layer in the “dark matter”. Frontiers Genet. 2, 107 (2012).

    Article  CAS  Google Scholar 

  36. Trapnell, C. et al. Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat. Biotechnol. 28, 511–515 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Kong, L. et al. CPC: assess the protein-coding potential of transcripts using sequence features and support vector machine. Nucleic Acids Res. 35, W345-9 (2007).

    PubMed  Google Scholar 

  38. Garber, M. et al. Identifying novel constrained elements by exploiting biased substitution patterns. Bioinformatics 25, i54–i62 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Guttman, M. et al. Ab initio reconstruction of transcriptomes of pluripotent and lineage committed cells reveals gene structures of thousands of lincRNAs. Nat. Biotechnol. 28, 503–510 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Wells, D. & Patrizio, P. Gene expression profiling of human oocytes at different maturational stages and after in vitro maturation. Am. J. Obstet. Gynecol. 198, 455.e1–455.e11 (2008).

    Article  CAS  Google Scholar 

  41. Roode, M. et al. Human hypoblast formation is not dependent on FGF signalling. Dev. Biol. 361, 358–363 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Brons, I.G.M. et al. Derivation of pluripotent epiblast stem cells from mammalian embryos. Nature 448, 191–195 (2007).

    Article  CAS  PubMed  Google Scholar 

  43. Tesar, P.J. et al. New cell lines from mouse epiblast share defining features with human embryonic stem cells. Nature 448, 196–199 (2007).

    Article  CAS  PubMed  Google Scholar 

  44. Boyer, L.A. et al. Core transcriptional regulatory circuitry in human embryonic stem cells. Cell 122, 947–956 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Suzuki, R. & Shimodaira, H. Pvclust: an R package for assessing the uncertainty in hierarchical clustering. Bioinformatics 22, 1540–1542 (2006).

    Article  CAS  PubMed  Google Scholar 

  46. Huang, J. et al. Characteristics of embryo development in Robertsonian translocations' preimplantation genetic diagnosis cycles. Prenat. Diagn. 29, 1167–1170 (2009).

    Article  PubMed  Google Scholar 

  47. Tang, F. et al. RNA-seq analysis to capture the transcriptome landscape of a single cell. Nat. Protoc. 5, 516–535 (2010).

    Article  CAS  PubMed  Google Scholar 

  48. Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25, 1754–1760 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Bu, D. et al. NONCODE v3.0: integrative annotation of long noncoding RNAs. Nucleic Acids Res. D210-5 (2012).

  50. Grabherr, M.G. et al. Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat. Biotechnol. 29, 644–652 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Haas, B.J. et al. Improving the Arabidopsis genome annotation using maximal transcript alignment assemblies. Nucleic Acids Res. 31, 5654–5666 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Kent, W.J. BLAT—The BLAST-Like Alignment Tool. Genome Res. 12, 656–664 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Baczkowski, T., Kurzawa, R. & Glabowski, W. Methods of embryo scoring in in vitro fertilization. Reprod. Biol. 4, 5–22 (2004).

    PubMed  Google Scholar 

  54. Madan, P., Rose, K. & Watson, A.J. Na/K-ATPase β1 subunit expression is required for blastocyst formation and normal assembly of trophectoderm tight junction-associated proteins. J. Biol. Chem. 282, 12127–12134 (2007).

    Article  CAS  PubMed  Google Scholar 

  55. Guo, G. et al. Resolution of cell fate decisions revealed by single-cell gene expression analysis from zygote to blastocyst. Dev. Cell 18, 675–685 (2010).

    Article  CAS  PubMed  Google Scholar 

  56. Bao, S. et al. Epigenetic reversion of post-implantation epiblast to pluripotent embryonic stem cells. Nature 461, 1292–1295 (2009).

    Article  CAS  PubMed  Google Scholar 

  57. Audic, S.p. & Claverie, J.-M. The significance of digital gene expression profiles. Genome Res. 7, 986–995 (1997).

    Article  CAS  PubMed  Google Scholar 

  58. Mortazavi, A., Williams, B.A., McCue, K., Schaeffer, L. & Wold, B. Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat. Methods 5, 621–628 (2008).

    Article  CAS  PubMed  Google Scholar 

  59. Eisen, M.B., Spellman, P.T., Brown, P.O. & Botstein, D. Cluster analysis and display of genome-wide expression patterns. Proc. Natl. Acad. Sci. USA 95, 14863–14868 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Saldanha, A.J. Java Treeview—extensible visualization of microarray data. Bioinformatics 20, 3246–3248 (2004).

    Article  CAS  PubMed  Google Scholar 

  61. Benjamini, Y. & Hochberg, Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J. Roy. Stat. Soc. B 57, 289–300 (1995).

    Google Scholar 

  62. Huang, D.W., Sherman, B.T. & Lempicki, R.A. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat. Protoc. 4, 44–57 (2008).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

F.T. was supported by grants from the National Basic Research Program of China (2012CB966704 and 2011CB966303) and National Natural Science Foundation of China (31271543). J.Q. was supported by grants from the National Basic Research Program of China (2011CB944500) and the National Natural Science Funds for Distinguished Young Scholar (30825038). L.Y. was supported by a grant from the National Science Foundation of China (81000275). We would like to thank S. Gao, Y. Zhang, P. Xu, S. Lin, X. Ren, Q. Zhang, Y. Jiang, M. Fan, J. Li, X. Zhuang, W. Song and Y. Chen for their great help.

Author information

Authors and Affiliations

Authors

Contributions

J.Q. and F.T. conceived and designed the project, and R.L. was in charge of the bioinformatic analysis. L. Yan, H.G., L. Yang, X.W. and L.W. conducted the majority of the experiments. M.Y. and J.W. did all of the data analysis. R.L., P.L., Y.L., X.Z., J.Y., J.H. and M.L. contributed to oocyte collection, sperm treatment and embryo culture in vitro. L. Yan, M.Y., K.L., R.L., J.Q. and F.T. prepared the manuscript. All authors contributed to the revision of the manuscript.

Corresponding authors

Correspondence to Ruiqiang Li, Jie Qiao or Fuchou Tang.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7 and Supplementary Note (PDF 9845 kb)

Supplementary Table 1

Expression (RPKM) of known RefSeq genes in 124 single cells from mature oocytes, preimplantation embryos and embryonic stem cells. (XLSX 20445 kb)

Supplementary Table 2

Expression (RPKM) of known RefSeq transcripts in 124 single cells from mature oocytes, preimplantation embryos and embryonic stem cells. (XLSX 28202 kb)

Supplementary Table 3

GO enrichment analysis for EPI-specific genes compared to all the other cells after the 8-cell stage (including 8-cell–stage embryos). (XLSX 62 kb)

Supplementary Table 4

Number of exon-exon junction reads that are unique to transcript isoforms of known RefSeq genes in 124 single cells from mature oocytes, preimplantation embryos and embryonic stem cells. (XLSX 4632 kb)

Supplementary Table 5

Expression (counts) of known lncRNA genes in 124 single cells from mature oocytes, preimplantation embryos and embryonic stem cells. (XLSX 11064 kb)

Supplementary Table 6

Expression (counts) of novel transcripts in 124 single cells from mature oocytes, preimplantation embryos and embryonic stem cells. (XLSX 3520 kb)

Supplementary Table 7

Expression (RPKM) of the differentially expressed genes among the epiblast (EPI), primitive endoderm (PE) and trophectoderm (TE) lineages of late blastocysts. (XLSX 6580 kb)

Supplementary Table 8

Expression (RPKM) of the differentially expressed genes between the epiblast (EPI) cells of late blastocysts and passage #0 hESCs. (XLSX 156 kb)

Supplementary Table 9

The rRNA contamination in the single cell RNA-Seq data for 124 single cells from mature oocytes, preimplantation embryos and embryonic stem cells. (XLSX 51 kb)

Supplementary Table 10

Summary of the sequencing exercise, quality control (Q20 and Q30 percentage of the sequencing reads) and mapped rates of RNA-Seq data to RefSeq, Ensemble, known lncRNAs, and genome of 124 single cells from mature oocytes, preimplantation embryos and embryonic stem cells. (XLSX 71 kb)

Supplementary Table 11

The distribution of all mapped reads in different features (exon, intron and intergenic) of the human genome in 124 single cells from mature oocytes, preimplantation embryos and embryonic stem cells. (XLSX 54 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Yan, L., Yang, M., Guo, H. et al. Single-cell RNA-Seq profiling of human preimplantation embryos and embryonic stem cells. Nat Struct Mol Biol 20, 1131–1139 (2013). https://doi.org/10.1038/nsmb.2660

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.2660

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing