The precise relationship of embryonic stem cells (ESCs) to cells in the mouse embryo remains controversial. We present transcriptional and functional data to identify the embryonic counterpart of ESCs. Marker profiling shows that ESCs are distinct from early inner cell mass (ICM) and closely resemble pre-implantation epiblast. A characteristic feature of mouse ESCs is propagation without ERK signalling. Single-cell culture reveals that cell-autonomous capacity to thrive when the ERK pathway is inhibited arises late during blastocyst development and is lost after implantation. The frequency of deriving clonal ESC lines suggests that all E4.5 epiblast cells can become ESCs. We further show that ICM cells from early blastocysts can progress to ERK independence if provided with a specific laminin substrate. These findings suggest that formation of the epiblast coincides with competence for ERK-independent self-renewal in vitro and consequent propagation as ESC lines.
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We thank C-É Dumeau and W. Mansfield for chimaera production, P. Humphreys for assistance with imaging, S. Jameson and staff for animal husbandry, the EMBL Genomics Core Facility for sequencing, and G. Martello for helpful discussion on the manuscript. This work was financially supported by the Wellcome Trust, Medical Research Council, BBSRC, the Louis Jeantet Foundation, EMBL and the University of Cambridge. A.S. is a Medical Research Council Professor.
The authors declare no competing financial interests.
Integrated supplementary information
Supplementary Figure 1 Increasing the amount of starting material improves the fidelity of whole-transcriptome preamplification.
(A) To evaluate the accuracy of whole-transcriptome preamplification37 from different amounts of starting material, total RNA from 2i and 2i-LIF ESC cultures was diluted to 200pg (equivalent to 10–20 cells) and 20pg (equivalent to 1–2 cells) and subsequently preamplified using 18, 20 and 22 preamplification cycles (2 technical replicates each). qPCR of conventional cDNA indicates that ESC respond to LIF by upregulation of Socs3 and Klf4, consistent with previous observations, while levels of other pluripotency factors did not change43. On average, preamplified samples corresponding to 10–20 cells recapitulated the expression pattern of the unamplified control more faithfully than preamplified samples equivalent to 1–2 cells. Error bars are s.d. between biological replicates. (B) Correlation of diluted and subsequently preamplified RNA of ESC in 2i-LIF compared to conventional cDNA of the same RNA. (C) Heatmaps of RNA levels of Notch and p38 signalling genes normalised to mean expression in embryonic samples E1.5–5.5. (D) Hierarchical clustering of embryonic samples from E1.5–5.5.
Supplementary Figure 2 ESC cultured in 2i and 2i-LIFcorrelate with the E4.5 epiblast based on 96 gene qRT-PCR arrays.
Correlation analysis between embryonic samples and ESC samples obtained from 2i and 2i-LIF cultures on gelatin. For the ESC samples, two independent biological replicates, each containing 20 randomly picked cells of the 4 ESC lines indicated in the graph, for both 2i and 2i-LIF, were processed and profiled by qRT-PCR arrays.
(A) Images of typical primary ESC colonies derived in 2i-LIF on gelatin from an E2.5 morula and the ICM of an E3.5 blastocyst after 6 and 5 days, respectively. (B) ESC derivation efficiencies from various developmental stages assayed by primary ES colony formation, as depicted in A. The numbers of embryos yielding an ESC colony per embryo are indicated. (C) Blastocoel formation of a morula 24 h after plating in 2i-LIF on gelatin. (D) PrE-like epithelial formation in an outgrowth from an E2.5 morula 4 days after plating in 2i-LIF on gelatin. We suspect that partial PrE specification is mediated by compensatory effects of LIF in 2i. (E) Surviving cells as judged by morphology at day 7 in percentage of wells per embryo after single ESC derivation in 2i on gelatin. The graph shows the mean and s.e.m. for the percentage of surviving cell-containing wells per embryo resulting from the analysis of n = ‘e’ embryos (with at least 3 wells analysed per embryo for each time point of embryonic development shown on the x axis, ‘w’ represents the total number of wells analysed per time point). (F) Images of surviving cells 7 days after single cell ESC derivation from E3.5 embryos in 2i-LIF on gelatin. (G,H) Single cell ESC derivation efficiency in serum-LIF (G) and under EpiSC conditions (ActA, bFGF) (H); Efficiency is displayed as the percentage of ESC-colony positive wells per embryo (ESC-col. pos. wells (%)/embryo) after 7 days. The total efficiency is further subdivided into ESC colonies arising from truly individual cells (ESC-col (single cell)) and ESC arising from small groups, usually between 2 and 5 cells (ESC-col (small group of cells)). The graphs show the mean and s.e.m. for the percentage of ESC colony-containing wells per embryo resulting from the analysis of n = ‘e’ embryos (with at least 3 wells analysed per embryo for each time point of embryonic development shown on the x axis, ‘w’ represents the total number of wells analysed per time point). Embryos were obtained from at least 3 individual litters on at least 2 different days.
Relative RNA levels of trophectoderm specific genes for the samples indicated. Error bars are s.d. between biological replicates.
Supplementary Figure 5 Correlation to previously published data and testing extracellular matrix components with stringent early ICM cells.
(A) Correlation of RNA levels as determined by RNA-seq of the E3.5-ICM samples analysed in this study. (B, C) Correlation of RNA levels as determined by RNA-seq of the E3.5-ICM samples 1 (B) and 2 (C) of the present study to the average of 9 individually processed and sequenced E3.5 ICM cells in Tang et al.38. (D) Average RNA levels at E3.5 for the same extracellular matrix genes shown in Fig5.B, as determined in Tang et al.by single-cell RNA-seq. (E) Single cell ESC derivation efficiency of stringent early ICM cells with a 24 h maturation step for the experimental conditions indicated on Fn-Lam511. The graph shows the mean and s.e.m. for the percentage of ESC-colony containing wells per embryo resulting from the analysis of n = ‘e’ embryos (with at least 4 wells analysed per embryo for each experimental condition shown on the x axis, ‘w’ represents the total number of wells analysed per experimental condition).
(A) Alkaline phosphatase staining of ESC colonies and PrE-like cells at day 7 after single ESC derivation in 2i-LIF on Fn-Lam511. (B, C) Immunofluorescence staining of ESC colonies and PrE-like cells at days 3 and 6 (B) and at day 7 (C) from single cell ESC derivation in 2i-LIF on Fn-Lam511. (D) Phase contrast images of the first 5 clonal ESC lines at passage 4 originally derived on either Fn-Lam511 (FL), Fibronectin (F) and Laminin511 (L). After manually picking and dissociating primary single cell ESC colonies at day 7, ESC lines were expanded in 2i-LIF on gelatin. Two of the FL lines were randomly selected (FL4 and FL11) and successfully tested for germline transmission. (E) PrE-like cells obtained after single cell ESC derivation in 2i-LIF on Fn-Lam511 of embryos cultured in the presence of FGF2 for 3 days from the 8-cell stage.
Supplementary Figure 7 Expression dynamics of Esrrb and Tcfcp2l1 and correlation analysis of primary ESC colonies to embryonic samples.
(A) Direct comparison of RNA levels in embryonic samples E3.5-ICM, E4.0-ICM and E4.5-EPI (epiblast), single cell ESC derivation samples day 2–6 and established ESC in 2i-LIF. Expression levels are shown relative to the preimplantation epiblast (E4.5-EPI) on a logarithmic scale for Esrrb and Tcfcp2l1. Error bars are s.d. between biological replicates. (B) Correlation analysis between embryonic samples, established ESC samples and primary ESC colony day 2, 4 and 6 samples. Samples were processed as described in Fig. 7c.
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Live imaging of a single cell isolated from an E4.5 ICM of a Pdgfra::GFP embryo during formation of an ESC colony. Green fluorescence identifies cells of the PrE lineage. In this example, a GFP-negative (epiblast) cell is filmed. (AVI 5306 kb)
Live imaging of a single cell isolated from an E4.5 ICM of a Rex1GFP embryo during formation of an ESC colony. Green fluorescence marks cells of the epiblast lineage. The chosen cell and its progeny exhibit GFP expression throughout the movie. (AVI 16628 kb)
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Boroviak, T., Loos, R., Bertone, P. et al. The ability of inner-cell-mass cells to self-renew as embryonic stem cells is acquired following epiblast specification. Nat Cell Biol 16, 513–525 (2014). https://doi.org/10.1038/ncb2965
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