IGF1-mediated human embryonic stem cell self-renewal recapitulates the embryonic niche

Abstract

Our understanding of the signalling pathways regulating early human development is limited, despite their fundamental biological importance. Here, we mine transcriptomics datasets to investigate signalling in the human embryo and identify expression for the insulin and insulin growth factor 1 (IGF1) receptors, along with IGF1 ligand. Consequently, we generate a minimal chemically-defined culture medium in which IGF1 together with Activin maintain self-renewal in the absence of fibroblast growth factor (FGF) signalling. Under these conditions, we derive several pluripotent stem cell lines that express pluripotency-associated genes, retain high viability and a normal karyotype, and can be genetically modified or differentiated into multiple cell lineages. We also identify active phosphoinositide 3-kinase (PI3K)/AKT/mTOR signalling in early human embryos, and in both primed and naïve pluripotent culture conditions. This demonstrates that signalling insights from human blastocysts can be used to define culture conditions that more closely recapitulate the embryonic niche.

Fig. 1. A chemically defined hESC medium comprising Activin and IGF.

a Boxplots of RPKM values for insulin (INS) and IGF ligands (IGF1, IGF2), and cognate receptors in human blastocyst lineages (EPI green, PE red, TE blue) determined by single-cell RNA-seq. Boxes symbolise first and third quartiles, horizontal lines the median, whiskers extend to 1.5 times the interquartile range, dots represent outliers. b Representative phase-contrast images of hESCs in KSR + FGF2 on MEFs, or mTeSR1 or AI medium on laminin-511. n = 2 or 3 biological and 3 technical replicates. Scale bar: 300 μm, top row; 200 μm bottom row. c Flow cytometry of AI-adapted Shef6 hESCs for the indicated proteins (blue); isotype control in grey. n = 3 technical replicates. d Representative immunofluorescence of AI-adapted Shef6 hESCs for the indicated proteins; DAPI (blue) nuclear stain. n = 3 biological replicates. Scale bar: 100 μm. e Upper panels: Representative image of a human day 6 blastocyst prior to ICM (circled) dissection and plating in AI medium on MEFs. The resulting ICM outgrowth (circled) was passaged onto laminin-511 to establish a stable hESC cell line (CH2). Scale bar: 150 μm. n = 3 biological replicates. Lower panels: Representative images of iPSCs derived following reprogramming of BJ fibroblasts with Sendai viruses. iPSC-like colonies (circled) could be detected in AI medium within 12 days of induction, and expanded for picking within 18 days to propagate an established iPSC line (CHiP1). n = 2 technical replicates. Scale bars, left to right: 300 μm, 100 μm, 100 μm. f–h Representative immunofluorescence of AI-cultured hESCs following directed differentiation. n = 3 biological replicates. DAPI in blue. f hESCs differentiated towards hepatocyte-like cells first express endoderm markers CXCR4 (green) and SOX17 (red), upregulate FOXA2 (green) as foregut endoderm, then AFP (red) and CK18 (green) as hepatocytes. Scale bar: 100 μm. g Expression of CTNT (green; scale bar 50 μm), α-Actin (green; scale bar 100 μm) and NKX2.5 (red; scale bar 100 μm) following directed differentiation towards cardiomyocyte-like cells. h Expression of neuronal markers OTX2, PAX6, TUJ1 (green) and NESTIN (red) following directed differentiation towards neuronal progenitor cells. Scale bar: 100 μm.

Fig. 2. hESCs cultured in AI medium are transcriptionally similar to conventional hESCs.

a Clusters detected after applying the unsupervised clustering tool griph to single-cell RNA-seq data from EPI, PE or TE cells of the human blastocyst^,, and hESCs cultured in AI, mTeSR1, KSR + FGF, 5iLA or t2iL + Gö media^,,,. A colour-coded sample key is provided. b, c Comparison of embryonic EPI cells to cells cultured in AI medium or naïve (5iLA or t2iL + Gö) media. The most enriched GO terms for genes associated with biological process (BP), cellular component (CC), molecular function (MF), and REACTOME pathways associated with differentially expressed genes are shown based on Benjamini-Hochberg corrected p-values. Red-dashed lines correspond to the significance level α = 0.05. d Quantification of RNA FISH in XXY fibroblast cells or in H9 XX hESC lines cultured in mTeSR1 or AI media for the transcription foci of XIST and ATRX, which is normally subject to X-chromosome inactivation. n = 51 mTeSR1 hESCs, 50 AI hESCs and 51 fibroblasts. Representative DAPI and RNA FISH images of hESCs or fibroblasts are shown. Scale bar: 10 μm. Source data are provided as a Source Data file.

Fig. 3. Insulin/IGF1R/PI3K/mTOR and MAPK/ERK signalling pathways are active in both conventional and naïve hESC culture conditions.

a Schematic of the crosstalk between the insulin/IGF1 and receptor tyrosine kinase (RTK) signalling pathways. Insulin receptor (IR), IGF1 receptor (IFG1R), IGF binding proteins (IGFBPs), receptor tyrosine kinase (RTK), IGFR and IR inhibitor OSI-906 (OSI), FGF receptor tyrosine kinase inhibitor PD173074 (PD17), PI3K inhibitor GDC-0941 (GDC), GSK3β inhibitor CHIR99021 (CHIR), MEK inhibitor PD0325901 (PD13), mTOR1 kinase inhibitor Everolimus (EV). b Representative western blot analysis for proteins related to PI3K/AKT/mTOR and MAPK/ERK signalling in hESCs cultured in AI medium supplemented with DMSO, PD17, PD03, OSI, GDC, EV, CHIR or the Activin/Nodal receptor inhibitor SB-431542 (SB43) for 24 h. n = 3 biological replicates. c Quantification of the total number of viable cells over 3 days in AI, mTeSR1 or t2iL + Gö media supplemented with DMSO, OSI, EV, PD17 or PD03. The value at each time point represents the mean of n = 2 biological and n = 4 technical replicates, and error bars represent s.e.m. d Representative immunofluorescence analysis for NANOG (green) expression in control or inhibitor-treated hESCs in AI, mTeSR1 or t2iL + Gö media, following 3 days of treatment. DAPI (blue) nuclear stain. Scale bar: 50 μm. Source data are provided as a Source Data file.

Fig. 4. IGF1 treatment of human embryos promotes ICM proliferation.

a Representative western blots for selected proteins related to MAPK/PI3K/AKT/mTOR signalling in mouse and human embryos. n = 6 human blastocysts at 6 days post fertilisation (dpf) per experiment, and 5 or 10 mouse blastocysts. Given the limited human material, after WB transfer the higher and lower molecular weight regions of the blot were cut and probed separately. The cut junction can be seen in the upper edge of pS6. Full-length western blots are shown in Supplementary Fig. 11. Data represents n = 5 replicate experiments. b Schematic of human embryo treatment schedule. c Immunofluorescence analysis for NANOG (EPI, green) and SOX17 (PE, red) with DAPI (blue) nuclear stain at 6.5 dpf in control embryos or embryos treated with 17 nM IGF1 from 2 dpf. Scale bar: 100 μm. d Immunofluorescence analysis for NANOG (green), KLF17 (red) and DAPI (blue) at 6.5 dpf following 17 nM IGF1 treatment from 2 dpf. Scale bar: 100 μm. e Quantification of total cell number (DAPI stained nuclei) and percentages of NANOG- or SOX17-expressing cells, and ICM (combined NANOG and SOX17 totals) in control or 17 nM IGF1-treated embryos. Data points represent the percentage per individual embryo; horizontal line indicates the mean. n = 5 control, 4 treated embryos. One-tailed t-test. *P < 0.05, **P < 0.01. f Immunofluorescence analysis for NANOG (red) and GATA6 (PE, green), with DAPI (blue) at 6.5 dpf in control embryos or embryos treated with 1 µg/ml FGF2 and 1 µg/ml heparin, or 100 ng/ml FGF2 from 2 dpf. Scale bar: 100 μm. g Immunofluorescence analysis for NANOG (green) and SOX17 (red) with DAPI (blue) nuclear stain at 6.5 dpf in control embryos or embryos treated with 100 ng/ml FGF2 from 5 dpf. h Quantification of total cell number (DAPI) and percentages of NANOG- or SOX17-expressing cells, and ICM in control embryos or embryos treated with 100 ng/ml FGF2 from 5 dpf. Data points represent the percentage per individual embryo; horizontal line indicates the mean. n = 10 control, 9 treated embryos. One-tailed t-test. *P < 0.05, **P < 0.01. Source data are provided as a Source Data file.