Early role for IL-6 signalling during generation of induced pluripotent stem cells revealed by heterokaryon RNA-Seq

Abstract

Molecular insights into somatic cell reprogramming to induced pluripotent stem cells (iPS) would aid regenerative medicine, but are difficult to elucidate in iPS because of their heterogeneity, as relatively few cells undergo reprogramming (0.1-1%; refs , ). To identify early acting regulators, we capitalized on non-dividing heterokaryons (mouse embryonic stem cells fused to human fibroblasts), in which reprogramming towards pluripotency is efficient and rapid, enabling the identification of transient regulators required at the onset. We used bi-species transcriptome-wide RNA-seq to quantify transcriptional changes in the human somatic nucleus during reprogramming towards pluripotency in heterokaryons. During heterokaryon reprogramming, the cytokine interleukin 6 (IL6), which is not detectable at significant levels in embryonic stem cells, was induced 50-fold. A 4-day culture with IL6 at the onset of iPS reprogramming replaced stably transduced oncogenic c-Myc such that transduction of only Oct4, Klf4 and Sox2 was required. IL6 also activated another Jak/Stat target, the serine/threonine kinase gene Pim1, which accounted for the IL6-mediated twofold increase in iPS frequency. In contrast, LIF, another induced GP130 ligand, failed to increase iPS frequency or activate c-Myc or Pim1, thereby revealing a differential role for the two Jak/Stat inducers in iPS generation. These findings demonstrate the power of heterokaryon bi-species global RNA-seq to identify early acting regulators of reprogramming, for example, extrinsic replacements for stably transduced transcription factors such as the potent oncogene c-Myc.

Figure 1. Heterokaryon global bi-species transcriptome analysis by RNA-Seq identifies differentially expressed genes during somatic cell reprogramming towards pluripotency

a, Scheme for heterokaryon generation. GFP mouse ES (mES) cells were co-cultured with DsRedpuro^R primary human fibroblasts (hFb) and then fused using PEG. b, Heterokaryon enrichment and isolation by FACS. Representative FACS plots for heterokaryon isolation. From left to right: GFP⁺ mouse ES cells, DsRed⁺ human fibroblasts, GFP⁺ DsRed⁺ day 2 heterokaryons first sort, second sort, enrichment. c, Histogram of heterokaryon nuclear content (total number of nuclei per FACS sorted heterokaryon assessed by single cell microscopy, n = 68). A fitted Gaussian curve reveals an average nuclear content of 4 nuclei per heterokaryon. d, Average mappable read summary for heterokaryon RNA-Seq libraries. e, Flowchart of heterokaryon RNA-Seq analysis. f, Comparison of biological replicates of human gene expression during heterokaryon reprogramming for the three days. Pearson's correlation values are displayed for heterokaryon biological replicates. Replicate 1 for each day is displayed on the x-axis, and Replicate 2 for each day on the y-axis. g, Hierarchical cluster of RNA-Seq data for heterokaryon as well as control samples, using the Euclidean distance metric and complete linkage method. h and i, Venn diagram of significantly upregulated and downregulated gene expression in heterokaryon reprogramming compared to the homokaryon, co-culture, and unfused human fibroblasts controls. Differential gene expression was identified using the NOISeq method with a cut-off probability threshold of 0.8. Source data are presented in Table SI 1.

Figure 2. Heterokaryon bi-species RNA-Seq identifies induction of GP130 signaling gene expression during somatic cell reprogramming

a, Heatmap of differentially expressed genes during heterokaryon reprogramming (day1-3 post fusion) as compared to unfused human fibroblasts, co-culture, and homokaryon (fibroblast-fibroblast fusion) controls. The gene expression (RPKM) values for each gene were normalized to the standard normal distribution to generate z-scores. The z-score color bar is shown with the minimum expression value for each gene in blue and the maximum value in red. b, Protein classification of the differentially expressed human genes (n=905 genes) using the PANTHER (Protein ANalysis THrough Evolutionary Relationships) classification system with default settings. Select protein classes encompassing at least 5% of the total number of upregulated genes (n=630) or downregulated genes (n=275) are shown. c, d, Heatmap of human pluripotency transcription factors and GP130 signaling gene pathway genes, respectively, during heterokaryon reprogramming. Normalized gene expression values were used (see above). e, Validation of heterokaryon RNA-Seq gene expression levels by qPCR for six human pluripotency genes and the lineage marker THY1 (Day 2 post fusion), relative to unfused human fibroblasts. Data are represented as mean ± s.d. for qPCR data (n=3) and mean with range (n=2) for RNA-Seq data.

Figure 3. IL6, but not LIF, enhances 4 Factor iPS generation

a, Relative transcript levels of LIF and IL6 during heterokaryon reprogramming, normalized to unfused human fibroblasts. Data are represented as mean RPKM withrange (n=2). b, Relative mRNA levels for Il6 and Lif gene expression during 4 Factor (4F) iPS generation in MEFs, normalized to mouse Hprt and mouse Gapdh, as determined by qPCR. Data are represented as mean ± s.e.m., n=4. c, Induction of pStat3 Tyr-705 in fibroblasts upon IL6 stimulation. A representative western blot over a 2.5 hour time course is shown. Full blot is shown in Supplementary Fig. 5. d, Quantification of pStat3 induction upon IL6 stimulation, normalized to α-Tubulin protein levels. Mean with range is shown (n=2). Maximal pStat3 is observed 30 minutes post-IL6 stimulation. e, Schematic of the 4Factorlentiviral vector used to generate 4F (OKSM) virus for mouse iPS induction. f, Phase and immunofluorescence images of a representative iPS colony stained for the mouse pluripotency marker Nanog (red) and nuclear Hoechst (blue). Scale bar = 200 μm. g, Representative iPS colonies, presented as in f with staining for the mouse pluripotency marker SSEA-1 (green). h, Nanog⁺ iPS colony formation from MEFs transduced with Mock or 4F virus in the absence of additional cytokines and cultured with or without IL6 and or LIF. Data are represented as mean ± s.e.m., n=3. P values were calculated using Student's unpaired two-tailed t-test. Source data are presented in Table SI 1.

Figure 4. Transient exposure to IL6 in medium replaces viral c-Myc in reprogramming to iPS and is only required at the onset of 3F reprogramming

a, Timecourse of relative mRNA expression levels for endogenous c-Myc in MEFs after stimulation with IL6, LIF, or mock (medium change only), normalized to mouse Actin and t=0 min. Data are represented as mean ±s.d., n=3. P values were calculated using Student's unpaired two-tailed t-test. Source data are presented in Table SI 1. b, Schematic of the 3F lentiviral vector. c-Myc was restored using a second virus when indicated. c, Nanog⁺ iPS colony formation from MEFs transduced with 3F virus with or without IL6 or co-transduction with Mock or c-Myc virus. Data are represented as mean ± s.e.m., n=6. P values were calculated using Student's unpaired two-tailed t-test. Source data are presented in Table SI 1. d, Nanog⁺ iPS colony formation from MEFs upon IL6 withdrawal from the medium on the day indicated. Maximum colony yield is obtained when IL6 is added to the medium for the first four days of reprogramming. Treatment beyond day 4 did not increase iPS colony number. e, Teratoma histological analysis from 3F + IL6 iPS clones 1 and 4 showing contribution to all three germ layers. Magnification is 200×, scale bar = 50 μm. f, Comparison of global gene expression between individually generated iPS (4F and 3F+IL6) clones and mES (average of n=2 for each). The Pearson's correlation values are displayed for iPS versus mES gene expression.

Figure 5. Pim1, a pro-survival gene, acts downstream of IL6 to enhance reprogramming to iPS

a, Timecourse of Pim1 mRNA expression levels in MEFs after stimulation with IL6, LIF, or mock (medium change only). Gene expression was normalized to Actin and t=0 min, as determined by qPCR (mean ±s.d., n=3). Data are presented in Table SI 1. b, Nanog⁺ iPS colony formation from MEFs transduced with 3F virus with or without addition of IL6, Pim1 inhibitor or DMSO (mean ± s.e.m., n=3, shown as a percent of control). Data are presented in Table SI 1. c, Nanog⁺ iPS colony formation from MEFs transduced with 4F virus, with or without IL6, Pim1 inhibitor or DMSO as indicated, shown as a percent of control (mean ± s.e.m., n=3). Data are presented in Table SI 1. d, Western blots of p-Stat3 induction in MEFs transduced with shRNAs against Il6r or shControl, assessed 30 minutes post stimulation with IL6 or mock. MEFs were stimulated 3 days after shRNA virus transduction. Total Stat3 levels and α-tubulin levels are shown as a reference and loading control, respectively. e, Mouse c-Myc, Pim1, and Mcl1 mRNA levels in MEFs transduced with shRNA virus targeting Il6r or control shRNA virus and stimulated with IL6, as assessed by qPCR. mRNA expression levels were normalized to mouse Actin and assessed at peak shControl levels post Il6 stimulation occurring at 60, 90, and 120min post stimulation, respectively (mean ±s.d., n=3). Data are presented in Table SI 1. f, Western blot for Pim1 overexpression in MEFs. g, Pim1 mRNA levels in MEFs transduced with Pim1 virus, as determined by qPCR, normalized to Actin and Pim1 levels in mES (mean ± s.d., n=3). Data are presented in Table SI 1. h, Nanog⁺ iPS colony formation from MEFs transduced with 4F and Pim1 virus or mock virus (mean ± s.e.m., n=3). Data are presented in Table SI 1. i, Western blot for p-NFκB protein levels in IL6 treated MEFs, 3 hours post-stimulation with IL6. NFκB and α-Tubulin are shown as controls. j, Model for early IL6-mediated enhancement of iPS generation. The yellow rectangle indicates the cell membrane containing the Gp130 and Il6 receptor. Il6 ligand is depicted as a diamond. Extracellular space and cytoplasm are indicated. P values were calculated using Student's unpaired two-tailed t-test. Full blots are shown in Supplementary Fig. 5.