Research ArticleStem Cells

Kynurenine signaling through the aryl hydrocarbon receptor maintains the undifferentiated state of human embryonic stem cells

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Science Signaling  25 Jun 2019:
Vol. 12, Issue 587, eaaw3306
DOI: 10.1126/scisignal.aaw3306
  • Fig. 1 Identification of secreted metabolites that are markers for undifferentiated or early differentiating ESCs and iPSCs.

    (A) The protocol for culture, induction of differentiation, and supernatant collection for the analysis of metabolites in human H9 ESCs and PFX#9 iPSCs. Cells were seeded in chemically defined medium (Es8 for H9 cells and TeSR-Es8 for PFX#9 cells) and a ROCK inhibitor on dishes coated with recombinant human (rh) VNT-N. The cells were untreated or treated with the indicated cocktails of differentiation factors specific for each germ layer. The medium was removed for analysis each day and replaced with fresh medium for 6 days. The cell numbers increased exponentially during the experiment because the cultures were not split during medium replacement. The harvested medium was subjected to LC-MS/MS analysis for 95 metabolites (table S1). (B) Time-course profiles of the amounts of tryptophan, kynurenine, lysine, 2-AAA, lactic acid, and methionine relative to an internal control in supernatants from H9 ESCs (H9/Es8) and PFX#9 iPSCs (PFX#9/TeSR-E8) in the absence of differentiation factors (Undiff) and in the presence of ectodermal (Ecto), mesodermal (Meso), or endodermal (Endo) differentiation cocktails. Blank, medium only. n = 3 independent experiments. (C) Comparison of two different quantification methods for four metabolites in undifferentiated or differentiated cultures of H9 ESCs in Es8 medium. Metabolites were measured relative to an internal standard (the ratio of the area under the peak for each metabolite to the area under the peak for 2-isopropyl malic acid) and by an external calibration method using a standard calibration curve of known concentrations of each metabolite. n = 3 independent experiments. (D) The average amounts of kynurenine and 2-AAA secreted from a single H9 cell per hour in Es8 medium were estimated by the external calibration method for days 1 to 3 and days 4 to 6 [in μM per cell per hour = nmol (in 2 ml) per cell per hour: normalization]. n = 3 independent experiments.

  • Fig. 2 Kynurenine generated by IDO1 mediates a signal that maintains the undifferentiated state.

    (A) Expression of IDO1, IDO2, and TDO2 in undifferentiated H9 cells cultured on VNT-N in Es8 medium as determined by qRT-PCR and shown as the fold increase compared with that of TDO2. n = 3 independent experiments. (B) Protocol for the addition of the IDO1 inhibitor INCB024360 analog (IDO1i) or vehicle [dimethyl sulfoxide (DMSO)] to the H9 culture system. Media were changed every day. (C) Gene expression profiles of H9 cells treated with IDO1i or DMSO as determined by qRT-PCR scorecard panel. n = 3 independent experiments. (D) Morphology of H9 cells that were untreated or treated with vehicle (DMSO) or IDO1i at day 3. Cell counts in one well of a six-well plate are noted in each image. n = 3 independent experiments. Scale bar, 200 μm. (E) Time-dependent changes in the concentration of kynurenine, 2-AAA, and tryptophan in culture medium of H9 cells treated with vehicle or IDO1i, as measured by LC-MS/MS. Blank, medium only. n = 3 independent experiments. (F) The amount of kynurenine secreted from a single H9 cell in a 1-hour period under the indicated conditions was estimated from the average of the normalized values and cell numbers from day 1 to day 3 (D1 to D3). A representative result is shown. n = 3 independent experiments.

  • Fig. 3 The IDO1-kynurenine-AhR loop sustains the undifferentiated state of ESCs.

    (A) Protocol for the addition of the AhR inhibitor CH223191 to the H9 culture system. DMSO, vehicle control. Medium was changed daily. (B) Gene expression profiles of H9 cells treated with CH223191 or DMSO at day 3. n = 3 independent experiments. (C) Morphology of H9 cells on day 3. The number in each image represents the cell count in one well of six-well plate. Scale bar, 200 μm. Data are representative of n = 3 independent experiments. (D) Changes in kynurenine and tryptophan concentrations over time in culture medium of H9 cells with or without CH223191. Blank, medium only. n = 3 independent experiments. (E) Expression of IDO1 and AHR in H9 cells 3 days after adding CH223191 and 6 days after adding the IDO1 inhibitor to the culture. Transcript abundances were determined by qRT-PCR and are shown as the fold change of expression in untreated cells. n = 3 independent experiments. (F) Expression of IDO1, IDO2, TDO2, and AHR, as determined by qRT-PCR, in undifferentiated H9 cells and at days 3 and 6 after induction of ectodermal differentiation. Data are shown as the fold change in IDO1 expression in H9 cells during ectoderm induction day 3 or AHR in untreated, undifferentiated H9 cells. n = 3 independent experiments. (G) Representative Western blot for IDO1, IDO2, and TDO2 in undifferentiated H9 cells. n = 3 independent experiments.

  • Fig. 4 The kynurenine-AhR complex mediates a signal that supports self-renewal.

    (A) Immunohistochemical detection of the intracellular distribution of kynurenine (red) in undifferentiated (Undiff.) H9 cells treated with vehicle only (DMSO) or with the AhR inhibitor CH223191 for 24 hours and at day 6 after ectoderm induction. Nuclei are stained with DAPI (4′,6-diamidino-2-phenylindole) (blue). Scale bars, 5.0 and 2.5 μm (zoom). (B) Immunohistochemical detection of the intracellular distribution of AhR (red) in undifferentiated H9 cells treated with DMSO alone or with the IDO1 inhibitor INCB024360 analog (IDO1i) for 24 hours. Nuclei are stained with DAPI (blue). n = 3 independent experiments. Scale bar, 5.0 μm. (C) Abundance of AhR in undifferentiated H9 cells or in H9 cells 6 days after ectoderm induction as determined by flow cytometry. Undifferentiated H9 cells stained with an isotype control antibody were used as control. n = 3 independent experiments. (D) Western blot for kynurenine in lysates of undifferentiated H9 cells before and after immunoprecipitation (IP) with an antibody against AhR or a control IgG antibody. n = 3 independent experiments. (E) GFP fluorescence in H9 cells expressing AhR-GFP or GFP alone (control) and treated with DMSO or the IDO1 inhibitor INCB024360 analog (IDO1i). n = 3 independent experiments. Scale bar, 10 μm. (F) ChIP-qPCR assay for POU5F1, NANOG, IDO1, AHR, CYP1A1, and EP300 in DNA immunoprecipitated from control and IDO1i-treated H9 cells using an antibody recognizing AhR or a control antibody and primers recognizing the promoters or downstream regions of each gene. qPCR values using primers for the promoter regions in the AhR immunoprecipitate were compared to those in the control immunoprecipitant and shown as fold enrichment (orange bars). qRCR values using primers for the downstream regions in the AhR immunoprecipitant were compared to those of the control immunoprecipitant and shown as fold enrichment (black bars). n = 3 independent experiments with three technical replicates per experiment. Gene expression profiling of the cells used in this experiment is also shown.

  • Fig. 5 Secretion of 2-AAA into the culture medium is a biomarker for ectoderm differentiation.

    (A) Protocol for testing the effect of the KAT2 inhibitor PF-04859989 (KI) on ectodermal differentiation of H9 cells. (B) Gene expression profiles of H9 cells treated with ectoderm stimuli in the presence or absence of KI were determined by qRT-PCR scorecard panel. n = 3 independent experiments. (C) The amount of 2-AAA secreted from a single H9 cell in 1 hour (in area ratio per cell per hour) under the indicated conditions was determined from the normalized values from day 1 to day 4 (D1 to D4) of culture. n = 3 independent experiments. (D) Quantification of the abundances of 2-AAA, kynurenine, tryptophan, lactate, and lysine in culture medium from day 1 to day 4 of ectodermal differentiation was determined by LC-MS/MS. Culture medium was replaced with fresh medium every day. n = 3 independent experiments.

  • Fig. 6 The kynurenine catabolic pathway is involved in the initiation of ectodermal differentiation.

    (A) Gene expression profiles (qRT-PCR scorecard panels) and expression of genes in the kynurenine catabolic pathway (HAAO, KMO, KYNU, ACMSD, OGDH, and KAT2) and the lysine catabolic pathway (ALDH7A1) in undifferentiated H9 cells and at days 3 and 6 of ectodermal differentiation. Gene expression was measured by qRT-PCR and shown as the fold change compared to the undifferentiated state. n = 3 independent experiments. (B) Photos and gene expression profiles of H9 cells at day 6 of ectodermal differentiation in the absence (Ecto diff.) or presence of 100 μM 2-AAA (Ecto diff. + 2-AAA). Undifferentiated H9 cells with no treatment were used as a control. The number of cells in one well of a six-well plate for each condition is noted in each image. n = 3 independent experiments. Scale bar, 200 μm. (C) Putative schema for metabolic pathways in undifferentiated ESCs and iPSCs and in ESCs and iPSCs committed to ectodermal differentiation. Kynurenine produced from tryptophan by IDO1 binds to AhR in the cytoplasm, and the kynurenine-AhR complex translocates to the nucleus, where it promotes the transcription of self-renewal factors, IDO1, and AHR to maintain self-renewal and the IDO1-kynurenine-AhR loop. Excess kynurenine not used for signaling is secreted into the culture medium, where it acts as a sustainable self-renewal signal. When cells commit to ectodermal differentiation, the kynurenine pool decreases through the activation of the kynurenine catabolic pathway. KAT2 converts AKA to 2-AAA, which is secreted instead of being used for additional pathways. Gene expression profiles and relevant time-lapse metabolic analyses of kynurenine and 2-AAA with LC-MS/MS are included in the schema.

  • Fig. 7 Kynurenine and 2-AAA in the culture medium are robust markers for the assessment of the differentiation state of cultured stem cells.

    (A) Protocol for EB formation. H9 cells were transferred to low-attachment plates in Essential 6 (Es6) medium at day 0. The medium was removed daily for LC-MS/MS analysis and replaced with fresh medium. (B) Phase contrast imaging of EBs at days 3 and 6. n = 3 independent experiments. Scale bar, 200 μm. (C) Gene expression profiles of EBs at days 3 and 6 as determined by a qRT-PCR scorecard panel. n = 3 independent experiments. (D) Quantification of kynurenine, 2-AAA, and lactic acid and the ratio of kynurenine to 2-AAA secreted into the culture medium by EBs over days 1 to 6 by LC-MS/MS. n = 3 independent experiments with three technical replicates per condition for each time point.

Supplementary Materials

  • stke.sciencemag.org/cgi/content/full/12/587/eaaw3306/DC1

    Fig. S1. Biomarkers for the differentiation status of H9 cells cultured in Es8 medium.

    Fig. S2. Biomarkers for the differentiation status of PFX#9 cells cultured in TeSR-E8.

    Fig. S3. Biomarkers for the differentiation status of PFX#9 cells cultured in mTeSR1.

    Fig. S4. Exogenous kynurenine partially rescues IDO1 inhibitor–induced suppression of cell proliferation.

    Fig. S5. Gene expression profiles of mouse Ahr and human AHR.

    Fig. S6. qRT-PCR scorecard panel for H9 cells under ectoderm induction.

    Fig. S7. KAT2 inhibition blocks ectodermal differentiation of H9 cells.

    Fig. S8. Characteristics of EBs.

    Fig. S9. TGF-β1 and FGF2 positively regulate the transcription of NANOG.

    Fig. S10. Kynurenic acid (kynurenate) in culture medium.

    Table S1. Primers used in this study.

    Table S2. Target metabolites and compounds analyzed by LC-MS/MS.

    Data file S1. Summary of LC-MS/MS data for each figure.

    Data file S2. Raw LC-MS/MS data.

  • The PDF file includes:

    • Fig. S1. Biomarkers for the differentiation status of H9 cells cultured in Es8 medium.
    • Fig. S2. Biomarkers for the differentiation status of PFX#9 cells cultured in TeSR-E8.
    • Fig. S3. Biomarkers for the differentiation status of PFX#9 cells cultured in mTeSR1.
    • Fig. S4. Exogenous kynurenine partially rescues IDO1 inhibitor–induced suppression of cell proliferation.
    • Fig. S5. Gene expression profiles of mouse Ahr and human AHR.
    • Fig. S6. qRT-PCR scorecard panel for H9 cells under ectoderm induction.
    • Fig. S7. KAT2 inhibition blocks ectodermal differentiation of H9 cells.
    • Fig. S8. Characteristics of EBs.
    • Fig. S9. TGF-β1 and FGF2 positively regulate the transcription of NANOG.
    • Fig. S10. Kynurenic acid (kynurenate) in culture medium.
    • Table S1. Primers used in this study.
    • Table S2. Target metabolites and compounds analyzed by LC-MS/MS.
    • Legends for data files S1 and S2

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Data file S1 (Microsoft Excel format). Summary of LC-MS/MS data for each figure.
    • Data file S2 (.pdf format). Raw LC-MS/MS data.

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