Mitochondrial dysfunction by glyoxalase 1 deficiency disrupts definitive endoderm and alveolar development of human pluripotent stem cells

Abstract

Normal mitochondrial function is essential for human induced pluripotent stem (hiPS) cell differentiation into definitive endoderm (DE). However, the underlying mechanisms that maintain mitochondrial homeostasis during DE differentiation are not fully elucidated. Here we report that glyoxalase 1 (GLO1) is a novel regulator of DE differentiation and subsequent alveolar development in hiPS cells via maintaining mitochondrial homeostasis. To determine the role of GLO1 in these processes, we first established GLO1-knockout hiPS cells using CRISPR-Cas9-mediated genome deletion and demonstrated that GLO1 deficiency significantly reduced the differentiation efficiency of DE, leading to defects in alveolar epithelial cell differentiation and alveolar organoid development. Moreover, GLO1 deficiency interfered with mitochondrial biogenesis and respiration during the early DE stage. Defects in DE differentiation due to dysfunctional mitochondria were effectively rescued by high-dose treatment with CHIR99021, a glycogen synthase kinase 3 inhibitor. Our study uncovered an essential role of GLO1 as a key regulator of mitochondrial homeostasis for early lineage specification of hiPS cells, moving away from its conventional role as a primary enzyme in methylglyoxal detoxification.

Fig. 1. GLO1 deficiency disturbs hiPS cell-derived DE differentiation.

a Scheme of the DE differentiation of hiPS cells. b qRT-PCR analysis of pluripotency (POU5F1 and SOX2) and DE-specific genes (SOX17, CXCR4, GATA6 and FOXA2) in WT and GLO1^−/− hiPS cells at day 4. Data are normalized to the mRNA levels from day 0. c Flow cytometric analysis to determine temporal induction efficiency between WT and GLO1^−/− hiPS cells. CXCR4⁺ cells represent DE cells. Isotype: an antibody that lacks specificity for the target proteins but matches the immunoglobulin class of the primary antibody. d Immunofluorescence staining of DE cells at day 4 of differentiation. CXCR4 (red) and SOX17 (red) served as DE markers. OCT4 served as a pluripotency marker. In the bright-field image, the white marking designates the area in which differentiation occurs, extending from the edge of the colony. Scale bars, 200 μm. e qRT-PCR analysis for DE-positive regulators in WT and GLO1^−/− hiPS cells. f Apoptosis analysis of WT and GLO1^−/− hiPS cells using Annexin V and PI staining. Apoptosis cells (%) were calculated by summing the Annexin V⁺PI⁻ (early apoptosis, red box) and Annexin V⁺PI⁺ (late apoptosis, blue box) populations, and the combined values are represented in the graph. g Western blot analysis of MG-H1 protein expression in undifferentiated WT and GLO1^−/− hiPS cells. h Western blot analysis of MG-H1 protein expression in WT and GLO1^−/− hiPS cells at DE day 4. The sum of the band densities from all detected bands was analyzed, and the resulting values are shown in the bar graph. All data are shown as the means ± s.d. n = 3. Statistical analysis was performed using a two-way ANOVA and Šídák’s multiple-comparisons test (b) or unpaired Student’s t-test (c, d, f and g). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 2. Effects of GLO1 efficiency on AEP differentiation, LO and AO development.

a Schematic of the stepwise induction protocol for generating AECs and AOs from hiPS cells. VAFE, ventralized alveolar foregut endoderm; ADAE, airway distal alveolar epithelium. b Transcript levels of pluripotency (OCT4 and NANOG)- and AEP (NKX2.1 and EPCAM)-related genes in WT and GLO1^−/− hiPS cells at day 14 of differentiation. c MFIs of NKX2.1⁺ and EPCAM⁺ cells in WT and GLO1^−/− hiPS cells at day 14 of differentiation. d Representative bright-field images of AO generated from WT and GLO1^−/− hiPS cells at day 28 of differentiation. Scale bars, 200 μm. e AO diameter was measured on day 28 of the organoid culture, and the mean was calculated (ten AOs per group). f Representative bright-field images at various days of differentiation, including day 0 (24 h after single-cell plating), days 6–10 (AF endoderm, AFE) and days 14–27 (collection of floating AF spheroids and induction to LOs). AF spheroids (white arrows) begin to form on approximately days 3–4 of AFE differentiation. Scale bars, 100 and 200 μm. hPSCs, hPS cells. All data are shown as the means ± s.d. n = 3 (b and c) and n = 10 (e). Statistical analysis was performed using a one-way ANOVA and Dunnett’s multiple-comparisons test (e) or unpaired Student’s t-test (b and c). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 3. GLO1 deficiency impairs mitochondrial homeostasis in hiPS cells.

a Relative mRNA expression levels of mitochondrial biogenesis-related genes (NRF1, TFAM and PGC1α) in undifferentiated WT and GLO1^−/− hiPS cells were determined by qRT-PCR. b Mitochondrial masses in WT and GLO1^−/− hiPS cells were measured by MitoTracker Green FM staining analysis. Scale bars, 100 μm. c Representative TEM images of mitochondrial structures in WT and GLO1^−/− hiPS cells. Scale bars, 500 nm. d Calculated mitochondrial C-MAX (long-axis length)/C-MIN (short-axis length) values in WT and GLO1^−/− hiPS cells. e The kinetic profile of the OCR was measured in undifferentiated WT and GLO1^−/− hiPS cells using the Seahorse XF Real-Time assay. Black lines show times of treatment with oligomycin, FCCP and rotenone/antimycin A (Rot/AA). f The basal respiration and maximal respiration in undifferentiated WT and GLO1^−/− hiPS cells. g The kinetic profile of the ECR was measured in WT and GLO1^−/− hiPS cells using the Seahorse XF Real-Time assay. Black lines show times of treatment with Rot/AA and 2-DG. h The basal and compensatory glycolysis in WT and GLO1^−/− hiPS cells. All data are shown as the means ± s.d. n = 3 (a and e–i), n = 7 (b), n = 51 (WT hiPS cells in c) and n = 69 (GLO1^–/– hiPS cells in c). Statistical analysis was performed using a two-way ANOVA (i) or unpaired Student’s t-test (a, b, d, f and h). *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 4. High-dose CHIR99021 restores mitochondrial function in GLO1^−/− hiPS cells.

a Schematic of DE differentiation using a range of doses of CHIR99021 (1, 2.5 and 5 μM) together with Activin A (100 ng/ml) in WT and GLO1^−/− hiPS cells. b Relative mRNA expression levels of NRF1, TFAM and PGC1α on day 4 were determined by qRT-PCR. c, Representative confocal images of mitochondria visualized by MitoTracker Green staining in WT and GLO1^−/− hiPS cells. Scale bars, 100 μm. d, Mitochondrial masses in WT and GLO1^−/− hiPS cells were measured by fluorescence intensity of MitoTracker Green FM staining. e Representative TEM images of the mitochondrial structure. f Calculated mitochondrial C-MAX/C-MIN values. g, The kinetic profile of the OCR was measured in WT and GLO1^−/− hiPS cells using the Seahorse XF Real-Time assay. Black lines show times of treatment with oligomycin, FCCP and Rot/AA. h, The basal respiration and maximal respiration in WT and GLO1^−/− hiPS cells. i Relative mRNA expression levels of mitochondrial fusion (MFN1, MFN2 and OPA1)-related genes were determined by qRT-PCR. All data are shown as the means ± s.d. n = 3 (b and g–i), n = 8 (d), n = 67 (WT 1 μM in f), n = 80 (GLO1^−/− 1 μM in f), n = 71 (GLO1^−/− 2.5 μM in f) and n = 45 (GLO1^−/− 5 μM in f). Statistical analyses were performed using a one-way ANOVA and Tukey’s multiple-comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 5. Rescue of the DE and AEC differentiation defects of GLO1^−/− hiPS cells by high-dose treatment with CHIR99021.

a Representative FACS dot plots showing the frequencies of SOX17⁺CXCR4⁺ DE cells (day 4) derived from GLO1^−/− hiPS cells treated with CHIR99021 (1, 2.5 and 5 μM). The WT hiPS cells were treated with 1 μM CHIR99021. b MFIs of SOX17⁺ and CXCR4⁺ cells in WT and GLO1^−/− hiPS cells at day 4 of differentiation. c qRT-PCR analysis for DE-positive regulators in WT and GLO1^−/− hiPS cells treated with CHIR99021. d, Representative FACS dot plots showing the effects of CHIR99021 treatment on the generation of EPCAM⁺NKX2.1⁺ alveolar progenitors from WT and GLO1^−/− hiPS cells at day 14 of differentiation. e, The graphs show the frequencies of EPCAM⁺NKX2.1⁺, EPCAM+ and NKX2.1 subpopulations. f, g, Relative mRNA expression levels of mitochondrial biogenesis- (f) and fusion- (g) related genes in AECs at day 14 of differentiation derived from WT and GLO1^−/− hiPS cells were determined by qRT-PCR. h, Representative histograms showing the frequencies of SFTPC+ type 2 AECs at day 21 of differentiation. i, Data are normalized to the frequency of the WT control treated with 1 μM CHIR99021. All data are shown as the means ± s.d. n = 3. Statistical analyses were performed using a one-way ANOVA and Tukey’s multiple-comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 6. Effects of high-dose CHIR99021 treatment on the expression and nuclear translocation of β-catenin in GLO1^−/− hiPS cells during DE induction.

a Non-phospho (active) β-catenin protein levels in WT and GLO1^−/− hiPS cells were analyzed by western blot analysis after 3 h of DE differentiation with 0, 1, 2.5 and 5 μM CHIR99021 treatment. b, Representative fluorescence images showing immunocytochemistry staining of non-phospho (active) β-catenin protein in WT and GLO1^−/− hiPS cells after 3 h of DE differentiation with 1, 2.5 and 5 μM CHIR99021. c, The bar graph represents the average fluorescence intensity of non-phospho (active) β-catenin in WT and GLO1^−/− hiPS cells. Western blot analysis was performed by collecting total protein lysates to compare non-phospho (active) β-catenin expression in response to different concentrations of CHIR99021 (0, 1, 2.5 and 5 μM) in WT and GLO1^−/− hiPS cells. All data are shown as the means ± s.d. n = 3. Statistical analyses were performed using a one-way ANOVA and Tukey’s multiple-comparisons test. ns not significant, *P < 0.05, **P < 0.01.