Journal: bioRxiv

Article Title: ER stress and lipid imbalance drive embryonic cardiomyopathy in a human heart organoid model of pregestational diabetes

doi: 10.1101/2023.06.07.544081

Figure Lengend Snippet: A , Immunofluorescence images of cardiomyocytes from NHOs and PGDHOs stained with the cardiomyocyte marker TNNT2 (red) and the nuclear marker DAPI (blue); scale bar: 10 µm. B , Quantification of cardiomyocyte area from NHOs and PGDHOs; n=4 organoids, n=145 cardiomyocytes for NHOs and n=193 cardiomyocytes for PGDHOs, nested t-test. C , Immunofluorescence of mitochondria using MitoTracker showing mitochondrial swelling in PGDHOs; arrowheads indicated swollen mitochondria, scale bar: 10 µm. D , Quantification of mitochondrial swelling in NHOs and PGDHOs; n=4; nested t-test. E , Live ROS imaging employing CellROX Green; scale bar: 10 µm. F , Quantification of ROS content in NHOs and PGDHOs; n=3; nested t-test. G–J , Time course qRT-PCR gene expression analysis of key developmental transcription factors from day 0 to day 14 of differentiation; n=9 (3 biological replicates of 3 pooled organoids). L , Calcium transient live imaging using Fluo-4 from NHOs (left) and PGDHOs (right), and M , Quantification of beats per minute in NHOs and PGDHOs; n=7; value = mean ± SD, unpaired t-tests.

Article Snippet: Organoids were dissociated into a single-celled suspension using a modified protocol using the STEMdiff Cardiomyocyte Dissociation Kit (STEMCELL Technologies).

Techniques: Immunofluorescence, Staining, Marker, Imaging, Quantitative RT-PCR, Gene Expression

Journal: bioRxiv

Article Title: ER stress and lipid imbalance drive embryonic cardiomyopathy in a human heart organoid model of pregestational diabetes

doi: 10.1101/2023.06.07.544081

Figure Lengend Snippet: A , t-SNE plot showing 5,951 cells from 4 pooled dissociated NHO organoids, B , t-SNE plot of 7,463 cells from 4 pooled dissociated PGDHO organoids, distributed by k-means clustering. Each cluster was named based on the expression of key genes in the respective cell populations. C, Cardiac cell composition in NHOs and PGDHOs expressed as percentage of cells. D , t-SNE plots showing key markers for cardiomyocytes and ( E ) and epicardial cells. F , Bubble plot with top DEGs specific for epicardial cells and ( G ) cardiomyocytes. Size of bubble corresponds to p-value between NHOs and PGDHOs. H , Gene ontology analysis of biological processes associated with significantly expressed genes in the epicardial cluster and ( I ) the epicardial cluster ( H ).

Article Snippet: Organoids were dissociated into a single-celled suspension using a modified protocol using the STEMdiff Cardiomyocyte Dissociation Kit (STEMCELL Technologies).

Techniques: Expressing

Journal: bioRxiv

Article Title: ER stress and lipid imbalance drive embryonic cardiomyopathy in a human heart organoid model of pregestational diabetes

doi: 10.1101/2023.06.07.544081

Figure Lengend Snippet: A , Immunofluorescence images of day 14 NHOs and PGDHOs showing colocalization of ROS (CellROX, green) and ER marker (ER Tracker, red); nuclear marker DAPI (blue); scale bar=10 µm. B , Quantification ROS localized in the ER; n=10 organoids per condition; value = mean ± SD, unpaired t-tests. C , Immunofluorescence images of day 14 NHOs and PGDHOs stained for phosphorylated IRE1 (IRE1p, green), cardiomyocyte marker TNNT2 (red) and nuclear marker DAPI; n=6; scale bar=20 µm. D , Quantification of the ratio of phosphorylated IRE1 to unphosphorylated IRE1 compared to NHOs, measured by immunofluorescence image analysis; n=7 for NHOs, n=8 for PGDHOs; value = mean ± SD, unpaired t-tests. E, LC-MS lipidomic analysis for VLCFA concentrations from day 15 organoids and their corresponding medium; n=9 organoids per condition, value = mean ± SD, unpaired t-test, *p<0.05. F , Heatmap representing expression level of key enzymes involved in LCFA and VLCFA biosynthesis. G , Predicted secondary FADS2 mRNA structure. H , qRT-PCR gene expression analysis of FADS2 ; n=6 and n=3 biological replicates of 3 pooled organoids for ERN1 knockdown and KIRA8 respectively; value = mean ± SD. I , FADS2 protein measurement by ELISA; n=8 organoids per condition; value = mean ± SD.

Article Snippet: Organoids were dissociated into a single-celled suspension using a modified protocol using the STEMdiff Cardiomyocyte Dissociation Kit (STEMCELL Technologies).

Techniques: Immunofluorescence, Marker, Staining, Liquid Chromatography with Mass Spectroscopy, Expressing, Quantitative RT-PCR, Gene Expression, Knockdown, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: ER stress and lipid imbalance drive embryonic cardiomyopathy in a human heart organoid model of pregestational diabetes

doi: 10.1101/2023.06.07.544081

Figure Lengend Snippet: A, Immunofluorescence images of day 14 NHOs and PGDHOs treated with TUDCA, BH4, or omega-3 fatty acids, stained with phosphorylated IRE1 (IRE1p, green), cardiomyocyte marker TNNT2 (red) and nuclear marker DAPI (blue); n=6; scale bar: 25 µm. B , Quantification of the ratio phosphorylated IRE1 to unphosphorylated IRE1, measured by immunofluorescence image analysis; n=6; value = mean ± SD, one-way ANOVA. C , Immunofluorescence images of ROS (CellROX, green) and ER marked (ER Tracker, red) in day 14 NHOs and PGDHOs; n=7; scale bar = 10 µm. D , Quantification of ROS content compared to NHOs; n=6; value = mean ± SD, one-way ANOVA. E , Immunofluorescence images of cardiomyocytes dissociated from day 14 NHOs, PGDHOs; n=4; scale bar = 10 µm. F , Quantification of cardiomyocyte area compared to NHOs; n=4; value = mean ± SD, one-way ANOVA. G , qRT-PCR gene expression analysis of FADS2 in NHOs and PGDHOs; n=6 biological replicates of 3 pooled organoids; value = mean ± SD. H , Schematic diagram summarizing the mechanism of PGD-induced CHD in human heart organoids.

Article Snippet: Organoids were dissociated into a single-celled suspension using a modified protocol using the STEMdiff Cardiomyocyte Dissociation Kit (STEMCELL Technologies).

Techniques: Immunofluorescence, Staining, Marker, Quantitative RT-PCR, Gene Expression