Journal: International Journal of Molecular Sciences

Article Title: Toward Xeno-Free Differentiation of Human Induced Pluripotent Stem Cell-Derived Small Intestinal Epithelial Cells

doi: 10.3390/ijms23031312

Figure Lengend Snippet: Differentiation toward small intestinal epithelium ( a ) Intracellular localization of small intestinal epithelial cell markers in SIEC-iPSCs cultured either on Geltrex or laminin 511. Caco-2 cells grown on Geltrex served as a control. Enterocyte marker proteins PEPT1 (green) and villin (green), and enteroendocrine cell marker chromogranin A (CGA, green) were visualized with indirect immunofluorescence and confocal microscopy. In samples stained with Chromogranin A, the arrowhead in Geltrex, and arrow in laminin iPSC-SIEC samples point to extracellular matrix. Number of biological replicates n = 4, number of technical replicates n = 2. Scalebar 20 µm. ( b ) The expression of enterocyte marker genes PEPT1, villin and CYP3A4 was analyzed with qRT-PCR. The expression of iPSC-SIECs were calibrated against iPSC-SIEC cultured on Geltrex. Number of biological replicates n = 3, number of technical replicates in every biological replicate n = 3. Data expressed as mean ± SD. The statistical significance: *** ( p ≤ 0.001).

Article Snippet: During the time of the experiment, the karyotypes of iPSC line was normal (Fimlab Laboratories, Tampere, Finland). iPSCs were maintained on Geltrex (Gibco; Thermo Fisher Scientific, Waltham, MA, USA; diluted 1:100) coated Cell-Bind treated 6-well plates (Corning, Corning, NY, USA) in mTeSR1 medium (StemCell Technologies, Vancouver, BC, Canada) with penicillin–streptomycin (P/S; Gibco, Paisley, UK).

Techniques: Cell Culture, Control, Marker, Immunofluorescence, Confocal Microscopy, Staining, Expressing, Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: Toward Xeno-Free Differentiation of Human Induced Pluripotent Stem Cell-Derived Small Intestinal Epithelial Cells

doi: 10.3390/ijms23031312

Figure Lengend Snippet: Small intestinal epithelial cell specific functionality assessed with dipeptide uptake assay. Posterior definitive endoderm differentiated iPSCs were cultured on Geltrex or Laminin511 and differentiated toward small intestinal epithelial cells. Caco-2 cells cultured on Geltrex served as control. ( a ) The intensity of fluorescence analyzed from micrographs after dipeptide (D-Ala-Leu-Lys-AMCA) uptake analyses +/− ibuprofen treatment. ( b ) iPSC-SIEC cultured on Geltrex, CTRL i.e., without ibuprofen, ( c ) iPSC-SIEC cultured on laminin, CTRL, i.e., without ibuprofen, ( d ) Caco-2 cultured on Geltrex, CTRL, i.e., without ibuprofen, ( e ) iPSC-SIEC cultured on Geltrex, with ibuprofen, ( f ) iPSC-SIEC cultured on laminin, with ibuprofen, ( g ) Caco-2 cultured on Geltrex, with ibuprofen. Number of biological replicates n = 3–4, number of technical replicates n = 3. Data expressed as mean ± SD. The statistical significance: ** ( p ≤ 0.005) and *** ( p ≤ 0.001) indicating the significance between the indicated samples.

Article Snippet: During the time of the experiment, the karyotypes of iPSC line was normal (Fimlab Laboratories, Tampere, Finland). iPSCs were maintained on Geltrex (Gibco; Thermo Fisher Scientific, Waltham, MA, USA; diluted 1:100) coated Cell-Bind treated 6-well plates (Corning, Corning, NY, USA) in mTeSR1 medium (StemCell Technologies, Vancouver, BC, Canada) with penicillin–streptomycin (P/S; Gibco, Paisley, UK).

Techniques: Cell Culture, Control, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Toward Xeno-Free Differentiation of Human Induced Pluripotent Stem Cell-Derived Small Intestinal Epithelial Cells

doi: 10.3390/ijms23031312

Figure Lengend Snippet: The functionality of efflux transport proteins. The functionality of efflux transporters was assessed with calcein retention assay in the absence (=CTRL) or presence of efflux protein inhibitors Cyclosporin A or verapamil from the small intestinal epithelial cells differentiated from posterior definitive endoderm on Geltrex or laminin511. ( a ) Functionality, which is seen as retention, is expressed as a percentage of fluorescence relative to the control (control = 100%, is marked with the red line. ( b – j ) micrographs taken to quantitate retention efficiency ( b ) iPSC-SIEC cultured on Geltrex, without inhibitors (CTRL), ( c ) iPSC-SIEC cultured on laminin, without inhibitors (CTRL), ( d ) Caco-2 cultured on Geltrex, without inhibitors (CTRL), ( e ) iPSC-SIEC cultured on Geltrex, with Cyclosporin A, ( f ) iPSC-SIEC cultured on laminin, with Cyclosporin A, ( g ) Caco-2 cultured on Geltrex, with Cyclosporin A. ( h ) iPSC-SIEC cultured on Geltrex, with Verapamil, ( i ) iPSC-SIEC cultured on laminin, with Verapamil, ( j ) Caco-2 cultured on Geltrex, with Verapamil. Scalebar 100 µm. Number of biological replicates n = 3–4, number of technical replicates n = 3. Data expressed as mean ± SD. The statistical significance: * ( p ≤ 0.05), ** ( p ≤ 0.005) and *** ( p ≤ 0.001) indicating the significance between the indicated samples.

Article Snippet: During the time of the experiment, the karyotypes of iPSC line was normal (Fimlab Laboratories, Tampere, Finland). iPSCs were maintained on Geltrex (Gibco; Thermo Fisher Scientific, Waltham, MA, USA; diluted 1:100) coated Cell-Bind treated 6-well plates (Corning, Corning, NY, USA) in mTeSR1 medium (StemCell Technologies, Vancouver, BC, Canada) with penicillin–streptomycin (P/S; Gibco, Paisley, UK).

Techniques: Fluorescence, Control, Cell Culture

Journal: Frontiers in Pharmacology

Article Title: Gene editing improves endoplasmic reticulum-mitochondrial contacts and unfolded protein response in Friedreich’s ataxia iPSC-derived neurons

doi: 10.3389/fphar.2024.1323491

Figure Lengend Snippet: Generation of isogenic FRDA iPSC cohort using gene editing. (A) Four FRDA iPSC cell lines were generated with varying pathogenic GAA repeat numbers. (B) Schematic of dual CRISPR guide gene editing approach for GAA repeat deletion and PCR primer locations for validation (C) Pluripotency marker expression of resulting cell lines (D) Gel electrophoresis of PCR products from the GE and NGE primers identified in (B) . (E) Quantification of FXN mRNA expression analyzed with ddPCR. Human TBP (TATA-box binding protein) was used as reference gene. (F) Western blot for mature, mitochondrial FXN protein expression with β-actin as loading control. (G) Quantification of FXN protein expression normalized to β-actin. 2-3 biological replicates of all cell lines were used for both mRNA and protein expression studies where ●corresponds to 264 control cell line, ■ represents FF1 FRDA and E35 gene corrected cell line in the respective columns, ▲ FF2 FRDA and FF2 Edited, in respective columns, ♦ 223 FRDA and 223 Edited, in respective columns and ★ corresponds to 850 FRDA and 850 Edited lines in the respective columns. Data are represented as Mean +SEM and groups are analyzed with one-way ANOVA where * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The iPSC FRDA FF2 line (denoted as in graphs) was purchased from Coriell Institute (#GM23913).

Techniques: Generated, CRISPR, Biomarker Discovery, Marker, Expressing, Nucleic Acid Electrophoresis, Binding Assay, Western Blot, Control

Journal: Frontiers in Pharmacology

Article Title: Gene editing improves endoplasmic reticulum-mitochondrial contacts and unfolded protein response in Friedreich’s ataxia iPSC-derived neurons

doi: 10.3389/fphar.2024.1323491

Figure Lengend Snippet: CRISPR/Cas9 gene editing prevents apoptosis of FRDA iPSC-derived neurons. (A) Representative confocal images of control, FRDA and gene edited neurons at different time points of differentiation (1, 2, 3 and 4 weeks) immunolabelled for neuronal marker ß-tubulin III. (B) Quantification of neuronal blebs across different time points; 5-8 neurons and 10 fields per cell line were used for quantification. (C) Representative confocal images of control, FRDA and gene edited neurons showing increasing caspase 3-positive (green) neurons from 1 week to 4 weeks. Cells were stained with ß-tubulin III (red) as neuronal marker and DAPI for nucleus. (D) Quantification of caspase 3, ß-tubulin positive neurons; 5-8 neurons and 10 fields per cell line were used for quantification. 2-3 biological replicates of all cell lines were used where ●corresponds to 264 control cell line, ■ represents FF1 FRDA and E35 gene corrected cell line in the graph, ▲ FF2 FRDA and FF2 Edited, in respective columns, and ♦ 223 FRDA and 223 Edited, in respective columns. Data are represented as Mean ± SEM and groups are analyzed with one-way ANOVA where * p > 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Scale bar, 50 μm.

Article Snippet: The iPSC FRDA FF2 line (denoted as in graphs) was purchased from Coriell Institute (#GM23913).

Techniques: CRISPR, Derivative Assay, Control, Marker, Staining

Journal: Frontiers in Pharmacology

Article Title: Gene editing improves endoplasmic reticulum-mitochondrial contacts and unfolded protein response in Friedreich’s ataxia iPSC-derived neurons

doi: 10.3389/fphar.2024.1323491

Figure Lengend Snippet: CRISPR/Cas9 mediated FXN gene editing improves mitochondrial structure and function in 2-week differentiated iPSC-derived FRDA neurons. (A) Representative images of transmission electron microscopy of mitochondria in control, FRDA and gene edited neurons at 2-week of differentiation (Scale bar 500 nm). (B) Quantification of mitochondrial numbers per cell and mitochondria area in control, FRDA and gene edited neurons. n = 3-4 cells for each cell line were used for quantification. (C) Representative quantification of mitochondrial superoxide levels analyzed with MitoSOX Red. Nuclei were stained with Hoechst 33342. (D) Quantification of MitoSOX Red fluorescence intensity per cell; 5-8 neurons per field were analyzed for each cell line and data was normalized to fluorescence intensity per cell. (E) Representative immunoblotting for expression of the mitochondrial electron transport chain proteins NDUFB8 and SDHB. (F) Quantification of expression of NDUFSB8 and SDHB as seen by Western blot analysis (E) and normalized to β-actin as a loading control. 2-3 biological replicates of all cell lines were used where ●corresponds to 264 control cell line, ■ represents FF1 FRDA cell line in the graph, ▲ FF2 FRDA and FF2 Edited, in respective columns, ♦ 223 FRDA and 223 Edited, in respective columns and ★ corresponds to 850 FRDA and 850 Edited lines in the respective columns. Data are represented as Mean +SEM and groups are analyzed with one-way ANOVA where * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The iPSC FRDA FF2 line (denoted as in graphs) was purchased from Coriell Institute (#GM23913).

Techniques: CRISPR, Derivative Assay, Transmission Assay, Electron Microscopy, Control, Staining, Fluorescence, Western Blot, Expressing

Journal: Frontiers in Pharmacology

Article Title: Gene editing improves endoplasmic reticulum-mitochondrial contacts and unfolded protein response in Friedreich’s ataxia iPSC-derived neurons

doi: 10.3389/fphar.2024.1323491

Figure Lengend Snippet: Gene editing to remove the detrimental FRDA GAA intronic repeat alters the transcriptome profile of FRDA neurons. (A) Volcano plot of differential gene expression delineating genes that are increased (DEG, red) and decreased (DEG, blue) in FRDA as compared to gene edited neurons from the same individuals (916 DEGs). (B) Gene ontology of DEGs decreased in FRDA neurons compared to gene edited neurons. (C) Ingenuity pathway analysis (IPA) of upstream regulators decreased in FRDA neurons (D) Gene ontology of genes increased in FRDA neurons compared to gene edited controls. (E) IPA analysis of genes increased in FRDA neurons. (F) Bar charts of differentially expressed genes related to XBP1 signaling and (G) ER associated protein degradation (ERAD) showing correction with CRISPR repeat deletion (Edited) when compared to isogenic control with homology directed GAA repeat repair (one way ANOVA, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). (H) Venn diagram comparing DEGs from FRDA neurons and isogenic repeat corrected controls with the DEGs here showing statistically significant overlap. (I) Gene ontology of overlap genes from (G) .

Article Snippet: The iPSC FRDA FF2 line (denoted as in graphs) was purchased from Coriell Institute (#GM23913).

Techniques: Gene Expression, CRISPR, Control

Journal: Frontiers in Pharmacology

Article Title: Gene editing improves endoplasmic reticulum-mitochondrial contacts and unfolded protein response in Friedreich’s ataxia iPSC-derived neurons

doi: 10.3389/fphar.2024.1323491

Figure Lengend Snippet: Dysregulation of endoplasmic reticulum stress pathway genes. Ingenuity Pathway Analysis of the ER stress pathway highlighting differentially expressed genes in FRDA neurons (labeled in blue) representing extensive dysregulation of ER stress in FRDA neurons.

Article Snippet: The iPSC FRDA FF2 line (denoted as in graphs) was purchased from Coriell Institute (#GM23913).

Techniques: Labeling

Journal: Frontiers in Pharmacology

Article Title: Gene editing improves endoplasmic reticulum-mitochondrial contacts and unfolded protein response in Friedreich’s ataxia iPSC-derived neurons

doi: 10.3389/fphar.2024.1323491

Figure Lengend Snippet: Restoration of mitochondrial-ER contacts in CRISPR-Cas9 gene edited iPSC-neurons. (A) qRT-PCR based validation of XBP1 expression where Total XBP1(XBP1-t) was normalized to GAPDH as internal control. Unspliced XBP1 (XBP1-u) and active, spliced XBP1 (XBP1-s) are normalized to (XBP1-t). (B) Thapsigargin (1 µM) mediated ER-calcium release analyzed with Cal Red R525/650 a.m. Quantification was performed on 10–20 neurons in 3-4 different batches of neuronal differentiation per cell line. All cell lines were used for both studies and in the graph, ●corresponds to 264 control cell line, ■ represents FF1 FRDA and E35 cell line, in respective columns, ▲ FF2 FRDA and FF2 Edited, in respective columns, ♦ 223 FRDA and 223 Edited, in respective columns and ★ corresponds to 850 FRDA and 850 Edited lines in respective columns. (C) Representative confocal images from proximity ligation assay (PLA) depicting VDAC and IP3R interactions in control, FRDA and gene-edited neurons. (D) Quantitative analysis of PLA puncta per nuclei shows a strong decrease of mitochondria-ER contacts in FRDA neurons compared to control line. Quantification was performed on 60–80 neurons per cell line from three independent experiments where E35 was used as control (■), FF2 as representative of FRDA and FF2 Edited as representative of CRISPR/Cas9 edited cell line (▲). PLA puncta are shown as the number of puncta/nuclei. (E) Representative transmission electron microscopic images from control, FRDA and gene edited neurons showing no mitochondrial-ER contact sites and lack of ER structures in FRDA neurons. ER-Endoplasmic reticulum; Scale bar 500 nm. Data are represented as Mean +SEM and groups are analyzed with one-way ANOVA where * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The iPSC FRDA FF2 line (denoted as in graphs) was purchased from Coriell Institute (#GM23913).

Techniques: CRISPR, Quantitative RT-PCR, Biomarker Discovery, Expressing, Control, Proximity Ligation Assay, Transmission Assay

Journal: Molecular psychiatry

Article Title: DPYSL2/CRMP2 isoform B knockout in human iPSC-derived glutamatergic neurons confirms its role in mTOR signaling and neurodevelopmental disorders

doi: 10.1038/s41380-023-02186-w

Figure Lengend Snippet: a DPYSL2 isoforms A (top) and B (bottom). The first exon of DPYSL2-B (boxed in red) was targeted to avoid impacting other transcripts. Image adapted from the GTEx portal. b Top panel shows sgRNA3 (green) and PAM (orange) used to create the frameshift mutation in the coding sequence (CDS, yellow) in exon 1. Bottom two panels show Sanger sequencing traces of DNA from control and frameshift iPSCs. c qRT-PCR results showing significantly reduced expression of DPYSL2-B in frameshift iPSC clones (n = 4) compared to control clones (n = 6 including 5 cell lines) (p = 0.001, t-test). Data were normalized to the geometric mean of GAPDH and β-actin loading controls, then to the average of the control clones. d Western blot comparing CRMP2-B (~64 kD, green) abundance in control (n = 5) and frameshift (n = 4) iPSC clones with GAPDH ( ~ 37 kD, red) as a loading control. e Quantification of (d) showing significantly reduced abundance of CRMP2-B in frameshift iPSCs compared to controls (p = 4.5 × 10−6, t-test). Data were normalized to GAPDH and then to the average of the control clones.

Article Snippet: Plasmids were isolated (Qiagen; Maryland, USA; 27104) and screened for correct insertion via Sanger sequencing with specialized primers (IDT, Supplementary Table 1 ). iPSC quality testing, culture and maintenance The iPSC line used for this study (MH0185922 aka RU13, African male control) was obtained from the NIMH/RUCDR repository ( https://www.nimhgenetics.org/ ).

Techniques: Mutagenesis, Sequencing, Control, Quantitative RT-PCR, Expressing, Clone Assay, Western Blot

Journal: Stem cell research

Article Title: Generation of an induced pluripotent stem cell line (TRNDi030-A) from a patient with Farber disease carrying a homozygous p. Y36C (c. 107 A>G) mutation in ASAH1

doi: 10.1016/j.scr.2021.102387

Figure Lengend Snippet: Resource Table

Article Snippet: This study has established a human iPSC line (TRNDi030-A) from the fibroblasts of a male patient with Farber disease (GM20015, Coriell Institute) carrying a homozygous mutation, p. Y36C (c.107 A>G), in the second exon of ASAH1 ( , ).

Techniques: Mutagenesis