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