Journal: Stem Cell Research & Therapy
Article Title: One-step genetic correction of hemoglobin E/beta-thalassemia patient-derived iPSCs by the CRISPR/Cas9 system
Figure Lengend Snippet: Hematopoietic differentiation of iPSCs using the feeder-free system. a Schematic of the feeder-free hematopoietic differentiation protocol used in this study. b Morphological changes of the corrected C46 cells during hematopoietic differentiation. c, d Numbers of CD34, CD43, CD235a and CD71-expressing cells in adherent and nonadherent systems at day 12 of differentiation. e Representative images of CFU from HDF-iPSCs. Differentiated cells at day 12 were harvested and seeded in MethoCult media. f Numbers of CFU colonies counted on day 14 of culture in MethoCult media. Data obtained from two independent experiments. Scale bars = 200 μm. IMDM Iscove modified Dulbecco medium, BMP-4 bone morphogenetic protein 4, VEGF vascular endothelial growth factor, KOSR knockout serum replacement, bFGF basic fibroblast growth factor, BSA bovine serum albumin, SCF stem cell factor, TPO thrombopoietin, IL interleukin, FICZ 6-formylindolo[3,2-b]carbazole, HDF human dermal fibroblasts, iPSC induced pluripotent stem cell, Eβ-iPSC2 iPSC lines derived from a patient with HbE/β - thalassemia, CFU-E colony-forming unit erythroid, EPO erythropoietin, BFU-E burst-forming unit erythroid, GM granulocyte, macrophage, GEMM granulocyte, erythrocyte, macrophage, megakaryocyte
Article Snippet: After 24 hours of culture (day 0), the medium was replaced with differentiation medium: day 0–1 medium, RPMI (Gibco) supplemented with 5 ng/ml of human bone morphogenetic protein 4 (hBMP4) (Peprotech), 50 ng/ml of human vascular endothelial growth factor (hVEGF) (Peprotech), 25 ng/ml of hWnt3a (R & D) and 5% Knockout serum replacement (KOSR) (Gibco); day 2 medium, RPMI supplemented with 5 ng/ml of hBMP4, 50 ng/ml of hVEGF, 20 ng/ml basic fibroblast growth factor (bFGF) (Peprotech) and 5% KOSR; day 3 medium, StemPro-34 (Invitrogen), 5 ng/ml of hBMP4, 50 ng/ml of hVEGF and 20 ng/ml bFGF; day 4–5 medium, StemPro-34, 15 ng/ml of hVEGF and 5 ng/ml bFGF; day 6 medium, 74% Iscove modified Dulbecco medium (IMDM) (Gibco), 24% Ham's F12 (Gibco), 1% B27 supplement (Gibco), 0.5% N2 supplement (Gibco), 0.5% bovine serum albumin (BSA), 50 ng/ml of hVEGF, 100 ng/ml of bFGF, 100 ng/ml of human stem cell factor (SCF) (R & D) and 25 ng/ml of hFlt3 ligand (R & D); and day 7 medium, 74% IMDM, 24% Ham's F12, 1% B27 supplement, 0.5% N2 supplement, 0.5% BSA, 50 ng/ml of hVEGF, 100 ng/ml of bFGF, 100 ng/ml of hSCF, 25 ng/ml of hFlt3 ligand, 50 ng/ml of human thrombopoietin (TPO) (R & D), 10 ng/ml of IL-6 (Peprotech), 0.5 U/ml of hEPO (Eprex) and 0.2 μM of 6-formylindolo[3,2-b]carbazole (FICZ) (Abcam).
Techniques: Expressing, Modification, Knock-Out, Derivative Assay