Elimination of Reprogramming Transgenes Facilitates the Differentiation of Induced Pluripotent Stem Cells into Hepatocyte-like Cells and Hepatic Organoids

Abstract

Hepatocytes and hepatic organoids (HOs) derived from human induced pluripotent stem cells (hiPSCs) are promising cell-based therapies for liver diseases. The removal of reprogramming transgenes can affect hiPSC differentiation potential into the three germ layers but not into hepatocytes and hepatic organoids in the late developmental stage. Herein, we generated hiPSCs from normal human fibroblasts using an excisable polycistronic lentiviral vector based on the Cre recombinase-mediated removal of the loxP-flanked reprogramming cassette. Comparing the properties of transgene-carrying and transgene-free hiPSCs with the same genetic background, the pluripotent states of all hiPSCs were quite similar, as indicated by the expression of pluripotent markers, embryonic body formation, and tri-lineage differentiation in vitro. However, after in vitro differentiation into hepatocytes, transgene-free hiPSCs were superior to the transgene-residual hiPSCs. Interestingly, the generation and hepatic differentiation of human hepatic organoids (hHOs) were significantly enhanced by transgene elimination from hiPSCs, as observed by the upregulated fetal liver (CK19, SOX9, and ITGA6) and functional hepatocyte (albumin, ASGR1, HNF4α, CYP1A2, CYP3A4, and AAT) markers upon culture in differentiation media. Thus, the elimination of reprogramming transgenes facilitates hiPSC differentiation into hepatocyte-like cells and hepatic organoids with properties of liver progenitor cells. Our findings thus provide significant insights into the characteristics of iPSC-derived hepatic organoids.

An Alternate Approach to Generate Induced Pluripotent Stem Cells with Precise CRISPR/Cas9 Tool

“…We overcome these factors by eliminating the c-MYC gene and controlling the expression with doxycycline induction, respectively. The constant expression of transcription factors present in the reprogramming cassette may interfere with the reprogramming process or functional properties of cells obtained from the differentiation of iPS cells [91][92][93]. Despite drug-inducible reprogramming cassette, there are chances for the leaky expression of reprogramming factors at the stage of differentiation [94].…”

Generation of multilineage liver organoids with luminal vasculature and bile ducts from human pluripotent stem cells via modulation of Notch signaling

“…2 A. BG01-derived HEs, ECs, and HscLCs were dissociated into single-cell suspensions by treatment with TrypLE (12604-021, Gibco) for 3 min and collected using DMEM (12100-046, Gibco) containing 10% FBS (16000-044, Gibco). 1.5 × 10 4 HE, 3 × 10 4 ECs, and 5 × 10 3 HscLCs were seeded together in ultra-low attachment 96-well plates (ULA PrimeSurface® 96U, MS-9096UZ, SIMADZU) in cold multilineage liver organoid differentiation medium [ 37 – 40 ] [DM, advanced DMEM/F12 media supplemented with 1 × HEPES (15630-080, Gibco), 1 × GlutaMAX (35050-061, Gibco), 1 × penicillin/streptomycin (15140-122, Gibco), 0.1 mg/ml BSA (A7096, Sigma), 1 × B27 supplement minus vitamin A (12587-010, Gibco), 25 ng/ml BMP7 (120-03p, Peprotech), 100 ng/ml FGF19 (100-32, Peprotech), 25 ng/ml HGF (CYT-244, Prospec), 10 nM (Leu15)-Gastrin I (G9145, Sigma), 1.25 mM N-acetyl-L-cysteine (A9165, Sigma), 0.5 μM A83-01 (2939, Tocris Bioscience), 10 μM DAPT (ab120633, Abcam), 3 μM dexamethasone (D4902, Sigma), 0.566 mg/ml growth factor-reduced Matrigel (M354230, Corning), 100 ng/ml VEGF-165 (100-20, Peprotech), 50 ng/ml bFGF (100-18b, Peprotech), and 10 μM ROCK inhibitor Y27632 (1254, Tocris Bioscience)]. On days 1 and 3, cold DM was added to the organoid culture.…”