In vitro and in vivo imaging and tracking of intestinal organoids from human induced pluripotent stem cells

Abstract

Human intestinal organoids (hIOs) derived from human pluripotent stem cells (hPSCs) have immense potential as a source of intestines. Therefore, an efficient system is needed for visualizing the stage of intestinal differentiation and further identifying hIOs derived from hPSCs. Here, 2 fluorescent biosensors were developed based on human induced pluripotent stem cell (hiPSC) lines that stably expressed fluorescent reporters driven by intestine-specific gene promoters Krüppel-like factor 5 monomeric Cherry (KLF5) and intestine-specific homeobox enhanced green fluorescence protein (ISX). Then hIOs were efficiently induced from those transgenic hiPSC lines in which mCherry- or eGFP-expressing cells, which appeared during differentiation, could be identified in intact living cells in real time. Reporter gene expression had no adverse effects on differentiation into hIOs and proliferation. Using our reporter system to screen for hIO differentiation factors, we identified DMH1 as an efficient substitute for Noggin. Transplanted hIOs under the kidney capsule were tracked with fluorescence imaging (FLI) and confirmed histologically. After orthotopic transplantation, the localization of the hIOs in the small intestine could be accurately visualized using FLI. Our study establishes a selective system for monitoring the differentiation and for tracking the localization of hIOs and contributes to further improvement of cell-based therapies and preclinical screenings in the intestinal field.-Jung, K. B., Lee, H., Son, Y. S., Lee, J. H., Cho, H.-S., Lee, M.-O., Oh, J.-H., Lee, J., Kim, S., Jung, C.-R., Kim, J., Son, M.-Y. and imaging and tracking of intestinal organoids from human induced pluripotent stem cells.

Interleukin-2 induces the in vitro maturation of human pluripotent stem cell-derived intestinal organoids

“…Three hIO lines were derived from hPSCs following a previously described 13 , 14 , stepwise hIO differentiation protocol: one hIO line was derived from a human embryonic stem cell (hESC) line and two lines were obtained from fully characterized, integration-free human induced pluripotent stem cell (hiPSC) lines reprogrammed from human fibroblasts (Supplementary Fig. 1 ).…”

Comparative analysis of human embryonic stem cell‑derived neural stem cells as an in vitro human model

“…Total RNA was extracted from cells with an RNeasy kit (Qiagen, Inc. Hilden, Germany) and reverse transcribed using a Superscript IV First-Strand Synthesis System kit (Invitrogen; Thermo Fisher Scientific, Inc.) as previously described ( 21 ). The resulting cDNA was diluted 1:10 with deionized water, and 1 µ l of the diluted cDNA was added to Accupower™ PCR PreMix (Bioneer Corp., Daejeon, Korea), 10 pmol/l of specific primers and deionized water to a final volume of 20 µ l. The RT-PCR analysis was performed under the following conditions: 5 min at 95°C; 30–40 cycles of 30 sec at 95°C, 30 sec at 60°C, 30 sec at 72°C, and 5 min extension at 72°C.…”

SETDB1 regulates SMAD7 expression for breast cancer metastasis

“…Quantitative real-time PCR was performed on cDNA samples using Brilliant III Ultra-Fast SYBR® Green QPCR Master Mix (Agilent Technologies), and the signal was detected by AriaMx Real-time PCR System (Agilent Technologies). The fluorescence threshold value was calculated using Agilent Aria 1.6 software (32)(33)(34).…”