Generation of Mouse and Human Organoid-Forming Intestinal Progenitor Cells by Direct Lineage Reprogramming

Miura, Shizuka; Suzuki, Atsushi

doi:10.1016/j.stem.2017.08.020

Cited by 78 publications

(85 citation statements)

References 38 publications

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“…Human HNF4A and FOXA3 cDNAs and human HNF1A cDNA were obtained by RT‐PCR. The cDNAs were subcloned into pGCDNsam‐IRES‐EGFP (a gift from M. Onodera, National Center for Child Health and Development, Tokyo, Japan), a retroviral vector with a long terminal repeat derived from murine stem cell virus .…”

Section: Methodsmentioning

confidence: 99%

“…The cDNAs were subcloned into pGCDNsam‐IRES‐EGFP (a gift from M. Onodera, National Center for Child Health and Development, Tokyo, Japan), a retroviral vector with a long terminal repeat derived from murine stem cell virus . Recombinant retroviruses were produced as described . Briefly, plasmid DNA was transfected into Plat‐GP cells (Cell Biolabs, San Diego, CA, USA) using linear polyethylenimine (PEI) (Polysciences, Taipei, Taiwan).…”

Section: Methodsmentioning

confidence: 99%

“…18 Recombinant retroviruses were produced as described. 17 Briefly, plasmid DNA was transfected into Plat-GP cells (Cell Biolabs, San Diego, CA, USA) using linear polyethylenimine (PEI) (Polysciences, Taipei, Taiwan). At 3 days before transfection, Plat-GP cells (1.8 × 10 6 ) were plated on poly-L-lysine-coated 10-cm dishes.…”

Section: Retrovirus Production and Transduction Of Cellsmentioning

confidence: 99%

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Prolonged inhibition of hepatocellular carcinoma cell proliferation by combinatorial expression of defined transcription factors

et al. 2018

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Hepatocellular carcinoma (HCC) accounts for a large proportion of liver cancer cases and has an extremely poor prognosis. Therefore, novel innovative therapies for HCC are strongly desired. As gene therapy tools for HCC, 2 hepatic transcription factors (TF), HNF4A and HNF1A, have been used to suppress proliferation and to extinguish cancer‐specific characteristics of target cells. However, our present data demonstrated that single transduction of HNF4A or HNF1A had only a limited effect on suppression of HCC cell proliferation. Thus, in this study, we examined whether combinations of TF could show more effective antitumor activity, and found that combinatorial transduction of 3 hepatic TF, HNF4A, HNF1A and FOXA3, suppressed HCC cell proliferation more stably than single transduction of these TF. The combinatorial transduction also suppressed cancer‐specific phenotypes, such as anchorage‐independent growth in culture and tumorigenicity after transplantation into mice. HCC cell lines transduced with the 3 TF did not recover their proliferative property after withdrawal of anticancer drugs, indicating that combinatorial expression of the 3 TF suppressed the growth of all cell subtypes within the HCC cell lines, including cancer stem‐like cells. Transcriptome analyses revealed that the expression levels of a specific gene set involved in cell proliferation were only decreased in HCC cells overexpressing all 3 TF. Moreover, combined transduction of the 3 TF could facilitate hepatic differentiation of HCC cell lines. Our strategy for inducing stable inhibition and functional differentiation of tumor cells using a defined set of TF will become an effective therapeutic strategy for various types of cancers.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Retrovirus Production and Transduction Of Cellsmentioning

confidence: 99%

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Prolonged inhibition of hepatocellular carcinoma cell proliferation by combinatorial expression of defined transcription factors

et al. 2018

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“…For example, myoblasts, neurons, cardiomyocytes, and hepatocytes can be generated directly from fibroblasts using this technology (Davis, Weintraub, & Lassar, 1987;Huang et al, 2011;Ieda et al, 2010;Sekiya & Suzuki, 2011;Vierbuchen et al, 2010). In our previous study, mouse embryonic fibroblasts (MEFs) could be converted into induced FIPC (iFIPCs) following the overexpression of the defined transcription factors hepatocyte nuclear factor 4 alpha (Hnf4α), forkhead box protein A3 (Foxa3), GATA-binding factor 6 (Gata6), and caudal-related homeobox protein 2 (Cdx2) in MEFs (Figure 2d; Miura & Suzuki, 2017). Similar to FIPCs, iFIPCs can form SOs in culture medium supplemented with EGF, noggin, R-spondin1, Wnt3a, and CHIR99021 (an inhibitor of glycogen synthase kinase 3β), and iFIPC-derived SOs can develop into BOs after serial passaging without exogenous Wnt stimulation ( Figure 2d; Miura & Suzuki, 2017).…”

Section: G Ener Ati On Of Mous E and H Uman Inte S Tinal Org Anoidsmentioning

confidence: 99%

“…In our previous study, mouse embryonic fibroblasts (MEFs) could be converted into induced FIPC (iFIPCs) following the overexpression of the defined transcription factors hepatocyte nuclear factor 4 alpha (Hnf4α), forkhead box protein A3 (Foxa3), GATA-binding factor 6 (Gata6), and caudal-related homeobox protein 2 (Cdx2) in MEFs (Figure 2d; Miura & Suzuki, 2017). Similar to FIPCs, iFIPCs can form SOs in culture medium supplemented with EGF, noggin, R-spondin1, Wnt3a, and CHIR99021 (an inhibitor of glycogen synthase kinase 3β), and iFIPC-derived SOs can develop into BOs after serial passaging without exogenous Wnt stimulation ( Figure 2d; Miura & Suzuki, 2017). The global gene expression patterns of iFIPC-derived SOs and BOs are similar to those of SOs and BOs derived from tissue fragments of fetal mouse intestine and the crypts of adult mouse intestine, respectively (Miura & Suzuki, 2017).…”

Section: G Ener Ati On Of Mous E and H Uman Inte S Tinal Org Anoidsmentioning

confidence: 99%

Brief summary of the current protocols for generating intestinal organoids

Miura

Suzuki

2018

Dev Growth Differ

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The intestine has fundamental functions for the maintenance of homeostasis, including food digestion and nutrient/water absorption. Although the lumen of the intestine is always exposed to pathogens, intestinal epithelial cells form monolayer sheets that act as an epithelial barrier to prevent the invasion of pathogens. Thus, disruption of the intestinal epithelial barrier causes inflammatory bowel diseases. To investigate the details of these intractable intestinal diseases, it is necessary to analyze the characteristics of intestinal epithelial cells in vitro. However, it is difficult to maintain and propagate intestinal epithelial cells in culture. Recently, intestinal organoid culture systems have been established, in which differentiated intestinal epithelial lineage cells can be continuously produced from intestinal stem cells and form epithelial organoids with crypt-like structures in long-term culture. Moreover, intestinal epithelial organoids can be generated not only from intestinal tissue-derived cells, embryonic stem cells, and induced pluripotent stem cells, but also by inducing direct conversion of nonintestinal somatic cells into intestinal epithelial cells. These intestinal organoids can be used in basic studies for understanding the mechanisms underlying intestinal development and diseases and will be applied in future transplantation therapy and drug discovery to treat intestinal diseases.

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Intestinal Organoid Culture in Polymer Film‐Based Microwell Arrays

et al. 2020

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compartment occupied by continuously dividing cells at the bottom, which after differentiation migrate upward toward the villus. In the villus, six different mature cell types are found, including secretory cells, such as goblet, Paneth, enteroendocrine, and tuft cells, and also absorptive cells, such as enterocytes and microfold cells. [1-3] The intestinal epithelium serves two main functions, nutrient uptake and protection against harmful substances and pathogens. Τhe presence of strong apical-basolateral compartmentalization is of critical importance for proper intestinal function. [3-7] Despite various advances in cell and tissue culture technologies [8-11] and organ-on-chip models [12] that have been used to study intestinal physiology over the past years, numerous morphological and functional aspects remain unknown. Miniaturized 3D, multicellular, stem cell-derived constructs that mimic in vivo tissue, called organoids, along with organ-on-chip systems represent culture systems able to recapitulate the complexity of an organ closer than any previously utilized technique. [13,14] Organoids have high self-organization capacity and hold great potential as a research tool to explore unknown aspects of organ development, tissue regeneration, disease pathology, cell biology, and as drug-screening platforms. In the past few years, numerous protocols have been described to grow organoids that resemble various organs, such as liver, brain, intestine, and lung. [15-21] Intestinal organoid culture is a relatively simple system, that typically involves the embedding of small multicellular fragments (containing LGR5 + cells) in Matrigel, which serves as an extracellular matrix mimic, supplemented with the right cocktail of growth factors. [16] This results in growing and self-sustaining intestinal organoids, which contain all the above mentioned cell types found in vivo and are organized in a crypt-villus structure that surrounds a central lumen, with strong apical-basolateral polarity. [13,22] Hence, these organoids have contributed significantly to the understanding of normal intestinal function and dysfunction in the last years. Even though organoids are a powerful tool, their use still faces numerous limitations. Bioengineering approaches hold great potential in overcoming some constraints. [23-27] More specifically, mass transport in organoids is usually restricted by their size, a limitation which researchers hope to overcome with either the use of bioreactors, microfluidic chips or integration of vascular

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Generation of Mouse and Human Organoid-Forming Intestinal Progenitor Cells by Direct Lineage Reprogramming

Cited by 78 publications

References 38 publications

Prolonged inhibition of hepatocellular carcinoma cell proliferation by combinatorial expression of defined transcription factors

Prolonged inhibition of hepatocellular carcinoma cell proliferation by combinatorial expression of defined transcription factors

Brief summary of the current protocols for generating intestinal organoids

Intestinal Organoid Culture in Polymer Film‐Based Microwell Arrays

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