Human pluripotent stem cell (hPSC)-derived intestinal organoids (hIOs) form 3D structures organized into crypt and villus domains, making them an excellent in vitro model system for studying human ...intestinal development and disease. However, hPSC-derived hIOs still require in vivo maturation to fully recapitulate adult intestine, with the mechanism of maturation remaining elusive. Here, we show that the co-culture with human T lymphocytes induce the in vitro maturation of hIOs, and identify STAT3-activating interleukin-2 (IL-2) as the major factor inducing maturation. hIOs exposed to IL-2 closely mimic the adult intestinal epithelium and have comparable expression levels of mature intestinal markers, as well as increased intestine-specific functional activities. Even after in vivo engraftment, in vitro-matured hIOs retain their maturation status. The results of our study demonstrate that STAT3 signaling can induce the maturation of hIOs in vitro, thereby circumventing the need for animal models and in vivo maturation.
Abstract
Human pluripotent stem cell (hPSC)-derived organoids and cells have similar characteristics to human organs and tissues. Thus, in vitro human organoids and cells serve as a superior ...alternative to conventional cell lines and animal models in drug development and regenerative medicine. For a simple and reproducible analysis of the quality of organoids and cells to compensate for the shortcomings of existing experimental validation studies, a quantitative evaluation method should be developed. Here, using the GTEx database, we construct a quantitative calculation system to assess similarity to the human organs. To evaluate our system, we generate hPSC-derived organoids and cells, and detected organ similarity. To facilitate the access of our system by researchers, we develop a web-based user interface presenting similarity to the appropriate organs as percentages. Thus, this program could provide valuable information for the generation of high-quality organoids and cells and a strategy to guide proper lineage-oriented differentiation.
We report on a molecularly tailored 1:1 donor–acceptor (D‐A) charge‐transfer (CT) cocrystal that manifests strongly red‐shifted CT luminescence characteristics, as well as noteworthy reconfigurable ...self‐assembling behaviors. A loosely packed molecular organization is obtained as a consequence of the noncentrosymmetric chemical structure of molecule A1, which gives rise to considerable free volume and weak intermolecular interactions. The stacking features of the CT complex result in an external stimuli‐responsive molecular stacking reorganization between the mixed and demixed phases of the D‐A pair. Accordingly, high‐contrast fluorescence switching (red↔blue) is realized on the basis of the strong alternation of the electronic properties between the mixed and demixed phases. A combination of structural, spectroscopic, and computational studies reveal the underlying mechanism of this stimuli‐responsive behavior.
To mix or not to mix: A donor–acceptor (D‐A) charge‐transfer (CT) crystalline film was designed to realize stimuli‐responsive reversible fluorescence switching media. Owing to the loosely packed CT state, reorganization between the red‐emissive mixed CT phase and blue‐emissive demixed self‐sorted phase can be demonstrated by external triggers such as solvent, thermal, and piezomechanical stimuli.
Diclofenac effectively reduces pain and inflammation; however, its use is associated with hepato- and nephrotoxicity. To delineate mechanisms of injury, we investigated a clinically relevant (3 ...mg/kg) and high-dose (15 mg/kg) in minipigs for 4 weeks. Initially, serum biochemistries and blood-smears indicated an inflammatory response but returned to normal after 4 weeks of treatment. Notwithstanding, histopathology revealed drug-induced hepatitis, marked glycogen depletion, necrosis and steatosis. Strikingly, the genomic study revealed diclofenac to desynchronize the liver clock with manifest inductions of its components CLOCK, NPAS2 and BMAL1. The > 4-fold induced CRY1 expression underscored an activated core-loop, and the dose dependent > 60% reduction in PER2mRNA repressed the negative feedback loop; however, it exacerbated hepatotoxicity. Bioinformatics enabled the construction of gene-regulatory networks, and we linked the disruption of the liver-clock to impaired glycogenesis, lipid metabolism and the control of immune responses, as shown by the 3-, 6- and 8-fold induced expression of pro-inflammatory CXCL2, lysozyme and ß-defensin. Additionally, diclofenac treatment caused adrenocortical hypertrophy and thymic atrophy, and we evidenced induced glucocorticoid receptor (GR) activity by immunohistochemistry. Given that REV-ERB connects the circadian clock with hepatic GR, its > 80% repression alleviated immune responses as manifested by repressed expressions of CXCL9(90%), CCL8(60%) and RSAD2(70%). Together, we propose a circuitry, whereby diclofenac desynchronizes the liver clock in the control of the hepatic metabolism and immune response.
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 Kriippel‐like factor 5 monomeric Cherry (KLF5mCherry) and intestine‐specific homeobox enhanced green fluorescence protein (ISXeGFP). 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 in vitro differentiation and for tracking the in vivo 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. In vitro and in vivo imaging and tracking of intestinal organoids from human induced pluripotent stem cells. FASEB J. 32,111‐122 (2018). www.fasebj.org
Focal cortical dysplasia (FCD) is a major cause of the sporadic form of intractable focal epilepsies that require surgical treatment. It has recently been reported that brain somatic mutations in ...MTOR account for 15%–25% of FCD type II (FCDII), characterized by cortical dyslamination and dysmorphic neurons. However, the genetic etiologies of FCDII-affected individuals who lack the MTOR mutation remain unclear. Here, we performed deep hybrid capture and amplicon sequencing (read depth of 100×–20,012×) of five important mTOR pathway genes—PIK3CA, PIK3R2, AKT3, TSC1, and TSC2—by using paired brain and saliva samples from 40 FCDII individuals negative for MTOR mutations. We found that 5 of 40 individuals (12.5%) had brain somatic mutations in TSC1 (c.64C>T p.Arg22Trp and c.610C>T p.Arg204Cys) and TSC2 (c.4639G>A p.Val1547Ile), and these results were reproducible on two different sequencing platforms. All identified mutations induced hyperactivation of the mTOR pathway by disrupting the formation or function of the TSC1-TSC2 complex. Furthermore, in utero CRISPR-Cas9-mediated genome editing of Tsc1 or Tsc2 induced the development of spontaneous behavioral seizures, as well as cytomegalic neurons and cortical dyslamination. These results show that brain somatic mutations in TSC1 and TSC2 cause FCD and that in utero application of the CRISPR-Cas9 system is useful for generating neurodevelopmental disease models of somatic mutations in the brain.
Abstract
Defect engineering is one of the key technologies in materials science, enriching the modern semiconductor industry and providing good test-beds for solid-state physics. While homogenous ...doping prevails in conventional defect engineering, various artificial defect distributions have been predicted to induce desired physical properties in host materials, especially associated with symmetry breakings. Here, we show layer-by-layer defect-gradients in two-dimensional PtSe
2
films developed by selective plasma treatments, which break spatial inversion symmetry and give rise to the Rashba effect. Scanning transmission electron microscopy analyses reveal that Se vacancies extend down to 7 nm from the surface and Se/Pt ratio exhibits linear variation along the layers. The Rashba effect induced by broken inversion symmetry is demonstrated through the observations of nonreciprocal transport behaviors and first-principles density functional theory calculations. Our methodology paves the way for functional defect engineering that entangles spin and momentum of itinerant electrons for emerging electronic applications.
Alternative cell sources, such as three‐dimensional organoids and induced pluripotent stem cell–derived cells, might provide a potentially effective approach for both drug development applications ...and clinical transplantation. For example, the development of cell sources for liver cell–based therapy has been increasingly needed, and liver transplantation is performed for the treatment for patients with severe end‐stage liver disease. Differentiated liver cells and three‐dimensional organoids are expected to provide new cell sources for tissue models and revolutionary clinical therapies. However, conventional experimental methods confirming the expression levels of liver‐specific lineage markers cannot provide complete information regarding the differentiation status or degree of similarity between liver and differentiated cell sources. Therefore, in this study, to overcome several issues associated with the assessment of differentiated liver cells and organoids, we developed a liver‐specific gene expression panel (LiGEP) algorithm that presents the degree of liver similarity as a “percentage.” We demonstrated that the percentage calculated using the LiGEP algorithm was correlated with the developmental stages of in vivo liver tissues in mice, suggesting that LiGEP can correctly predict developmental stages. Moreover, three‐dimensional cultured HepaRG cells and human pluripotent stem cell–derived hepatocyte‐like cells showed liver similarity scores of 59.14% and 32%, respectively, although general liver‐specific markers were detected. Conclusion: Our study describes a quantitative and predictive model for differentiated samples, particularly liver‐specific cells or organoids; and this model can be further expanded to various tissue‐specific organoids; our LiGEP can provide useful information and insights regarding the differentiation status of in vitro liver models. (Hepatology 2017;66:1662–1674).
This year, France banned the application of titanium dioxide nanoparticles as a food additive (hereafter, E171) based on the insufficient oral toxicity data. Here, we investigated the subchronic ...toxic responses of E171 (0, 10, 100, and 1,000 mg/kg) and tried to elucidate the possible toxic mechanism using AGS cells, a human stomach epithelial cell line. There were no dose‐related changes in the Organisation for Economic Cooperation and Development test guideline‐related endpoints. Meanwhile, E171 deeply penetrated cells lining the stomach tissues of rats, and the IgM and granulocyte‐macrophage colony‐stimulating factor levels were significantly lower in the blood from rats exposed to E171 compared with the control. The colonic antioxidant protein level decreased with increasing Ti accumulation. Additionally, after 24‐h exposure, E171 located in the perinuclear region of AGS cells and affected expression of endoplasmic reticulum stress‐related proteins. However, cell death was not observed up to the used maximum concentration. A gene profile analysis also showed that immune response‐related microRNAs were most strongly affected by E171 exposure. Collectively, we concluded that the NOAEL of E171 for 90 days repeated oral administration is between 100 and 1,000 mg/kg for both male and female rats. Additionally, further study is needed to clarify the possible carcinogenesis following the chronic accumulation in the colon.
In this study, we found that E171 did not induce any dose‐related changes in the OECD test guideline‐related endpoints. Meanwhile, E171 deeply penetrated cells lining the stomach tissues of rats, and the colonic antioxidant protein level decreased with increasing Ti accumulation. After 24‐h exposure, E171 also located in the perinuclear region of AGS cells and affected expression of ER stress‐related proteins. Additionally, a gene profile analysis showed that immune response‐related microRNAs were most strongly affected by E171 exposure.
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