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•Pluripotent stem cell (PSC)-derived expandable human hepatocyte-like liver organoids were generated.•PSC-derived human hepatic organoids are capable of long-term expansion with ...competent liver functionality.•PSC-derived human hepatic organoids provide a robust hepatic model for toxicity prediction and drug screening.
The development of hepatic models capable of long-term expansion with competent liver functionality is technically challenging in a personalized setting. Stem cell-based organoid technologies can provide an alternative source of patient-derived primary hepatocytes. However, self-renewing and functionally competent human pluripotent stem cell (PSC)-derived hepatic organoids have not been developed.
We developed a novel method to efficiently and reproducibly generate functionally mature human hepatic organoids derived from PSCs, including human embryonic stem cells and induced PSCs. The maturity of the organoids was validated by a detailed transcriptome analysis and functional performance assays. The organoids were applied to screening platforms for the prediction of toxicity and the evaluation of drugs that target hepatic steatosis through real-time monitoring of cellular bioenergetics and high-content analyses.
Our organoids were morphologically indistinguishable from adult liver tissue-derived epithelial organoids and exhibited self-renewal. With further maturation, their molecular features approximated those of liver tissue, although these features were lacking in 2D differentiated hepatocytes. Our organoids preserved mature liver properties, including serum protein production, drug metabolism and detoxifying functions, active mitochondrial bioenergetics, and regenerative and inflammatory responses. The organoids exhibited significant toxic responses to clinically relevant concentrations of drugs that had been withdrawn from the market due to hepatotoxicity and recapitulated human disease phenotypes such as hepatic steatosis.
Our organoids exhibit self-renewal (expandable and further able to differentiate) while maintaining their mature hepatic characteristics over long-term culture. These organoids may provide a versatile and valuable platform for physiologically and pathologically relevant hepatic models in the context of personalized medicine.
A functionally mature, human cell-based liver model exhibiting human responses in toxicity prediction and drug evaluation is urgently needed for pre-clinical drug development. Here, we develop a novel human pluripotent stem cell-derived hepatocyte-like liver organoid that is critically advanced in terms of its generation method, functional performance, and application technologies. Our organoids can contribute to the better understanding of liver development and regeneration, and provide insights for metabolic studies and disease modeling, as well as toxicity assessments and drug screening for personalized medicine.
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.
Current cerebral organoid technology provides excellent in vitro models mimicking the structure and function of the developing human brain, which enables studies on normal and pathological brain; ...however, further improvements are necessary to overcome the problems of immaturity and dearth of non-parenchymal cells. Vascularization is one of the major challenges for recapitulating processes in the developing human brain. Here, we examined the formation of blood vessel-like structures in cerebral organoids induced by vascular endothelial growth factor (VEGF) in vitro. The results indicated that VEGF enhanced differentiation of vascular endothelial cells (ECs) without reducing neuronal markers in the embryonic bodies (EBs), which then successfully developed into cerebral organoids with open-circle vascular structures expressing an EC marker, CD31, and a tight junction marker, claudin-5, characteristic of the blood-brain barrier (BBB). Further treatment with VEGF and Wnt7a promoted the formation of the outer lining consisting of pericyte-like cells, which surrounded the vascular tubes. RNA sequencing revealed that VEGF upregulated genes associated with tube formation, vasculogenesis, and the BBB; it also changed the expression of genes involved in brain embryogenesis, suggesting a role of VEGF in neuronal development. These results indicate that VEGF treatment can be used to generate vessel-like structures with mature BBB characteristics in cerebral organoids in vitro.
•VEGF induced vascularization without inhibiting neural differentiation of EBs.•VEGF promoted the generation of CD31-positive blood vessels in cerebral organoids.•Long-term culture of cerebral organoids with VEGF and Wnt7a produces vascular tubes with a two-layer structure.•In cerebral organoids, VEGF upregulated genes associated with vascular development, Neurogenesis, and blood-brain barrier.
The human microbiome plays an essential role in the human immune system, food digestion, and protection from harmful bacteria by colonizing the human intestine. Recently, although the human ...microbiome affects colorectal cancer (CRC) treatment, the mode of action between the microbiome and CRC remains unclear. This study showed that propionate suppressed CRC growth by promoting the proteasomal degradation of euchromatic histone-lysine N-methyltransferase 2 (EHMT2) through HECT domain E3 ubiquitin protein ligase 2 (HECTD2) upregulation. In addition, EHMT2 downregulation reduced the H3K9me2 level on the promoter region of tumor necrosis factor α-induced protein 1 (TNFAIP1) as a novel direct target of EHMT2. Subsequently, TNFAIP1 upregulation induced the apoptosis of CRC cells. Furthermore, using Bacteroides thetaiotaomicron culture medium, we confirmed EHMT2 downregulation via upregulation of HECTD2 and TNFAIP1 upregulation. Finally, we observed the synergistic effect of propionate and an EHMT2 inhibitor (BIX01294) in 3D spheroid culture models. Thus, we suggest the anticancer effects of propionate and EHMT2 as therapeutic targets for colon cancer treatment and may provide the possibility for the synergistic effects of an EHMT2 inhibitor and microbiome in CRC treatment.
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
Summary Background Stricturing and penetrating complications account for substantial morbidity and health-care costs in paediatric and adult onset Crohn's disease. Validated models to predict risk ...for complications are not available, and the effect of treatment on risk is unknown. Methods We did a prospective inception cohort study of paediatric patients with newly diagnosed Crohn's disease at 28 sites in the USA and Canada. Genotypes, antimicrobial serologies, ileal gene expression, and ileal, rectal, and faecal microbiota were assessed. A competing-risk model for disease complications was derived and validated in independent groups. Propensity-score matching tested the effect of anti-tumour necrosis factor α (TNFα) therapy exposure within 90 days of diagnosis on complication risk. Findings Between Nov 1, 2008, and June 30, 2012, we enrolled 913 patients, 78 (9%) of whom experienced Crohn's disease complications. The validated competing-risk model included age, race, disease location, and antimicrobial serologies and provided a sensitivity of 66% (95% CI 51–82) and specificity of 63% (55–71), with a negative predictive value of 95% (94–97). Patients who received early anti-TNFα therapy were less likely to have penetrating complications (hazard ratio HR 0·30, 95% CI 0·10–0·89; p=0·0296) but not stricturing complication (1·13, 0·51–2·51; 0·76) than were those who did not receive early anti-TNFα therapy. Ruminococcus was implicated in stricturing complications and Veillonella in penetrating complications. Ileal genes controlling extracellular matrix production were upregulated at diagnosis, and this gene signature was associated with stricturing in the risk model (HR 1·70, 95% CI 1·12–2·57; p=0·0120). When this gene signature was included, the model's specificity improved to 71%. Interpretation Our findings support the usefulness of risk stratification of paediatric patients with Crohn's disease at diagnosis, and selection of anti-TNFα therapy. Funding Crohn's and Colitis Foundation of America, Cincinnati Children's Hospital Research Foundation Digestive Health Center.
The future of safe cell-based therapy rests on overcoming teratoma/tumor formation, in particular when using human pluripotent stem cells (hPSCs), such as human embryonic stem cells (hESCs) and human ...induced pluripotent stem cells (hiPSCs). Because the presence of a few remaining undifferentiated hPSCs can cause undesirable teratomas after transplantation, complete removal of these cells with no/minimal damage to differentiated cells is a prerequisite for clinical application of hPSC-based therapy. Having identified a unique hESC signature of pro- and antiapoptotic gene expression profile, we hypothesized that targeting hPSC-specific antiapoptotic factor(s) (i.e., survivin or Bcl10) represents an efficient strategy to selectively eliminate pluripotent cells with teratoma potential. Here we report the successful identification of small molecules that can effectively inhibit these antiapoptotic factors, leading to selective and efficient removal of pluripotent stem cells through apoptotic cell death. In particular, a single treatment of hESC-derived mixed population with chemical inhibitors of survivin (e.g., quercetin or YM155) induced selective and complete cell death of undifferentiated hPSCs. In contrast, differentiated cell types (e.g., dopamine neurons and smooth-muscle cells) derived from hPSCs survived well and maintained their functionality. We found that quercetin-induced selective cell death is caused by mitochondrial accumulation of p53 and is sufficient to prevent teratoma formation after transplantation of hESC- or hiPSC-derived cells. Taken together, these results provide the “proof of concept” that small-molecule targeting of hPSC-specific antiapoptotic pathway(s) is a viable strategy to prevent tumor formation by selectively eliminating remaining undifferentiated pluripotent cells for safe hPSC-based therapy.
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.
Osteosarcoma is a highly aggressive cancer for which treatment has remained essentially unchanged for more than 30 years. Osteosarcoma is characterized by widespread and recurrent somatic copy-number ...alterations (SCNA) and structural rearrangements. In contrast, few recurrent point mutations in protein-coding genes have been identified, suggesting that genes within SCNAs are key oncogenic drivers in this disease. SCNAs and structural rearrangements are highly heterogeneous across osteosarcoma cases, suggesting the need for a genome-informed approach to targeted therapy. To identify patient-specific candidate drivers, we used a simple heuristic based on degree and rank order of copy-number amplification (identified by whole-genome sequencing) and changes in gene expression as identified by RNA sequencing. Using patient-derived tumor xenografts, we demonstrate that targeting of patient-specific SCNAs leads to significant decrease in tumor burden, providing a road map for genome-informed treatment of osteosarcoma. SIGNIFICANCE: Osteosarcoma is treated with a chemotherapy regimen established 30 years ago. Although osteosarcoma is genomically complex, we hypothesized that tumor-specific dependencies could be identified within SCNAs. Using patient-derived tumor xenografts, we found a high degree of response for "genome-matched" therapies, demonstrating the utility of a targeted genome-informed approach.
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