Project description:In this study, we established human hepatocyte organoids (HHOs) that were expanded from primary human hepatocytes (PHH) in a defined medium. Briefly, the cryopreserved primary human hepatocytes (purchased) were thawn in advanced DMEM/F12 (Thermo) or Cryopreserved hepatocyte Recovery Medium (Gibco). Then, the cells were embedded in Matrigel (Corning) and cultured with the expansion medium (EM). For the eHHOs, expansion medium (EM) was prepared with a basal medium (Advanced DMEM/F12 was supplemented with penicillin/streptomycin, 10 mM HEPES, 2 mM GlutaMAX, 1 ×(Thermo Fisher Scientific), 10 nM gastrin I (Sigma), and 1 mM N-acetylcysteine (Wako, Japan) ) containing the following niche factors: 50 ng/ml mouse recombinant EGF (Thermo Fisher Scientific), 25 ng/mL human recombinant HGF (Peprotech), 100 ng/mL human recombinant FGF-10 (Peprotech), 10 uM Forskolin (Cayman Chemical), 25 ng/ml mouse recombinant noggin (Peprotech), 1 mg/ml human recombinant R-spondin1 (R; R&D), 20% Afamin-Wnt-3A serum-free conditioned medium (W; Mihara et al., 2016), 5 uM A83-01 (Tocris), and 20ng/ml human Oncostatin M (OSM; Peprotech). For dHHOs, eHHOs were cultured for 10-14 days in differentiation medium (DM), which was EM without OSM and WR, and containing a hormone cocktail (10ng/ml Growth Hormone, 10ng/ml Prolactin, and 100 ng/ml Cortisol) and 10uM DAPT (differentiation medium: DM).
Project description:Human liver organoids are expected to be a hepatocyte source for preclinical in vitro studies. Although these organoids show long-term proliferation, their hepatic functions remain low. Here, we propose a novel method for two dimensional (2D)-cultured hepatic differentiation from human liver organoids. When cultured under a 2D condition, the single cells from human liver organoids were seeded on collagen type I-coated plates. Then, optimal conditions for hepatic differentiation were screened using several reagents. We determined the 2D-cultured hepatocyte differentiation method from human liver organoids. Hepatic gene expressions in human liver organoids-derived hepatocytes (Org-HEPs) were greatly increased, compared to those in human liver organoids. The metabolic activities of cytochrome P450 (CYP) 1A2, CYP2C8, CYP2E1 and CYP3A4 were at levels comparable to those in primary human hepatocytes (PHHs). These results suggested that human liver organoids could be differentiated into highly functional hepatocytes in 2D culture. We also treated Org-HEPs and PHHs with hepatotoxic drugs. The cell viability of Org-HEPs was almost the same as that of PHHs, suggesting that Org-HEPs could be used for hepatotoxicity tests. Thus, Org-HEPs will be useful for pharmaceutical research.
Project description:WDR6 was a scaffolding/ regulatory protein that interacts with diverse signaling molecules. Our findings showed that WDR6 might be involved in post-translational modifications (PTMs). Then we performed Proteomic analysis of primary hepatocytes from WDR6-WKO and control mice to analyze the functions of WDR6. In addition, the proteomic analysis was also performed as an internal control.
Project description:Knowledge on tooth epithelial stem cells and corresponding biology in humans is sparse. Here, we developed an organoid model, typically replicating epithelial tissue stem cell biology, starting from human tooth. Dental follicle (DF), isolated from extracted wisdom teeth, efficiently generated long-term expandable epithelial organoids. The organoids displayed a tooth stemness phenotype as present in DF’s Epithelial Cell Rests of Malassez, a compartment deemed to contain tooth epithelial stem cells. To further decode the organoids in more granular detail, we applied scRNA-seq analysis on DF-derived organoids (passage 1 (P1) and P4) together with their primary tissue. Additionally, to decipher the amelogenesis-resembling process that takes place in the tooth organoids upon differentiation, we performed scRNA-seq analysis of P4 organoids switched to mineralization-inducing medium for 8 days.
Project description:Hepatocellular carcinoma (HCC) and cholangiocarcinoma (ICC) are two main forms liver cancers with poor prognosis. Models for studying HCC and ICC development using human liver cells are urgently needed. Organoids serve as in vitro models for cancer studies as it recapitulates in vivo structures and microenvironment of solid tumors. Herein, we established liver cancer organoid models by introducing specific mutations into human induced hepatocyte (hiHep)-derived organoids. c-MYC and hRASG12V overexpression in hiHep organoids with repressed p53 activation by large T led to distinct HCC and ICC signatures. With these oncogenic mutations, the neoplastic hiHep organoids formed cancerous structures and possessed cancer-specific hallmarks. Comprehensive transcriptional analysis of liver cancer organoids revealed genes and pathways with disease-stage-specific alterations. Notably, with RAS mutations, hiHep organoids acquired biliary trans-differentiation, and showed a process of conversion from hepatocytes to ICC. To sum up, we have established a useful and convenient in vitro human organoid systems modeling liver cancer development.
Project description:Liver is a central organ for drug and xenobiotics metabolism in human body. Due to the differences in interspecies metabolism, human model system for liver metabolism is necessary. However, the hepatic model systems derived from human pluripotent stem cells for metabolic screening and assays did not recapitulate the most of metabolic capabilities of human liver or primary human hepatocytes to date. In this study, we developed human hepatic endoderm organoids which are expandable and subsequently differentiated human hepatic organoids having metabolic capabilities. To investigate the transcriptome of human hepatic organoids, we performed RNA-sequencing.
Project description:Recently, we have shown that after partial hepatectomy (PHx), an increased hepatic blood flow initiates liver growth in mice by vasodilation and mechanically-triggered release of angiocrine signals. Here, we use mass spectrometry to identify a mechanically-induced angiocrine signal in human hepatic endothelial cells, that is, myeloid-derived growth factor (MYDGF). We show that it induces proliferation and promotes survival of primary human hepatocytes derived from different donors in two-dimensional cell culture, via activation of mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription 3 (STAT3). MYDGF also enhances proliferation of human hepatocytes in three-dimensional organoids. In vivo, genetic deletion of MYDGF decreases hepatocyte proliferation in the regenerating mouse liver after PHx; conversely, adeno-associated viral delivery of MYDGF increases hepatocyte proliferation and MAPK signaling after PHx. We conclude that MYDGF represents a mechanically-induced angiocrine signal and that it triggers growth of, and provides protection to, primary mouse and human hepatocytes
Project description:Genome editing has demonstrated its utility in generating isogenic cell-based disease models, enabling the precise introduction of genetic alterations into wildtype cells to mimic disease phenotypes and explore underlying mechanisms. However, its application in liver-related diseases has been limited by challenges in genetic modification of mature hepatocytes in a dish. Here we conducted a systematic comparison of various methods for primary hepatocyte culture and gene delivery to achieve robust genome editing of hepatocytes ex vivo. Our efforts yielded editing efficiencies of up to 80% in primary murine hepatocytes cultured in monolayer and 20% in organoids. To model human hepatic tumorigenesis, we utilized hepatocytes differentiated from human pluripotent stem cells (hPSCs) as an alternative human hepatocyte source. We developed a series of cellular models by introducing various single or combined oncogenic alterations into hPSC-derived hepatocytes. Our findings demonstrated that distinct mutational patterns led to phenotypic variances, affecting both overgrowth and transcriptional profiles. Notably, we discovered that the PI3KCA E542K mutant, whether alone or in combination with exogenous c-MYC, significantly impaired hepatocyte functions and facilitated cancer metabolic reprogramming. In conclusion, our study demonstrates genome-engineered hepatocytes as valuable cellular models for understanding hepatocarcinoma (HCC), especially highlighting PIK3CA and c-MYC—key oncogenes often amplified or upregulated in HCC—and providing insights into early tumorigenesis mechanisms.