Biology subjects

Kamei, K.-i.

Publications and source records attributed to Kamei, K.-i..

26 records · Page 2Linked to original sources

bioRxiv2020

Integrated gut-liver-on-a-chip platform as an in vitro human model of non-alcoholic fatty liver disease

Non-alcoholic fatty liver disease (NAFLD) afflicts a large percentage of the population, but no effective treatments have been established so far because of the unsuitability of in vitro assays and experimental models using animals. By co-culturing human gut and liver cell lines interconnected via microfluidics for a closed circulation loop, we created a gut-liver-on-a-chip (iGLC) platform as an in vitro human model of the gut-liver axis (GLA) for the initiation and progression of NAFLD. Microscopic high-content analysis followed by mRNA sequencing showed that co-culturing the gut and liver cells significantly affected each cell type compared to culturing them separately. NAFLD-inducing free fatty acids (FFAs) accumulated in the gut cells and elevated gene expressions associated with retinol metabolism and glucuronidation. The FFA-treated liver cells accumulated intracellular lipid droplets and showed an increase in gene expressions associated with a cellular response to copper ions and endoplasmic reticulum stress. As an in vitro human GLA model, the iGLC platform may serve as an alternative to animal experiments for investigating NAFLD mechanisms.

bioengineering↗

bioRxiv2020

Recapitulation of human embryonic heart beating to promote differentiation of hepatic endoderm to hepatoblasts

A microfluidic platform recapitulating human embryonic heart beat improves the functionalization state of hepatocytes derived from hepatic endoderm (HE). Mechanical stretching of mimicked heart beats was applied to HE cells cultured on the microfluidic platform. Stimulated HE-derived hepatoblasts increased cytochrome P450 3A (CYP3A) metabolic activities and hepatoblast functional markers expression, leading for advancement of regenerative medicine and drug screening.

bioengineering↗

bioRxiv2020

Genetic signatures of evolution of the pluripotency gene regulating network across mammals

Mammalian pluripotent stem cells (PSCs) have distinct molecular and biological characteristics, but we lack a comprehensive understanding of regulatory network evolution in mammals. Here, we applied a comparative genetic analysis of 134 genes constituting the pluripotency gene regulatory network across 48 mammalian species covering all the major taxonomic groups. We found evolutionary conservation in JAK-STAT and PI3K-Akt signaling pathways, suggesting equivalent capabilities in self-renewal and proliferation of mammalian PSCs. On the other hand, we discovered rapid evolution of the downstream targets of the core regulatory circuit, providing insights into variations of characteristics. Our data indicate that the variations in the PSCs characteristics may be due to positive selections in the downstream targets of the core regulatory circuit. We further reveal that positively selected genes can be associated with species unique adaptation that is not dedicated to regulation of PSCs. These results provide important insight into the evolution of the pluripotency gene regulatory network underlying variations in characteristics of mammalian PSCs.

evolutionary biology↗

bioRxiv2020

39°C-in vitro culture facilitates hepatic functions of hepatocyte-like cells derived from human embryonic stem cells

Hepatocyte-like cells derived from human pluripotent stem cells (hPSC-HLCs) offer an alternative to primary hepatocytes commonly used for drug screenings and toxicological tests. Although tremendous efforts have been made to facilitate hepatic functions of hPSC-HLCs using growth factors and chemicals, these cells have not yet reached hepatic functions comparable to hepatocytes in vivo. Therefore, there exists a critical need to use an alternative trigger to facilitate hepatic functions in hPSC-HLCs. We noted that human liver temperature (around 39{degrees}C) is higher than normal human body temperature (around 36.5{degrees}C), yet hepatocytes are generally cultured at 37{degrees}C in-vitro. Here we showed that hepatic functions of hPSC-HLCs would be facilitated under physiological liver temperatures. We identified the optimal temperature by treating HLCs derived from H9 human embryonic stem cells (hESC-HLCs) at 39{degrees}C and 42{degrees}C. 42{degrees}C-treatment caused significantly greater cell death compared to 39{degrees}C. We also confirmed the increases of hepatic functions, such as secretion of albumin, cytochrome P450 3A4 (CYP3A4) activities, and collagen productions, without severe cell damages. To elucidate the underlying mechanisms of heat-induced hepatic functions, RNA-seq was to identify gene expression signatures due to 39{degrees}C-treated hESC-HLCs. This study also showed the possible mechanisms of heat-induced hepatic function via glucocorticoid receptor pathway and molecular chaperons. In combination with existing hepatic differentiation protocols, the method proposed here may further improve hepatic functions for hPSCs, and lead to the realization of drug discovery efforts and drug toxicological tests. Significance statementHepatocyte-like cells derived from human pluripotent stem cells (hPSC-HLCs) offer an alternative to primary hepatocytes commonly used for drug screenings and toxicological tests. We noted that human liver temperature (around 39{degrees}C) is higher than normal human body temperature (around 36.5{degrees}C), affecting the in-vitro hepatic functions of hPSC-HLCs, such as metabolic activities. Here we showed that hepatic functions of hPSC-HLCs, albumin secretion, CYP3A4 activities, and collagen production would be facilitated under physiological liver temperatures at 39{degrees}C, without severe cell damages. RNA-seq was used to elucidate the underlying mechanisms of heat-induced hepatic functions. This study also showed the possible mechanisms of heat-induced hepatic function via glucocorticoid receptor pathway and molecular chaperons.

bioengineering↗

bioRxiv2019

Multi-corneal barriers-on-a-chip to recapitulate eye blinking shear stress forces

Human corneal epithelium coexists tear fluids and shows its barrier functionality under the dynamic conditions of eye blinking. However, the current in vitro cell culture settings for corneal epithelial cells lack the dynamic flow conditions to recapitulate shear stress of eye blinking, hindering corneal function evaluation. We developed a microfluidic platform enabling the dynamic culture of the human corneal barrier with recapitulation of eye blinking. The device consisted of upper and lower channels separated by a porous membrane. Human corneal epithelial cells (HCE-T) were seeded on a porous membrane in an upper channel and cultured for ten days. The cells formed a barrier with high expression of zonula occludens 1 (ZO-1) tight junction protein on day seven, and the translocation of fluorescein sodium across the barrier in the microfluidic device was comparable to that in the transwell system. Then, bidirectional and unidirectional flows were applied in the upper and lower channels, respectively, and the cells in the upper channels were stimulated with 0.6 dyn s cm-2 of shear stress. While the fluid stimuli after 24 h did not affect cell adhesion, the flow stimuli facilitated the expression of cytokeratin 19 (CK-19) intermediate filaments in cells after 24 h, indicating strengthening of the barrier function. Furthermore, the morphological single-cell analysis revealed an increase in cell body area rather than nuclei. We envision that this multicorneal barriers-on-a-chip device will unlock new possibilities in ophthalmic drug development and will be useful for studying the mechanobiology of the ocular surface.

bioengineering↗

bioRxiv2019

Direct synthesis of self-organized blastocyst-like cysts derived from human pluripotent stem cells

We introduce a simple, robust and scalable method to generate self-organized blastocyst-like cysts (soBLCs) from human pluripotent stem cells (hPSCs). We use a copolymer hydrogel of poly(N-isopropylacrylamide) and poly(ethylene glycol) (PNIPAAm-PEG). hPSC aggregates with a diameter of approximately 117.2 {+/-} 5.1 {micro}m are cultured in a medium supplemented with a hydrogel and a serum for three days. Molecular signatures in the medium revealed the generation of trophoblasts and inner cell mass at specific positions in the soBLCs.

bioengineering↗

bioRxiv2018

Genetic Signatures of Lipid Metabolism Evolution in Cetacea

In mammalian evolutionary history, Cetacea (whales, dolphins, and porpoises) achieved astonishing success by adapting to an aquatic environment. One unique characteristic of cetaceans, contributing to this adaptive success, is efficient lipid utilization. Here we report comparative genetic analysis of aquatic and terrestrial Cetartiodactyla using 144 genes associated with lipid metabolism. We analyzed mutation rate, amino acid substitution, and metabolic pathways using genetic data publicly available. Our test detected 18 positively selected genes in Cetacea compared to 13 in Bovidae with little overlap between the lineages. We identified lineage-specific patterns of amino acid substitutions and functional domain that were mutually exclusive between cetaceans and bovids, supporting divergent evolution of lipid metabolism since the divergence of these taxa from a common ancestor. Moreover, a pathway analysis showed that the identified genes in cetaceans were associated with lipid digestion, lipid storage, and energy producing pathways. This study emphasizes the evolutionary context of lipid metabolism modification of cetaceans and provides a foundation for future studies of elucidating the adapted biological mechanisms of cetacean lipid metabolism and a framework for incorporating ecological context into studies aimed at investigating adaptive evolution.

evolutionary biology↗

bioRxiv2017

Robust direct differentiation and maturation of human pluripotent stem cells to hepatocyte-like cells

Liver-on-a-Chip technology holds considerable potential for applications in drug screening and chemical-safety testing. To establish such platforms, functional hepatocytes are required; however, primary hepatocytes are commonly used, despite problems involving donor limitations, lot-to-lot variation, and unsatisfactory two-dimensional culture methods. Although human pluripotent stem cells (hPSCs) may represent a strong alternative contender to address the aforementioned issues, remaining technological challenges include the robust, highly efficient production of high-purity hepatic clusters. In addition, current Liver-on-a-Chip platforms are relatively complicated and not applicable for high-throughput experiments. Here, we develop a very simple Liver-on-a-Chip platform with mature and functional hepatocyte-like cells derived from hPSCs. To establish a method for hepatic differentiation of hPSCs, cells were first treated by inhibiting the phosphoinositide 3-kinase- and Rho-associated protein kinase-signaling pathways to stop self-renewal and improve survival, respectively, which enabled the formation of a well-defined endoderm and facilitated hepatocyte commitment. Next, a simple microfluidic device was used to create a three-dimensional (3D) culture environment that enhanced the maturation and function of hepatocyte-like cells by increasing the expression of both hepatic maturation markers and cytochrome P450. Finally, we confirmed improvements in hepatic functions, such as drug uptake/excretion capabilities, in >90% of 3D-matured hepatocyte-like cells by indocyanin green assay. These results indicated that the incorporation of hPSC-derived hepatocytes on our Liver-on-a-Chip platform may serve to enhance the processes involved in drug screening and chemical-safety testing.

bioengineering↗