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 significant percentage of the population; however, no effective treatments have yet been established because of the unsuitability of in vitro assays and animal experimental models. Here, we present an integrated-gut-liver-on-a-chip (iGLC) platform as an in vitro human model of the gut-liver axis (GLA) by co-culturing human gut and liver cell lines interconnected via microfluidics in a closed circulation loop, for the initiation and progression of NAFLD by treatment with free fatty acids (FFAs) for 1 and 7 days, respectively. Co-cultured Caco-2 gut-mimicking cells and HepG2 hepatocyte-like cells demonstrate the protective effects from apoptosis against FFAs treatment, whereas mono-cultured cells exhibit induced apoptosis. Phenotype and gene expression analyses reveal that the FFAs-treated gut and liver cells accumulated intracellular lipid droplets and show an increase in gene expression 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 the mechanisms of NAFLD.

Generation and Gene Expression Profiles of Grevy's Zebra Induced Pluripotent Stem Cells.

Induced pluripotent stem cells (iPSCs) can serve as a biological resource for functional and conservation research for various species. This realization has led to the generation of iPSCs from many species, including those identified as endangered. However, the understanding of species variation in mammalian iPSCs remains largely unknown. To gain insight into species variation in iPSCs, we generated iPSCs from a new species Grevy's zebra (Equus grevyi; gz-iPSCs), which has been listed as endangered in the IUCN (International Union for Conservation of Nature) Red List. We isolated primary fibroblast cells from an individual and successfully reprogrammed them into iPSCs. The generated gz-iPSCs continued to grow under primed-type culture condition and showed pluripotency and differentiation potential. To describe the molecular characteristics of gz-iPSCs, we performed RNA sequencing analysis. The gz-iPSC transcriptome showed robust expression of pluripotency-associated genes reported in human and mouse, suggesting evolutionary conservation among the species. This study provides insight into the iPSCs from a rare species and helps the understanding of the gene expression basis underlying mammalian pluripotent stem cells.

In vitro culture at 39 °C during hepatic maturation of human ES cells facilitates hepatocyte-like cell functions.

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. However, these cells do not have hepatic functions comparable to those of hepatocytes in vivo due to insufficient hepatic differentiation. Here we showed that the hepatic functions of hPSC-HLCs were facilitated by applying physiological liver temperatures during hepatic differentiation. We identified the optimal temperature by treating HLCs derived from H9 human embryonic stem cells (hESC-HLCs) at 39 °C; the 42 °C treatment caused significantly greater cell death than the 39 °C treatment. We confirmed the improvement of hepatic functions, such as albumin secretion, cytochrome P450 3A activity, and collagen production, without severe cell damage. 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.

Cyclo olefin polymer-based solvent-free mass-productive microphysiological systems.

A microphysiological system (MPS) holds great promise for drug screening and toxicological testing as an alternative to animal models. However, this platform faces several challenges in terms of the materials used (e.g. polydimethylsiloxane; PDMS). For instance, absorption of drug candidates and fluorescent dyes into PDMS, as well as the effect elicited by materials on cultured cells, can cause inaccurate or misleading results in cell assays. The use of PDMS also poses challenges for mass production and long-term storage of fabricated MPSs. Hence, to circumvent these issues, herein we describe the development of a cyclo olefin polymer (COP)-based MPS using photobonding processes and vacuum ultraviolet (VUV), designated as COP-VUV-MPS. COP is an amorphous polymer with chemical/physical stability, high purity and optical clarity. Due to the thermostability and high modulus of COP, the metal molding processes was applied for mass production of MPSs without deformation of microstructures and with quick fabrication cycle time (approx. 10 min/cycle). Moreover, VUV photobonding process with an excimer light at a 172nm wavelength allowed assembling COP materials without the use of additional solvents and tapes, which might cause cell damages. In comparison with the conventional MPS made of PDMS (PDMS-MPS), COP-VUV-MPS showed improved chemical resistance without causing molecule absorption. Moreover, COP-VUV-MPS maintained the stemness of environmentally sensitive human-induced pluripotent stem cells without causing undesired cellular phenotypes or gene expression. These results suggest that COP-VUV-MPS may be broadly applicable for the advancement of MPS and applications in drug development, as well asin vitrotoxicological testing.

Large Field Enhancement of Nanocoral Structures on Porous Si Synthesized from Rice Husks.

Silicon (Si) is a highly abundant, environmentally benign, and durable material and is the most popular semiconductor material; and it is used for the field enhancement of dielectric materials. Porous Si (PSi) exhibits high functionality due to its specific structure. However, the field enhancement of PSi has not been clarified sufficiently. Herein, we present the field enhancement of PSi by the fluorescence intensity enhancement of a dye molecule. The raw material used for producing PSi was rice husk, a biomass material. A nanocoral structure, consisting of spheroidal structures on the surface of PSi, was observed when PSi was subjected to chemical processes and pulsed laser melting, and it demonstrated large field enhancement with an enhancement factor (EF) of up to 545. Confocal microscopy was used for EF mapping of samples before and after laser melting, and the maps were superimposed on nanoscale scanning electron microscope images to highlight the EF effect as a function of microstructure. Nanocoral Si with high EF values were also evaluated by analyzing the porosity from gas adsorption measurements. Nanocoral Si was responsible for the high EF, according to thermodynamic calculations and agreement between experimental and calculation results as determined by Mie scattering theory.

Terahertz pulse-altered gene networks in human induced pluripotent stem cells.

Terahertz (THz) irradiation has been exploited in biomedical applications involving non-invasive manipulation of living cells. We developed an apparatus for studying the effects of THz pulse irradiation on living human induced pluripotent stem cells. The THz pulse of the maximum electric field reached 0.5 MV/cm and was applied for one hour with 1 kHz repetition to the entire cell-culture area, a diameter of 1 mm. RNA sequencing of global gene-expression revealed that many THz-regulated genes were driven by zinc-finger transcription factors. Combined with a consideration of the interactions of metal ions and a THz electric field, these results imply that the local intracellular concentration of metal ions, such as Zn2+, was changed by the effective electrical force of our THz pulse.

Recapitulation of Human Embryonic Heartbeat to Promote Differentiation of Hepatic Endoderm to Hepatoblasts.

Hepatic development requires multiple sequential physicochemical environmental changes in an embryo, and human pluripotent stem cells (hPSCs) allow for the elucidation of this embryonic developmental process. However, the current in vitro methods for hPSC-hepatic differentiation, which employ various biochemical substances, produce hPSC-derived hepatocytes with less functionality than primary hepatocytes, due to a lack of physical stimuli, such as heart beating. Here, we developed a microfluidic platform that recapitulates the beating of a human embryonic heart to improve the functionality of hepatoblasts derived from hepatic endoderm (HE) in vitro. This microfluidic platform facilitates the application of multiple mechanical stretching forces, to mimic heart beating, to cultured hepatic endoderm cells to identify the optimal stimuli. Results show that stimulated HE-derived hepatoblasts increased cytochrome P450 3A (CYP3A) metabolic activity, as well as the expression of hepatoblast functional markers (albumin, cytokeratin 19 and CYP3A7), compared to unstimulated hepatoblasts. This approach of hepatic differentiation from hPSCs with the application of mechanical stimuli will facilitate improved methods for studying human embryonic liver development, as well as accurate pharmacological testing with functional liver cells.

Three-dimensional cultured liver-on-a-Chip with mature hepatocyte-like cells derived from human pluripotent stem 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.

Fabrication of a Multiplexed Artificial Cellular MicroEnvironment Array.

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are well orchestrated and are crucial in regulating cell functions in a living system. Although a number of researchers have attempted to investigate the correlation between environmental factors and desired cellular functions, much remains unknown. This is largely due to the lack of a proper methodology to mimic such environmental cues in vitro, and simultaneously test different environmental cues on cells. Here, we report an integrated platform of microfluidic channels and a nanofiber array, followed by high-content single-cell analysis, to examine stem cell phenotypes altered by distinct environmental factors. To demonstrate the application of this platform, this study focuses on the phenotypes of self-renewing human pluripotent stem cells (hPSCs). Here, we present the preparation procedures for a nanofiber array and the microfluidic structure in the fabrication of a Multiplexed Artificial Cellular MicroEnvironment (MACME) array. Moreover, overall steps of the single-cell profiling, cell staining with multiple fluorescent markers, multiple fluorescence imaging, and statistical analyses, are described.

Microfluidic-Nanofiber Hybrid Array for Screening of Cellular Microenvironments.

Cellular microenvironments are generally sophisticated, but crucial for regulating the functions of human pluripotent stem cells (hPSCs). Despite tremendous effort in this field, the correlation between the environmental factors-especially the extracellular matrix and soluble cell factors-and the desired cellular functions remains largely unknown because of the lack of appropriate tools to recapitulate in vivo conditions and/or simultaneously evaluate the interplay of different environment factors. Here, a combinatorial platform is developed with integrated microfluidic channels and nanofibers, associated with a method of high-content single-cell analysis, to study the effects of environmental factors on stem cell phenotype. Particular attention is paid to the dependence of hPSC short-term self-renewal on the density and composition of extracellular matrices and initial cell seeding densities. Thus, this combinatorial approach provides insights into the underlying chemical and physical mechanisms that govern stem cell fate decisions.

Nano-on-micro fibrous extracellular matrices for scalable expansion of human ES/iPS cells.

Human pluripotent stem cells (hPSCs) hold great potential for industrial and clinical applications. Clinical-grade scaffolds and high-quality hPSCs are required for cell expansion as well as easy handling and manipulation of the products. Current hPSC culture methods do not fulfill these requirements because of a lack of proper extracellular matrices (ECMs) and cell culture wares. We developed a layered nano-on-micro fibrous cellular matrix mimicking ECM, named "fiber-on-fiber (FF)" matrix, which enables easy handling and manipulation of cultured cells. While non-woven sheets of cellulose and polyglycolic acid were used as a microfiber layer facilitating mechanical stability, electrospun gelatin nanofibers were crosslinked on the microfiber layer, generating a mesh structure with connected nanofibers facilitating cell adhesion and growth. Our results showed that the FF matrix supports effective hPSC culture with maintenance of their pluripotency and normal chromosomes over two months, as well as effective scaled-up expansion, with fold increases of 54.1 ± 15.6 and 40.4 ± 8.4 in cell number per week for H1 human embryonic stem cells and 253G1 human induced pluripotent stem cells, respectively. This simple approach to mimick the ECM may have important implications after further optimization to generate lineage-specific products.

The Arabidopsis ortholog of the DEAH-box ATPase Prp16 influences auxin-mediated development.

In animals and yeasts, the DEAH-box RNA-dependent ATPase Prp16 facilitates pre-mRNA splicing. However, in Chlamydomonas reinhardtii and Caenorhabditis elegans, Prp16 orthologs are not important for general pre-mRNA splicing, but are required for gene silencing and sex determination, respectively. The CLUMSY VEIN (CUV) gene, which encodes a unique Prp16 ortholog in Arabidopsis thaliana, influences auxin-mediated development. A loss-of-function cuv-1 mutation tells us that CUV does not facilitate splicing of pre-mRNA substrates indiscriminately, but differentially effects splicing and expression of genes. Here we show that CUV influences root-meristem maintenance and planar polarity of root-hair positioning, both of which are processes regulated by auxin. We propose that Arabidopsis PRP16/CUV differentially facilitates the expression of genes, including genes involved in auxin biosynthesis, transport, perception and signaling, and that in this way it influences auxin-mediated development.

CLUMSY VEIN, the Arabidopsis DEAH-box Prp16 ortholog, is required for auxin-mediated development.

Pre-messenger RNA (pre-mRNA) splicing is essential in eukaryotic cells. In animals and yeasts, the DEAH-box RNA-dependent ATPase Prp16 mediates conformational change of the spliceosome, thereby facilitating pre-mRNA splicing. In yeasts, Prp16 also plays an important role in splicing fidelity. Conversely, PRP16 orthologs in Chlamydomonas reinhardtii and nematode do not have an important role in general pre-mRNA splicing, but are required for gene silencing and sex determination, respectively. Functions of PRP16 orthologs in higher plants have not been described until now. Here we show that the CLUMSY VEIN (CUV) gene encoding the unique Prp16 ortholog in Arabidopsis thaliana facilitates auxin-mediated development including male-gametophyte transmission, apical-basal patterning of embryonic and gynoecium development, stamen development, phyllotactic flower positioning, and vascular development. cuv-1 mutation differentially affects splicing and expression of genes involved in auxin biosynthesis, polar auxin transport, auxin perception and auxin signaling. The cuv-1 mutation does not have an equal influence on pre-mRNA substrates. We propose that Arabidopsis PRP16/CUV differentially facilitates expression of genes, which include genes involved in auxin biosynthesis, transport, perception and signaling, thereby collectively influencing auxin-mediated development.