Kenneth Ka Ho Lee

Background & Aims: Liver cirrhosis is a major cause of death worldwide, and its prevalence is growing rapidly due to the growth of obesity and diabetes population with non-alcoholic fatty liver disease (NAFLD). Yet, no effective therapeutics have been developed to treat NAFLD or its more advanced stage, non-alcoholic steatohepatitis (NASH). This has raised great concern for a representative liver model to be developed so that novel drugs could be screened, identified and developed. Presently, we aim to develop a liver organoid entirely from human induced pluripotent stem cells (hiPSC) to model liver fibrogenesis and NAFLD. Methods: Hepatoblasts (HBs), mesenchymal stem cells (MSCs), hepatic stellate cell (HSCs) and endothelial cells (ECs) were derived from hiPSCs, allowed to self-organized and differentiated into liver organoids. Liver functions, transcriptomic and protein expression of liver organoids were characterized and validated. Liver organoids were exposed to thioacetamide (TAA) and free fatty acids (FFA) to be induced into liver disease model. Results: The liver organoids we fabricated were highly vascularized, exhibited liver-specific functions and hepatic cellular spatial organization. The presence of liver specific ECs, macrophages and cholangiocytes were found within our organoids. TAA induced fibrosis in our liver organoids that exhibited diminished liver functions, elevated pro-inflammatory cytokines and fibrosis-related gene expression, as well as extensive collagen deposit. Organoids treated with FFA developed steatosis, inflammation and fibrosis. Conclusions: We generated a novel method, that is Matrigel-independent and size-controllable, for making human liver organoids. These organoids can potentially be utilized as tissue-mimetic in vitro model for high throughput screening to identify drugs that can be used to treat liver fibrosis and NAFLD.

BRISC and BRCA1-A complex member 2 (Babam2) plays an essential role in promoting cell cycle progression and preventing cellular senescence. Babam2-deficient fibroblasts show proliferation defect and premature senescence compared with their wild-type (WT) counterpart. Pluripotent mouse embryonic stem cells (mESCs) are known to have unlimited cell proliferation and self-renewal capability without entering cellular senescence. Therefore, studying the role of Babam2 in ESCs would enable us to understand the mechanism of Babam2 in cellular aging, cell cycle regulation, and pluripotency in ESCs. For this study, we generated Babam2 knockout (Babam2^−/−) mESCs to investigate the function of Babam2 in mESCs. We demonstrated that the loss of Babam2 in mESCs leads to abnormal G1 phase retention in response to DNA damage induced by gamma irradiation or doxorubicin treatments. Key cell cycle regulators, CDC25A and CDK2, were found to be degraded in Babam2^−/− mESCs following gamma irradiation. In addition, Babam2^−/− mESCs expressed p53 strongly and significantly longer than in control mESCs, where p53 inhibited Nanog expression and G1/S cell cycle progression. The combined effects significantly reduced developmental pluripotency in Babam2^−/− mESCs. In summary, Babam2 maintains cell cycle regulation and pluripotency in mESCs in response to induced DNA damage.

Background: The functions of microRNA-1 (miR-1) in cardiac hypertrophy, and cardiomyocyte differentiation have been investigated. However, the mechanism on how miR-1 could repress cardiomyocyte proliferation has not been fully elucidated. Methods: We address this issue by investigating whether miR-1 affected the proliferation of neonatal cardiomyocyte and identify some of the genes targeted by miR-1. miR-1 was over-expressed in neonatal cardiomyocytes and the effect on cell cycle and growth were analyzed by flow cytometry and Brdu-incorporation assay. Relevant vectors carrying the luciferase reporter were constructed for validation of miR-1 binding to its matching sites on the 3’-untranslated region of the predicated target mRNAs. Cardiomyocytes were co-transfected with the vectors and miR-1 mimics, then luciferase reporter assay was performed. Lastly, we examined the expression of target genes in cardiomyocytes after transfection with miR-1 mimics, as well as their normal expression pattern in 2- and 13-day-old mice hearts. Results: We have demonstrated that miR-1 was the most significantly upregulated miRNA in 13-day-old mouse hearts compared with 2-day-old hearts. We also showed that miR-1 could repress cardiomyocyte G1/S phase transition, proliferation and viability. IGF1 and CCND1 were identified as candidate target genes regulated by miR-1. In addition, overexpression of miR-1 could suppress the expression of these two genes at the mRNA level. It could also correspondingly inhibit CCND1 expression at the protein level but not for IGF1. Conclusions: Our results suggest that miR-1 plays an important role in inhibiting cardiomyocyte proliferation in the developing neonatal mouse heart by directly suppressing the cell-cycle regulator, CCND1.

Suspension spheroid cultures of anchorage‐dependent cell types have been widely used in cancer and stem cell research, as well as for producing organoids. It is believed that the 3‐dimensional spheroid presents cells with a more physiological microenvironment to grow so that they behave more like cells in vivo, which is lacking in conventional 2‐dimensional monolayer cultures. Recently, it has been reported that cancer cells grown as spheroids could express stem cell‐associated genes. Hence, it is investigated whether normal mouse and human fibroblasts cultured as spheroids could also be induced to express stem cell‐associated genes. The transcriptomes of human fibroblasts cultured as a monolayer and spheroids are compared and analyzed using real‐time RT‐qPCR, RNA‐sequencing, and bioinformatics. The results reveal that the spheroid transcriptome resemble somatic cells being reprogramed into stem cells, including the induced expression of stemness‐associated genes, increased expression of mesenchymal‐to‐epithelial transition‐associated genes, and decreased expression of epithelial‐to‐mesenchymal transition‐associated genes. In this context, it is hypothesized that during the process of spheroid formation, matrix‐cell signaling is lost in favor of cell–cell contact signaling and that this subsequently increases the activity of the PI3K/Akt pathway that then upregulates Tbdx3 and stemness‐associated genes.

BRE (Brain and Reproductive Organ-Expressed) is an anti-apoptotic protein and a core component of DNA-repair BRCA1-A complex. Microarray-detected high BRE gene expression has been found to be associated with better patient survival in AML (acute myeloid leukemia) with MLL-AF9 translocation, and radiotherapy-treated non-familial breast cancer. A recent finding suggests that the high BRE gene expression in MLL-AF9 AML could be attributed to the additional expression of a transcript variant encoding a novel C-terminal BRE isoform. Using THP-1 as the MLL-AF9 AML cell model, we found that ectopic expression of the C-terminal BRE, which could not form an intact BRCA1-A complex, indeed increased cellular sensitivity to chemotherapeutic drugs and inhibited cell proliferation, while the complete opposite was achieved by the ectopic expression of full-length BRE. Our findings suggest that the C-terminal BRE-encoding transcript could be responsible for better patient survival and may have therapeutic potential for cancer.

Summary: Fibroblast growth factor (FGF) signalling acts as one of modulators that control neural crest cell (NCC) migration, but how this is achieved is still unclear. In this study, we investigated the effects of FGF signalling on NCC migration by blocking this process. Constructs that were capable of inducing Sprouty2 (Spry2) or dominant-negative FGFR1 (Dn-FGFR1) expression were transfected into the cells making up the neural tubes. Our results revealed that blocking FGF signalling at stage HH10 (neurulation stage) could enhance NCC migration at both the cranial and trunk levels in the developing embryos. It was established that FGF-mediated NCC migration was not due to altering the expression of N-cadherin in the neural tube. Instead, we determined that cyclin D1 was overexpressed in the cranial and trunk levels when Sprouty2 was upregulated in the dorsal neural tube. These results imply that the cell cycle was a target of FGF signalling through which it regulates NCC migration at the neurulation stage.

Glioblastoma multiforme (GBM), a malignant tumor of the central nervous system, has a high mortality rate. No curative treatment is presently available, and the most commonly used chemotherapeutic drug, the alkylating agent temozolomide (TMZ), is only able to increase life expectancy and is often associated with drug resistance. Therefore, an urgent need does exist for novel drugs aimed at treating gliomas. In the present study, we obtained three major results using corilagin: (a) demonstrated that it inhibits the growth of U251 glioma cells through activation of the apoptotic pathway; (b) demonstrated that it is also active on TMZ-resistant T98G glioma cells; and (c) demonstrated that when used in combination with TMZ on T98G glioma cells, a higher level of proapototic and antiproliferative effects is observed. Our study indicates that corilagin should be investigated in more detail to determine whether it can be developed as a potential therapeutic agent. In addition, our results suggest that corilagin could be used in combination with low doses of other standard anticancer chemotherapeutic drugs against gliomas (such as TMZ) with the aim of obtaining enhanced anticancer effects.

A detailed analysis of zoological and botanical engineering structures reveals remarkable ideas for developing new healthcare technologies. One eclectic example is the surface microprotrusions of Gecko lizard skins, featuring nanoscale tips that engage in bacterial repulsion and killing. However, building biomimetic synthetic nanostructures in long range for technological use is hampered by the lack of concentrated digital information for 3D nano‐fabrication. For instance, standard micro‐computed tomography (µCT), confocal laser scanning microscopy (CLSM) and atomic force microscopy (AFM) imaging failed to replicate gecko spinules at the nanoscale. The authors now show that the latest generation of high‐power X‐ray microscopy (Zeiss X‐Radia 810) builds high contrast images of the gecko skin surface, and following 3D digital conversion the individual spinules, in the large array, are perfectly delineated at 150 nm and clearly featured the 50 nm nanotips crucial for bacterial cell rupturing. Consequently, the authors generate stenographic (STL.) digital file formats loaded with the instructions for 3D printing and the design blueprints for soft lithography replication. A further reconstruction of the digital data is undertaken into a 3D solid object using MeshLabs, yielding biologically realistic, virtual‐world spinule arrays. In this form, the authors are able to edit the spinules for optimal killing performance against bacterial cells.