A Systematic Approach to Time-series Metabolite Profiling and RNA-seq Analysis of Chinese Hamster Ovary Cell Culture.

Chinese hamster ovary (CHO) cells are the primary host used for biopharmaceutical protein production. The engineering of CHO cells to produce higher amounts of biopharmaceuticals has been highly dependent on empirical approaches, but recent high-throughput "omics" methods are changing the situation in a rational manner. Omics data analyses using gene expression or metabolite profiling make it possible to identify key genes and metabolites in antibody production. Systematic omics approaches using different types of time-series data are expected to further enhance understanding of cellular behaviours and molecular networks for rational design of CHO cells. This study developed a systematic method for obtaining and analysing time-dependent intracellular and extracellular metabolite profiles, RNA-seq data (enzymatic mRNA levels) and cell counts from CHO cell cultures to capture an overall view of the CHO central metabolic pathway (CMP). We then calculated correlation coefficients among all the profiles and visualised the whole CMP by heatmap analysis and metabolic pathway mapping, to classify genes and metabolites together. This approach provides an efficient platform to identify key genes and metabolites in CHO cell culture.

Chondrogenic differentiation of human mesenchymal stem cells on fish scale collagen.

Fish collagen has recently been reported to be a novel biomaterial for cell and tissue culture as an alternative to conventional mammalian collagens such as bovine and porcine collagens. Fish collagen could overcome the risk of zoonosis, such as from bovine spongiform encephalopathy. Among fish collagens, tilapia collagen, the denaturing temperature of which is near 37°C, is appropriate for cell and tissue culture. In this study, we investigated chondrogenic differentiation of human mesenchymal stem cells (hMSCs) cultured on tilapia scale collagen fibrils compared with porcine collagen and non-coated dishes. The collagen fibrils were observed using a scanning electronic microscope. Safranin O staining, glycosaminoglycans (GAG) expression, and real-time PCR were examined to evaluate chondrogenesis of hMSCs on each type of collagen fibril. The results showed that hMSCs cultured on tilapia scale collagen showed stronger Safranin O staining and higher GAG expression at day 6. Results of real-time PCR indicated that hMSCs cultured on tilapia collagen showed earlier SOX9 expression on day 4 and higher AGGRECAN and COLLAGEN II expression on day 6 compared with on porcine collagen and non-coated dishes. Furthermore, low mRNA levels of bone gamma-carboxyglutamate, a specific marker of osteogenesis, showed that tilapia collagen fibrils specifically enhanced chondrogenic differentiation of hMSCs in chondrogenic medium, as well as porcine collagen. Accordingly, tilapia scale collagen may provide an appropriate collagen source for hMSC chondrogenesis in vitro.

Detection of initial angiogenesis from dorsal aorta into metanephroi and elucidation of its role in kidney development.

Reconstruction of blood vessels is considered the most difficult part for the complicated organs, therefore, blood vessel construction is regarded as a key point for kidney regeneration in vitro. Vasculogenesis and angiogenesis are the two mechanisms to form blood vessels in embryonic organs, and most studies resided in vaculogenesis. Angiogenesis resided mostly in adult diseases such as wound healing, growth of tumors, and psoriasis diseases. However, renal angiogenesis is simply attributed to the sprouting of pre-existing blood vessel from dorsal aorta into metanephroi, and its occurrence is considered to be at a late stage of metanephric development. Since no techniques are available for delicate detection, the initial angiogenesis from dorsal aorta into metanephroi as well as its role in kidney development still remained unclear. In this study, we developed a method to detect the initial angiogenesis of dorsal aorta into metanephroi, and firstly clarified that dorsal aorta angiogenesis occurred at an early stage of metanephric development. We also elucidated the role of dorsal aorta angiogenesis in promoting the early blood vessel formation, tubule formation and glomeruli maturation. It is suggested that blood flow and dynamic circulation of various factors at the early developing stage may be prerequisite to a successful construction of blood vessels in the complicated organs either in vitro or in vivo. These findings contribute to a better understanding of dorsal aorta angiogenesis during kidney development and shed light on its significant value for the application of tissue engineering to complicated organs.

Type V collagen fibrils in mouse metanephroi.

Type V collagen (Col V) molecule, a minor component of kidney connective tissues, was found in adult cornea, and has been considered as a regulatory fibril-forming collagen that emerges into type I collagen to trigger the initiation of Col I fiber assembly. Col V was also found in injured, wound healing tissues or placenta, and was considered as a dysfunctional extracellular matrix (ECM). Reconstituted Col V fibril was characterized as an ECM to detach cells in vitro, and our previous study showed that the reconstituted Col V fibril facilitated the migration of glomerular endothelial cells and induced ECM remodeling, whereas Col V molecules stabilized cells. These facts suggest that not only the structure but also the function of Col V fibril are different from Col V molecule. Recently, Col V molecule has been reported existing in various developing tissues such as bone and lung, but Col V fibril has not been reported yet. In this study, we firstly explored the existence of Col V fibril in metanephroi, and found it distributed in the immature kidney tissues whereas disappeared when the tissues reached mature. It is likely that Col V fibril may form a prototype of pericellular microenvironment and the transient existence of Col V fibril may play a role as the pioneering ECM during metanephric tissue morphogenesis.