Kazunari Igawa

Biological Safety of Fish (Tilapia) Collagen

Marine collagen derived from fish scales, skin, and bone has been widely investigated for application as a scaffold and carrier due to its bioactive properties, including excellent biocompatibility, low antigenicity, and high biodegradability and cell growth potential. Fish type I collagen is an effective material as a biodegradable scaffold or spacer replicating the natural extracellular matrix, which serves to spatially organize cells, providing them with environmental signals and directing site-specific cellular regulation. This study was conducted to confirm the safety of fish (tilapia) atelocollagen for use in clinical application. We performed in vitro and in vivo biological studies of medical materials to investigate the safety of fish collagen. The extract of fish collagen gel was examined to clarify its sterility. All present sterility tests concerning bacteria and viruses (including endotoxin) yielded negative results, and all evaluations of cell toxicity, sensitization, chromosomal aberrations, intracutaneous reactions, acute systemic toxicity, pyrogenic reactions, and hemolysis were negative according to the criteria of the ISO and the Ministry of Health, Labour and Welfare of Japan. The present study demonstrated that atelocollagen prepared from tilapia is a promising biomaterial for use as a scaffold in regenerative medicine.

Effects of hypoxia on pluripotency in murine iPS cells

Retroviral transduction of four transcription factors (Oct4, Sox2, Klf4 and c‐Myc) or three factors, excluding c‐Myc, has been shown to initiate a reprogramming process that results in the transformation of murine fibroblasts to induced pluripotent stem (iPS) cells, and there has been a rapid increase in the number of iPS cell‐based preclinical trials. In this study, the effects of these transcription factors were evaluated regarding the growth and differentiation of murine iPS cells under hypoxia. Based on the results of RT‐PCR and alizarin red S staining, there were no statistical differences in the growth and differentiation of iPS cells or the induction of iPS cells to osteoblasts under hypoxia between the transcription factor groups. Furthermore, the function of hypoxia inducible factors (HIFs) in murine iPS cells under hypoxia was investigated in relation to the morphology and expression of transcription factors using RT‐PCR and Western blotting. The HIF‐2α knockdown group exhibited a decrease in the colony size of the iPS cells. The HIF‐2α or ‐3α knockdown group demonstrated a statistically significant decrease in the transcription factor expression compared to that observed in the control group. These results demonstrate that HIF‐2α among HIFs is the most influential candidate for the maintenance of the pluripotency of murine iPS cells. Microsc. Res. Tech., 76:1084–1092, 2013.

Mitochondrial DNA analysis of Yayoi period human skeletal remains from the Doigahama site

We analyzed the mitochondrial DNA extracted from 14 human skeletal remains from the Doigahama site in Japan to clarify the genetic structure of the Doigahama Yayoi population and the relationship between burial style and kinship among individuals. The sequence types obtained in this study were compared with those of the modern Japanese, northern Kyushu Yayoi and ancient Chinese populations. We found that the northern Kyushu Yayoi populations belonged to the groups that include most of the modern Japanese population. In contrast, most of the Doigahama Yayoi population belonged to the group that includes a small number of the modern Japanese population. These results suggest that the Doigahama Yayoi population might have contributed less to the formation of the modern Japanese population than the northern Kyushu Yayoi populations. Moreover, when we examined the kinship between individuals in the Doigahama site, we found that the vicinal burial of adult skeletons indicated a maternal kinship, although that of juvenile skeletons did not. The vicinal burial style might have been influenced by many factors, such as paternal lineages, periods and geographical regions, as well as maternal lineages. In addition, skeletons considered to be those of shamans or leaders had the same sequence types. Their crucial social roles may have been inherited through maternal lineage.

D-Glucosamine Conjugation Accelerates the Labeling Efficiency of Quantum Dots in Osteoblastic Cells

Quantum dots (QDs) are useful imaging tools in the medical and biological fields due to their optical properties, such as a high fluorescence intensity, remarkable resistance to photobleaching, broad absorption spectra, and narrow emission spectra. This is the first study to investigate the uptake of carboxylated QDs conjugated with D-glucosamine (core size: approximately 3 nm, final modified size: 20–30 nm) into cultured osteoblastic cells. The QDs attached to the cell surface and were transported into the cytoplasm within approximately three hours of culture, whose process was clearly demonstrated using specific fluorescent staining of the cell membrane. Although the intranuclear distribution was not observed, a dramatic decrease in the transfer of quantum dots into the cytoplasm was recognized after approximately seven days of culture. Other interesting phenomena include the escape of the quantum dots from lysosomes in the cytoplasm, as confirmed by the merging of both QD fluorescence and specific fluorescent staining of lysosomes in the cytoplasm. These findings suggest that D-glucosamine conjugation enhances proton absorption in acid organelles and promotes the lysosomal escape of QDs.

D-Glucosamine Promotes Transfection Efficiency during Electroporation

D-Glucosamine is a useful medicament in various fields of medicine and dentistry. With respect to stability of the cell membrane, it has been reported that bradykinin-induced nociceptive responses are significantly suppressed by the direct application of D-glucosamine. Electroporation is usually used to effectively introduce foreign genes into tissue culture cells. Buffers for electroporation with or without D-glucosamine are used in experiments of transfection vectors. This is the first study to indirectly observe the stability and protection of the osteoblast membrane against both electric stress and gene uptake (the proton sponge hypothesis: osmotic rupture during endosomes prior to fusion with lysosomes) in electroporation with D-glucosamine application. The transfection efficiency was evaluated as the fluorescence intensity of the transfected green fluorescent protein (GFP) in the cultured cells (osteoblasts; NOS-1 cells). The transfection efficiency increased over 30% in the electroporation samples treated with D-glucosamine-supplemented buffer after one day. The membrane absorption of D-glucosamine is the primary mechanism of membrane stress induced by electric stress. This new function of D-glucosamine is useful and meaningful for developing more effective transformation procedures.

Effects of hypoxia inducible factors on pluripotency in human iPS cells

A hypoxic condition is known to contribute to pluripotency. In the present article, the effects of transcription factors were first assessed regarding the proliferation and differentiation of human induced pluripotent stem (iPS) cells under hypoxic conditions using cell morphology and real‐time polymerase chain reaction (RT‐PCR). Morphology evaluations and RT‐PCR revealed that the colony formation was promoted and the expression of pluripotent markers was increased under hypoxic conditions. In addition, the function of hypoxia inducible factors (HIFs) in human iPS cells under hypoxic conditions was evaluated in relation to the morphology and the expression of pluripotency markers by siRNA and RT‐PCR. The HIF‐2α silencing group showed a reduction in the colony size of human iPS cells and a statistically significant reduction in the expression of undifferentiation markers compared to the control group. Furthermore, the expression of HIF‐2α was decreased when signal transducer and activator of transcription 3 (STAT3) was suppressed by its inhibitor, Stattic or S31 201. The inhibition using Stattic did not produce colony formation. The expression of pluripotent markers was also decreased using Stattic or S31 201. This study indicates that the HIF‐2α expression in human iPS cells was activated under hypoxic conditions, similarly to that in murine iPS cells, and that HIF‐2α among HIFs is the most effective compound for maintaining the pluripotency of human iPS cells. Furthermore, the STAT3 signal pathway regulates the expression of HIF‐2α.

Effects of hypoxia on pluripotency in murine iPS cells: Pluripotency in iPS Cells Under Hypoxia

Retroviral transduction of four transcription factors (Oct4, Sox2, Klf4 and c‐Myc) or three factors, excluding c‐Myc, has been shown to initiate a reprogramming process that results in the transformation of murine fibroblasts to induced pluripotent stem (iPS) cells, and there has been a rapid increase in the number of iPS cell‐based preclinical trials. In this study, the effects of these transcription factors were evaluated regarding the growth and differentiation of murine iPS cells under hypoxia. Based on the results of RT‐PCR and alizarin red S staining, there were no statistical differences in the growth and differentiation of iPS cells or the induction of iPS cells to osteoblasts under hypoxia between the transcription factor groups. Furthermore, the function of hypoxia inducible factors (HIFs) in murine iPS cells under hypoxia was investigated in relation to the morphology and expression of transcription factors using RT‐PCR and Western blotting. The HIF‐2α knockdown group exhibited a decrease in the colony size of the iPS cells. The HIF‐2α or ‐3α knockdown group demonstrated a statistically significant decrease in the transcription factor expression compared to that observed in the control group. These results demonstrate that HIF‐2α among HIFs is the most influential candidate for the maintenance of the pluripotency of murine iPS cells. Microsc. Res. Tech., 76:1084–1092, 2013.