Establishing a deeper understanding of the osteogenic differentiation of monolayer cultured human pluripotent stem cells using novel and detailed analyses.

BACKGROUND: Derivation of osteoblast-like cells from human pluripotent stem cells (hPSCs) is a popular topic in bone tissue engineering. Although many improvements have been achieved, the low induction efficiency because of spontaneous differentiation hampers their applications. To solve this problem, a detailed understanding of the osteogenic differentiation process of hPSCs is urgently needed. METHODS: Monolayer cultured human embryonic stem cells and human-induced pluripotent stem cells were differentiated in commonly applied serum-containing osteogenic medium for 35 days. In addition to traditional assays such as cell viability detection, reverse transcription-polymerase chain reaction, immunofluorescence, and alizarin red staining, we also applied studies of cell counting, cell telomerase activity, and flow cytometry as essential indicators to analyse the cell type changes in each week. RESULTS: The population of differentiated cells was quite heterogeneous throughout the 35 days of induction. Then, cell telomerase activity and cell cycle analyses have value in evaluating the cell type and tumourigenicity of the obtained cells. Finally, a dynamic map was made to integrate the analysis of these results during osteogenic differentiation of hPSCs, and the cell types at defined stages were concluded. CONCLUSIONS: Our results lay the foundation to improve the in vitro osteogenic differentiation efficiency of hPSCs by supplementing with functional compounds at the desired stage, and then establishing a stepwise induction system in the future.

Toxic effects of 56Fe ion radiation on the zebrafish (Danio rerio) embryonic development.

All living organisms and ecosystems are permanently exposed to ionizing radiation. Of all the types of ionizing radiation, heavy ions such as 56Fe have the potential to cause the most severe biological effects. We therefore examined the effects and potential mechanisms of iron ion irradiation on the induction of developmental toxicity and apoptosis in zebrafish embryos. Zebrafish embryos at 4h post-fertilization (hpf) were divided into five groups: a control group; and four groups irradiated with 0.5, 1, 2, and 4Gy radiation, respectively. Mortality and teratogenesis were significantly increased, and spontaneous movement, heart rate, and swimming distance were decreased in the irradiated groups, accompanied by increased apoptosis. mRNA levels of genes involved in the apoptotic pathway, including p53, bax, bcl-2, and caspase-3, were significantly affected by radiation exposure. Moreover, protein expression levels of P53 and Bcl-2 changed in accordance with the corresponding mRNA expression levels. In addition, we detected the protein expression levels of γ-H2AX, which is a biomarker for radiation-induced DNA double-strand breaks, and found that γ-H2AX protein levels were significantly increased in the irradiated groups. Overall, the results of this study improve our understanding of the mechanisms of iron ion radiation-induced developmental toxicity and apoptosis, potentially involving the induction of DNA damage and mitochondrial dysfunction. The findings of this study may aid future impact assessment of environmental radioactivity in fish.

Effects of Ru(CO)3Cl-glycinate on the developmental toxicities induced by X-ray and carbon-ion irradiation in zebrafish embryos.

The inhibitory effects of carbon monoxide (CO), generated by Ru(CO)3Cl-glycinate [CO-releasing molecule (CORM-3)], on developmental toxicity in zebrafish embryos induced by ionizing radiation with different linear energy transfer (LET) were studied. Zebrafish embryos at 5h post-fertilization were irradiated with X-ray (low-LET) and carbon-ion (high-LET) with or without pretreatment of CORM-3 1h before irradiation. CORM-3 pre-treatment showed a significant inhibitory effect on X-ray irradiation-induced developmental toxicity, but had little effect on carbon-ion irradiation-induced developmental toxicity. X-ray irradiation-induced significant increase in ROS levels and cell apoptosis could be modified by CORM-3 pretreatment. However, embryos exposed to carbon-ion irradiation showed significantly increase of cell apoptosis without obvious ROS generation, which could not be attenuated by CORM-3 pretreatment. CORM-3 could inhibit apoptosis induced by ionizing radiation with low-LET as an effective ROS scavenger. The expression of pro-apoptotic genes increased significantly after X-ray irradiation, but increased expression was reduced markedly when CORM-3 was applied before irradiation. Moreover, the protein levels of P53 and γ-H2AX increased markedly after X-ray irradiation, which could be modified by the presence of CORM-3. The protective effect of CORM-3 on X-ray irradiation occurred mainly by suppressing ROS generation and DNA damage, and thus inhibiting the activation of P53 and the mitochondrial apoptotic pathway, leading to the attenuation of cell apoptosis and consequently alleviating X-ray irradiation-induced developmental toxicity at lethal and sub-lethal levels.

Effects of Exogenous Carbon Monoxide Releasing Molecules on the Development of Zebrafish Embryos and Larvae.

The use of exogenous carbon monoxide releasing molecules (CORMs) provides promise for clinical application; however, the hazard potential of CORMs in vivo remains poorly understood. The developmental toxicity of CORM-3 was investigated by exposure to concentrations ranging from 6.25 to 400 µmol/L during 4-144 h post fertilization. Toxicity endpoints of mortality, spontaneous movement, heart rate, hatching rate, malformation, body length, and larval behavior were measured. CORM-3 disrupted the progression of zebrafish larval development at concentrations exceeding 50 µmol/L, resulting in embryonic developmental toxicity.

Toxicity of Graphene Quantum Dots in Zebrafish Embryo.

OBJECTIVE: To evaluate the bio-safety of graphene quantum dots (GQDs), we studied its effects on the embryonic development of zebrafish. METHODS: In vivo, biodistribution and the developmental toxicity of GQDs were investigated in embryonic zebrafish at exposure concentrations ranging from 12.5-200 µg/mL for 4-96 h post-fertilization (hpf). The mortality, hatch rate, malformation, heart rate, GQDs uptake, spontaneous movement, and larval behavior were examined. RESULTS: The fluorescence of GQDs was mainly localized in the intestines and heart. As the exposure concentration increased, the hatch and heart rate decreased, accompanied by an increase in mortality. Exposure to a high level of GQDs (200 µg/mL) resulted in various embryonic malformations including pericardial edema, vitelline cyst, bent spine, and bent tail. The spontaneous movement significantly decreased after exposure to GQDs at concentrations of 50, 100, and 200 µg/mL. The larval behavior testing (visible light test) showed that the total swimming distance and speed decreased dose-dependently. Embryos exposed to 12.5 µg/mL showed hyperactivity while exposure to higher concentrations (25, 50, 100, and 200 µg/mL) caused remarkable hypoactivity in the light-dark test. CONCLUSION: Low concentrations of GQDs were relatively non-toxic. However, GQDs disrupt the progression of embryonic development at concentrations exceeding 50 µg/mL.