Microphysiological systems (MPS) are making advances to provide more standardized and predictive physiologically relevant responses to test articles in living tissues and organ systems. The excitement surrounding the potential of MPS to better predict human responses to medicines and improving clinical translation is overshadowed by their relatively slow adoption by the pharmaceutical industry and regulators. Collaboration between multiorganizational consortia and regulators is necessary to build an understanding of the strengths and limitations of MPS models and closing the current gaps. Here, we review some of the advances in MPS research, focusing on liver, intestine, vascular system, kidney and lung and present examples highlighting the context of use for these systems. For MPS to gain a foothold in drug development, they must have added value over existing approaches. Ideally, the application of MPS will augment in vivo studies and reduce the use of animals via tiered screening with less reliance on exploratory toxicology studies to screen compounds. Because MPS support multiple cell types (e.g. primary or stem-cell derived cells) and organ systems, identifying when MPS are more appropriate than simple 2D in vitro models for understanding physiological responses to test articles is necessary. Once identified, MPS models require qualification for that specific context of use and must be reproducible to allow future validation. Ultimately, the challenges of balancing complexity with reproducibility will inform the promise of advancing the MPS field and are critical for realization of the goal to reduce, refine and replace (3Rs) the use of animals in nonclinical research.
The importance of glucose is well known as an energy source in testes. In order to evaluate the effects of long-lasting hypoglycemia on testes, a novel glucokinase activator, TMG-123, was dosed to rats at 5, 20 and 100 mg/kg for 13 weeks. As a result, plasma glucose levels decreased for several hours with increasing doses over the dose range of 5 to 100 mg/kg. No toxicological findings attributable to the test article were observed in clinical observation, measurements of body weight and food consumption, necropsy, and organ weight measurement. Histopathology showed scattered degeneration of seminiferous tubules in testes, and exfoliation of germ cells related to the degeneration of seminiferous tubules was observed in the lumen of both epididymides in the same animals at the end of the dosing period. Similar histopathological findings were noted at the end of the recovery period. In addition, a fertility study was conducted at the same doses for 13 weeks for males and 5 weeks for females. Sperm analysis showed decreases in the sperm concentration and the motility index and an increase in the incidences of sperm malformations. However, there were no abnormalities in the copulation or fertility rate. These results suggest that long-lasting hypoglycemia in rats is harmful to spermatogenesis and the testicular damage does not recover.
Cadmium (Cd) is a toxic heavy metal, long-term exposure to which causes renal damage associated with disruption in gene expression. Transcription factors whose activities were altered in the kidneys of mice exposed to Cd for 3 months were assessed using protein/DNA-binding assays. Female C57BL/6J mice were exposed to 300 ppm Cd in the diet for 3 months. Nuclear extracts of kidney were used for protein/DNA-binding assays. The concentration of Cd was approximately 100 ppm in mouse kidney, a level that did not induce renal toxicity. Among the 345 transcription factors evaluated, five transcription factors showed over a two-fold increase in their activities and 14 transcription factors showed a half-fold change in their activities after Cd exposure. These findings may provide new information about the causative transcription factors associated with Cd renal toxicity.
Pluripotent stem cells (PSCs) possess unique characteristics that distinguish them from other cell types. Human embryonic stem (ES) cells are recently gaining attention as a powerful tool for human toxicity assessment without the use of experimental animals, and an embryonic stem cell test (EST) was introduced for this purpose. However, human PSCs have not been thoroughly investigated in terms of drug resistance or compared with other cell types or cell states, such as naïve state, to date. Aiming to close this gap in research knowledge, we assessed and compared several human PSC lines for their resistance to drug exposure. Firstly, we report that RIKEN-2A human induced pluripotent stem (iPS) cells possessed approximately the same sensitivity to selected drugs as KhES-3 human ES cells. Secondly, both ES and iPS cells were several times less resistant to drug exposure than other non-pluripotent cell types. Finally, we showed that iPS cells subjected to naïve-state induction procedures exhibited a sharp increase in drug sensitivity. Upon passage of these naïve-like cells in non-naïve PSC culture medium, their sensitivity to drug exposure decreased. We thus revealed differences in sensitivity to drug exposure among different types or states of PSCs and, importantly, indicated that naïve-state induction could increase this sensitivity.
Lysosomes are degradative organelles essential for cell homeostasis. However, various internal and external stimuli, including L-leucyl–L-leucine methyl ester (LLOMe), which is one of the common lysosomotropic agents, permeabilize the lysosomal membrane, leading to lysosome-dependent cell death because of leakage of lysosomal contents to the cytosol. The microphthalmia/transcription factor E (MiT/TFE) family members, which include transcription factor EB (TFEB), transcription factor E3 (TFE3), and microphthalmia-associated transcription factor (MITF), are master regulators of lysosomal biogenesis and are known to be involved in the lysosomal stress response. However, their protective effects against cell death associated with lysosomal-membrane damage are still poorly understood. In this study, we confirmed that LLOMe-induced lysosomal damage triggered nuclear translocation of TFEB/TFE3/MITF and increased the mRNA levels of their target genes encoding lysosomal hydrolases and lysosomal membrane proteins in HeLa cells. Furthermore, we revealed that TFEB/TFE3/MITF knockdown exacerbated LLOMe-induced cell death. However, TFEB overexpression only slightly attenuated LLOMe-induced cell death, despite enhanced LLOMe-induced increase in CTSD mRNA levels, implying that the endogenous levels of MiT/TFE family members might be sufficient to promote lysosomal biogenesis in response to lysosomal-membrane damage. Our results suggest that MiT/TFE family members suppress the cell death associated with lysosomal-membrane damage.