Preparation of mechanically patterned hydrogels for controlling the self-condensation of cells.

Synthetic protocols providing mechanical patterns to culture substrate are essential to control the self-condensation of cells for organoid engineering. Here, we present a protocol for preparing hydrogels with mechanical patterns. We describe steps for hydrogel synthesis, mechanical evaluation of the substrate, and time-lapse imaging of cell self-organization. This protocol will facilitate the rational design of culture substrates with mechanical patterns for the engineering of various functional organoids. For complete details on the use and execution of this protocol, please refer to Takebe et al. (2015) and Matsuzaki et al. (2014, 2022).1,2,3.

Mechanical guidance of self-condensation patterns of differentiating progeny.

Spatially controlled self-organization represents a major challenge for organoid engineering. We have developed a mechanically patterned hydrogel for controlling self-condensation process to generate multi-cellular organoids. We first found that local stiffening with intrinsic mechanical gradient (IG > 0.008) induced single condensates of mesenchymal myoblasts, whereas the local softening led to stochastic aggregation. Besides, we revealed the cellular mechanism of two-step self-condensation: (1) cellular adhesion and migration at the mechanical boundary and (2) cell-cell contraction driven by intercellular actin-myosin networks. Finally, human pluripotent stem cell-derived hepatic progenitors with mesenchymal/endothelial cells (i.e., liver bud organoids) experienced collective migration toward locally stiffened regions generating condensates of the concave to spherical shapes. The underlying mechanism can be explained by force competition of cell-cell and cell-hydrogel biomechanical interactions between stiff and soft regions. These insights will facilitate the rational design of culture substrates inducing symmetry breaking in self-condensation of differentiating progeny toward future organoid engineering.

Threshold dose of cow's milk in sensitization to casein higher than those of casein and ß-lactoglobulin in children with cow's milk allergy.

BACKGROUND: Recent treatment for food allergies involves the intake of allergy-causing foods at doses lower than the threshold dose determined by the oral food challenge (OFC). For a more successful treatment, it is necessary to identify a biomarker to establish safer doses of allergens in foods consumed at home. OBJECTIVE: In this study, we aim to investigate whether the pattern of sensitization to cow's milk (CM) is related to the threshold dose of CM. METHODS: Fifty patients with sensitization to casein (casein-specific IgE titer ≥ 0.7 UA/ml) and who have undergone the CM OFC test from July 2013 to July 2015 were enrolled. They were examined for the presence or absence of sensitization to ß-lactoglobulin (BLG) (BLG-specific IgE ≥ 0.7 UA/ml). They were divided into two groups, namely, the only-casein-specific IgE-positive (C) group, and both casein- and BLG-specific IgE-positive (C + B) group. RESULTS: The C group had 26 patients and the C + B group had 24. Both the CM- and casein-specific IgE titers were higher in the C + B group than in the C group. The positivity rates determined from OFC test results were 53.8 and 87.5%, and the threshold doses of CM were 88.7 and 31.1 ml in the C and C + B groups, respectively. In patients with low casein-specific IgE titers (≤ 10 UA/ml), the C + B group showed a significantly lower threshold dose of CM than the C group. CONCLUSIONS: Our results suggest that children with CM allergy sensitized to casein alone have a higher threshold dose than those sensitized to both casein and BLG.

Organizational effects of estrogen on male-type vulnerability to early weaning.

We previously reported that early-weaned (postnatal day 14) male ICR mice, compared to normally weaned animals, exhibited a persistent increase in anxiety-related behavior in the elevated plus maze test. In this study, we examined whether steroid hormone manipulations on postnatal day 0 and at the ages of 2 or 3 weeks affected male-type vulnerability to early weaning. Neither castration nor ovariectomy at the age of 3 weeks affected male-type vulnerability. However, in males, castration at the age of 2 weeks attenuated the increased anxiety levels induced by early weaning, and the implantation of testosterone or estradiol, but not of dihydrotestosterone, restored the effects of early weaning. In contrast, in females, neonatal treatment with testosterone propionate together with testosterone at the age of 2 weeks, which reversed sexual behavior to the male type, did not affect anxiety levels in response to early weaning. When pregnant females were repeatedly treated with testosterone propionate on embryonic days 14, 17, and 19, in addition to testosterone treatment at the age of 2 weeks, the anxiety levels in female were increased by early weaning. Furthermore, the prenatal treatment of estradiol benzoate, but not dihydrotestosterone, induced enhanced anxiety levels by early weaning in females. These results suggest that neural systems are masculinized by estrogen from the embryonic phase to the early postnatal period and are responsible for the high levels of anxiety elicited by early weaning.

Effects of sex and rearing environment on imipramine response in mice.

RATIONALE: Antidepressant treatments are commonly prescribed, but few studies have been published, even in animal models, on differences in reactivity to antidepressants with respect to rearing environment and sex. OBJECTIVES: Using a mouse model, we investigated the hypothesis that rearing environment (weaning time) and sex could impact responses to imipramine treatment. METHODS: ICR mice were assigned to four groups for each sex: early weaned saline or imipramine treated, normally weaned saline or imipramine treated. Early weaning was conducted on postnatal day 14. All groups were injected intraperitoneally with drug or vehicle for 2 weeks from the age of 6 weeks and tested in the elevated plus maze to estimate anxiety levels. Hippocampal neurogenesis was also assessed with immunostaining for calretinin and calbindin because it has suggested that neurogenesis is required for the behavioral effects of antidepressants. RESULTS: Imipramine treatment decreased anxiety levels in females, but not in males, in the normal weaning condition. By contrast, the same treatment decreased anxiety levels in males, but not females, in the early weaning condition. Hippocampal changes, which correlated these behavioral responses to imipramine, were seen in the extragranule cell layer: the number of calretinin-positive cells was increased by imipramine in females, but not in males, in the normal weaning condition. In the early weaning condition, however, the treatment was associated with similar changes in males but not in females. CONCLUSIONS: The results suggest that rearing environment and sex differences are implicated in responses to imipramine treatment with respect to anxiety behavior and neurogenesis in the hippocampus.