Summary Human embryonic stem cells (hESCs) hold great promise for cell therapy as a source of diverse differentiated cell types. One key bottleneck to realizing such potential is allogenic immune rejection of hESC-derived cells by recipients. Here, we optimized humanized mice (Hu-mice) reconstituted with a functional human immune system that mounts a vigorous rejection of hESCs and their derivatives. We established knock-in hESCs that constitutively express CTLA4-Ig and PD-L1 before and after differentiation, denoted CP hESCs. We then demonstrated that allogenic CP hESC-derived teratomas, fibroblasts, and cardiomyocytes are immune protected in Hu-mice, while cells derived from parental hESCs are effectively rejected. Expression of both CTLA4-Ig, which disrupts T-cell co-stimulatory pathways, and PD-L1, which activates T-cell inhibitory pathway, is required to confer immune protection as neither was sufficient on their own. These findings are instrumental for developing a strategy to protect hESC-derived cells from allogenic immune responses without requiring systemic immune suppression.

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3',6'-Bis(diphenylphosphinyl)fluorescein (PF-1) was synthesized as a highly selective and sensitive fluorescent probe for imaging O(2) (.-) in living cells. The design strategy for the probe was based on the nucleophilic mechanism of O(2) (.-) to mediate deprotection of this probe to give fluorescein. Upon reaction with O(2) (.-), the probe exhibits a strong fluorescence response and high selectivity for O(2) (.-) over other reactive oxygen species and some biological compounds. The phosphinate-based probe, as a new fluorescent reagent, is cell-permeable and can detect micromolar changes of O(2) (.-) concentrations by using confocal microscopy in living cells. The unique combination of good selectivity, high sensitivity, good water solubility, and rapid reactivity establishes the potential value of the probe for facilitating investigations of the generation, metabolism, and mechanisms of superoxide-mediated cellular homeostasis and injury.

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