MN•CS•IO System: Cross-Species Study for HBB Function in High-Altitude Hypoxia Adaptation.

The genetic basis of high-altitude hypoxia adaptation remains largely elusive, particularly the role of HBB. Here, we developed an integrative mesh-nanorod (MN) cell sorting, chitosan nanoparticle (CS) delivery, and in vivo intraosseous (IO) injection (MN•CS•IO) system to functionally investigate HBB in cross-species high-altitude hypoxia adaptation. Using this MN•CS•IO platform, we transplanted high/low-altitude species HBB-modified lineage-negative (Lin-) cells into murine models, achieving 95.59% transfection efficiency and 8.15% HBB relative expression, and subjected them to simulated and natural Qinghai-Tibet hypoxia conditions. High-altitude HBB recipients exhibited downregulated Rragd expression, activated oxygen transport and hematopoietic lineage pathways, improved behavioral performance, and neuroprotection against hypoxia-induced damage, compared with low-altitude animals. MN•CS•IO system data demonstrate that HBB determines high-altitude hypoxia adaptation. For validation, we generated bone marrow chimeras using CD45.1 hematopoietic cells with Pika-HBB integrated at the Rosa26 locus. The reconstituted mice exhibited consistent hypoxic behavioral and phenotypic alterations with those observed via the MN•CS•IO system, validating the reliability and stability of HBB-mediated high-altitude hypoxia adaptation. Together, our findings demonstrate that HBB is a key genetic determinant of high-altitude hypoxia adaptation, and also introduce a versatile gene-function validation tool for evolutionary and biomedical research.