Deguchi, K. | bioRxiv Science

bioRxiv2025

Scaffold-free cryopreservable cartilage grafts obtained from hiPSC-derived chondroprogenitor cells for airway reconstruction with growth adaptability

Pediatric tracheal reconstruction remains a major clinical challenge because of limited graft availability and the need for growth-adaptive materials. Current approaches, such as costal cartilage grafting and use of scaffold-based constructs, often suffer from complications including graft resorption, donor site morbidity, and poor integration. Here, we present scaffold-free cartilage grafts (chondro-plates) derived from expandable limb-bud mesenchymal cells generated from human leukocyte antigen-homozygous human induced pluripotent stem cells. These grafts are cryopreservable and maintain their hyaline cartilage phenotype after a brief pre-culture. In both rat and rabbit tracheal defect models, chondro-plates supported robust cartilage regeneration, epithelial reconstitution, and neovascularization. Importantly, in a pediatric-like growing rat model, chondro-plates preserved luminal patency and structural integrity, outperforming autologous costal cartilage. This study demonstrates a clinically viable, off-the-shelf strategy for tracheal reconstruction using scalable, immunocompatible, and growth-adaptive cartilage grafts.

bioengineering↗

bioRxiv2022

Characterisation of an Escherichia coli line that completely lacks ribonucleotide reduction yields insights into the evolution of obligate intracellularity.

All life requires ribonucleotide reduction for de novo synthesis of deoxyribonucleotides. A handful of obligate intracellular species are known to lack ribonucleotide reduction and are instead dependent on their host for deoxyribonucleotide synthesis. As ribonucleotide reduction has on occasion been lost in obligate intracellular parasites and endosymbionts, we reasoned that it should in principle be possible to knock this process out entirely under conditions where deoxyribonucleotides are present in the growth media. We report here the creation of a strain of E. coli where all three ribonucleotide reductase operons have been fully deleted. Our strain is able to grow in the presence of deoxyribonucleosides and shows slowed but substantial growth. Under limiting deoxyribonucleoside levels, we observe a distinctive filamentous cell morphology, where cells grow but do not appear to divide regularly. Finally, we examined whether our lines are able to adapt to limited supplies of deoxyribonucleosides, as might occur in the evolutionary switch from de novo synthesis to dependence on host production during the evolution of parasitism or endosymbiosis. Over the course of an evolution experiment, we observe a 25-fold reduction in the minimum concentration of exogenous deoxyribonucleosides necessary for growth. Genome analysis of replicate lines reveals that several lines carry mutations in deoB and cdd. deoB codes for phosphopentomutase, a key part of the deoxyriboaldolase pathway, which has been hypothesised as an alternative to ribonucleotide reduction for deoxyribonucleotide synthesis. Rather than synthesis via this pathway complementing the loss of ribonucleotide reduction, our experiments reveal that mutations appear that reduce or eliminate the capacity for this pathway to catabolise deoxyribonucleotides, thus preventing their loss via central metabolism. Mutational inactivation of both deoB and cdd is also observed in a number of obligate intracellular bacteria that have lost ribonucleotide reduction. We conclude that our experiments recapitulate key evolutionary steps in the adaptation to intracellular life without ribonucleotide reduction.

microbiology↗