Single-cell analysis of lizard blastema fibroblasts reveals phagocyte-dependent activation of Hedgehog-responsive chondrogenesis.

Lizards cannot naturally regenerate limbs but are the closest known relatives of mammals capable of epimorphic tail regrowth. However, the mechanisms regulating lizard blastema formation and chondrogenesis remain unclear. Here, single-cell RNA sequencing analysis of regenerating lizard tails identifies fibroblast and phagocyte populations linked to cartilage formation. Pseudotime trajectory analyses suggest spp1+-activated fibroblasts as blastema cell sources, with subsets exhibiting sulf1 expression and chondrogenic potential. Tail blastema, but not limb, fibroblasts express sulf1 and form cartilage under Hedgehog signaling regulation. Depletion of phagocytes inhibits blastema formation, but treatment with pericytic phagocyte-conditioned media rescues blastema chondrogenesis and cartilage formation in amputated limbs. The results indicate a hierarchy of phagocyte-induced fibroblast gene activations during lizard blastema formation, culminating in sulf1+ pro-chondrogenic populations singularly responsive to Hedgehog signaling. These properties distinguish lizard blastema cells from homeostatic and injury-stimulated fibroblasts and indicate potential actionable targets for inducing regeneration in other species, including humans.

Lizard Blastema Organoid Model Recapitulates Regenerated Tail Chondrogenesis.

(1) Background: Lizard tail regeneration provides a unique model of blastema-based tissue regeneration for large-scale appendage replacement in amniotes. Green anole lizard (Anolis carolinensis) blastemas contain fibroblastic connective tissue cells (FCTCs), which respond to hedgehog signaling to create cartilage in vivo. However, an in vitro model of the blastema has not previously been achieved in culture. (2) Methods: By testing two adapted tissue dissociation protocols and two optimized media formulations, lizard tail FCTCs were pelleted in vitro and grown in a micromass blastema organoid culture. Pellets were analyzed by histology and in situ hybridization for FCTC and cartilage markers alongside staged original and regenerating lizard tails. (3) Results: Using an optimized serum-free media and a trypsin- and collagenase II-based dissociation protocol, micromass blastema organoids were formed. Organoid cultures expressed FCTC marker CDH11 and produced cartilage in response to hedgehog signaling in vitro, mimicking in vivo blastema and tail regeneration. (4) Conclusions: Lizard tail blastema regeneration can be modeled in vitro using micromass organoid culture, recapitulating in vivo FCTC marker expression patterns and chondrogenic potential.