GSE118454
Brief Local Application of Progesterone via a Wearable Bioreactor Induces Long-Term Regenerative Response in Adult Xenopus Hindlimb
The induction of limb repair depends on signals that initiate regeneration and the successful transmission of those signals in vivo. Here, we characterize the effects of an integrated device-based in vivo delivery of drug compounds in adult Xenopus laevis, which normally regenerate only a cartilaginous spike after limb amputation. A wearable bioreactor containing a silk protein-based hydrogel containing progesterone that was applied to the wound site immediately after hind-limb amputation for 24 hours induced regeneration of flattened paddle-like structures in adult frogs. Molecular assays (ELISA and NGS), Morphometric analysis (soft-tissue patterning), X-ray imaging (bone regrowth), immunofluorescence (cell proliferation, immune response, neuro/vascular tissues), and behavioral data (functional use of regenerates) were used to characterize the differences between the paddle-like structures and the hypomorphic spike observed in untreated animals. Initially, treated animals exhibited an extensive wound epithelium, and increased nerve supply compared to controls. Subsequently, flattened structures with robust bone remodeling were formed, resembling comparable tissues of adult limbs at the early and middle stages of regeneration. The limbs ceased regeneration at the position where the digits usually form. Functional tests revealed that the response to the paddle-like structures was similar to that of uncut limbs. Analysis of the blastema transcriptome identified a number of key genes related to the combined intervention, and also revealed regulated transcripts related to hormonal and neural pathways, representing novel targets for emerging regenerative therapies. Our experiments (1) establish a new model for testing of therapeutic cocktails in vertebrate limb regeneration; (2) identify pro-regenerative activities of progesterone, and (3) provide proof-of-principle of using integrated device-based delivery of small molecule drugs as a viable strategy to induce and maintain a regenerative response. Overall design: Samples from 3 treatment groups were RNA sequenced (Control, Sham or Silk, and Prog) via NGS, n= 9 per group, n=3 for three tissues (brain, stump, and blastema)