Axolotls (urodele amphibians) have the unique ability, among vertebrates, to perfectly regenerate many parts of their body including limbs, tail, jaw and spinal cord following injury or amputation. The axolotl limb is the most widely used structure as an experimental model to study tissue regeneration. The process is well characterized, requiring multiple cellular and molecular mechanisms. The preparation phase represents the first part of the regeneration process which includes wound healing, cellular migration, dedifferentiation and proliferation. The redevelopment phase represents the second part when dedifferentiated cells stop proliferating and redifferentiate to give rise to all missing structures. In the axolotl, when a limb is amputated, the missing or wounded part is regenerated perfectly without scar formation between the stump and the regenerated structure. Multiple authors have recently highlighted the similarities between the early phases of mammalian wound healing and urodele limb regeneration. In mammals, one very important family of growth factors implicated in the control of almost all aspects of wound healing is the transforming growth factor-beta family (TGF-β). In the present study, the full length sequence of the axolotl TGF-β1 cDNA was isolated. The spatio-temporal expression pattern of TGF-β1 in regenerating limbs shows that this gene is up-regulated during the preparation phase of regeneration. Our results also demonstrate the presence of multiple components of the TGF-β signaling machinery in axolotl cells. By using a specific pharmacological inhibitor of TGF-β type I receptor, SB-431542, we show that TGF-β signaling is required for axolotl limb regeneration. Treatment of regenerating limbs with SB-431542 reveals that cellular proliferation during limb regeneration as well as the expression of genes directly dependent on TGF-β signaling are down-regulated. These data directly implicate TGF-β signaling in the initiation and control of the regeneration process in axolotls.

Urodele amphibians, such as the axolotl (Ambystoma mexicanum), have the unique faculty among vertebrates to regenerate lost appendages (limbs and tail) and other body parts (apex of the heart, forebrain and jaw) after amputation. Interestingly, axolotls never seem to form scar tissue at the site of amputation once regeneration is completed. Before now, very few studies were directly focused on the description of the events happening during wound healing after a skin injury in salamanders. In this paper, we directly investigated skin wound healing after excisional wounding which removed the epidermis, dermis and basement membrane in the axolotl. Axolotls were wounded with a 1.5-mm skin biopsy punch. Results show rapid re-epithelialization of the wound within 8 hrs after wounding. Histological analysis of wound healing confirmed the absence of tissue fibrosis throughout the process and shows that skin integrity is re-established by 90 days after wounding. Results also reveal the absence of neutrophils in the wound area, suggestive of a lack of or low inflammatory response. The expression of proteins central to wound healing seemed different than in mammals as α-smooth muscle actin was absent and transforming growth factor β-1 was only transiently expressed during wound healing in the axolotl. Finally, subcutaneous injections of bleomycin were performed to verify whether the induction of scar tissue was possible in axolotls. Surprisingly, results show that axolotls are not resistant to bleomycin-induced tissue fibrosis, but the resulting scar tissue does not seem to contain significant amounts of collagen.

Objective In diabetics, hyperglycemia results in deficient endothelial progenitors and cells, leading to cardiovascular complications. We aim to engineer three-dimensional (3D) vascular networks in synthetic hydrogels from type-1 diabetes (T1D) patient-derived human induced pluripotent stem cells (hiPSCs), to serve as a transformative autologous vascular therapy for diabetic patients. Approach and Results We validated and optimized an adherent, feeder free differentiation procedure to derive early vascular cells (EVCs) with high portions of VEcad+ cells from hiPSCs. We demonstrate similar differentiation efficiency from hiPSCs derived from healthy donor and T1D patients. T1D-hiPSC-derived VEcad+ cells can mature to functional endothelial cells (ECs) expressing mature markers: von Willebrand factor and eNOS, are capable of lectin binding and acetylated low density lipoprotein uptake, form cords in Matrigel and respond to tumor necrosis factor alpha. When embedded in engineered hyaluronic acid (HA) hydrogels, T1D-EVCs undergo morphogenesis and assemble into 3D networks. When encapsulated in a novel hypoxia-inducible (HI) hydrogel, T1D-EVCs respond to low oxygen and form 3D networks. As xenografts, T1D-EVCs incorporate into developing zebrafish vasculature. Conclusion Using our robust protocol, we can direct efficient differentiation of T1D-hiPSC to EVCs. Early ECs derived from T1D-hiPSC are functional when mature. T1D-EVCs self-assembled into 3D networks when embedded in HA and HI hydrogels. The capability of T1D-EVCs to assemble into 3D networks in engineered matrices and to respond to a hypoxic microenvironment is a significant advancement for autologous vascular therapy in diabetic patients and has broad importance for tissue engineering.

BackgroundAxolotls have the unique ability, among vertebrates, to perfectly regenerate complex body parts, such as limbs, after amputation. In addition, axolotls pattern developing and regenerating autopods from the anterior to posterior axis instead of posterior to anterior like all tetrapods studied to date. Sonic hedgehog is important in establishing this anterior-posterior axis of limbs in all tetrapods including axolotls. Interestingly, its expression is conserved (to the posterior side of limb buds and blastemas) in axolotl limbs as in other tetrapods. It has been suggested that BMP-2 may be the secondary mediator of sonic hedgehog, although there is mounting evidence to the contrary in mice. Since BMP-2 expression is on the anterior portion of developing and regenerating limbs prior to digit patterning, opposite to the expression of sonic hedgehog, we examined whether BMP-2 expression was dependent on sonic hedgehog signaling and whether it affects patterning of the autopod during regeneration.ResultsThe expression of BMP-2 and SOX-9 in developing and regenerating axolotl limbs corresponded to the first digits forming in the anterior portion of the autopods. The inhibition of sonic hedgehog signaling with cyclopamine caused hypomorphic limbs (during development and regeneration) but did not affect the expression of BMP-2 and SOX-9. Overexpression of BMP-2 in regenerating limbs caused a loss of digits. Overexpression of Noggin (BMP inhibitor) in regenerating limbs also resulted in a loss of digits. Histological analysis indicated that the loss due to BMP-2 overexpression was the result of increased cell condensation and apoptosis while the loss caused by Noggin was due to a decrease in cell division.ConclusionThe expression of BMP-2 and its target SOX-9 was independent of sonic hedgehog signaling in developing and regenerating limbs. Their expression correlated with chondrogenesis and the appearance of skeletal elements has described in other tetrapods. Overexpression of BMP-2 did not cause the formation of extra digits, which is consistent with the hypothesis that it is not the secondary signal of sonic hedgehog. However, it did cause the formation of hypomorphic limbs as a result of increased cellular condensation and apoptosis. Taken together, these results suggest that BMP-2 does not have a direct role in patterning regenerating limbs but may be important to trigger condensation prior to ossification and to mediate apoptosis.

The downstream consequences of inflammation in the adult mammalian heart are formation of a non-functional scar, pathological remodelling and heart failure. In zebrafish, hydrogen peroxide (H2O2) released from a wound is the initial instructive chemotactic cue for the infiltration of inflammatory cells, however, the identity of a subsequent resolution signal(s), to attenuate chronic inflammation, remains unknown. Here we reveal that Thymosin β4-Sulfoxide inhibits interferon-γ, and increases monocyte dispersal and cell death, lies downstream of H2O2 in the wounded fish and triggers depletion of inflammatory macrophages at the injury site. This function is conserved in the mouse and observed after cardiac injury, where it promotes wound healing and reduced scarring. In human T cell/CD14+ monocyte co-cultures, Tβ4-SO inhibits IFN-γ and increases monocyte dispersal and cell death, likely by stimulating superoxide production. Thus, Tβ4-SO is a putative target for therapeutic modulation of the immune response, resolution of fibrosis and cardiac repair.