Reproducible strategy for excisional skin-wound-healing studies in mice.

Wound healing is a complex physiological process involving various cell types and signaling pathways. The capability to observe the dynamics of wound repair offers valuable insights into the effects of genetic modifications, pharmaceutical interventions or other experimental manipulations on the skin-repair process. Here, we provide a comprehensive protocol for a full-thickness, excisional skin-wound-healing assay in mice, which can easily be performed by any scientist who has received an animal welfare course certificate and can be completed within ~3 h, depending on the number of animals. Crucially, we highlight the importance of considering key aspects of the assay that can dramatically contribute to the reliability and reproducibility of these experiments. We thoroughly discuss the experimental design, necessary preparations, wounding technique and analysis. In addition, we discuss the use of lineage-tracing techniques to monitor cell migration, differentiation and the contribution of different cell populations to the repair process. Overall, we explore key aspects of the skin-wound-healing assay, supplying a detailed procedure and guidelines essential for decreasing variability and obtaining reliable and reproducible results.

Successful Introduction of Human Renovascular Units into the Mammalian Kidney.

BACKGROUND: Cell-based therapies aimed at replenishing renal parenchyma have been proposed as an approach for treating CKD. However, pathogenic mechanisms involved in CKD such as renal hypoxia result in loss of kidney function and limit engraftment and therapeutic effects of renal epithelial progenitors. Jointly administering vessel-forming cells (human mesenchymal stromal cells [MSCs] and endothelial colony-forming cells [ECFCs]) may potentially result in in vivo formation of vascular networks. METHODS: We administered renal tubule-forming cells derived from human adult and fetal kidneys (previously shown to exert a functional effect in CKD mice) into mice, alongside MSCs and ECFCs. We then assessed whether this would result in generation of "renovascular units" comprising both vessels and tubules with potential interaction. RESULTS: Directly injecting vessel-forming cells and renal tubule-forming cells into the subcutaneous and subrenal capsular space resulted in self-organization of donor-derived vascular networks that connected to host vasculature, alongside renal tubules comprising tubular epithelia of different nephron segments. Vessels derived from MSCs and ECFCs augmented in vivo tubulogenesis by the renal tubule-forming cells. In vitro coculture experiments showed that MSCs and ECFCs induced self-renewal and genes associated with mesenchymal-epithelial transition in renal tubule-forming cells, indicating paracrine effects. Notably, after renal injury, renal tubule-forming cells and vessel-forming cells infused into the renal artery did not penetrate the renal vascular network to generate vessels; only administering them into the kidney parenchyma resulted in similar generation of human renovascular units in vivo. CONCLUSIONS: Combined cell therapy of vessel-forming cells and renal tubule-forming cells aimed at alleviating renal hypoxia and enhancing tubulogenesis holds promise as the basis for new renal regenerative therapies.