Background: Due to their self-renewal, proliferation, differentiation, and angiogenesis-inducing capacity, human adipose mesenchymal stem cells (AMSC) have potential clinical applications in the treatment of limb ischemia. AMSC from healthy donors have been shown to induce neovascularization in animal models. However, when cells were obtained from donors suffering from any pathology, their autologous application showed limited effectiveness. We studied whether liposuction niche and obesity could determine the regenerative properties of cells meaning that not all cell batches are suitable for clinical practice. Methods: AMSC obtained from 10 donors, obese and healthy, were expanded in vitro following a good manufacturing practice-like production protocol. Cell viability, proliferation kinetics, morphological analysis, phenotype characterization, and stemness potency were assessed over the course of the expansion process. AMSC selected for having the most suitable biological properties were used as an experimental treatment in a preclinical mouse model of hind limb ischemia. Result: All cell batches were positively characterized as mesenchymal stem cells, but not all of them showed the same properties or were successfully expanded in vitro, depending on the characteristics of the donor and the extraction area. Notably, AMSC from the abdomen of obese donors showed undesirable biological properties. AMSC with low duplication times and multilineage differentiation potential and forming large densely packed colonies, were able, following expansion in vitro, to increase neovascularization and repair when implanted in the ischemic tissue of mice. Conclusion: An extensive AMSC biological properties study could be useful to predict the potential clinical efficacy of cells before in vivo transplantation. Thus, peripheral ischemia and possibly other pathologies could benefit from stem cell treatments as shown in our preclinical model in terms of tissue damage repair and regeneration after ischemic injury.