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Summary: Human mesenchymal stem cells (hMSCs) are an appealing cell type for therapeutic applications but remain limited by poor efficacy in clinical trials. Here, we describe a conditioning technique that enhances the vascular regenerative properties of hMSCs and increases their expression of endothelial cell and pericyte markers. We also describe an alginate gel encapsulation protocol for delivering the conditioned cells.For complete details on the use and execution of this protocol, please refer to Lee et al. (2021).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.