Matrigel Plug In Vivo Angiogenesis Assay

To test the ability of iECs to form blood vessels in vivo, we performed an in vivo gel plug assay.¹⁹ Nonobese diabetic/severe combined immunodeficiency (NOD-SCID) mice were used to avoid the rejection of human cells. Anesthesia was induced using a mixture of xylazine (10 mg/kg) and ketamine (100 mg/kg) in saline IP. A total of 1×10⁶ cells (either CD34⁺ progenitors, fibroblasts, or aortic ECs) were suspended in 200 μl of cold growth factor-reduced Matrigel Matrix (Corning, 356234). We then injected the cell-containing Matrigel solutions subcutaneously in the abdomen. The mice were euthanized after 1 week implantation, and the Matrigel plugs were removed by a wide excision of the skin, fixed in 10% formalin overnight, embedded in paraffin, and sectioned. Five sections from each plug were randomly selected to quantify human vessels. Quantification of vessel density in angiogenesis plugs was shown per section per plug. Each mouse was injected with 3 plugs containing iECs, fibroblasts, or mature ECs. Each data point represents 1 plug from 1 mouse.