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Epidermal stem cells (ESCs) play a critical roles in wound healing. But the biological characteristics of ESCs derived from diabetes mellitus are not fully understood. In this chapter, we compared the biological characteristics between diabetes and normal skin derived ESCs in vitro. The diabetes mellitus (DM) rat models were made by intraperitoneal injected streptozotocin. ESCs were extracted from the dorsal skin samples of rats by trypsin digesting method and further purified by collagen adhering methods. The morphologic change and cell colony formation were observed. The expression of K19, β1-integrin were identified by immunocytochemical stain and image analysis. The results showed that the number of primary anchorage-dependent ESCs in DM group was decreased. The clone forming efficiency of ESCs in DM group was significantly lower than that in the normal control group. ESCs of two groups both express K19, P 1-integrin. The integral absorbance values of positive cells for K19, β1-integrin in ESCs of DM group were significantly lower than those in the normal control group. This suggested that the multiplication capacity in vitro of rat ESCs derived from diabetes skin was lower than that in normal skin, which may be one of important factors that diabetes mellitus wound is hard to heal.