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Previously, we had addressed the issues of shape control/maintenance of in vitro engineered human-ear-shaped cartilage. Thus, lack of applicable cell source had become a major concern that blocks clinical translation of this technology. Autologous microtia chondrocytes (MCs) and bone marrow stromal cells (BMSCs) were both promising chondrogenic cells that did not involve obvious donor site morbidity. However, limited cell availability of MCs and ectopic ossification of chondrogenically induced BMSCs in subcutaneous environment greatly restricted their applications in external ear reconstruction. The current study demonstrated that MCs possessed strong proliferation ability but accompanied with rapid loss of chondrogenic ability during passage, indicating a poor feasibility to engineer the entire ear using expanded MCs. Fortunately, the co-transplantation results of MCs and BMSCs (25% MCs and 75% BMSCs) demonstrated a strong chondroinductive ability of MCs to promote stable ectopic chondrogenesis of BMSCs in subcutaneous environment. Moreover, cell labeling demonstrated that BMSCs could transform into chondrocyte-like cells under the chondrogenic niche provided by co-cultured MCs. Most importantly, a human-ear-shaped cartilaginous tissue with delicate structure and proper elasticity was successfully constructed by seeding the mixed cells (MCs and BMSCs) into the pre-shaped biodegradable ear-scaffold followed by 12 weeks of subcutaneous implantation in nude mouse. These results may provide a promising strategy to construct stable ectopic cartilage with MCs and stem cells (BMSCs) for autologous external ear reconstruction., (Copyright © 2014 Elsevier Ltd. All rights reserved.)