AbstractOur previous studies indicated that a coculture system containing human amnion‐derived mesenchymal stem cells (HAMSCs) and human bone marrow mesenchymal stem cells (HBMSCs) has the potential of application for bone regeneration. However, there is currently no enough comparative investigation between HAMSCs/HBMSCs transwell and mixed coculture systems. This study aimed to assess the phenotype and mechanisms regulated by indirect and direct coculture systems, respectively. Two in vitro models were employed with HAMSCs and HBMSCs at a ratio of 3:1, and then were analyzed by a series of processes, including flow cytometry, alkaline phosphatase (ALP) substrate assays, Alizarin red S staining, quantitative reverse transcription polymerase chain reaction (RT‐qPCR), and Western blot analysis. We found that cell proliferation, ALP activity, mineralized matrix formation, and osteoblast‐related mRNA expression were accelerated in transwell coculture system compared with mixed coculture system. Conditioned medium from transwell coculture system achieved an elevated level of vascular endothelial growth factor and induced more vascular structures in human umbilical vein endothelial cells than those of mixed coculture system. Moreover, we observed that transwell coculture system, promoted osteogenesis and angiogenesis by maintaining stemness through extracellular regulated protein kinases 1/2 (ERK1/2) mitogen‐activated protein kinase (MAPK) signaling pathway. U0126, a selective inhibitor of ERK1/2 MAPK signaling, significantly suppressed maintaining of the stemness‐based effects on transwell coculture system. Taken together, our results compared the merits of two different models and clarified the role of HAMSCs/HBMSCs transwell coculture system in the development of bone tissue engineering. © 2019 IUBMB Life, 2019