Multipotent stern cells are one of the most powerful tools available for the bone regeneration. However, owing to various limitations, including a lack of tissue-specific stem cell identification, reconstruction of large cranial bone defects remains challenging. In the current study, we isolated a population of Sca-1(+)CD105(+)CD140a(+) stem cells from adult mouse calvarium and cultured them as three-dimensional spheroids. Although these cells shared similar surface antigens when grown in either monolayers or spheroids, the cranial stem cells grown in spheroids possessed enhanced multipotency and proliferation capacity. In addition, the cranial stem cells in spheroids were found to express high levels of the self-renewal transcription factors Nanog, Oct-4, and Sox-2. Mechanistically, we found that three-dimensional spheroid culture suppressed NF-kappa B pathways, but activated the PI3K/AKT pathway in cranial stem cells. More importantly, activation of NF-kappa B pathways or specific inhibition of the PI3K/AKT pathway partially impaired spheroid formation and suppressed expression of self-renewal transcription factors. In summary, these findings reveal a novel effect of spheroid culture in promoting the maintenance of cranial stem cell sternness and indicate that NF-kappa B and PI3K/AKT pathways might be involved in the sternness maintenance. (C) 2017 Elsevier Inc. All rights reserved.