Collection and Culture of Alveolar Bone Marrow Multipotent Mesenchymal Stromal Cells From Older Individuals Juan Han, 1,2 Hironori Okada, 3 Hideki Takai, 2,4 Youhei Nakayama, 2,4 Takahide Maeda, 1,2 and Yorimasa Ogata 2,4 * 1 Department of Pediatric Dentistry, Nihon University School of Dentistry at Matsudo, Matsudo, Chiba, Japan 2 Research Institute of Oral Science, Nihon University School of Dentistry at Matsudo, Matsudo, Chiba, Japan 3 Two Cells Co., Ltd., Hiroshima, Hiroshima, Japan 4 Department of Periodontology, Nihon University School of Dentistry at Matsudo, Matsudo, Chiba, Japan ABSTRACT In this work, we examined the culture condition of alveolar bone marrow multipotent mesenchymal stromal cells (ABMMSCs), aiming to apply regenerative therapy to older periodontitis patients. To better understand the character of cultured cells from alveolar bone marrow, the expression profiles of well-known genes and their responses to the induction of osteogenic, chondrogenic, or adipogenic differentiation were examined. Using aMEM-based culture, ABMMSCs could be obtained from older individuals than in previous reports. Interestingly, ABMMSCs expressing Klf4 were able to differentiate into osteoblasts. The prediction of differentiation potential by Klf4 could be a useful guide for further improvement of the culture conditions required to culture ABMMSCs derived from older individuals. J. Cell. Biochem. 107: 1198– 1204, 2009. ß 2009 Wiley-Liss, Inc. KEY WORDS: REGENERATIVE MEDICINE; ORAL; ALVEOLAR; BONE MARROW; STROMA; OLDER INDIVIDUALS T he choice of stem cell source is a matter of great importance because it directly influences the success of regenerative therapy. While various stem cell sources have been reported [Barrilleaux et al., 2006; Bjerknes and Cheng, 2006; Dominici et al., 2006; Burke et al., 2007; Yu et al., 2008], iliac bone marrow [Matsubara et al., 2005], alveolar bone marrow [Matsubara et al., 2005], dental pulp [Gronthos et al., 2000], and periodontal ligament [Seo et al., 2004] have been examined as stem cells for oral regenerative therapy. Alveolar bone marrow is a realistic candidate because tooth extraction is needed to obtain stem cells from dental pulp and the periodontal ligament. In a previous report, the success of the human alveolar bone marrow multipotent mesenchymal stromal cells (ABMMSCs) culture was relatively limited to young patients [Matsubara et al., 2005]. In this study, we examined the culture condition of ABMMSCs, aiming to apply regenerative therapy to older periodontitis patients. To better understand the character of ABMMSCs, the expression profiles of well-known genes associated with the maintenance of undifferentiated embryonic stem cells [Ulloa-Montoya et al., 2007; Jiang et al., 2008], and associated with the induction of pluripotent stem cells from somatic cells [Takahashi et al., 2007], osteogenesis [Song and Tuan, 2004; Delorme et al., 2008; Ogata, 2008], chondrogenesis [Song and Tuan, 2004; Delorme et al., 2008], and adipogenesis [Song and Tuan, 2004; Delorme et al., 2008] were Journal of Cellular Biochemistry ARTICLE Journal of Cellular Biochemistry 107:1198–1204 (2009) 1198 Abbreviations Used: ABMMSCs, alveolar bone marrow multipotent mesenchymal stromal cells; BSP, bone sialo- protein; ESCs, embryonic stem cells; FGF2, fibroblast growth factor 2; IBMMSCs, iliac bone marrow multipotent mesenchymal stromal cells; MAPK, mitogen-activated protein kinase; MSCs, mesenchymal stem cells; a-MEM, a-minimum essential medium; Runx2, runt homeodomain protein 2; SMC, smooth muscle cell. Grant sponsor: Japan Society for the Promotion of Science (JSPS); Grant number: 18390563; Grant sponsor: Supporting Project for Strategic Research by the Ministry of Education, Culture, Sports, Science and Technology; Grant number: 2008-2012. *Correspondence to: Prof. Yorimasa Ogata, DDS, PhD, Department of Periodontology, Nihon University School of Dentistry at Matsudo, Chiba 271-8587, Japan. E-mail: ogata.yorimasa@nihon-u.ac.jp Received 11 October 2008; Accepted 30 April 2009  DOI 10.1002/jcb.22224  ß 2009 Wiley-Liss, Inc. Published online 8 June 2009 in Wiley InterScience (www.interscience.wiley.com).