Basic fibroblast growth factor supports expansion of mouse compact bone-derived mesenchymal stem cells (MSCs) and regeneration of bone from MSC in vivo.
Some progress has been made in development of methods to regenerate bone from cultured cells, however no method is put to practical use. Here, we developed methods to isolate, purify, and expand mesenchymal stem cells (MSCs) from mouse compact bone that may be used to regenerate bone in vivo. These cells were maintained in long-term culture and were capable of differentiating along multiple lineages, including chondrocyte, osteocyte, and adipocyte trajectories. We used standard cell isolation and culture methods to establish cell cultures from mouse compact bone and bone marrow. Cultures were grown in four distinct media to determine the optimal composition of culture medium for bone-derived MSCs. Putative MSCs were subjected to flow cytometry, alkaline phosphatase assays, immunohistochemical staining, and several differentiation assays to assess cell identity, protein expression, and developmental potential. Finally, we used an in vivo bone formation assay to determine whether putative MSCs were capable of regenerating bone. We found that compact bone of mice was a better source of MCSs than the bone marrow, that growth in plastic flasks served to purify MSCs from hematopoietic cells, and that MSCs grown in basic fibroblast growth factor (bFGF)-conditioned medium were, based on multiple criteria, superior to those grown in leukemia inhibitory factor-conditioned medium. Moreover, we found that the MSCs isolated from compact bone and grown in bFGF-conditioned medium were capable of supporting bone formation in vivo. The methods and results described here have implications for understanding MSC biology and for clinical purpose.
Department of Oral and Maxillofacial Reconstructive Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-Cho, Kitaku, Okayama, 7008558, Japan. eikiyama@md.okayama-u.ac.jp
Eiki
E
Yamachika
Hidetsugu
H
Tsujigiwa
Masakazu
M
Matsubara
Yasuhisa
Y
Hirata
Kenichiro
K
Kita
Kiyofumi
K
Takabatake
Nobuyoshi
N
Mizukawa
Yoshihiro
Y
Kaneda
Hitoshi
H
Nagatsuka
Seiji
S
Iida
D017667:Adipocytes / Q000166:cytology* / Q000378:metabolism; D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology / Q000187:drug effects; D001842:Bone and Bones / Q000166:cytology* / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002469:Cell Separation; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology* / Q000378:metabolism; D017077:Culture Media, Conditioned; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology*; D005434:Flow Cytometry; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000187:drug effects; D051379:Mice; D008822:Mice, Transgenic; D010011:Osteocytes / Q000166:cytology* / Q000378:metabolism; D010012:Osteogenesis / Q000187:drug effects* / Q000502:physiology; D012038:Regeneration / Q000187:drug effects / Q000502:physiology; D023822:Tissue Engineering
D017077:Culture Media, Conditioned; D016222:Fibroblast Growth Factor 2