Comparison of the cytokine-induced migratory response between primary and subcultured populations of rat mesenchymal bone marrow cells.
BACKGROUND
The aim of this study was to investigate the phenotypic differences of primary rat mesenchymal bone marrow cells (MBMCs) and subcultured cells, the influence of subculture and cell density on the cellular phenotypes, and the difference in the migratory responses of these cells to cytokines.


METHODS
MBMCs were isolated from 8-week-old Wistar rats, and the cells were cultured for 1 week (passage 0, P0) or 3 weeks (P0-3W). P0 cells were subcultured for 1 week (P1). P1 cells were subcultured at several cell densities for 1 week (P2). Cell size and granularity were analyzed by flow cytometry. The gene expression characteristics of these cells were analyzed by reverse transcription polymerase chain reaction. Cell migration to bone morphogenetic protein-2 (BMP-2), fibroblast growth factor-2 (FGF-2), and platelet-derived growth factor-bb (PDGF-bb) was evaluated using a Boyden chamber.


RESULTS
Three morphologically distinct populations in P0 and two in P2 were detected. The levels of human rapidly self-renewing cell-related marker genes in P0 were more highly expressed than in P2. Mesenchymal stem cell-associated markers were expressed at the same level in P0 and P2. The gene expression levels of immature oligodendrocyte precursor cell markers in P0 were higher than those in P2, whereas those of smooth muscle cell markers and osteoblastic cell markers in P0 were lower than those in P2. Subculture decreased the gene expression levels of human rapidly self-renewing cell-associated markers. Cell migration of P0 cells was stimulated by PDGF-bb but not by BMP-2 or FGF-2. In contrast, PDGF-bb, BMP-2, and FGF-2 all stimulated cell migration of P2.


CONCLUSIONS
The types of cells in populations of primary and subcultured rat MBMCs were different, and the distribution of each cell population appeared to be changed by the culture conditions. The cell migration effect by PDGF-bb, BMP-2, and FGF-2 differed between the primary and subcultured MBMCs.
Uchida
K
K
Department of Biomedical Engineering, Graduate School of Medical Sciences, Kitasato University, Sagamihara, Kanagawa, Japan.
Urabe
K
K
Naruse
K
K
Ujihira
M
M
Mabuchi
K
K
Itoman
M
M
D000818:Animals; D000077214:Becaplermin; D015415:Biomarkers / Q000378:metabolism; D001854:Bone Marrow Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins / Q000494:pharmacology*; D002452:Cell Count; D002465:Cell Movement / Q000187:drug effects*; D048429:Cell Size / Q000187:drug effects; D002478:Cells, Cultured; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology*; D015870:Gene Expression / Q000187:drug effects; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology*; D020574:Proto-Oncogene Proteins c-sis; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016212:Transforming Growth Factor beta / Q000494:pharmacology*
C525718:BMP2 protein, human; D015415:Biomarkers; C525720:Bmp2 protein, rat; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D010982:Platelet-Derived Growth Factor; D020574:Proto-Oncogene Proteins c-sis; D012333:RNA, Messenger; D016212:Transforming Growth Factor beta; D016222:Fibroblast Growth Factor 2; D000077214:Becaplermin