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. 2008 Nov;58(3):163-71.
doi: 10.1007/s10616-009-9184-1. Epub 2009 Feb 17.

Isolation of osteoprogenitors from murine bone marrow by selection of CD11b negative cells

A Dumas  1 , M A Le Drévo, M F Moreau, C Guillet, M F Baslé, D Chappard
Affiliations

Isolation of osteoprogenitors from murine bone marrow by selection of CD11b negative cells

A Dumas et al. Cytotechnology. 2008 Nov.

Abstract

Selection of cells having the most osteogenic potential is a strategy used in bone tissue engineering. Preclinical studies using murine bone marrow cells must consider the large amount of hematopoietic cells in the adherent fraction. The aim of this study was to enrich a murine bone marrow cell population with osteoprogenitors by using a simple and reliable method. Bone marrow from C57Bl/6 mice was extracted and cells which adhered onto plastic were expanded in primary culture for 14 days. Immunolabeling of the CD11b surface antigen was performed and the CD11b(-) cell fraction was isolated by FACS. Sorted and unsorted populations were analyzed for gene expression of osteoblast differentiation, alkaline phosphatase (AlkP) activity and matrix mineralization capacities. Selection of CD11b(-) cells increased the number of AlkP(+) cells from the plastic adherent fraction from 6.3% +/- 0.8 to 56% +/- 3.3 with a sevenfold increase in AlkP activity. mRNA analysis revealed a significant increase in the CD11b(-) fraction for Osterix (41-fold), RANKL (17-fold), M-CSF (8-fold) and Runx-2 (8-fold). An osteogenic population was obtained with improved capacities to produce a mineralized extracellular matrix in vitro, independently of the presence of glucocorticoids in the culture medium.

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Figures

Fig. 1
Fig. 1
FACS analysis of plastic adherent murine marrow cells prior to (a, isotype control and b, CD11b labeled cells) and after immunodepletion (c). CD11b− cells are in the lower area. Only 8.2% of plastic adherent bone marrow cells were CD11b−. This percentage reached 96% after immunodepletion
Fig. 2
Fig. 2
a Microphotographs of unsorted and CD11b− cells, at day 1 of subculture after cytochemical staining of the AlkP. Same cultures were observed at ×100 magnification under transmitted and fluorescent light. Note the selection of large flattened and AlkP+ cells in the CD11b− population. b Number of AlkP+ cells among the unsorted or CD11b− cell populations, at day 1 of subculture, counted after cytochemical staining. c AlkP activity assay of unsorted and CD11b− cell populations at day 1 of subculture
Fig. 3
Fig. 3
mRNA expression profile of the CD11b− cells compared to the unsorted population. Values are ratios of mRNA levels (CD11b− cells/unsorted cells) as determined by real-time PCR, averaged ± SEM for three independent experiments. * p < 0.05 from unsorted cells (value = 1)
Fig. 4
Fig. 4
Calcein labeling of calcium deposits in unsorted (a, at magnification ×100) and CD11b− cell populations (b, at magnification ×25 and c, at magnification ×100), after 21 days of subculture in medium containing ascorbic acid and β-glycerophosphate
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