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First published online 2 February 2005
doi: 10.1242/dev.01615

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Mesenchymal progenitor cells localize within hematopoietic sites throughout ontogeny

Department of Cell Biology and Genetics, Erasmus University Medical Center, PO Box 1738, 3000 DR, Rotterdam, The Netherlands

View larger version (33K): [in a new window]   Fig. 1. (A) Schematic drawing of a mouse embryo at E11. Yolk sac (YS), head, somites, limb buds (Lb), heart (H), liver (L), umbilical (U) and vitelline (V) arteries, and the aorta-gonad-mesonephros (AGM) region were dissected and cells isolated for further testing. (B) AGM region, divided into the aorta (A), with the adjacent mesenchyme (M), and the gonads and mesonephros (GM) that constitute the urogenital ridges.

View larger version (88K): [in a new window]   Fig. 2. Differentiation potential of AGM cells. (A-C) Osteogenic cell differentiation. After 10 days in osteogenic permissive medium, cell colonies stained positive for ALP activity (A), and cultures expressed the osteoblastic markers osteopontin and osteocalcin (B, lane 2). Neither marker was expressed in freshly isolated AGM cells (B, lane1). Differentiated cultures also showed an upregulation of the osteogenic marker Cbfa1 (B, lane 2). At day 21, mineralization of the cultures was observed with Alizarin Red (C). (D) Adipogenic differentiation and colony formation. After stimulation with adipogenic-inductive medium for one week, Pparg was upregulated (B, lane 2) when compared with the expression of this marker in freshly isolated AGM cells (B, lane 1). (E-H) Chondrogenic differentiation. After 21 days in chondrogenic medium, cells formed several cartilaginous foci (E) with an extracellular matrix rich in proteoglycans, as detected by Toluidine Blue staining (F). Foci were also strongly positive for collagen type II, as shown by the brown DAB staining (G). A secondary antibody control is shown in H.

View larger version (12K): [in a new window]   Fig. 3. Frequency of (A) osteogenic, (B) adipogenic and (C) chondrogenic progenitors in E11 AGM and its subregions, the dorsal aorta with its surrounding mesenchyme (AoM) and the urogenital ridges (UGRs). Osteogenic and adipogenic progenitor frequencies were determined based on the number of colonies displaying ALP activity and refractile, lipid-containing vesicles, respectively. Chondrogenic progenitor frequency was determined based on the number of cartilagenous foci after 21 days of aggregate culture. Data are expressed as mean±s.d. (n=3 to five independent experiments). Because the AGM region also contains primordial germ cells expressing ALP, the specificity of this marker with regard to osteogenic commitment was addressed. Oct4-GFP transgenic AGM cells were sorted and evaluated in osteogenic cultures for ALP-expressing colony frequency. The frequency of osteogenic progenitors in the primordial germ cell depleted (sorted Oct4-GFP negative) fraction was identical to unsorted AGM cells (1.32±0.41/104 vs 1.30±0.14/104, respectively). Thus, primordial germ cells are not contributing to the ALP-expressing colony frequency after culture in osteogenic-inductive conditions.

View larger version (23K): [in a new window]   Fig. 4. Frequency and absolute number of (A) osteogenic, (B) adipogenic and (C) chondrogenic progenitors in hematopoietic sites through development. Osteogenic and adipogenic progenitor frequencies and numbers were determined based on the number of colonies displaying the appropriate phenotype. Chondrogenic progenitor frequency and number was determined based on the number of cartilage foci after 21 days of aggregate culture. Data are expressed as mean±s.d. (n=3 to six independent experiments). Absolute number determinations in the neonatal and adult BM used the following values: newborn BM, 9.8 x107 cells; adult BM, 30.0 x107 cells (Harrison, 1993). These values were extrapolated from those of Stewart et al. (Stewart et al., 1993).

View larger version (48K): [in a new window]   Fig. 5. Presence of stem/progenitor cells in cultures of E12 blood. (A) Osteogenic cell differentiation after 10 days of treatment in osteogenic permissive medium. Colonies stained positive for ALP. (B) Adipogenic differentiation after stimulation with adipogenic-inductive medium for 10 days; adipocytes are shown. (C) Chondrogenic differentiation after 21 days in chondrogenic medium. E12 blood cells formed a small cartilage-like pellet, in which the extracellular matrix was rich in proteoglycans, as detected by Toluidine Blue staining.