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First published online 19 October 2004
doi: 10.1242/jcs.01488

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In vivo contribution of murine mesenchymal stem cells into multiple cell-types under minimal damage conditions

Hematopoietic Stem Cell Laboratory, Cancer Research UK, London Research Institute, 44 Lincoln's Inn Fields, London, WC2A 3PX, UK

View larger version (44K): [in a new window]   Fig. 1. Determination of muMSC frequency in the bone marrow (A) and persistence of hematopoietic cell contamination in MSC cultures (B). (A) Representative FACS plots analysis of NOD/SCID BMMNCs (top plots) stained with CD45/CD11b/TER119/CD31 antibodies; the frequency of muMSCs in the bone marrow can be considered as the negative fraction for CD45 (R1) with the combination of the other cell surface antigens used here. Alternatively, the same frequencies can also be determined by using the following antibody combination: CD45/Lin/CD31 (bottom plots). By using this criterion no differences in the frequency was found between the three mouse strains studied. (B) The presence of CD45+/CD11b+ cells in different passages (P1-P3) of MSC cultures is shown (top plots). Percentages indicate the CD45–/CD11b– populations, which were considered as the MSC fraction. The CD45+/11b+ hematopoietic cell population expressed SCA-1 and different levels of CD34 and CD90 (middle and bottom plots, respectively). Results shown are mean±s.d from three independent experiments.

View larger version (29K): [in a new window]   Fig. 2. In vitro eGFP expression by muMSCs following lentiviral transduction. A purified highly enriched muMSC population (puP4) was transduced with lentivirus for 20 hours. Analysis for eGFP expression was performed 1, 3 and 5 days post-transduction. (A) A representative overlay FACS plot from day 1 with m.o.i.=5 (solid line), 10 (blue dotted line), 30 (red dashed line), 50 (dark grey area) and untransduced control MSCs (light grey area) is shown. Cells gated in R1 expressing high levels of eGFP from different time points of analysis were plotted in the graph (B). Results shown are mean±s.d. from two independent experiments, and each time point was performed in duplicate.

View larger version (71K): [in a new window]   Fig. 3. In vitro differentiation of eGFP-MSCs. Unstimulated eGFP-MSCs were viewed under UV light (A). After 14 days of induction, cells were fixed and stained with Oil Red O (B,C). Chondrogenic differentiation was revealed with Safranin O staining (E) with a representative section of the micropellet viewed under the fluorescent microscope before the staining (D). The osteogenic potential of MSCs was determined by staining for alkaline-phosphatase (F) and calcium production (G): stimulated eGFP-MSCs (black bar), non-stimulated eGFP-MSCs (grey bar), control-water (light grey bar). Results shown are mean±s.d. from two independent experiments, each one performed in triplicate. eGFP expression alone on myocyte-like cells (H). Myogenic differentiation was confirmed by staining with dystrophin-Cy3 (I,J) and FTM-Cy3 (K). eGFP expression alone on astrocyte-(L) and neuronal-like (O) cells. Neuronal differentiation was confirmed by staining with GFAP-Cy3 (M,N) and Tau-TRITC (O). Overlay of eGFP and dystrophin (J), eGFP and FTM (K) and eGFP with GFAP (N). Cells were counterstained with hematoxylin (B,C,F), DAPI (H-J, L-N) or methyl green (E). Magnifications: x100 (A,B,F); x200 (D,L-N); x400 (C,E,H-K,O).

View larger version (135K): [in a new window]   Fig. 4. Transplanted eGFP-MSCs are circulating and/or trapped in the different tissues. Immnunohistochemistry revealed the presence of infused eGFP-MSCs in different tissues. Circulating or trapped (arrows to brown cells) MSCs were found in the spleen (A), gut (B) and lung (C). In the lung, trapped cells stained neither for -sma [(red-pink) indicated by the right-hand arrow] (C) nor for the lectin Lycopersicon esculentum-Cy3 (D, overlay with eGFP). Trapped eGFP-MSCs have caused severe lung damage in some of the mice. Representative sections of an undamaged lung (E) and severely damaged lung (F) from two different mice that were injected with eGFP-MSCs (brown cells) are shown. Sections were counterstained with hematoxylin (A-C,E,F) or DAPI (D). Magnifications: x25 (E,F); x100 (C); x200 (D); x400 (A,B).

View larger version (139K): [in a new window]   Fig. 5. In vivo contribution of eGFP-MSCs into bronchiolar epithelial, hepatocyte and renal tubular-like cells. (A) Representative paraffin sections of lung stained either with rabbit immunoglobulins (isotype control) (A1) or with rabbit anti-pan-CK antibodies (A2). Serial sections (A3,4) show that a group of pan-CK+ cells (red) (A4) also stained positive for eGFP (A3). (B) Representative liver sections with hepatocytes staining positive for eGFP (B1,2; brown). eGFP expression coincided with albumin (red) giving a brown-purple staining (B2, inset). Serial paraffin sections from another liver showing purple cells with hepatocyte morphology, as a result of co-expression of eGFP (red) and CK18 (blue) (B4). Isotype control sections shown in B3. (C) Differentiation of eGFP-MSCs into renal tubular cells. Paraffin-embedded sections stained with isotype control antibodies show no background staining (C1). Arrows indicate eGFP+ cells after staining with rabbit anti-eGFP antibody (C2,3) co-expressing both lectins Ricinus communis (C2, bottom-center inset; red staining) and Lotus tetragonolobus (C4; pink-red staining) as shown in serial sections. A frozen section of a kidney demonstrating eGFP+ cells by direct visualization using a fluorescent microscope (C5). All sections were counterstained with hematoxylin (except for B4) or DAPI (C5). Magnifications: x100 (A1,2); x200 (C1,2,5); x400 (A3,4, B1-4, C3,4).

View larger version (122K): [in a new window]   Fig. 6. Contribution of eGFP-MSCs into myofibroblast and myofiber-like cells in vivo. (A) eGFP positive cells with myofibroblast morphology were located among skeletal muscle fibers (A1,4; brown). No expression of desmin was observed (A2; red staining) showing mutual exclusion of expression when double staining for desmin and eGFP was performed (A3). These same eGFP+ cells (brown) stained positive for -sma (red) in serial sections (A5) resulting in a red-brown color. (A6) The same section stained for both rabbit and mouse immunoglubulins (isotype control) (A7). (B) Representative section of the skin from the ear with myofibers staining positive for eGFP (B2,3; brown cells). The same group of myofibers demonstrating expression of desmin (B4,5; pink-red staining) and FTM (B6,7; red) alone or in combination with an eGFP antibody (blue) giving a purple color (B5,7). Sections were counterstained with hematoxylin (except for B5,7). Magnifications: x200 (A7, B1,2,4-7); x400 (A1-6, B3).

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