Isolation and perivascular localization of mesenchymal stem cells from mouse brain

Abstract

Background: Although originally isolated from the bone marrow, mesenchymal stem cells (MSCs) have recently been detected in other tissues. However, little is known about MSCs in the brain.

Objective: To determine the extent to which cells with the features of MSCs exist in normal brain tissue and to determine the location of these cells in the brain.

Methods: Single-cell suspensions from mouse brains were cultured according to the same methods used for culturing bone marrow-derived MSCs (BM-MSCs). These brain-derived cells were analyzed by fluorescence-activated cell sorting for surface markers associated with BM-MSCs (stem cell antigen 1 [Sca-1+], CD9+, CD45-, CD11b-, and CD31-). Brain-derived cells were exposed to mesenchymal differentiation conditions. To determine the locations of these cells within the brain, sections of normal brains were analyzed by immunostaining for Sca-1, CD31, and nerve/glial antigen 2.

Results: Cells morphologically similar to mouse BM-MSCs were identified and called brain-derived MSCs (Br-MSCs). Fluorescence-activated cell sorting indicated that the isolated cells had a surface marker profile similar to BM-MSCs, ie, Sca-1V+, CD9+, CD45-, and CD11b-. Like BM-MSCs, Br-MSCs were capable of differentiation into adipocytes, osteocytes, and chondrocytes. Immunostaining indicated that Sca-1+ Br-MSCs are located around blood vessels and may represent progenitor cells that serve as a source of mesenchymal elements (eg, pericytes) within the brain.

Conclusion: Our results indicate that cells similar to BM-MSCs exist in the brain. These Br-MSCs appear to be located within the vascular niche and may provide the mesenchymal elements of this niche. Because MSCs may be part of the cellular response to tissue injury, Br-MSCs may represent targets in the therapy of pathological processes such as stroke, trauma, and tumorigenesis.

FIGURE 1

The morphology of brain-derived cells at different passages. A, after 1 week, isolated cells from the brain were subconfluent and had heterogeneous morphology. Tube-forming cells were seen (arrow) (magnification × 200). B, at passage 28, cells had a more homogenous appearance (magnification × 200). C, the morphology of bone marrow–derived mouse mesenchymal stem cells is shown for comparison (original magnification × 200).

FIGURE 2

Flow cytometric analysis for mesenchymal (stem cell antigen 1 [Sca-1], CD9) and hematopoietic (CD45, CD11b) surface markers of brain-derived cells obtained at increasing passage. Each column represents the specific marker tested. Each row represents the labeled passage number. Within each graph, the control antibody is shown as the filled curve, and the tested antibody is shown as the unfilled curve. The percentage of cells expressing the marker is also shown in each graph. Cells were positive for Sca-1 and CD9 and negative for CD45 and CD11b, consistent with known markers of bone marrow–derived mesenchymal stem cells. The marker profile remained stable over time.

FIGURE 3

Photomicrographs of brain-derived mesenchymal stem cells (Br-MSCs) demonstrating trilineage mesenchymal differentiation. A, differentiation of Br-MSCs into adipocytes was confirmed by Oil Red O staining (orange) for lipid formation (magnification × 320). B, corresponding control for A in which cells were grown in noninducing medium. Control cultures were negative for adipogenesis (magnification × 320). C, differentiation of Br-MSCs into osteocytes was confirmed by staining with Alizarin Red (red color) for calcium formation (magnification × 200). D. corresponding control for B in which noninduced Br-MSCs were negative for Alizarin Red staining (original magnification × 200). E, differentiation of Br-MSCs into chondrocytes was confirmed by pellet culture with chondrogenic medium as described in Materials and Methods. Pellets were positive for Safranin O staining (reddish or purple), which revealed glycosaminoglycan (magnification × 100). F, corresponding control for E in which cells were grown in pellet culture noninduced medium. Only small, poorly organized pellets (consistent with lack of cartilage matrix) that were negative for Safranin staining were evident (magnification × 100).

FIGURE 4

Photomicrographs of immunohistochemical staining with anti-stem cell antigen 1 (Sca-1) antibody in the normal mouse brain. A, Sca-1–positive cells (brown) were distributed throughout the brain and were located almost exclusively in the vasculature (magnification × 200). B, high-powered view of Sca-1⁺ cells verifying location within the vasculature (magnification × 400).

FIGURE 5

Photomicrographs of normal mouse brain after double-immunofluorescent labeling. A, C, and E were stained with anti-stem cell antigen 1 (Sca-1) antibody (secondary antibody Alexa Fluor 488, green) and anti-CD31 antibody (secondary antibody Alexa Fluor 594, red). A and C are unmerged images, and E is the merged image. Arrow indicated Sca-1⁺/CD31⁺ cells. B, D, and F were stained with anti-Sca-1 antibody (secondary antibody Alexa Fluor 488, green) and anti-nerve/glial antigen 2 (NG2) antibody (secondary antibody Alexa Fluor 594, red). B and D are unmerged images, and F is the merged image. Arrow indicates Sca-1⁺/NG2⁺ cells. Merged images also reveal Sca-1⁺/NG2⁻/CD31⁻ cells (green only).

FIGURE 6

Flow cytometric analysis of cultured brain-derived mesenchymal stem cells (Br-MSCs) after labeling with antibodies to stem cell antigen 1 (Sca-1) and nerve/glial antigen 2 (NG2) or CD31. A, plot showing isotype control for IgG phycoerythrin and IgG allophycocyanin, which is control for graph of C. B, plot showing isotype control for IgG phycoerythrin and IgG FITC, which is control for graph of D. C, plot showing result after simultaneous staining of Br-MSCs for Sca-1 and CD31. Sca-1⁺ cells are negative for CD31. D, plot showing result after simultaneous staining of Br-MSCs for Sca-1 and NG2. A population of Sca-1⁺ cells also stain for NG-2 (10%).