Mesenchymal stem cell-like properties of CD133+ glioblastoma initiating cells

Abstract

Glioblastoma is composed of dividing tumor cells, stromal cells and tumor initiating CD133+ cells. Recent reports have discussed the origin of the glioblastoma CD133+ cells and their function in the tumor microenvironment. The present work sought to investigate the multipotent and mesenchymal properties of primary highly purified human CD133+ glioblastoma-initiating cells. To accomplish this aim, we used the following approaches: i) generation of tumor subspheres of CD133+ selected cells from primary cell cultures of glioblastoma; ii) analysis of the expression of pluripotency stem cell markers and mesenchymal stem cell (MSC) markers in the CD133+ glioblastoma-initiating cells; iii) side-by-side ultrastructural characterization of the CD133+ glioblastoma cells, MSC and CD133+ hematopoietic stem cells isolated from human umbilical cord blood (UCB); iv) assessment of adipogenic differentiation of CD133+ glioblastoma cells to test their MSC-like in vitro differentiation ability; and v) use of an orthotopic glioblastoma xenograft model in the absence of immune suppression. We found that the CD133+ glioblastoma cells expressed both the pluripotency stem cell markers (Nanog, Mush-1 and SSEA-3) and MSC markers. In addition, the CD133+ cells were able to differentiate into adipocyte-like cells. Transmission electron microscopy (TEM) demonstrated that the CD133+ glioblastoma-initiating cells had ultrastructural features similar to those of undifferentiated MSCs. In addition, when administered in vivo to non-immunocompromised animals, the CD133+ cells were also able to mimic the phenotype of the original patient's tumor. In summary, we showed that the CD133+ glioblastoma cells express molecular signatures of MSCs, neural stem cells and pluripotent stem cells, thus possibly enabling differentiation into both neural and mesodermal cell types.

Keywords: CD133+; TEM; neurospheres; tumorigenesis in vivo; MSC immunophenotype.

Figure 1

A, B. The establishment of human glioblastoma primary cell culture (A-a). Isolation of tumor neurospheres derived from glioblastoma primary cell culture. (A-b, c) Purification of glioblastoma cells from tumor subspheres using CD133 microbeads. Immunophenotypic characterization by using flow cytometry to evaluate the efficiency of magnetic cell separation for the antigenic marker, CD133 (78.0%). (B) CD133⁺ glioblastoma cells were able to further generate subspheres. Culture of glioblastoma subspheres (A-d, e) compared with the absence of subspheres obtained from CD133⁻ fractions. (A-f) Representative figure of five glioblastoma samples at 400X magnification. C-F. Immunophenotyping of CD133⁺ glioblastoma cells by using flow cytometry. (C) First plot shows the isotype control. The second and third plots show the staining for CD44 and SSEA-3 (99.8%) and Nanog and MSh-1 (96.7%), respectively. (D) The first plot is an unstained sample, and the second plot shows the staining for CD44 and CD133 (94.0%). (E) The first plot is an unstained sample, and the second plot shows the staining for CD90 and CD133 (94.4%). (F) Red (isotype control); Blue (stained sample).

Figure 2. The increased expression of the mesenchymal markers (CD29, CD44, CD73, CD90, CD105 and CD166) and low or no expression of the MHC class I antigens, HLA-DR and the hematopoietic/vascular cells markers (CD14, CD31, CD34, CD45 and CD106) on the CD133+ glioblastoma cells; red: isotype control; blue: stained sample

A-F. The adipogenic differentiation of the MSCs and glioblastoma CD133⁺ cells as detected by the formation of intra-cytoplasmic lipid droplets stained with Oil Red O (black arrow). (A) CD133⁻ (control). (B) Adipogenic differentiation of the MSCs. (C-F) The adipogenic differentiation of the CD133⁺ glioblastoma cells. (A, B) Magnification: 200X. (C-F) Magnification: 600X.

Figure 3. (A-F) TEM of the CD133⁺ hematopoietic stem cells (UCB)

n = nucleus, c = cytoplasm, mi = mitochondria, rer = rough endoplasmic reticulum, ser = smooth endoplasmic reticulum, pv = pinocytic vesicles, li = lipid droplets, gc = Golgi complex, ly = lysosomes, arrow = electron-dense granules or magnetic beads. TEM of the CD133⁺ glioblastoma stem cells. n = nucleus, c = cytoplasm, mi = mitochondria, rer = rough endoplasmic reticulum, pv = pinocytic vesicles, arrow = electron-dense granules or magnetic beads. Scale: A-C, G-J. 5.0 μm; F, K. 2.0 μm; D, E. 1.0 μm; L, M. 0.5 μm.

Figure 4. TEM micrographs of the UC-MSCs

n = nucleus, nu = nucleolus, c = cytoplasm, mi = mitochondria, rer = rough endoplasmic reticulum, v = vacuoles, ce = cytoplasmic expansions. Scale: A-C, E. 5.0 μm; D, F. 0.5 μm.

Figure 5

A. Fluorescence detection of MION-Rh labeling in the CD133⁺ glioblastoma cells. Magnification: 600X. B. Stereotaxic implantation of the MION-Rh labeled CD133⁺ glioblastoma cells labeled in the tumor experimental models. C. Ex vivo brain imaging; arrow = region of the tumor. D. In vivo detection of the glioblastoma using combined fluorescence and X-ray tomography. D1. Fluorescence image detail. E. Immunohistochemical analysis for Prussian blue staining of the tumor. F. Fluorescence assay of the tumor. (E) Magnification: 400X. (F) Magnification: 600X.

Figure 6

A, C, G. Hematoxylin and eosin staining. B, D, H. Immunohistochemical analysis for GFAP. E, F. Immunohistochemical analysis for Ki67. Arrow = vascular proliferation. Arrow a = tumor area. (A, B, C, E, G, H) Magnification: 400X. (D, F) Magnification: 600X.