CD133-LV does not transduce normal mouse brain cells, hESC-derived neurons and primary human astrocytes <i>in vitro</i>.

<p><b>A</b>. Injection of CD133-LV expressing mCherry into the mouse basal ganglia did not lead to transduction of normal brain tissue, as opposed to VSVG-LV (BG: basal ganglia, Cx: cortex, CC: corpus callosum, LV: lateral ventricle). <b>Bi,ii</b>. hESC-derived neurons were transduced with either CD133-LV or VSVG-LV expressing mCherry. VSVG-LV led to transduction of MAP2A+ neurons, as opposed to CD133-LV. <b>Ci,ii</b>. Primary human astrocytes were transduced with either CD133-LV or VSVG-LV expressing mCherry (MOI = 10). VSVG-LV led to transduction of GFAP+ astrocytes, as opposed to CD133-LV. (NeuN: neural nuclei, MAP2A: microtubule associated protein 2A, GFAP: glial fibrillary acidic protein). Nuclei were counterstained with DAPI.</p

Stem-like properties of GBM cells transduced with CD133-LV.

<p><b>A</b>. Cells transduced with CD133-LV expressing TagBFP are clonogenic and produce spheres comprised of TagBFP+ cells. <b>B,C</b>. <i>In vitro</i> tumorsphere formation assays for 3 serial passages did not show any difference in the clonogenic potential of FACS-isolated CD133-LV transduced cells and untransduced CD133+ cells in terms of the number (<b>B</b>) and size (<b>C</b>) of spheres formed.</p

CD133-LV transduces CD133+ cells in primary GBM xenografts in the mouse brain.

<p><b>A</b>. Intracranial xenograft tumors were generated using injection of GBML20 cells (5×10⁵ cells/animal) and tumor formation (red circle) was confirmed with small animal MRI 1.5 months after injection. High titer stocks of CD133-LV or VSVG-LV expressing TagBFP were injected into the tumor and animals were sacrificed 7 days later for immunofluorescence analysis. <b>B</b>. CD133-LV - transduced cells expressing TagBFP (red) show cell surface immunoreactivity for CD133 (green). <b>C</b>. CD133+ cells were significantly more enriched among TagBFP+ transduced cells in the case of CD133-LV compared to VSVG-LV.</p

Selectivity of transduction by CD133-LV.

<p><b>A</b>. Flow cytometry analysis with GBML20 (CD133 content is 68.4±9.8%) shows that CD133-LV - transduced cells (TagBFP+) are also positive for CD133 (right top). In contrast, CD133+ cells are not enriched in the cohort transduced with VSVG-LV (right bottom). Untransduced cells show no TagBFP expression as expected (left panel) <b>B</b>. <b>i</b>. Percent CD133 positivity of cells transduced with either CD133-LV or VSVG-LV. <b>ii</b>. Population statistics for enrichment of CD133+ cells within populations transduced by either CD133-LV or VSVG-LV (MOI = 1). <b>C</b>. Schematic representation of epitopes on the second extracellular loop of CD133 recognized by antibodies predicted to block (recognizing 293C3 epitope) or not block (recognizing AC133 epitope) the interaction of CD133-LV's envelope with CD133 on the cell surface. Primary GBM cells were treated with varying amounts of blocking or non-blocking antibody prior to transduction with CD133-LV (MOI = 0.5). Transduction efficiency of CD133-LV was significantly reduced with blocking antibody. In contrast, non-blocking antibody did not show any significant effect (*, p<10⁻⁷). <b>D,E</b>. Primary GBM lines were modified with lentiviral constructs to either knockdown (<b>D</b>) or overexpress CD133 (<b>E</b>). GBML20 (CD133 content 68.9±9.8%) was used for shRNA-mediated knockdown of CD133. GBML27 (CD133 content 1.4±0.4%) was used for CD133 overexpression after transduction with lentiviral vector CD133-OE. <b>i-ii</b>. Flow cytometric analysis showing the CD133 content of primary lines expressing shRNA against CD133 or overexpressing CD133. <b>iii</b>. qRT-PCR analysis confirmed knockdown and overexpression of <i>PROM1</i> mRNA. <b>iv</b>. Western Blotting confirmed knockdown and overexpression of CD133 in these lines. β-actin was used as loading control. <b>v</b>. CD133 knockdown in GBML20 led to reduced transduction with CD133-LV (MOI = 5). Conversely, CD133 overexpression in GBML27 increased the rate of transduction by CD133-LV (MOI = 5).</p

CD133-LV transduces CD133+ cells in primary human GBM cultures <i>in vitro</i>.

<p><b>A</b>. Plasma membrane topology of CD133. CD133-LV recognizes an epitope on the second extracellular loop of the glycoprotein. <b>B</b>. Envelope protein of CD133-LV (Hmut: mutant hemagglutinin, 141.7Fv: single chain antibody against 293C3 epitope on second extracellular loop of CD133). Hmut and 141.7Fv are separated by a linker peptide. <b>C</b>. Observations collected from 3 primary GBM cultures. <b>i</b>. Primary GBM cultures are maintained as tumorspheres in suspension using media supplemented with EGF and bFGF. <b>ii</b>. Cell surface CD133 expression varies among different GBM lines, as assayed by flow cytometry. <b>iii,iv</b>. Flow cytometric analysis of transduction efficiency with CD133-LV (<b>iii</b>) and VSVG-LV (<b>iv</b>) expressing TagBFP. <b>D</b>. CD133-LV transduction efficiency correlates with the CD133 content of three primary GBM cultures (MOI = 5). <b>E</b>. Fluorescent microscopy shows a human GBM tumorsphere with scattered cells transduced with CD133-LV and thereby expressing TagBFP. This image was taken three days after incubation of cells with CD133-LV. <b>F</b>. Transduction of GBML3 cultures (CD133 content is 1.7±0.1%) with CD133-LV is dose-dependent and transduction efficiency is significantly lower than with pantropic VSVG-LV.</p