Robust in vivo gene transfer into adult mammalian neural stem cells by lentiviral vectors - PubMed (original) (raw)

Robust in vivo gene transfer into adult mammalian neural stem cells by lentiviral vectors

Antonella Consiglio et al. Proc Natl Acad Sci U S A. 2004.

Abstract

Stable genetic modification of adult stem cells is fundamental for both developmental studies and therapeutic purposes. Using in vivo marking studies, we showed that injection of lentiviral vectors (LVs) into the subventricular zone of the adult mouse brain enables efficient gene transfer into long-term self-renewing neural precursors and steady, robust vector expression in their neuronal progeny throughout the subventricular zone and its rostral extension, up to the olfactory bulb. By clonal and population analysis in culture, we proved that in vivo-marked neural precursors display self-renewal and multipotency, two essential characteristics of neural stem cells (NSCs). Thus, LVs efficiently target long-term repopulating adult NSCs, and the effect of the initial transduction is amplified by the continuous generation of NSC-derived, transduced progeny. LVs may thus allow novel studies on NSCs' physiology in vivo, and introduction of therapeutic genes into NSCs may allow the development of novel approaches for untreatable CNS diseases.

PubMed Disclaimer

Figures

Fig. 1.

Long-term marking of SVZ cells by LV. (a_–_c) GFP-expressing LV were unilaterally injected in the forebrain SVZ; brain sections were analyzed 3 (a and b) and 6 (c) months later. GFP expression was evident at the site of injection at both time points (b, high-power magnification of a). (d and e) Three months after injection confocal analysis of double-labeled sections showed GFP+ cells (d and e, green) expressing markers of glial (d, GFAP, red) or neural (e, Tuj1, red) cells. (f_–_i) Many GFP+ cells with the typical morphology of granule neurons (g and h) were detected in the OB 2 (f_–_h) and 6 (i) months after LV injection in the SVZ. Injection of GFP-expressing LV in the lateral ventricle resulted in intense labeling of the ependymal layer (j), but no GFP-expressing cells were detected in the OB of these animals 2 months after injection (k, DAPI staining). (l and m) Double-staining confocal immunofluorescence using antibodies against cell differentiation markers showed that the majority of GFP-expressing cells found in the OB 3 months after LV injection into the SVZ expressed NeuN (l, red), whereas no GFP+ cells colabeled with the astrocyte marker GFAP (m, red) could be detected. Arrows identify the position of representative cells in the fields. Representative pictures from three mice analyzed per time point.

Fig. 2.

GFP-positive neuroblasts were observed in the RMS 3 and 6 months after LV-GFP injection. Photograph shows migrating GFP+ cells in the RMS 3 months after injection in the SVZ (a, DAPI staining, blue; b, GFP, green). Confocal immunofluorescence microscopy showed that many of the GFP+ migrating cells expressed the early neuronal markers Tuj1(c, red) and doublecortin (DCX; d, red). Insets show higher magnification pictures of the double-labeled cells. e and f show RMS and SVZ from mice administered BrdUrd for 8 days, starting 3 months after LV-GFP injection in the SVZ and stained for BrdUrd (red) and GFP (green). Cells stained for both BrdUrd and GFP appear yellow in the merged picture. Inset in f shows nuclei staining for BrdUrd. Representative pictures from three mice analyzed per time point.

Fig. 3.

SVZ cells targeted in vivo by LV display features of multipotent NSCs in culture. (a) Growth curves for cell lines (passages 6–15) derived from the injected SVZ (GFP+, squares) or controlateral SVZ (GFP–, circles). The slope values ± SE (b = 0.07389 ± 0.00588 and 0.07132 ± 0.0039 for GFP+ and GFP–cells, respectively) showed no significant difference (P = 0.8). Data shown are from one of three independent cultures with similar results. DIV, days in vitro. (b and c) Cytofluorimetric analysis of NSCs from LV-injected SVZ (passage 29; b) and from noninjected SVZ after in vitro transduction with LV-GFP (6 × 106 transducing units (T.U.)/ml) (c). The frequency and MFI of GFP+ cells are indicated. (d) Southern analysis of DNA extracted from ex vivo expanded GFP+ NSCs showing a unique integration pattern (lanes: 0, negative control; 1 and 3, plasmid standards for one and three vector copies per genome; p13 and p33, GFP+ cells from injected SVZ, at passages 13 and 33; C, noninfected NSCs). Each band corresponds to one vector copy per genome. The same analysis performed on in vitro transduced NSCs (same cells shown in c) showed random LV integration (lanes: + and –, transduced and nontransduced cells, respectively). (e_–_m) Stable GFP expression and multipotency of NSC cultures from LV-injected SVZ; upon mitogens removal, clonally derived cells gave rise to GFAP+ astrocytes (e_–_g), TUJ1+ neurons (h_–_j), and O4+ oligodendrocytes (k_–_m). GFP in green (e, h, and k), GFAP-(f), TUJ1– (i), and O4-positive cells (l) in red; nuclei stained with DAPI (g, j, and m) in blue. Arrows identify the same cells in the field. (Magnification, e_–_g, ×200; h_–_m, ×400.) [Bars, e_–_g, 30 μm (in g); h_–_m, 20 μm (in m)].

References

1. Altman, J. (1962) Science 135, 1127–1128. - PubMed
1. Altman, J. (1969) J. Comp. Neurol. 137, 433–457. - PubMed
1. Eriksson, P. S., Perfilieva, E., Bjork-Eriksson, T., Alborn, A. M., Nordborg, C., Peterson, D. A. & Gage, F. H. (1998) Nat. Med. 4, 1313–1317. - PubMed
1. Gage, F. H. (2000) Science 287, 1433–1438. - PubMed
1. Alvarez-Buylla, A., Seri, B. & Doetsch, F. (2002) Brain Res. Bull. 57, 751–758. - PubMed

Robust in vivo gene transfer into adult mammalian neural stem cells by lentiviral vectors - PubMed (original) (raw)