Survival of transplanted neural progenitor cells enhanced by brain irradiation

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Object

Authors of previous studies have reported that adult transplanted neural progenitor cells (NPCs) are suitable for brain cell replacement or gene delivery. In this study, the authors evaluated survival and integration of adult rat–derived NPCs after transplantation and explored the potential impact on transplant survival of various mechanical and biological factors of clinical importance.

Methods

Adult female Fischer 344 rats were used both as a source and recipient of transplanted NPCs. Both 9L and RG2 rat glioma cells were used to generate in vivo brain tumor models. On the 5th day after tumor implantation, NPCs expressing green fluorescent protein (GFP) were administered either intravenously (3.5 × 107 cells) or by stereotactic injection (1 × 104–1 × 106 cells) into normal or tumor-bearing brain. The authors evaluated the effect of delivery method (sharp compared with blunt needles, normal compared with zero-volume needles, phosphate-buffered saline compared with medium as vehicle), delivery sites (intravenous compared with intratumoral compared with intraparenchymal), and pretreatment with an immunosuppressive agent (cyclosporin) or brain irradiation (20–40 Gy) on survival and integration of transplanted NPCs.

Results

Very few cells survived when less than 105 cells were transplanted. When 105 cells or more were transplanted, only previously administered brain irradiation significantly affected survival and integration of NPCs. Although GFP-containing NPCs could be readily detected 1 day after injection, few cells survived 4 days to 1 week unless preceded by whole-brain radiation (20 or 40 Gy in a single fraction), which increased the number of GFP-containing NPCs within the tissue more than fivefold.

Conclusions

he authors' findings indicate that most NPCs, including those from a syngeneic autologous source, do not survive at the site of implantation, but that brain irradiation can facilitate subsequent survival in both normal and tumor-bearing brain. An understanding of the mechanisms of this effect could lead to improved survival and clinical utility of transplanted NPCs.

Abbreviations used in this paper: CNS = central nervous system; GFP = green fluorescent protein; HBSS = Hanks balanced salt solution; MASC = multipotent astrocytic stem cell; NPC = neural progenitor cell; NSC = neural stem cell; PBS = phosphate-buffered saline; SEZ = subependymal zone.

Article Information

Address reprint requests to: Glenn T. Gobbel, D.V.M., Ph.D., Department of Neurological Surgery, B-400, University of Pittsburgh Medical Center-Presbyterian, 200 Lothrop Street, Pittsburgh, Pennsylvania 15213. email: gobbelg@upmc.edu.

© AANS, except where prohibited by US copyright law.

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Figures

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    Photomicrographs demonstrating the presence of neuronal, astrocytic, and oligodendroglial markers in cultures of NPCs that express GFP. When NPCs were cultivated on poly-d-lysine in medium containing fetal calf serum, the cells differentiated into multiple phenotypes and expressed markers of neurons (β-tubulin III, B), astrocytes (glial fibrillary acidic protein, D), and oligodendroglia (2,3-cyclic nucleotide 3-phosphohydrolase; F), which were detected by immunocytochemical analysis and fluorescent microscopy. Differences between the nonspecific, GFP expression to the left (A, C, and E) of each of the fluorescent, immunocytochemical stains (bright white areas) demonstrate that only subsets of the population expressed a particular phenotypic marker. Bar = 50 μm.

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    Flow chart showing the experimental plan to test factors that may affect transplant survival including cell type (A), injection technique (B), and recipient tissue microenvironment (C). Hanks = HBSS; IV = intravenous; NPGM = neural progenitor growth medium.

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    Illustrations (upper) and photomicrographs (lower) showing the impact of syringe type on intracerebral implantation of NPCs. Both open needle (upper left) and positive-displacement (upper center and right) syringes were used as well as beveled (left and center) and blunt (upper right) needle tips. At 1 day after administration, plugs of bright white, GFP-positive cells could be seen by epifluorescent microscopy in the tissue directly adjacent to the injection track for all of the three needle types (lower). Although the blunt-ended needle did appear to produce larger injection tracks in the tissue (lower right), there were no clear differences among the three syringe types in terms of the number or viability of NPCs delivered. Each photomicrograph demonstrates a region of the rat caudate nucleus that was injected with NPCs by using the syringe type shown directly above (upper). Original magnification × 400.

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    Photomicrographs demonstrating the presence of GFP-positive NPCs in the normal brain tissue at 1 day (upper) and 1 week (center and lower) after administration. In previously nonirradiated brain, no cells were visible beyond 1 day after NPC administration (upper and center). In contrast, numerous cells could be found at the site of injection up to 1 week after 20 Gy of whole-brain irradiation (lower).

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    Photomicrographs demonstrating the presence of GFP-positive NPCs in intracerebral tumors 8 days after implantation. In previously nonirradiated brain, as in normal brain, very few cells could be detected at 1 week after implantation, even after injecting as many as 106 NPCs (upper). In contrast, numerous cells could be found within and around the intracerebral tumor in brains pretreated with 20 Gy of whole-brain irradiation followed 7 days later by injection of 105 (center) or 106 (lower) NPCs.

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    Results of a quantitative analysis of the impact of irradiation on the number of NPCs detected following implantation into normal (left) or tumor-bearing (right) brain. No or extremely few NPCs could be located following implantation of either 1 × 105 (small symbols) or 1 × 106 (large symbols) cells into the four animals that had not been treated with previous brain irradiation (open symbols). In contrast, GFP-positive (GFP+), NPCs could readily be detected in the six animals treated with 20 Gy of whole-brain irradiation (closed symbols). Each symbol represents one animal.

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