Survival and differentiation of neural progenitor cells derived from embryonic stem cells and transplanted into ischemic brain

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Object. Cell replacement therapy including the use of embryonic stem cells (ESCs) may represent a novel treatment for damage from stroke. In this study, the authors transplanted neural progenitor cells (NPCs) derived from ESCs into ischemic brain and analyzed their survival and differentiation.

Methods. Multipotential NPCs were generated from ESCs by using the stromal cell—derived inducing activity method. These cells could differentiate in vitro into neurons, glia, and oligodendrocytes, thus revealing them to be neural stem cells. The NPCs were then transplanted into ischemic brain. At 2 weeks postischemia, the transplanted cells occupied 18.8 ± 2.5% of the hemispheric area; by 4 weeks postischemia, 26.5 ± 4% of the hemisphere. At 4 weeks after transplantation, green fluorescent protein (GFP)—positive transplanted cells showed mature neuronal morphological features. The authors also investigated the expression of differentiation markers and various neurotransmitters. Transplanted cells were immunopositive for neuronal nuclei, β-tubulin-III, and glial fibrillary acidic protein. Of the GFP-positive cells, 33.3 ± 11.5% were positive for glutamate decarboxylase, 13.3 ± 5.8% for glutamate, 2.1 ± 2.5% for tyrosine hydroxylase, 1.8 ± 2% for serotonin, and 0.4 ± 0.2% for choline acetyltransferase.

Conclusions. The authors confirmed the survival and differentiation of ESC-derived NPCs transplanted into the ischemic brain. Surviving transplanted cells expressed several neural markers and neurotransmitters. These findings indicate that these cells can function in the brain.

Article Information

Address reprint requests to: Yasushi Takagi, M.D., Ph.D., Department of Neurosurgery, Graduate School of Medicine, Kyoto University, 54 Kawahara-cho, Shogoin, Sakyo, Kyoto 606–8507, Japan. email: ytakagi@kuhp.kyoto-u.ac.jp.

© AANS, except where prohibited by US copyright law.

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Figures

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    a: Phase-contrast image of undifferentiated mouse ESCs cultured on a gelatin-coated dish. Fluorescence microscopy images of cells. b: Confoca microscope image of neural cell adhesion molecule expression (red) in differentiated mouse ESCs, which were cultured on PA6 cells for 10 days. c: Phase-contrast image of neurospheres derived from mouse ESCs. The differentiated ESCs become detached from the PA6 cell monolayer and form spheres. d and e: Confocal microscope images of differentiation from neurospheres derived from mouse ESCs. Neurospheres derived from ESCs were cultured on ornithine laminin—coated dishes for 7 days in the presence of LIF, fibroblast growth factor 2, and epidermal growth factor. Both TuJ1 (neuronal marker, green) and GFAP (glial marker, red) are expressed in neuroprogenitors differentiated from ESCs. f: Oligodendrocyte differentiation from neurospheres formed from ESCs. The CNPase (oligodendrocyte marker, red; TuJ1, blue) is expressed in differentiated neuroprogenitors. Original magnification × 40 (c), × 100 (a, b, and d), × 400 (e and f).

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    Images demonstrating survival in the ischemic brain of transplanted neurospheres made from mouse ESCs 2 weeks after transplantation (a–c), 4 weeks after transplantation (d–f), and in sham controls (g–i). Red indicates NeuN (a, d, and g); green, GFP (b, e, and h). The GFP-positive cells occupied the ischemic areas (a–f), especially 4 weeks after ischemia (d–f). Interference differential microscopic images (c, f, and i) demonstrate the area of infarction, which was located in the lateral striatum after ischemia (c and f). Original magnification × 40.

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    Fluorescence microscopy images revealing differentiated transplanted NPCs derived from ESCs. The GFP-positive cells demonstrated mature neuron-like morphological features (a). Note that NeuN-positive cells (b), GFAP-positive cells (c), GAD-positive cells (d), glutamate-positive cells (e), ChAT-positive cells (f), TH-positive cells (g), and serotonin-positive cells (h) were found in the graft. Original magnification × 400.

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