May 29, 2002 - weeks. Transplantation was with suspensions of cells or whole neurospheres; these were studied four weeks after transplantation into the ...
Neuroscience and Behavioral Physiology, Vol. 34, No. 7, 2004
Development of Neural Stem/Progenitor Cells from Human Brain by Transplantation into the Brains of Adult Rats M. A. Aleksandrova, R. A. Poltavtseva, A. V. Revishchin, L. I. Korochkin, and G. T. Sukhikh
UDC 611.813.013.018.8:599.323.4
Translated from Morfologiya, Vol. 123, No. 3, pp. 17–20, May–June, 2003. Original article submitted May 29, 2002. The aim of the present work was to study human neural stem/progenitor cells (SPC) cultured in vitro and their potential to survive, migrate, and differentiate after transplantation into adult rat brain. SPC were extracted from the brains of nine-week human embryos and were cultured in selective medium for three weeks. Transplantation was with suspensions of cells or whole neurospheres; these were studied four weeks after transplantation into the hippocampus, striatum, and lateral ventricles of adult rats. Analysis of transplanted cells was based on various histological and immunohistological staining methods: bisbenzimide, bromodeoxyuridine, and antibodies to human nuclei, vimentin, β-tubulin, neurofilaments, and glial fibrillar acidic protein, which allowed us to make independent assessments of their state and differentiation. Transplanted SPC from human brains survived well for one month in all areas of adult rat brain without immunosuppression. Cells from suspension transplants migrated intensely and differentiated into neurons and gliocytes. At the same time, transplants of whole neurospheres showed limited or no migration because of the development of a glial barrier. Key words: neural stem cells, transplantation, survival, differentiation, migration.
The last decade was marked by revolutionary discoveries in neurobiology, demonstrating the existence of stem cells in the brains of mammalian animals and humans [4, 11, 12]. This discovery and further studies showed that the processes of neuro- and gliogenesis are not blocked, but occur in the hippocampus and the periventricular area of the brain throughout life, because of division of these cells [4, 5, 14]. Recent studies have established the methodological principles for extracting stem cells from the brains of embryonic and adult mammals and for culturing and growing them in vitro [1, 2, 5, 12, 13, 16]. A number of labora-
tories have produced long-lived clones of neural stem cells from rodents and humans and have identified factors for their guided differentiation for use in cell therapy in a variety of types of CNS pathology [9, 10, 12]. There is great hope for the transplantation of neural stem cells for the treatment of a variety of neurodegenerative brain disorders [3, 6–8, 15]. The aim of the present work was to study neural stem/progenitor cells (SPC) from humans, which we grew and multiplied in in vitro tissue culture, and their ability to survive, migrate, and differentiate in the brains of adult rats after transplantation as suspensions and whole neurospheres.
Laboratory of Experimental Neurobiology (Director: Doctor of Biological Sciences M. A. Aleksandrova), Institute of Developmental Biology, Russian Academy of Sciences; Laboratory of Experimental Neurogenetics (Director: Corresponding Member of the Russian Academy of Sciences Professor L. I. Korochkin), Institute of Gene Biology, Russian Academy of Sciences; Laboratory of Clinical Immunology (Director: Corresponding Member of the Russian Academy of Medical Sciences Professor G. T. Sukhikh), Institute of Biological Medicine, Moscow.
MATERIALS AND METHODS Multiplication of Human Neural Progenitor Cells in Culture. Isolation and cultivation of human brain progenitor cells have been described in detail elsewhere [1, 2]. Brain tissue from nine-week embryos was collected, dissociated, 659 0097-0549/04/3407-0659 ©2004 Plenum Publishing Corporation
660 and seeded into growth medium at a concentration of 2·106 cells/ml. Cultivation was with NPBM (Neural Progenitor Basal Medium, Clonetics) supplemented with a standard set of growth factors including nerve cell growth factor (NSP), human fibroblast growth factor (hFGF), human epidermal growth factor (hEGF), and gentamicin/amphotericin (NPMM, Clonetics). Cells were grown in suspension culture at a density of 2·106 cells/ml. Half the medium was replaced every four days. As neurospheres formed, cultures were repeatedly pipetted and re-seeded to a density of about 100,000 cells/ml. Cells were cultured for four weeks and were then ready for transplantation. Preparation of Cells for Transplantation. Two different methods were used to visualize cells prior to transplantation: bromodeoxyuridine (BrdU, Sigma) and a nuclear luminescent stain (Hoechst 33342, Sigma). Neurospheres were cultured in medium containing BrdU at a concentration of 1 µM for 48 h and then for 30 min after addition of stain to a concentration of 20 µg/ml, after which they were thoroughly washed and prepared for transplantation. In one case, cell suspensions were made from a mixture of single cells and small neurospheres, diluted to a concentration of about 100,000 cells/µl; in another case, native neurospheres of diameter 150–200 µm were used. Transplantation of Cultured Cells. Recipients were adult Wistar rats weighing 250 g. Animals were anesthetized with chloral hydrate (300 mg/kg) and placed in a stereotaxic apparatus. Cultured cells in 3 µl were injected with a microsyringe according to the following coordinates: striatum – A = –0.5 mm, L = 2.0 mm, V = 4.2 mm; hippocampus – A = –3.5 mm, L = 2.1 mm; V = 2.5–3.0 mm; lateral ventricle – A = +1.5 mm, L = 1.5 mm, V = 4.3 mm. One series of rats received transplants of suspensions of dissociated cells at these coordinates, while the other series received whole native neurospheres. Tissue Processing and Immunohistochemistry. Animals were subjected to transcardiac perfusion with 4% paraform in phosphate buffer (1 M, pH 7.4) 30 days after transplantation. Brains were removed and placed in fixative for 24 h, followed by 30% sucrose at 4°C. A cryomicrotome was used to cut sections of thickness 20–40 µm. The luminescence op bisbenzimide-stained cells was used to select sections containing transplanted cells. These sections were stained with cresyl violet and immunohistochemical methods using primary antibodies to BrdU (Sigma, 1:100), neurofilament-70 (Serotec, 1:500), human nuclei (h-nuc; Chemicon, 1:30), glial fibrillar acidic protein (GFAP; DAKO, 1:250), β-tubulin (ICN, 1:100), and vimentin (NeoMarkers, 1:100). The methods used for processing sections have been described previously [2, 13]. Sections were kept overnight in solutions of primary antibodies to human nuclei with addition of 0.3% Triton X-100 and 2% normal animal serum for the host used for preparation of secondary antibodies. After washing, specimens were processed by treatment with biotinylated secondary antibodies to mouse
Aleksandrova, Poltavtseva, Revishchin, et al.
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Fig. 1. In vitro culture stem-progenitor cells from human brain before transplantation into adult rat brains. a) Cell suspension; b) whole native neurospheres. a) Stained with bisbenzimide; b) stained with antibody to BrdU. Magnification: a) ×160; b) ×200.
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Fig. 2. Suspended stem cells from human brain four weeks after transplantation into adult rat brain. a) Migration of human neural stem/progenitor cells (SPC) along fibers of the internal capsule; b) SPC differentiated into neurons expressing neurofilaments. a) Stained with antibodies to humnuc; b) stained with antibodies to hum-neurofilament. Magnification: a) ×160; b) ×1000.
immunoglobulins (Vector Laboratories), and were then stained with a solution of streptavidin labeled with the fluorescent stain Texas Red (Jackson) or with avidin-biotinperoxidase complex (Vector Laboratories) with subsequent detection of peroxidase with diaminobenzidine. Double immunocytochemical staining used secondary antibodies labeled with the fluorescent stains Texas Red and Cy-2. After washing three times, slides were covered with 50% glycerol and examined under a fluorescence lamp or with combined illumination.
RESULTS Distribution and Migration of Transplanted Cells in the Recipient Brain. Human progenitor neural cells implanted as cell suspensions and whole neurospheres (Fig. 1a, b) were seen in all recipients in the transplantation areas four weeks after transplantation. Analysis of brain sections after transplantation of suspensions involved treatment of tissues with antibodies to h-nuc, which specifically
Development of Neural Stem/Progenitor Cells from Human Brain
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Fig. 3. Neurospheres of human stem/progenitor cells four weeks after transplantation into adult rat brain. a) A whole neurosphere on the surface of the fringe of the hippocampus; b) individual cells migrate from a neurosphere in the lateral ventricle; c) a neurosphere from brain tissue separated by a glial barrier. a) Stained with cresyl violet; b) stained with antibodies to BrdU; c) stained with antibodies to GFAP. Magnification ×160.
stains only human nuclei, and with antibodies to BrdU. These experiments showed that immunopositive cells were located in the hippocampus (partially in the cerebral cortex and thalamus), and within the lateral ventricle and striatum. Stained cells formed dense accumulations in the area of the injection track and in the migration zone within the recipient brain tissue. h-nuc-positive cells migrated intensely along the fibers of the corpus callosum and internal capsule (Fig. 2a). In the hippocampus, cell migration was clearly marked into the suprapyramidal areas. Cultured cells transplanted as whole neurospheres (150–200 µm in diameter) were seen in the hippocampus, lateral ventricle, and striatum. Transplants in the hippocampus and lateral ventricle retained the neurosphere shape, while in the striatum they consisted of individual cells. Neurospheres located in the hippocampus consisted of hollow cell aggregates consisting of several rows of cells (Fig. 3a). Migration of cells from neurosphere transplants into recipient brain tissue was never seen. Neurospheres seen in the lateral ventricle were always attached to its wall. Unlike intra-tissue transplants, these consisted of spherical aggregates with centers filled with densely packed cells. The surfaces of these neurospheres consisted of quite loose collections of cells. Cells of the ependymal pavement were absent at the sites at which neurospheres were attached to the ventricular wall and zones of direct contact with recipient brain tissue were seen. Occasional BrdU-immunopositive transplant cells migrated into recipient brain tissue at intergrowth zones (see Fig. 3b). Differentiation of Transplant Cells. Differentiation of implanted cells was studied by double staining with antibodies to h-nuc and the specific differentiation markers vimentin, β-tubulin III, neurofilament-70, and GFAP four weeks after transplantation. Significant numbers of immature, undifferentiated cells were seen on double labeling for h-nuc and vimentin. Nearly all cells of intraparenchymal suspension transplants located in the most distant migration
zones among the fibers of the white matter and internal capsule were intensely stained with h-nuc and were simultaneously vimentin-positive. These cells often had extended nuclei, which is characteristic of migrating cells. Areas of dense groupings of transplant cells showed separate groups of h-nuc/vimentin-positive cells. Neuronal differentiation was detected in cells located in the zones of denser groupings of transplanted cells using double labeling for h-nuc and β-tubulin III or neurofilament-70. Antibodies to β-tubulin III revealed an insignificant number of transplanted cells of the unipolar type, while antibodies to neurofilament-70 resulted in staining of many cells. These had uni- or bipolar shapes with long processes and very large growth cones (see Fig. 2b), which is evidence for differentiation and active growth of human nerve cells in the microenvironment of the recipient brain. Staining of cells in transplanted neurospheres was weak and did not allow detection of specific neuronal differentiation in these structures. Differentiation via the glial pathway was revealed by antibodies to GFAP. Individual cells from suspension transplants doubly stained for h-nuc and GFAP revealed cells in the distant migration zones as well as in areas of dense groupings of implanted cells, though GFAP-positive cells never formed structures of the barrier type. Conversely, the whole surface of intraparenchymal neurospheres was covered with GFAP-positive cells (see Fig. 3c), which blocked cell migration and also, possibly, normal cell differentiation.
DISCUSSION The results obtained here showed that neural human stem/progenitor cells grown and multiplied in tissue culture can successfully be transplanted into the brains of adult rats for four weeks without immunosuppression, which is in agreement with previously obtained results [3, 9, 10]. After
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transplantation of suspension cultures of SPC, microenvironmental factors in the brains of adults are responsible for cell migration and differentiation via the neuronal or glial pathways, as noted by other authors [9, 10, 12]. At the same time, after transplantation of large whole neurospheres of SPC, migration and differentiation of cells was significantly blocked because of the formation of a glial barrier. This study was supported by the Russian Fund for Basic Research (Grant No. 02-04-48/53) and the Main Schools of the Russian Fund for Basic Research and the PNG.
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