Biology Bulletin, Vol. 28, No. 6, 2001, pp. 553–560. Translated from Izvestiya Akademii Nauk, Seriya Biologicheskaya, No. 6, 2001, pp. 656–665. Original Russian Text Copyright © 2001 by Aleksandrova, Saburina, Korochkin, Revishchin, Repin, Rzhaninova, Sukhikh.

SYMPOSIUM

Behavior and Differentiation of the Neural Stem Cells in vivo M. A. Aleksandrova1, I. N. Saburina1, L. I. Korochkin1, 2, A. V. Revishchin2, V. S. Repin3, A. A. Rzhaninova3, and G. T. Sukhikh3 1

Kol’tsov Institute of Developmental Biology, Russian Academy of Sciences, ul. Vavilova 26, Moscow, 119991 Russia 2 Institute of Biology of the Gene, Russian Academy of Sciences, ul. Vavilova 34/5, Moscow, 117984 Russia 3 Institute of Biological Medicine, ul. Oparina 4, Moscow, 117815 Russia e-mail: [email protected] Received February 22, 2001

Abstract—We studied the behavior and differentiation of human and rat neural stem cells after transplantation in the adult rat brain without immunosuppression. The rat stem cells were isolated from the presumptive neocortex of 15-day-old embryos. The human cells were isolated from the ventricular brain zone of 9-week-old embryos and cultivated for two weeks before transplantation. The results of histomorphological studies suggest that the microenvironment factors did not suppress the growth or development of transplanted stem cells. Both rat and human embryonic multipotent neural cells showed similar behavior and differentiation into neurons and glial cells. After transplantation, they continued to mitotically divide and migrated from the graft area to the surrounding tissue of a recipient brain. The presumptive glial cells migrated preferentially along the capillaries and fibrous structures of the recipient brain. Similar behavior of the rat and human neural stem cells in the microenvironment of the recipient adult rat brain and the absence of immune reaction suggest that the transplantation into the rat brain may serve as a model for studying the developmental biology of the human stem cells.

The central nervous system of mammals has a very limited capacity for structural restoration upon various lesions (Ramon and Cajal, 1928; Carlson, 1986). This partly due to the incapacity of the adult brain to generate new cellular elements in response to trauma. The transplantation of neuronal cells allows for the replenishment of the lost populations of neurons and glial cells. It was repeatedly shown on various mammals that the transplanted neurons could not only survive, grow, and differentiate, but also structurally and functionally replace the lost neurons. At the same time, the committed neuronal precursors had a low capacity for migration, did not reproduce the architectonics, and were not practically integrated with the recipient brain (Fisher and Gage, 1993; Dunnett and Bjorklund, 1994; Aleksandrova, 1999). In this respect, the attention was drawn to studying the potencies of multipotent neural stem cells (Gage et al., 1995; Brustle and McKay, 1996; Sukhikh, 1998). All cell populations of the central nervous system (neurons, astrocytes, and oligodendrocytes) are derived from precursors located in the germinative areas of the developing brain: ventricular and subventricular zones. Multipotent neural stem cells are present in this zone during embryonic and early postnatal development (Walsh and Cepko, 1992; Johe et al., 1996; Qian et al., 1998; Fricker et al., 1999; Gage, 2000). In addition, recent studies showed that neurogenesis and gliogenesis proceeded during the entire life of mammals and neural stem cells were present not only during development but also in the adult mammalian and human brain

(Lois and Alvarez-Buylla, 1994; Gage et al., 1995; McKay, 1997; Kukekov et al., 1999; Temple and Alvarez-Buylla, 1999). Neural stem cells can be isolated from the embryonic or adult mammalian brain, and their differentiation may be studied both in vitro and, after transplantation, in vivo. Here, we studied the behavior and differentiation of the rat and human neural stem cells isolated from the embryos after their transplantation in the adult rat brain. The aim of this study was to compare the behavior and differentiation of native rat cells and cultured human cells in the adult rat brain microenvironment. MATERIALS AND METHODS Transplantation of the rat neural stem cells in the adult rat brain. Experiments were carried out on 30 Wistar rats. The females weighing 100–150 g served as recipients and 15-day-old embryos (day 1 of gestation was determined by the appearance of sperm in a vaginal smear) as donors. The embryonic tissues were labeled by 3H-thymidine: females were injected with 3H-thymidine at 10 µCi/kg weight (specific activity 52.1 µCi/mol) twice a day on days 12, 13, and 14 of gestation. Transplantation was performed under chloral hydrate anesthetization as described elsewhere (Aleksandrova, 1998). In recipients, a region of the occipital cortex was removed and a mechanically suspended tissue of presumptive embryonic cortex with multipotent stem cells of the ventricular and subventricular zones was trans-

1062-3590/01/2806-0553$25.00 © 2001 MAIK “Nauka /Interperiodica”

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planted in the cavity. Within 4, 10, and 20 days and 1 and 3 months, the animals were sacrificed by a high chloral hydrate dose and the brains were fixed. Immunohistochemical studies were carried out using antibodies against glial fibrillar protein (GFAP), GABA, and neuropeptides parvalbumin (PARV), calbindin D-28K (CALB), and NPY. The brain tissue fixed by 4% paraformaldehyde was cut on a vibratome or cryostat and the coronary brain section 40 µm thick was incubated with murine primary monoclonal antibodies against GABA (1 : 100, Chemicon), CALB D-28K (1 : 8000, Sigma, C8666), PARV (1 : 8000, Sigma, P3171), and NPY (1 : 700, Amersham AB) diluted in PBS with a detergent (0.1% Triton X-100) and normal horse serum. The sections were then incubated in a solution of secondary antibodies at 1 : 100 and transferred in a solution of avidin-biotin-peroxidase complex (Vectastain ABS Kit, Vector Laboratories BA2000). The treatment was completed by the visualization of the peroxidase ABC complex, dehydration in alcohols and embedding in Depex. A part of the materials was embedded in paraffin and processed using the Golgi method or its sections were covered by type M photoemulsion of radioautography and stained by cresyl violet. Transplantation of human neural stem cells in the adult rat brain. Tissues of the human embryonic brain were obtained from medically aborted 9-week-old embryos. Tissue fragments were isolated from the brain periventricular area, placed in medium F-12, and suspended by long-term pipeting. The resulting suspension was cultivated in the neural progenitor basal medium (NPBM, Clonetics) complemented with the basal fibroblastic growth (bFGF) and epidermal growth (EGF) and neural survival (NSF) factors. The cells were grown in a suspension culture at a density of 2 million cells/ml and the medium was replaced every four days. As the cultured cells formed neurospheres, the latter were carefully pipeted and plated. The cells were used for transplantation after four passages (the total duration of cultivation was 12–16 days). For visualization of the cells after transplantation, they were stained by the nuclear fluorescent dye bisbenzimide (Hoechst 33342, Serva), The culture of stem cells was incubated in a medium with 20 µg/ml bisbenzimide for 30 min., The cells were then carefully washed and a suspension was prepared in a dilution of 4 × 10000/µl. Before transplantation, the cells were stained by trypan blue to estimate their viability. In the rest of the culture, the cells were stained by antibodies against nestin and tubulin (B-Tub III). During transplantation, the suspension was kept in the cold and carefully pipeted before every injection. Wistar rats served as recipients: eight 10-day-old pups and eight adult 2-month-old pups. A suspension of the human neural stem cells (4 µl) was introduced in lateral ventricle area of the recipient brain.Before transplantation, the rats were anesthetized by chloral hydrate

at 300 mg/kg weight. The animals were placed in stereotaxis, a hole was drilled in the skull, and the cells were transplanted according to the coordinates +1.5 mm from bregma and 1.5. mm laterally (see Pellegrino et al., 1981 and Dunnett and Bjorklund, 1992). A suspension of cells was sucked in a Hamilton syringe, the syringe was fixed in the stereotaxis holder, and the needle was introduced in the middle of the hole at a depth of 4–5 mm. The suspension was introduced for 3 min, slowly lifting the needle with a step of about 0.5 mm, and the needle was then kept in the brain for 2–3 min. To be transplanted into pups, they were placed on ice for immobilization. A suspension (4 µl) was sucked in a syringe and the syringe was fixed in the stereotaxis holder. The immobilized animals were carried in hand and the needle was introduced in the lateral ventricle area directly across the skin and skull and the cell suspension was injected. After transplanted, no immunosuppression was performed either on the adult rats or on pups. Within 10 and 20 days after transplantation, all animals were transcardially perfused with 4% paraformaldehyde on 1 M phosphate buffer, pH 7.3. After perfusion, the brain was removed and placed in a fixative for 24 h and then transferred in 30% sucrose. The tissue was cut on a cryostat and sections 40 µm thick were placed on gelatinized slides. The sections were examined under a fluorescent microscope and then stained by cresyl violet. The cultured cells precipitated on the dish bottom were fixed using a similar method. Immunohistochemical studies were carried out using primary antibodies: anti nestin (Biogenesis, 1 : 20), anti GFAP (DAKO, 1 : 250), anti B-tubulin (ICN, 1 : 100), and anti vimentin (NeoMarkers, 1 : 100). After washing, the cells and cryostat sections were treated with 0.3% Triton X-100 and kept overnight in a solution of primary antibodies added with 2% normal horse serum and 0.01% sodium azide. After careful washing, the materials were treated by a solution of biotinilated secondary antibodies (Vector Laboratories) raised in a horse against murine immunoglobulin (in the case of monoclonal primary antibodies) or in a goat against rabbit immunoglobulin (in the case of polyclonal primary antibodies) at 1 : 200 at the room temperature for 1 h. The materials were then stained by a solution of streptavidine labeled by the fluorescent dye Dil (Molecular probes) and, after washing three times, cleared by 50% glycerol in PBS. The cells were examined and photographed under a fluorescent microscope and opaqueillumination by green light in red fluorescence. RESULTS Behavior and differentiation of the rat neural stem cells after transplantation in the adult rat brain. A histological study has shown that the multipotent stem cells that form the ventricular and subventricular zones BIOLOGY BULLETIN

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of the presumptive neocortex continued to develop after being transplanted into the adult rat brain. In all cases, transplanted cells were found in the recipient brain, which were located as compact groups along the transplantation track and were practically not separated from the recipient brain cells. The multipotent cells retained their potencies for proliferation, as follows from the presence of mitotic cells in the transplants within four days after transplantation (Figs. 1a, 1b). The migration of multipotent cells was studied on radioautographs within one month after transplantation, when the transplanted cells differentiated into neurons and glial cells. The results obtained suggest that precursor cells migrated over the recipient brain tissue. In the adult rat brain, the 3H-thymidine-labeled cells were found at different distances from the place of transplantation. The labeled cells were located radially in the cortical gray matter, crossed corpus callosum and, in come cases, came to the upper striatum regions. The migration of the cells that differentiated into astrocytes was very extensive. They were found in glia limitans, corpus callosum, and all layers of the recipient cerebral cortex. The labeled astrocytes were located singly or in groups and were often associated with vessels (Fig. 2). The astrocytes migrating along the fibrous structures moved the greatest distance: up to 3 mm. Trajectories of the movement of donor cells were quite diverse. Sometimes, a limited migration was observed: rare labeled neurons were found at a distance if up to 100 µm from the transplants, while astrocytes predominated at a distance of up to 230 µm. In other cases, penetration of the cells from a transplant to the recipient brain was much more expressed. The cells differentiated as neurons migrated over a distance of up to 680 µm and those differentiated as astrocytes, up to 2– 3 mm. From a radially located transplant, the labeled cells migrated in the 1st, 2nd, 3rd, and 4th layers of the host cerebral cortex. Penetration of the migrated glial cells in the recipient cerebral cortex was in all cases much wider than that of neurons. The astrocytes migrated in the 1st–4th layers over distance of 230 to 960 µm. In all cases, the labeled glial cells were located in the recipient brain tissue singly or in groups (Fig. 3). The number of migrated astrocytes decreased in a gradient way from the area of transplantation to the periphery. In the 1st layer of the cerebral cortex, individual labeled astrocytes and their groups were located which were observed only around the lesion zone, where the astrocytes formed a glial scar. The astrocytes migrated most intensely in the 2nd and 3rd layers, and an insignificant number of these cells was found in the 4th layer. In these cases, the labeled astrocytes migrated along the recipient brain vessels as well, but they went especially far (2–3 mm) among the white matter fibers. The cells migrating from the transplants and differentiating into neurons were of average size (13–17 µm in diameter). The highest density of labeled neurons was observed at a distance of 200 µm from the area of BIOLOGY BULLETIN

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(a)

SvZ

VZ (b) T

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Fig. 1. Stem cells in an embryonic rat brain and after transplantation: (a) stem cells in the presumptive cerebral cortex of a 15-day rat embryo. (VZ) Ventricular zone, (SvZ) subventricular zone; (b) division of stem cells 4 days after transplantation in the adult rat brain. (T) Transplanted tissue, (r) recipient brain. Semithin sections, Staining by toluidine blue. Scale: 50 (a) and 30 (b) µm.

transplantation, although some donor cells migrated in the recipient brain tissue over a distance of up to 680 µm. The labeled neurons were most frequent in the 2nd and 3rd layers of the recipient cerebral cortex (Fig. 4). Single labeled neurons were found at the boundary between the 1st and 2nd layers. The migrated donor cells did not affect the recipient brain structure and were distributed in a natural way among the host cells. The histotypical structure of the transplants of multipotent cells was analyzed one and three months after transplantation. The results obtained suggest that, the layered organization of cells wasn’t detected, in any of

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The potencies of multipotent cells or neurochemical differentiation were studied on the population of inhibitory neurons widely represented in the rat neocortex. In the transplants, the antibodies against GABA visualized the neurons with the average immunoreactivity, which were distributed singly or in groups. Since the

GABA neocortical neurons are characterized by colocalization of some neuropeptides, whose expression is an important feature of their differentiation, we studied the expression of the calcium-binding proteins PARV and CALB and neuropeptide NPY. The results obtained suggest that the neurons in the transplants expressed PARV, CALB, and NPY. The distribution of immunoreactive neurons in the transplants differed markedly from that in the surrounding tissue of the recipient brain. The PARV immunoreactive neurons were distributed singly or in groups in all parts of the transplants. Many PARV immunoreactive processes crossed the boundary between the transplant and recipient brain tissues, although it was impossible to establish the affiliation of these neurons (transplant or host). The CALB neurons with average immunoreactivity were visualized all over the transplant area, and single cells with a strong immunoreactivity also occurred (Fig. 5). When studying the NPY expression in the transplants, we found chaotically distributed immunoreactive cells of average size, multi- or bipolar shape, and with weakly stained processes. The transplantation of the multipotent precursor cells from the ventricular and subventricular zones of the embryonic rat brain in the adult rat brain has shown that the adult brain microenvironment did not block their development or behavior. In a new microenvironment, the multipotent cells divided, migrated, and formed astrocytes and neurons, which expressed specific neurotransmitters and neuropeptides. Development of human neural stem cells after transplantation in the adult rat brain. The cultured human neural multipotent cells were stained by antibodies against nestin before transplantation. A great amount of nestin-positive neurospheres and individual cells suggested that the culture contained many neural stem cells (Fig. 6a). One part of the cultured cells was stained by bis-benzimide and prepared for subsequent transplantation, and another was placed in a medium

Fig. 3. Migration of a group of astrocytes labeled by 3H-thymidine to the boundary between the 1st and 2nd layers of a recipient brain. Radioautography. Staining by cresyl violet. Scale: 15 µm.

Fig. 4. Migration of single neural cells labeled by 3H-thymidine in the recipient brain tissue. Radioautography. Staining by cresyl violet. Scale: 10 µm.

Fig. 2. The astrocytes labeled by 3H-thymidine migrate from the transplanted tissue along the recipient brain vessels. Radioautography, Staining by cresyl violet. Scale: 20 µm.

the transplants, which is inherent in the normal cerebral cortex. The morphological characteristics of the donor neurons were described on the preparations stained after Golgi. These neurons were morphologically close to those of the normal cerebral cortex. In the transplants, the neurons were always located chaotically and their processes often crossed the boundary between the transplant and recipient tissues, although they could run along the boundary and not cross it. In those cases, when the transplanted cells were separated from the recipient brain by a glial scar, multipolar neurons with a similar phenotype were located within the transplants, many of which were hypertrophied.

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for further differentiation. The latter were stained by antibodies against β-Tubulin-III for marking the early neuroblasts: about 30% of cultured cells start their neuronal differentiation (Fig. 6b). The results of light microscope studies carried out within 10 and 20 days after transplantation suggest that the brains of all recipient rats contained the transplants of cultured human stem cells. Brightly fluorescing transplanted cells stained by bis-benzimide were quite distinct in the surrounding tissue. Clusters of transplanted cells were localized in the caudate nucleus, lateral ventricle, white matter, and cerebral cortex, i.e., they were located along the injection track. Individual macrophages and, sometimes, small necrotic zones could be seen among the transplanted cells but a pronounced immune reaction wasn’t observed in any of the cases. The density of transplanted cells gradually decreased from the area of injection to the periphery. The migrated cells were distributed among the recipient brain neurons and were never separated from the surrounding tissue by a glial barrier. Mitotic cells occurred among the stem cells, which suggests that their proliferative potencies are preserved in a new microenvironment. The transplanted stem cells were clearly divided in two populations: cells with small elongated brightly fluorescing nuclei and larger cells with paler oval nuclei (Fig. 7). Both groups had insignificant size: 9 to 12 µm. The cells of the first group had, as a rule, a bipolar shape and often had elongated processes. These cells were located amidst the transplant cellular mass and widely migrated beyond its limits to the recipient brain parenchyma. When the transplants were located inside or near the lateral ventricle, the migration of small cells over the ventricle epithelial layer to a distance of 1.5–2 mm could be seen. The longest migration (2–3 mm) of small cells was observed in the white matter area. The labeled cells widely migrated along the white matter fibrous layer and, in some cases, reached the opposite hemisphere. In the cortex cellular layer, the migration was much weaker. Small cells often migrated along the recipient brain capillaries and over the external surface of fibrous bundles, as in the case of transplantation in the caudate nucleus area (Figs. 8a, 8b). The cells of the second group did not demonstrate such a wide migration in the recipient brain tissue. They were concentrated, as a rule, in rather compact groups in the central zones of transplantation. At the same time, some cells migrated in the recipient brain parenchyma over a distance o 150–300 µm. They were observed in the lateral ventricle marginal zone, among the caudate nucleus cells, and in the cerebral cortex of recipients. The localization of small cells in a satellite position with respect to the recipient neurons or closely to the blood vessels suggested that these cells could be astrocytes. The immunochemical staining for GFAP has shown that some small cells differentiated as astroBIOLOGY BULLETIN

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Fig. 5. Individual neurons differentiate in the transplants that express the calcium binding protein calbindin. Antibodies against calbindin. Scale: 10 µm.

(a)

(b) Fig. 6. Neural stem cells of a human embryo in vitro: (a) a culture of stem cells from a 9-week human embryo before transplantation. Staining by antibodies against nestin. Scale: 25 µm; (b) cultured stem cells that start differentiation form many cells of the neuronal type. Staining by antibodies against β-tubulin. Scale: 25 µm.

cytes. The results of light microscopy studies suggest that, in some cases, the nuclei of small cells stained by bis-benzimide were surrounded by a dense crown of GFAP-positive fibers.

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Fig. 7. Stem cells of the human brain form two populations after transplantation in the rat brain. Stem cells are stained by fluorescent nuclear dye bis-benzimide. Scale: 100 µm.

At the same time, the staining them with antibodies against vimentin showed the presence in the transplants of the cells that express this protein, characteristic of the early noncommitted cells. Hence, the transplants contained the embryonic type cells which did not yet begin their differentiation (Fig. 9). No differences in the morphology of transplanted cells, their behavior, or differentiation were found after transplantation in the postnatal or adult rat brain. In both cases, the transplanted cells were equally capable of migration and differentiation and survived during the entire period of experiment without visible immune or glial reaction of the recipient brain. The data obtained suggest that the cultured human neural stem cells transplanted in the adult rat brain survived, retained their capacities for division and differentiation, and could differentiate along the glial or neural pathway. DISCUSSION

(a)

(b) Fig. 8. Migration of human neural stem cells in the rat brain: (a) along the capillaries and (b) along the fibrous bundles in the recipient brain striatum. Scale: 100 µm.

The differentiation of multipotent stem cells was shown by staining them with antibodies against CALB. Double staining demonstrated that the antibodies were bound to some transplanted cells of the neuronal type, whose nuclei contained bis-benzimide.

The results obtained suggest that the multipotent neural cells from the rat and human brain transplanted in the adult rat brain demonstrate similar behavior and differentiation into neurons and glia. Dissociated cells of the ventricular and subventricular zones of the embryonic rat cerebral cortex continued their development after allotransplantation in the adult brain. The factors of the recipient brain microenvironment did not suppress the growth and differentiation of multipotent cells. At the early stages after transplantation, the multipotent cells continued mitotic divisions and actively migrated from the area of transplantation in the recipient brain tissue. The transplanted embryonic cells with a wide potency for migration (McConnell, 1988; Aleksandrova et al., 1993; Olsson et al., 1998; Desai and McConnell, 2000) were found in practically all layers of the recipient cerebral cortex along the transplantation track and in the white matter. The migration of neural cells was always weaker (up to 680 µm) than that of glial cells (up to 3 mm). Blood vessels and fibrous structures of the brain served as pathways for the migration of astrocytes, as was noted also by other authors (Goldberg and Bernstein, 1988; Jacque et al., 1992; Andersoon et al., 1993; Okoye et al., 1995). It was proposed that the clustering of astrocytes in the region of the recipient cerebral cortex lesion was related to the formation of a glial barrier between the transplant and recipient tissues (Smith and Silver, 1988). Structural studies of the compactly arranged transplants have shown that their cytoarchitectonics are chaotic and the cells do not form layered structures. The transplanted cells are close in structure to the cells of the normal cerebral cortex only in the absence of glial barrier between the donor and recipient tissues. Otherwise, the cell structure is atypical and they are often hypertrophied (Aleksandrova and Girman, 1995). NeuBIOLOGY BULLETIN

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rochemical differentiation of the multipotent cells demonstrated the formation of inhibitory GABA-ergic neurons and expression of the proteins PARV, CALB, and NPY in the transplants. This suggests that the adult brain retains the factors of microenvironment supporting division, migration, and differentiation of the multipotent cells (Zigova et al., 1996; Snyder et al., 1997; Aleksandrova, 1998; Fricker et al., 1999). Studies of the fate of human multipotent neural cells gave the following results. In the culture of human stem cells isolated from the periventricular brain zone of 9-week-old embryos, many nestin-positive multipotent cells were preserved after the fourth passage, some of them developed by the neuronal pathway, in accordance with the published results (Carpenter et al., 1999; Fricker et al., 1999). After transplantation in the adult rat brain, the cultured human stem cells demonstrated the behavior similar to that of freshly isolated rat multipotent cells in the same microenvironment. Stem cells of both types divided mitotically and migrated in the recipient brain tissue. No significant differences were found in the pathways of their migration, although their trajectories were limited, as compared to those after transplantation in the brain of embryos or newborn animals (Sabate et al., 1995; Flax et al., 1998; Brustle, 1999). Two cell populations were distinguished in the transplants of human cells: small and larger cells. The latter migrated in the parenchyma and along the fibrous structures of the recipient brain over insignificant distances: up to 300 µm. The longest migrations (up to 3 mm) were characteristic of the small cells, some of which differentiated into astrocytes, as was shown using the antibodies against GFAP. Cells of both types were found in the lateral ventricle wall. This suggested incorporation of the transplanted cells in the rostral pathway of migration, as was shown while studying the transplantation of human stem cells in the brain of adult rodents (Lois et al., 1996; Suhonen et al.,1996). Note that the human and rat multipotent cells that differentiated into astrocytes had the same preferential substrates for migration: blood capillaries and fibrous structures of the recipient brain. This was also described by many other authors (Goldberg and Bernstein, 1988; Jacque et al., 1992; Andersoon et al., 1993; Brustle, 1999; Fricker et al., 1999). Studies of differentiating human stem cells in vivo using antibodies against GFAP, CALB, and vimentin demonstrated the formation of both astrocytes and neurons. At the same time, many cells were vimentin-positive, unlike those in the rat transplants (Aleksandrova and Kozlova, 1993). This means that some human multipotent cells do not begin differentiation within 10 or 20 days after transplantation confirmed the data on a slower, as compared to rodents, development of the human stem cells (Fricker et al., 1999). The results of our experiments suggest that the human stem cells survived for at least 20 days after BIOLOGY BULLETIN

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Fig. 9. In a transplant of human stem cells some cells express vimentin. Staining by antibodies against vimentin. Scale: 25 µm.

transplantation without immunosuppression and demonstrated a significant tolerance to the adult brain microenvironment. Thus, not only embryos (Flax et al., 1998; Brustle, 1999) but also adult rats may serve as a convenient model for studying the development of human neural stem cells. The data obtained suggest that the rat and human neural stem cells transplanted in the adult rat brain showed similar behavior and differentiation. The human embryonic stem cells survive quite well in a culture, retain their multipotency, and may develop in the adult rat brain microenvironment without immunosuppression. The knowledge accumulated in numerous studies of neurotransplantation on rodents and primates could serve as a basis for studying the developmental biology of human stem cells in vivo. ACKNOWLEDGMENTS This study was supported by the Russian Foundation for Basic Research, project no. 99-04-48490. REFERENCES Aleksandrova, M.A., Differentiation of Embryonic Neocortex upon Transplantation in Rats, Byull. Eksper. Biol. Med., 1998, vol. 126, append. 1, pp. 88–92. Aleksandrova, M.A., Mechanisms of Neural Tissue Differentiation and Cell Interactions upon Neurotransplantation in Mammals, Doctoral (Biol.) Dissertation, Moscow: Inst. Biol. Razv. Ross. Akad. Nauk, 1999. Aleksandrova, M.A. and Girman, S.V., Correlation between Morphological and Electrophysiological Characteristics of Neocortical Transplants Placed in the Optic Cortex of Adult Rats, Dokl. Ross. Akad. Nauk, 1995, vol. 340, no. 5, pp. 705– 708. Aleksandrova, M.A. and Kozlova, E.N., Differentiation of Neural and Glial Cells upon Transplantation of Embryonic Neural Tissue, Transplantatsiya tkani mozga v biologii i

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BIOLOGY BULLETIN

Vol. 28

No. 6

2001