Human fetal cortical and striatal neural stem cells generate region-specific neurons in vitro and differentiate extensively to neurons after intrastriatal transplantation in neonatal rats

Journal of Neuroscience Research 84:1630–1644 (2006)

Human Fetal Cortical and Striatal Neural Stem Cells Generate Region-Specific Neurons In Vitro and Differentiate Extensively to Neurons After Intrastriatal Transplantation in Neonatal Rats There´se Kallur,1,3 Vladimer Darsalia,1,3 Olle Lindvall,2,3 and Zaal Kokaia1,3* 1 Laboratory of Neural Stem Cell Biology, Section of Restorative Neurology, Stem Cell Institute, University Hospital, Lund, Sweden 2 Laboratory of Neurogenesis and Cell Therapy, Section of Restorative Neurology, Wallenberg Neuroscience Center, University Hospital, Lund, Sweden 3 Lund Strategic Research Center for Stem Cell Biology and Cell Therapy, Lund, Sweden

Human fetal brain is a potential source of neural stem cells (NSCs) for cell replacement therapy in neurodegenerative diseases. We explored whether NSCs isolated from cortex and striatum of human fetuses, aged 6–9 weeks post-conception, maintain their regional identity and differentiate into specific neuron types in culture and after intrastriatal transplantation in neonatal rats. We observed no differences between cortex- and striatum-derived NSCs expanded as neurospheres in proliferative capacity, growth rate, secondary sphere formation, and expression of neural markers. After 4 weeks of differentiation in vitro, cortical and striatal NSCs gave rise to similar numbers of GABAergic and VMAT2- and parvalbumin-containing neurons. However, whereas cortical NSCs produced higher number of glutamatergic and tyrosine hydroxylase- and calretininpositive neurons, several-fold more neurons expressing the striatal projection neuron marker, DARPP-32, were observed in cultures of striatal NSCs. Human cortical and striatal NSCs survived and migrated equally well after transplantation. The two NSC types also generated similar numbers of mature NeuN-positive neurons, which were several-fold higher at 4 months as compared to at 1 month after grafting. At 4 months, the grafts contained cells with morphologic characteristics of neurons, astrocytes, and oligodendrocytes. Many of neurons were expressing parvalbumin. Our data show that NSCs derived from human fetal cortex and striatum exhibit region-specific differentiation in vitro, and survive, migrate, and form mature neurons to the same extent after intrastriatal transplantation in newborn rats. VC 2006 Wiley-Liss, Inc.

and functional recovery. Clinical trials with intrastriatal transplantation of human fetal mesencephalic tissue, rich in postmitotic dopaminergic neurons, in Parkinson patients have provided proof-of-principle that neuronal replacement can work also in the human brain (Lindvall and Bjorklund, 2004). There is also some evidence that neuronal replacement using human fetal striatal tissue is possible and may give symptomatic relief in Huntington patients (Bachoud-Levi et al., 2000). These promising results support neuronal replacement as a potential future therapeutic strategy, but limitations in availability and difficulties in standardization severely restrict the usefulness of human fetal tissue for transplantation. The major challenge for the further development of neuronal replacement therapies for brain diseases is the identification of reliable sources of easily expandable cells having the capacity to generate those specific neuron types that are lost in the different disorders. In the past decade, self-renewing and multipotential neural stem cells (NSCs) have been isolated from developing and adult rodent CNS and expanded, both short and long term, in culture as freefloating aggregates known as neurospheres (Reynolds and Weiss, 1992, 1996; Svendsen et al., 1997, 1998; Carpenter

Key words: cortex; striatum; differentiation; migration; survival; graft; neurosphere

*Correspondence to: Zaal Kokaia, Lund Strategic Research Center for Stem Cell Biology and Cell Therapy, University Hospital BMC B10, Klinikgatan 26, SE-221 84 Lund, Sweden. E-mail: [email protected]

In animal models of human neurodegenerative diseases, cell transplantation can lead to neuronal replacement, partial reconstruction of damaged neural circuitry, ' 2006 Wiley-Liss, Inc.

T.K. and V.D. contributed equally to this article. Contract grant sponsor: Swedish Research Council; Contract grant number: EU project LSHBCT-2003-503005 (EUROSTEMCELL); Contract grant sponsor: King Gustav V and Queen Victoria Foundation; Contract grant sponsor: So¨derberg Foundation; Contract grant sponsor: Crafoord Foundation; Contract grant sponsor: Kock Foundation.

Received 3 May 2006; Revised 7 July 2006; 22 July 2006; Accepted 26 July 2006 Published online 16 October 2006 in Wiley InterScience (www. interscience.wiley.com). DOI: 10.1002/jnr.21066

Neural Stem Cells From Human Fetal Brain

et al., 1999; Vescovi et al., 1999). Neurospheres consist of a heterogeneous population of different cell types at various stages of lineage commitment. Cells within a neurosphere, which are responsive to the cytokines epidermal growth factor (EGF), basic fibroblast growth factor (FGF) or both, are able to survive and expand when supported by these growth factors under culture conditions (Reynolds and Weiss, 1996; Gritti et al., 1999, 2002; Vescovi et al., 1999, Reynolds and Rietze, 2005). Neural stem cells in neurosphere cultures derived from distinct subregions of the fetal rodent forebrain maintain certain aspects of their molecular specification and ability for differentiation even after expansion in vitro and intracerebral transplantation (Nakagawa et al., 1996; Eagleson et al., 1997; Zappone et al., 2000; Yamamoto et al., 2001; Hitoshi et al., 2002; Parmar et al., 2002; Horiguchi et al., 2004). In a clinical setting, cells of human origin are most likely needed for transplantation. Detailed characterization of the properties of human NSCs of different origins and their progeny is required before considering application in patients. Currently, the expansion capacity and differentiation potential of NSCs isolated from human fetal brain are incompletely understood. Horiguchi et al. (2004) reported region-specific differences for human fetal NSCs expanded as neurospheres in vitro. First, neurospheres from the rostral part of the brain showed higher proliferation rate than those from the caudal part. Second, NSCs from diencephalon and mesencephalon generated large, multipolar, tyrosine hydroxylase (TH)-immunoreactive neurons, whereas those from telencephalon and rhombencephalon gave rise to small, bipolar TH+ cells that were also GABA+. Ostenfeld et al. (2002) showed that neurosphere cells generated from different forebrain structures proliferated similarly, but that cells of cortical and striatal origin migrated more extensively and generated more neurons as compared to cells from other regions. It is unclear how much the region of origin, site of implantation, and characteristics of the host environment influence the survival, migration and differentiation of human fetal NSCs after transplantation. In previous studies, neurospheres were generated either from the whole forebrain and grafted in neonatal rats (Englund et al., 2002b) and mice (Uchida et al., 2000), or intact (Englund et al., 2002a) and lesioned CNS of adult rats (Svendsen et al., 1997; Kelly et al., 2004) and mice (Tamaki et al., 2002). Moreover, NSCs have been also isolated from human fetal cortex and implanted in hippocampus, striatum and substantia nigra of adult rats (Caldwell et al., 2001; Burnstein et al., 2004; Le Belle et al., 2004). To generate cortical and striatal neurons, which are the ones degenerating in Huntington’s disease, traumatic brain injury and ischemic stroke, we isolated NSCs from the cortex and striatum of aborted human fetuses and expanded them in vitro as neurospheres. The specific objectives were two-fold: first, to compare the ability of cortical and striatal NSCs to expand and on differentiation in vitro develop into region-specific neuronal phenotypes; and second, to explore to what extent the cortical and striatal NSCs survive at various time-points after intrastriatal transplantation into the striatum of newborn Journal of Neuroscience Research DOI 10.1002/jnr

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rats, migrate from the implantation site, and differentiate into neurons, astrocytes, and oligodendrocyte-like cells. MATERIALS AND METHODS Culturing of Human Neural Stem Cells Forebrain tissue from dead, aborted human fetuses aged 6– 9 weeks post-conception, was obtained from Lund and Malmo¨ University Hospitals according to the guidelines approved by Lund/Malmo¨ Ethical Committee. Cortical and striatal tissues were microdissected under a stereo microscope (Leica, Germany), incubated for 30 min in expansion medium at +378C, and then mechanically dissociated to single cell suspension. Expansion medium (DMEM/F12 [1:1; Gibco, Grand Island, NY], 2.92 g/ 100 ml L-glutamine, 23.8 mg/100 ml HEPES, 7.5% NaHCO3, 0.6% glucose, and 2% heparin [all from Sigma-Aldrich, St. Louis, MO]) contained N-2 supplement (1%; Gibco), human leukemia inhibitory factor (LIF; 10 ng/ml; Sigma-Aldrich), EGF, and FGF (20 ng/ml and 10 ng/ml, respectively; both from R&D Systems, Minneapolis, MN). Live cells were counted by trypan blue exclusion method before plating in culture flasks. Cells were maintained at +378C in a humidified atmosphere with 5% CO2. All experiments were carried out on one cortical and one striatal NSC line. Assessment of Neurosphere Formation and Size Neurospheres derived from human fetal cortex and striatum were passaged 11–15 times. To determine the capacity of the cells to form secondary spheres, the neurospheres were first passaged and then plated for 1 week. The newly formed neurospheres were randomly (in equal volumes) distributed in wells in a 24-well plate and the number of spheres in each well was counted (a sphere was considered an aggregate containing 5 cells or more). Digital images were acquired from all wells and the diameter of the spheres was measured using Adobe Photoshop (Adobe Systems Inc., San Jose, CA) software. The neurospheres were then transferred back to the culture flasks and the same procedure was repeated after 1 week. In Vitro Differentiation Small (50–75 lm in diameter) neurospheres were plated on poly-L-lysine-coated 4-well chamber slides. After 24 hr, the medium was changed to basic medium (without growth factors and heparin) containing 1% fetal bovine serum (FBS) (differentiation medium). Neurospheres were maintained under differentiation conditions for 1 day, or 2 or 4 weeks. Immunocytochemistry Free-floating, undifferentiated neurospheres or attached differentiated cultures were fixed in ice-cold 4% paraformaldehyde (with addition of 0.2% glutaraldehyde for glutamate and GABA stainings) for 15 min at room temperature followed by three rinses in potassium phosphate-buffered saline (KPBS). Neurospheres were spun down, and mounted in Tissue-Tek (Sakura Finetek Europe B.V., Zoeterwoude, Netherlands), frozen and cut on a cryostat in 9 lm thick sections. Before immunostaining, the neurosphere sections and differentiated cultures were pre-incubated in 5% normal serum and 0.025% Triton X-100 in KPBS for 45 min at room temperature. For staining of

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Fig. 1. Size (A–E) and growth rate (F) of human cortical and striatal NSCs in neurosphere cultures. Note similar appearance and growth of cortical and striatal neurospheres (A–D). Size is shown as sphere diameter (lm; E), and growth rate is expressed as cell numbers at sequential passages from Passage 3 up to Passage 16 (F). Data in (E) are means 6 SEM for 50 spheres per region. *P < 0.05, one-way ANOVA and Fischer’s post-hoc test. Scale bar ¼ 200 lm.

tissue sections, animals were deeply anaesthetized with sodium pentobarbital and perfused transcardially with saline followed by 4% ice-cold paraformaldehyde. Brains were post-fixed overnight and then kept in 20% sucrose solution until they sunk. Thirtyfive micrometer thick sections were cut in the coronal plane using a freezing microtome (Leica, Germany), and kept at
208C in cryo-protective solution. Incubation in primary antiserum was carried out overnight at room temperature. Primary antibodies were: rabbit anti-nestin (1:1,000, provided by Dr. R.G. McKay, NIH, Bethesda, MA), mouse-anti human cytoplasm (SC121, 1:3,000, provided by Dr. N. Uchida, StemCells, Inc., Palo Alto, CA), mouse anti-bIII-tubulin (1:333, SigmaAldrich), rabbit anti-GFAP (1:500, DakoCytomation, Glostrup, Denmark), mouse anti-GFAP (1:400, DakoCytomation), mouse anti-Ki67 (1:200, Novocastra, Newcastle, UK), goat anti-DCX (1:200, Santa Cruz Biotechnologies, Santa Cruz, CA), rabbit anti-VMAT2 (1:500, Chemicon, Temecula, CA), mouse antiTH (1:200, Chemicon), goat anti-calretinin (1:200, Chemicon), rabbit anti-parvalbumin (1:1,000, provided by Prof. P. Emson, Babraham Institute, Cambridge, UK), rabbit anti-GABA (1:2,000, Sigma-Aldrich), rabbit anti-glutamate (1:100, Chemicon), mouse anti-DARPP-32 (1:200, provided by Dr. P. Greengard), biotinylated mouse anti-NeuN (1:100 Chemicon), and mouse anti-human nucleus (HuNu, 1:100 Chemicon). Primary antibodies were detected by using appropriate fluorescent (Cy3) or biotin-conjugated secondary antibodies (1:200). Biotin-conjugated antibodies were detected with Alexa 488-conjugated streptavidin (1:200, Molecular Probes, Eugene, OR). For

double labeling, only one biotinylated secondary antibody was used at a time. With two monoclonal antibodies (NeuN and HuNu), sections were first incubated with HuNu antibody followed by Cy3-conjugated secondary antibody. After consecutive rinsing, sections were then incubated with biotinylated NeuN antibody followed by Alexa-488 conjugated streptavidin. Sections were mounted on gelatin-coated slides and both sections and cultures were cover-slipped with PVA (DABCO) mounting medium. For nuclear staining of cultured cells, 10 mg/ml Hoechst 33342 (Molecular Probes) was added during final incubation with secondary antibodies. Preparations for Transplantation Small (&50 lm in diameter) cortical and striatal nedrospheres were infected with recombinant VSV-G pseudotyped retrovirus (RV) carrying enhanced green fluorescent protein (GFP) under control of CMV promoter (transfer plasmid was pLNIT/GFP (van Praag et al., 2002). Virus was removed from medium in the flasks 24 hr after infection and 24 hr later, expression of GFP protein was already detectable in individual cells. At the day of transplantation, neurospheres (diameter 100 lm) were centrifuged and re-suspended in HBSS (Gibco) to reach a final concentration of about 100,000 cells/ll. As control for cell density, the number of cells in 1 ll of neurosphere solution was counted by the trypan blue exclusion method. The neurosphere suspension was kept on ice during the whole transplantation procedure. Journal of Neuroscience Research DOI 10.1002/jnr

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Transplantation in Neonatal Rats Twenty-two neonatal (postnatal days 2–3) Sprague-Dawley rats (Scanbur BK AB, Sollentuna, Sweden) were anaesthetized by lowering body temperature to +1
38C. Animals were placed in cooled Cunningham’s stereotaxic frame and scull surface was aligned in the horizontal plane. The neurosphere suspension (1.5 ll) was injected unilaterally using a Hamilton syringe into the right striatum at the following coordinates: 0.5 mm posterior and 2 mm lateral from bregma and 3 mm from brain surface. After injection, the needle was kept in place for 5 min. The wound was closed with surgical suture and animals were resuscitated by slowly increasing body temperature to normal level. Litters were kept together with mother during weaning period and were housed under 12 hr light–12 hr dark cycle with ad lib access to food and water. Because survival of human-derived cells in neonatal rodent brain usually is very good without signs of rejection, rats were not immunosuppressed. Experiments were conducted according to polices on the use of animals of the Society for Neuroscience and guidelines approved by Lund/Malmo¨ Ethical Committee. Twelve pups received neurospheres from cortex and 10 pups from striatum. The rats were perfused at 1 (n ¼ 7) and 4 (n ¼ 15) months after transplantation. Quantification and Statistical Analysis Cell counting was carried out using a computerized setup for stereology driven by the Computer Assisted Stereological Toolbox (C.A.S.T.-GRID) software (Olympus, Denmark) using a 403 objective. A CCD IRIS color video camera displayed the acquired images from the epifluorescence microscope (Olympus BX-61) live on a monitor screen. Counting frame areas and stepping distances were chosen to sample approximately 100–200 cells per well. Each well was evaluated separately, counting both total cell number by Hoechst nuclei stain, and cells immunopositive for the protein of interest. Counting of grafted cells located in the striatum was carried out in all brain slices with visible grafts under 403 objective. Double immunoreactivity was later confirmed by laser-scanning confocal microscopy. One-way ANOVA followed by Fischer post-hoc test were used to assess differences between groups. Data are expressed as means 6 SEM and differences considered significant at P < 0.05.

RESULTS Proliferation of Human Cortical and Striatal NSCs Grown as Neurospheres In Vitro In 1 week, both human cortical and striatal NSCs, when plated as single cells in growth medium supplemented with EGF, FGF, and LIF, formed spheres with a diameter of about 80 lm (Fig. 1A,B,E). At 2 weeks after plating, the size of the spheres almost doubled (Fig. 1C–E), reaching a diameter of about 160 lm. Thus, the human cortical and striatal neurospheres were growing with similar speed (Fig. 1E). To build proliferation curves between passages 3 and 16 (Fig. 1F), cortical and striatal neurospheres triturated to single cell suspensions were plated with the same density (100,000 cells/ml), based on counting of living cells with the trypan blue exclusion method. After 3– 4 weeks, neurospheres were harvested and again triturated Journal of Neuroscience Research DOI 10.1002/jnr

Fig. 2. A-F: Photomicrographs of 2–3-week-old human cortical (A– C) and striatal (D–F) neurospheres sectioned and double-immunostained with antibodies against the NSC marker nestin and the mitosis marker Ki67, and counterstained with the nuclear marker Hoechst. Note that in both cortical and striatal neurospheres virtually all cells express nestin (A and D, green) and many cells are also positive for the proliferation marker Ki67 (B and E, red); these cells are distributed evenly throughout the spheres. G–N: Photomicrographs of cortical and striatal NSCs plated for 1 day (G–J) or differentiated for 4 weeks (K–N) and stained with Ki67 (G,I,K,M) and counterstained with Hoechst (H,J,L,N). O: Number of proliferating, Ki67+ cells in the human cortical and striatal NSC cultures expressed as percentage of total number of Hoechst+ cells. NSCs were either plated for 1 day, or differentiated for 2 and 4 weeks. Data are expressed as means 6 SEM. *P < 0.05, one-way ANOVA and Fischer’s post-hoc test. Scale bar ¼ 50 lm (A–F); 20 lm (G–N).

into single cell suspensions and re-counted. The proliferation curves confirmed that the human cortical and striatal NSCs were expanding with the same velocity. The selfrenewing capacity of the cortical and striatal NSCs in the

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Fig. 3. Photomicrographs of human cortical and striatal neurospheres double-immunostained with nestin (A–L, green) and vimentin (A–D, red), GFAP (E–H, red), or bIII-tubulin (I–L, red). The insets in (A,B,E,F,I,J) are enlarged in (C,D,G,H,K,L), respectively. Arrows indicate double-labeled cells and arrowheads depict single-labeled cells. Scale bar ¼ 50 lm for neurospheres; 10 lm for magnified insets.

cultures was also evaluated by counting the number of spheres generated both 1 week and 2 weeks after plating at defined density. The number of secondary spheres was the same for cortical and striatal NSCs, and did not differ between one (622 6 205 and 669 6 79, respectively; n ¼ 10) and 2 weeks (389 6 139 and 740 6 95, respectively; n ¼ 10). To determine the spatial location of the proliferating cells, the cortical and striatal neurospheres were collected at 2 weeks after passage, sectioned, and immunostained with antibodies against the neural stem cell and mitotic markers, nestin and Ki67, respectively (Fig. 2A–F). The vast majority of cells in the neurospheres were positive for nestin (Fig. 2A,D). Cells expressing Ki67 were evenly distributed with no apparent differences between cortical and striatal neurospheres (Fig. 2B,C,E,F). To assess whether cortical and striatal NSCs stop proliferation under differentiation conditions, neurospheres were seeded onto coated chamber-slides with medium supplemented with 1% FBS

but without EGF, FGF, and LIF. After 1 day of differentiation, both cortical and striatal NSCs cultures contained similar proportion of Ki67+ cells (about 30–35%) (Fig. 2O). At 2 weeks, the proportion of Ki67+ cells had decreased significantly in both cultures but most markedly in striatal cultures (to 17% and 9% in cortical and striatal cultures, respectively). At this time point, the number of proliferating cells in the striatal cultures was significantly lower than in cortical cultures. After 4 weeks of differentiation, the proportion of proliferating Ki67+ cells in striatal cultures was unchanged, whereas it had decreased further in cortical cultures, reaching the same level as in striatal cultures (Fig. 2O). In both cortical and striatal cultures, Ki67+ cells were found within or in close proximity to the attached neurospheres after one day of differentiation, whereas 2 weeks and especially 4 weeks later, proliferating cells had migrated out from the neurospheres and were distributed randomly throughout the whole chamber area. Journal of Neuroscience Research DOI 10.1002/jnr

Neural Stem Cells From Human Fetal Brain

Fig. 4. Photomicrographs of human cortical (A–F) and striatal (G–L) NSCs at 1 day (A,C,E,G,I,K) and 4 weeks (B,D,F,H,J,L) after plating under differentiation conditions and stained with antibodies against nestin (A,B,G,H), GFAP (E,F,K,L) and bIII-tubulin (C,D,I,J). Number of nestin+ (M), bIII-tubulin+ (N), and GFAP+ (O) in cortical and striatal NSC cultures are expressed as percentage of total number of Hoechst+ cells after 1 day, and 2 and 4 weeks of differentiation. Data are given as means 6 SEM. *P < 0.05, one-way ANOVA and Fischer’s post-hoc test. Scale bar ¼ 20 lm.

Journal of Neuroscience Research DOI 10.1002/jnr

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Phenotype of Cells in Human Cortical and Striatal Neurospheres The neurospheres were cryosectioned and stained for nestin, vimentin, GFAP, and bIII-tubulin. Neurospheres of both cortical and striatal origin were found to contain different cell types, expressing more than one of the cellspecific markers. Virtually all cells were nestin+ without any regional differences within the neurospheres or between cortical and striatal neurospheres (Fig. 3). Nearly all of the nestin+ cells also expressed vimentin (Fig. 3A–D). Many cells in the cortical and striatal neurospheres were GFAP+ and co-expressed nestin (Fig. 3E–H). Interestingly, in every cortical and striatal neurosphere examined, a few cells expressing the neuron-specific marker bIIItubulin were found. These cells exhibited a somewhat differentiated neuronal morphology and were never located in the periphery of the spheres (Fig. 3I–L). Differentiation of Human Cortical and Striatal Neurospheres At 1 week after passage, the neurospheres were plated on coated chamber-slides in medium without growth factors and LIF and supplemented with 1% FBS. Neurospheres were allowed to attach to the surface and fixed and immunostained one day later. Quantification of the phenotype in the cortical and striatal cultures at this time point showed high numbers of nestin+ and GFAP+ cells, whereas very few cells were immunoreactive for bIII-tubulin (Fig. 4). These numbers were in good agreement with the observations in the sectioned neurospheres. Interestingly, the bIII-tubulin+ cells in the cortical and striatal neurosphere cultures exhibited typical neuronal morphology already after 1 day of differentiation, being predominantly bipolar with well-developed neurites (Fig. 4). At 2 weeks of differentiation, the number of nestin+ cells decreased in both cortical and striatal cultures. A similar reduction was observed in the number of GFAP+ cells. In contrast, the number of bIII-tubulin+ cells increased in cortical (to 18.4 6 1.3%) as well as striatal (to 20.6 6 1.8%) cultures (Fig. 4). At this time point, cortical and striatal cultures also contained a small number of cells (3.1 6 0.7 and 3.4 6 0.2%, respectively) immunoreactive for doublecortin (DCX), a marker of migrating neuroblasts (Fig. 5). After 4 weeks of differentiation, the number of nestin+ cells was further decreased in cortical cultures but in striatal cultures remained at the same level as at 2 weeks. The number of GFAP+ cells was lower at 4 weeks in both cortical and striatal cultures and represented only 9.9 6 1.7 and 9.2 6 1.4%, respectively, of the total number of cells. At this time point, the GFAP+ cells had mature astrocytic morphology with long processes and relatively large, round nuclei (Fig. 4F,L). The number of bIII-tubulin+ cells in the cortical cultures at 4 weeks did not differ compared to 2 weeks, whereas in the striatal cultures, we observed a further increase of bIII-tubulin+ cells, which were now more abundant in the striatal than cortical cul-

Fig. 5. Photomicrographs of human cortical (A,C) and striatal (B,D) NSCs at 2 weeks after plating under differentiation conditions and double-stained with antibody against doublecortin (DCX) (A,B) and Hoechst (C,D). Scale bar ¼ 20 lm.

tures. The differentiated bIII-tubulin+ cells in the cortical and striatal cultures exhibited a mature neuronal morphology, almost exclusively bipolar. The cells completely covered the surface, forming an extensive network of dendrites and axons (Fig. 4D,J). Phenotypic Expression in Long-Term Differentiated Cortical and Striatal Neurospheres After 4 weeks of differentiation, cells in cortical and striatal cultures were stained with antibodies against GABA, glutamate, and DARPP-32, a marker for striatal projection neurons. The majority of neurons in both cortical and striatal cultures were of bipolar, GABAergic phenotype (Fig. 6A,B,G). Virtually all GABAergic cells co-expressed bIII-tubulin (data not shown). Unfortunately, it was not possible to carry out double immunostainings with bIII-tubulin and glutamate or DARPP-32. Interestingly, cortical NSCs gave rise to significantly more glutamate+ cells as compared to striatal NSCs (Fig. 6C,D,H). Conversely, we found 3.6 6 0.4% DARPP32+ cells in striatal cultures but detected only few such cells in cortical cultures (Fig. 6E,F,I). About 10% of the differentiated cells showed immunoreactivity to the neuronal markers parvalbumin and VMAT2 (Fig. 7A–D) with no differences between the cortical and striatal cultures. Small numbers of differentiated neurons were calretinin+, being more abundant in cortical as compared to striatal cultures (Fig. 7E,F). Some of the cortical, but not striatal VMAT2+ neurons coexpressed TH (Fig. 7G–J). Taken together, these data indicate that human fetal cortical and striatal NSCs maintain the capacity to generate multiple types of neurons in vitro, and retain some region-specificity despite the fact that they have been expanded as neurospheres for over 1 year. Journal of Neuroscience Research DOI 10.1002/jnr

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Fig. 6. Photomicrographs of human cortical (A,C,E) and striatal (B,D,F) NSCs at 4 weeks after plating under differentiation conditions and stained with antibodies against GABA (A,B), glutamate (C,D) and DARPP-32 (E,F). Arrows depict examples of immunopositive cells. Numbers of GABA+, glutamate+ and DARPP-32+ cells in cortical and striatal NSC cultures are expressed as percentage of total number of Hoechst+ cells at 4 weeks of differentiation. Data are given as means 6 SEM. *P < 0.05, one-way ANOVA and Fischer’s post-hoc test. Scale bar ¼ 20 lm.

Survival and Migration of Grafted Human Cortical and Striatal NSCs We transplanted 1–2-week-old neurospheres derived from human fetal cortex and striatum into the striatum of 2–3-day-old rat pups. Quantification of the total number of cells positive for HuNu, a marker of cells of human origin at 4 months after transplantation showed that 6–10% of the grafted cells had survived. We observed no differences between cortical and striatal neurospheres in cell survival, migration from the implantation site (up to 1 mm rostrally and 1.5 mm caudally) (Fig. 8), or distribution in forebrain structures. After 4 months, most HuNu+ cortical and striatal NSCs were located throughout striatum (42% and 61%, respectively), whereas others had migrated to corpus callosum (39% and 25%), cerebral cortex (3% and 4%), or globus pallidus (16% and 10%). Differentiation of Human Cortical and Striatal NSCs After Intracerebral Transplantation All quantification of the phenotype of grafted NSCs was carried out in the striatum of host brain. To assess the number of undifferentiated cells in the grafts, sections were stained with an antibody specific for human nestin, not cross-reacting with rodent nestin. At 1 month after implantation, the nestin+ cells represented about 30% of all HuNu+ cells (Fig. 9A–D). No difference was observed between animals implanted with cortical and striatal neurospheres. At 4 months after implantation, the number of Journal of Neuroscience Research DOI 10.1002/jnr

HuNu+/nestin+ cells had decreased to 21.5 6 4.4% in human striatal NSC implanted animals, whereas in animals grafted with human cortical NSCs, the number of HuNu+/ nestin+ cells remained at the same level as at 1 month. At 1 month after implantation, 44.6 6 5.3% of HuNu+ cells in striatum co-expressed DCX (Fig. 9E–H), 10.3 6 3.6% were also positive for GFAP (Fig. 9I–L) and 10.0 6 2.0% were immunoreactive for the mature neuronal marker NeuN (Fig. 9M–P). At 4 months, the number of HuNu+/DCX+ cells had decreased 9-fold and, at the same time, the number of HuNu+/NeuN+ cells was increased 6-fold. The number of HuNu+/GFAP+ cells did not change between 1 and 4 month after transplantation (Fig. 9L). We did not observe any significant differences between the grafted cortical and striatal NSCs in the generation of DCX+, NeuN+, and GFAP+ cells. Although the neurospheres were retrovirally labeled with a GFP reporter gene, at 1 and 4 months after transplantation, only few HuNu+ cells were still expressing GFP. Some GFP+ cells in striatum had morphology characteristic of mature neurons, with long branching processes and visible dendritic spines (Fig. 10I–K), or of astrocytes and oligodendrocytelike cells (Fig. 10H). To further characterize the morphological profiles of the grafted cells, sections were stained with an antibody specific to human cytoplasm (SC121). The SC121 antibody has been used previously to detect human NSCs isolated from 16–21-week-old fetal forebrains and grafted in stroke-lesioned rat cortex (Kelly et al., 2004) and injured spinal cord of NOD-SCID mice

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(Cummings et al., 2005). This antibody was reported to label >90% of human neural cells and not to cross-react with rat cells (Kelly et al., 2004). Because both HuNu and SC121 are mouse monoclonal antibodies, we were not able to carry out reliable double-immunostaining. Counting of cells immunopositive to these markers in separate stainings showed that under our experimental conditions, the number of SC121+ cortical and striatal NSCs corresponded to about 25–30% of the HuNu+ cells. The SC121+ cells displayed morphologic characteristics of migrating neuroblasts (Fig. 10B), or mature neurons (Fig. 10D,E) and astrocytes (Fig. 10F). There were no clear differences in the number of cells with specific morphological features between cortical and striatal NSCs at the two time-points after intrastriatal transplantation. After 1 month, 42–71% of SC121+ cells had the morphology of migrating or undifferentiated cells. About 1–3% of cells had astrocytelike appearance with multiple branches symmetrically arising from the soma, and 27–54% of SC121+ cells exhibited neuronal morphology. At 4 months after transplantation, the percentage of SC121+ cells with migrating or undifferentiated morphology had decreased to 24–43%, whereas the proportion of astrocytes and neurons was increased (to 6–9% and 48–69%, respectively). No differences in the total number of SC121+ cells were observed between 1 and 4 months. To characterize the phenotype of the differentiated human neurons, sections were double-stained for SC121 and parvalbumin (a marker for a subpopulation of GABAergic interneurons). The majority (90.0 6 5.1%) of SC121+ neurons were co-labeled with parvalbumin (Fig. 9Q–S). The soma size of human cortical and striatal NSC-generated parvalbumin+ cells was somewhat smaller with fewer neurites as compared to parvalbumin+ interneurons in rat brain (Fig. 9R). We did not observe any differences in percentage of SC121+/parvalbumin+ neurons between rats grafted with cortical and striatal NSCs. At 4 months after transplantation, the percentage of SC121 and parvalbumin co-expressing cells remained the same as at 1 month. DISCUSSION We show that human NSCs, isolated from two distinct subregions of the fetal forebrain, cortex and striatum, have similar capacity to expand in neurosphere cultures without senescence for an extended period of time and, on differentiation in vitro, develop into region-specific neuronal phenotypes. We also show that the human NSCs of cortical and striatal origin survive and migrate to the same extent after intrastriatal transplantation into newborn rats. The grafted NSCs generate neuroblasts, which gradually develop into mature neurons, as well as astrocytes and oligodendrocyte-like cells with no clear differences related to the region of origin. Neural stem cells derived from the human fetal brain have previously been expanded in vitro in the presence of EGF, FGF, and LIF (Carpenter et al., 1999; Horiguchi et al., 2004; Tarasenko et al., 2004). We observed no difference between our cortical and striatal NSC lines as

Fig. 7. A–F: Photomicrographs of human cortical (A,C,E) and striatal (B,D,F) NSCs at 4 weeks after plating under differentiation conditions and stained with antibodies against parvalbumin (A,B), VMAT2 (C,D) and calretinin (E,F). Note that approximately 10-fold more differentiated cortical NSCs were positive for calretinin as compared to striatal NSCS. G-J: Photomicrographs showing differentiated human cortical NSCs positive for VMAT2 (G) or TH (H), as well as coexpression of VMAT2 and TH (I), and distribution of cells with nuclear marker Hoechst (J). Arrow indicates double-labeled cell and arrowheads single-labeled cells. Scale bar ¼ 20 lm.

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Fig. 8. Number and rostro-caudal distribution of human cortical and striatal NSCs at 4 months after intrastriatal transplantation in newborn rats. Top panel illustrates the coronal levels at which counting of cells stained with human specific antibody (HuNu) was carried out. The NSCs were implanted at level ‘‘0.’’ Data are expressed as means 6 SEM.

regard proliferation capacity in vitro assessed by neurosphere size and total number of living cells in the neurospheres. In accordance, Ostenfeld et al. (2002) found no significant differences in [3H] thymidine uptake between neurospheres generated from human cortex, striatum, thalamus, ventral mesencephalon, and cerebellum. Virtually all cells in both the cortical and striatal neurospheres expressed the stem/progenitor cell markers nestin and vimentin (Hockfield and McKay, 1985; Lendahl et al., 1990; Liem, 1993; Go¨tz et al., 1998). A high proportion of the neurosphere cells were also GFAP+, coexpressing nestin. This finding agrees well with the notion that GFAP expression identifies stem cells in the adult subventricular zone (Doetsch et al., 1999), and shows that the vast majority of cells in the human neurospheres of both cortical and striatal origin are immature stem/progenitor cells. We also detected a population of bIII-tubulin+ cells, indicating some neuronal differentiation within the neurospheres. In contrast, Svendsen et al. (1998) found no Tuj1 immunoreactive cells within growing human neurospheres. Our data are consistent with those in a previous study (Parmar et al., 2002) in which bIII-tubulin+ cells were observed within rodent-derived neurospheres. Taken together, these findings provide further evidence that cells in human-derived neurospheres are heterogeneous and at different levels of commitment (Svendsen and Caldwell, 2000; Martinez-Serrano et al., 2001). Differentiation by removal of EGF and FGF and addition of FBS lead to a gradual increase of bIII-tubulin+ cells and a concomitant reduction of the numbers of nestin Journal of Neuroscience Research DOI 10.1002/jnr

positive cells. The number of GFAP+ cells also decreased dramatically over time, most likely reflecting that GFAP immunoreactivity depicted progenitor cells (Doetsch et al., 1999) that underwent maturation. After 1 day of differentiation, the cells had not acquired any specific morphology and the GFAP+ cells probably comprised both committed glia cells and uncommitted progenitors. In contrast, after 2 and 4 weeks of differentiation, the GFAP immunoreactive cells had astrocytic morphology. The marked increase in bIII-tubulin+ cells and the slight decrease in GFAP+ cells between 2 and 4 weeks of differentiation could suggest that part of the remaining pool of undifferentiated progenitor cells in the cortical and striatal cultures became lineage restricted during this time period, preferentially adopting a neuronal rather than a glial fate. Cerebral cortex normally contains mainly glutamatergic pyramidal neurons as well as GABAergic interneurons, which constitute 25–30% of all cortical neurons (Jones, 1993). The majority of striatal neurons are medium-sized spiny projection neurons, which are positive for DARPP32 (Deacon et al., 1994; Olsson et al., 1998) and produce the inhibitory neurotransmitter GABA. Striatum contains no glutamatergic neurons but several types of interneurons, which are either cholinergic or GABAergic and contain parvalbumin, calretinin, somatostatin, neuropeptide Y or neuronal nitric oxide synthase (Kawaguchi et al., 1995). After 4 weeks of in vitro differentiation, the NSCs derived from cerebral cortex had generated a population of glutamate-containing neurons. Most neurons were GABAergic and a significant number expressed calretinin. The presence

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Fig. 9. A–P: Photomicrographs of confocal images with orthogonal reconstruction of human striatal (A–C and E–G) and cortical (I–K and M–O) NSCs at 4 months after intrastriatal transplantation in newborn rats. Cells were double-immunostained with antibodies against human-specific antibody HuNu (A,E,I,M), and nestin (B), DCX (F), GFAP (J), or NeuN (N). Number of nestin+ (D), DCX+ (H), GFAP+ (L) and NeuN+ (P) cells are expressed as percentage of total number of HuNu+ grafted cells at 1 and 4 months after intracerebral

transplantation in newborn rats. Data are expressed as mean 6 SEM. *P < 0.05, one-way ANOVA and Fischer’s post-hoc test. Q–S: Photomicrographs of grafted human striatal cells showing SC121 (Q) and parvalbumin (R) immunoreactivity separately or as merged image (S). Arrows depict a SC121+/parvalbumin+ cell of human origin and arrowheads indicate either a SC121-/parvalbumin+ interneuron of the host brain or a grafted human SC121+/parvalbumin- cell. Scale bar for (Q,R,S) ¼ 40 lm. Journal of Neuroscience Research DOI 10.1002/jnr

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Fig. 10. Photomicrographs of human striatal NSCs at 4 months after intrastriatal transplantation in newborn rats and stained with the human-specific antibody SC121 (A–F) or exhibiting GFP autofluorescence (G–K). B,D,E,K: Enlargements of corresponding areas indicated by squares on (A) and (J). A: Overview of the implantation site showing distribution of grafted cells. Some cells are exhibiting morphology of neuroblasts (B,G), oligodendrocyte-like cells (C,H), mature neurons (D,E,I,J) and astrocytes (F). Note the well-developed spines on (K). Scale bar A ¼ 300 lm (A); 20 l (B–J); 5 lm (K).

of a low number of glutamatergic neurons also in the striatal cultures was due probably to inclusion of overlying fetal cortical tissue during dissection, and subsequent expansion and differentiation of a small subset of cortical NSCs (Parmar et al., 2002). However, our finding also raises the possibility that striatal NSCs, at least in vitro, can differentiate into glutamatergic neurons by some unknown mechanism. Journal of Neuroscience Research DOI 10.1002/jnr

The NSCs derived from striatum generated significantly higher numbers of mature neurons expressing DARPP-32 as compared to cortical NSCs. Several-fold more cells were positive for GABA than for DARPP-32 in the striatal NSC cultures, indicating that only a subdivision of the GABA+ cells were medium spiny neurons. The remaining GABA+ cells were probably parvalbumin and calretinin+. Taken together, our findings provide evidence

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that the human striatal and cortical NSC lines have an intrinsic fate specification consistent with their region of origin. After 4 weeks of differentiation, both cortex- and striatum-derived NSCs expressed vesicular monoamine transporter-2 protein (VMAT2), which suggests the occurrence of catecholaminergic neurons. In support, the VMAT2+ cells generated by the cortical NSCs co-expressed the catecholamine-synthesizing enzyme TH. Carpenter et al. (1999) reported that human forebrain NSCs cannot express TH unless the hematopoietic cytokine IL-1b was added to the differentiation protocol. However, a more recent study (Horiguchi et al., 2004) indicated that forebrain NSCs can give rise to TH+ cells, but to much lesser extent than mesencephalic or rhombencephalic NSCs. In the adult human striatum, very few neurons are positive for TH (Cossette et al., 2005a) and virtually all of them are GABAergic (Cossette et al., 2005b). Although NSCs isolated from mouse embryonic striatum can generate a limited number of TH+ of cells in vitro (Daadi and Weiss, 1999), it is unknown whether human fetal striatum is also capable of producing TH+ neurons. Neurospheres isolated from cerebral cortex and striatum were transplanted into the neonatal rat striatum. Despite the ectopic location of the cortical NSCs, we observed no differences in their survival or migration within the striatum or to other forebrain structures as compared to the striatal NSCs. Also, the generation of DCX+ neuroblasts at 1 month and the subsequent differentiation to mature NeuN+ neurons at 4 months did not differ between the cortical and striatal NSCs. Thus, we obtained no evidence that the region of origin influenced the survival, migratory capacity, or neuronal maturation of the human fetal NSCs. We were not able to double stain the human mature SC121+ neurons with antibodies for the various subtypes of cortical and striatal neurons. Previous studies have also been unable to confirm double-labeling with markers for human neurons and either DARPP-32, calbindin or GAD67 in grafts of human NSCs implanted into striatum of neonatal or adult rats (Englund et al., 2002b; Parmar et al., 2003). We found that about 90% of the SC121+ neurons in both the cortical and striatal grafts were co-labeled with parvalbumin, which identifies a population of GABAergic interneurons. In the striatal environment surrounding the grafts, parvalbumin+ GABAergic neurons constitute only about 0.7% (Luk and Sadikot, 2001) of the total neuronal population, whereas >90% are medium-sized spiny neurons not containing parvalbumin (Kawaguchi et al., 1995). Thus, the phenotype of neurons generated by the human NSCs after transplantation into the neonatal rat brain did not reflect the normal neuronal composition in the host striatum. One explanation could be that the human cells are unable to respond to the differentiation signals in the rat striatum due to the xenograft situation. Alternatively, these signals may not be operating efficiently in the intact striatum. It is interesting in this context that after striatal injury in rats, the neuroblasts that are generated from endogenous NSCs in the subventricular zone migrate to the damaged area and mature to the

phenotype of most of the neurons which have died, i.e., those expressing DARPP-32 (Arvidsson et al., 2002). The SC121 antibody only stained a fraction (about 25–30%) of the human-derived grafted cells, as judged by the number of HuNu+ cells. Most likely, the SC121 antibody marked only a subpopulation of the HuNu+ cells. The difference between HuNu+/SC121+ and HuNu+/ SC121
cells and the reason why the antigen recognized by SC121 antibody is not expressed in the majority of HuNu+ cells are currently unclear. In contrast to our data, when cells were isolated from 16–21-week-old human forebrains, grown as neurospheres and transplanted in NOD-SCID mouse brains, virtually all (about 99%) SC121+ cells also expressed human nuclear marker (S.J. Tamaki, personal communication). One major hypothetical explanation is that the expression of the antigen recognized by SC121 is ontogeny-related. Also, the methods of isolation and in vitro expansion of the human NSCs, as well as the characteristics of the host brain environment may influence the expression of the SC121 antigene. In contrast to the HuNu antibody, which stains only the nucleus, SC121 is a cytoplasmic marker showing the morphology image of the immunopositive cell. Therefore, this antibody was particularly useful for demonstrating that grafted cortical and striatal NSCs not only expressed neuronal and glial markers but also differentiated into morphologically distinct cell types. Interestingly, the proportion of different types of cells detected by double-staining with HuNu and the phenotypical antibodies such as NeuN, GFAP and DCX at both 1 and 4 months after transplantation resembled to that of mature neurons, astrocytes and neuroblasts, respectively, based on morphologic classification of SC121+ cells. This indicates that SC121 does not discriminate between the different types of human neural cells and that its expression level is not changed over time. Both in vitro and after transplantation in vivo, a substantial proportion of the cells generated from both cortical and striatal NSCs expressed the neural progenitor marker nestin. Although there was a slight decrease of the percentage of nestin+ cells between 1 and 4 months post-transplantation in the grafts of striatal NSCs, >20% of the cells from both sources remained in the undifferentiated state at the later time-point. Our findings extend previous observations that a large fraction of grafts of neurospheres isolated from human fetal forebrain co-expressed nestin (Englund et al., 2002b). The population of nestin+ cells may lack fate commitment or be unable to respond to environmental cues in the cultures or the neonatal brain. Before any clinical application, it will be necessary to identify those factors that will make it possible to induce differentiation also of these nestin+ cells. It cannot be ruled out that the species differences in the present experiments may have influenced the fate of the human cells grafted in the rat brain. However, previous studies with human fetal mesencephalic tissue in Parkinson’s disease have documented the value of human-to-rat xenograft studies in predicting the outcome in human-tohuman transplantations. The grafted human fetal mesencephalic cells survived and differentiated into dopaminergic Journal of Neuroscience Research DOI 10.1002/jnr

Neural Stem Cells From Human Fetal Brain

neurons and improved neurological deficits after implantation in a rat model of Parkinson’s disease (Brundin et al., 1988; Clarke et al., 1988). These findings later became the basis for clinical trials in Parkinson’s patients demonstrating functional integration of grafted cells and improvement of impaired motor skills (Lindvall et al., 1989a,b; 1990a,b; Widner et al., 1991a,b). It is conceivable, therefore, that if human-derived NSCs survive and differentiate into neurons after transplantation in the rodent brain, that these cells will also integrate when grafted in the human brain. In conclusion, we show that NSC lines that have been generated from human fetal cerebral cortex and striatum exhibit regio-specificity in their neuronal differentiation in vitro and give rise to mature neurons after intracerebral transplantation. However, our data also point to two major problems from the perspective of a neuronal replacement strategy, namely the presence of a substantial proportion of undifferentiated cells in the NSC grafts, and the failure by direct intracerebral NSC implantation to generate the appropriate repertoire of neuron types required to repair a structure like the striatum in a neurodegenerative disorder. These findings indicate that in a possible future clinical scenario, controlled in vitro differentiation will be necessary, followed by cell sorting and transplantation of neurons and glia with specific phenotype into the diseased brain area. ACKNOWLEDGMENTS We thank M. Lundahl and U. Sparrhult-Bjo¨rk for technical help, Dr. N. Uchida for SC121 antibody, and Dr. F.H. Gage and Dr. H. van Praag for GFP retrovirus. The Lund Stem Cell Center is supported by a Center of Excellence grant in Life Sciences from the Swedish Foundation for Strategic Research. REFERENCES Arvidsson A, Collin T, Kirik D, Kokaia Z, Lindvall O. 2002. Neuronal replacement from endogenous precursors in the adult brain after stroke. Nat Med 8:963–970. Bachoud-Levi AC, Remy P, Nguyen JP, Brugieres P, Lefaucheur JP, Bourdet C, Baudic S, Gaura V, Maison P, Haddad B, Boisse MF, Grandmougin T, Jeny R, Bartolomeo P, Dalla Barba G, Degos JD, Lisovoski F, Ergis AM, Pailhous E, Cesaro P, Hantraye P, Peschanski M. 2000. Motor and cognitive improvements in patients with Huntington’s disease after neural transplantation. Lancet 356:1975–1979. Brundin P, Strecker RE, Widner H, Clarke DJ, Nilsson OG, Astedt B, Lindvall O, Bjo¨rklund A. 1988. Human fetal dopamine neurons grafted in a rat model of Parkinson’s disease: immunological aspects, spontaneous and drug-induced behavior, and dopamine release. Exp Brain Res 70: 192–208. Burnstein RM, Foltynie T, He X, Menon DK, Svendsen CN, Caldwell MA. 2004. Differentiation and migration of long term expanded human neural progenitors in a partial lesion model of Parkinson’s disease. Int J Biochem Cell Biol 36:702–713. Caldwell MA, He X, Wilkie N, Pollack S, Marshall G, Wafford KA, Svendsen CN. 2001. Growth factors regulate the survival and fate of cells derived from human neurospheres. Nat Biotechnol 19:475–479. Carpenter MK, Cui X, Hu ZY, Jackson J, Sherman S, Seiger A, Wahlberg LU. 1999. In vitro expansion of a multipotent population of human neural progenitor cells. Exp Neurol 158:265–278. Journal of Neuroscience Research DOI 10.1002/jnr

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Journal of Neuroscience Research DOI 10.1002/jnr