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High-level expression of functional chemokine receptor CXCR4 on human neural precursor cells
Developmental Brain Research 152 (2004) 159 – 169 www.elsevier.com/locate/devbrainres
Research report
High-level expression of functional chemokine receptor CXCR4 on human neural precursor cells Hsiao T. Nia, Shuxian Hub, Wen S. Shengb, Judy M. Olsona, Maxim C.-J. Cheeranb, Anissa S.H. Chana, James R. Lokensgardb, Phillip K. Petersonb,* b
a Stem Cell Group, R&D Systems, Inc., Minneapolis, MN 55413, USA Minneapolis Medical Research Foundation and the University of Minnesota Medical School, Minneapolis, MN 55404, USA
Accepted 6 June 2004 Available online 8 August 2004
Abstract Neural precursor cells (NPCs) are self-renewing, multipotent progenitors that give rise to neurons, astrocytes and oligodendrocytes in the central nervous system (CNS). Fetal NPCs have attracted attention for their potential use in studying normal CNS development. Several studies of rodent neural progenitors have suggested that chemokines and their receptors are involved in directing NPC migration during CNS development. In this study, we established a consistent system to culture human NPCs and examined the expression of chemokine receptors on these cells. NPCs were found to express the markers nestin and CD133 and to differentiate into neurons, astrocytes and oligodendrocytes at the clonal level. Flow cytometry and RNase protection assay (RPA) indicated that NPCs express high levels of CXCR4 and low levels of several other chemokine receptors. When examined using a chemotaxis assay, NPCs were able to respond to CXCL12/SDF-1a, a ligand of CXCR4. Treatment with anti-CXCR4 antibody or HIV-1 gp120 abolished the migratory response of NPCs towards CXCL12/SDF-1a. These findings suggest that CXCR4 may play a significant role in directing NPC migration during CNS development. D 2004 Elsevier B.V. All rights reserved. Theme: Development and regeneration Topic: Cell differentiation and migration Keywords: Neural precursor cell; Differentiation; Chemokine receptor
1. Introduction Chemokines and their cognate receptors were initially associated with the trafficking of leukocytes in physiological immune surveillance and with inflammatory cell recruitment in different diseases [45,48]. Subsequently, they have been found to play a critical role in hematopoietic development by regulating migration, proliferation, differentiation and survival of human and murine hematopoietic stem and progenitor cells [1,3,24,27,37,56]. The involve* Corresponding author. Department of Medicine, Medical School and Hennepin County Medical Center, University of Minnesota, 701 Park Avenue South, Minneapolis, MN 55415, United States. Tel.: +1 612 873 2877; fax: +1 612 904 4299. E-mail address: [email protected] (P.K. Peterson). 0165-3806/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.devbrainres.2004.06.015
ment of the chemokine/chemokine receptor family in central nervous system (CNS) development has recently been identified, but is still poorly understood and remains to be elucidated. Neural precursor cells (NPCs) are self-renewing, multipotent cells that give rise to neurons, astrocytes and oligodendrocytes in the CNS [5,10,33]. In recent years, in vitro methods have been developed which permit the expansion and differentiation of multipotent fetal NPCs in culture [5,7,19,43,44,50,54]. NPCs can be grown in the presence of mitogen, as either monolayer cultures or as free floating spherical aggregates termed dneurospheres.T Following expansion, NPCs can be induced to differentiate by withdrawal of mitogenic agent or by exposure of the cells to other factors that cause them to develop along specific lineages. While much work is required to characterize NPCs
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derived from in vitro systems, these systems have served as a valuable resource for studies of normal nervous system development. Much of our understanding of NPCs has been derived from animal models. Early studies of human NPCs suggest that, like rodent NPCs, they can be isolated from fetal tissues and propagated in culture [7,50]. However, results of these studies are preliminary, and in some cases, the findings appear to be divergent when compared to their rodent counterparts. Several studies using rodent neural progenitors have suggested the potential involvement of chemokines in directing NPC migration during CNS development [18,30,31,41,57]. In particular, the interaction of CXCR4 and its ligand CXCL12/SDF-1a has been strongly suggested to be important in neuronal patterning of cerebellum and hippocampus as well as astrocyte development in the rat brain [4,25]. In the present study, we established an in vitro system to consistently culture human NPCs derived from 8to 11-week-old fetal brain tissue. These in vitro propagated NPCs have demonstrated their capability for proliferation in culture while retaining the potential to produce highly differentiated descendants: neurons, oligodendrocytes and astrocytes. Expression of chemokine receptors on these human NPCs was examined by flow cytometry and RNase protection assay (RPA), and migratory activity was assessed by in vitro and in vivo systems.
2. Materials and methods 2.1. Cell preparation and culture 2.1.1. NPCs NPC cultures were prepared from the telencephalon of 8- to 11-week-old human fetal brain using previously described methods [52,53] with modifications. Human fetal brain tissues obtained under a protocol approved by the Human Subjects Research Committee at Hennepin County Medical Center were mechanically dissociated, resuspended in DMEM/F12 (Invitrogen, Carlsbad, CA) media (containing 8 mM glucose and glutamine [Sigma, St. Louis, MO], N2 plus supplement [R&D Systems, Minneapolis, MN], penicillin and streptomycin [Sigma] and 20 ng/ml human fibroblast growth factor-basic (hFGFb)/20 ng/ml human epidermal growth factor (hEGF) [R&D Systems]) and plated onto poly-O-lysine (Sigma) and bovine fibronectin (FN, R&D Systems) coated 10-cm tissue culture dishes or 24-well plates when indicated. This stage is considered as passage 0. When cell cultures reached 60% confluence, they were subcultured by trypsin (0.0125%, Sigma) at a density of 2105 cells per 10-cm culture dish and considered as passage 1. Medium was replaced every other day and hFGFb was supplemented daily. For studies of clonality, the method of Tsai and McKay [52] was modified for our purpose. Briefly, NPCs obtained from passage 0 were diluted to a single cell per well in 48-well culture
plates to give rise to a single clone (passage 1) for further experiments. NPCs from the single clone (passage 1) were plated onto one well of four-well chamber slides and after 1 week, were subjected to differentiation conditions (1% FBS, no hEGF/ hFGFb) for 3 weeks followed by immunocytochemical staining to identify differentiated neurons and astrocytes. 2.1.2. Highly enriched cortical neuronal cells Neuronal cultures were prepared as previously described [17]. In brief, 16-week-old fetal brain cortical tissues were dissociated after 30 min of incubation at 37 8C with trypsin (0.25%). After washing, cells were resuspended in DMEM (Sigma) containing 10% heat-inactivated fetal bovine serum (FBS, Hyclone, Logan, UT) with penicillin (100 U/ml) and streptomycin (100 Ag/ml). Dispersed cells were plated onto collagen-coated plates (5105, 106 or 3106 cells/well for 24-, 12- or 6-well plates, respectively) or four-well chamber slides (5105 cells/well). On day 5, cell cultures were treated with uridine (33.6 A/ml, Sigma) and fluorodeoxyuridine (13.6 Ag/ml, Sigma) followed by DMEM replacement with 10% heat-inactivated FBS at day 6 and every 4 days thereafter. By day 12, these neuronal cultures contain approximately 60–70% neurons (stained by anti-NeuN antibody, Chemicon, Temecula, CA), 20–30% astrocytes (stained by anti-GFAP antibody, Sternberger Monoclonals, Lutherville, MD) and 2–5% microglial cells (stained by anti-CD68, BD Pharmingen, San Diego, CA). Using specific antibodies to distinguish the different types of neurons, we found that human cerebrocortical neurons are comprised of approximately 20–30% cholinergic, 20–25% GABAergic, 20–30% glutamatergic and 15% unidentified neurons. To obtain greater enrichment of neurons, at day 6, the culture medium was replaced with neurobasal medium (Gibco, Carlsbad, CA) containing B-27 supplement (Gibco). By day 12, these highly enriched neuronal cultures contain approximately 90–95% neurons, 5–10% astrocytes and b2% microglia. 2.2. NPC proliferation assays Cells were plated onto 24-well plates at a density of 1102 cells per well with hFGFb/hEGF. At 0, 24, 72 and 120 h after plating, 3H-thymidine (Amersham, Piscataway, NJ) was added to the wells at 0.25 ACi per well and incubated for 8 h before being harvested and measured for 3 H-thymidine incorporation in a beta-counter. NPC proliferation was also assayed by 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT, Sigma) or alamarBluek (Serotec, Raleigh, NC) uptake by live cells. After adding MTT (final concentration of 1 mg/ml, 4 h) followed by lysis buffer (20% SDS [w/v] in 50% N,Ndimethyl formamide, pH 4.7 adjusted with 2.5% acetic acid and 1 N HCl [32:1]) (16 h), NPC cell lysates were collected and read in a microplate reader (Molecular Devices, Sunnyvale, CA) at 600 nm, or NPCs were read
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at 544 nm (excitation) and 590 nm (emission) after addition of alamarBluek (equal to 10% of the culture volume) for 16 h. 2.3. Flow cytometry Staining of cells with mouse anti-human CD133-phycoerythrin and anti-human chemokine receptor-phycoerythrin antibodies (R&D Systems) was performed according to the manufacturer’s suggested procedures. For nestin staining, cells were fixed with 4% paraformaldehyde in PBS at room temperature for 20 min and washed twice with PBS. After washing, cells were resuspended in SAP buffer (2% FCS, 0.5% saponin and 0.1% sodium azide in PBS), and mouse anti-human nestin or mouse IgG1 isotype control antibody (R&D Systems) was added at the final concentration of 10 Ag/ml to 2.5105 cells in a total reaction volume of 200 Al. Samples were then incubated for 20 min at room temperature. Following incubation, excess nestin or isotype control antibody was removed by washing cells twice with SAP buffer. After washing, cells were resuspended in 200 Al of SAP buffer and 1 Ag of goat anti-mouse IgG-FITC (Caltag, San Francisco, CA) was added. The samples were incubated for 20 min in the dark at room temperature, and cells were then washed once with SAP buffer, once with PBS and then resuspended in 400 Al of PBS and analyzed on a FACScan flow cytometer (BectonDickinson, Mountain View, CA). Five thousand events were collected and analyzed using CELL Quest software.
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2.5. RNase protection assay For assessments of chemokine receptor mRNA expression, total RNA was used in the RiboQuantk multiprobe RNase Protection Assay (RPA) according to the manufacturer’s instructions (BD Pharmingen). Total RNA was collected from NPCs with Qiagen RNeasy Mini kit (Qiagen, Valencia, CA). After hybridization with 32Plabeled template, samples were resolved on denaturing (5%) polyacrylamide urea gels (19:1 40% acrylamide/bis, 10 Tris–Borate–EDTA and urea) and analyzed with a phosphorimager (Molecular Dynamics, Sunnyvale, CA). 2.6. Chemotaxis assay NPCs were added to upper chambers of a 96-well chemotaxis device (Neuro Probe, Gaithersburg, MD) (106 cells/well) separated from the lower chambers with an 8-Am pore size of polyvinylpyrrolidone-free polycarbonate filter. The lower chambers were filled with chemoattractants. After 6 h of incubation, NPCs that had migrated from upper chambers into lower chambers were quantified by DiffQuik staining (Dade Diagnostics, Aguada, PR). To determine the effect of blockade of CXCR4, NPCs were treated with anti-CXCR4 antibody (10 Ag/ml) (R&D Systems) or human immunodeficiency virus type 1 (HIV-1) gp120 (10 9 and 10 8 M) (Protein Sciences, Meriden, CT) for 30 min before being used in chemotaxis assay towards CXCL12/SDF-1a.
2.4. Immunocytochemical staining
2.7. In vitro differentiation
Anti-nestin, A2B5 and Tuj1 antibodies (R&D Systems) were used at the concentration of 10 Ag/ml in fixed cells. Anti-GFAP antibody was used at 1:200 dilutions. Cells were fixed with 4% paraformaldehyde and 0.15% picric acid in PBS at room temperature for 20 min and were then permeated and blocked with 0.1% Triton X-100, 1% BSA and 10% normal donkey serum in PBS at room temperature for 45 min. After blocking, cells were incubated with diluted primary antibody overnight at 4 8C and sequentially with fluorescence-coupled anti-mouse IgG Ab (Jackson Laboratory, Bar Harbor, ME) at room temperature in the dark for 1 h. Between each step, cells were washed with 0.1% BSA in PBS. Anti-oligodendrocyte marker O4 antibody (R&D Systems) was used at a concentration of 1 Ag/ml. Rabbit anti-human GABA antibody (Sigma) was used at 1:10,000 dilutions. Cells were fixed with 4% paraformaldehyde in PBS at room temperature for 20 min and then blocked with 10% normal donkey serum and 1% BSA in PBS at room temperature for 45 min. After blocking, cells were incubated with diluted primary antibody overnight at 4 8C and sequentially with fluorescence-coupled anti-mouse IgM Ab (Jackson Laboratory) at room temperature in the dark for 1 h. Between each step cells were washed with 0.1% BSA in PBS.
Media of NPC cultures at passages 1–5 after 7 days of plating were replaced with 10 % FBS in DMEM/F-12 medium without hFGFb/hEGF. After 7 days, the differentiated NPCs were stained with anti-human NeuN, GFAP or O4 antibodies to identify differentiated neurons, astrocytes or oligodendrocytes, respectively. 2.8. In vivo differentiation To characterize in vivo differentiation of NPCs, cultured cells at passage 2 were transplanted into the subventricular zone (SVZ) and hippocampus of 8- to 10-week-old immunodeficient C.B.17 SCID/bg mice. Anesthetized mice were aligned on a stereotaxic apparatus (David Kopf Instruments, Tujunga, CA) equipped with a neonatal rat/ mouse adapter (Stoelting, Wood Dale, IL). Openings were created on the skull at predetermined coordinates relative to the bregma. NPCs were deposited into the left SVZ and right hippocampus using these coordinates: rostrocaudal vector (AP)=0 mm, interaural vector (mid)=+1.0 mm, dorsoventral vector (DV)= 3.0 mm and AP= 2.0 mm, mid= 0.8 mm, DV= 2.0 mm, respectively. All ventral coordinates were measured from the surface of the skull. Injections (1104 NPCs/2 Al) were administered using a 10-
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Al Hamilton syringe attached to the stereotaxic apparatus over a period of 2–3 min at each site. At days 10 and 21 post-transplant, animals were anesthetized and perfused with 4% paraformaldehyde. Mouse brains were fixed overnight in 4% paraformaldehyde and transferred into 30% sucrose solution for 48 h. Longitudinal brain sections (30 Am) were obtained by using a microtome (Bright Instrument, Huntingdon, England). To detect the transplanted human NPCs, brain sections were immunostained with specific anti-human nestin, neuron specific enolase (NSE, Dako, Carpinteria, CA) and GFAP antibodies. Immunohistochemical staining procedures were similar to immunocytochemical staining as described above.
3. Results 3.1. Generation of continuously proliferating NPCs NPCs derived from 8- to 11-week-old human fetal brain tissues were cultured and expanded in DMEM/F12 and N2 medium supplied with hEGF and hFGFb on FN-coated plates. Continuous proliferation of these cells was indicated by 3H-thymidine incorporation assay (Fig. 1A). The cultured cells were also examined at the clonal level. Single clones derived from single cells (one cell per well in a 48well plate) showed clonal proliferation. NPCs were plated at a density of 1103 cells per 10-cm culture dish and well-
Fig. 2. Expression of human NPC markers. NPCs obtained from 9-weekold fetal brain specimens were analyzed by immunocytochemical staining and flow cytometry for cellular markers characteristic of these cells. Immunocytochemical staining of undifferentiated NPCs by (A) anti-nestin, (B) anti-A2B5 (red) antibodies with nuclear staining with DAPI (blue) was shown. (C) PE-conjugated anti-human CD133 (green) or isotype-matched control (black) antibodies was used to analyze NPC by flow cytometry.
Fig. 1. Proliferation of human NPCs. NPCs (passages 1–3) obtained from 8- to 9-week-old fetal brain specimens were (A) temporally assayed by 3H-thymidine incorporation and photographed under phase-contrast microscopy at the clonal level at (B) day 1 and (C) day 4. Data presented in (A) are meansFS.E.M. of triplicates of three separate experiments.
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Fig. 3. Flow cytometry and immunocytochemical staining analysis of human NPCs. Human NPCs obtained from 9-week-old fetal brain specimens at passages 1, 3 and 5 were analyzed by flow cytometry: (A) plots displaying FSC versus SSC (R1 indicated the gated live population), (B, C) nestin-positive cells, and by (D) immunocytochemical double staining of nestin-positive (red) and BrdU-incorporated cells (green).
separated colonies were tracked. The proliferation of cultured cells was also demonstrated at the clonal level (Fig. 1B and C).
their ability to incorporate bromodeoxyuridine (BrdU) (Fig. 3D). The expression of nestin suggests that a majority of cells in these cultures during expansion remain as NPCs.
3.2. Expression of selective cell markers in cultured NPCs Cultured cells showed positive immunoreactivity to the NPC markers nestin [16], A2B5 [34] and CD133 [53] (Fig. 2A–C). No expression of the cell differentiation markers GFAP (a marker for astrocytes) and of MAP2 (a neuronal cell marker) was detected (data not shown). Since even small methodological variation may influence whether cells remain as NPCs, we used flow cytometry to analyze undifferentiated NPCs during in vitro passages. FACS analysis of undifferentiated NPCs revealed a relatively homogenous population of cells on plots displaying forward scatter (FSC), a measure of cell size, against side scatter (SSC), a measure of granularity (Fig. 3A). This homogenous pattern of in vitro expanded NPCs was seen consistently. FACS analysis was performed on the gated live population discounting the dead cell bodies and cellular debris. Results obtained on NPCs at passages 1, 3 and 5 showed that a range of 85–95% of cells highly expressed nestin during expansion (Fig. 3B and C). When examined by immunocytochemistry, N90% of the cells expressed nestin and remained proliferative as judged by
3.3. Multipotency of cultured NPCs indicated by clonal differentiation To determine if these in vitro expanded NPCs maintain their multipotency, we examined their capability to differentiate at the clonal level. For these experiments, NPCs were plated at low density (1103 cells per 10-cm culture dish) and well-isolated single cells were marked and tracked by a 2-mm circle on the bottom of the plates. After clonal expansion, cells were induced to differentiate by withdrawal of the mitogenic effect of hFGFb and hEGF and culturing in 10% FBS. After differentiation, cells displayed at least three different cell fates (Fig. 4A–D). After 7 days of differentiation, 15–46% of cells were immunoreactive to Tuj1 (Fig. 4A), a monoclonal antibody that recognizes a neuronspecific subtype III h-tubulin [26]. The expression of GFAP, a marker for astrocytes [35], could also be detected in 52– 80% of cells with a complex fibrous morphology (Fig. 4B). Following differentiation, immunoreactivity to O4, an oligodendrocyte marker antibody [49], was observed in 0.5–2% of cells (Fig. 4C). A high percentage of GABAergic
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the SCID mouse brain (Fig. 5C and D). A specific mouse anti-human nuclei (Chemicon, Cat. no. MAB1281) antibody was also used to identify the differentiated cells being derived from transplanted human NPCs (data not shown). Therefore, the differentiation potential of cultured NPCs was clearly demonstrated in vivo. In addition, human NPCs were found in the olfactory bulb at 10 and 21 days posttransplant. Detection of human NPCs in an area distal to the initial injection site suggests that these cells possess the ability to migrate. 3.5. Examination of chemokine receptor expression in NPCs
Fig. 4. Differentiation of human NPCs. Following in vitro differentiation of NPCs in medium containing 10% FBS for 7 days, cells were immunostained with (A) anti-Tuj1 Ab (green), an early neuronal cell marker, contrasted with propidium iodide nuclear staining (red), (B) anti-GFAP Ab (red), an astrocyte marker, contrasted with DAPI nuclear staining (blue), (C) anti-O4 Ab (FITC labeled, became white due to high intensity), an oligodendrocyte marker, contrasted with DAPI nuclear staining (blue), and (D) anti-GABA Ab (green), a GABAergic neuronal cell marker, contrasted with DAPI nuclear staining (blue). (E) NPCs derived from a single clone (one cell per well) were differentiated in 1% FBS for 3 weeks and stained with anti-Tuj1 Ab (green) and anti-GFAP Ab (red), contrasted with DAPI nuclear staining (blue).
The observation that transplanted human NPCs migrated away from the injection site suggests these cells have the ability to respond to guidance cues and signals which direct their migration in vivo. Given the well-known roles of chemokines in directing migration of leukocyte subsets, expression of a large panel of chemokine receptors on human NPCs was investigated. To measure levels of various chemokine receptors, we performed FACS analysis of NPCs from different brain specimens at passages 1, 3 and 5. Expression of 13 chemokine receptors was examined, including CCR1, 2, 3, 5, 6, 7, 9 and CXCR1 to CXCR6. When FACS analysis was performed on NPCs from 11week-old fetal brain tissue, we found that 10–30% of NPCs expressed low levels of CCR7, 40–70% of NPCs expressed CCR9, 20–40% of NPCs expressed a minimal level of CXCR1, 20–40% of NPCs expressed CXCR5 and over 85% of NPCs consistently expressed high levels of CXCR4
neurons stained by anti-GABA antibody (green) was also observed (Fig. 4D). When NPCs from single clones were cultured in 1% FBS for 3 weeks, differentiation into neurons and astrocytes was also seen (Fig. 4E). The identification of all three differentiated cell phenotypes in most of the colonies demonstrate the same precursors could give rise to multi-neural cell lineages. Therefore, we conclude that up to passage 5, the multipotency of NPCs is preserved during the process of in vitro expansion. 3.4. Differentiation of cultured NPCs indicated in vivo To characterize in vivo differentiation of NPCs, cultured cells at passage 2 were transplanted into the SVZ and hippocampus of immunodeficient C.B.17 SCID/bg mice. At days 10 and 21 post-transplant, the presence of human NPCs in the murine host was observed by immunohistochemical staining using human specific nestin antibody (Fig. 5A and B). Using antibodies specific to the human cell markers NSE and GFAP, we found that transplanted human NPCs differentiated into neurons and GFAP-positive cells in
Fig. 5. Characterization of human NPCs in SCID/beige mice. NPCs (1104 cells/2 Al) were injected into the left SVZ and right hippocampus of SCID/ beige mice using a stereotaxic apparatus. The transplanted NPCs cells were identified by immunohistochemical staining in longitudinal sections (30 Am) (A, B) 10 days or (C, D) 21 days post-transplantation using humanspecific antibodies against nestin, GFAP and NSE (Texas red). The brain sections were counterstained with DAPI (blue nuclear stain). Nestinpositive human NPCs were detected in the (A) olfactory bulb and (B) hippocampus, and GFAP- and NSE-positive NPCs were detected in the (C, D) olfactory bulb.
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Fig. 6. Expression of chemokine receptors on human NPCs. NPCs obtained from 8- to 11-week-old fetal brain specimens at passages (P) 1, 3 and 5 were analyzed by flow cytometry for expression of CCR, and CXCR chemokine receptors.
(Fig. 6). When NPCs from 8- to 9-week-old fetal brain tissues were assayed, a similar pattern of chemokine receptor expression was observed (data not shown). To verify the results of FACS, we also evaluated chemokine receptor expression of NPCs at the mRNA level by RPA. As expected, significant levels of CXCR4 and a very low level of CCR7 mRNA were observed, while CCR2, CCR5, CCR6 and CXCR1 mRNA were not detectable (Fig. 7). Although CCR3 was detectable by FACS (Fig. 6), no CCR3 mRNA was detected by RPA (Fig. 7). This difference may be attributed to low amount of total RNA (5 Ag) used and/or the stability/regulation of CCR3 mRNA. Thus, the results of RPA confirmed that human NPCs express high levels of CXCR4. 3.6. Association of nestin and CXCR4 expression Nestin has been the most frequently used marker for defining NPCs. To ascertain CXCR4 expression on NPCs, FACS analysis was performed using Abs to CXCR4 and nestin to examine the association of their expression. We found that all of the cells that stained positively for nestin also were shown to be CXCR4 positive (Fig. 8A and B), suggesting a strong association of nestin and CXCR4 expression. For comparison, we performed the same assay on cortical neuronal cultures which were derived from 16week-old fetal brain tissue. These neuronal cultures were comprised of 90–95% neurons (NeuN positive, a neuronal marker) and 5–10% astrocytes (GFAP positive), but b20% of the cells in these cultures were nestin-positive. However, all nestin-positive cells in these neuronal cultures coexpressed CXCR4 (Fig. 8C). Whereas approximately 30% of the cells in these neuronal cultures that were CXCR4 positive were nestin-negative.
3.7. Chemotaxis of NPCs To demonstrate the functional significance of high-level expression of CXCR4, we first examined the effect of CXCL12/SDF-1a on human NPC proliferation in the presence of hFGFb and hEGF. No significant effect of CXCL12/SDF-1a on NPC proliferation was found by MTT or alamarBluek assay (data not shown). Next, the migratory response of NPCs to CXCL12/SDF-1a, the natural ligand of CXCR4, was investigated by chemotaxis assay. CCL2, a ligand of CCR2 that was not detected in NPCs by FACS or RPA, was included in this experiment as a negative control, as were the chemokines CXCL13 and CCL20, the receptors for which, i.e., CXCR5 and CCR6, respectively, were found to be expressed at low levels (Fig. 6). NPCs were found to migrate towards CXCL12/SDF-1a but not towards other chemokines (Fig. 9A). The migratory response to CXCL12/SDF-1a was found to be concentration-dependent (Fig. 9B). Next, NPCs treated with antiCXCR4 antibody (10 Ag/ml) or HIV-1 gp120 for 30 min were used for migration studies. Anti-CXCR4 antibody and gp120, which is known to bind to CXCR4, blocked the migratory response of NPCs towards CXCL12/SDF-1a (Fig. 9C) supporting that CXCR4 is involved in the migratory response. These results demonstrated that a functional response of NPCs to CXCL12/SDF-1a is associated with the high-level expression of CXCR4.
4. Discussion NPC proliferation and migratory activity are fundamental processes responsible for generating the correct number and destination of cells during normal brain development. The
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Fig. 7. Expression of CXCR4 mRNA in NPCs. Five micrograms of total RNA obtained from 9- to 11-week-old fetal brain specimens at passage 1 or 2 was used in RPA to identify mRNA expression of CXCR4. Lane 1: RPA template; lanes 2, 3 and 4 are NPCs isolated from three different brain specimens.
biological importance of these processes is maintained in the mature brain where NPCs play a key role in new memory formation [6,38]. The processes of NPC proliferation and migration are highly regulated in vivo by extrinsic factors, such as growth factors, integrins, extracellular cell matrix components, neurotransmitters and chemokines [6,9,55]. During proliferation, NPCs also give rise to differentiated progeny through a process of progressive lineage restriction and the formation of intermediate progenitor cells that have different response profiles to environmental cues [29]. In this study, we have established an in vitro system to consistently culture human NPCs derived from 8- to 11week-old fetal brain tissue. These cells were examined for
NPC attributes, mainly through selective cell marker expression [39] and clonal analysis. We found that cultured cells express high levels of the NPC markers, nestin and CD133. Also, a high level of A2B5 expression (over 90% of cells) was observed in these cultures. This finding contradicts the previous belief that A2B5-positive cells are glialrestricted precursor cells, since we found that an average of 40% of the NPCs that co-express A2B5-antigen and nestin could differentiate into neurons. This result suggests that the nature of NPCs is better identified by the expression of multiple markers. The capability of multilineage differentiation of cultured NPCs was demonstrated by clonal differentiation experiments. Cells displayed at least three different fates, indicated by the staining of Tuj1, GFAP and O4 antibodies. We observed that the frequency of these three cell types generated from individual clones varied among clones, and O4-positive cells exist in the lowest frequency compared to Tuj1- or GFAP-positive cells. These data indicate that, during ex vivo expansion, this NPC population exists as a heterogeneous population, which may consist of multipotent NPCs and a group of neural progenitors. In this study, we also showed that transplanted human NPCs survived and differentiated into neurons (NSE-positive) and GFAP-positive cells in the SCID mouse brain. However, the GFAP-positive cells did not appear to have normal astrocyte morphology which may either reflect the microenvironment in the mouse CNS which fails to support the development of mature human astrocytes or a requirement of longer than 21 days to have human astrocytes develop their distinctive morphology in the mouse CNS. In addition to their well-known role in immune system function, chemokines and their receptors have also been shown to be important in angiogenesis and tumor metastasis [12,13,42,47]. More recently, it has become evident that interactions between chemokines and their receptors contribute to regulation of developmental processes through their effects on cell growth and migration [8,11,22,28,40,46]. In particular, the interaction of CXCL12/SDF-1a with CXCR4 appears to play a pivotal role in influencing stem cell proliferation, differentiation, and migration [2,22,46]. The role of CXCL12/SDF-1a in CNS development was initially suggested by the description of selective and regulated expression of this chemokine during brain development [51]. Deletion of the gene encoding either CXCL12/SDF-1a or CXCR4 results in the premature migration of granule cell precursors out of the external granule cell layer [32,58] suggesting that they play a role in localizing granule cell precursors in the proliferative environment of the external granule cell layer. Expression of CXCR4 on various neural progenitors has been recently reported [23,25,31,36]. In this study, we also showed by flow cytometry that majority of our cultured human NPCs express high levels of CXCR4. When the effect of CXCL12/SDF-1a on human NPC proliferation
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Fig. 8. Association of nestin expression with CXCR4 in human NPCs. Flow cytometry analysis with PE-conjugated anti-human CXCR4 (green) or isotypematched control antibodies (black) in (A) NPCs and PE-conjugated anti-human CXCR4, and FITC-conjugated anti-nestin antibodies in (B) NPCs or (C) cortical neuronal cultures was shown.
was examined, no significant effect on growth was observed. Instead, a migratory response of NPCs to CXCL12/SDF-1a was seen in a chemotaxis assay, a finding
that corroborates the recent report of Peng et al. [36]. In our study, we also demonstrated that treatment of NPCs with gp120, which plays an important role in HIV-1 neuropatho-
Fig. 9. Migration of human NPCs towards chemokines. Using a chemotaxis chamber, the number of NPCs migrating towards (A) medium (Control) or a panel of chemokines (100 ng/ml) and (B) different concentrations of CXCL12/SDF-1a were counted. (C) NPCs were treated with medium alone or medium containing gp120 or anti-CXCR4 antibody for 30 min prior to assessing migration towards CXCL12/SDF-1a (10 ng/ml). Cells (A–C) were stained with DiffQuik and quantified by cell count of five fields. Data presented are meansFS.E.M. of triplicates samples from three separate brain specimens.
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genesis [20,21] and is known to bind CXCR4 [14,15], abolished the migratory response towards CXCL12/SDF1a. These results suggest that the functional significance of CXCR4 in NPCs is to regulate cell migration during CNS development. Recently, CD133 has been shown to be a useful cell surface marker in isolating live human fetal NPCs [53], although the functional significance of CD133 in human NPCs remains unknown. In this study, expression of nestin, the most widely recognized NPC marker, was shown to be highly associated with CXCR4 expression. Similar to CD133, CXCR4 expression appears to be a common characteristic among a variety of stem cell populations [40,56]. Nevertheless, unlike CD133, the function of CXCR4 is better characterized and understood. In the present study, we found that virtually all nestin-positive cells also co-expressed CXCR4.
Acknowledgements We are grateful to Drs. R. McKay and E. Major for their valuable advice and assistance. This study was supported in part by United States Public Health Service Grants NS38836 and DA-09924.
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Research report
High-level expression of functional chemokine receptor CXCR4 on human neural precursor cells Hsiao T. Nia, Shuxian Hub, Wen S. Shengb, Judy M. Olsona, Maxim C.-J. Cheeranb, Anissa S.H. Chana, James R. Lokensgardb, Phillip K. Petersonb,* b
a Stem Cell Group, R&D Systems, Inc., Minneapolis, MN 55413, USA Minneapolis Medical Research Foundation and the University of Minnesota Medical School, Minneapolis, MN 55404, USA
Accepted 6 June 2004 Available online 8 August 2004
Abstract Neural precursor cells (NPCs) are self-renewing, multipotent progenitors that give rise to neurons, astrocytes and oligodendrocytes in the central nervous system (CNS). Fetal NPCs have attracted attention for their potential use in studying normal CNS development. Several studies of rodent neural progenitors have suggested that chemokines and their receptors are involved in directing NPC migration during CNS development. In this study, we established a consistent system to culture human NPCs and examined the expression of chemokine receptors on these cells. NPCs were found to express the markers nestin and CD133 and to differentiate into neurons, astrocytes and oligodendrocytes at the clonal level. Flow cytometry and RNase protection assay (RPA) indicated that NPCs express high levels of CXCR4 and low levels of several other chemokine receptors. When examined using a chemotaxis assay, NPCs were able to respond to CXCL12/SDF-1a, a ligand of CXCR4. Treatment with anti-CXCR4 antibody or HIV-1 gp120 abolished the migratory response of NPCs towards CXCL12/SDF-1a. These findings suggest that CXCR4 may play a significant role in directing NPC migration during CNS development. D 2004 Elsevier B.V. All rights reserved. Theme: Development and regeneration Topic: Cell differentiation and migration Keywords: Neural precursor cell; Differentiation; Chemokine receptor
1. Introduction Chemokines and their cognate receptors were initially associated with the trafficking of leukocytes in physiological immune surveillance and with inflammatory cell recruitment in different diseases [45,48]. Subsequently, they have been found to play a critical role in hematopoietic development by regulating migration, proliferation, differentiation and survival of human and murine hematopoietic stem and progenitor cells [1,3,24,27,37,56]. The involve* Corresponding author. Department of Medicine, Medical School and Hennepin County Medical Center, University of Minnesota, 701 Park Avenue South, Minneapolis, MN 55415, United States. Tel.: +1 612 873 2877; fax: +1 612 904 4299. E-mail address: [email protected] (P.K. Peterson). 0165-3806/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.devbrainres.2004.06.015
ment of the chemokine/chemokine receptor family in central nervous system (CNS) development has recently been identified, but is still poorly understood and remains to be elucidated. Neural precursor cells (NPCs) are self-renewing, multipotent cells that give rise to neurons, astrocytes and oligodendrocytes in the CNS [5,10,33]. In recent years, in vitro methods have been developed which permit the expansion and differentiation of multipotent fetal NPCs in culture [5,7,19,43,44,50,54]. NPCs can be grown in the presence of mitogen, as either monolayer cultures or as free floating spherical aggregates termed dneurospheres.T Following expansion, NPCs can be induced to differentiate by withdrawal of mitogenic agent or by exposure of the cells to other factors that cause them to develop along specific lineages. While much work is required to characterize NPCs
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derived from in vitro systems, these systems have served as a valuable resource for studies of normal nervous system development. Much of our understanding of NPCs has been derived from animal models. Early studies of human NPCs suggest that, like rodent NPCs, they can be isolated from fetal tissues and propagated in culture [7,50]. However, results of these studies are preliminary, and in some cases, the findings appear to be divergent when compared to their rodent counterparts. Several studies using rodent neural progenitors have suggested the potential involvement of chemokines in directing NPC migration during CNS development [18,30,31,41,57]. In particular, the interaction of CXCR4 and its ligand CXCL12/SDF-1a has been strongly suggested to be important in neuronal patterning of cerebellum and hippocampus as well as astrocyte development in the rat brain [4,25]. In the present study, we established an in vitro system to consistently culture human NPCs derived from 8to 11-week-old fetal brain tissue. These in vitro propagated NPCs have demonstrated their capability for proliferation in culture while retaining the potential to produce highly differentiated descendants: neurons, oligodendrocytes and astrocytes. Expression of chemokine receptors on these human NPCs was examined by flow cytometry and RNase protection assay (RPA), and migratory activity was assessed by in vitro and in vivo systems.
2. Materials and methods 2.1. Cell preparation and culture 2.1.1. NPCs NPC cultures were prepared from the telencephalon of 8- to 11-week-old human fetal brain using previously described methods [52,53] with modifications. Human fetal brain tissues obtained under a protocol approved by the Human Subjects Research Committee at Hennepin County Medical Center were mechanically dissociated, resuspended in DMEM/F12 (Invitrogen, Carlsbad, CA) media (containing 8 mM glucose and glutamine [Sigma, St. Louis, MO], N2 plus supplement [R&D Systems, Minneapolis, MN], penicillin and streptomycin [Sigma] and 20 ng/ml human fibroblast growth factor-basic (hFGFb)/20 ng/ml human epidermal growth factor (hEGF) [R&D Systems]) and plated onto poly-O-lysine (Sigma) and bovine fibronectin (FN, R&D Systems) coated 10-cm tissue culture dishes or 24-well plates when indicated. This stage is considered as passage 0. When cell cultures reached 60% confluence, they were subcultured by trypsin (0.0125%, Sigma) at a density of 2105 cells per 10-cm culture dish and considered as passage 1. Medium was replaced every other day and hFGFb was supplemented daily. For studies of clonality, the method of Tsai and McKay [52] was modified for our purpose. Briefly, NPCs obtained from passage 0 were diluted to a single cell per well in 48-well culture
plates to give rise to a single clone (passage 1) for further experiments. NPCs from the single clone (passage 1) were plated onto one well of four-well chamber slides and after 1 week, were subjected to differentiation conditions (1% FBS, no hEGF/ hFGFb) for 3 weeks followed by immunocytochemical staining to identify differentiated neurons and astrocytes. 2.1.2. Highly enriched cortical neuronal cells Neuronal cultures were prepared as previously described [17]. In brief, 16-week-old fetal brain cortical tissues were dissociated after 30 min of incubation at 37 8C with trypsin (0.25%). After washing, cells were resuspended in DMEM (Sigma) containing 10% heat-inactivated fetal bovine serum (FBS, Hyclone, Logan, UT) with penicillin (100 U/ml) and streptomycin (100 Ag/ml). Dispersed cells were plated onto collagen-coated plates (5105, 106 or 3106 cells/well for 24-, 12- or 6-well plates, respectively) or four-well chamber slides (5105 cells/well). On day 5, cell cultures were treated with uridine (33.6 A/ml, Sigma) and fluorodeoxyuridine (13.6 Ag/ml, Sigma) followed by DMEM replacement with 10% heat-inactivated FBS at day 6 and every 4 days thereafter. By day 12, these neuronal cultures contain approximately 60–70% neurons (stained by anti-NeuN antibody, Chemicon, Temecula, CA), 20–30% astrocytes (stained by anti-GFAP antibody, Sternberger Monoclonals, Lutherville, MD) and 2–5% microglial cells (stained by anti-CD68, BD Pharmingen, San Diego, CA). Using specific antibodies to distinguish the different types of neurons, we found that human cerebrocortical neurons are comprised of approximately 20–30% cholinergic, 20–25% GABAergic, 20–30% glutamatergic and 15% unidentified neurons. To obtain greater enrichment of neurons, at day 6, the culture medium was replaced with neurobasal medium (Gibco, Carlsbad, CA) containing B-27 supplement (Gibco). By day 12, these highly enriched neuronal cultures contain approximately 90–95% neurons, 5–10% astrocytes and b2% microglia. 2.2. NPC proliferation assays Cells were plated onto 24-well plates at a density of 1102 cells per well with hFGFb/hEGF. At 0, 24, 72 and 120 h after plating, 3H-thymidine (Amersham, Piscataway, NJ) was added to the wells at 0.25 ACi per well and incubated for 8 h before being harvested and measured for 3 H-thymidine incorporation in a beta-counter. NPC proliferation was also assayed by 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT, Sigma) or alamarBluek (Serotec, Raleigh, NC) uptake by live cells. After adding MTT (final concentration of 1 mg/ml, 4 h) followed by lysis buffer (20% SDS [w/v] in 50% N,Ndimethyl formamide, pH 4.7 adjusted with 2.5% acetic acid and 1 N HCl [32:1]) (16 h), NPC cell lysates were collected and read in a microplate reader (Molecular Devices, Sunnyvale, CA) at 600 nm, or NPCs were read
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at 544 nm (excitation) and 590 nm (emission) after addition of alamarBluek (equal to 10% of the culture volume) for 16 h. 2.3. Flow cytometry Staining of cells with mouse anti-human CD133-phycoerythrin and anti-human chemokine receptor-phycoerythrin antibodies (R&D Systems) was performed according to the manufacturer’s suggested procedures. For nestin staining, cells were fixed with 4% paraformaldehyde in PBS at room temperature for 20 min and washed twice with PBS. After washing, cells were resuspended in SAP buffer (2% FCS, 0.5% saponin and 0.1% sodium azide in PBS), and mouse anti-human nestin or mouse IgG1 isotype control antibody (R&D Systems) was added at the final concentration of 10 Ag/ml to 2.5105 cells in a total reaction volume of 200 Al. Samples were then incubated for 20 min at room temperature. Following incubation, excess nestin or isotype control antibody was removed by washing cells twice with SAP buffer. After washing, cells were resuspended in 200 Al of SAP buffer and 1 Ag of goat anti-mouse IgG-FITC (Caltag, San Francisco, CA) was added. The samples were incubated for 20 min in the dark at room temperature, and cells were then washed once with SAP buffer, once with PBS and then resuspended in 400 Al of PBS and analyzed on a FACScan flow cytometer (BectonDickinson, Mountain View, CA). Five thousand events were collected and analyzed using CELL Quest software.
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2.5. RNase protection assay For assessments of chemokine receptor mRNA expression, total RNA was used in the RiboQuantk multiprobe RNase Protection Assay (RPA) according to the manufacturer’s instructions (BD Pharmingen). Total RNA was collected from NPCs with Qiagen RNeasy Mini kit (Qiagen, Valencia, CA). After hybridization with 32Plabeled template, samples were resolved on denaturing (5%) polyacrylamide urea gels (19:1 40% acrylamide/bis, 10 Tris–Borate–EDTA and urea) and analyzed with a phosphorimager (Molecular Dynamics, Sunnyvale, CA). 2.6. Chemotaxis assay NPCs were added to upper chambers of a 96-well chemotaxis device (Neuro Probe, Gaithersburg, MD) (106 cells/well) separated from the lower chambers with an 8-Am pore size of polyvinylpyrrolidone-free polycarbonate filter. The lower chambers were filled with chemoattractants. After 6 h of incubation, NPCs that had migrated from upper chambers into lower chambers were quantified by DiffQuik staining (Dade Diagnostics, Aguada, PR). To determine the effect of blockade of CXCR4, NPCs were treated with anti-CXCR4 antibody (10 Ag/ml) (R&D Systems) or human immunodeficiency virus type 1 (HIV-1) gp120 (10 9 and 10 8 M) (Protein Sciences, Meriden, CT) for 30 min before being used in chemotaxis assay towards CXCL12/SDF-1a.
2.4. Immunocytochemical staining
2.7. In vitro differentiation
Anti-nestin, A2B5 and Tuj1 antibodies (R&D Systems) were used at the concentration of 10 Ag/ml in fixed cells. Anti-GFAP antibody was used at 1:200 dilutions. Cells were fixed with 4% paraformaldehyde and 0.15% picric acid in PBS at room temperature for 20 min and were then permeated and blocked with 0.1% Triton X-100, 1% BSA and 10% normal donkey serum in PBS at room temperature for 45 min. After blocking, cells were incubated with diluted primary antibody overnight at 4 8C and sequentially with fluorescence-coupled anti-mouse IgG Ab (Jackson Laboratory, Bar Harbor, ME) at room temperature in the dark for 1 h. Between each step, cells were washed with 0.1% BSA in PBS. Anti-oligodendrocyte marker O4 antibody (R&D Systems) was used at a concentration of 1 Ag/ml. Rabbit anti-human GABA antibody (Sigma) was used at 1:10,000 dilutions. Cells were fixed with 4% paraformaldehyde in PBS at room temperature for 20 min and then blocked with 10% normal donkey serum and 1% BSA in PBS at room temperature for 45 min. After blocking, cells were incubated with diluted primary antibody overnight at 4 8C and sequentially with fluorescence-coupled anti-mouse IgM Ab (Jackson Laboratory) at room temperature in the dark for 1 h. Between each step cells were washed with 0.1% BSA in PBS.
Media of NPC cultures at passages 1–5 after 7 days of plating were replaced with 10 % FBS in DMEM/F-12 medium without hFGFb/hEGF. After 7 days, the differentiated NPCs were stained with anti-human NeuN, GFAP or O4 antibodies to identify differentiated neurons, astrocytes or oligodendrocytes, respectively. 2.8. In vivo differentiation To characterize in vivo differentiation of NPCs, cultured cells at passage 2 were transplanted into the subventricular zone (SVZ) and hippocampus of 8- to 10-week-old immunodeficient C.B.17 SCID/bg mice. Anesthetized mice were aligned on a stereotaxic apparatus (David Kopf Instruments, Tujunga, CA) equipped with a neonatal rat/ mouse adapter (Stoelting, Wood Dale, IL). Openings were created on the skull at predetermined coordinates relative to the bregma. NPCs were deposited into the left SVZ and right hippocampus using these coordinates: rostrocaudal vector (AP)=0 mm, interaural vector (mid)=+1.0 mm, dorsoventral vector (DV)= 3.0 mm and AP= 2.0 mm, mid= 0.8 mm, DV= 2.0 mm, respectively. All ventral coordinates were measured from the surface of the skull. Injections (1104 NPCs/2 Al) were administered using a 10-
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Al Hamilton syringe attached to the stereotaxic apparatus over a period of 2–3 min at each site. At days 10 and 21 post-transplant, animals were anesthetized and perfused with 4% paraformaldehyde. Mouse brains were fixed overnight in 4% paraformaldehyde and transferred into 30% sucrose solution for 48 h. Longitudinal brain sections (30 Am) were obtained by using a microtome (Bright Instrument, Huntingdon, England). To detect the transplanted human NPCs, brain sections were immunostained with specific anti-human nestin, neuron specific enolase (NSE, Dako, Carpinteria, CA) and GFAP antibodies. Immunohistochemical staining procedures were similar to immunocytochemical staining as described above.
3. Results 3.1. Generation of continuously proliferating NPCs NPCs derived from 8- to 11-week-old human fetal brain tissues were cultured and expanded in DMEM/F12 and N2 medium supplied with hEGF and hFGFb on FN-coated plates. Continuous proliferation of these cells was indicated by 3H-thymidine incorporation assay (Fig. 1A). The cultured cells were also examined at the clonal level. Single clones derived from single cells (one cell per well in a 48well plate) showed clonal proliferation. NPCs were plated at a density of 1103 cells per 10-cm culture dish and well-
Fig. 2. Expression of human NPC markers. NPCs obtained from 9-weekold fetal brain specimens were analyzed by immunocytochemical staining and flow cytometry for cellular markers characteristic of these cells. Immunocytochemical staining of undifferentiated NPCs by (A) anti-nestin, (B) anti-A2B5 (red) antibodies with nuclear staining with DAPI (blue) was shown. (C) PE-conjugated anti-human CD133 (green) or isotype-matched control (black) antibodies was used to analyze NPC by flow cytometry.
Fig. 1. Proliferation of human NPCs. NPCs (passages 1–3) obtained from 8- to 9-week-old fetal brain specimens were (A) temporally assayed by 3H-thymidine incorporation and photographed under phase-contrast microscopy at the clonal level at (B) day 1 and (C) day 4. Data presented in (A) are meansFS.E.M. of triplicates of three separate experiments.
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Fig. 3. Flow cytometry and immunocytochemical staining analysis of human NPCs. Human NPCs obtained from 9-week-old fetal brain specimens at passages 1, 3 and 5 were analyzed by flow cytometry: (A) plots displaying FSC versus SSC (R1 indicated the gated live population), (B, C) nestin-positive cells, and by (D) immunocytochemical double staining of nestin-positive (red) and BrdU-incorporated cells (green).
separated colonies were tracked. The proliferation of cultured cells was also demonstrated at the clonal level (Fig. 1B and C).
their ability to incorporate bromodeoxyuridine (BrdU) (Fig. 3D). The expression of nestin suggests that a majority of cells in these cultures during expansion remain as NPCs.
3.2. Expression of selective cell markers in cultured NPCs Cultured cells showed positive immunoreactivity to the NPC markers nestin [16], A2B5 [34] and CD133 [53] (Fig. 2A–C). No expression of the cell differentiation markers GFAP (a marker for astrocytes) and of MAP2 (a neuronal cell marker) was detected (data not shown). Since even small methodological variation may influence whether cells remain as NPCs, we used flow cytometry to analyze undifferentiated NPCs during in vitro passages. FACS analysis of undifferentiated NPCs revealed a relatively homogenous population of cells on plots displaying forward scatter (FSC), a measure of cell size, against side scatter (SSC), a measure of granularity (Fig. 3A). This homogenous pattern of in vitro expanded NPCs was seen consistently. FACS analysis was performed on the gated live population discounting the dead cell bodies and cellular debris. Results obtained on NPCs at passages 1, 3 and 5 showed that a range of 85–95% of cells highly expressed nestin during expansion (Fig. 3B and C). When examined by immunocytochemistry, N90% of the cells expressed nestin and remained proliferative as judged by
3.3. Multipotency of cultured NPCs indicated by clonal differentiation To determine if these in vitro expanded NPCs maintain their multipotency, we examined their capability to differentiate at the clonal level. For these experiments, NPCs were plated at low density (1103 cells per 10-cm culture dish) and well-isolated single cells were marked and tracked by a 2-mm circle on the bottom of the plates. After clonal expansion, cells were induced to differentiate by withdrawal of the mitogenic effect of hFGFb and hEGF and culturing in 10% FBS. After differentiation, cells displayed at least three different cell fates (Fig. 4A–D). After 7 days of differentiation, 15–46% of cells were immunoreactive to Tuj1 (Fig. 4A), a monoclonal antibody that recognizes a neuronspecific subtype III h-tubulin [26]. The expression of GFAP, a marker for astrocytes [35], could also be detected in 52– 80% of cells with a complex fibrous morphology (Fig. 4B). Following differentiation, immunoreactivity to O4, an oligodendrocyte marker antibody [49], was observed in 0.5–2% of cells (Fig. 4C). A high percentage of GABAergic
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the SCID mouse brain (Fig. 5C and D). A specific mouse anti-human nuclei (Chemicon, Cat. no. MAB1281) antibody was also used to identify the differentiated cells being derived from transplanted human NPCs (data not shown). Therefore, the differentiation potential of cultured NPCs was clearly demonstrated in vivo. In addition, human NPCs were found in the olfactory bulb at 10 and 21 days posttransplant. Detection of human NPCs in an area distal to the initial injection site suggests that these cells possess the ability to migrate. 3.5. Examination of chemokine receptor expression in NPCs
Fig. 4. Differentiation of human NPCs. Following in vitro differentiation of NPCs in medium containing 10% FBS for 7 days, cells were immunostained with (A) anti-Tuj1 Ab (green), an early neuronal cell marker, contrasted with propidium iodide nuclear staining (red), (B) anti-GFAP Ab (red), an astrocyte marker, contrasted with DAPI nuclear staining (blue), (C) anti-O4 Ab (FITC labeled, became white due to high intensity), an oligodendrocyte marker, contrasted with DAPI nuclear staining (blue), and (D) anti-GABA Ab (green), a GABAergic neuronal cell marker, contrasted with DAPI nuclear staining (blue). (E) NPCs derived from a single clone (one cell per well) were differentiated in 1% FBS for 3 weeks and stained with anti-Tuj1 Ab (green) and anti-GFAP Ab (red), contrasted with DAPI nuclear staining (blue).
The observation that transplanted human NPCs migrated away from the injection site suggests these cells have the ability to respond to guidance cues and signals which direct their migration in vivo. Given the well-known roles of chemokines in directing migration of leukocyte subsets, expression of a large panel of chemokine receptors on human NPCs was investigated. To measure levels of various chemokine receptors, we performed FACS analysis of NPCs from different brain specimens at passages 1, 3 and 5. Expression of 13 chemokine receptors was examined, including CCR1, 2, 3, 5, 6, 7, 9 and CXCR1 to CXCR6. When FACS analysis was performed on NPCs from 11week-old fetal brain tissue, we found that 10–30% of NPCs expressed low levels of CCR7, 40–70% of NPCs expressed CCR9, 20–40% of NPCs expressed a minimal level of CXCR1, 20–40% of NPCs expressed CXCR5 and over 85% of NPCs consistently expressed high levels of CXCR4
neurons stained by anti-GABA antibody (green) was also observed (Fig. 4D). When NPCs from single clones were cultured in 1% FBS for 3 weeks, differentiation into neurons and astrocytes was also seen (Fig. 4E). The identification of all three differentiated cell phenotypes in most of the colonies demonstrate the same precursors could give rise to multi-neural cell lineages. Therefore, we conclude that up to passage 5, the multipotency of NPCs is preserved during the process of in vitro expansion. 3.4. Differentiation of cultured NPCs indicated in vivo To characterize in vivo differentiation of NPCs, cultured cells at passage 2 were transplanted into the SVZ and hippocampus of immunodeficient C.B.17 SCID/bg mice. At days 10 and 21 post-transplant, the presence of human NPCs in the murine host was observed by immunohistochemical staining using human specific nestin antibody (Fig. 5A and B). Using antibodies specific to the human cell markers NSE and GFAP, we found that transplanted human NPCs differentiated into neurons and GFAP-positive cells in
Fig. 5. Characterization of human NPCs in SCID/beige mice. NPCs (1104 cells/2 Al) were injected into the left SVZ and right hippocampus of SCID/ beige mice using a stereotaxic apparatus. The transplanted NPCs cells were identified by immunohistochemical staining in longitudinal sections (30 Am) (A, B) 10 days or (C, D) 21 days post-transplantation using humanspecific antibodies against nestin, GFAP and NSE (Texas red). The brain sections were counterstained with DAPI (blue nuclear stain). Nestinpositive human NPCs were detected in the (A) olfactory bulb and (B) hippocampus, and GFAP- and NSE-positive NPCs were detected in the (C, D) olfactory bulb.
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Fig. 6. Expression of chemokine receptors on human NPCs. NPCs obtained from 8- to 11-week-old fetal brain specimens at passages (P) 1, 3 and 5 were analyzed by flow cytometry for expression of CCR, and CXCR chemokine receptors.
(Fig. 6). When NPCs from 8- to 9-week-old fetal brain tissues were assayed, a similar pattern of chemokine receptor expression was observed (data not shown). To verify the results of FACS, we also evaluated chemokine receptor expression of NPCs at the mRNA level by RPA. As expected, significant levels of CXCR4 and a very low level of CCR7 mRNA were observed, while CCR2, CCR5, CCR6 and CXCR1 mRNA were not detectable (Fig. 7). Although CCR3 was detectable by FACS (Fig. 6), no CCR3 mRNA was detected by RPA (Fig. 7). This difference may be attributed to low amount of total RNA (5 Ag) used and/or the stability/regulation of CCR3 mRNA. Thus, the results of RPA confirmed that human NPCs express high levels of CXCR4. 3.6. Association of nestin and CXCR4 expression Nestin has been the most frequently used marker for defining NPCs. To ascertain CXCR4 expression on NPCs, FACS analysis was performed using Abs to CXCR4 and nestin to examine the association of their expression. We found that all of the cells that stained positively for nestin also were shown to be CXCR4 positive (Fig. 8A and B), suggesting a strong association of nestin and CXCR4 expression. For comparison, we performed the same assay on cortical neuronal cultures which were derived from 16week-old fetal brain tissue. These neuronal cultures were comprised of 90–95% neurons (NeuN positive, a neuronal marker) and 5–10% astrocytes (GFAP positive), but b20% of the cells in these cultures were nestin-positive. However, all nestin-positive cells in these neuronal cultures coexpressed CXCR4 (Fig. 8C). Whereas approximately 30% of the cells in these neuronal cultures that were CXCR4 positive were nestin-negative.
3.7. Chemotaxis of NPCs To demonstrate the functional significance of high-level expression of CXCR4, we first examined the effect of CXCL12/SDF-1a on human NPC proliferation in the presence of hFGFb and hEGF. No significant effect of CXCL12/SDF-1a on NPC proliferation was found by MTT or alamarBluek assay (data not shown). Next, the migratory response of NPCs to CXCL12/SDF-1a, the natural ligand of CXCR4, was investigated by chemotaxis assay. CCL2, a ligand of CCR2 that was not detected in NPCs by FACS or RPA, was included in this experiment as a negative control, as were the chemokines CXCL13 and CCL20, the receptors for which, i.e., CXCR5 and CCR6, respectively, were found to be expressed at low levels (Fig. 6). NPCs were found to migrate towards CXCL12/SDF-1a but not towards other chemokines (Fig. 9A). The migratory response to CXCL12/SDF-1a was found to be concentration-dependent (Fig. 9B). Next, NPCs treated with antiCXCR4 antibody (10 Ag/ml) or HIV-1 gp120 for 30 min were used for migration studies. Anti-CXCR4 antibody and gp120, which is known to bind to CXCR4, blocked the migratory response of NPCs towards CXCL12/SDF-1a (Fig. 9C) supporting that CXCR4 is involved in the migratory response. These results demonstrated that a functional response of NPCs to CXCL12/SDF-1a is associated with the high-level expression of CXCR4.
4. Discussion NPC proliferation and migratory activity are fundamental processes responsible for generating the correct number and destination of cells during normal brain development. The
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Fig. 7. Expression of CXCR4 mRNA in NPCs. Five micrograms of total RNA obtained from 9- to 11-week-old fetal brain specimens at passage 1 or 2 was used in RPA to identify mRNA expression of CXCR4. Lane 1: RPA template; lanes 2, 3 and 4 are NPCs isolated from three different brain specimens.
biological importance of these processes is maintained in the mature brain where NPCs play a key role in new memory formation [6,38]. The processes of NPC proliferation and migration are highly regulated in vivo by extrinsic factors, such as growth factors, integrins, extracellular cell matrix components, neurotransmitters and chemokines [6,9,55]. During proliferation, NPCs also give rise to differentiated progeny through a process of progressive lineage restriction and the formation of intermediate progenitor cells that have different response profiles to environmental cues [29]. In this study, we have established an in vitro system to consistently culture human NPCs derived from 8- to 11week-old fetal brain tissue. These cells were examined for
NPC attributes, mainly through selective cell marker expression [39] and clonal analysis. We found that cultured cells express high levels of the NPC markers, nestin and CD133. Also, a high level of A2B5 expression (over 90% of cells) was observed in these cultures. This finding contradicts the previous belief that A2B5-positive cells are glialrestricted precursor cells, since we found that an average of 40% of the NPCs that co-express A2B5-antigen and nestin could differentiate into neurons. This result suggests that the nature of NPCs is better identified by the expression of multiple markers. The capability of multilineage differentiation of cultured NPCs was demonstrated by clonal differentiation experiments. Cells displayed at least three different fates, indicated by the staining of Tuj1, GFAP and O4 antibodies. We observed that the frequency of these three cell types generated from individual clones varied among clones, and O4-positive cells exist in the lowest frequency compared to Tuj1- or GFAP-positive cells. These data indicate that, during ex vivo expansion, this NPC population exists as a heterogeneous population, which may consist of multipotent NPCs and a group of neural progenitors. In this study, we also showed that transplanted human NPCs survived and differentiated into neurons (NSE-positive) and GFAP-positive cells in the SCID mouse brain. However, the GFAP-positive cells did not appear to have normal astrocyte morphology which may either reflect the microenvironment in the mouse CNS which fails to support the development of mature human astrocytes or a requirement of longer than 21 days to have human astrocytes develop their distinctive morphology in the mouse CNS. In addition to their well-known role in immune system function, chemokines and their receptors have also been shown to be important in angiogenesis and tumor metastasis [12,13,42,47]. More recently, it has become evident that interactions between chemokines and their receptors contribute to regulation of developmental processes through their effects on cell growth and migration [8,11,22,28,40,46]. In particular, the interaction of CXCL12/SDF-1a with CXCR4 appears to play a pivotal role in influencing stem cell proliferation, differentiation, and migration [2,22,46]. The role of CXCL12/SDF-1a in CNS development was initially suggested by the description of selective and regulated expression of this chemokine during brain development [51]. Deletion of the gene encoding either CXCL12/SDF-1a or CXCR4 results in the premature migration of granule cell precursors out of the external granule cell layer [32,58] suggesting that they play a role in localizing granule cell precursors in the proliferative environment of the external granule cell layer. Expression of CXCR4 on various neural progenitors has been recently reported [23,25,31,36]. In this study, we also showed by flow cytometry that majority of our cultured human NPCs express high levels of CXCR4. When the effect of CXCL12/SDF-1a on human NPC proliferation
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Fig. 8. Association of nestin expression with CXCR4 in human NPCs. Flow cytometry analysis with PE-conjugated anti-human CXCR4 (green) or isotypematched control antibodies (black) in (A) NPCs and PE-conjugated anti-human CXCR4, and FITC-conjugated anti-nestin antibodies in (B) NPCs or (C) cortical neuronal cultures was shown.
was examined, no significant effect on growth was observed. Instead, a migratory response of NPCs to CXCL12/SDF-1a was seen in a chemotaxis assay, a finding
that corroborates the recent report of Peng et al. [36]. In our study, we also demonstrated that treatment of NPCs with gp120, which plays an important role in HIV-1 neuropatho-
Fig. 9. Migration of human NPCs towards chemokines. Using a chemotaxis chamber, the number of NPCs migrating towards (A) medium (Control) or a panel of chemokines (100 ng/ml) and (B) different concentrations of CXCL12/SDF-1a were counted. (C) NPCs were treated with medium alone or medium containing gp120 or anti-CXCR4 antibody for 30 min prior to assessing migration towards CXCL12/SDF-1a (10 ng/ml). Cells (A–C) were stained with DiffQuik and quantified by cell count of five fields. Data presented are meansFS.E.M. of triplicates samples from three separate brain specimens.
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genesis [20,21] and is known to bind CXCR4 [14,15], abolished the migratory response towards CXCL12/SDF1a. These results suggest that the functional significance of CXCR4 in NPCs is to regulate cell migration during CNS development. Recently, CD133 has been shown to be a useful cell surface marker in isolating live human fetal NPCs [53], although the functional significance of CD133 in human NPCs remains unknown. In this study, expression of nestin, the most widely recognized NPC marker, was shown to be highly associated with CXCR4 expression. Similar to CD133, CXCR4 expression appears to be a common characteristic among a variety of stem cell populations [40,56]. Nevertheless, unlike CD133, the function of CXCR4 is better characterized and understood. In the present study, we found that virtually all nestin-positive cells also co-expressed CXCR4.
Acknowledgements We are grateful to Drs. R. McKay and E. Major for their valuable advice and assistance. This study was supported in part by United States Public Health Service Grants NS38836 and DA-09924.
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