Cell Mol Neurobiol DOI 10.1007/s10571-015-0216-4

ORIGINAL RESEARCH

A Novel Monoclonal Antibody Against Neuroepithelial and Ependymal Cells and Characteristics of Its Positive Cells in Neurospheres Masaharu Kotani1 • Yasunori Sato2 • Akemichi Ueno2 • Toshinori Ito3 Kouichi Itoh4 • Masato Imada5

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Received: 10 April 2015 / Accepted: 20 May 2015 Ó Springer Science+Business Media New York 2015

Abstract There are still few useful cell membrane surface antigens suitable for identification and isolation of neural stem cells (NSCs). We generated a novel monoclonal antibody (mAb), designated as mAb against immature neural cell antigens (INCA mAb), which reacted with the areas around a lateral ventricle of a fetal cerebrum. INCA mAb specifically reacted with neuroepithelial cells in fetal cerebrums and ependymal cells in adult cerebrums. The recognition molecules were O-linked 40 and 42 kDa glycoproteins on the cell membrane surface (gp40 INCA and gp42 INCA). Based on expression pattern analysis of the recognition molecules in developing cerebrums, it was concluded that gp42 INCA was a stage-specific antigen expressed on undifferentiated neuroepithelial cells, while gp40 INCA was a cell lineage-specific antigen expressed at the stages of differentiation from neuroepithelial cells to ependymal cells. A flow cytometric analysis showed that fetal and young adult neurospheres were divided into INCA mAb- CD133 polyclonal antibody (pAb)-, INCA mAb? & Masaharu Kotani [email protected] 1

Department of Molecular and Cellular Biology, Faculty of Pharmaceutical Sciences, Ohu University, Fukushima 963-8611, Japan

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Department of Health Chemistry, Faculty of Pharmaceutical Sciences, Ohu University, Fukushima 963-8611, Japan

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Department of English Language Technology, Faculty of Pharmaceutical Sciences, Ohu University, Fukushima 963-8611, Japan

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Laboratory for Pharmacotherapy and Experimental Neurology, Kagawa School of Pharmaceutical Sciences, Tokushima Bunri University, Kagawa 769-2193, Japan

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Department of Functional Morphology, Nihon University School of Medicine, Tokyo 173-8610, Japan

CD133 pAb-, and INCA mAb? CD133 pAb? cell populations based on the reactivity against INCA mAb and CD133 pAb. The proportion of cells having the neurosphere formation capability in the INCA mAb? CD133 pAb? cell population was significantly larger than that of undivided neurospheres. Neurospheres formed by clonal expansion of INCA mAb? CD133 pAb? cells gave rise to neurons and glial cells. INCA mAb will be a useful immunological probe in the study of NSCs. Keywords Cell membrane surface antigen
Ependymal cell
Monoclonal antibody
Neural stem cell
Neuroepithelial cell
Neurosphere Abbreviations Ab BSA CNE CP ECL EGF FACS b-FGF GFAP O-Glycosidase LIF LMS LV mAb NSC pAb PBS PNGase F PVDF

Antibody Bovine serum albumin Cortical neuroepithelium Caudate putamen Ependymal cell layer Epidermal growth factor Fluorescence-activated cell sorting Basic-fibroblast growth factor Glial fibrillary acidic protein End-a-N-acetylgalactosaminidase Leukemia inhibitory factor Lateral migratory stream Lateral ventricle (s) Monoclonal antibody Neural stem cell Polyclonal antibody Phosphate-buffered saline Peptide-N-glycosidase F Polyvinylidene difluoride

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SDS-PAGE SeNE StNE SVZ VZ VZ/SZV

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis Septal neuroepithelium Striatal neuroepithelium Subventricular zone Ventricular zone VZ plus SVZ

Introduction Neural stem cells (NSCs) are defined as having long-term self-renewal capability and multilineage potential and giving rise to neural cells such as neurons and glial cells. It has been reported that NSCs inhabit the areas surrounding a lateral ventricle (LV) in fetal mammalian brains as well as in adult brains (Temple 1989; Reynolds and Weiss 1992). The LV in a fetal brain consists of the ventricular zone (VZ), which is composed mainly of symmetrically dividing cells, and the subventricular zone (SVZ), which is composed mainly of asymmetric dividing cells (Takahashi et al. 1996; Kosodo et al. 2004). Since the VZ and the SVZ are histologically undistinguishable, they are sometimes considered as a single zone and referred to as VZ plus SVZ (VZ/SVZ) (Schambra 2008). On the other hand, the LV in an adult brain consists of a single layer of ependymal cells called an ependymal cell layer (ECL) and the SVZ, in which some different kinds of cells such as type B cells (GFAP? NSCs), type C cells (neural progenitor cells), and type A cells (neuroblasts) constitute a thin layer with comparatively high cell density (Doetsch et al. 1999). NSCs have been expected to help the development of new medicines that would serve for effective medical treatment against neurological disorders, many of which are incurable. However, there remain some difficulties that must be surmounted before the realization of NSC-based treatments. Quality and safety control of NSCs is one of them. Although there have been some notable achievements in identification and isolation of NSCs under the physiological condition, they are yet to be refined. Cellular identity and loci of NSCs in an adult LV are still unclear (Chojnacki et al. 2009). Additionally, it remains unknown whether the cellular identity of NSCs in fetal and adult cerebrums is the same or not. In contrast, the concept that NSCs inhabiting the VZ/SVZ of a fetal LV are neuroepithelial cells is strongly supported by numerous reports regarding the developmental process of brains (Smart 1973; Chenn and McConnell 1995; Takahashi et al. 1996; Haubensak et al. 2004; Kosodo et al. 2004; Konno et al. 2008).

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Currently, there are two different views regarding the cellular identity of NSCs in an adult LV: One is that NSCs are a subset of glial fibrillary acidic protein (GFAP) positive (GFAP?) astrocytes in the SVZ (Doetsch et al. 1997, 1999; Alvarez-Buylla and Garcia-Verdugo 2002; Doetsch 2003; Garcia et al. 2004; Pastrana et al. 2009). The other is that NSCs are a subset of CD133? ependymal cells in the ECL (Johansson et al. 1999; Corti et al. 2007; Pfenninger et al. 2007; Coskun et al. 2008; Pfenninger et al. 2011). For the resolution of these controversies, it is necessary to find cell membrane surface antigens which enable the accurate identification and isolation of NSCs in adult cerebrums. Cell membrane surface antigens and antibodies (Abs) against these antigens are crucial to the isolation of a specific cell population under the physiological conditions and the elucidation of its cellular identity. Antigens that fulfill the above-mentioned requirements should be expressed on the cells in adult cerebrums that correspond to the neuroepithelial cells in the fetal VZ/SVZ. On the basis of this idea, we generated one novel monoclonal antibody (mAb) that reacts with cell membrane surface antigens of neuroepithelial cells by immunizing rats with membrane fraction prepared from telencephalons at embryonic day 14.5 (E14.5). E14.5 telencephalons were used as an antigen source for the following reasons; since asymmetrical division of neuroepithelial cells reaches its peak in E14.5 brains (Takahashi et al. 1996), a sufficient number of target cells can be collected, and the sample handling of E14.5 brains is comparatively easy. We designated the Ab as INCA mAb (mAb against immature neural cell antigens). In this study, we show that INCA mAb is a novel mAb to react with O-linked cell membrane surface glycoproteins expressed on neuroepithelial cells and ependymal cells in mouse fetal and young adult cerebrums. Moreover, we point out that INCA mAb? cells isolated from fetal and young adult neurospheres had self-renewal capability and multilineage potential. Thus, INCA mAb will be effective for the cellular identity and isolation of NSCs in fetal and adult cerebrums.

Materials and Methods Animals Three-week-old (3W) F344 female rats, 8W male ICR nude mice, pregnant ICR female mice, and 6W ICR male mice were purchased from Charles River Laboratories Japan (Tokyo, Japan). All the animals were housed in the Ohu University Animal Care Facility, and the experiments were performed according to the guidelines of Ohu University Animal Research Committee.

Cell Mol Neurobiol

Cells and Cell Cultures

Generation of mAb

PAI mouse myeloma cells (PAI cells) were cultured in RPMI-1640 medium containing 10 mM HEPES, 2 mM Lglutamine, 1 mM non-essential amino acids, 1 mM sodium pyruvate, and 10 % fetal calf serum. Hybridoma cells were cultured in the medium which was made by adding HAT (Invitrogen, Carlsbad, CA) into the PAI cell culture medium. Fetal and young adult neurospheres derived from telencephalons in mouse brains at E14.5 and from surrounding tissues of LVs in 6W brains were prepared as follows: These tissues were taken as single cell suspension by trypsin-EDTA treatment and pipetting. These cells were cultured in the neurosphere culture medium which added 20 nM epidermal growth factor (EGF), 20 nM basicfibroblast growth factor (b-FGF), 20 nM leukemia inhibitory factor (LIF), and B27 supplement without vitamin A (Invitrogen) into the neurosphere basic medium, which contained 300 lM sodium selenite (50 ll), 600 mM putrescine (50 ll), 100 lM progesterone (100 ll), transferrin (50 mg), insulin (12 mg), 3 mM sodium bicarbonate (0.126 g), D-glucose (3 g), 1 M HEPES (2.5 ml), 200 mM glutamine (5 ml) in 500 ml of F12/D-MEM (1:1) medium. A medium change and subculture for expanding the number of neurospheres were performed at 4 day intervals as follows: The neurospheres were dispersed to single cells by mechanical pipetting, and then, these cells were plated onto new dishes with the neurosphere culture medium as described above.

Four 3W female F344 rats were immunized in footpads twice on day 5 and day 7 intervals with 100 ll emulsion prepared by mixing 50 ll immunogen (10 lg/ml) and 50 ll TiterMax Gold (CytRx, Norcross, GA). Three days after the booster-immunization, which followed the two immunizations, the popliteal lymph node cells of immunized rats were fused with PAI myeloma cells using polyethylene glycol 1500 (Roche, Mannheim, Germany) according to Kotani et al (1993). Hybridoma cells which secreted the target Ab were sorted out by immunohistochemical staining as described below. Ascites containing the desired mAb was produced in pristine-primed nude mice, and the mAb in ascites was purified by caprylic acid precipitation (Russo et al. 1983). The protein concentration of the purified mAb was measured by BCA (Thermo Fisher Scientific).

Preparation of Cytoplasmic Fraction and Cell Membrane Fraction Fetal cerebrums, young adult organs, and tissues and neurospheres were homogenized in 10 volumes of phosphate-buffered saline (PBS) containing 1 mM PMSF, 1 mM EDTA, 10 lM aprotinin, and 1 mM iodoacetoamide with a Politoron homogenizer. The homogenates were centrifuged at 800 rpm for 10 min at 4 °C. The supernatants were centrifuged at 25,000 rpm for 20 min at 4 °C. The precipitates were used as cell membrane fraction, and a part of the cell membrane fraction prepared from E14.5 brains was used as an immunogen. On the other hand, the supernatant was used as cytoplasmic fraction solution. The cell membrane lysates were prepared as follows: The cell membrane fractions were treated on ice for 20 min with 2 volumes of lysis buffer, PBS containing 1 % NP-40, and centrifuged at 25,000 rpm for 20 min at 4 °C. The supernatants were used as cell membrane fraction lysate. The protein concentration of the cell membrane fraction lysates and the cytoplasmic fraction solution was measured by BCA (Thermo Fisher Scientific, Waltham, MA).

Immunohistochemical and Immunocytochemical Stainings Frozen tissue sections (thickness 10 lm) and cells were fixed with 4 % paraformaldehyde-PBS for 10 min, and then blocked and incubated with primary mAbs and/or polyclonal antibodies (pAbs) followed by incubation with FITC- or Cy3-labeled secondary pAbs (Jackson ImmunoResearch, West Grove, PA). The stained sections and cells were observed under a microscope (Axiovert 100M; Carl Zeiss, Oberkochen, Germany) equipped with a confocal laser scanning system (LSM510; Carl Zeiss). The existing Abs used in this study were b-catenin pAb (rabbit IgG; Sigma, St. Louis, MO), CD133 pAb (rabbit IgG; Abnova, Taipei, Taiwan), glia fibrillary acidic protein (GFAP) mAb (mouse IgG2b; Sternberger, Berkeley, CA), Nestin mAb (mouse IgG1; CHEMICON, Temecula, CA), Neurofilament (NF)-200 mAb (mouse IgG1; Sigma), Numb pAb (rabbit IgG; Upstate, Lake Placid, NY), bPhospho-histone3 (H3) pAb (rabbit IgG; Santa Cruz Biochemistry, Santa Cruz, CA), S100b mAb (mouse IgG1; Sigma), Rip mAb (mouse IgG1; Sigma), and b-tubulin IV mAb (mouse IgG1; Sigma). Hematoxylin–eosin (H–E) staining was performed routinely. Western Blotting The cell membrane fraction lysates (5 lg/lane) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in 4–20 % acrylamide gradient gel (Cosmobio, Tokyo), and then electroblotted onto polyvinylidene difluoride (PVDF) membranes (Immobilon; GE Healthcare, Buckinghamshire, UK) according to Towbin et al (1979). The PVDF membranes were blocked with 5 % skim milk in PBS followed by incubation with

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primary Abs for 1 h. After washed with 1 % skim milk in PBS containing 0.05 % Tween 20, the membranes were incubated with peroxidase-conjugated secondary pAb (Jackson ImmunoResearch) for 45 min. The bands were visualized with a chemiluminescence detection system (GE Healthcare) according to the manufacturer’s protocol. Epitope Identification For metaperiodate oxidation of the antigens, the electroblotted PVDF membranes were treated with or without 25 mM NaIO4 in 100 mM acetate buffer, pH 4.0, for 30 min in the dark followed by immunological detection described in ‘‘Western Blotting.’’ To determine whether the carbohydrate chains of glycoproteins were N- or O-linked, the membrane lysates were treated with Peptide-N-Glycosidase F (PNGase F; New England BioLabs, Ipswich, MA) and End-a-N-Acetylgalactosaminidase (O-glycosidase; New England BioLabs) according to the manufacturer’s protocol. The treated samples were separated by SDS-PAGE followed by Western blotting and then chemiluminescence detection described in ‘‘Western Blotting.’’ Flow Cytometric Analysis The neurospheres were prepared as single cell suspension by mechanical pipetting. The cells (5 9 105 living cells/tube/sample) were incubated on ice with primary Abs for 45 min. After washed 3 times with 1 % bovine serum albumin (BSA) in PBS, the cells were incubated on ice with FITC- or Cy3-labeled secondary pAbs (Jackson ImmunoResearch) for 45 min. The stained cells were applied to Epics-XL flow cytometry (Beckman Coulter, Brea, CA). Dead cells stained with propidium iodide were excluded from the analysis by an appropriate scatter gating. Cell Isolation with pluriBead Cell Separation Kit Isolation of INCA mAb? cells was performed with pluriBead Cell Separation Kit (pluriSelect, Spring Valley, CA) according to the manufacturer’s protocol. In brief, INCA mAb binding pluriBeads were incubated for 45 min at 4 °C with neurospheres ([1 9 107 living cells) prepared as single cell suspension. The reaction solutions were applied to pluriStrainer. After washed gently 5 times with PBS, the cells which remained on pluriStrainer were collected into a new sterile tube by elution buffer. The collected cells were used as INCA mAb? cell population in the following assays. The isolation of INCA mAb? CD133 pAb? cells was performed as follows: Isolated INCA mAb? cells were incubated for 45 min at 4 °C with CD133 pAb binding pluriBeads. The reaction solutions were applied to

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pluriStrainer. After washed gently 5 times with PBS, the cells which remained on pluriStrainer were collected into a new sterile tube by elution buffer. The collected cells were used as INCA mAb? CD133 pAb? cell population in the following assays. Neurosphere Formation and Neural Differentiation Assays A neurosphere formation assay for the evaluation of selfrenewable capability was executed as follows: Single cell suspension of neurospheres prepared by mechanical pipetting was cultured on 96-well plates under the condition of 1 living cell/200 ll of neurosphere culture medium/ well (see ‘‘Cells and cell cultures’’ for the neurosphere culture medium). On day 16, the number of neurospheres that clonally expanded was counted. Half of the neurosphere culture medium was changed at 4-day intervals. A neural differentiation assay for the evaluation of multilineage potential was performed as follows: Neurospheres clonally expanded by the neurosphere formation assay were cultured for 3 days on 8-well chamber slides, which precoated with 0.2 % polyethyleneimine in 0.15 M boric-acid buffer, under the condition of 10 or less neurospheres/200 ll/well of neurosphere basic medium (see ‘‘Cells and cell cultures’’). On day 3, the slides were fixed and stained with some biomarkers for neurons and glial cells. The stained cells were observed under a microscope (Axiovert 100 M; Carl Zeiss) equipped with a confocal laser scanning system (LSM510; Carl Zeiss).

Results Immunoreactivity of INCA mAb Against Embryonic Cerebrums and Fetal Neurospheres Among hundreds of hybridoma cells obtained by cell fusion of PAI myeloma cells and popliteal lymph node cells immunized with the cell membrane fraction prepared from E14.5 telencephalons, we selected a hybridoma secreting mAb, INCA mAb (rat IgG2a isotype), which reacted with some cells in the VZ/SVZ of LVs in E14.5 cerebrums (Fig. 1A). However, in the case of 6W cerebrums, it reacted mainly in the ECL (Fig. 1A). INCA mAb? areas were determined based on the reports of Schambra (2008) and Johansson et al (1999). INCA mAb reacted with unfixed fetal neurospheres, demonstrating that its recognition molecules are cell membrane surface antigens (Fig. 1B, white arrows). Additionally, some INCA mAb negative (INCA mAb-) cells were also observed in those neurospheres (Fig. 1B, yellow arrows). Therefore, there might be at least two different

Cell Mol Neurobiol Fig. 1 Immunofluorescence staining of fetal and young adult cerebrums and fetal neurospheres with INCA mAb. A Frozen coronal sections of E14.5 (a, c) and 6W brains (d, f) underwent H–E staining (a, d) or indirect immunofluorescence staining with INCA mAb (green) (c, f). Diagrams in the middle column (b, e) are drawn based on the H– E stained sections (a, b). Scale bar 100 lm. B Unfixed fetal neurospheres dissociated by mechanical pipetting and stained with INCA mAb. Both INCA mAb? and INCA mAbcells were observed in these neurospheres. The white arrows are pointing to INCA mAb? cells (green). The yellow arrows are pointing to INCA mAbcells. Scale bar 10 lm. CC corpus callosum, CP caudate putamen, ECL ependymal cell layer, LS lateral septal area, LV lateral ventricle, VZ/SVZ ventricular zone plus subventricular zone (Color figure online)

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cell populations in fetal neurospheres, i.e., INCA mAb? cells and INCA mAb- cells. Biochemical Analyses of INCA mAb Recognition Molecules To clarify that INCA mAb recognition molecules are cell membrane surface antigens, we performed Western blot analysis using the cell membrane fraction lysate and the cytoplasmic fraction solution prepared from E14.5 telencephalons. INCA mAb detected a broad band (from 40 kDa to 45 kDa) of proteins in the cell membrane fraction lysate under reducing conditions (Fig. 2A), but it did

Phase contrast

not detect any bands in the cytoplasmic fraction solution (Fig. 2A). The detected proteins were designated as INCA. Together with the results in Fig. 1B, INCA was concluded to be cell membrane surface antigens. Next, we examined an INCA mAb recognition epitope on INCA. The PVDF membranes on which the cell membrane fraction lysates prepared from E14.5 telencephalons and fetal neurospheres were blotted were treated with NaIO4. As shown in Fig. 2B, INCA mAb lost the reactivity with INCA. In the case of fetal neurospheres without NaIO4, however, INCA mAb detected two separate bands (40 and 42 kDa), while its detection bands in E14.5 telencephalons were not as clear. These results demonstrate

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Fig. 2 Specificity of INCA mAb. A Cytoplasmic fraction solution and membrane fraction lysate prepared from E14.5 whole brain isolated by SDS-PAGE were electroblotted onto PVDF membranes followed by Western blotting using INCA mAb. INCA mAb detected a broad band (from 40 to 45 kDa) in the membrane fraction lysate. The band is indicated as INCA. B Recognition epitopes on INCA. Whole E14.5 brain membrane fraction lysates and fetal neurosphere membrane fraction lysates separated by SDS-PAGE were electroblotted onto PVDF membranes. The blots were treated with or without NaIO4 followed by incubation with INCA mAb. INCA mAb

recognition epitopes were sensitive to NaIO4 oxidation. INCA mAb detected two bands (40 and 42 kDa) in the fetal neurospheres without NaIO4. The two recognized molecules are designated as gp40 INCA and gp42 INCA. C Whole E14.5 brain membrane fraction lysates and fetal neurosphere membrane fraction lysates digested with or without PNGase F or with or without O-glycosidase were separated by SDSPAGE followed by electroblotting onto PVDF membranes. The blots were incubated with INCA mAb. The reactivity of INCA mAb against gp40 INCA and gp42 INCA disappeared with the digestion with O-glycosidase

that INCA mAb reacted with the carbohydrate portion of INCA and suggest that its recognition molecule is two different molecules, designated as gp40 INCA and gp42 INCA. Then, we examined the reactivity of INCA mAb with the cell membrane fraction lysates treated with enzymes which cut of O- or N-linked carbohydrate chains from

glycoproteins. In the case of lysates treated with O-glycosidase, INCA mAb did not detect any antigens (Fig. 2C). In the lysates treated with PNGase F, however, INCA mAb clearly detected gp40 INCA and gp42 INCA (Fig. 2C). These results demonstrate that the INCA mAb recognition epitope of gp40 INCA and gp42 INCA is an O-linked carbohydrate chain.

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Expression of INCA mAb Recognition Molecules in Young Adult Organs, Developing Cerebrums, and Neurospheres Expression of gp40 INCA and gp42 INCA in 6W mouse organs and tissues was examined by Western blot analysis. gp40 INCA and gp42 INCA were undetectable in any organs or tissues (Fig. 3A). However, a broad band was detected near 38 kDa only in lung (Fig. 3A), which was designated as gp38 INCA. We do not have any satisfactory explanation for this locality. In 6W cerebrums, neither gp40 INCA nor gp42 INCA was detected by INCA mAb. We thought that the amount of gp40 INCA and gp42 INCA in this lysate might be below detectable limits. So, we carried out Western blot analysis using lysates of individual zones in 6W cerebrums. As shown in Fig. 3B, gp40 INCA was detected in the lysate prepared from the area containing the VZ and the SVZ but not in the lysates prepared from the areas containing the caudate putamen (CP) or the cerebral cortex. gp42 INCA was totally undetectable in any lysate, suggesting that the reactivity of INCA mAb in the VZ/SVZ might be against gp40 INCA and gp42 INCA and that the reactivity in the ECL might be against gp40 INCA. The expression of gp40 INCA and gp42 INCA in cerebrums in the developmental process was examined by Western blot analysis using the lysates prepared from whole

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cerebrums at E14.5, E17.5, and postnatal (P1). At E14.5, gp40 INCA and gp42 INCA were clearly detected (Fig. 3C). At E17.5, the expression of gp42 INCA was weak compared with that of gp40 INCA (Fig. 3C). At P1, only gp40 INCA was expressed (Fig. 3C). In fetal and young adult neurospheres, both gp40 INCA and gp42 INCA were expressed, and the expression levels were approximately the same (Fig. 3C). These results indicate that whereas the expression of gp42 INCA decreases in the course of cerebrum development, that of gp40 INCA does not undergo any significant changes during the development process. Immunoreactivity of INCA mAb Against Developing Cerebrums To clarify the reactivity of INCA mAb against developing cerebrums, we performed indirect immunofluorescence staining of cerebrums at different developmental stages. At E14.5, INCA mAb strongly reacted with the septal neuroepithelium (SeNE) and the lateral migratory stream (LMS) areas in the VZ/ SVZ, and weekly reacted with the striatal neuroepithelium (StNE) and the cortical neuroepithelium (CNE) areas in the VZ/SVZ (Fig. 4). At E17.5, the reactivity was basically similar to that at E14.5. However, it was observed that one area showed distinctly different reactivity. This area was the medial preoptic area (MPOA) (Fig. 4), which is one of the areas that show

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Fig. 3 Expression of gp40 INCA and gp42 INCA in organs, tissues, and neurospheres. A Expression of gp40 INCA and gp42 INCA in different organs at 6W. Membrane fraction lysates from the organs were subjected to Western blotting using INCA mAb. A broad band was detected near 38 kDa only in lung, designated as gp38 INCA. B Expression of gp40 INCA and gp42 INCA in different areas in 6W cerebrums. Membrane fraction lysates prepared from the tissues containing the VZ and SVZ, the CP tissues, and the cerebral cortex

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tissues were subjected to Western blotting using INCA mAb. gp40 INCA was detected in the tissues containing the VZ and SVZ. C Expression of gp40 INCA and gp42 INCA in developing cerebrums and neurospheres. Membrane fraction lysates prepared from whole brains at E14.5, E17.5, and P1 and from fetal and young adult neurospheres were subjected to western blotting using INCA mAb. gp42 INCA decreased as development of cerebrum

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Fig. 4 Immunofluorescence staining of cerebrums at different developmental stages with INCA mAb. The lower stand shows the indirect immunofluorescence staining of frozen coronal sections of E14.5 (A), E17.5 (B), P1 (C), and 6W (D) cerebrums with INCA mAb (green). On the upper stand are shown the diagrams that correspond to the frozen

sections. CC corpus callosum, CNE cortical neuroepithelium, CP caudate putamen, ECL ependymal cell layer, LMS lateral migratory stream, LV lateral ventricle, MPOA medial preoptic area, SeNE septal neuroepithelium, StNE striatal neuroepithelium, VZ/SVZ ventricular zone plus subventricular zone. Scale bar 100 lm (Color figure online)

active neurogenesis in fetal cerebrums. At P1, INCA mAb reacted with the VZ/SVZ, the LMS, and the narrow area of the third ventricular side (Fig. 4). Interestingly, the reactivity of INCA mAb in the VZ/SVZ was not uniform. It had strong reactivity on the inside of the VZ/SVZ, whereas the reactivity was weak or negative on the outside. These results demonstrate that the VZ/SVZ of developing cerebrums consists of neuroepithelial cells of different gp40 INCA and gp42 INCA expression levels. In 6W cerebrums, its reactivity was observed exclusively in the ECL (Fig. 4). INCA mAb never reacted with terminal differentiated cells in developing cerebrums such as neurons and glial cells (Fig. 4). These results indicate that INCA mAb? areas are restricted from the VZ/SVZ to the ECL and also suggest that INCA mAb? cells in fetal and young adult cerebrums may be neuroepithelial cells or ependymal cells.

fetal and adult cerebrums. We examined the reactivity of Numb pAb, which reacts with Numb, a transcription factor in neuroepithelial cells (Zhong et al. 1996), against INCA mAb? areas in E14.5 cerebrums. As shown in Fig. 5A, Numb pAb reacted with INCA mAb? areas in the VZ/SVZ. More specifically, INCA mAb? Numb pAb? areas were mainly observed in the SeNE, the LMS, and the areas surrounding the ECL. This result demonstrates that the INCA mAb? cells in the VZ/SVZ are neuroepithelial cells. We also examined the reactivity of b-catenin pAb, btubulin IV mAb, and S100b mAb, which are used for the identifying ependymal cells (Hirota et al. 2010; Renthal et al. 1993; Gleason et al. 2008), against INCA mAb? areas in 6W cerebrums. As shown in Fig. 5B, almost all the INCA mAb? areas in the ECL overlapped with b-catenin pAb?, b-tubulin IV mAb?, or S100b mAb? areas, indicating that the cells in the INCA mAb? areas in the ECL are ependymal. In contrast, when the INCA mAb? cell areas were examined at high magnification, a small number of INCA mAb? cells were observed in the SVZ (Fig. 5B, white arrows). The reactivity of INCA mAb against the cells, however, was weak compared with that against

Immunohistochemical Identification of INCA mAb1 Cells in Fetal and Young Adult Cerebrums Indirect double immunofluorescence staining with INCA mAb and one of Abs against cellular biomarkers was performed to identify the species of INCA mAb? cells in

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Fig. 5 Double immunofluorescence staining of fetal and young adult cerebrums with INCA mAb and Abs against biomarkers of neuroepithelial or ependymal cells. Frozen coronal sections of E14.5 (A) and 6W (B) cerebrums were stained by indirect double immunofluorescence staining with INCA mAb (green) (A, a, d and g; B, a, e and i) and Numb pAb (red) (A, b, e and h), b-catenin pAb (red) (B, b), b-

tubulin IV mAb (red) (B, f), or S100b mAb (red) (B, j). The white arrows in the high-magnification photographs point to INCA mAb? cells (green) (h and l) and INCA mAb? b-catenin Ab? cells (yellow) (d) in the SVZ. ECL ependymal cell layer, SVZ subventricular zone. Scale bars A; a–c and B; a–c, e–g, i–k 100 lm: A; d–h 20 lm, B; d, h and l 10 lm (Color figure online)

ependymal cells, and although those cells reacted with bcatenin pAb (Fig. 5B, d), they reacted with neither btubulin IV mAb nor S100b mAb (Fig. 5B, h and l). These results indicate that INCA mAb reacts not only with ependymal cells in the ECL but with b-catenin Ab? cells in the SVZ.

SVZ (Fig. 6A). In 6W cerebrums, INCA mAb? b-PhosphoH3 pAb? cells were mainly observed in the ECL, and some INCA mAb- b-Phospho-H3 pAb? cells were observed in the SVZ, which is located outside the ECL (Fig. 6B). Moreover, we examined in fetal and young adult neurospheres whether INCA mAb? cells were dividing cells. As shown in Fig. 6C, some INCA mAb? cells in fetal and young adult neurospheres were reacted with b-Phospho-H3 pAb. These results demonstrate that some INCA mAb? cells in cerebrums and neurospheres are in the G2- and M-phases of the cell cycle.

Immunoreactivity of b-Phospho-H3 pAb Against INCA mAb1 Cells in Cerebrums and Neurospheres Based on the results above, we assumed that INCA mAb? cells in the VZ/SVZ were dividing cells. Then, we examined the reactivity of b-Phospho-H3 pAb, which reacts with dividing cells in the gap 2 (G2)- and mitotic (M)-phases (von Bohlen Halbach 2007), against INCA mAb? areas. In the VZ/ SVZ of E14.5 cerebrums, b-Phospho-H3 pAb reacted with INCA mAb? cells (Fig. 6A). However, it did not react with INCA mAb- cells and INCA mAbweak? cells outside the VZ/

Immunoreactivity of CD133 pAb, GFAP mAb, and Nestin mAb Against INCA mAb1 Cells in Developing Cerebrums We further examined the reactivity of CD133 pAb, GFAP mAb, and Nestin mAb, which are used to identify NSCs (Johansson et al. 1999; Doetsch et al. 1999; Lendahl et al.

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Fig. 6 Double immunofluorescence staining of fetal and young adult cerebrums and neurospheres with INCA mAb and phosphor-H3 pAb. Frozen coronal sections of E14.5 (A) and 6W (B) cerebrums and neurospheres (C) were fixed and stained by indirect double

immunofluorescence staining with INCA mAb (green) and phosphoH3 pAb (red). Scale bars A; a–c and B; a–c, 100 lm: A; d–i and C; a and b 20 lm: B; d–l 10 lm (Color figure online)

1990), against INCA mAb? cells in fetal and young adult cerebrums by indirect double immunofluorescence staining. The reactivity of CD133 pAb and Nestin mAb in the cerebrums at any developmental stage was basically similar to each other, while the reactivity of GFAP mAb was obviously different. As shown in Fig. 7, at E14.5, CD133 pAb and Nestin mAb reacted with INCA mAb? cells in the SeNE and the LMS in the VZ/SVZ and the surrounding areas of the LV. At E17.5, they reacted with INCA mAb? cells in the SeNE, the LSM, the CNE, and the areas around the LV. At P1.0, they reacted with INCA mAb? cells in the SeNE and the areas around the LV. On the other hand, GFAP mAb reacted with INCA mAb? cells in the LV side of the SeNE and the LMS at all the development stages. INCA mAb? GFAP mAb? cell areas, however, were extremely narrow compared with INCA mAb? CD133 pAb? or INCA mAb? Nestin mAb? cell areas. In 6W cerebrums, CD133 pAb, Nestin mAb, and GFAP mAb reacted with INCA mAb? cells in the ECL and the LMS. When the INCA mAb? cell areas were examined at high magnification, a small number of INCA mAb? cells were observed in the SVZ (Fig. 7T, V and X, white arrows) like the results in Fig. 5B. Although those cells reacted with GFAP mAb, they reacted with neither CD133 pAb nor Nestin mAb. These results indicate that NSC biomarkers express with some INCA mAb? cells in the ECL and with a few small

numbers of INCA mAb? cells in the SVZ some in cerebrums after middle fetal periods.

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Flow Cytometric Analysis of INCA mAb1 Cells in Neurospheres As shown in Fig. 1B, it was suggested that there might be at least two different cell populations (INCA mAb? and INCA mAb-) in fetal neurospheres. To confirm that neurosphere cells could be classified into different populations with INCA mAb, we performed flow cytometric analysis of fetal and young adult neurospheres. As shown in Fig. 8A, neurosphere cells were classified into INCA mAb? and INCA mAb- cell populations. The proportion of INCA mAb? cells to INCA mAb- cells was 56.4–43.5 % in fetal neurospheres and 70.7–29.3 % in young adult neurospheres. Moreover, INCA mAb? cell populations in both neurospheres were further divided into INCA mAbweak? and INCA mAbstrong? cell populations. The proportion of INCA mAbweak? to INCA mAbstrong? cells was 24.4–19.1 % in fetal INCA mAb? cell populations and 21.3–8.0 % in young adult INCA mAb? cell populations. While the proportion of INCA mAbweak? cell populations was similar in fetal and young adult neurospheres, the population of INCA mAbstrong? cell populations in fetal neurospheres was more than two times larger than in young adult

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Fig. 7 Double immunofluorescence staining of cerebrums at different developmental stages with INCA mAb and Abs against biomarkers of NSCs. Frozen coronal sections of E14.5 (A–F), E17.5 (G–L), P1 (M–R), and 6W (S–X) cerebrums were stained by indirect double immunofluorescence staining with INCA mAb (green) and CD133 pAb (red) (A, B, G, H, M, N, S, and T), Nestin mAb (red) (C, D, I, J, O, P, U, and V) or GFAP mAb (red) (E, F, K,

L, Q, R, W, and X). The white arrows in the high-magnification photographs point to INCA mAb? cells (green) (T and V) and INCA mAb? GFAP mAb? cells (yellow) (X) in the SVZ. ECL ependymal cell layer, SVZ subventricular zone. Scale bars A, C, E, G, I, K, M, O, Q, S, U, and W 100 lm: B, D, F, H, J, and L 20 lm: N, P, and R 50 lm: T, V, and X 10 lm (Color figure online)

neurospheres. These results indicate that neurospheres are classified into three cell populations: INCA mAb-, INCA mAbweak?, and INCA mAbstrong?. We further examined the cell populations in the neurospheres by two-color flow cytometric analyses using INCA mAb and CD133 pAb. As shown in Fig. 8B, fetal and young adult neurospheres were classified into three cell populations: INCA mAbstrong? CD133 pAb?, INCA mAbweak? CD133 pAb-, and INCA mAb- CD133 pAb-. The proportion of INCA mAbweak? CD133 pAb- cells and that of INCA mAb- CD133 pAb- cells was not significantly different between fetal and young adult neurospheres. However, the proportion of INCA mAbstrong? CD133 pAb? cells in fetal neurospheres was twice as large

as the proportion of those cells in young adult neurospheres. INCA mAb- CD133 pAb? cells were not observed in this experiment. Cellular Characteristics of INCA mAb1 Cells Isolated from Neurospheres From the results reported above, it was speculated that INCA mAb? cells might have the potentials that NSCs have. To test their self-renewable capability and multilineage potential, we performed a neurosphere formation assay and a neural differentiation assay using INCA mAb? and INCA mAb? CD133 pAb? cell populations isolated from fetal and young adult neurospheres with pluriBead Cell Separation Kit.

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Fig. 8 Flow cytometric analysis of INCA mAb? cells in fetal and young adult neurospheres. A Single cell suspensions of fetal (upper right) and young adult neurospheres (lower right) were stained by indirect immunofluorescence staining with INCA mAb followed by flow cytometric analysis. Fetal and young adult neurospheres were divided into INCA mAband INCA mAb? cell populations. The INCA mAb? cell population was further divided into INCA mAbweak? and INCA mAbstrong? cell populations. The proportion of each cell population is indicated in the upper right corner. B Single cell suspensions of fetal (left) and young adult neurospheres (right) were stained by indirect double immunofluorescence staining with INCA mAb and CD133 pAb followed by flow cytometric analysis. Fetal and young adult neurospheres were divided into at least three cell populations: INCA mAbCD133 pAb-, INCA mAb? CD133 pAb-, and INCA mAb? CD133 pAb?. The proportion of each cell population is indicated in each quadrant

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The total number of cells in isolated fetal and young adult INCA mAb? cell populations was 0.5–1 9 106, and the purity of INCA mAb? cells in fetal and young adult INCA mAb? cell populations was 73 and 76 %, respectively (Fig. 9A). On the other hand, the purity of INCA mAb? CD133 pAb? cells in fetal and young adult INCA mAb? CD133 pAb? cell populations was not able to be confirmed by two-color flow cytometric analysis, because too few cells (4–8 9 104 cells) were isolated as INCA mAb? CD133 pAb? cells. As an examination of self-renewable capability, we performed a neurosphere formation assay under the conditions of clonal expansion. As shown in Fig. 9B, the proportions of the cells having the neurosphere formation capability in fetal and young adult INCA mAb? cell populations were 46 and 44 %, respectively. However, when these values were corrected with the purity (73 and

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76 %) in Fig. 9A, they became 63.01 and 57.89 %. Moreover, in the case of fetal and young adult INCA mAb? CD133 pAb? cell populations, these values were 61 and 64 %. Thus, the proportion of the cells having neurosphere formation capability in the two isolated cell populations was significantly high compared with that of the cells in the fetal and young adult neurospheres before isolation. These results indicate that many cells in INCA mAb? and INCA mAb? CD133 pAb? cell populations have the self-renewable capability. Next, we performed a neural differentiation assay with the neurospheres obtained in the neurosphere formation assay in order to clarify whether they would differentiate into neurons and glial cells. As shown in Fig. 9C, the neurospheres formed from fetal and young adult INCA mAb? CD133 pAb? cells differentiated into neurons and glial cells. The neurospheres from fetal and young adult

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Fig. 9 Cellular characteristics of the cells in INCA mAb? and INCA mAb? CD133 pAb? cell populations isolated from neurospheres. A Flow cytometric analysis of INCA mAb? cell population isolated by pluriBead Cell Separation Kit. The proportion of INCA mAb? cells in fetal (left) and young adult (right) INCA mAb? cell population was 68 and 70 %, respectively. B Neurosphere formation assay under the condition of clonal expansion of 1 cell/well. Black and red columns indicate fetal and young adult neurospheres, respectively. The

proportion was represented as percentage. The data are the mean ± SD (n = 3). C Neural differentiation assay of neurospheres clonally expanded from cells in fetal (left) and young adult (right) INCA mAb? CD133 pAb? cell populations. Neurospheres cultured for 3 days into neurosphere basic medium were fixed and stained by indirect double immunofluorescence staining with NF-200 mAb (green) and GFAP mAb (red) (upper stand) and with Rip mAb (green) and GFAP mAb (red) (lower stand). Scale bars 20 lm (Color figure online)

INCA mAb? cells provided the same results (data not shown). These results demonstrate that neurospheres formed from the INCA mAb? and INCA mAb? CD133 pAb? cells have the multilineage potential. In sum, INCA mAb? and INCA mAb? CD133 pAb? cells isolated from neurospheres have the self-renewable capability and the multilineage potential.

Discussion In the present study, our results showed that INCA mAb reacted specifically with cell membrane surface antigens (gp40 INCA and gp42 INCA) expressed on the cell membranes of neuroepithelial and ependymal cells and neurospheres. Moreover, INCA mAb? and INCA mAb?

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CD133 Ab? cell populations isolated from neurospheres contained a large number of cells with self-renewable capability and multilineage potential. Based on the following two findings, it can be concluded that INCA mAb? areas were localized in the region from the VZ/SVZ to the ECL in developing cerebrums (Fig. 4): (1) Dimensional changes of the VZ/SVZ in developing cerebrums (Privat and Leblond 1972; Kaplan and Hinds 1977; Takahashi et al. 1996) were extremely similar to those of INCA mAb? areas. (2) INCA mAb? cells in the VZ/SVZ and the ECL were immunologically identified as neuroepithelial and ependymal cells (Fig. 5). It is well known that neuroepithelial and ependymal cells constitute the VZ/SVZ of fetal cerebrums and the ECL of young adult cerebrums, respectively. Moreover, INCA mAb? areas were also observed in the MPOA and the third ventricles. Recently, the MPOA in mouse fetal brains was reported as a novel source of cortical GABAergic interneurons (Gelman et al. 2009). Thus, it was suggested that in fetal cerebrums, INCA mAb reacted exclusively with the areas that contain neuroepithelial cells which qualify as NSCs, though it is less clear whether the third ventricle also counts as another similar area. Additionally, the MPOA observed in Fig. 4 was not found in Fig. 7 probably because the stained coronal sections used in Fig. 7 originated from areas which did not contain the MPOA because the MPOA should be already formed in E14.5 cerebrums (Gelman et al. 2009). In fetal cerebrums, the reactivity of INCA mAb was weak or negative outside the VZ/SVZ while it was strong inside, and these INCA-expressing areas shrank as a cerebrum develops (Fig. 4). Additionally, the reactivity of NSC Ab and/or mAb in the INCA mAbstrong? areas was also strong (Fig. 6). These results suggest that INCA mAbstrong? areas contain symmetrically dividing NSCs and asymmetrically dividing NSCs giving rise to neural cells and NSCs. In contrast, INCA mAbweak? and INCA mAb- areas contain both differentiating and differentiated neural cells because the reactivity of INCA mAb against neurons or glial cells was not clearly observed in this experiment (Figs. 1A, 4 and 9C). This idea is supported by the reports that almost all neuroepithelial cells in E10 brains give rise to NSCs by symmetric cell division and that neurogenesis by asymmetric cell division of neuroepithelial cells reaches its peak at E14 to E15 (Takahashi et al. 1996; Konno et al. 2008). In young adult cerebrums, the conclusion that INCA mAb? cells are mainly ependymal (Fig. 5) was supported not only by the results in Fig. 5 but also by the results in Fig. 7 because it has already been known that CD133 and GFAP are expressed in ependymal cells as well as in NSCs (Kasper et al. 1987; Pfenninger et al. 2007). In particular, the reactivity of CD133 pAb in this study was consistent with the report that CD133 pAb reacts with cell membrane

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areas on the apical side of ependymal cells (Weigmann et al. 1997; Marzesco et al. 2005). Ependymal cells line the ventricular system such as LVs, third ventricles, cerebral aqueducts, fourth ventricles, and central canals of the spinal cord, and their morphology widely differs depending on their loci (Manthrope et al. 1977). Their main functions in adult brains are secretion and/or absorption of cerebrospinal fluid components (Manthrope et al. 1977). A great deal of recent literatures has reported that ependymal cells work as NSCs in adult rodent brains (Pfenninger et al. 2007; Coskun et al. 2008; Gleason et al. 2008; Pfenninger et al. 2011) and in the chordate larval nervous system (Horie et al. 2011). There is no direct evidence that ependymal cells in this study are NSCs in young adult brains. However, given that INCA mAb? cells were ependymal (Figs. 5, 7) and that INCA mAb? and INCA mAb? CD133 pAb? cells isolated from young adult neurospheres had self-renewable capability and multilineage potential (Fig. 9), it is not reasonable to rule out the possibility that INCA mAb? cells in the ECL in adult cerebrums might be NSCs. Unfortunately, there are no sufficient data which defend our discussion regarding INCA mAb? cells (green) in the SVZ (Figs. 5, 7). However, it is plausible to assume that INCA mAb? b-catenin pAb? and INCA mAb? GFAP mAb? cells (yellow) in the SVZ might be active NSCs because it is reported that GFAP? astrocytes in the SVZ are active NSCs (Pastrana et al. 2009). Nevertheless, it is not obvious whether INCA mAb? cells are astrocytes, neural progenitor cells, NSCs, or ependymal since they were reacted with GFAP mAb and bcatenin pAb. It was suggested that gp40 INCA and gp42 INCA are novel biomarkers for neuroepithelial and ependymal cells, mainly because the molecular weights and the cellular localizations of gp40 INCA and gp42 INCA are significantly different from those of the other cell biomarkers used in this study, though the final conformation awaits molecular cloning. Expression patterns of gp40 INCA and gp42 INCA were different from each other (Fig. 3). gp42 INCA down-regulated as a cerebrum develops and it disappeared at postnatal stages. In contrast, gp40 INCA was expressed at the investigated developmental stages. These results suggest that gp42 INCA is a stage-specific antigen which is expressed on NSCs such as symmetrically and asymmetrically dividing neuroepithelial cells, whereas gp40 INCA is a cell lineage-specific antigen expressed in a cell lineage which differentiates from neuroepithelial cells to ependymal cells. From these differences, it was suggested that the core proteins of gp40 INCA and gp42 INCA differ from each other. The function of gp40 INCA and gp42 INCA is still unknown. However, we assume that these antigens play a role in cell–cell or cell–extracellular matrix interactions, lectin binding, and in signal transduction, since they are cell membrane surface antigens.

Cell Mol Neurobiol

There are a number of reports regarding the identification and isolation of NSCs and neural progenitor cells by flow cytometric analysis and fluorescence-activated cell sorting (FACS) using Abs against NSC biomarkers. Among those NSC biomarkers, CD133, which is also known as prominin-1 and is a membrane glycoprotein having molecular weight of 117 kDa with five transmembrane domains (Miraglia et al. 1997), is frequently used for the isolation of NSCs (Uchida et al. 2000; Lee et al. 2005; Corti et al. 2007; Peh et al. 2009; Fisher et al. 2011). This antigen was first reported as a marker for hematopoietic stem cells (Miraglia et al. 1997) but its function is still unresolved. We isolated INCA mAb? and INCA mAb? CD133 pAb? cell populations from neurospheres by pluriBead Cell Separation Kit because INCA mAb reacts with cell membrane surface antigens (Figs. 2, 3) like CD133 pAb. (We do not have FACS, which would be a better tool for precise isolation of Ab? cells from cell populations under the physiological conditions.) In this study, a majority of INCA mAb? and INCA mAb? CD133 pAb? cells had selfrenewable capability (Fig. 9). More specifically, if we assume that the purities of INCA mAb? CD133 pAb? cells in INCA mAb? CD133 pAb? cell populations are equivalent to those in INCA mAb? cell populations, the proportions of the cells with the neurosphere formation capability in fetal and young adult neurospheres will become 83.56 and 84.64 %, respectively. Based on the results in Fig. 8, it was considered that INCA mAb? cells with neurosphere formation capability might be INCA mAbweak? CD133 pAb- and INCA mAbstrong? CD133 pAb? cells and that INCA mAb? CD133 pAb? cells might actually be INCA mAbstrong? CD133 pAb? cells. It has been reported that the proportion of NSCs in young adult neurospheres is less than 10 % (Johansson et al. 1999; Uchida et al. 2000; Kawaguchi et al. 2001; Rietze et al. 2001; Capela and Temple 2002; Barraud et al. 2005; Lee et al. 2005; Corti et al. 2007; Pastrana et al. 2009). The results shown in Fig. 8 are consistent with the conclusions of these reports. Therefore, these results show that INCA mAbstrong? cells in fetal and young adult neurospheres might be NSCs according to their histological and cellular localizations and their cellular characteristics. Acknowledgments This study was partially supported by Ohu University research funding. Conflict of interest peting interests.

The authors declare that they have no com-

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