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Cellular and Molecular Neurobiology ISSN 0272-4340Volume 36Number 1 Cell Mol Neurobiol (2016) 36:11-26DOI 10.1007/s10571-015-0216-4
A Novel Monoclonal Antibody AgainstNeuroepithelial and Ependymal Cellsand Characteristics of Its Positive Cells inNeurospheres
Masaharu Kotani, Yasunori Sato,Akemichi Ueno, Toshinori Ito, KouichiItoh & Masato Imada
1 23
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ORIGINAL RESEARCH
A Novel Monoclonal Antibody Against Neuroepithelialand Ependymal Cells and Characteristics of Its Positive Cellsin Neurospheres
Masaharu Kotani1 • Yasunori Sato2 • Akemichi Ueno2 • Toshinori Ito3 •
Kouichi Itoh4 • Masato Imada5
Received: 10 April 2015 / Accepted: 20 May 2015 / Published online: 27 May 2015
� Springer Science+Business Media New York 2015
Abstract There are still few useful cell membrane sur-
face 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?
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 neuro-
sphere 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 im-
munological probe in the study of NSCs.
Keywords Cell membrane surface antigen � Ependymal
cell � Monoclonal antibody � Neural stem cell �Neuroepithelial cell � Neurosphere
Abbreviations
Ab Antibody
BSA Bovine serum albumin
CNE Cortical neuroepithelium
CP Caudate putamen
ECL Ependymal cell layer
EGF Epidermal growth factor
FACS Fluorescence-activated cell sorting
b-FGF Basic-fibroblast growth factor
GFAP Glial fibrillary acidic protein
O-Glycosidase End-a-N-acetylgalactosaminidase
LIF Leukemia inhibitory factor
LMS Lateral migratory stream
LV Lateral ventricle (s)
mAb Monoclonal antibody
NSC Neural stem cell
pAb Polyclonal antibody
PBS Phosphate-buffered saline
PNGase F Peptide-N-glycosidase F
PVDF Polyvinylidene difluoride
& Masaharu Kotani
1 Department of Molecular and Cellular Biology, Faculty of
Pharmaceutical Sciences, Ohu University,
Fukushima 963-8611, Japan
2 Department of Health Chemistry, Faculty of Pharmaceutical
Sciences, Ohu University, Fukushima 963-8611, Japan
3 Department of English Language Technology, Faculty of
Pharmaceutical Sciences, Ohu University,
Fukushima 963-8611, Japan
4 Laboratory for Pharmacotherapy and Experimental
Neurology, Kagawa School of Pharmaceutical Sciences,
Tokushima Bunri University, Kagawa 769-2193, Japan
5 Department of Functional Morphology, Nihon University
School of Medicine, Tokyo 173-8610, Japan
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DOI 10.1007/s10571-015-0216-4
Author's personal copy
SDS-PAGE Sodium dodecyl sulfate-polyacrylamide
gel electrophoresis
SeNE Septal neuroepithelium
StNE Striatal neuroepithelium
SVZ Subventricular zone
VZ Ventricular zone
VZ/SZV 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 achieve-
ments 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 neuroep-
ithelial 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).
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) posi-
tive (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 ex-
pressed 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 di-
vision 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 Ja-
pan (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.
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Cells and Cell Cultures
PAI mouse myeloma cells (PAI cells) were cultured in
RPMI-1640 medium containing 10 mM HEPES, 2 mM L-
glutamine, 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 sur-
rounding 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 basic-
fibroblast growth factor (b-FGF), 20 nM leukemia in-
hibitory factor (LIF), and B27 supplement without vitamin
A (Invitrogen) into the neurosphere basic medium, which
contained 300 lM sodium selenite (50 ll), 600 mM pu-
trescine (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 num-
ber 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 de-
scribed above.
Preparation of Cytoplasmic Fraction and Cell
Membrane Fraction
Fetal cerebrums, young adult organs, and tissues and
neurospheres were homogenized in 10 volumes of phos-
phate-buffered saline (PBS) containing 1 mM PMSF,
1 mM EDTA, 10 lM aprotinin, and 1 mM iodoace-
toamide with a Politoron homogenizer. The homogenates
were centrifuged at 800 rpm for 10 min at 4 �C. The su-
pernatants were centrifuged at 25,000 rpm for 20 min at
4 �C. The precipitates were used as cell membrane frac-
tion, 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 pre-
pared 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 con-
centration of the cell membrane fraction lysates and the
cytoplasmic fraction solution was measured by BCA
(Thermo Fisher Scientific, Waltham, MA).
Generation of mAb
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 llTiterMax Gold (CytRx, Norcross, GA). Three days after
the booster-immunization, which followed the two immu-
nizations, 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 stain-
ing 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).
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 Im-
munoResearch, West Grove, PA). The stained sections and
cells were observed under a microscope (Axiovert 100M;
Carl Zeiss, Oberkochen, Germany) equipped with a con-
focal 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), b-Phospho-histone3 (H3) pAb (rabbit IgG; Santa Cruz Bio-
chemistry, 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 gra-
dient 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 elec-
troblotted 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-Gly-
cosidase F (PNGase F; New England BioLabs, Ipswich,
MA) and End-a-N-Acetylgalactosaminidase (O-glycosi-
dase; New England BioLabs) according to the manufac-
turer’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 cell-
s/tube/sample) were incubated on ice with primary Abs for
45 min. After washed 3 times with 1 % bovine serum al-
bumin (BSA) in PBS, the cells were incubated on ice with
FITC- or Cy3-labeled secondary pAbs (Jackson Im-
munoResearch) 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 plur-
iBead 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 �Cwith 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 as-
says. 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
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 self-
renewable capability was executed as follows: Single cell
suspension of neurospheres prepared by mechanical
pipetting was cultured on 96-well plates under the condi-
tion 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 neuro-
sphere culture medium was changed at 4-day intervals.
A neural differentiation assay for the evaluation of
multilineage potential was performed as follows: Neuro-
spheres 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 neuro-
spheres/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 fu-
sion 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 re-
acted 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). Ad-
ditionally, 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
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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 telen-
cephalons. INCA mAb detected a broad band (from
40 kDa to 45 kDa) of proteins in the cell membrane frac-
tion lysate under reducing conditions (Fig. 2A), but it did
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 telen-
cephalons 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
A
6W
E14.5
H-E
a
d
INCA mAb
c
f
ECLLV
CC
LV
VZ/SVZ
Diagram
CP
LS
b
e
SVZ
BINCA mAb Phase contrast
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 mAb-
cells were observed in these
neurospheres. The white arrows
are pointing to INCA mAb?
cells (green). The yellow arrows
are pointing to INCA mAb-
cells. 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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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-gly-
cosidase, 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.
A
250
150
10075
50
37
252015
(kDa) CBB
INCA
: INCA mAb+ +B : NaIO4- +
2520
250
150
10075
50
37
15
(kDa)
- +
gp40 INCAgp42 INCA
++++ : INCA mAb
- + - + - + - +PNGase F O-glycosidaseO-glycosidase
gp40 INCAgp42 INCA
PNGase FC
2520
250
150
10075
50
37
15
(kDa) ++++++++ : INCA mAb
E14.5 brain membrane lysate
Fetal neurosphere membrane lysate
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 electroblot-
ted 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 SDS-
PAGE 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
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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 or-
gans or tissues (Fig. 3A). However, a broad band was de-
tected 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 cere-
brums in the developmental process was examined by
Western blot analysis using the lysates prepared from whole
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 neuro-
spheres, 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 devel-
opment, 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 stain-
ing of cerebrums at different developmental stages. At E14.5,
INCA mAb strongly reacted with the septal neuroepithelium
(SeNE) and the lateralmigratory stream (LMS) areas in theVZ/
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 reactivitywas basically similar
to that at E14.5.However, itwas observed that one area showed
distinctly different reactivity. This areawas themedial preoptic
area (MPOA) (Fig. 4), which is one of the areas that show
A
(kDa)25015010075
503725201510
gp38 INCA
INCA mAb B
(kDa)250150
10075
50
37252015
gp40 INCA
INCA mAb
gp40 INCAgp42 INCA
(kDa)250150
10075
503725201510
C INCA mAbINCA mAb
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
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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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
wasweak or negative on the outside. These results demonstrate
that the VZ/SVZ of developing cerebrums consists of neu-
roepithelial cells of different gp40 INCA and gp42 INCA ex-
pression levels. In 6W cerebrums, its reactivity was observed
exclusively in the ECL (Fig. 4). INCAmAb never reactedwith
terminal differentiated cells in developing cerebrums such as
neurons and glial cells (Fig. 4). These results indicate that
INCAmAb? areas are restricted from the VZ/SVZ to the ECL
and also suggest that INCAmAb? cells in fetal and young adult
cerebrums may be neuroepithelial cells or ependymal cells.
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
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, b-tubulin 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-cateninpAb?, b-tubulin IV mAb?, or S100b mAb? areas, indi-
cating 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
LVVZ/SVZ(CNE)
VZ/SVZ(StNE)
E14.5
LMS
CP
(SeNE)VZ/SVZ
6W
LMS
CP
LV
ECL and SVZ
CCLV
LMS
CPVZ/SVZ(StNE)
VZ/SVZ(CNE)
E17.5
SeNEVZ/SVZ
MPOA
LMSCP
LVVZ/SVZ(CNE)
P1
VZ/SVZ(StNE)
(SeNE)VZ/SVZ
Third ventricle
A INCA mAb B INCA mAb C INCA mAb D INCA mAb
Fig. 4 Immunofluorescence staining of cerebrums at different devel-
opmental 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 INCAmAb (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)
18 Cell Mol Neurobiol (2016) 36:11–26
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ependymal cells, and although those cells reacted with b-catenin pAb (Fig. 5B, d), they reacted with neither b-tubulin 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.
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 di-
viding cells in the gap 2 (G2)- and mitotic (M)-phases (von
BohlenHalbach 2007), against INCAmAb? 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
INCAmAb- cells and INCAmAbweak? cells outside the VZ/
SVZ (Fig. 6A). In 6W cerebrums, INCA mAb? b-Phospho-H3 pAb? cells were mainly observed in the ECL, and some
INCAmAb- 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 INCAmAb? 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 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.
c
f
i
a
d
g
b
e
h
A
fed
hhg
B
INCA mAb -tubulin IV mAbg
h
INCA mAb S100 mAb
lkl
cd
f
j
b
e
i
a
h
dINCA mAb -catenin pAb Merge MergeNumb pAb MergeINCA mAb
ECL SVZ
ECL SVZ
ECL SVZ
β
β
β
Fig. 5 Double immunofluorescence staining of fetal and young adult
cerebrums with INCA mAb and Abs against biomarkers of neuroep-
ithelial or ependymal cells. Frozen coronal sections of E14.5 (A) and6W (B) cerebrums were stained by indirect double immunofluores-
cence 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)
Cell Mol Neurobiol (2016) 36:11–26 19
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1990), against INCA mAb? cells in fetal and young adult
cerebrums by indirect double immunofluorescence stain-
ing. The reactivity of CD133 pAb and Nestin mAb in the
cerebrums at any developmental stage was basically simi-
lar 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 ex-
tremely 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 magnifi-
cation, 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 cere-
brums after middle fetal periods.
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 neu-
rosphere 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 neuro-
spheres. 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
Fetal-neurospheresYoung adult-neurospheres
INCA mAb/Phospho-H3 pAb
a
b
INCA mAb Phospho-H3 pAb Merge
d e f
f
i
g h i
d
g
a
e
h
b c
E14.5A B C
6WINCA mAb Phospho-H3 pAb Merge
d e f
g h i
j k l
dg
j
a e
h
k
bf
i
l
c
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 phospho-
H3 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)
20 Cell Mol Neurobiol (2016) 36:11–26
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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 neuro-
spheres 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 sig-
nificantly different between fetal and young adult neuro-
spheres. 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 neuro-
spheres. INCA mAb- CD133 pAb? cells were not ob-
served in this experiment.
Cellular Characteristics of INCA mAb1 Cells
Isolated from Neurospheres
From the results reported above, it was speculated that INCA
mAb? cellsmight 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.
E14
.5E
17.5
P1.0
6WINCA mAb/CD133 pAb
B
H
N
T
B
H
N
T
A
G
M
S
INCA mAb/Nestin mAb
D
P
V
D
J
P
V
C
JI
O
U
INCA mAb/GFAP mAb
F
L
R
X
F
L
R
X
E
K
Q
W
ECL SVZECL SVZECL SVZ
Fig. 7 Double immunofluorescence staining of cerebrums at differ-
ent 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), NestinmAb (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, andR 50 lm: T, V, and X 10 lm (Color figure online)
Cell Mol Neurobiol (2016) 36:11–26 21
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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 %, respec-
tively (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 con-
ditions 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
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 neuro-
sphere 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
Green fluorescence intensity (log)
Red
fluo
resc
ence
inte
nsity
(log
)
INCA mAb
Fetal neurospheres Young adult neurospheres
CD
133A
b
15%0%
29%56%
7%0%
22%61%
B
A
Num
ber
of c
ells
Negative INCA mAb
Fluorescence intensity (log)
Fetal neurospheres43.5%
INCAstrong+
INCAweak+
Young adult
neurospheresINCAstrong+
INCAweak+
29.3%0.0%
0.0%
(24.4%)
(19.1%)
(22.3%)
(8.0%)
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 mAb-
and 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 mAb-
CD133 pAb-, INCA mAb?
CD133 pAb-, and INCA mAb?
CD133 pAb?. The proportion of
each cell population is indicated
in each quadrant
22 Cell Mol Neurobiol (2016) 36:11–26
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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?
Rip GFAP merge
NF-200 GFAP merge
CNeural differentiation of neurospheres clonal expanded from
fetal INCA mAb+ CD133 mAb+ cells
Rip GFAP merge
NF-200 GFAP merge
Neural differentiation of neurospheres clonal expanded fromyoung adult INCA mAb+ CD133 mAb+ cells
AR
ed fl
uore
scen
ce in
tens
ity (l
og)
CD
133
Ab
Green fluorescence intensity (log)INCA mAb
INCA mAb+ cell population isolatedfrom fetal neurospheres
6%
70%
0%
24%
5%
68%
0%
27%
INCA mAb+ cell population isolated from young adult neurospheres
B
10
20
30
40
50
60
70
0
Neu
rosp
here
form
atio
n ra
tio
(%)
9.0
±±2.
1619.3
3 ±
4.03
46.6
6 ±
3.40
44.3
3 ±
3.68
61.0
±3.
27
64.3
3 ±
3.30
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 INCAmAb? 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)
Cell Mol Neurobiol (2016) 36:11–26 23
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CD133 Ab? cell populations isolated from neurospheres
contained a large number of cells with self-renewable ca-
pability 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 (Gel-
man 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 be-
cause 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 cere-
brum 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 asymmet-
rically dividing NSCs giving rise to neural cells and NSCs.
In contrast, INCA mAbweak? and INCA mAb- areas con-
tain both differentiating and differentiated neural cells be-
cause 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 asym-
metric 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
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 capa-
bility 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
INCAmAb? 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 b-catenin 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 lo-
calizations 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 asymmet-
rically dividing neuroepithelial cells, whereas gp40 INCA
is a cell lineage-specific antigen expressed in a cell lineage
which differentiates from neuroepithelial cells to ependy-
mal 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 inter-
actions, lectin binding, and in signal transduction, since
they are cell membrane surface antigens.
24 Cell Mol Neurobiol (2016) 36:11–26
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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 neuro-
spheres by pluriBeadCell SeparationKit because INCAmAb
reacts with cell membrane surface antigens (Figs. 2, 3) like
CD133 pAb. (We do not have FACS, whichwould be a better
tool for precise isolation of Ab? cells from cell populations
under the physiological conditions.) In this study, a majority
of INCAmAb? and INCAmAb?CD133 pAb? cells had self-
renewable capability (Fig. 9).More specifically, ifwe 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 %, respec-
tively. 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 INCAmAbstrong?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 The authors declare that they have no com-
peting interests.
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