Wnt-mediated self-renewal of neural
M. Yashar S. Kalania,b,c,1, Samuel H. Cheshierc,d,1, Branden J. Cordd, Simon R. Bababeygyb, Hannes Vogele,
Irving L. Weissmanb,c,d,e,2, Theo D. Palmerd, and Roel Nussea,b,c,2
aHoward Hughes Medical Institute, Departments ofbDevelopmental Biology,dNeurosurgery andePathology, andcInsitute of Stem Cell Biology and
Regenerative Medicine, Stanford University School of Medicine, Stanford, CA 94305
Contributed by Irving L. Weissman, September 17, 2008 (sent for review June 16, 2008)
In this work we have uncovered a role for Wnt signaling as an
We identified Wnt-responsive cells in the subventricular zone of the
developing E14.5 mouse brain. Responding cell populations were
enriched for self-renewing stem cells in primary culture, suggesting
that Wnt signaling is a hallmark of self-renewing activity in vivo. We
also tested whether Wnt signals directly influence neural stem cells.
Using inhibitors of the Wnt pathway, we found that Wnt signaling is
required for the efficient cloning and expansion of single-cell derived
populations that are able to generate new stem cells as well as
neurons, astrocytes, and oligodendrocytes. The addition of exoge-
nous Wnt3a protein enhances clonal outgrowth, demonstrating not
only a critical role for the Wnt pathway for the regulation of neuro-
genesis but also its use for the expansion of neural stem cells in cell
culture and in tissue engineering.
neurons, astrocytes, and oligodendrocytes that make up the func-
tioning brain. Several studies have suggested that these precursor
cells are able to self-renew, a hallmark of stem cells, and that
renewal maintains a reservoir of stem cells throughout life (1). In
maintenance, proliferation, and neuronal fate commitment of the
local stem cell populations. These signals and the microenviron-
for limiting concentrations of growth factors, thereby maintaining
a balance between self-renewal and differentiation of the cells.
Factors that regulate renewing versus differentiating cell divisions
strongly influence the stem cell pool size.
While much effort has been devoted to understanding the
development of the central nervous system in both the embryonic
and adult settings, the identity of the signals regulating stem cell
activity and neurogenesis is largely unknown. Identifying these
factors may increase opportunities to regulate neurogenesis and
stem cells in culture, a prerequisite for tissue engineering.
Wnt signaling and Wnt proteins are important for the mainte-
nance of stem cells of various lineages. The classic example is in the
digestive tract, where in the crypt of the colon the loss of transcrip-
tion factor TCF4 leads to depletion of stem cells (2, 3). The Wnt
pathway has also been implicated as a self-renewal signal in the
hematopoietic system (4, 5). Alternatively, loss of the tumor sup-
pressor APC or gain of ?-catenin activity leads to deregulated
self-renewal and cancer (6, 7).
In the nervous system, the anatomical phenotypes of mouse Wnt
mutants suggest that Wnts are involved in regulating neural stem
and progenitor cell activity. Loss of Wnt1 results in malformation
of most of the midbrain and some rostral metencephalon (8), and
because of lack of proliferation (9). Recent work demonstrating
enhanced neurogenesis in vivo via exogenous expression of Wnt3a
via lentiviral vectors strengthens the model that the Wnt signaling
pathway is a major regulator of adult stem cell activity and fate in
uring the development of the nervous system, primitive
neurectodermal stem cells act as a source for the specialized
and Walsh shows that continuous Wnt signaling results in marked
and generalized hypercellularity of the brain (11).
While these studies have indicated an important role for Wnt
signaling in the control over stem cells, they bring up a number of
important questions. Where are the Wnt responsive cells located
relative to the known neurogenic zones? Is Wnt responsiveness a
hallmark of neural stem cells that enables prospective enrichment
for self-renewal? What is the direct effect of Wnt signals on neural
stem cells: is it mitogenic or does Wnt control the symmetry of fate
in two daughter cells (e.g., self-renewal)? Are Wnt proteins by
themselves sufficient to act as a signal for single stem cells in
isolation or does Wnt act through indirect mechanisms? Herein we
on the fate decision of neural stem cells both in vitro and in vivo,
and use purified soluble Wnts as tools to expand and manipulate
neural stem cells in culture.
Results and Discussion
The Axin2-LacZ Reporter Visualizes Wnt Signaling in the Developing
is expressed in response to Wnt signaling (12). Insertion of a
?-galactosidase gene into the Axin2 locus (Axin2-LacZ) provides a
useful tool for visualizing cells that are actively responding to Wnt
does not lead to a detectable phenotype in the heterozygous state
(13). The pattern of endogenous Wnt pathway activation in the
developing mammalian CNS has not been previously reported.
Thus, we isolated embryos from heterozygous Axin2-LacZ mice at
embryonic day 14.5 (E14.5) and stained tissues with an anti-?-
Wnt-responsive LacZ-positive cells were found scattered
throughout the cortex and white matter tracks, consistent with
known Wnt signaling in differentiated cells. In addition, a
small subpopulation of cells (1–5%) in the subventricular zone
(SVZ) expressed the reporter gene (Fig. 1). Morphologically,
Axin-2 expressing cells in the SVZ resembled radial glial cells
with bipolar morphology and end feet contacting the ventric-
ular and pial surfaces (see Fig. 1 C). Cells with radial glial
morphology within the SVZ have been proposed as the central
nervous system stem cells (1).
Wnt Proteins and Neural Colony Formation. Anexvivoapproachwas
used to directly determine if and how Wnt signaling influences
Author contributions: M.Y.S.K., S.H.C., T.D.P., and R.N. designed research; M.Y.S.K., S.H.C.,
analytic tools; M.Y.S.K., S.H.C., B.J.C., H.V., I.L.W., T.D.P., and R.N. analyzed data; and
M.Y.S.K., S.H.C., T.D.P., and R.N. wrote the paper.
The authors declare no conflict of interest.
Freely available online through the PNAS open access option.
1M.Y.S.K. and S.H.C. contributed equally to this work.
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2008 by The National Academy of Sciences of the USA
November 4, 2008 ?
vol. 105 ?
neural stem cells. Primary cultures of neural stem and progenitor
cell types, ranging from the most immature self-renewing neural
stem cell to fully committed neuronal and glial progenitor cells. In
cell culture, the most stringent test for the presence of stem cells is
to ask whether single cells can expand into colonies and whether
these colonies include progeny that retain the ability to form new
clonally derived colonies, each of which can produce neurons,
astrocytes, and oligodendrocytes. The formation of secondary or
tertiary colonies indicates that self-renewing stem cells were pro-
were multipotent, with the capacity to generate progeny that
differentiate into neurons, astrocytes, and oligodendrocytes.
A limitation of such colony-forming assays is the inability to
distinguish between colonies derived from a single cell versus
aggregation of multiple cells. To avoid this caveat, we used
mixtures of cells isolated from the forebrains of E14.5 mice
from a ?-actin GFP (where GFP is expressed in every cell) and
a non-GFP mouse of the same background strain to determine
the plating density required to produce spheres of clonal
origin. As expected, mixed colonies became less common as
cell density was decreased. At cell densities greater than 2
cells/?l within a well of a 96-well plate (200 cells per well), we
found that 85% of the colonies contained either green or white
cells and ?15% contained mixed phenotype. At cell densities
equal to or less than 2 cell/?l (1–200 cells per well), fewer than
5% of the colonies were mixed, suggesting that the majority of
colonies formed under these conditions were clonally derived
Endogenous Wnt Signaling Is Present in Clonally Derived Colonies.
Cells were isolated from Axin2 reporter mice and plated at clonal
density (0.5–1 cells/?l) and the resulting colonies evaluated for
expression of the Wnt reporter. Using quantitative PCR, we
detected the RNA transcript of several members of the Wnt family
(data not shown). Even without the addition of exogenous Wnt,
2 D), and that upon the formation of a multicellular colony some
cells commit to producing Wnts while others gain the ability to
respond to the signal. Addition of exogenous Wnt resulted in many
more cells responding to the Wnt signal (see Fig. 2 E), while
inhibition of the pathway with Dkk abolished the Axin2-reporter
expression, suggesting complete inhibition of Wnt signaling within
these colonies (see Fig. 2 F).
Wnt Protein Increases Stem Cell Cloning Efficiency in Vitro.Tofurther
explore the effects of exogenous Wnt protein on colony formation,
unsorted primary cells were isolated from the forebrain of E14.5
mice and plated at three cellular densities ranging from limiting
dilutions, where many wells failed to generate colonies (0.2 cell/?l
one colony. Wnt and Dkk proteins were added to determine the
effect of elevated or reduced Wnt signaling on efficiency of colony
formation. Adding Wnt protein (1 ng/?l) increased the number of
colonies at all cell densities relative to the control condition (Fig. 3
A). The addition of soluble Wnt protein resulted in an average
Using linear regression to estimate the abundance of clonogenic
cells in the total population (Fig. 3 B), we found that under control
conditions, roughly one colony was formed for every 395 cells
plated (a frequency of 0.003 clones per plated cell, R2? 0.99).
When Wnt was added to the medium, the incidence increased to
0.005 or one colony in 205 cells (R2? 0.99), and when endogenous
Wnt signaling was blocked by the addition of Dkk1c, colony
frequency dropped to 0.001 or roughly one colony per 1,650 cells
(R2? 0.98). This indicates that endogenous Wnt signaling is
necessary for efficient colony formation and that exogenously
added Wnt can further increase cloning efficiency.
Axin2-Expression in Vivo Is Predictive of Self-Renewing Properties in
Axin2-LacZ mice and enriched by flow cytometry for the presence
or absence of expression of the Axin2-LacZ reporter (14). Roughly
9% of the total cell population from the E14.5 brain expressed the
Axin2-LacZ reporter (Fig. 3 C). Sorted LacZ-positive cells, LacZ-
colony formation (0.5–2 cells/?l). Cultures were maintained for 10
to 14 days and the number of primary colonies formed was scored.
Primary colonies were then dissociated and replated into identical
conditions to assay potential change in the abundance of colony
forming cells per total cell population. There was no significant
effect of in vivo Axin2-expression on the frequency of colony-
forming cells in primary culture (f ? 0.6, P ? 0.6 for effect of
Axin2-phenotype over the combined experiments by MANOVA,
with no significant interaction between phenotype and Wnt or Dkk
treatment, f ? 1.3, P ? 0.3). Furthermore, the addition of Wnt had
only slight effects on primary colony formation for each Axin2
phenotype, with an increase approaching significance only in un-
sorted primary colony cultures (P ? 0.06, t-test, n ? 3). Otherwise,
Wnt effects did not reach significance for any of the sorted primary
Low magnification view (10?) of the lateral ventricle of the reporter mouse (Scale bar, 100 ?m); (B) DAPI marking the nuclei of cells lining the ventricle (40?
shows the population of cells that are Wnt-responsive.
A small subpopulation of cells (1–5%) in the ventricular and subventricular zone of E14.5 Axin2-LacZ embryos show expression of the Wnt reporter. (A)
Kalani et al.
November 4, 2008 ?
vol. 105 ?
no. 44 ?
3 D). Upon secondary plating, however, Axin2? cells had an
increased ability to generate colonies when plated in the presence
of Wnt protein relative to Axin2? cells plated in the presence of
Wnt (P ? 0.09). Even in the absence of exogenous Wnt protein,
Axin2? cells show increased secondary colony formation relative
to Axin2? cells (P ? 0.09).
Contrasting the specific effects of Wnt or Dkk on colony forma-
tion in primary versus secondary cultures suggests that the gener-
ation of colonies in the presence of Wnt encourages
the accumulation of Wnt-responsive neural stem cells (Fig. 3 E).
The efficiency of secondary colony formation (colonies per cell
plated) in vehicle- versus Wnt-treated cultures is substantially
increased when primary cultures were treated with Wnt and
substantially decreased when primary cultures were treated with
Dkk. Primary cultures generate colonies roughly two times more
efficiently in the presence of Wnt (see Fig. 3 A and E), while
secondary colonies from these Wnt-treated cultures show nearly
10-fold increases in colony formation in the presence of Wnt versus
vehicle (P ? 0.03, n ? 3, t-test), suggesting a nearly 5-fold increase
in the abundance of Wnt-responsive neural stem cells. Dkk-treated
cultures showed nearly 10-fold depletion of colony-forming cells
(P ? 0.01, n ? 3, t-test), suggesting that Wnt signaling is both
the selective increase in the fraction of neural stem cells present in
the resulting cell populations.
Wnt Treatment Results in the Generation of Morphologically Unique
Primary Clones. Wnt treatment also influenced the size of colonies
formed in primary culture. Contrary to expectation, Wnt-treated
cells generated primary and secondary colonies that were morpho-
logically smaller and more homogenous than the colonies treated
with vehicle or Dkk (Fig. 3 F–H). The average size of Wnt-treated
colonies was 177 ?/? 15 ?m (sem, n ? 3), nearly half the size of
the average vehicle-treated colony. The Dkk colonies were not
significantly different from vehicle-treated colonies, with an aver-
age size of 300 ?/? 25 ?m (sem, n ? 3) (see Fig. 3 F–G).
Vehicle-treated colonies had a variable range of sizes, containing
both small and large colonies. Cells that were already expressing
Axin2 in vivo (e.g., those sorted for Axin2-LacZ expression) also
or by virtue of the addition of exogenous Wnt, limited colony
growth in the 10 to 14 days after plating. Colony size and number
plated directly, presumably because of the physiological and shear
stresses of the sorting process itself.
Colonies Formed in the Presence of Wnt Contain Cells that Differen-
tiate into Neurons, Astrocytes, or Oligodendrocytes. To confirm that
colonies generated under Wnt conditions retained the ability to
generate both neuronal and glial progeny, cells were isolated from
the E14.5 forebrain, harvested, and cultured at clonal density (1
cell/?l) for 10 to 14 days in the presence of Wnt. BrdU was added
at a concentration of 5 ?moles per liter overnight to label dividing
cells. Colonies were then transferred to laminin-coated chamber
slides and cultured for an additional 1 to 2 weeks in differentiation
neurons (doublecortin), oligodendrocytes (NG2), and astrocytes
(glial fibrillary acidic protein, GFAP), as well as nuclei labeled with
BrdU. Cells treated at clonal density with the Wnt protein were
indeed capable of generating neurons, astrocytes, and oligoden-
drocytes, and each was labeled with BrdU, confirming that the
differentiated cells were derived from proliferative progenitor cells
e i n
o l o
0.51210cells / ul
c l tc l t
c l t
c l t
?-actin GFP and a non-GFP mouse, we ascertained that at cell densities of 2 cells/?l or less, the colonies formed are predominantly a single phenotype resulting from
clonal expansion of a single cells. (B) At higher cell densities (?5 cells/?l) the colonies are formed from cells of each phenotype and represent oligoclonal rather than
cell density. Treatment with Wnt or Dkk did not alter incidence of mixed colonies. At cell densities equal to or less than 2 cells/?l, the colonies generated are less than
to the Wnt signal. (F) Addition of purified Dkk, a Wnt inhibitor, abolishes the intrinsic Wnt signal leading to an absence of detectable Lac-Z-positive cells.
Cell mixing experiments were used to determine cell densities at which clonal colony formation occurs. (A) By mixing equal numbers of cells from an E14.5
www.pnas.org?cgi?doi?10.1073?pnas.0808616105Kalani et al.
The Axin2-LacZ mouse strain is a line in which the Wnt target
gene Axin2 is mutated by insertion of LacZ. Axin2 is a negative-
feedback regulator of Wnt signaling and, like negative feedback
regulators in other pathways (Patched), Axin2 expression is a
faithful readout of the Wnt signal in numerous tissues. Using this
reporter in the heterozygous state, we show that there is a small
subpopulation of Wnt-responsive cells lining the neurogenic zone
(SVZ) in the developing (E14.5) mouse brain. The location of the
Wnt-responsive cells correlates well with established neurogenic
zones (1). We further note that these cells at the SVZ have the
morphology of radial glia, with foot processes lining the ventricle.
A great deal of literature supports the role of the radial glia cell as
the stem cell population in the central nervous system (1).
Wnt Signaling in Vivo.ThepresenceofWntsignalingwithintheSVZ
suggests a role for Wnt in neural development, and in this work we
have shown that exogenous Wnt protein promotes the clonal
expansion of stem cell populations that can generate neurons,
astrocytes, and oligodendrocytes. Moreover, using a Wnt inhibitor,
we established that Wnt is required for self-renewal and expansion
of stem-like cells in primary culture. The Axin2 reporter system
shows that stem cell colonies generated in vitro spontaneously
activate endogenous Wnt signaling and that Dkk can abrogate this
activation. The addition of Wnt protein resulted in more cells
responding to the Wnt signal. The duration of Wnt treatment was
too short to allow for specific expansion of Wnt-responsive cells,
thus suggesting that many cells are capable of responding to a Wnt
signal but that few cells are presented with the signal. It is well
known that primary neural stem cell cultures are heterogeneous,
containing both stem cells and differentiated cells. In this respect,
neural stem cell colonies and neurospheres can be viewed as
self-organizing structures, with multiple cell types sustaining each
other by intercellular signaling. We show here that Wnt protein is
important to maintain stem cells, the self-renewing subpopulation
of progenitor cells that make up these complex structures.
r e t t a
d r a
n i - k
r o t n
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c e l c i h
v o t e
v i t a l e r s
e i n
o l o
c # (
V W D
V W D
V W D
V W D
V W D
V W D
e i c i f f e g
r o f y
o l o
) l l e
c / s
e i n
o l o
0 200 400 600
l l e
e i n
o l o
cells / well
Ctl Wnt3A Dkk1c
l l e
c / s
e i n
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isolates of neural stem cells shows that Wnt treatment increased the efficiency of colony formation at all cell densities. Dkk inhibited the production of colonies at all
forming ability (Wnt3a), while Dkk results in a fivefold depletion in the ability to form primary colonies. Relative abundance of stem cells in populations is enriched
of cells in the preparation were Wnt-responsive. (D) Cells were sorted and plated into 96-well plates and colony-forming efficiency (number of primary colonies per
cell plated) was measured in each population under control (vehicle, V)-, Wnt (W)-, or Dkk (D)-treated conditions. Primary colonies were dissociated and replated to
control conditions. Dkk-treated cells generated fewer colonies, which were similar in size to the larger colonies in the control condition. (G) Estimation of colony
diameter shows significant differences in colony size between control- and Wnt-treated cells in primary and secondary colonies (**, P ? 0.0001). The size difference
between vehicle and Dkk-treated colonies were not statistically significant for either primary or secondary colonies (P ? 0.2, 0.7 respectively). (H) Cells that expressed
showed an intermediate mean colony size.
Kalani et al.
November 4, 2008 ?
vol. 105 ?
no. 44 ?
The Role of Wnt Protein in Neural Stem Cell Cultures. Our experi-
mental approach was based on neural stem cell colonies initiated
from single cells. Under conditions that favor the generation of
clonal colonies, we find that the addition of Wnt protein caused an
could be because of: (i) increased survival of neural stem cells and
more efficient colony initiation; (ii) proliferative amplification of
neural stem cells to increase their abundance; or (iii) decreased
intercellular adhesion of cells that are dividing within the newly
forming colony and seeding of secondary colonies. The fact that
stem cells cultured in the presence of Wnt increase the abundance
10-fold (see Fig. 3 E) indicates that Wnt does indeed stimulate an
increase in the abundance of neural stem cells through self-
Studies of hematopoesis suggest that a hierarchy may exist
between self renewal and proliferative amplification, with the most
primitive subset of stem cells cycling slowly but with the greatest
self-renewal capacity and the more differentiated progenitors hav-
It should be noted in the present studies, Wnt by itself is not
sufficient for amplifying self-renewal in neural stem cells because
FGF and EGF are also required in the cell-culture assays. Wnt
cells by roughly 10-fold; however, the colonies formed under Dkk
conditions were just as large as those generated in control condi-
tions, suggesting that inhibition of Wnt affects self-renewing divi-
sions but does not hinder transiently amplifying divisions of the
more differentiated progenitors.
Observing Wnt reporter-positive cells in vivo, we asked whether
these cells are enriched for stem cells by FACS isolation and by
culturing them in the presence of the factors. In primary colony
expressing versus nonexpressing cells. Axin2? cells grown in the
presence of Wnt protein and replated in the presence of Wnt
protein did exhibit increased self-renewal, suggesting that Wnt
protein can be used to enrich self-renewing stem cells in culture. In
colony formation relative to the Axin2? counterparts, again sug-
that these Wnt-responsive cells formed small colonies, consistent
with the effects of exogenously added Wnt on the population as a
Wnt proteins have diverse effects on different stem cell popu-
lations; while they inhibit neural differentiation and maintain
pluripotency in embryonic stem cells (ESCs) (7, 22), they can also
promote differentiation in other progenitor populations (17). Ex-
periments using a TCF-dependent reporter gene in differentiating
cortical neurons during development suggests that canonical Wnt
signaling is involved in the differentiation process (18), and our
observations with the Axin2 reporter mouse show that mature
a continuing role for Wnt in adult neural function [supporting
information (SI) Fig. S1]. Wnt3a has been reported to promote
differentiation into the neural and astrocyte lineage by inhibiting
neural stem cell maintenance (19). Additionally, Wnt7a signaling
has been shown to induce differentiation in neural stem cells of the
neocortex (20), reducing the neural progenitor cell pool at late
developmental stages (E13.5), whereas neural stem cells at earlier
developmental stages (E10.5) are not affected by the Wnt treat-
Wnt pathway in neural stem cell maintenance and expansion. The
a Wnt signal is necessary for the maintenance of stem cell popu-
lations giving rise to the midbrain (21). Chenn and Walsh used a
transgenic mouse expressing a stabilized ?-catenin in neural pre-
cortical surface area and folds resembling sulci and gyri of higher
mammals. Brains from transgenic animals have enlarged lateral
ventricles lined with neuroepithelial precursor cells, reflecting an
expansion of the precursor population (11). A recent article from
Lie et al. implicates Wnt3a in promoting neurogenesis in the adult
The observation that the pathway is active in radial cells of the
ventricular zone at E14.5, the ability of the factor to result in
expansion of self-renewing cells in vitro, and the observation that
blockage of the pathway results in depletion of neural stem cells in
in central nervous system stem cells. Interestingly, although Wnt-
treated neural stem cells formed a greater number of colonies with
greater replating efficiency, the absolute number of cells produced
was lower than vehicle or Dkk-treated cells (data not shown),
suggesting that Wnt3a acts at the level of self-renewal rather than
as a mitogen. The ability to manipulate these cells in culture with
essential signaling molecules allows for a strategy to both dissect
niche effects and selectively manipulate cells in culture to produce
selective cell profiles useful in cell transplantation and other
Materials and Methods
All research was approved by the Stanford University committee on animal
medium: Neurobasal-A, penicillin/streptomycin (BioWhittaker), and 250 U/ml
conditions on laminin-coated chamber slides. After 1 to 2 weeks, the differentiated cells were fixed and stained to detect neurons, oligodendrocytes, and astrocytes.
Clonal-density colonies yielded cells capable of generating astrocytes (GFAP, green in A), neurons (doublecortin, white in A) and oligodendrocytes (NG2, green in B).
BrdU incorporation (red) is detected in the nuclei (blue) of the differentiated neurons and glia (arrows for each inset), indicating that the differentiated cells were
derived from proliferative stem cells that were dividing in vitro, and not simply contaminating postmitotic cells from the fetal brain.
www.pnas.org?cgi?doi?10.1073?pnas.0808616105 Kalani et al.
DNase I (DNAse1, Sigma D-4527), 2.5 U/ml papain, and 1 U/ml dispase II (Worth-
ington Chemicals). After centrifuging at 500 ? g, the cells were triturated with
pipettes of various calibers, filtered through nylon screen (40-?m filter) (BD
Falcon), counted by hemocytometer, and plated.
Cell Culture. Nonadherent cultures of CNS stem cells were performed by plating
cells on ultra nonadherent 96-well plates (Corning Incorporated). In all cases the
culture medium was based on a Neurobasal-A medium. The medium was sup-
plemented with 20-ng/ml recombinant human bFGF (R&D Systems), 20-ng/ml
recombinant mouse EGF (R&D Systems), 2% B27 without vitamin-A supplement
penicillin/streptomycin (Biowhittaker). All cultures were maintained at 37°C in
5% CO2/balance air.
Assay for Neural Stem Cell Colony Formation. Cells derived from forebrain
cultures as described above or sorted from the forebrains of reporter mice were
plated into 96-well plates at various cell densities (0.1–5 cell/?l) to evaluate
directly the clonal frequency of precursors that initiate colonies. Each well con-
tained 100 ?l of the above mentioned media. EGF, bFGF, B27, and vehicle, Wnt,
original concentrations by adding the appropriate concentrations of EGF, FGF,
and B27 in a small volume to the well. Halfway through the experiment, half of
the media was carefully removed from each well and replenished with fresh
media, with the growth factors adjusted to the proper concentrations. Plates
were scored for neurosphere growth blinded to each condition using phase-
of colony formation (22, 23).
Cell Mixing Experiments. Cells were isolated from the forebrain of E14.5 mice
embryos from ?-actin GFP:C57/BL6 and non-GFP:C57/BL6 mice as above. These
as described above. Cells were mixed at equal quantities to produce cell concen-
200 or 2 cells/?l (100 GFP?, 100 GFP?) and 1,000 or 10 cells/?l (500 GFP?, 500
GFP using phase-contrast/fluorescent and confocal microscopy.
Differentiation Conditions. Neural stem cells were harvested either clonally at a
tiation medium [N-acetylcsteine, brain-derived neurotrophic factor (10 ng/ml)
glial-derived neurotrophic factor (10 ng/ml), EFG and FGF (2 ng/ml)] on laminin-
coated chamber slides. After 1 to 2 weeks, chamber slides were fixed with 4%
paraformaldehyde in PBS and stained to detect differentiation into neurons,
oligodendrocytes, and astrocytes, and retention of any progenitors with mAbs
against doublecortin (1:800; Chemicon), NG2 (1:500, Chemicon), glial fibrillary
1,000, Promega). In all cases, cells were counterstained for 10 min at room
labeling experiments, 5 ?mol/liter final concentration of BrdU was added to the
cells overnight. It was subsequently washed out and the cells were allowed to
was accomplished by antigen retrieval by HCl treatment, followed by using an
antibody against BrdU (1:500; Chemicon) and a fluorescent secondary.
Protein Purification. Wnt3a Purification-Wnt3a protein was purified from 6.5
created in the laboratory as previously described (5).
by 293 cells stably over-expressing mouse Dkk1c protein as described in (24).
in 4% paraformaldehyde for 5 days followed by embedding in cryoprotectant
(1? PBS with 25% glycerin and 25% ethylene glycol at pH 6.7 and stored at
?20°C). They were subsequently sectioned and stained with the same antibody
concentrations as described above.
Cell Sorting. Cells were incubated with a fluorescent marker against ?-Galacto-
ogies, Inc.) at a dilution of 1:50 and incubated at 37°C for 30 min before sorting
on a Beckman Aria FACS-sorter. Background levels of staining were determined
by exposing neural stem cells from nonreporter mice of the same strain to the
ACKNOWLEDGMENTS. M.Y.S.K. is a fellow of Paul & Daisy Soros, the Howard
Hughes Medical Institute (HHMI), the Hanbery Society, and Stanford University
and California Institute of Regenerative Medicine (RC1–00133–1).
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