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
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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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