Stem Cell Reports
Ar ticle
Replicable Expansion and Differentiation of Neural Precursors from Adult
Canine Skin
Thomas Duncan,1 Aileen Lowe,1,2 Kuldip Sidhu,2 Perminder Sachdev,3,4 Trevor Lewis,5 Ruby C.Y. Lin,5
Vladimir Sytnyk,6 and Michael Valenzuela1,*
1Regenerative
Neuroscience Group, Brain and Mind Centre, University of Sydney, Sydney, NSW 2050, Australia
Cell Laboratory, University of New South Wales, Sydney, NSW 2031, Australia
3Centre for Healthy Brain Ageing, University of New South Wales, Sydney, NSW 2031, Australia
4School of Psychiatry, University of New South Wales, Sydney, NSW 2052, Australia
5School of Medical Sciences, University of New South Wales, Sydney, NSW 2052, Australia
6School of Biotechnology and Biomolecular Science, University of New South Wales, Sydney, NSW 2052, Australia
*Correspondence: michael.valenzuela@sydney.edu.au
http://dx.doi.org/10.1016/j.stemcr.2017.07.008
2Stem
SUMMARY
Repopulation of brain circuits by neural precursors is a potential therapeutic strategy for neurodegenerative disorders; however, choice of
cell is critical. Previously, we introduced a two-step culture system that generates a high yield of neural precursors from small samples of
adult canine skin. Here, we probe their gene and protein expression profiles in comparison with dermal fibroblasts and brain-derived neural stem cells and characterize their neuronal potential. To date, we have produced >50 skin-derived neural precursor (SKN) lines. SKNs
can be cultured in a highly replicable fashion and uniformly express a panel of identifying markers. Upon differentiation, they self-upregulate neural specification genes, generating neurons with basic electrophysiological functionality. This unique population of neural
precursors, derived from mature skin, overcomes many of the practical issues that have limited clinical translation of alternative cell
types. Easily accessible, neuronally committed, and patient specific, SKNs may have potential for the treatment of brain disorders.
INTRODUCTION
Profound neuronal and synaptic loss is a hallmark of neurodegenerative disorders and is the best pathological correlate
of cognitive impairment in Alzheimer’s disease (AD) (Terry
et al., 1991; Scheff and Price, 2006). Restoration of depleted
neuronal populations with stem cell therapy is a promising
treatment strategy (Daley, 2012; Lindvall et al., 2012; Duncan and Valenzuela, 2017), but the inaccessibility of endogenous neural stem cells within the brain’s neurogenic
niches means that alternative cell sources are required, preferably of autologous origin, to avoid long-term immunosuppression, which is harmful to both graft and host.
Reprogramming technology transformed the field, converting fibroblasts (FBTs) and other somatic cells into
induced pluripotent stem cells (iPSCs) (Takahashi and Yamanaka, 2006) capable of generating functional neurons in vivo
(Wernig et al., 2008). Alternatively, somatic cells can be
converted directly into induced neurons (iNs), in effect bypassing the pluripotent state by induced upregulation of
neuronal specification genes ASCL1, NEUROD, MYTL, and
BRN2 (Pang et al., 2011; Pfisterer et al., 2011). However, clinical translation of reprogramming technology remains
limited for a number of reasons. First, conversion efficiency
is typically very low, <1% of initial cells for iPSCs (Liao et al.,
2008) and <6% for iNs (Pang et al., 2011). Second, both cell
types suffer from unacceptably high line-to-line (and even
clone-to-clone) variability (Truong et al., 2016), a factor
that precludes clinical translation. Third, the unlimited ca-
pacity of iPSCs to self-renew presents an inherent risk of
uncontrolled cell growth in vivo, with high (60%) tumor
rates reported (Ring et al., 2012). Finally, genetic modification raises significant safety concerns, including activating
oncogenes and the risk of unanticipated mutagenesis.
The discovery of niches within adult skin that continue
to harbor multipotent stem cells raised the prospect of
overcoming some of these limitations. Cell populations
within these niches exhibit stem cell-like properties,
distinct from mesenchymal or hematopoietic stem cells,
and retain a neurogenic developmental potency without
genetic manipulation, including expression of neural
stem cell-related proteins such as NESTIN and SOX2 in
situ (Toma et al., 2001; Fernandes et al., 2004). Using a
neurosphere propagation method, these native stem celllike cells, termed skin-derived precursors (SKPs), can be
expanded for multiple passages (>50), and mature into
neural cell types when exposed to neurodifferentiation factors (Toma et al., 2001; Biernaskie et al., 2006; Lavoie et al.,
2009). However, the final neuronal yield achieved by this
approach has been very low (2%–10% across studies), and
the propensity for glial cell differentiation (Toma et al.,
2005; Hunt et al., 2008) has generally excluded translation
of SKP cells to neuronal therapeutic application.
Responding to this, we previously reported enhanced
cellular homogeneity and neurogenic potential using a
two-step neurosphere-adherent culture system that begins
with mature adult canine skin (Valenzuela et al., 2008).
The complex three-dimensional growth environment and
Stem Cell Reports j Vol. 9 j 557–570 j August 8, 2017 j ª 2017 The Authors. 557
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
uneven exposure to growth signals inherent in the neurosphere assay permits and promotes heterogeneous cell
growth (Bez et al., 2003; Babu et al., 2007), with neural
stem cells reported to represent less than 1% of this population (Reynolds and Rietze, 2005). By contrast, we used
neurospheres solely as a primary selection step, further
expanding the resultant cells as an exclusively epidermal
growth factor (EGF)/basic fibroblast growth factor
(bFGF)-dependent adherent monolayer culture. Adherent
expansion of human SKPs has been reported previously
(Joannides et al., 2004), and while better neuronal yields
were achieved using serum and astrocyte-conditioned
medium, glial and mesenchymal cell types remained
commonplace. Adherent culture systems have also been
employed to expand brain-derived neural stem cells, producing more homogeneous cell populations biased toward
GABAergic and glutamatergic neurons (Conti et al., 2005;
Pollard et al., 2006; Goffredo et al., 2008). Combining these
approaching in our two-step serum-free culture system,
a unique population of skin-derived neural precursors
(SKNs) can be routinely generated from adult canine skin,
maturing to produce greater than 90% neuronal yields
in vitro without genetic manipulation (Valenzuela et al.,
2008). Accordingly, SKNs represent a promising candidate
for autologous neural cell therapy.
Our choice of studying canine skin was intentional
because of the poor history of translation of rodent
research into effective human neurodegenerative treatment. Rodents do not naturally develop AD pathology or
neurobehavioural signs in late life, and transgenic models
have failed to predict outcomes in human clinical trials
(Cummings et al., 2014; Breitner, 2015). By contrast,
canine cognitive dysfunction (CCD) is a naturally occurring analogue of human AD; affected dogs display a progressive amnestic syndrome (Cummings et al., 1996; Salvin
et al., 2011) as well as AD pathology (Cummings et al.,
1996), and, as in humans, prevalence accelerates exponentially in old age (Salvin et al., 2010). CCD may therefore be
an ideal translational model to test regenerative therapies.
Yet prior to this, any candidate cell type needs thorough
characterization. Here, we therefore assess the line-to-line
replicability and neurogenic potential of canine SKNs in
comparison with both canine dermal FBTs and brainderived neural precursor cells (NPCs) isolated from the
canine neurogenic niche.
RESULTS
SKNs Are Isolated and Expanded Using a Clinically
Replicable Protocol
Our protocol for the culture of canine SKNs combines
initial neurosphere selection with passage as an adherent
558 Stem Cell Reports j Vol. 9 j 557–570 j August 8, 2017
monolayer for cellular homogenization (Figure 1A). From
an approximately 6 cm2 abdominal skin sample, under
proliferative conditions, floating neurospheres (z100 mm
in diameter) formed in culture within 7 days of isolation
(Figure 1B). Following selection of these neurospheres,
and their enzymatic dissociation, the resulting cells were
cultured as an adherent monolayer. A large amount of
cell death occurred within the first 2 days of this adherent
culture, but a selective population consisting of exclusively
EGF/bFGF-maintained SKNs survived. Following first passage, approximately 1,000,000 SKNs could be routinely
generated from over 50 individual skin donors (85%
donor-wise success rate). The clonal ability of SKNs was
demonstrated by seeding at single-cell density in a collagen
matrix that precluded cell fusion, with spheres approximately 50 mm in diameter forming after 7 days of culture
(Figure 1C).
SKNs Demonstrate Limited Proliferative Capacity
In Vitro
Under our adherent culture conditions, a homogeneous
SKN population could be routinely expanded for up to
five passages. At first and second passage, 80% confluence (passage density) was reached within 7 days, undergoing an average of 2.7 population doublings, defined as
[log(final cell count/starting cell count)]/log(2) (Greenwood et al., 2004). Following third passage, proliferative
rates began to decline, with only 1.4 population doublings occurring within an equivalent time frame. This
decline was also quantified by 5-ethynyl-20 -deoxyuridine
labeling of actively dividing cells. Initially comparable
levels of active proliferation in SKNs and brain-derived
NPCs significantly declined in the SKN cultures at passage 3 and beyond (Figure 1D). This observation was
also consistent with SOX2 gene expression, a regulator
for stem cell proliferation, where expression in SKNs
was significantly lower than in NPCs (*p = 0.01; Figure 1E). Under adherent proliferative conditions, SKNs
are therefore limited in terms of their proliferative
capacity.
SKNs Are a Distinct Population from Dermal FBTs
Global transcriptome analysis was carried out on canine
dermal FBTs, SKNs, and NPCs, in order to better understand
the neural potential of SKNs and the general relationship
between these three cell types. Principle-component analysis of 32,000 transcripts accounted for 60.1% of total transcriptomic variance, with three cell populations observed,
each with distinctive expression profiles (Figure 2A). Cell
lines within cell types were highly correlated (r R 0.98
SKN, r R 0.98 NPC, r R 0.98 FBT), while globally SKN
and FBT lines were more closely correlated (r range: 0.96–
0.97) than SKN and NPC lines (r range: 0.92–0.94).
Figure 1. SKNs Are Rate Limited In Vitro
(A) SKN culture protocol and timing of
differentiation.
(B) Representative morphology of adult
canine SKNs and NPCs at the neurosphere
stage (top) and during subsequent adherent
monolayer expansion (bottom). Scale bars,
50 mm.
(C) Clonal proliferation from a single SKN
cell. Scale bars, 50 mm.
(D) SKNs exhibit significantly lower expression of cell division marker 5-ethynyl-20 deoxyuridine (EDU) at passage 3 and beyond
compared with adult NPCs (P3, *p = 0.02; P4,
**p = 0.008; P5, **p = 0.003; n = 3 independent experiments; mean ± SEM).
(E) Gene expression of master neural stem
cell regulator SOX2 in SKNs at passage 3
is intermediate between adult fibroblasts
(FBT) and NPCs (*p = 0.01; n = 3 independent experiments; mean ± SEM).
Correlation coefficients for individual cell lines are displayed in Table S1.
To elucidate whether underlying molecular profiles were
similar between these three cell types, ANOVA was performed at a false discovery rate of <0.05 to identify genes
with a greater than 2-fold difference in normalized expression between cell types. Of the 32,000 genes analyzed,
291 genes were differentially expressed between SKNs
and FBTs, while 2,157 genes were differentially expressed
between SKNs and NPCs (Figure 2B). These univariate
results indicate that SKNs are closer in global gene
expression to FBTs than NPCs. A multivariate method of
hierarchical clustering on differentially expressed genes
also placed SKNs in a closer relationship to FBTs than
NPCs (Figure 2C). Nevertheless, qPCR examination of specific neural stem and precursor markers showed that SKNs
retain a distinct gene expression profile to FBTs, with NES,
P75NTR, DCX, and TUBB3 all significantly upregulated
(Figure 2D). Moreover, from the Venn diagram we can
infer that there are at least 40 genes specific to SKNs
(detailed in Table S2). Many of these genes are associated
with cell proliferation, adhesion, migration, and cytoskeletal organization. Of particular note were genes coding
for protocadherins. These cell-adhesion proteins are predominantly expressed by the developing CNS (Sano
et al., 1993), and have key roles in establishing neuronal
connectivity and dendrite arborization (Schalm et al.,
2010; Lefebvre et al., 2012).
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Figure 2. Gene Expression Patterns of
SKNs Are Distinguished from FBTs or NPCs
(A) Principal components analysis of
global gene expression of SKN, FBT, and
NPC lines showed three distinct expression
patterns. Further analysis indicated that
gene expression from SKNs correlate closer
to gene expression from FBTs than NPCs. See
also Table S1 (n = 3 independent experiments; mean ± SEM).
(B) Univariate analysis shows SKNs and FBTs
to have 291 differentially expressed genes
and the least dissimilarities between cell
types. Forty genes were expressed exclusively by SKNs. See also Table S2.
(C) Hierarchical clustering of significant
differentially expressed genes based on
Euclidean distance implicated that SKNs are
globally closer to FBTs than NPCs.
(D) qPCR showed that SKNs have significantly higher transcript expression of neural
stem cell-like marker gene NES (**p =
0.003), neural crest gene P75NTR (**p =
0.03), neuroblast gene DCX (***p = 0.0005),
and immature neuronal gene TUBB3 (*p =
0.04) compared with FBTs (n = 3 independent experiments; mean ± SEM).
Transcript lists containing the most commonly cited
genes for pluripotency, neurogenesis, SKPs, mesenchymal
stem cells (MSCs), growth factors, and growth factor receptors were compared for expression intensity between SKNs
and NPCs (Figure 3). A full list of the individual gene
expression fold changes between SKNs and NPCs can be
found in Table S3. The iPSC pluripotency reprogramming
factor KLF4 (Takahashi and Yamanaka, 2006; Takahashi
et al., 2007), and the TBX3 gene implicated in embryonic
stem cell proliferation and neuroepithelial differentiation
(Esmailpour and Huang, 2012), were found to be upregulated in SKNs. Pluripotency markers BUB1, TCL1A, and
PRC1 were all upregulated in NPCs, confirming their
greater proliferative capacity, as reported in Figure 1D. As
expected, NPCs also showed stronger expression of neurogenesis markers. However, early neurogenesis markers
MKI67, PAX6, NCAM1, NES, and SOX2 were also strongly
560 Stem Cell Reports j Vol. 9 j 557–570 j August 8, 2017
expressed in SKNs, indicative of a largely undifferentiated
population of neural precursors. SKNs expressed the
majority of SKP and MSC markers more intensely
than NPCs, with significant upregulation of PDGFRA,
HOXA5, HOXC4, HOXC6, TWIST2, EYA1, MAB21L1,
MSX1, RHOBTB3, and AP2A1.
Growth factor and receptor gene expression were
explored in order to gain insight into factors that SKNs
may produce, or be responsive to, if transplanted in
the brain. Interestingly, genes for insulin-like growth factors and their receptors were highly expressed in SKNs
compared with NPCs. Furthermore, expression of IL7 and
VEGFC were upregulated in SKNs relative to NPCs. These
growth factors, while expressed in adult skin, also have
key roles in both the adult and developing brain, including
neurogenesis and synaptogenesis, and as migratory cues
for neuronal precursors (Michaelson et al., 1996; D’Ercole
Figure 3. Gene Expression Heatmaps for
NPC and SKN Lines
Heatmap of (A) pluripotency, (B) neurogenesis, (C) SKP and MSC, and (D) growth
factor- and receptor-associated genes
comparing NPC with SKN cell lines. Gene
expression fold changes are color coded
to visualize highly expressed genes. High
relative expression of multiple genes associated with pluripotency and neurogenesis
was evident in both SKN and NPC lines,
while many SKP and MSC genes were expressed to a greater degree in SKNs, indicative of their dermal origins.
et al., 2002; Rosenstein et al., 2010). As such, they
may facilitate SKN survival and engraftment following
transplantation.
Pathway enrichment was then carried out on the 2,157
differentially expressed gene list (SKNs versus NPCs) and
scored based on Fisher’s exact test (p < 0.01) to identify
functional groups with more than two over-represented
genes from a specific pathway. Genes were referenced
against the Kyoto Encyclopedia of Genes and Genomes
C. familiaris genome database (Kanehisa et al., 2004). The
Stem Cell Reports j Vol. 9 j 557–570 j August 8, 2017 561
Table 1. Pathway Enrichment Analysis of Differentially Expressed Genes in SKN Cell Lines Based on the Kyoto Encyclopedia of
Genes and Genomes Pathways
Pathway Name
Enrichment Score
Enrichment
p Value
Percentage of Pathway
Genes Represented
Focal adhesion
17.29
0.00000003
24
Extracellular matrix-receptor interaction
16.47
0.00000007
33
PI3K-Akt signaling pathway
11.90
0.000006
19
Proteoglycans in cancer
9.82
0.00005
30
Dilated cardiomyopathy
9.81
0.00005
20
Cell-adhesion molecules
9.57
0.00006
26
Actin cytoskeleton regulation
8.95
0.0001
23
Hypertrophic cardiomyopathy
8.89
0.0001
19
Keratan sulfate glycosaminoglycan biosynthesis
7.69
0.0004
26
Axon guidance
7.33
0.0006
47
Mitogen-activated protein kinase signaling pathway
7.16
0.0007
21
Pathways in cancer
4.95
0.007
17
Glycosphingolipid biosynthesis
4.81
0.008
15
Prostate cancer
4.66
0.009
30
Melanoma
4.61
0.01
20
PI3K, phosphatidylinositol 3-kinase.
aim was to characterize and compare the biological pathways best represented in both cell types. Focal adhesion,
extracellular matrix (ECM)-receptor interaction and PI3AKT signaling pathway were the three most enriched pathways (Table 1). The relative gene expression in SKNs and
NPCs for components of these pathways and others
have been mapped in Figures S1–S6. The focal adhesion
pathway (Figure S1), incorporating upregulated expression
of p21-activated kinase proteins in SKNs, has an important
role in the regulation of the actin cytoskeleton and is
implicated in cell proliferation and migration, mediating
filopodia formation, and cell motility (Xia et al., 2008).
Interlinked with the focal adhesion pathway, phosphatidylinositol 3-kinase-Akt signaling (Figure S2) was also
highly enriched in SKNs. An important regulator of the
cell cycle, this pathway has been implicated in promoting
proliferation and inhibiting differentiation in adult hippocampal NPCs (Peltier et al., 2007). Axon guidance was
also identified as an enriched pathway between NPCs and
SKNs (Figure S3). Ephrins, upregulated in SKNs, have
been shown to mediate growth cone survival and collapse,
and play a large role in the neural crest migration during
gastrulation. EPH-A in particular has also been shown to
interact with P75 neurotrophin receptor (P75NTR) in
562 Stem Cell Reports j Vol. 9 j 557–570 j August 8, 2017
axon guidance and mapping (Lim et al., 2008). In addition,
the differentially expressed gene list also contained a
number of upregulated genes associated with glutamatergic and GABAergic synapse (Figure S4) pathways, including
AMP(A) and GABA(A) postsynaptic receptors, which were
both upregulated in SKNs compared with NPCs. Nevertheless, these synaptic pathways in their entirety were not
significantly enriched, likely due to the cells’ undifferentiated neural precursor phenotype.
SKNs Possess Phenotypic Stability across Cell Lines
As expected, embryonic pluripotency marker NANOG was
not expressed. KI67 and SOX2 proliferation markers were
present in a majority of SKNs (51% and 72%, respectively),
and while OCT4 was highly expressed, staining was perinuclear, consistent with reported expression in dermal cells
(Li et al., 2010) (Figures 4A and 4B). SKNs were homogeneous for stem cell-like marker CD133, neural stem/crest
cell-like marker NESTIN, and neural crest cell marker
P75NTR, a regulatory molecule implicated in both skin
stem cell biology (Adly et al., 2009) and neurotrophin
signaling (Roux and Barker, 2002) (Figures 4A and 4B).
In fact, across these three distinct markers, line-to-line
variance was remarkably low: fluorescence-activated cell
Figure 4. SKNs Homogenously Express
Neural Stem Cell-like and Neural Crest
Cell Markers
(A) Representative immunocytochemistry
images of an array of pluripotency (OCT4
and NANOG), proliferation (KI67 and
SOX2), neural stem/precursor cell (PAX6,
NESTIN, CD133, P75NTR, SCF, PSA-NCAM,
DOUBLECORTIN, BETAIII-TUBULIN), glial
cell (GFAP and O4), transmembrane receptors (BETA1-INTEGRIN and ALPHA4INTEGRIN), and mesenchymal cell markers
(PDGFRA, CD44, SMA, and TFR). Nuclei were
counterstained with DAPI (blue). Scale bars,
50 mm.
(B) Corresponding flow cytometric quantification showed SKNs were highly homogeneous for neural stem/precursor cell markers
(n = 3 independent experiments; mean ±
SEM).
sorting of three independent SKN lines (in triplicate) revealed >97% expression for each marker and a betweenline coefficient of variance of <1.8%. This excellent level
of line-to-line phenotypic replicability is therefore consistent with therapeutic usage.
SKNs also highly expressed an array of neural precursor
markers: polysialylated neuronal cell-adhesion molecule
(PSA-NCAM), DOUBLECORTIN, and immature neuronal
BETAIII-TUBULIN were present in over 85% of cells.
Of note, the astrocyte marker glial fibrillary acidic protein
Stem Cell Reports j Vol. 9 j 557–570 j August 8, 2017 563
(GFAP) and oligodendrocyte marker O4 were observed in
less than 1% of cells (Figure 4A). Expression of BETA1INTEGRIN, combined with the absence of ALPHA4INTEGRIN expression, may indicate a propensity of SKNs
to preferentially bind laminins. This supports upregulated
laminin-associated genes observed within the ECM-receptor-enriched pathway (Figure S5). Mesenchymal markers,
platelet-derived growth factor receptor alpha (PDGFRA)
and CD44 were found to be expressed in SKNs (Figures
4A and 4B). These proteins were found to be co-expressed
with NSC markers CD133 and P75NTR within single cells
(Figure S6). Of note, PDGFRA, while classically a mesenchymal marker, is also found in neuroepithelial cells (Andræ et al., 2001) and adult neural precursors. Moreover,
adult human PDGFRA-expressing neural precursors of the
periventricular region do not co-express GFAP (Chojnacki
and Weiss, 2004), and so SKNs may share this non-glial
propensity. Expression of mesenchymal myofibroblast
marker smooth muscle actin was minimal. Transferrin receptor, traditionally considered a haematopoetic stem cell
marker, was also observed in SKNs, although this receptor
is also associated with neural cell types throughout the
CNS (Erickson et al., 2008).
SKNs Are Neuronally Fate Restricted In Vitro
Following passage 3, the proliferative rate of SKNs
declines rapidly and their morphology increasingly
comes to resemble differentiating neurons. This spontaneous neuronal differentiation was enhanced through
the removal of mitogens from the culture medium and
supplementation with brain-derived neurotrophic factor
(BDNF), known to support neuronal differentiation. Over
a 28-day differentiation period, SKNs remained viable,
and developed increasingly more complex elongated
processes and mature neuronal morphology (Figure 5A),
significantly more frequent than in NPCs (p < 0.0001; Figure 5B). Post-differentiation SKNs were also a more homogeneous population than NPCs. SKNs expressed neuronal
marker genes TUBB3 and MAP2 but, unlike differentiated
NPCs, glial cell GFAP was not expressed (Figure 5C). Mature
neuronal gene ENO2 was expressed in both cultures, while
GABAergic gene GAD67 was only present in differentiated
SKNs. Protein expression corroborated these findings (Figure 5D). Mature neuronal proteins NEUN and neurofilament were seen in differentiated SKNs. BETAIII-TUBULIN
and MAP2 co-expression can also be seen in morphologically immature (Figure 5D; left) and mature stage differentiated SKNs (Figure 5D; right), while peripheral nervous
system (PNS) neuron markers peripherin and P75NTR
(when co-expressed with a neuronal cytoskeletal marker,
Fernandes et al., 2004) were not present. PNS glial protein
MBP (Schwann cells), and CNS glial proteins GFAP (astrocytes) and OLIG2 (oligodendrocytes) were also absent.
564 Stem Cell Reports j Vol. 9 j 557–570 j August 8, 2017
Positive control stains for these proteins can be seen in Figure S7. Presynaptic vesicular protein BASSOON and punctate accumulation of the FM4-64FX membrane-affinity
dye was also detected along the length of the cell processes
as well as internalized within the soma, indicative of synaptic vesicle turnover (Figure 5E).
qPCR analysis following differentiation revealed downregulation of proliferative markers NES (3-fold; ****p =
0.0001) and P75NTR (4.9-fold; **p = 0.001), complemented
by upregulation of the p21 gene CDKN1A (4.8-fold; **p =
0.007), signifying terminal differentiation (Figure 5F).
Notably, significant upregulation of neural specification
gene ASCL1 was observed (27.8-fold; *p = 0.04), as well as
trends toward increased expression for POU3F2, MYT1L,
and NEUROD. SKN differentiation was also accompanied
by upregulation of oligodendrocyte repressor gene DLX2,
and of several neurotransmitter-associated genes including
glutamate transporter SLC17A7, monoamine transporter
SLC18A2, GABA receptor GABBR2 (4.1-fold; *p = 0.01),
and dopamine transporter DAT1. Significant upregulation
of the BASSOON gene BSN (5-fold; *p = 0.03), and a trend
toward upregulation of the voltage-gated sodium channel
gene SCN8A, was also indicative of neuronal maturation
(Figure 5F). Overall, these protein and gene expression
patterns suggest that canine SKNs are highly neuronally
committed.
Differentiated SKNs Possess Basic Neuronal and
Synaptic Functionality In Vitro
Over the differentiation period, SKNs displayed a steady
hyperpolarization of resting membrane potential, with
the majority of cells exhibiting resting membrane potentials analogous to mature functional neurons (R55 mV)
at 25–28 days in vitro (Figure 6A). Functional voltage-gated
ion channels were observed under voltage-clamp conditions (Figure 6B), and sub-threshold depolarization and
graded regenerative responses were observed via currentclamp recordings as early as 14 days in vitro (Figure 6C).
However, under these conditions mature action potentials
were not recorded, and as such SKNs remained in an electrophysiologically immature neuronal state.
Calcium (Ca2+) influx via voltage-gated channels is
known to accompany action potentials. Fura-2-acetoxymethyl ester calcium-affinity dye was therefore used as a
higher-throughput method for detection of voltage-gated
events. Calcium entry was observed by change in the
340/380 florescence signal ratio over time (Figure 6D) and
focal shifts in pseudocolorized image intensity (Figure 6E),
when normalized to baseline T0. Differentiated SKNs
exposed to a depolarization solution of 129 mM KCl and
2 mM Ca2+ show rapid Ca2+ internalization (+). Interestingly, depolarization solution containing 100 mM ATP
(++) induced an increased influx of Ca2+, with repeated
Figure 5. SKNs Are Neuronally Fate
Restricted and Endogenously Upregulate
Neuronal Specification Genes Following
Differentiation
(A) Representative images of SKN and NPC
morphology at differentiation days 14 and
28. Scale bars, 50 mm.
(B) Quantitative data of multipolar cell
morphology in 28 day differentiated SKNs
and NPCs. (****p < 0.0001; n = 3 independent experiments; mean ± SEM).
(C) Gene expression analysis shows differentiated SKNs and NPCs both express mature
neuronal genes. Unlike NPC cultures, SKNs
also expressed GABAergic neuronal gene
GAD67, and did not express glial marker
gene GFAP. Canine brain isolate was used
as a positive control and water a negative
control.
(D) Representative immunocytochemistry
images of mature neuronal proteins
BETAIII-TUBULIN (green) and MAP2 (red),
NEUN, and NEUROFILAMENT. PNS neuronal
proteins PERIPHERIN and P75NTR (same
double stain as NEUROFILAMENT), and both
CNS and PNS glial proteins GFAP, OLIG2, and
MBP were not expressed in differentiated
SKNs. For positive controls stains of these
proteins see Figure S7. Scale bars, 50 mm.
(E) Punctate vesicle loading of membraneaffinity dye FM4-64FX within a differentiated SKN cell’s neurites and soma, and
expression of presynaptic vesicular protein
BASSOON. Scale bars, 25 mm.
(F) Differentiated SKNs downregulated
neural stem cell genes NES (****p = 0.0001)
and P75NTR (**p = 0.001), and upregulated
cell-cycle exit gene CDKN1A (**p =
0.007), known neuronal specification genes
ASCL1 (*p = 0.04), POU3F2, MYT1L, and
NEUROD1, oligodendrocyte repressor gene
DLX2, neurotransmitter-associated genes
SLC17A7, SLC18A2, GABBR2 (*p = 0.01), and
DAT1, BASSOON gene BSN (*p = 0.03), and
brain-specific voltage-gated sodium channel gene SCN8A (n = 3 independent experiments; mean ± SEM).
spikes of calcium entry observed consistent with repeated
voltage-gated events.
DISCUSSION
Adult skin is extraordinarily complex, comprised of several
interactive stem cell niches that originate from ectodermal,
mesenchymal, and neural crest cell lineages. Here, we show
that the canine dermis can be used to isolate and propagate
precursor cells that display an interesting combination of
neural crest, neural stem, and mesenchymal markers.
Further, SKN cell lines can be reliably produced from
mature dogs, display a high degree of line-to-line stability,
are homogeneous across multiple phenotypic markers,
and in vitro are highly neuronally committed. SKNs are
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Figure 6. Differentiated SKNs Develop
Basic Neuronal and Synaptic Functionality
(A) SKN resting membrane potential hyperpolarized and approached physiological
neuronal levels (R55 mV) during 28 days
of differentiation (each time point represents readings from >10 individual cells
across n = 3 independent experiments). From
post hoc analysis of multiple comparisons,
days 25–28 were significantly different to
days 12–14 only (F2, 39 = 4.28, *p = 0.02;
mean ± SEM).
(B) Example voltage-clamp recordings illustrating inward and outward voltage-gated
ion channel currents in differentiated SKNs.
(C) Current-clamp recordings of regenerative
action potentials in a differentiated SKN.
(D) Change in Fura-2AM calcium-affinity dye
fluorescence intensity over time, demonstrated functional calcium channel response
to (+) a depolarizing solution and potentiation of this response following (++) ATPbased co-activation.
(E) Pseudocolor visualization of (D).
therefore a promising candidate for neural cell therapeutic
applications.
Embryologically, brain and skin originate from the same
ectodermal lineage. Many adult tissues, including skin,
continue to harbor populations of multipotent stem cells.
Rather than being necessarily restricted to the tissuespecific lineages of origin, an increasing number of studies
suggest that their differentiation potential is diverse
and strongly influenced by environmental stimuli. For
example, under defined culture conditions, dental pulp
stem cells are capable of generating CNS neurons, yet are
neural crest-derived and accordingly developmentally
restricted to the PNS (Arthur et al., 2008). Similar plasticity
is seen in dermal-derived precursor cells. When SKPs
(closely related to SKNs) are isolated from neural crest566 Stem Cell Reports j Vol. 9 j 557–570 j August 8, 2017
derived skin tissue they generate not only PNS neural cell
types but also CNS neurons (De Kock et al., 2011), and
even mesenchymal osteogenic cells (Lavoie et al., 2009).
The converse is also possible: the dermal stem cell niche
in ventral abdominal skin is non-neural crest in origin
yet, despite this, SKPs derived from this niche are capable
of generating neural crest-derived cell types (Jinno et al.,
2010). SKNs in our study were isolated from ventral skin
and therefore are likely to originate from the same nonneural crest-derived multipotent stem cells as ventral SKP
cells.
Despite their similar origins, SKNs have several advantages over SKPs from a therapeutic perspective, including
reliable homogeneous culture and strong neurogenic bias.
Adherent culture is one potential reason for this increased
uniformity, as observed in studies of neurogenic cells (Joannides et al., 2004; Conti et al., 2005; Pollard et al., 2006;
Goffredo et al., 2008). In our case, uniformity was seen
both within and across lines. We observed less than 2%
coefficient of variance for three key phenotypic markers
(CD133, NESTIN, and P75NTR) across multiple cell SKN
lines and have, to date, produced more than 50 such lines
from different dogs with a greater than 85% success rate.
Furthermore, for several markers (P75NTR, CD133, and
NESTIN), the level of positive expression was >97%, confirming that our protocol produces a reliably homogeneous
SKN culture as required for clinical studies.
The pattern of SKN cell marker expression was unique,
reflecting both their NPC-like properties (NESTIN and
CD133) and dermal origins (PDGFRA and CD44). This
pattern has some precedence, with human MSCs
shown to spontaneously express neural linage markers
SOX2, NESTIN, BETAIII-TUBULIN, and NEUROFILAMENT
in vitro (Deng et al., 2006; Okolicsanyi et al., 2015). More
generally, SKNs also exhibit upregulated and even unique
expression of several genes associated with cell proliferation, cell adhesion, and cell migration. SKNs are hence an
enriched population of neurogenic cells that may be well
suited for survival and migration in vivo, a key factor that
now requires empirical testing. Importantly, the absence
of any genetic manipulation and observation that cell
doubling tends to self-limit after four to five passages alongside downregulation of SOX2 further supports the safe use
of these cells.
We found that SKNs are fate restricted by virtue of
almost exclusive neuronal differentiation. Differentiated
cells expressed a range of mature neuronal markers
including synaptic and neurotransmitter-associated proteins. While these protein markers can also be found in
PNS neurons, the absence of specific peripheral neuronal
and glial proteins suggests that SKNs can mature in vitro
into neurons compatible with the CNS. This neuronal
fate bias may be linked to the endogenous upregulation
of oligodendrocyte repressor gene, DLX2 (Petryniak et al.,
2007), and spontaneous upregulation of several neural
specification genes in response to a differentiation stimuli (high-dose BDNF). Indeed, these specification genes
encode for the same four transcription factors (ASCL1,
BRN2, MYTL1, and NEUROD) necessary for direct conversion of human somatic cells to iNs (Pang et al., 2011).
Differentiated SKNs also possessed basic neuronal functionality including voltage-gated ion channels and
ATP-potentiated calcium transients, as seen in CNS neurons (Lin et al., 2007). However, mature action potentials
remained elusive, suggesting that extended differentiation
may be necessary for complete functional maturation into
definitive neuronal subtypes. Indeed, embryonic stem cellderived neural precursors have reportedly required up to
7 weeks to develop mature action potentials (Johnson
et al., 2007). Nevertheless, global and focal gene expression
data suggested that SKNs may possess a GABAergic and
glutamatergic neuronal differentiation bias. This is in line
with reports that similar adherent expansion of neural
stem cells induces a bias toward GABAergic neurons (Goffredo et al., 2008), an intriguing finding that can be followed up in future work. In general, we hope that the
strong neurogenic potential of canine SKN cells can be exploited to overcome one of the more pressing deficits in the
cell replacement field: poor neuronal yields and an in vivo
bias toward glial differentiation (Hofstetter et al., 2005;
Blurton-Jones et al., 2009).
In summary, SKNs are capable of generating rich and uniform yields of mature neurons comparable with CNS neurons and may therefore have a role in replacement of lost
brain cells. Having thoroughly characterized canine SKNs
in vitro, we are now well placed to determine whether
they are affected by disease-related signals and capable of
generating structurally and functionally mature neurons
in vivo.
EXPERIMENTAL PROCEDURES
Cell Culture
Mature dogs up to 6 years of age had abdominal skin tissue
(z6 cm2) harvested following owner consent. Abdominal skin
has been shown to harbor neurogenic stem cells (Toma et al.,
2001; Fernandes et al., 2004; Valenzuela et al., 2008; Jinno et al.,
2010) and is a practical region for future human clinical study as
it is not frequently exposed to potentially mutagenic sunlight
(UV radiation), and would represent a less-invasive harvest procedure for aged patients than alternatives such as facial skin
(Fernandes et al., 2004). Canine tissue use was approved by the
Animal Care and Ethics Committee of the University of New
South Wales.
Skin biopsies were divided into 1–2 mm2 pieces and digested in
0.1% Trypsin (Thermo Fisher Scientific Australia Pty Ltd, Scorseby,
VIC, Australia) for 40 min at 37 C, followed by 0.1% DNAse (Roche
Applied Science, Castle Hill, NSW, Australia) for 1 min at room
temperature. The tissue was then mechanically dissociated and
passed through a 40 mm cell strainer (BD Bioscience, Sydney,
NSW, Australia), and centrifuged at 180 3 g for 5 min.
SKNs were then established using our published protocol
(Valenzuela et al., 2008). In brief, the dissociated cells were then
re-suspended in serum-free complete medium consisting of 3:1
DMEM/Ham’s F-12 Nutrient Mixture (DMEM/F12; Thermo Fisher
Scientific), 1% Penicillin Streptomycin (Thermo Fisher Scientific),
20 ng/mL EGF (BD Bioscience, Sydney, NSW, Australia), 40 ng/mL
bFGF (Thermo Fisher Scientific), and 2% B-27 supplement
(Thermo Fisher Scientific). The cells were then seeded at a density
of 100,000 cells/cm2. When resulting neurospheres reached 50–
100 mm in diameter, they were dissociated using TrypLE Select
(Thermo Fisher Scientific) for 5 min at 37 C. Cells were then re-suspended in complete medium and grown as an adherent monolayer
Stem Cell Reports j Vol. 9 j 557–570 j August 8, 2017 567
by seeding at 10,000 cells/cm2 on 0.1% gelatin-coated flasks, and
passaged when 80% confluent.
For FBT culture, the dissociated cells were re-suspended in 3:1
DMEM/F12 medium containing 10% fetal bovine serum (Thermo
Fisher Scientific), 1% GlutaMAX (Thermo Fisher Scientific), and
1% penicillin streptomycin. Hereafter referred to as FDMEM, and
seeded at a density of 100,000 cells/cm2.
NPCs were derived from subventricular zone brain isolates,
following previously published protocols (Duncan et al., 2016).
Statistical Analysis
Repeat experiments were conducted on biologically independent
cell lines. Statistical analysis was performed using GraphPad Prism
(GraphPad Software, La Jolla, CA, USA), analyzed by Student’s t test
or ANOVA for multiple comparisons and considered significant at
p < 0.05.
For further details of experimental procedures see Supplemental
Experimental Procedures.
SUPPLEMENTAL INFORMATION
Transcriptomic Analysis
RNA from three individual donor lines each of SKNs, NPCs, and
FBTs were subjected to genome-wide transcriptomic analysis using
an Affymetrix Canine Genechip Gene 1.0 ST array (Affymetrix,
Santa Clara, CA, USA). The complete microarray data are available
online at https://www.ncbi.nlm.nih.gov/geo/ (accession number
GEO: GSE74714).
Immunocytochemistry and Flow Cytometry
Cells fixed with 4% paraformaldehyde were permeabilized in 0.5%
Triton X-100 for 30 min, then blocked using 10% normal donkey
serum (Sigma-Aldrich, Castle Hill, NSW, Australia) for 1 hr prior to
overnight incubation in diluted primary antibodies. Alexo Fluor
488 or 594 secondary antibody (Thermo Fisher Scientific) incubation, and DAPI counterstaining was then carried out prior to imaging on a Leica DMI3000B Microscope, or quantification using a
MACSQuant Analyzer flow cytometer (Miltenyi Biotec Pty Ltd,
Sydney, NSW, Australia).
Gene Expression
RNA was extracted using an illustra RNAspin Mini Isolation Kit
(GE Healthcare, Little Chalfont, Buckinghamshire, UK) according
to the manufacturer’s instructions. cDNA was synthesized using
Superscript III RT First Strand Synthesis System (Thermo Fisher
Scientific). PCR amplification was then performed using Platinum
Taq DNA Polymerase (Thermo Fisher Scientific). qRT-PCR was carried out using the CFX96 Real-Time PCR/C1000 thermal cycler
(Bio-Rad Laboratories, Hercules, CA, USA) using SsoFast EvaGreen
Supermix (Bio-Rad Laboratories). Relative gene expression was
calculated using endogenous GAPDH housekeeping gene.
Electrophysiology
Whole-cell patch-clamp recordings were performed on differentiated SKN cells at room temperature (22.5 C ± 0.3 C). All membrane potentials were corrected for the liquid junction potential
of 15.4 mV. Recordings were performed using an Axopatch-1D
amplifier and digitized using pClamp 10.4 software and a Digidata
1440 interface (Molecular Devices, Sunnyvale, CA, USA) at 10 kHz,
and filtered with an 8 pole Bessel filter at 2 kHz.
Calcium Imaging
Fura2-AM dye (Thermo Fisher Scientific) was used as a ratiometric
calcium indicator. Fluorescence intensity was recorded while
perfused in a 5 mM KCl, 0 mM Ca2+ baseline solution, followed
by 129 mM KCl, 2 mM Ca2+, then 129 mM KCl, 2 mM Ca2+, and
100 mM ATP solutions.
568 Stem Cell Reports j Vol. 9 j 557–570 j August 8, 2017
Supplemental Information includes Supplemental Experimental
Procedures, seven figures, and three tables and can be found
with this article online at http://dx.doi.org/10.1016/j.stemcr.
2017.07.008.
AUTHOR CONTRIBUTIONS
T.D. collected data, analyzed and interpreted results, and prepared
the manuscript. A.L. and R.C.Y.L. designed the experiments,
collected data, analyzed and interpreted results, and prepared the
manuscript. K.S. and P.S. designed the experiments and analyzed
and interpreted results. T.L., V.S., and M.V. designed the experiments, provided research material/equipment, and analyzed and
interpreted results.
ACKNOWLEDGMENTS
This work was supported by the Rebecca L. Cooper Medical
Research Foundation, the ANZ Mason Foundation, the National
Health and Medical Research Council (NHMRC) of Australia
Program Grant (no. 568969), and philanthropic gifts from Ron
Sinclair and Alzheimer’s Australia. We appreciate the expert
contributions of Dr Sandra Fok, Professor Mirjana Maletic-Savatic,
and Professor Perry Bartlett.
Received: December 15, 2016
Revised: July 7, 2017
Accepted: July 10, 2017
Published: August 8, 2017
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