Vol.1, No.3, 29-43 (2011)
opyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/SCD/
Stem Cell Discovery
Lineage restriction of adult human olfactory-derived
progenitors to dopaminergic neurons*
Meng Wang, Chengliang Lu, Hong Li, Mengsheng Qiu, Welby Winstead, Fred Roisen#
Department of Anatomical Sciences and Neurobiology, University of Louisville, Louisville, USA.
#Corresponding Author: fjrois01@louisville.edu
Received 11 June 2011; revised 5 July 2011; accepted 30 July 2011.
Human adult olfactory epithelium contains neu-
ral progenitors (hONPs) which replace damaged
cellular components throughout life. Methods to
isolate and expand the hONPs which form
neuronspheres in vitro have been developed in
our laboratory. In response to morphogens, the
hONPs differentiate along several neural line-
ages. This study optimized conditions for the
differentiation of hONPs towards dopaminergic
neurons. The hONPs were treated with Sonic
hedgehog (Shh), in the presence or absence of
retinoic acid (RA) and/or forskolin (FN). Transcrip-
tion factors (Nurr1, Pitx3 and Lmx1a) that pro-
mote embryonic mouse or chicken dopaminergic
deve lopment w ere e mployed to determine if they
would modulate lineage restriction of these adult
human progenitors. Four expression vectors
(pIRES-Pitx3-Nurr1, pLN-CX2-Pitx3, pLN-CX2-Nurr1
and pLNCX2-Lmx1a) were transfected into the
hONPs, respectively. Transcription factor expre-
ssion and the rate-limiting enzyme in dopamine
synthesis tyrosine hydroxylase (TH) were de-
tected in the transfected cells after 4 month-se-
lection with G418, indicating transfected hONPs
were stably restricted towards a dopaminergic
li n ea g e. Fu r th ermore, a dopamine enzyme immu-
noassay (EIA) was employed to detect the syn-
thesis and release of dopamine. The most efficient
transfection paradigm was determined. Several
neurotrophic factors were detected in the pre-
transfected hONPs which have potential roles in
the maintenance, survival and proliferation of
dopaminergic neurons. Therefore the effect of
transfection on the neurotrophin synthesis was
examined. Transfection did not alter synthesis.
The use of olfactory progenitors as a cell-based
therapy for Parkinson’s disease (PD) would al-
low harve st without in vasive surgery, p rovide a n
autologous cell population, eliminate need for
immunosuppression and avoid the ethical con-
cerns associated with embryonic tissues. This
study suggests that specific transcription fac-
tors and treatment with morphogens can re-
strict human adult olfactory-derived progenitors
to a dopaminergic neuronal lineage. Future stud-
ies will evaluate the utility of these unique cells
in cell-replacement paradigms for the treatment
of PD like animal models.
Keywords: Human Olfactory Epithelium;
Progenitors; Dopaminergic Neurons;
Parkinson’ s D i s e ase
Parkinson’s disease (PD) remains one of the leading
causes of chronic degenerative neurological disability,
which affects more than 6,000,000 people world-wide,
with approximately 60,000 new cases diagnosed each
year in the United States [1]. The incidence rises with
age, being approximately 1:1000 overall and 1% of the
population over the age of 60 and 4% in those over 80
years. Unfortunately, the mortality rate of PD has in-
creased steadily in recent years [2,3]. PD is characterized
by the extensive loss of dopaminergic (DA) neurons in
the substantia nigra (SN) in the midbrain [4]. Currently
the principle treatment for PD is oral L-3, 4-dihydroxy-
phenylalanine (L-dopa) [5], which is the precursor of do-
pamine that can pass the blood-brain-barrier [6]. L-dopa
promotes symptomatic relief, but with time becomes less
effective for two reasons: 1) During the progression of
the disease the neurons become less sensitive to the drug
[7] and 2) L-DOPA does not prevent or rescue the DA
neurons from degeneration [8,9].
Recent research has attempted to find cell populations
that can be used to replace lost or degenerating dopaminer-
*This work was supported by the Dishman Family Foundation and
Funds from RhinoCyte™, Inc. (OICB070316).
M. Wang et al. / Stem Cell Discovery 1 (2011) 29-43
Copyright © 2011 SciRes. Openl y accessible at http://www.scirp.org/journal/SCD/
gic neurons [2,10,11]. The basic concept of cell replace-
ment therapy is to restore function lost as a result of the
disease in the central nervous system (CNS) by replacing
degenerating or lost cells with viable functional cells.
Recent studies also suggest that the engraftment of stem
cells or progenitors can up-regulate or enhance existing
endogenous progenitor populations [12-14]. Studies
have employed neural cell grafts obtained from the fetal
ventral mesencephalic (VM) dopaminergic neurons
[15-20]. However, this frequently resulted in significant
dyskinesia [21-24]. Even when clinical improvements
were achieved in the absence of dyskinesia, the amount
of tissue required for each PD patient necessitated a
minimum of 4-5 fetal brains [25]. This requirement in-
creased the possibility of viral or bacterial infection and
significantly limited the utility of this approach. In addi-
tion the number of surviving neurons was highly limited
as the majority of the engrafted cells died in the initial
days following transplantation [15,20,24]. The limited
supply of fetal VM cells coupled with their poor graft
viability severely limited the therapeutic utility of this
population for the treatment of PD. Therefore, an alter-
nate expandable source of dopamine cells has become a
major research focus [26-29].
Stem cells are undifferentiated cells with an unlimited
capacity for self-renewal and the potential for lineage re-
striction (maturation) into one or more specific cell types,
depending on their origin and the micro-environmental
signals that they receive [28,30]. These characteristics
make stem cells an attractive target population for PD cell
replacement therapy [31-34]. Human embryonic stem
cells (hESCs), lineage-restricted towards dopaminergic
neurons when transplanted into a rodent model of PD,
provide a significant relief of symptoms. However, with
time, animals engrafted with hESCs have frequently de-
veloped teratomas [35]. Clearly an alternate approach is
Human olfactory epithelium (OE) is a unique source
for neural progenitors that can be harvested by mini-
mally invasive endoscopic nasal surgery without a cra-
niotomy. Furthermore since no demonstrable olfactory
deficits result from OE biopsy [36], the tissue can be
used to generate an autologous progenitor population
from patients with PD. An autologous cell source pro-
vides total histocompatability and thus eliminates the
need for immunosuppressive therapy as well as long
waiting lists for available matched tissue. Previously our
laboratory developed methods for the isolation and cul-
ture of a neurosphere forming population [37]. To date
more than 150 patient-specific cell lines of human ol-
factory neural progenitors (hONPs) have been estab-
lished from primary cultures of human adult olfactory
epithelium isolated from cadavers [37] and patients un-
dergoing endoscopic sinus surgery [36]. Our studies have
shown that the hONPs have the potential to differentiate
along several neural lineages following exposure to en-
vironmental signals in vitro [38].
The objective of this study was to determine if hONPs
could be lineage restricted towards dopaminergic neu-
rons and if so to optimize the methodology. Molecular
techniques were applied for the transfection of Nurr1 [34,
39], Pitx3 [40,41] and Lmx1a [42], transcription factors
which promote dopaminergic differentiation. The trans-
fection effects of different paradigms were evaluated and
Several studies have shown that neurotrophic factors,
such as brain-derived neurotrophic factor (BDNF), cil-
iary neurotrophic factor (CNTF), neurotrophin-3 (NT-3),
etc. are important for the survival and function of dopa-
minergic neurons in CNS [43-47]. Recent studies also
indicate that the neurotrophins have the potential to op-
timize the local micro-environment of the damaged area,
and thereby induce endogenous stem cells to replace or
rescue degenerating neurons [48,49]. HONPs derived
from adult human olfactory epithelium have been shown
to produce and release neurotrophins [10,50,51], which
could further support their use in a cell-based therapy for
PD. Therefore, this study also evaluated the ability of
pre and post transfected hONPs to synthesize key neuro-
2.1. Cell Culture
The two patient-specific olfactory progenitor lines used
in this study were obtained from adult olfactory epithe-
lium harvested from a 42-year-old female patient and a
20-year-old male via endoscopic biopsy [37]. The tissues
were cultured to allow the emergence and harvest of
hONPs as previously described [36,52]. The hONPs were
thawed from frozen stock that was maintained in liquid
nitrogen and cultured in minimal essential medium (MEM)
with 10% heat inactivated fetal bovine serum (FBS,
GIBCO, Grand Island, NY) (10% OE) for one week.
The hONPs were adapted to serum-free growth media
via serial dilution of serum every day for 4 days until the
cells were finally cultured in DFBNM (DMEM/F12
supplemented with 1% B27 and 0.5% N2 and 100 μg/ml
gentamycin (GIBCO, Grand Island, NY)) [52]. Parallel
independent experiments were performed on hONP lines
from the two different patient lines. Since equivalent
results were achieved, data from only one line has been
2.2. Construction of Expression Plasmids
The mouse Nurr1 cDNA was cloned into the pLNCX2
expression vector (Clontech) between ClaI. Similarly,
M. Wang et al. / Stem Cell Discovery 1 (2011) 29-43
Copyright © 2011 SciRes. http://www.scirp.org/journal/SCD/
the rat Pitx3 and mouse Lmx1a cDNA were inserted into
pLNCX2 vector between ClaI. For the Nurr1 and Pitx3
co-expression vector, Nurr1 cDNA was cloned into
pIRES (Clontech) between XbaI and SalI, and pitx3 was
inserted between EcoRIs. The pLNCX2 and pIRES ex-
pression vectors served as controls (Figure 1). All ex-
pression vectors were verified by extensive DNA se-
Openly accessible at
2.3. Transfection and Selection
All plasmid constructs were introduced into the hONPs
by liposomal transfection. The cells were plated on glass
coverslips in six-well plates (5 × 104 cells per 35 mm well)
in DFBNM without antibiotics 1 day before transfection.
HONPs were transfected with each plasmid (4 μg/well)
for 24 hours according to the manufacture’s protocol (Li-
pofectamine 2000, Invitrogen, Carlsbad, California). One
day after transfection, the cells were fed with 10% FBS
in MEM and selected with G418 (400 μg/ml; Invitrogen,
Carlsbad, California). The selection pressure was kept
for up to 4 months to insure a purified stably transfected
cell population. Immunocytochemistry and Western blot
analysis were applied to detect several dopaminergic
neuronal markers. After a four-month selection, the trans-
fected hONPs were frozen in liquid nitrogen for addi-
tional four-six months of storage. After removal from cry-
ostorage and several days’ recovery in MEM with 10%
FBS at 37˚C, the dopaminergic lineage restriction was
probed with immunocytochemistry and Western blot analy-
2.4. Treatment with Morphogens
The hONPs were treated with Sonic hedgehog (Shh)
in the presence or absence of retinoic acid (RA, 1 μM)
and/or forskolin (FN, 5 μM) [52]. Highly purified Shh
(kindly provided under a Material Transfer Agreement with
Curis and Wyeth, Inc.) was applied to hONPs and com-
pared to a commercially available control sample obtained
from Sigma to determine the extent to which purification of
Shh can affect the expression of tyrosine hydroxylase (TH).
The hONPs were plated on glass coverslips in six-well
plates (5 × 104 cells/35 mm well) in DFBNM and treated
with medium containing various concentrations and com-
binations of RA, FN, and Shh for 7 days (CO2 atmo-
sphere at 5% and temperature of 37˚C). Treatment with
Shh included several concentrations: 0.25 mg/ml
(Shh0.25), 0.1 mg/ml (Shh0.1), 0.05 mg/ml (Shh0.05),
0.025 mg/ml (Shh0.025) in the presence or absence of
Figure 1. Construction of expression plasmids.
M. Wang et al. / Stem Cell Discovery 1 (2011) 29-43
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1 μM retinoic acid (RA1) and/or 5 μM forskolin (FN5).
After treatment, the TH expression was determined at 1 -
7 days in vitro by immunocytochemical analysis. Once
the optimized environment for inducing dopaminergic
neurons was determined, the medium containing the
optimized combination was applied to stably transfected
hONPs to further improve the yield of these neurons.
2.5. Immunocytochemistry
The hONPs (5 × 104 cells/well) were plated on 35 mm
round glass coverslips in six-well plates (Becton, Dick-
inson and Co.) and incubated at 37˚C in 5% CO2/95%
air for 24 hours and treated with RA, FN, and Shh or
transfected and selected for different periods of time
prior to fixation for immunofluorescence. 4, 6-diamidi-
no-2-phenylindole dihydrochloride (DAPI) (1:1000, 2
mg/ml, Molecular Probes, Eugene, OR) was applied in
culture for 30 minutes at 37˚C for vital nuclear staining.
The coverslips were rinsed with cytoskeletal buffer (CB)
twice and fixed in 3% paraformaldehyde in CB (10 min-
utes). 0.2% Triton X-100 (10 minutes, Sigma) in tris buf-
fered saline (TBS) was applied and cells were incubated
(1 hour) in 3% bovine serum albumin (BSA) in TBS. Pri-
mary antibodies were applied overnight (4˚C). After 30
minutes washing (10 minutes each, 3 times) in TBS, the
cells were incubated with secondary antibodies: Texas-
red conjugated goat anti-rabbit immunoglobulin G (IgG),
Texas-red-conjugated goat anti-mouse IgG, Cy2-conju-
gated goat anti-mouse IgG and/or Cy2-conjugated goat
anti-rabbit IgG (all diluted 1:600, Cy2, Jackson Immunol-
ogy Research Laboratories; Texas red, Molecular Probes).
The coverslips were rinsed in TBS for 30 minutes (10
minutes each, 3 times) and mounted on slides. The slides
were examined with confocal microscopy. All experi-
ments were repeated a minimum of two times to ensure
the specificity of staining; only one set of data has been
presented since similar results were obtained.
2.6. Western Blot Analysis
Western blot analysis was used to further examine and
confirm the immunofluorescence studies. Proteins from
hONPs transfected with control vectors, as well as
hONPs transfected with the vectors plus each combina-
tion of transcriptions factors (pLNCX2-Pitx3, pLNCX2-
Nurr1, pLNCX2-Lmx1a, pIRES-Pitx3-Nurr1), cultured
in DFBNM, selected in all groups were collected in cell
lysis buffer (Sigma, St. Louis, MO). After 15 minutes of
incubation on ice, samples were centrifuged for 30 min-
utes (4˚C) and the protein concentration of each super-
natant was determined. The protein samples (20 μg/well)
were electrophoresed on 10% SDS-polyacrylamide gels
along with standardized-molecular-size marker proteins
in an adjacent lane and transferred from gel to nitrocel-
lulose paper. Nonspecific binding was blocked (1 hour)
with 5% nonfat dry milk in TBS-Tween (TBST) buffer.
Blots were incubated (4˚C overnight) in primary anti-
bodies (anti-TH, MAB; anti-actin, MAB). Blots were
washed three times for 10 minutes in TBST, after which
they were incubated (1 hour, room temperature) mono-
clonal horseradish peroxidase-labeled anti-mouse IgG
(1:2000). ECL Western blotting detection reagents (A-
mersham Biosciences) were used to identify bound an-
tibodies. Densitometry of the protein bands was carried
out on a high performance chemiluminescence film (A-
mersham Biosciences). Data was analyzed using the
Image-J software programs supplied by the NIH official
website (http://rsb.info.nih.gov/ij/ ).
2.7. Dopamine Assay
Stably transfected hONPs were plated into flasks (25
cm2, Corning) at 105 per flask before they were adapt- ed
to the absence of serum via serial dilution of serum
every day for 4 days until the cells were finally cultured
in DFBNM, which was collected daily after the serum
was totally eliminated from the medium. The DFBNM
collected from each restricted hONP line was then con-
centrated to 1/50 volume respectively by centrifugal filters
(Amicon Ultra-15, Millipore). The differentiated hONPs
were then collected and lysed (lysis buffer, Sigma). Do-
pamine expression was analyzed quantitatively in the
concentrated medium as well as in the cell lysates with a
dopamine enzyme immunoassay kit (Dopamine EIA,
Immuno Biological Laboratories, Inc.), according to the
manufacture’s protocol.
2.8. Neurotrophin Assay
Pre- and post-transfected hONPs were plated into
flasks (25 cm2, Corning) at 5 × 105 per flask and cultured
in 10% OE media for two days before they were adapted
to the absence of serum via serial dilution of serum e-
very day until they were finally cultured in DFBNM.
The differentiated hONPs were then collected and lysed
(lysis buffer, Sigma). Neurotrophins were detected in the
extracted protein with different enzyme-linked immu-
nosorbent assay (ELISA) kits (BDNF, Chemicon; CNTF,
Quantikine; NT-3, Chemicon) respectively, according to
the manufacture’s protocol. The ELISA absorbance (OD)
was obtained with a microplate spectrophotometer (Spec-
tra-max Plus), and the results were plotted and calcu-
lated with the compatible software (Softmax Pro).
Cryopreserved vials of the two representative hONP
M. Wang et al. / Stem Cell Discovery 1 (2011) 29-43
Copyright © 2011 SciRes. Openl y accessible at http://www.scirp.org/journal/SCD/
lines were obtained from storage and grown for 1 - 2
weeks prior to the start of these experiments to insure
equivalent passage (4 - 8) and sufficient cell numbers for
the following studies.
3.1. Transfection of Olfactory-Derived
Progenitors (hONPs) to Achieve
Dopaminergic Lineage Restriction
HONPs were obtained from previously frozen stock
with low passage number (4 - 8) and maintained in
MEM 10 medium during their recovery period. These
mitotically active cells divided every 18 - 20 hour which
typically required passage three times per week as previ-
ously described. The heterogeneous nature of the hONP
population prior to transfection was determined by im-
munocytochemistry. No reactivity was observed for
Pitx3, Nurr1, Lmx1a with pre-transfected hONPs and
only a few (5 - 10%) of them were positive for the do-
pamine precursor, TH, when treated conditionally [53].
Low passages (Passage 4 - 8) of hONPs from 2 different
patient-specific cell lines were employed in parallel trans-
fection experiments. To examine the phenotypic expres-
sion of hONPs after transfection and selection, repre-
sentative cultures as well as their respective pre-trans-
fection controls were evaluated. Non-transfected hONPs
or those transfected with lipofectamine alone died within
1 week after selection with 400 µg/ml G418. In contrast,
30% of the transfected cells (both with the concerned
genes and the control vectors) survived under the selec-
tion pressure. Transfection with control vectors, single
genes, or Pitx3-Nurr1 combined resulted in no morpho-
logic changes compared to the typical pretreated hONPs.
However, the transfected hONPs divided more slowly,
with a new doubling time of three to four days, which
required a feeding schedule of only twice a week and
necessitated passage every 9 - 10 days. Immunofluores-
cent analysis of the transfected populations demonstrated
that hONPs were stably transfected and TH expressed.
Human olfactory derived hONPs were transfected
by pIRES-Pitx3-Nurr1 to restrict them towards DA
neurons. The vector alone was employed as a con-
trol. To obtain a purified population of restricted
cells the transfected population was maintained in
G418 for selection. Although only several weeks of
selection produced relatively pure populations, an
interval of four months was employed to insure sta-
bility and purity. HONPs remained TH positive after
transfection of pIRES-Pitx3-Nurr1, whereas the trans-
fection of control vectors exhibited no phenotypic
changes, demonstrating that hONPs can be restricted
towards dopaminergic neurons (Figure 2).
HONPs were transfected with pLNCX2-Nurr1, pL-
NCX2-Pitx3, pLNCX2-Lmx1a or the vector alone
as a control. The transfected cells were exposed to
G-418 for selection for periods up to 4 months. HONPs
were TH positive after transfection of pLNCX2-Nurr1
and pLNCX2-Pitx3, whereas the transfection of con-
trol vectors resulted in no phenotypic changes. There-
fore pLNCX2-Nurr1 or pLNCX2-Pitx3 can be em-
ployed to lineage restrict the hONPs towards dopa-
minergic neurons. In contrast, the hONPs trans-
fected with pLNCX2-Lmx1a remained unreactive
for TH, although positive of myc, which demonstra-
ted the successful incorporation of the plasmid (Fig-
ure 2).
a b d
e f gh
Figure 2. Immunocytochemical analysis. HONPs transfected with pIRES-Pitx3-Nurr1, pLNCX2-
Pitx3 or pLNCX2-Nurr1 were tyrosine hydroxylase (TH) positive after 4 months selection with G418
(c, d, f, g), while the lines transfected with pIRES or pLNCX2 were TH negative (b, e). HONPs
transfected with pLNCX2-Lmx1a were Myc positive, demonstrating that the plasmid was transfected
into the nucleus (h).
M. Wang et al. / Stem Cell Discovery 1 (2011) 29-43
Copyright © 2011 SciRes. Openl y accessible at http://www.scirp.org/journal/SCD/
Western blot analysis was employed to confirm quan-
titatively the immunocytochemical studies of the tran-
sfected hONP populations. The following transfected
lines were analyzed for TH expression: hONPs trans-
fected with pIRES-Pitx3-Nurr1, pLNC-X2-Nurr1, pL-
NCX2-Ptx3 and pLN-CX2-Lmx1a all of which were
TH positive, which indicated their potential to release
dopamine. In contrast, the hONP populations’ trans-
fected with the control vectors (pIRES and pLNCX2)
did not express TH. β-actin, a protein that is widely
expressed in all mammalian and avian cells was used
as a reference protein for the comparison of TH ex-
pression by the various lines. Image-J was applied for
the data analysis. Each curve from B to M in Figure 3
illustrates the density of bands evident on the western
gel (Figure 3a), and the area that each curve was
measured. The bars in picture N represent the ratio of
TH expression and ACTIN expression in the cell line.
HONPs transfected with pIRES-Pitx3- Nurr1 exhibited
the highest ratio for the TH and ACTIN expression,
while the cells transfected with the control vector
(pLNCX2 or pIRES) had the least TH staining (Figur e
3(b-n)). These results demonstrate that individual tran-
scription factors have unique abilities in promoting the
dopaminergic restriction of hONPs.
3.2. Transfected hONPs Remain Restricted
to Dopaminergic Lineage after Removal
from Cryostorage
After a 4-month selection, the dopaminergic lineage re-
stricted cells were cryopreserved in liquid nitrogen for
additional 4 - 6 months. Following their removal from cry-
ostorage and several days’ recovery in MEM10 at pIRES-
Pitx3-Nurr1 to restrict them towards DA neurons. The
vector alone was employed as a 37˚C, all but one of the
transfected hONP populations survived under the selec-
tion pressure of 400 µg/ml G418, demonstrating that
these cells were stably transfected and retained their po-
tential for long term storage and clinical application.
Immuno-cytochemistry and Western blot analysis was
applied to these previously stored populations to exam-
ine their TH expression. The hONPs transfected with
pLNCX2-Pitx3, pLNCX2-Nurr1 and pIRES-Pitx3-Nurr1
remained healthy and TH positive under the pressure of
selection, while the pLNCX2-Lmx1a transfected line did
not (Figure 4).
3.3. Lineage Restricted hONPs Produced
and Released Dopamine
After removal from the cryostorage, dopamine produc-
tion was detected in the hONP lines which were stably
transfected with concerned genes, while the cells tranfected
Figure 3. a. Western blot analysis. b-g. Scanning densitometry
demonstrates ACTIN-expression in a hONP line of pL-NCX2,
pLNCX2-Pitx3, pLNCX2-Nurr1, pIRES and pIRES-Pitx3-
Nurr1 respectively. h-m. Densitometry of TH-expression as
shown in A. N. Histogram demonstrating the ratio of TH/ac-
tion produced by each population.
with control vectors and the non-transfected hONPs didn’t
produce dopamine. The dopamine level of each sample
was then divided by the concentration of protein in each
specific hONP line to calculate the efficiency of dopa-
mine production. Among all the 4 gene transfected lines,
hONPs transfected with pIRES-Pitx3-Nurr1 exhibited
the most efficient dopamine formation (Figure 5(a)).
Spent medium was collected 4 days after culturing the
lineage restricted hONPs. This medium was then concen-
trated to 1/50 volume respectively, and dopamine E. I. A.
was applied to detect the dopamine release (extracellular
levels). Data were calculated in the same manner as the
intracellular dopamine analysis. Lower levels of dopa-
mine were detected in the concentrated media compared
to the corresponding analysis of the cell lysis. The grea-
test level of dopamine release was detected in pIRES-
Pitx3-Nurr1 transfected hONPs compared to the other
restricted cell lines (Figure 5(b)).
M. Wang et al. / Stem Cell Discovery 1 (2011) 29-43
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Figure 4. Immunocytochemistry (a-g) and western blot analysis
(i) demonstrating that hONPs transfected with pLNCX2-Pitx3,
pLNCX2-Nurr1 and pIRES-Pitx3-Nurr1 remain healthy and TH
positive following removal from cryostorage under selection
pressure (d, f, g). In contrast, the Lncx2-Lmx1a transfected line
no longer expressed TH (h).
Figure 5. Histograms demonstrating the ratio of dopamine for-
mation (pg/100 µl) to total protein concentration (mg/ml) of cells
transfected with pIRES-Pitx3-Nurr1, pLNCX2-Pitx3, pLNCX2-
Nurr1, pLNCX2-Lmx1a, pIRES, pLNCX2 and non-transfected
hONPs. HONPs transfected with pIRES-Pitx3-Nurr1 exhibited the
highest levels of intracellular and extracellular dopamine produc-
tion. Dopamine production and release were enhanced in hONPs
treated with the morphogens.
3.4. The Effect of Morphogens on Tyrosine
Hydroxylase (TH) Expression,
Dopamine Formation and Release
HONPs were cultured in DFBNM along with RA (1
µM), FN (5 µM) and either of two different sources (puri-
ties) of Shh for four days. Both Shh treatments resulted in
greater expression than in those cultured solely in DFBNM.
TH expression was greater in the cells that were treated
with highly purified Shh than the commercial product
obtained from SIGMA when applied for same period of
time (Figure 6).
HONPs treated with RA1FN5 and highly purified Shh
expressed seemingly more intensive TH reactivity in the
positive cells (Figure 7(a)). Therefore, the concentration
of Shh was reduced to determine the lowest concentra-
tion of Shh that could drive the hONPs towards dopa-
minergic neurons. In contrast to the response when a
high level of Shh was applied, the reduction of the Shh
to 0.025 mg/ml applied with RA (1 µM) & FN (5 µM)
did not produce an immediate response. The hONPs be-
came TH positive only after 18 hours of treatment with
highly purified Shh; however, they were healthy and
maintained TH expression for longer periods. The ap-
plication of RA and FN promoted an even greater ex-
pression of TH (Figure 6 A). Therefore, the optimal con-
ditions for restricting the hONP lineage to dopaminergic
neurons (under these defined conditions) was determined
to be DFBNM supplemented with RA1FN5Shh0.025 (Fig-
ure 7(b)).
Stably transfected hONPs were treated with a cock-
tail of RA1FN5Shh0.025 to determine if a combination
of genetic modification and morphogen exposure would
increase intracellular and intercellular dopamine levels.
Spent medium was collected four days after morphogenic
treatment and concentrated to a 1/50 volume. The treated
lineage restricted hONPs were also collected. Dopamine E.
I. A. was applied to both cell lysis sample and concentrated
medium. Dopamine formation efficiency was calculated as
pre- viously described. HONPs transfected with pIRES-
Pitx3-Nurr1 were the most efficient population with re-
spect to dopamine formation and release after morpho-
genic treatment (Figures 5(a)-(b)). Compared to intracel-
lular and extracellular dopamine levels of the lineage re-
stricted hONPs in the absence of morphogens, dopaminer-
gic expression was greatly enhanced in the stably trans-
fected hONPs in the presence of the combination of Shh,
RA and FN (Figures 5(a)-(b)). These studies suggest that
treatment with morphogens can play an important role in
dopamine formation and release by the lineage restricted
ONPs. h
M. Wang et al. / Stem Cell Discovery 1 (2011) 29-43
Copyright © 2011 SciRes. http://www.scirp.org/journal/SCD/
(a) (b) (c)
Figure 6. HONPs treated in DFBNM with a highly purified Shh(c) exhibited greater reactivity to tyrosine hydroxy-
lase (TH) than those treated with commercially available Shh (b) for 3 days in the presence of RA and FN.
(a) (b)
Figure 7. (a) HONPs cultured in DFBNM supplemented with 0.025 mg/ml of Shh, in the presence or absence of reti-
noic acid (RA) (1 µM) and forskolin (FN)(5 µm) for days indicated; (b) HONPs were tyrosine hydroxylase (TH) posi-
tive following 7 days treatment with RA1FN5Shh.
3.5. Stably Transfected and Pre-transfected
hONPs Produce Neurotrophins (BDNF,
CNTF and NT-3) at Equivalent Levels
The non(pre)-transfected hONPs were found to pro-
duce neurotrophic factors such as BDNF (56.09 ± 10.24
pg/ml), CNTF (18.72 ± 1.43 pg/ml) and NT-3 (24.87 ±
6.53 pg/ml). The stably transfected lines were examined to
determine if lineage restriction to dopaminergic neurons
alters the synthetic capacity and activity of these neuro-
trophins; no significant differences in intracellular neuro-
trophin (BDNF, CNTF, NT-3) levels between transfected
and non-transfected hONP lines were observed (P >
0.01), indicating that transfection did not alter neurotro-
phin synthesis (Figure 8).
Parkinson’s disease, as a neurodegenerative disease, is
characterized by loss of specific dopaminergic neurons in
substantia nigra [4]. Although a variety of pharmaco-
logical agents have been employed in the treatment of
PD their effects are transient. “Proof of Concept Studies”
with embryonic adrenal medulla cells [54] although end-
ing in failure demonstrated the potential of cell-based re-
placement therapy. Recently substantial effort has been
Openly accessible at
M. Wang et al. / Stem Cell Discovery 1 (2011) 29-43
Copyright © 2011 SciRes. Openl y accessible at http://www.scirp.org/journal/SCD/
Figure 8. Histogram demonstrating the neurotrophin levels in
hONPs (pg/ml) transfected with pIRES-Pitx3-Nurr1, pLNCX2-
Pitx3, pLNCX2-Nurr1, pLNCX2-Lmx1a, pIRES, pLNCX2 and
non-transfected NSFCs. Lineage restriction did not alter neuro-
trophin production.
devoted to the search for a suitable cell source for a cell
replacement strategy for the treatment of PD. Many stud-
ies have focused on the use of embryonic stem cells;
studies utilizing embryonic cells derived from mice or
porcine were found to be functional in relieving PD like
symptoms in PD animal models [33,55], and positive
results obtained from human oriented ES cells further
advanced the use and promise of stem cells as a potential
source for cell therapy for PD [56-58]. However, these
studies were all generally hampered by the significant
side effects due to the transplantation of ES cells, such as
dyskinesias and/or the formation of teratomas [35,56, 59].
Unfortunately, low cell viability following transplanta-
tion, tissue compatibility, a limited of source and ethical
concerns further diminish the therapeutic utility of ES
cells. In contrast, the use of adult human olfactory epi-
thelium derived progenitors, as a unique autologous cell
source, which can be obtained with minimally invasive
surgery can avoid these negative factors and also elimi-
nate the need for immunosuppression. The studies de-
scribed in this manuscript demonstrate that hONPs can
be stably lineage restricted under an optimized paradigm,
so that they produce and release dopamine, which makes
them potential candidates for cell-based therapy for PD.
Additionally, the genetic modification didn’t alter the
capability of hONPs to produce and release key neuro-
trophic factors, which have the potential to support neu-
ronal survival, as well as rescue degenerating neurons.
These factors can also provide permissive micro-envi-
ronments that may induce endogenous stem cell gener-
ation and differentiation [60-62].
In the present study, several conditions have been uti-
lized to optimize the environment for hONPs and facili-
tate their differentiation to dopaminergic neurons, includ-
ing genetic modification and treatment with morpho-
gens. Furthermore, hONPs have the unique potential to
synthesize and release key neurotrophic molecules which
can have beneficial effects on the survival of dopaminer-
gic neurons as well as the proliferation and differentia-
tion of endogenous stem cell populations. These will all
be discussed individually below.
4.1. Pitx3 and Nurr1 Induce the DA Neuron
Maturation Synergistically
The Pitx3 gene belongs to the Pitx family of transcrip-
tion factor genes and has been shown to be required for
the expression of TH, the precursor of dopamine, both in
vitro and in mice from E11.5 [40]. It has been reported
that Pitx3 is crucial to the formation of SN and the spec-
ification and/or the survival of the subpopulation of the
DA neurons in striatum [63-65]. The earlier studies sug-
gest that Pitx3 increased TH promoter induction in
mouse and rat cell lines, but not in human cell lines [63,
66]. However, human embryonic stem cells were em-
ployed in experiments to demonstrate the regulation of
TH expression by Pitx3 [67-69]. These studies suggested
that pitx3 is a key transcriptional regulator of genes re-
quired specifically for the mesencephalic dopaminergic
(mesDA) phenotype [69,70] and for TH expression [40,
64]. Nurr1 is a member of the nuclear receptor super
family of transcription factors that is expressed in both
developing and mature dopaminergic neurons in the cen-
tral nervous system in mice [71]. Previous studies have
shown that Nurr1 is essential to both survival and differ-
entiation of the ventral mesencephalic dopaminergic
precursor neurons [34,72]. Nurr1 has also been reported
to be essential in the expression of TH, which is required
for DA synthesis; and for vesicular monoamine trans-
porter 2 (VM-AT2), which is related to DA storage; and
dopamine transporter (DAT), which is crucial for DA
re-uptake [72]. In addition, a recent study has shown that
Nurr1 plays a previously unexpected role in protecting
TH positive neurons from neurotoxicity [73]. Further-
more, Nurr1 is the only known transcription factor that is
associated with the dopaminergic neurotransmitter iden-
tity in mesDA neurons [71]. Therefore, both Pitx3 and
Nurr1 have been shown to be crucial to the formation of
SN and the specification and/or the survival of the DA
neurons in midbrain in rodents [39,74,75]. The results
obtained in the present study indicate that overexpression
of Pitx3 and/or Nurr1 promotes the expression of DA
neuron marker, TH in human adult olfactory epithe-
lial-derived progenitors in vitro. HONP lines that were
stably transfected with Pitx3 and/or Nurr1 and selected
for 4 months, remained healthy and TH positive follow-
ing 6 months cryostorage in liquid nitrogen. Furthermore,
the direct detection of dopamine production was also
evaluated. Lysates of Pitx3 or Nurr1 transfected hONPs
M. Wang et al. / Stem Cell Discovery 1 (2011) 29-43
Copyright © 2011 SciRes. Openl y accessible at http://www.scirp.org/journal/SCD/
were dopaminergic as determined by dopamine E.I.A.
These results suggest that the transcription factors, Pitx3
and Nurr1, not only function as a dopaminergic promot-
ers in chick, mouse, or human embryonic cells [41,68,
71,76], but also can participate in dopamine production
in adult human olfactory-derived progenitors. Based on
previous studies which focused on the regulatory func-
tion of Pitx3 and Nurr1 in dopaminergic neuron promo-
tion [63,68,70,72,74,77] and the studies described in this
manuscript, we hypothesized that Pitx3 and Nurr1 may
collaborate to induce a higher efficiency of dopamine
production in midbrain DA neuron maturation. Previ-
ously a synergistic effect between Pitx3 and Nurr1 on TH
expression has been reported, which appeared to be spe-
cies dependent occurring in human but not in embryonic
murine stem cells [66-78]. The current studies demon-
strate that the simultaneous transfection of Pitx3 and
Nurr1 into the hONPs produced higher levels of TH ex-
pression and dopamine production than transfection of
either of the individual genes. We evaluated the effect of
transfection on the level of the precursor (TH) and final
intracellular and extracellular dopamine levels to confirm
and compare the efficiency of the different transfected
hONP lines. Therefore, our data, in combination with
published reports in rodents [79,80] and human embry-
onic stem cells [67,81], indicate that Pitx3 and Nurr1
cooperatively induce the maturation of DA neurons. We
extend the previous studies to show the feasibility of
genetic modification of adult human olfactory-derived
progenitors to promote the generation of DA neurons.
These studies demonstrate that the co-expression of Pitx3
and Nurr1 will enhance significantly the lineage restric-
tion of adult human progenitors toward dopaminergic
neurons which can be employed in cell-replacement
paradigms for the treatment of PD.
4.2. Treatment of hONPs with Morphogens
Enhances Intracellular and
Extracellular Dopamine Levels
Human adult epithelial derived progenitors have the
potential to differentiate along several neural lineages in
response to morphogenic signals in vitro [82]. For exam-
ple, 11.6 (±1.5) % of hONPs expressed TH following a 7
day treatment of RA1FN5Shh (1 µM RA, 5 µM FN and
15 nM Shh), indicating that a dopaminergic lineage can
be driven by exposure to these morphogens [53]. Sonic
hedgehog (Shh), (RA) and Forskolin (FN) have all been
shown to be crucial developmental factors that regulate
neuronal specification and differentiation [83-88]. Shh
has been shown to be required for the generation of ven-
tral midbrain motor neurons [89,90] as well as dopa-
minergic neurons in rodents [56,58,75] and chick em-
bryos [59]. This study suggests that Shh increases the
expression of TH and that the purity of Shh is an impor-
tant determinant of TH expression. RA regulates neu-
ronal differentiation in embryonic stem cells [91,92] and
adult human neuronal progenitors [93, 94]. RA has sev-
eral pathways through which it can effect cellular differ-
entiation [95,96]. FN is an adenyl cyclase activator that
increases intercellular levels of cAMP that can stimulate
axonal elongation [85,86] and induce embryonic rat/mouse
motor neuron survival [97,98]. Following the treatment
of RA and FN, the progenitor nature of hONPs is dimin-
ished, as characterized by a loss of nestin expression, and
the presence of more mature neuronal markers. In this
study, a combination of highly purified Shh, RA and FN
was applied to the lineage restricted hONPs. The intra-
cellular level of dopamine was demonstrated to be sig-
nificantly increased by this treatment. This result con-
firms and extends the published data by showing that
these morphogens can increase TH expression by pro-
genitors obtained from adult humans [53]. Furthermore,
following a 4 day treatment of RA1FN5-Shh, the dopa-
mine level of the spent conditioned medium was signifi-
cantly enhanced, indicating that the morphogens pro-
moted the release of dopamine, which is important for
future studies transplanting lineage restricted hONPs into
PD animal models. Among all 4 lineage restricted hONP
lines, those cells transfected with pIRES-Pitx3-Nurr1
produced and released the highest levels of dopamine in
the presence of Shh, RA and FN. This result is consistent
with the analysis of the lineage restricted cells in the ab-
sence of treatment with the morphogens. This data fur-
ther supports the conclusion that hONPs transfected with
pIRES-Pitx3-Nurr1 are the most efficient line in dopa-
mine production studies to date, and therefore are likely
candidates for engraftment into an animal model of PD.
Shh is secreted by the notochord and floor plate at early
stage of development [99], RA is detectable in the mid-
brain of chick and mice embryos [100], and FN is highly
concentrated in the rat substantia nigra [101]. The local
distribution of these morphogens in situ should influence
the engrafted hONPs and may further support their sur-
vival and dopamine release following transplantation.
The higher level of dopamine released following Shh,
RA and FN treatment suggests their potential utility for
cell-replacement therapy for PD. Previous studies on the
non-human primate PD models, demonstrated that the
transplanted responsive human embryonic progenitor
cells were still capable of differentiation to DA pheno-
type within the micro-environment around the lesioned
adult host SN, an unexpected finding was that the en-
graftment also up-regulated an endogenous progenitor
population [12]. The results of our studies utilizing a
paradigm that combines transfection and morphogen in-
duced lineage modulation highlight the potential therapeu-
tic utility of olfactory epithelial-derived neural progenitors
M. Wang et al. / Stem Cell Discovery 1 (2011) 29-43
Copyright © 2011 SciRes. Openl y accessible at http://www.scirp.org/journal/SCD/
as an autologous cell source for cell-based replacement
and regenerative strategies for patients with Parkinson’s
4.3. Lineage Restricted hONPs Retain Their
Capability to Produce Neurotrophic
It’s been reported that neurotrophins such as BDNF,
CNTF and NT-3 are crucial in the recovery of primate
and rodent models of Parkinson’s disease [12,102].
BDNF is a member of the neurotrophin family which
supports the maturation and survival of dopaminergic
neurons in substantia nigra [44,103]. In the presence of
BDNF, more TH positive cells can be found in cultures
of ventral mecensephalic tissue than in the absence of the
neurotrophin [103,104]. NT-3 belongs to the same family
of neurotrophins as BDNF, and has been shown to play a
protective role in the degeneration of adult central nora-
drenergic neurons in vivo [105,106]. CNTF has been
reported to rescue the degenerating striatal neurons in
primate and rodent models [45,107]. Furthermore, the
absence of CNTF leads to the apoptosis of motor neurons
in adult mice [46,108]. Collectively these studies strong-
ly suggest an important role for these neurotrophins in
future therapeutic strategies for neurodegenerative dis-
eases, including PD, Alzheimer’s disease and Huntington
disease. Therefore, a cell population that can produce
neurotrophins could be an ideal for therapy for these dis-
eases. They can provide protective micro-environments in
vivo and prevent, rescue and or replace neuronal degen-
eration. The pre-transfected hONPs were found to pro-
duce several neurotrophins including BDNF, NT-3, and
even nerve growth factor (NGF) when in a serum en-
riched medium [10]. The stably transfected lines were
examined to determine if lineage restriction to dopa-
minergic neurons or absence of serum alters the syn-
thesis of these neurotrophins since they play a role in
neuronal survival, differentiation and maturation. As
shown in the results, the transfection of hONPs did not
alter neurotrophin production. The post-transfected hONPs
produce BDNF, NT-3 and CNTF at equivalent levels with
the pre-transfected progenitors. Therefore, genetically
modified hONPs can not only serve as replacements of
the dead or dysfunctional dopaminergic neurons but also
can provide protective micro-environments to help res-
cue dying or damaged neurons from further degeneration
and to enhance the endogenous progenitor populations.
The stably lineage restricted hONPs are unique popula-
tions with high potential for cell transplantation for ani-
mal models of Parkinson’s disease.
The long term goal of this study is to develop restrict-
ed hONP lines that will have therapeutic utility in cell
replacement strategies for patients with PD. The in vivo
viability and stability are important variables, especially
considering the likelihood that with time the engrafted
population may die and require replacement. Therefore,
experiments were undertaken to determine the stability
and viability of frozen stocks of transfected cells. HONPs
survived under the pressure of selection after removal
from cryostorage and retained their ability to express TH,
as well as produce and release dopamine and neurotro-
phins, which further demonstrates the unique potential of
these progenitors to perhaps serve as an autologous cell
source for cell-based strategies for the long-term treat-
ment of Parkinson’s disease.
Human adult olfactory epithelial-derived progenitors
may provide a unique autologous cell population for cell-
based therapy of Parkinson’s disease, because of their
potential to become dopaminergic neurons which pro-
duce and release dopamine and their capability to pro-
vide neurotrophic factor enriched micro-environments
which support cell survival, protect cells from degenera-
tion and activate endogenous stem cell populations. In
vivo studies are in progress to determine the ability of
hONPs to diminish Parkinson like locomotory deficits in
a rodent model.
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