Lineage restriction of adult human olfactory-derived progenitors to dopaminergic neurons

doi:10.4236/scd.2011.13004

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Vol.1, No.3, 29-43 (2011)

doi:10.4236/scd.2011.13004

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.

ABSTRACT

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

1. INTRODUCTION

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

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

warranted.

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

compared.

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-

trophins.

2. MATERIAL AND METHODS

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

presented.

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

3131

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-

quencing.

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-

sis.

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

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).

3. RESULTS

Cryopreserved vials of the two representative hONP

M. Wang et al. / Stem Cell Discovery 1 (2011) 29-43

3333

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

 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

3535

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).

(a)

(b)

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

(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).

4. DISCUSSION AND CONCLUSIONS

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

3737

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

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

3939

as an autologous cell source for cell-based replacement

and regenerative strategies for patients with Parkinson’s

disease.

4.3. Lineage Restricted hONPs Retain Their

Capability to Produce Neurotrophic

Factors

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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