Stem Cell Applications for Parkinson’s disease
Shih-Ping Liu1,2,*, Ru-Huei Fu1,3, Yu-Chuen Huang4,5, Shih-Yin Chen4,5, Woei-Cherng Shyu1,3,
Shinn-Zong Lin1,3,6,7,*
1Center
for Neuropsychiatry, China Medical University and Hospital, Taichung, Taiwan
2Graduate
Institute of Basic Medical Science, China Medical University, Taichung, Taiwan
3Graduate
Institute of Immunology, China Medical University, Taichung, Taiwan
4Genetics
Center, Department of Medical Research, China Medical University Hospital,
Taichung, Taiwan
5Graduate
Institute of Chinese Medical Science, College of Chinese Medicine, China Medical
University, Taichung, Taiwan
6Department
of Neurosurgery, China Medical University Beigan Hospital, Yunlin, Taiwan
7Department
of Neurosurgery, Tainan Municipal An-Nan Hospital-China Medical University,
Tainan, Taiwan
Shih-Ping Liu and Shinn-Zong Lin contributed equally to this study.
Address correspondence to Shih-Ping Liu, PhD, No. 2, Yuh-Der Road, Taichung, Taiwan 40447,
Republic of China. Phone: 886-4-2205-2121 ext. 7828; Fax: 886-4-2205-2121 ext. 7810;
spliu@mail.cmu.edu.tw.
A Conflict of interest statement: The authors declare no conflict of interest.
Running Title: Stem cell Applications for Parkinson’s disease
1
ABSTRACT
Parkinson’s disease (PD) is a neurodegenerative disorder that results from the degeneration
of dopaminergic neurons. To date, effective therapeutic methods for PD are unavailable.
Therefore, stem cell therapy has provided hope for treatment of PD and for improving PD
symptoms. Stem cells have the ability of self-renewal and they can differentiate into a wide
variety of cell types with a potential for multiple therapeutic applications. Many animal
models of PD have been established, including toxin-induced and transgenic models, and
they are widely used to study the pathogenesis and cell therapeutic evaluation. Many types
of stem cells have been reported that can be used to improve the PD symptoms in animal
models. Embryonic stem (ES) cells and induced pluripotent stem (iPS) cells have been
observed to differentiate into functional dopamine neurons for cell therapy in a rat PD
model. Neural stem cells and mesenchymal stem cells are also being used in PD therapeutic
evaluation. Stem cell therapy has the potential to become a powerful tool for PD therapy in
the future. In this manuscript, we have discuss the current research on the potential
application of neural stem cells, mesenchymal stem cells, ES cells, and iPS cells for PD.
Keywords: stem cell therapy, Parkinson’s disease, embryonic stem cells, induced pluripotent
stem cells
2
Introduction
Parkinson’s disease (PD) is characterized by the progressive loss of terminals of the
neuromelanin (NM)-containing dopamine neurons in the substantia nigra (SNc), thereby
leading to reduced dopamine levels in the striatum (25). Current treatments for the
symptoms include levodopa (L-DOPA, the precursor to the neurotransmitters dopamine)
administration, neural lesion surgery, and deep brain stimulation (15,58). Unfortunately,
these treatments are not the effective therapeutic methods. For this reason, stem cells
therapy provides the hope for PD.
The existence of genetic forms of PD has been recognized in nearly 10–15% of PD
patients (9). Gene mutations in the α-synuclein (35), leucine-rich repeat serine ⁄ threonine
kinase 2 (LRRK2) (36), Parkin (42), and DJ-1 (3) have been reported to be associated with PD.
Many animal models of PD have been established, including toxin-induced
and
transgenic groups, and they are widely used to study the pathogenesis and evaluation of cell
therapy (9). The most widely used toxin-induced animal model to mimic PD is the mouse and
rat
model
administered
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
(MPTP),
6-hydroxydopamine (6-OHDA) Rotenone and Paraquat. Transgenic mice with mutations in
the α-synuclein (SNCA) (35), LRRK2 (36), and Parkin (42) genes are established and used to
study the pathogenesis of PD and also to evaluate cell therapy.
Toxin-induced PD mouse models
3
MPTP induced PD mouse model: Administration of MPTP is a classic systemic model
with selective toxicity towards dopaminergic neurons (33). Earlier reports have shown that
after MPTP was administered into the mice, it crosses the blood-brain barrier (BBB) and it is
transformed into the active metabolite, 1-methyl-4-phenylpyridinium ion (MPP+). MPP+ is
carried into dopaminergic neurons of the SNc to block the mitochondrial complex I activity
by the dopamine (DA) transporter (DAT) (33). These findings indicate that the mitochondria
of the dopaminergic neurons are the preferred target for this toxin.
6-OHDA-induced PD mouse model: Administration of 6-OHDA is also a classic systemic
model that shows selective toxicity for dopaminergic neurons (66).Compared to MPTP,
6-OHDA cannot cross the BBB and therefore a local injection is required (9). The structure of
6-OHDA is similar to dopamine and it binds with high affinity to DAT, which transports the
toxin inside dopaminergic neurons (9). At 12 h after 6-OHDA administration into SNc,
dopamine neurons begin to die (9). This resulted in a marked lesion of the dopaminergic
terminals in the striatum within 2–3 days, thereby leading to PD symptoms.
Rotenone-induced PD model: the Rotenone-induced model has two advantages to be
used for PD study. The first one is that this model reproduces most of the movement
disorder and Lewy bodies accumulation (5). The second reason is that Rotenone is a
powerful inhibitor of mitochondrial respiration (54).
Paraquat-induced PD model: The structure of Paraquat is similar to MPP+. Paraquat
4
enters animal brain by the neutral amino acid transporter (59). After entering cells, Paraquat
results in mitochondrial toxicity in dopamine secretion cells to lead to PD symptoms (47).
Transgenic mice model for PD
Over-expression of SNCA in families with multiplication types mutation of this gene
resulted in parkinsonism and subsequent dementia, characterized by diffuse Lewy body
disease, as assessed in autopsy cases (35). The importance of SNCA in the pathogenesis of
PD was reported by the presence of the missense mutation resulting in a A53T amino acid
change in SNCA in subjects with familial Parkinsonism (48). In addition, other mutations of
the SNCA, resulting in substitution of A30P and E46K amino acids, were also found to cause
familial Lewy body Parkinsonism (31,73). To date, several SNCA transgenic mice have been
established, for example, A53T SNCA transgenic mice (34) and A30P SNCA transgenic mice
(16). The A53T SNCA transgenic mice demonstrate an age-related phenotype, including
progressive motor deficits, presence of intraneuronal inclusion bodies, and neuronal cell loss
(34). The transgenic mice expressing the A53T missense mutant form of human SNCA,
spontaneously developed PD between 9–16 months of age (34). Brain regions also show
SNCA-dependent neural degeneration associated with increased SNCA aggregation.
Compared to A30P mutant or wild-type, the A53T SNCA transgenic mice display significantly
greater neurotoxicity in vivo (34). However, the A30P SNCA transgenic mice are associated
5
with the development of familial PD. At around 12 months of age, the mice develop
complications with the hind limb mobility because of loss of motor neurons and presence of
SNCA aggregates. These SNCA mutant transgenic mice model may be useful for studying PD
(16).
Substitutions in the LRRK2 gene have been linked to familial PD (46). Mutations in
LRRK2 are the most common genetic cause of PD (38). Li et al. developed an LRRK2 (R1441G)
transgenic mouse model that recapitulates the cardinal features of the disease:
age-dependent degeneration of SNc, levodopa-responsive slowness of movement,
diminished dopamine release and an axonal pathology of nigrostriatal dopaminergic
projection (38). LRRK2-G2019S transgenic mice also showed an age-dependent decrease in
striatal DA content, as well as decreased striatal DA release and uptake (36,55). LRRK2
mutant transgenic mouse models are also a useful model for the study of PD.
Parkin mutations are the most common cause of familial PD (42). Parkin-Q311X
mutation transgenic mice were established in 2009 (42). Parkin-Q311X mutation transgenic
mice exhibit several phenotypes, including multiple late-onset and progressive hypokinetic
motor deficits (42). Parkin-Q311X mutation transgenic mice also exhibit age-dependent
accumulation of SNCA in substantia nigra and colocalized with 3-nitrotyrosine, a marker for
oxidative protein damage (42).
6
Stem Cell Applications for Parkinson’s disease
Stem cells are capable of self-renewal and have the potential to become a part of
powerful clinical techniques. For example, hematopoietic stem cells (HSCs) isolated from the
bone marrow can be used to treat leukemia, hemophilia, and anemia. Mesenchymal stem
cells (MSCs), as well as their role in revascularization and tissue repair, are being studied in
terms of their response to ischemia and other injuries (39). For PD, cell therapy in the form
of dopamine neuron or neural stem cell transplants have been reported as alternative
treatment strategies (18). Stem cell therapy is still in its infancy, and most studies are based
on animal models. In this manuscript, we have provided an overview of the research
involving MSCs, neural stem cells (NSCs), embryonic stem cells (ESCs) and induced
pluripotent stem cells (iPSc) that have potential for application in PD therapy, in animal
models.
Mesenchymal stem cells (MSCs)
MSCs can be isolated from the bone marrow, synovial fluid, umbilical cord blood,
amniotic fluid, deciduous teeth, placenta, adipose tissue, and dermal tissues (32,60). MSCs
are capable of differentiating into adipocytes, osteoblasts, chondrocytes, and stromal cells.
MSCs are currently being used for many cell therapy applications (6,26). Recent studies have
shown the ability of MSCs to differentiate into connective tissue cell types (12,13,20).
In 2005, Lu et al. reported the therapeutic benefit of tyrosine hydroxylase
7
(TH)-engineered MSCs for PD (41). In their study, they over-expressed TH in MSCs and
transplanted TH-MSCs in to the striatum of 6-OHDA-induced PD rat. They found a significant
improved in the rotational behavior test scores in these PD rat models transplanted with
TH-MSCs. This study established that MSCs could be a useful gene delivery vehicle for
transferring genes to treat PD (41). A study by Hellmann et al. (24) also demonstrated that
MSCs could increase survival and migration in 6-OHDA-lesioned rodents. In 2007, Pisati et al.
reported that MSCs could implicate cell therapy in PD by induction of neurotrophin
expression (53). Bouchez et al. demonstrated that grafting of adult mesenchymal stem cells
in a 6-OHDA rat model of PD could partially recover the dopaminergic pathway (11). In vitro
experiments showed that MSC treatment also significantly decreased LPS-induced microglial
activation, and tumor necrosis factor (TNF)-alpha and inducible nitric oxide synthase (iNOS)
gene expression levels indicating that MSCs turn on the neuroprotective effects on
dopaminergic neurons via the anti-inflammatory pathway (30). It is also known that MSC
transplantation attenuates BBB damage and neuroinflammation and protects dopaminergic
neurons against MPTP toxicity in the SNc in an animal model of PD (17). VEGF-expressing
human umbilical cord MSCs also improved the therapy strategy for PD (70). In 2011, Delcroix
et al. reported the therapeutic potential of MSCs combined with pharmacologically active
microcarriers transplanted in hemi-parkinsonian rats correlated with the increased survival
of MSCs that secreted a wide range of growth factors and chemokines (19). Hypoxia has also
8
been reported to promote dopaminergic differentiation of MSCs and has shown to benefit
transplantation in a rat model of PD (68). In addition, MSCs also reported that could
differentiate into glial-like phenotype, as shown by immunoreactivity for glial fibrillary acid
protein (GFAP) and toward a neuronal (dopaminergic) phenotype was observed in vivo (10).
Based on the promising data obtained from animal studies, clinical trials on the
transplantation of MSCs have been initiated. MSCs were transplanted into PD patients to
evaluate the therapeutic effect; partial improvement in symptoms were noted and there was
no tumor formation (67). We believe that MSCs can be used to improve PD symptoms in the
future.
Neural stem cells (NSCs)
NSCs are capable of differentiating into neuronal and glial lineages (50). Using
flow-cytometry-based size separation (forward scattering), and surface antigens (e.g., CD24,
CD133) (27,65), NSCs can be isolated based on their physical properties such as granularity
(44,49). NSCs are being studied as candidates for neural transplantation in response to
neurological disorders (64).
In 1999, the application of human NSCs in the treatment of PD was reported (21). In
Phase I clinical trials, stem cells harvested from patients with PD were injected into their
own brains. Nearly 30% of the transplanted dopaminergic cells survived in the first two
9
months (21). Li et al. evaluated the transplanted NSC survival and differentiation rate in mice
treated with MPTP-induced PD in 2003 and they reported a low efficiency (37). Ahn et al.
also reported the survival and migration of transplanted neural stem cell-derived dopamine
cells in the brain of a parkinsonian rat (2). While some cells migrated into the striatum, no
significant improvement in the behavioral test was seen (2). In 2005, the transplantation of
neurosphere cell suspensions from aged mice was functional in the mouse model of PD that
induced by 6-OHDA (45). The results obtained in this study indicated that the subventricular
zone (SVZ) in the aged mouse brain contains cells that can be expanded in the form of
neurospheres and then transplanted into the damaged dopamine system to generate
functional cells (45).
A genetically modified NSC approach has also been tested. Neurturin-expressing NSCs
confer dopaminergic neuroprotection in a rat model of 6-OHDA induced PD (40). A
combination treatment of neurotrophin-3 gene and NSCs improved behavioral recovery in
PD rat models (22). Results obtained in this study indicate that NSCs expressing
neurotrophin-3 endogenously would be a better graft candidate for the treatment of PD.
Further, it had also been observed that transplantation of genetically modified NSCs
expressing TH gene into a PD rat model enhanced treatment efficacy (74). Xu et al. (71) used
the same model and approach in hemiparkinsonian rhesus monkeys and observed the
survival of modified NSCs at the transplantation sites.
10
Embryonic stem cells (ESCs)
ESCs are pluripotent stem cells isolated from the inner cell masses of mammalian
blastocysts. ESCs are capable of differentiating into all the three embryonic germ layers i.e.
the endoderm, mesoderm, and ectoderm cells (8). ESCs are useful in clinical cell therapies
because of their ability of self-renewal and capacity to differentiate into multiple cell types
(43). Most of the studies thus far have demonstrated that ESCs must differentiate into
specific stem cells or mature cell types, and then they must be used in cell therapy (4,7,51).
However, only few papers have shown the use of undifferentiated ESCs in cell therapy (72).
ESCs are capable of differentiating into functional dopamine neurons, and they are being
tested for use in cell therapy (28).
In 2000, Arnhold et al. showed that green fluorescent protein-labeled ESCs-derived
neural precursor cells differentiated into Thy-1-positive neurons and glia after
transplantation into adult rat striatum (4). These findings indicate that ESC-derived neural
precursor cells could appropriately differentiate into dopamine secreting cells in vivo. In
2002, Bjorklund et al. demonstrated that ESCs develop into functional dopaminergic neurons
after transplantation in a PD rat model (7). In this study, the authors directly transplanted
the undifferentiated ESCs into the rat brain and demonstrated that ESCs can spontaneously
develop into dopamine neurons. The dopamine neurons can restore cerebral function and
11
behavior in a rat model with PD (7). In the same year, Kim et al. (29) used an animal model to
show that ESC-generated dopamine neurons exhibit electrophysiological and behavioral
properties typical of midbrain neurons—a finding that might support the development of
treatment therapies for PD. This was further confirmed by Nishimura et al. who observed
improved behavioral recovery in mouse parkinsonian models upon transplantation of in vitro
differentiated dopaminergic neurons derived from ESCs (51). Monkey ESCs can differentiate
into dopamine neurons and function in a PD primate model (61). Recently, Yang et al. (72)
used porcine ESCs to direct differentiation into neural lineages to study the therapeutic
potential in a rat model of PD, and the data obtained revealed that these cells exhibit stably
decreasing asymmetric rotations (72). However, they did not mention about the safety of
the ESC direct transplanted into brain. ESC is the pluripotent cells that mean they have the
ability to differentiate into multiple cell types. Direct transplanted ESC into animal body need
to check the teratoma formation in the organ. It is safety to use the ESC-derived somatic
stem cell for cell therapy. Together, these results suggest that ESCs that differentiate into
neural lineages may help towards the development of therapies involving stem cell
transplantation.
Use of genetically modified ESCs has also been developed. Genetically modified human
ESCs (over-expression of TH and GTP cyclohydrolase I genes) relieve symptomatic motor
behavior in the rat model of PD (52). Abasi et al. also reported the synergistic effect of
12
beta-boswellic acid and Nurr1 over-expression on dopaminergic programming of antioxidant
glutathione peroxidase-1-expressing murine ESCs (1).
Induced pluripotent stem (iPS) cells
iPS cells are pluripotent stem cells that were originally generated by using fibroblasts
transfected with the transcriptional factors Oct4, Sox2, c-Myc, and Klf4 in 2006 (63). iPS cells
are similar to ESCs in terms of proliferation, morphology, gene expression, surface antigens,
epigenetic status of pluripotent cell-specific genes, and telomerase activity (62), but they
overcome hurdles associated with ESCs because of their generation from mature somatic
cells. iPS cells are viewed as having significant potential in cell therapy.
With recent developments in iPS cell technology, the most important aspect is if the
iPS cell-derived specific cells are functional in vivo. In studies on iPS cell research and PD, the
6-OHDA rat model of PD transplanted with the neurons derived from iPS cells showed an
alleviation in the symptoms of PD (69). 6-OHDA-lesioned rat PD model was also used to test
the therapeutic effect of these cells. The data obtained indicated that dopaminergic neurons
derived from human iPS cells could survive and integrate into 6-OHDA-lesioned rats (14). The
Parkinson patient-derived iPS cells were assessed for their ability to be used in therapy. After
transplantation of the differentiated Parkinson patient-derived iPS cells into 6-OHDA PD rat
models, these cells could grow in the adult rodent brain and reduce motor asymmetry (23).
13
Rhee et al. (57) reported that protein-based human iPS cells were capable of generating
functional dopamine neurons, which were used to treat PD rat model. The protein-based
human iPS cells differentiated into neural progenitor cells that were highly expandable
without senescence, and significant improvement in motor deficits were observed in
6-OHDA PD rats (57).
A genetically modified iPS cell approach was developed. The LRRK2 mutation human
iPS cells used for genetic correction of the LRRK2 G2019S mutation induced dysregulation of
CPNE8, MAP7, UHRF2, ANXA1, and CADPS2 (56). The data showed that genetic correction of
LRRK2 mutation in human iPS cells links PD neurodegeneration to ERK-dependent changes in
the gene expression levels (56).
In summary, iPS cell technology is the powerful method for cell therapy in the future. It
could overcome the hurdles of ESC and other adult stem cells, including ethic issue, the
amount of cells and immune rejection. However, the safeties of iPS cells need to be
demonstrated in the body even after differentiating into somatic stem cells. After checking
the safeties of iPS cells, there will become the powerful cell therapy material not only for PD.
CONCLUSION AND RESEARCH DIRECTIONS
Stem cell therapy is still in its infancy. However, stem cell therapy has the potential to
become a powerful therapeutic approach in the future, especially for diseases that lack
14
effective therapy methods, such as PD. The potential for stem cell-based therapies is
providing hope for many diseases. There are many hurdles in cell therapy that must be
overcome before its clinical applications. These aspects will be at the center of the research
conducted in this decade. There are still some problems for clinical application, ex: 1.
Immune rejection: the iPS cell technology may overcome this hurdle. 2. Cell number: MSC
and NSC population isolated from human body are not enough for clinical application. The
ESC and iPS cells needed to differentiate into somatic stem cells. However, the ESC and iPS
cell derived somatic stem cells is hard to purify and generate a big population. 3. Safeties:
Although stem cells therapy is considered correlate with tumorigenicity, there are few study
mentioned about the tumor formation in animal bodies. It is very important to check the
tumor formation potential in animal bodies for long term monitor in the future study for
clinical application. 4. Culture contaminations: Each of the stem cells needed to culture with
culture medium in vitro to increase the cell numbers. However, the source of the serum
(Bovine) is the very important xenogeneic contaminations for clinical application. We hope
stem cell therapy could overcome these hurdles and used for clinical application as soon as
possible.
ACKNOWLEDGMENTS
The authors wish to thank the Taiwan Department of Health Clinical Trial and Research
15
Center of Excellence (DOH102-TD-B-111-004) and China Medical University (CMU100-N2-10)
for their financial support.
16
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23
Table 1. The animal models used for PD study.
Type
Toxin
Toxin-induced
PD models
MPTP
Gene
mutaiotn
6-OHDA
Rotenone
Paraquat
Transgenic
mouse models
α-synuclein
(SNCA)
A53T, A30P
and E46K
LRRK2
R1441G and
G2019S
Parkin
Q311X
Phenotype
References
Block the mitochondrial complex I activity by
the dopamine (DA) transporter (DAT)
At 12 h after 6-OHDA administration into SNc,
dopamine neurons begin to die.
This model reproduces most of the movement
disorder and Lewy bodies accumulation
Paraquat results in mitochondrial toxicity in
dopamine secretion cells to lead to PD
symptoms
Over-expression of SNCA in families with
multiplication types mutation of this gene
resulted in parkinsonism and subsequent
dementia, characterized by diffuse Lewy body
disease, as assessed in autopsy cases
Age-dependent degeneration of SNc,
levodopa-responsive slowness of movement,
diminished dopamine release and an axonal
pathology of nigrostriatal dopaminergic
projection
Parkin mutants may exert dominant toxic
effects to cultured cells and such dominant
toxicity can lead to progressive dopaminergic
(DA) neuron degeneration
(33)
24
(9)
(5)
(47)
(35)
(38)
(42)
Table 2. Stem Cell Applications for Parkinson’s disease
Stem cell Type
Mesenchymal
stem cells
(MSCs)
Neural stem
cells (NSCs)
Embryonic stem
cells (ESCs)
Mouse model
6-OHDA-induced PD
rat
6-OHDA- induced
rodents
6-OHDA-induced PD
rat
MPTP-induced PD rat
Rotenone-induced
PD rat
MPTP-induced PD
mice
6-OHDA-induced PD
mice
Modified
TH-expressed
6-OHDA-induced PD
rat
6-OHDA-induced PD
rat
6-OHDA-induced PD
rat
Adult Wistar rats
Neurturinexpressing
Neurotrophin-3
expressing
TH-expressed
6-OHDA-induced PD
rat
6-OHDA-induced PD
rat
6-OHDA-induced PD
mice
6-OHDA-induced PD
rat
Induced
pluripotent stem
(iPS) cells
6-OHDA-induced PD
rat
6-OHDA-induced PD
rat
6-OHDA-induced PD
rat
6-OHDA PD rats
VEGF-expressing
ESC-derived
neural precursor
cells
ESC
ESC-generated
dopamine
neurons
Dopaminergic
neurons derived
from ESC
Over-expression
of TH and GTP
cyclohydrolase I
Neurons derived
from iPS cells
Dopaminergic
neurons derived
from human iPS
cells
Differentiated
Parkinson
patient-derived
iPS cells
iPS cells –
derived neural
progenitor cells
25
Therapeutic effect
Improved in the rotational
behavior test
Increase survival and migration
References
(41)
Partially recover the dopaminergic
pathway
Protects dopaminergic neurons
Improved the therapy strategy
(11)
Low efficiency
(37)
NSC transplanted into the
damaged dopamine system could
generate functional cells
Dopaminergic neuroprotection
(45)
SCs improved behavioral recovery
(22)
Enhanced treatment efficacy
(74)
Differentiated into Thy-1-positive
neurons and glia
(4)
Develop into functional
dopaminergic neurons
Electrophysiological and
behavioral properties typical of
midbrain neurons
Improved behavioral recovery
(7)
Relieve symptomatic motor
behavior
(52)
Alleviation in the symptoms of PD
(69)
Could survive and integrate into
6-OHDA-lesioned rats
(14)
Could grow in the adult rodent
brain and reduce motor
asymmetry
(23)
Expandable without senescence,
and significant improvement in
motor deficits
(57)
(24)
(17)
(70)
(40)
(29)
(51)