Download Adjusted from Momčilović et al., 2012

From Fibroblast to neuron:
the use of stem cells for Parkinson’s disease
Iris Wever
Iris Wever
Student number: 3258319
Supervisor: M.P. Smidt and J. Veenvliet
Master Thesis in the master programme
Neuroscience&Cognition, Utrecht University
December 2011- January 2012
Abstract
One of the characterizations of Parkinson’s disease (PD) is the motor symptoms caused by the loss of
the dopaminergic neurons of the substantia nigra pars compacta. The disease affects about 1% of the
world population above 60 years old and is second most common neurodegenerative disorder, after
Alzheimers disease. The mechanisms underlying the pathology of PD are not well understood and
there is no available cure up tot this date. The rather selective loss of the dopaminergic neurons of the
substantia nigra pars compacta makes the disease suitable for cell-replacement therapies. Currently
pluripotent stem cells represent the most promising source for the derivation of dopaminergic neurons.
Human embryonic stem cells are the best studied type of pluripotent stem cells and have been
successfully transplanted into animal models for PD, however another type of pluripotent stem cells,
namely induced pluripotent stem cells, hold a greater potential for personalized patient-specific stem
cells therapy and disease-specific disease modeling.
Introduction
Pluripotent stem cells (PSCs) represent a potential unlimited source of cells for applications in
developmental biology, pathological processes and regenerative medicine. PSCs are characterized by
prolonged undifferentiated proliferation and stable differential potential to form derivatives of all three
germ layers and can be derived from several different sources, including the inner cell mass of
blastocysts and the reprogramming of somatic cells to a pluripotent state (Evans&Kaufman, 1981;
Martin, 1981; Thomas et al., 1998; Takahashi et al., 2007). The derivation of midbrain dopaminergic
neurons (mDN) from PSCs forms a promising strategy for therapy in PD. PD is characterized by
progressive loss of the dopaminergic (DA) neurons of the substantia nigra pars compacta, which
project to the striatum and are responsible for movement control (Arenas, 2010; Momčilović et al.,
2012), making the disease suitable for cell replacement therapy (Kriks&Studer, 2009). First attempts
were made to replace the degenerated DA-producing neurons by using fetal mesencephalic tissue
(Meyer et al., 2012; Ribeiro et al., 2012). In the last 20 years between 300 and 400 PD patients have
had a graft of fetal mesencephalic tissue transplanted with varying results (Arenas, 2010). While
successful transplantations verified that PD was suitable for cell-replacement therapy, failure marked
issues in need of alterations. The source and availability of the donor tissue are strongly associated
with ethical concerns. In addition, the poor survival of the grafts, the low percentage of mDN and the
risk of teratoma formation, indicated that an alternative cell source was required for clinical translation
(Kriks&Studer, 2009; Arenas, 2010; Momčilović et al., 2012). The most promising source of cells
were PSCs and several protocols to generate mDN neurons in vitro were developed (Lee et al., 2000;
Barberi et al., 2003; Ying et al., 2003; Perrier et al., 2004). The protocols can be divided into three
main categories: i) the default pathway methods, which relies on spontaneous differentiation of PSCs
due to an intrinsic program; ii) a feeder-based system, where PSCs are co-cultured with stromal cells
which promote differentiation and survival; and iii) differentiation of PSCs in a chemically defined
medium, promoting differentiation by recapitulating embryonic development ( Lee et al., 2000;
Tropepe et al., 2001; Barberi et al., 2003; Ying et al., 2003; Perrier et al., 2004 Smukler et al., 2006;
Kriks et al., 2011). Thus far DA cells have been successfully generated using protocols from all three
categories (Li et al., 2008; Laguna-Goya&Barker, 2009). Neural cells derived from mouse embryonic
stem cells (mESCs) were shown to survive transplantation in rodent models and supported behavioral
recovery. The same was observed with monkey ESCS that were grafted into monkeys (Li et al., 2008).
However, grafts with human ESCs (hESCs)-derived neurons contained relatively few surviving
dopaminergic neurons after transplantation and generated tumors more frequently (Wang et al., 2007;
Li et al., 2008; Hwang et al., 2010). Before hESCs can be used for cell therapy, it needs to be
determined which method is most suitable to generate DA neurons, how to promote survival after
transplantation and prevent tumor formation in vivo. In addition to determining the correct method of
how to generate the neurons it also remains undefined as to which PSCs type can be best used to
derive the neurons, as all types have advantages and disadvantages in respect to their source,
differential potential and tendency to form teratomas (Momčilović et al., 2012; Robintong&Daley,
2012). This review provides an overview of the methods that are currently used to generate
dopaminergic neurons and of the two main types of PSCS used; ESCs and induced pluripotent stem
cells (iPSCs), and it will discuss which method and cell type is most suitable for clinical applications.
Developmental program of midbrain dopaminergic neurons
Committing to a neuronal fate
The acquisition of neural fate by embryonic ectodermal cells is a critical event in the morphogenesis
of developing embryos. The initial model for neural induction suggests that the forming organizer/
node emits diffusible inhibitors of Bone morphogenetic protein (BMP), permitting ectodermal cells to
acquire a neuronal fate by default (Streit et al., 2000; Wilson&Edlund, 2001; Delaune et al., 2005;
Stern, 2005). Although down-regulation of BMP signaling in the prospective neural plate seems to be
required, it is not sufficient (Streit et al., 2000; Wilson et al., 2001; Delaune et al., 2005; Marchal et
al., 2009), as mouse embryos lacking a functional node still developed a neural plate and ectopic
expression of BMP antagonist in the epiblast did not lead to the induction of neural markers
(Ang&Rossant, 1994; Wilson&Edlund, 2001; Stern, 2005). The induction of neural tissue appeared to
be more complicated than initially thought. Other instructive signals were found to be important for
the specification of neural cells, among which WNTs and Fibroblast growth factors (FGFs) (Alvarez
et al., 1998; Streit et al., 2000; Wilson et al., 2000; Wilson et al., 2001; Delaune et al., 2005; HeegTruesdall&LaBonne, 2006; La Vaute et al., 2009). The integration of WNT, FGF and BMP signaling
specify cells towards a neuronal or epidermal fate before the onset of gastrulation (FIG. 1)
(Wilson&Edlund, 2001).
Figure 1. Schematic overview of neural induction. The state of WNT signaling determines whether primitive
ectodermal cells respond to FGF or BMP, by inhibiting FGF signaling. With the activation of FGFs a
transcriptional program is initiated which actively inhibits alternatve cell fates and induces genes involved in
neural induction.
Depending on the state of WNT signaling epiblast cells acquire either neuronal or epidermal fate.
WNT signaling prohibits the response of epiblast cells to FGF signaling, allowing the expression and
signaling of BMPs to redirect the cells towards an epidermal fate, while the lack of exposure to WNT
signaling allows FGFs to induce a neural fate (Wilson et al., 2000; Heeg-Truesdell&LaBonne, 2006).
Different FGFs were found to be involved in several stages of the phased progression of pluripotent
cells towards neural differentiation (Sterneckert et al., 2010). The initial step of neural induction is the
formation of primitive ectoderm from epiblast cells, which was found to be dependent on the high
activation of the ERK-members of the MAP kinase family by either FGF2 or FGF4 (Denaule et al.,
2005; Stavridis et al., 2007; Kunath et al., 2007; Sterneckert et al., 2010). The primitive ectoderm is
susceptible to inductive signals that redirect to cells to form neural tissue. FGF8 is implicated to be
the endogenous inducer of neuronal fate by enabling cellular differentiation and cross-reacting and
inhibiting BMP and Activin signaling (Sterneckert et al., 2010). In addition to the suppression of
alternative cell lineages, FGF8 also acts as a positive inducing signal for several neural fate regulators,
like Poly (ADP-ribose) polymerase-1 and Zic genes (Marchal et al., 2009; Dang&Tropepe, 2010;
Zhang et al., 2010; Yoo et al., 2011), and is involved in the induction of Sox2 and the maintenance of
Sox3, two important early neuronal genes (Rogers et al., 2011). Although FGF signaling is required
for neural induction, to further promote neural differentiation FGF-mediated ERK activity needs to be
decreased (Delaune et al., 2005; Stavridis et al., 2010; Jaeger et al., 2012). In ESC the attenuation of
FGF signaling is regulated by Retionic Acid (RA). ESCs require an initial period of ERK signaling,
caused by a RA-mediated increase in Fgf8 expression, after which a decrease is necessary to further
promote differentiation. The decrease in ERK signaling is mediated by the inhibition of Oct4 by RA,
causing Fgf4 levels to gradually reduce (Stavridis et al., 2010). In addition to its role in FGF signaling
RA was also found to be involved in the inhibition of mesoderm formation from ESC, by inhibiting
Nodal/Activin signaling (Engberg et al., 2010), suggesting that RA might also play an important role
in inducing neural tissue.
Formation of the midbrain dopaminergic progenitor domain
Coincident with the initial specification of the neuroectoderm, anterior-posterior (AP) patterning is
initiated by posteriorizing signals derived from adjacent non-neuronal tissue (Foley et al., 2000;
Wilson&Edlund, 2001; Stern, 2002; Stavridis et al., 2007; Yoo et al., 2011). The prospective forebrain
cells are protected from these signals by antagonists secreted by the anterior visceral endoderm (AVE)
and in later stages by the anterior definitive endoderm and the anterior axial mesoderm (Muhr et al.,
2000; Foley et al., 2000; Kimura et al., 2000; Wilson&Edlund, 2001; Stern, 2002; Lewis&Tam,
2006). Three factors have been suggested to impose posterior character upon anterior neural tissue,
FGFs, Retinoic acid (RA) and WNTs (Nordström et al., 2002; Kudoh et al., 2002; OliveraMartinez&Storey, 2007; Ribes et al., 2009). These three pathways are important during several
temporal and spatial aspects of neural patterning, suggesting that they interact differently and use
distinct co-factors depending on the time and position where they are active (Kudoh et al., 2002;
Olivera-Martinez&Storey, 2007; Ribes et al., 2009). The initial regionalization subdivides the neural
plate into four distinct territories comprising the prosencephalon, mesencephalon, rhombencephalon
and the spinal cord. During the course of development the AP pattern becomes progressively more
refined by signal derived from secondary organizer and distinct neural progenitor domains are formed
at specific positions along the AP and the dorsoventral (DV) axes (Shamin et al., 1999; Wurst&BallyCuif, 2001; Prakash&Wurst, 2004; Olander et al., 2006). The isthmic organizer (IsO) is the secondary
organizing centre required for the proper specification of mesencephalic and metencephalic structures
(Wurst&Bally-Cuif, 2001). The IsO is induced at the junction between the midbrain and hindbrain
under the influence of signals derived from the node and the primitive streak (Olander et al., 2006),
identified in chick as WNT and FGF (Crossley et al., 1996; Shamin et al., 1999; Wurst&Bally-Cuif,
2001; Nordstrom et al., 2002; Olander et al., 2006). The location of the IsO is determined by the
mutual repression of two opposing transcription factors Otx2 and Gbx2. Otx2 is already expressed
prior to the onset gastrulation in the AVE and in the induced neuroectoderm. During gastrulation,
however, its expression becomes progressively restricted to the anterior region of the mouse embryo in
all three germ layers. Conversely, Gbx2 is initially expressed throughout the germ layers in the
posterior part of the embryo and becomes limited to the anterior hindbrain later in development (Millet
et al., 1996; Millet et al., 1999; Kimura et al., 2000; Prakash&Wurst, 2004; Alavian et al., 2008).
Coincident with the formation of the sharp border by Otx2 and Gbx2, the expression patterns of Wnt1,
Fgf8, Pax2/5, Lmx1b and En1/2 become refined to the areas within and adjacent to this border,
forming the complex polarized pattern of transcription factors characteristic for the IsO (FIG. 2)
(Crossley et al., 1996; Reifers et al., 1998; Shamin et al., 1999; Adams et al., 2000; Alavian et al.,
2008). FGF8 was identified as one of the key mediators of IsO functioning. Ectopic application of
FGF8 to the rostral mesencephalon and caudal diencephalon showed the same refinement of
expression patterns of Wnt1, Pax2/5, Lmx1b and En1 as observed at the MHB, indicating that FGF8 is
sufficient to maintain and restrict the expression of these genes to areas within and adjacent to the IsO
(Crossley et al., 1996; Shamim et al., 1999). Despite the fact that the relationship between FGF8 and
the transcription factors it regulates is only partially understood, experiments using targeted gene
disruption in transgenic mice imply that the factors might contribute to a regulatory loop maintaining
each other’s expression (Crossley et al., 1996; Reifers et al., 1998; Shamim et al., 1999; Adams et al.,
2000; Alavian et al., 2008). While the IsO determines the AP position of the mes- and metencephalic
structures, the DV axis is regionalized by BMP, WNT and Sonic hedgehog (SHH) secreted by the
notochord, the floor plate, the roof plate and the non-neuronal ectoderm (Hynes et al., 1995a;
Wurst&Bally-Cuif, 2001). SHH is an important ventral signaling molecule initially expressed in the
notochord and later in the floor plate along the neural axis. A series of elegant experiments in the
midbrain showed that the efficient induction of mDN progenitors is mediated by a contact-dependent
mechanism for which SHH is required (Hynes et al., 1995a; Hynes et al., 1995b). The integration of
the signals from both the IsO and the floor plate is required to define the domain in which the mDN
progenitors will further develop and also to maintain and form the progenitor-populations
(Wurst&Bally-Cuif, 2001).
Figure 2. Transcriptional regulation of the formation and maintance of the Isthmus. The mutual repression of
GBX2 and OTX2 determine the location of the Istmic Organizer. The expression pattern of Fgf8, Wnt1, En1,
Lmx1b and Pax2, -5 become restricted to areas adjacent to the Isthmic Organizer and are important for the
specification of the mesencephalon and rhombomere. The factors form a regulatory loop to maintain each other’s
expression
Transcriptional regulation of neurogenesis
Cell mapping studies suggest that mDN progenitors originate from floor plate cells that develop at the
ventricular zone (VZ) of the ventral mesencephalon. Although floor plate cells were characterized as
non-neurogenic cells, recent studies showed that mesencephalic floor plate cells can acquire a mDN
progenitor identity under the influence of Otx2 and Wnt1 (Andersson et al., 2006a; Ono et al., 2007;
Chung et al., 2009). OTX2 together with WNT1 and SHH induce the expression of Lmx1a in the cells
(Andersson et al., 2006a; Ono et al., 2007; Chung et al., 2009; Momčilović et al., 2012). Lmx1a was
found to control the timing of mDN neurogenesis in chicks and mice by activating Msx1, which
activates Neurogenin2 (Ngn2), a proneural transcription factor that promotes neurogenesis (Puelles et
al., 2004; Vernay et al., 2005; Andersson et al., 2006b; Ono et al., 2007; Omodei et al., 2008). In
addition to the control of neurogenesis, the Wnt1/Otx2/Lmx1a pathway is also involved in the
developmental programming of mDN progenitors, by suppressing alternative cell fates and regulating
the expression of transcription factors involved in the induction of the DA neurotransmitter phenotype,
like Nurr1 and Pitx3 (Puelles et al., 2004; Vernay et al., 2005; Andersson et al., 2006b; Ono et al.,
2007; Omodei et al., 2008; Chung et al., 2009; Lin et al., 2009). However, the phenotype inducing
activity of this pathway is context dependent, as ectopic expression of Lmx1a could only generate
ectopic mDN in the ventral mesencephalon. In addition, Lmx1a is only competent to induce mDN in
stem cells that have been ventralized by SHH (Anderson et al., 2006b), indicating that cooperation
with other factors is required to specify mDN progenitors and to initiate the differentiation program.
Figure 3. Schematic representation of the transcriptional regulation of midbrain dopaminergic neurons (mDN).
Under the influence of SHH, ventral mesencephalic progenitors arise in the floor plate, which are FOXA1, -2
positive. Further exposure to WNT1 and OTX2 induces Lmx1a in these cells. FOXA1, -2 together with LMX1A
further promote neurogenesis and maturation of the mDN progenitors and inhibit alternative cells fates.
Members of the Foxa subfamily of forkhead/winged helix transcription factors are induced by SHH in
a wide domain in ventral mesencephalic progenitors from an early time point on and co-expression of
FOXA1/2 with LMX1A confirmed that both FOXA proteins are also expressed in mDN progenitors.
The analysis of gain-of-function and loss-of-function studies demonstrated that Foxa1/2 are required
for the maintenance of Shh and the inhibition of Nkx2.2, thus playing a critical role in regulating
neuronal subtype identity in the mesencephalon. Furthermore, Foxa1/2 is necessary to maintain
Lmx1a/b expression in ventral mDN progenitors and it functions cooperatively with Lmx1a to
coordinate specification and differentiation of mDN progenitors (FIG 3.) (Ferri et al., 2007; Lin et al.,
2009; Nakatani et al., 2010).During neurogenesis, mDN progenitors in the VZ enter the G0 phase of
the cell cycle and give rise to post mitotic mDN precursor that start migrating to their specific ventral
position via radial and tangential migration (Shults et al., 1990; Kawano et al., 1995). Just below the
VZ, mDN precursors start expressing the DA synthesizing enzyme AADC and the orphan nuclear
receptor Nurr1. In the absence of Nurr1, mDN precursors are arrested in a developmental state and die
as development progresses to the neonatal stage (Saucedo-Cardenas et al., 1998). Nurr1 is required for
neurotransmitter phenotype determination, by regulating tyrosine hydroxylase (Th), vesicular
monoamine transporter 2 (Vmat2) and the dopamine transporter (Dat) expression (Saucedo-Cardenas
et al., 1998; Smits et al., 2003). Paired-like homeobox gene Pitx3 was also identified as an important
factor for the development of mDN. The expression of Pitx3 in the brain is restricted to mDN and is
induced at the same developmental stage as Th (Smidt et al., 1997). Recent studies have demonstrated
a relationship between Pitx3 functioning and Nurr1 transcriptional activity. It was shown that PITX3
protein interacts with the NURR1 transcriptional complex and that recruitment of PITX3 leads to the
dissociation of co-repressors SMRT and the activation of NURR1 (Jacobs et al., 2009a). Further
confirmation of this interaction between Nurr1 and Pitx3 was given when a co-regulatory effect of
NURR1 and PITX3 was observed for novel targets of NURR1 (Jacobs et al., 2009b). The factors
induced by Pitx3 and Nurr1 are crucial for the establishment of fully differentiated mDN.
In vitro derivation of mDN from PSCs
Knowledge of the signaling pathways underlying midbrain specification and mDN differentiation has
been employed to develop promising strategies for the in vitro derivation of DA neurons from PSCs.
All protocols can be subdivided in several stages: (i) neural induction (ii) exposure to midbrain
patterning factors, e.g. Shh and Fgf8, to promote mid- and hindbrain identity of the neural precursors
(iii) terminal differentiation and maturation in the absence of mitogens but in the presence of survivalpromoting factors, e.g. BDNF, GDNF and TGF-beta3 (Lee et al., 2000; Rolletschek et al., 2001).
Most available protocols use the same patterning factors followed by terminal differentiation in the
presence of survival-promoting factors, however, considerable different strategies are used to induce
the neural lineage (Table 1). The first successful demonstration of the generation of mDNs from PSCs
was based on the formation of Embryonic bodies (EBs) (Lee et al., 2000). EBs are thought to mimic
the environment of the peri-implanted embryo in which cell-cell interactions facilitate the formation of
all three germ layers (Kriks&Studer, 2009). To select for neuronal lineage a defined medium is used,
which is later enriched with SHH and FGF8 to promote DA neuron differentiation (Lee et al., 2000;
Swistowski et al., 2010). Although this method allows for the study of the function of factors and the
mechanisms behind neural differentiation, function and survival, the method requires extended in vitro
culturing and has a low efficiency, as only 30-34% of the total amount of neurons are also TH+ (Lee et
al., 2000; Swistowski et al., 2010). A faster and more efficient method of the in vitro derivation of
DA neurons from PSCs is a single feeder-based system (Barberi et al., 2003; Perrier et al., 2004). By
co-cultivating ESCs with bone marrow stromal cells and a sequential treatment with morphogens and
survival-promoting factors, 65-80% of the total amount of generated Tuj+ cells also expressed tyrosine
Table 1. Methods for the derivation of DA neurons
Key features
Efficiency
References
52% neurons, 30% of which where
TH+ cells
30-50% of the TUJ1+ neurons, of
which 64-79% were TH+
Kawasaki et al., 2002
22% of all cells were TH+
50-60% of the TUJ+ cells are also
TH+
Lee et al., 2000
Swistowski et al., 2010
Co-culture methods
Neural induction by co-culturing mESCs
with stromal feeders.
Neural induction by co-culturing mESCs
with stromal feeders, followed by
treatment with morphogenes and growth
factors
EB-based methods
Five step protocol from mouse ESCs.
Neural induction in EBs followed by
application of morphogens and growth
factors.
Default pathway
Serum-free, EB-free and without
morphogens.
In the presence of SMAD inhibitors,
followed by treatment with morphogens
> 90% of NESTIN and SOX1+ cells
> 80% PAX6 and SOX2+ cells
Perrier et al., 2004
Smukler et al., 2006
Chamber et al., 2009 and
Zhou et al., 2010
* Adjusted from Momčilović et al., 2012
hydroxylase (TH) (Perrier et al., 2004). In addition, the derived mDNs were successfully transplanted
into parkinsonian mice, which showed an alleviation of the behavioral deficits. The grafts consisted of
at least 70% TH+ cells and extended over a large portion of the striatum (Barberi et al., 2003).
However, although the feeder-based seems very successful, other studies showed that the feeder-based
protocol did not have such high survival rates of the mDNs 8 weeks after transplantations (Perrier et
al., 2004). Other studies have thus focused on developing protocols based on default-like mechanisms.
ESCs grown in monolayers gave rise to SOX1+ cells upon withdrawal of LIF and cultured in serum
free medium (Tropepe et al., 2001; Ying et al., 2003; Smukler et al., 2006). To promote mDN
differentiation, cells were replated and cultured in the presence of SHH and FGF8, resulting in a
significant amount of TH+ neurons (Ying et al., 2003). To decrease the heterogeneity in the type of
neurons created by these methods and to increase the yield of mDNs, genetic strategies were
developed. Several transcription factors, like Nurr1, Pitx3 and Lmx1a, were over-expressed in mESCs
and all lines showed enhanced yield, survival and expression of the appropriate set of mDN markers
after differentiation was induced (Chung et al., 2002; Andersson et al., 2006; Hedlund et al., 2008;
Friling et al., 2009). Next to genetic strategies to enhance the yield and decrease the heterogeneity of
the population, chemical compounds were utilized to control stem cell fate. The synergistic action of
two SMAD inhibitors, Noggin and SB431542, was found to be sufficient to induce rapid and complete
neural conversion in over 80% of the hPSCs (Chambers et al., 2009). Recently, compound C was
identified as a potent regulator of fate decision in hESCs and hiPSCs (Zhou et al., 2010). Compound C
targets at least seven of the receptors of the TGF-beta super family, thereby blocking Activin and
BMP signaling pathways. The dual inhibition of Activin and BMP probably accounts for the rapid and
highly efficient neural conversion of hPSCs (Zhou et al., 2010). Most available methods have been
used for both ESCs and iPSCs and result so far show that irrespective to their source, the cells behave
virtually similar to each other (Chin et al., 2009).
Embryonic stem cells
ESCs were successfully isolated from the inner cell mass of pre-implanted or peri-implanted mouse
embryos for the first time in 1981 (Evans&Kaufman, 1981; Martin, 1981). With the successful
isolation and the establishment of ESC lines several new experimental approaches became available
for multiple applications including elucidating early development, functioning of genes, toxins and
pharmaceutical screenings and even cell-replacement therapy (Kellet et al., 1995; Zhang et al., 2006
Muguruma&Sasai, 2012). The different strategies used to direct neural differentiation were adapted
for the generation of neural progenitors and DA neurons from ESCs from different sources, e.g. mouse
and human (Kriks&Studer, 2009). Although the signals that induce neural lineage, midbrain
specification and DA differentiation appear similar, there are some remarkable differences in the
signals that regulate maintenance of the pluripotency of ESCs from different species. The maintenance
of the pluripotent state of mESC in vitro is dependent on the presence of a gp130 agonist, e.g.
leukaemia inhibitory factor (LIF) (Keller et al., 1995; Rathjen et al., 2002), while the maintenance of
the undifferentiated state of hESC requires active FGF2 to promote self-renewal and TGF-beta
signaling to repress BMP-induced differentiation (Oh&Choo, 2006; Xu et al., 2008). In addition to
differences in the maintenance of pluripotentcy, the response to BMP inhibition also seems to vary
between humand and mouse ESCs. The addition of BMP signaling inhibitors like noggin seem
dispensable for neural differentiation in mESCs, however differentiation towards neural fate is greatly
enhanced in hESC by active BMP inhibition (Chambers et al., 2009; Kriks&Studer, 2009; La-Vaute et
al., 2009; Zhou et al., 2010). Another difference can be observed in the timeline of in vitro
differentiation. Most mESC based protocols yield DA neurons within about 2 weeks of differentiation,
while hESC based protocols require 1-2 months to obtain DA neurons (Kriks&Studer, 2009).
Furthermore, mESCs-derived DA neurons supported behavioral recovery after transplantation into
Parkinsonian mice, while hESCs-derived DA neurons generally show poor in vivo performance (Wang
et al., 2007; Li et al., 2008; Hwang et al., 2010; Kriks et al., 2011). Initially it was thought that low
frequency of TH+ cells was caused by apoptosis, however recent work showed that TH+ cells has a
instable phenotype which is not maintained in vivo, suggesting that hESCs-derived neurons might not
be properly specified (Li et al., 2008). The loss of DA phenotype is not the only concern, since the
presence of other cell types, e.g. serotonergic neurons, undefined neural progenitors or
undifferentiated hESCs, in the transplant can cause off-medication dyskninesias, neural overgrowth
and teratoma formation (Li et al., 2008; Lindvall&Kokaia, 2010). For the development of a safe cell
replacement therapy for PD it is necessary to overcome the risks and the challenges associated with
the generation of DA neurons, including the immune responses that are triggered by the
transplantation. To overcome immune rejection several approaches have been considered, including
the generation of hESCs that genetically match the host by somatic cell nuclear transfer. However this
strategy has came across a lot of ethical controversy driven by the opposition to human cloning. In
addition to the ethical concerns, the oocyte nucleus needs to remain intact to derive pluripotent stem
cells, resulting in cells that are triploid making them unsuitable for therapeutic use (Li et al., 2008;
Robinton&Daley, 2012).
Induced pluripotent stem cells
An alternative for somatic nuclear transferring for the generation of personalized patient-specific stem
cells was developed after the discovery that somatic cells could be reprogrammed to an ESC-like
pluripotent state (Table 2) (Takahashi&Yamanaka, 2006). Retrovirus-mediated transfection of just
four transcription factors, namely Oct3/4, Sox2, c-Myc and Klf4, could generate iPSCs from mouse
embryonic fibroblasts and mouse tall-tip fibroblasts (Takahashi&Yamanaka, 2006). The same factors
could be used to reprogram human somatic cells, including dermal fibroblasts of a male suffering from
multifactorial PD (Takahashi et al., 2008; Park et al., 2008). However, iPSC-derived chimeras
Table 2. Methods to obtain induced pluripotent stem cells.
Vector type Factors*
Advantages
Disadvantages
Retrovirus OSKM+
Efficient
Genomic integration
OSK
and incomplete viral
silencing
Lentivirus
OSNL
Inducible
lentivirus
Efficient
OSKM+OSK Efficient and
controlled
expression of genes
Adenovirus OSKM+OSK No genomic
integration
Sendaivirus OSKM
No genomic
integration
Inducible
OSKM
Sendaivirus
Genomic integration
and incomplete viral
silencing
Genomic integration
and need for
transactivator
Low efficiency
Sequence-sensitive
RNA replicase and
difficult to remove
virus
Sequence-sensitive
RNA replicase
Reference
Takahashi&Yamanaka,
2006; Takahashi et al.,
2007; Nakagawa et al.,
2008; Wernig et al., 2008
Yu et al., 2007
Soldner et al., 2009
Okita et al., 2008
Fusaki et al., 2009
No genomic
Ban et al., 2011
integration, virus
easy to remove
Protein
OSKM
No genomic
Low efficiency,
Zhou et al., 2009
integration, direct
requires large
delivery of the
quantities of highly
factor without
purified protein
DNA-related
complications
mRNA
OSKM +
No genomic
Labor intensive,
Warren et al., 2010
OSKML
integration, fast,
require multiple
controllable and
round of
efficient production administration
K; Klf4, L; Lin28, M; c-Myc, N; Nanog, O; Oct3/4, S; Sox2 (Adjusted from Robinton&Daley, 2012).
frequently developed tumors resulting from the reactivation of the oncogene c-Myc (Okita et al.,
2007). To overcome this issue, the protocol was adjusted to derive iPSCs in the absence of c-Myc
(Nakagawa et al., 2008; Wernig et al., 2008). However the use of integrating viral vectors to
transduce the reprogramming vectors limits the use of these cells for clinical application, since even
the lowest viral expression may affect differentiation and can still cause malignancies in animal
models, even in the absence of c-Myc (Okita et al., 2007; Yu et al., 2007). Protocols were explored
based on nonintegrating strategies, including the use of cell-penetrating reprogramming protein (Zhou
et al., 2009), an adenovirus-mediated delivery system (Okita et al., 2008) and doxycycline-inducible
lentiviral vectors (Soldner et al.,2009). However these approaches results in a lower efficiency in
iPSC production than methods with integrating viruses (Fusaki et al., 2009; Momčilović et al., 2012).
The use of synthetis messenger RNA as a non-integrating, transgene-free strategy showed enhanced
efficiencies and kinetics, but required daily administration of the RNA (Warren et al., 2010). An
alternative method that is simpler than the use of synthetic messenger RNA and also has a high
efficiency in creating iPSC involves the use of Sendai viruses (Fusaki et al., 2009). To overcome any
safety concerns about sustained cytoplasmic replication of the viral vector, a temperature-sensitive
sendai viral vector was created. Although the level of factor-carrying virus was rapidly decreased
during cell expansion, the virus could also easily be removed by using a temperature-shift protocol
(Ban et al., 2011). iPSCs possess the same characteristics as ESCs, like prolonged proliferated and a
stable differential potential to form all somatic cells ( Takahashi&Yamanak, 2006). Adaption of ESC
growth and differentiation protocols for iPSCs has led to the succesfull derivation of TH+ cells from
hiPSCs (Chambers et al., 2009; Swistowski et al., 2010). Recently it was shown that DA neurons
could be obtained from hiPSCs using a xenogenic-free defined medium and differentiation protocol,
which were successfully transplanted into a Parkinsonian rat, leading to recovery of the behavioral
symptoms (Swistowski et al., 2010). However, before hiPSC are suitable for clinical applications the
same problems as observed with hESC need to be overcome, in addition to the optimization of the
production method (Hwang et al., 2010; Momčilović et al., 2012; Robinton&Daley, 2012).
Discussion
PD affects about 1% of the world population over 65 (Laguna-Goya&Barker, 2009). The main
features of the disease are resting tremor, rigidity and bradykinesia/akinesia (Hwang et al., 2010;
Momčilović et al., 2012). Although there is medication available to alleviate the symptoms of PD,
like L-DOPA, the drugs have a limited affect and are associated with numerous side effect, including
hallucinations, anxiety, nausea, disorientation and confusion (Nutt et al., 2005). An alternative
approach to treat the motor symptoms of PD would be the substitution of the lost DA neurons.
PSCs represent a potential unlimited source of defined DA neurons at any stage of
differentiation and although recent developments in growth and differentiation protocols have
demonstrated effective ways to acquire DA neurons, considerable challenges remain in translating
these strategies into safe and efficient cell therapy for PD (Kriks&Studer, 2009; Lewis, 2012). A
highly efficient strategy to derive DA neurons from hESCs is co-cultivation of hESCs with stromal
cells and a sequential treatment with morphogens and growth factors (Barberi et al., 2003; Perrier et
al., 2004). However the presence of animal-derived products in the media prohibits the use of these
protocols for clinical applications. To overcome this, hESCs were cocultured with human fetal
mesencephalic astrocytes and sequentially treated with SHH and FGF8. Although DA neurons were
derived with high efficiency, grafts exhibited phenotypic instability and persistent proliferation of
undifferentiated cells (Roy et al., 2006). The formation of EBs is another method commonly used to
induce neural lineage. With this method DA neurons were successfully derived from both hESCs and
hiPSCs (Swistowski et al., 2009; Swistowski et al., 2010). These neurons could be transplanted into
Parkinsonian rats, which showed a behavioral recovery of the motor symptoms (Swistowski et al.,
2009; Swistowski et al., 2010). However, it is important that DA neurons are generated by methods
that are simple and easily scaled for production of large numbers of neurons and do not require
extensive in vitro culturing. In addition, cell culture conditions should preferably mimic embryonic
development, in that cells should respond similar to developmental modulators observed in vivo. So
far the best differentiation protocol for hESCs seems to be a floor-plate-strategy that follows
embryonic development. The strategy uses dual SMAD-inhibition to induce FOXA+ floor plate
precursors , which are than further specified by WNT to induce Lmx1a (Chamber et al., 2009)..
Sequential treatment with SHH and FGF8 led to the formation of DA neurons. These floor platederived DA neurons co-expressed LMX1A, FOXA2 and NURR1 and were successfully transplanted
into two rodent models for PD, leading to complete behavioral recovery. To test the scalability of the
approach, a pilot graft study was done in two adult Parkinsonian monkeys. The grafts showed good
survival rates and generated TH+ fibers that extended into the surrounding host tissue. In addition
hardly any contamination with other cell types, e.g. serotonergic, GABAergic and unspecified, was
observed, reducing the change of unwanted side affects, neural overgrowth and tumor formation.
Currently the two most used sources of PSCs are ESCs and iPSCs. The major advantages of
iPSCs over ESCs are the lack of ethical concerns and the possibilities to derive personalized patientand disease-specific stem cells. Both stem cell types suffer from relatively the same issues prohibiting
their use in clinical therapy. Differential problems related to the generation of large quantities of well
defined populations of DA neurons, which have a high survival rate and maintain a stable phenotype
after grafting, need to be solved, in addition to adverse surgical affects, including tumors formation
and immune reactions. Although iPSCs are less likely to trigger an immune response, they have a
higher tendency to form tumors (Nishikawa et al., 2008; Robinton&Daley, 2012). To overcome this
issue, safer methods for the generation of iPSCs were developed that did not require the use
integrating viruses (Hwang et al., 2010). In addition to overcoming the use of integrating viruses,
iPSCs seem to retaine a transcriptional memory of its cell of origin (Robintong&Daley, 2012).
Somatic memory can affect the autonomous differential potential of iPSCs and can bias the cell
towards the cell of origin (Robinton&Daley, 2012). Therefore iPSC lines can only be used for clinical
applications after a proper understanding is obtained of the genetic alterations caused by the
reprogramming. Although iPSCs can not yet be successfully used for cell replacement therapies,
recent studies established that iPSCs can be used to model disease, which can be used to identify toxic
effects of drugs, potential pharmalogical agents and optimal drug dosage (Lee et al., 2009; Moretti et
al., 2010).
Promising strategies have been developed to derive DA neurons in vitro that are transplantable
in vivo (Swistowski et al., 2010; Kriks et al., 2011). These promising early steps towards the
development of a treatment for PD also represent a broad range of other biomedical applications, such
as basic developmental studies, drugs screening and PD-iPSC based disease modeling. Although
hESCs currently represent the most promising source for DA neurons, significant progress has been
made in the iPSC field. Due to the development of noninvasive methods to obtain iPSCs and
generation of better assays to identify iPSCs line that most closely approximate the generic state of the
naïve genome (Swistowski et al., 2010 ;Ban et al., 2011, Robinton&Daley, 2012), iPSCs represents
the PSCs with the most extended applications possibilities, including personalized patient-specific
stem cells and disease-specific modeling.
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