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First published online April 10, 2009
doi: 10.1242/10.1242/dev.029447
Departments of Anatomy and Neurology, School of Medicine and Public Health, Waisman Center, University of Wisconsin-Madison, 1500 Highland Avenue, Madison, WI 53705, USA.
Author for correspondence (e-mail:
zhang{at}waisman.wisc.edu)
Accepted 23 February 2009
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Glia, Myelination, Neuron-glial switch, Purmorphamine, Transplantation
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Zhou et al., 2000). In the
spinal cord, the OLIG2-expressing progenitors give rise to motoneurons during
the neurogenic phase. Thereafter, OLIG2-expressing progenitors downregulate
neurogenic transcription factors such as neurogenin 2 (NGN2) and PAX6, begin
to express the oligodendroglial transcription factors NKX2.2 and SOX10
(Lee et al., 2005;
Marquardt and Pfaff, 2001;
Qi et al., 2001;
Stolt et al., 2004;
Sugimori et al., 2007;
Zhou et al., 2001), and become
migratory oligodendrocyte precursor cells (OPCs) that also express the surface
markers platelet-derived growth factor receptor alpha (PDGFR
) and
membrane proteoglycan NG2 [also known as chrondroitin sulfate proteoglycan 4
(CSPG4)]. In addition to the ventral source, a smaller population of
oligodendrocytes is generated independently of SHH from the dorsal neural tube
(Cai et al., 2005;
Fogarty et al., 2005;
Vallstedt et al., 2005).
Nevertheless, expression of OLIG2 is a prerequisite step for the dorsal
precursor cells to become OPCs (Cai et al.,
2005). OLIG2 is expressed in human OPCs, as shown in limited
studies on human fetal brain tissues
(Jakovcevski and Zecevic,
2005). Whether OLIG2 expression is necessary for human OPC
specification and how neural progenitors differentiate into OPCs in humans
remain elusive.
Embryonic stem cells (ESCs), isolated from a blastocyst embryo, can
differentiate into all cell lineages of the organism
(Evans and Kaufman, 1981;
Thomson et al., 1998). They
thus offer an in vitro model system for studying early mammalian development,
including oligodendrocyte specification. As in the developing mouse neural
tube, SHH induces OLIG2 expression and promotes the generation of OPCs from
mouse ESC (mESC)-derived neural progenitors
(Billon et al., 2002). The
SHH-induced OPC differentiation from mESCs in vitro retains the correct timing
seen in embryonic development (Billon et
al., 2002; Du et al.,
2006; Samanta and Kessler,
2004), which may be accounted for by activation of the intrinsic
transcriptional networks. Hence, the nature of in vitro OPC differentiation
from mESCs is consistent with what has been learned from in vivo development.
Nevertheless, insight into the molecular mechanism underlying OPC
specification in vertebrates and the ease of OPC generation from mESCs have
not yet been translated to human primates. Several groups have reported the
production of oligodendrocytes from hESCs
(Izrael et al., 2007;
Kang et al., 2007;
Nistor et al., 2005); however,
in none of the reports was SHH used for differentiation nor SHH signaling
analyzed. It is not known whether the SHH-dependent transcriptional network is
required for human OPC specification, as in other vertebrates.
Besides SHH signaling, fibroblast growth factor 2 (FGF2) promotes OPC
generation from rodent neural stem/progenitor cells
(Avellana-Adalid et al., 1996;
Zhang et al., 1998). This
effect may be SHH-dependent (Gabay et al.,
2003) or -independent (Chandran
et al., 2003; Kessaris et al.,
2004). Similar to OPC differentiation from rodent neural
progenitors, OPCs can be efficiently differentiated from mESCs in response to
FGF2 followed by PDGF (Brustle et al.,
1999). However, attempts from several independent laboratories to
generate OPCs from FGF2-expanded human neural stem/progenitor cells using
similar approaches as for their mouse counterparts have been consistently
unsuccessful (Chandran et al.,
2004; Roy et al.,
1999; Zhang et al.,
2000). Similarly, hESC-derived neural progenitors, following
expansion in the presence of FGF2 or EGF, appear to give rise to OPCs at low
efficiency (Izrael et al.,
2007; Kang et al.,
2007). It seems that stem/progenitor cells from human and rodents
respond to FGF2 differently in oligodendrocyte differentiation.
Using a chemically defined oligodendrocyte differentiation system, we
present evidence that SHH-dependent induction of OLIG2, co-expression of OLIG2
and NKX2.2, and activation of SOX10 and PDGFR, are essential for OPC
specification from hESCs, indicating that a conserved transcriptional network
underlies OPC specification in human as in other vertebrates. The transition
from the OLIG2 and NKX2.2-expressing pre-OPCs to OPCs in human is a uniquely
protracted process. FGF2 blocks the transition of pre-OPCs to OPCs by
disrupting SHH-dependent co-expression of OLIG2 and NKX2.2.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). Differentiated colonies were marked and removed physically
with a pipette tip and the stem cell state was monitored by expression of OCT4
(POU5F1) and SSEA4 (Pankratz et al.,
2007). hESCs were differentiated to primitive NE in an adherent
colony culture for 10 days in a neural induction medium consisting of
DMEM/F12, N2 supplement and non-essential amino acids (Invitrogen, Carlsbad,
CA, USA), as detailed elsewhere (Pankratz
et al., 2007; Zhang et al.,
2001). To pattern the primitive NE to ventral spinal progenitors,
differentiation cultures at day 10 were treated with retinoic acid (RA, 100
nM; Sigma, St Louis, MO, USA) followed by RA and SHH (100 ng/ml; R&D
Systems, Minneapolis, MN, USA) (Li et al.,
2005) or purmorphamine (1 µM; Calbiochem, San Diego, CA, USA)
(Li et al., 2008;
Sinha and Chen, 2006) at day
14 for 2 weeks. NE cells grew rapidly and formed neural tube-like rosettes at
day 14-17 of differentiation. The NE colonies were gently blown off the
surface using a pipette and grown as floating clusters in suspension in the
same medium. OLIG2-expressing neural progenitors usually appear at 
day 24
and differentiate to motoneurons at the fifth week (day 28-35)
(Li et al., 2005). At day 35,
the culture was switched to a glial differentiation medium consisting of DMEM,
N1 supplement, T3 (60 ng/ml), biotin (100 ng/ml) and cAMP (1 µM) (all from
Sigma). A cocktail of cytokines consisting of the PDGF-AA isoform,
insulin-like growth factor 1 (IGF1) and neurotrophin 3 (NT3; NTF3) (all at 10
ng/ml from R&D Systems) were added to promote the expansion of the
progenitor population. The cultures were fed every other day and the
progenitor clusters disaggregated into smaller cell aggregates with a polished
glass Pasteur pipette every week. FGF2 (10 ng/ml), EGF (10 ng/ml), noggin (100
ng/ml) (all from R&D Systems), SHH or purmorphamine was added to examine
their effect on motoneuron differentiation, progenitor proliferation and OPC
specification. To differentiate OPCs to oligodendrocytes, progenitor clusters
were incubated in ACCUTASE (Innovative Cell Technologies, San Diego, CA, USA)
at 37°C for 5 minutes. After removal of ACCUTASE, the loosened clusters
were triturated and plated onto glass coverslips coated with polyornithine and
laminin in the glial differentiation medium without SHH/purmorphamine. The
concentration of PDGF-AA, IGF1 and NT3 was 5 ng/ml each.
Lentivirus-mediated RNAi transfection
OLIG2 and non-silencing control RNAi constructs were made using the pGIPZ
vector (Open Biosystems, Huntsville, AL, USA). Lentivirus was produced by
co-transfecting the pGIPZ RNAi plasmid and packaging plasmid into 293T cells
by the calcium-phosphate method, as described
(Xia et al., 2008). Neural
progenitors at day 35 were transfected by directly adding the concentrated
lentivirus to the cell cultures at a multiplicity of infection of 10.
RNA extraction and RT-PCR
Total RNA was extracted from cells using Trizol reagent (Invitrogen) and
reverse-transcribed using the SuperScript III First-Strand Synthesis System
(Invitrogen). RT-PCR was performed as described
(Pankratz et al., 2007).
Primers are listed in Table
1.
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; Zhang et al.,
2001). The primary antibodies used are listed in
Table 2. The immunostained
samples were visualized with a Nikon TE600 fluorescence microscope (Nikon
Instruments, Melville, NY, USA) fitted with a SPOT camera (Diagnostic
Instruments, Sterling Heights, MI, USA) or with a Nikon C1 laser-scanning
confocal microscope. For counting immunostained cells, fields of fixed area
were randomly selected from biological replicates using ImageJ (W. Rasband,
NIMH, Bethesda, MD, USA). Statistical analyses were performed using one-way
ANOVA (Tukey or Dunnett for multiple comparisons) in the R environment (R
Development Core Team, Vienna, Austria).
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). FACS
was performed with a FACSCalibur flow cytometer and analyzed using CellQuest
Pro (BD Biosciences, San Diego, CA, USA).
Transplantation of OPCs into the shiverer mouse brain and tissue preparation
Animal experiments were performed following protocols approved by the
Animal Care and Use Committees at the University of Wisconsin-Madison.
Progenitors enriched for OPCs after 10-12 weeks of hESC differentiation were
dissociated with ACCUTASE and prepared in artificial cerebral spinal fluid at
50,000 cells/µl. About 2 µl of cell suspension (100,000 cells in total)
was transplanted into the lateral ventricle of a newborn shiverer mouse with a
glass pipette (Zhang et al.,
2001). The transplantation site was on the right-hand side, 1 mm
from the midline between the Bregman and Lambda. Cells were injected 1 mm deep
so as to target the ventricle and future corpus callosum. Animals were
anesthetized and perfused with 4% paraformaldehyde 3 months after
transplantation. Brain tissues were processed for immunohistochemistry and
electron microscopy as described (Zhang et
al., 1998; Zhang et al.,
2001).
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Pankratz et al., 2007). To
examine the role of OLIG2 in human oligodendrocyte differentiation, we treated
the primitive NE with retinoic acid (RA; 0.1 µM) from day 10 to promote the
specification of spinal progenitors. By day 14, multiple neural tube-like
rosettes formed in each colony, which were detached and grown in suspension in
the same neural medium with or without SHH (100 ng/ml)
(Fig. 1A). At day 24 (or 10
days following SHH treatment), a subset of NE cells began to express OLIG2,
which peaked at week 4 to 5 (Fig.
1B,C). In the absence of SHH, very few OLIG2-expressing cells (6%)
were observed (Fig. 1B). To
determine whether the generation of these few OLIG2+ cells depends
on endogenous SHH, cyclopamine (5 µM), an inhibitor of hedgehog signaling,
was added at day 10 together with RA. The presence of cyclopamine nearly
eliminated the presence of OLIG2+ cells
(Fig. 1B). Together, these
results indicate that SHH, especially exogenous SHH, is required for OLIG2
expression in the hESC-derived NE.
To determine whether OLIG2 expression is essential for human OPC
specification, at day 35, after the peak of OLIG2 progenitor generation, the
cultures were switched to glial differentiation medium
(Fig. 1A). OPCs co-expressing
PDGFR and OLIG2 began to appear at the tenth week of differentiation,
reaching a peak in the fourteenth week
(Fig. 1D). In the cultures with
few OLIG2 progenitors (the control and cyclopamine groups), there were few
PDGFR+ OPCs. (Fig.
1B,E). Therefore, there was a clear correlation between the OLIG2
expression at week 5 and the presence of OPCs at week 14.
The requirement of OLIG2 expression for human OPC generation was verified
by knocking down OLIG2 expression from day 35 with lentivirus-delivered RNAi.
The expression of OLIG2 RNAi, indicated by the co-expression of GFP, inhibited
the generation of PDGFR+ cells at week 14, whereas the
expression of OLIG2 and PDGFR was not altered in the control
RNAi-transfected cultures (compare the GFP+ and GFP-
cells in Fig. 1F,G; see Fig.
S1A,B in the supplementary material). Thus, OLIG2 expression is required for
human OPC specification.
To further confirm the necessity of SHH signaling in OLIG2 expression and
OPC specification, we replaced SHH with purmorphamine, a small molecule that
activates the hedgehog signaling pathway
(Sinha and Chen, 2006), in the
above differentiation cultures. Treatment with 1 µM purmorphamine
(Li et al., 2008) resulted in
earlier (by 4-5 days) and more potent induction of OLIG2 expression
(Fig. 1B,C). At week 14, a
similar population of PDGFR+ OPCs was generated as in the
SHH-treated cultures (Fig. 1E).
These results support the proposal that OLIG2 expression induced by SHH
signaling is essential for human OPC specification. Since purmorphamine
replicates the effect of SHH in OPC generation, yet is chemically stable, we
have presented the data that were generated using purmorphamine.
SHH signaling is required for inducing and maintaining OLIG2 and NKX2.2-expressing pre-OPCs
At the time of OLIG2 progenitor generation (day 24), examination of OPC
markers including SOX10, NKX2.2 and PDGFR indicated that only NKX2.2
was present, but it did not co-express with OLIG2
(Fig. 2A,B). Following neuronal
differentiation at the fifth week, OLIG2 progenitors from hESCs no longer
produced motoneurons (Li et al.,
2005). We asked whether these OLIG2 progenitors were now becoming
OPCs. At day 35, 40% of the OLIG2+ cells co-expressed NKX2.2
(Fig. 2B). In the chick and
mouse brainstem it is thought that OLIG2 and NKX2.2 co-expression defines the
region for OPC generation, and OLIG2+ NKX2.2+
progenitors quickly acquire the expression of OPC markers including
PDGFR, NG2 and SOX10 (Finzsch et
al., 2008; Liu et al.,
2007; Richardson et al.,
2006). However, the human OLIG2+ NKX2.2+
cells were negative for PDGFR, NG2 and SOX10 (see below). We refer to
these cells as pre-OPCs for convenience of description.
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To determine whether the maintenance of pre-OPCs is dependent on SHH, the
pre-OPC cultures (day 35) were grown in the absence or presence of
purmorphamine (1 µM) or cyclopamine (5 µM)
(Fig. 3A). At day 50, cells
expressing OLIG2 and NKX2.2 were present in the cultures with or without
continued purmorphamine, although 40% more OLIG2+
NKX2.2+ cells were seen in the purmorphamine-treated cultures as
compared with cells without purmorphamine
(Fig. 3B,C). However, the
cultures treated with cyclopamine had very few cells expressing OLIG2 and/or
NKX2.2, even though the cultures grew similarly to those of the other groups
(Fig. 3B,C). At week 14,
PDGFR+ OPCs were present in the cultures with or without
purmorphamine, but not in the cyclopamine-treated cultures
(Fig. 3B,D). Thus, SHH
signaling is necessary to maintain the co-expression of OLIG2 and NKX2.2 in
pre-OPCs, and it subsequently promotes OPC generation even though exogenous
SHH is no longer needed.
FGF2 increases pre-OPCs by inhibiting motoneuron differentiation
In rodents, OLIG2-expressing spinal progenitors are a source of both
motoneurons and OPCs (Lu et al.,
2002; Zhou and Anderson,
2002). We hypothesized that repressing neurogenic potential might
result in more OLIG2 progenitors for OPC generation during the gliogenic
phase. Two mitogens, FGF2 (10 ng/ml) and EGF (20 ng/ml), were applied to the
cultures along with purmorphamine from day 24 (before motoneuron
differentiation) (Fig. 4A).
FGF2 completely blocked HB9 (MNX1) expression at day 35, unlike the cultures
without FGF2, which contained 38% (38.4±5.3%) HB9-expressing
motoneurons. Whereas cultures with FGF2 alone had very few OLIG2+
or HB9+ cells, those with both purmorphamine and FGF2 had a much
larger population (55%) of OLIG2+ cells
(Fig. 4B,C; see Fig. S2 in the
supplementary material). These OLIG2+ cells, after removal of FGF2
and addition of RA and SHH, a condition for efficient motoneuron
differentiation (Li et al.,
2005), no longer generated HB9+ cells (see Fig. S3 in
the supplementary material). EGF had a moderate effect on motoneuron
differentiation, but did not significantly alter the OLIG2+
population (Fig. 4B,C).
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70% of the total population and FGF2 caused a drastic reduction of
post-mitotic motoneurons from 38% to less than 5%
(Fig. 4C), the increased
Ki67+ population following FGF2 treatment can be largely attributed
to the shrinking population of non-dividing motoneurons. Indeed, EGF had less
effect in inhibiting motoneuron differentiation and did not alter the
proportion of OLIG2+ or Ki67+ cells significantly
(Fig. 4C,D). TUNEL labeling
showed no obvious differences in cell death among the three groups (not
shown). The increased OLIG2+ cell population is likely to be
contributed by the accumulation of OLIG2 progenitors that failed to
differentiate to motoneurons in the presence of FGF2, rather than simply by
FGF2-induced proliferation of OLIG2 progenitors. Thus, FGF2 might increase the
pre-OPCs by preventing OLIG2 progenitors from differentiating to
motoneurons.
FGF2 inhibits the transition of pre-OPCs to OPCs by disrupting the SHH-dependent co-expression of OLIG2 and NKX2.2
FGF2 promotes OPC generation from mESCs and rodent neural stem/progenitor
cells (Brustle et al., 1999;
Chandran et al., 2003;
Gabay et al., 2003;
Kessaris et al., 2004). We
asked whether FGF2 could similarly enhance the generation of OPCs from human
pre-OPCs. hESCs were differentiated for 35 days in the presence of
purmorphamine and FGF2 to enrich for pre-OPCs
(Fig. 4A). The enriched
pre-OPCs from day 35 were continued with purmorphamine until day 50. From day
35, the cells were cultured in the presence or absence of FGF2
(Fig. 5A). Weekly examination
of the differentiation cultures revealed that by the fourteenth week, the
majority of the cells (84%) in the group without FGF2 exhibited a typical
bipolar or tripolar OPC morphology. These cells expressed multiple OPC markers
including PDGFR, SOX10 and NG2 (Fig.
5B,C). Very few cells were positive for the neuronal protein
βIII-tubulin (5.1±4.4%) or the astrocytic antigen GFAP
(4.7±2.7%) (Fig. 5B,D).
By contrast, the FGF2-treated cultures possessed few PDGFR+
OPCs (4.3±3.1%), whereas the majority of the cell populations were
βIII-tubulin+ neurons (53±6.8%) and GFAP+
progenitors/astrocytes (34.7±5.2%)
(Fig. 5B-D). Thus, FGF2
inhibits OPC generation and instead favors that of neurons or astrocytes.
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), were significantly upregulated by FGF2 alone
or FGF2 plus purmorphamine (Fig.
5G). This might account for the loss of OLIG2 and NKX2.2
co-expression in pre-OPCs, hence inhibiting subsequent transition to OPCs.
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or NG2 on the membrane and processes
(Fig. 6E-G). Over the course of
the subsequent 2-4 weeks, these bipolar cells became multipolar and were
positive for the immature oligodendrocyte marker O4
(Fig. 6H,I). The O4+
cells were no longer positive for PDGFR (see Fig. S4 in the
supplementary material), similar to our previous finding using fetal brain
tissue-derived progenitor cultures (Zhang
et al., 2000). The expression of another commonly used OPC marker,
NG2, often overlapped with that of O4 (see Fig. S4 in the supplementary
material). With further culturing for 6-8 weeks, most cells died in our
oligodendroglia-enriched cultures under the serum-free conditions.
Nevertheless, surviving cells exhibited more-elaborated processes,
occasionally with membrane sheaths. These cells were positively labeled for
myelin basic protein (MBP) (Fig.
6J), which is expressed by mature oligodendrocytes. The myelination potential of the in vitro-produced human OPCs was investigated by transplanting the OPCs (differentiated from hESCs for 12 weeks) into the ventricle of neonatal shiverer mice. Shiverer mice do not produce endogenous compact myelin sheaths owing to a complete lack of MBP. At 3 months following transplantation, grafted human cells, identified by specific human nuclear antigen (hNA) expression, were present preferentially in the corpus callosum (Fig. 6K). Virtually every hNA-expressing cell was also positive for OLIG2 (Fig. 6L). Most of the human cells displayed numerous processes and immunostained positively for MBP (Fig. 6M). These MBP-expressing processes connected to multiple neurofilament-positive axons in the corpus callosum (Fig. 6N). No human cells co-labeled for GFAP (Fig. 6O). Toluidine Blue staining on the semi-thin sections showed numerous myelin sheaths in grafted animals, but not in non-transplanted shiverer mice (not shown). Electron microscopy analyses confirmed the presence of compact myelin sheaths in the corpus callosum of the transplanted mice (Fig. 6P,Q), but not in the non-transplanted shiverer mice (Fig. 6R).
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Li et al., 2005;
Pankratz et al., 2007;
Roy et al., 2006;
Watanabe et al., 2007;
Yan et al., 2005). In the
present study, we have revealed that human oligodendrocyte development
involves a transcriptional network of nearly identical sequence to that
observed in vertebrate models. Following expression of OLIG2 and genesis of
motoneurons, the human OLIG2 progenitors become pre-OPCs by co-expressing
NKX2.2, and finally differentiate into OPCs by activation of SOX10 and
PDGFR. Blocking OLIG2 expression inhibits OPC production, confirming
the requirement of OLIG2 for human OPC specification. The vast majority of
human OLIG2 progenitors are generated in a SHH-dependent manner, because
cultures without exogenous SHH, or those in which endogenous SHH signaling has
been blocked with cyclopamine, have few OLIG2 progenitors and OPCs. We also
found that SHH is not only crucial for efficiently inducing pre-OPCs, but it
is also required for the transition from pre-OPCs to OPCs, for which
endogenous SHH signaling is sufficient
(Fig. 7). Thus, the
SHH-dependent signaling network underlying vertebrate OPC development is
conserved in humans (Fig.
7).
Our study also reveals unique aspects of human OPC generation. In our human
OPC differentiation cultures, NKX2.2 is the first OPC-related transcription
factor that is co-expressed with OLIG2 at the fifth week, preceding the
expression of PDGFR. This expression pattern resembles that in the
chick and in the mouse hindbrain
(Vallstedt et al., 2005;
Zhou et al., 2001), but
differs from that in the mouse spinal cord, where NKX2.2 expression is induced
after PDGFR+ OPCs are formed
(Fu et al., 2002;
Qi et al., 2001). This might
be partly related to the hindbrain/cervical spinal identity of our human
progenitors patterned by RA (Li et al.,
2005), although species differences cannot be excluded. We have
also found a protracted transition period from pre-OPCs at the fifth week to
human OPCs at the fourteenth week. In chick and mouse brainstem, the
OLIG2+ NKX2.2+ progenitors quickly express PDGFR
and become migrating OPCs (Vallstedt et
al., 2005; Zhou et al.,
2001). OLIG2 progenitors differentiated from mESCs also co-express
PDGFR and NG2 shortly following motoneuron differentiation
(Billon et al., 2002;
Du et al., 2006). It is
possible that our serum-free culture conditions are not optimal for
acquisition of the OPC identity at an earlier time. The addition of FGF2, EGF,
SHH or noggin, however, did not accelerate the transition of pre-OPCs to OPCs
(data not shown). Similarly, removal of PDGF, NT3 and/or IGF1 did not alter
the time course of OPC generation either (data not shown). Alternatively, this
protracted transition might be intrinsically controlled. The long
OPC-specification process (3 months) appears to match the earliest appearance
of OPCs in human embryos at the end of the first trimester
(Grever et al., 1997;
Jakovcevski and Zecevic, 2005;
Weidenheim et al., 1996). One
potential explanation for the late appearance of OPCs is a need for expanding
neurogenic progenitors, as large numbers of neurons are needed for the
evolutionarily enlarged human brain and spinal cord.
FGF2 is a mitogen for rodent and human neural stem/progenitor cells and
enhances the generation of OPCs in rodents
(Avellana-Adalid et al., 1996;
Brustle et al., 1999;
Gabay et al., 2003;
Zhang et al., 1998). In the
present study, it is interesting that following the induction of OLIG2, FGF2
nearly completely blocked motoneuron differentiation and preferentially
increased the proportion of OLIG2 progenitors. The inhibition of motoneuron
differentiation is not attributable to the mitogenic effect of FGF2 preventing
the OLIG2 progenitors from exiting the cell cycle. Nor does FGF2
preferentially promote the survival of the OLIG2 progenitors. It is likely
that FGF2 enhances the transition from the neurogenic OLIG2 progenitors to
pre-OPCs (Fig. 7). Although
fine dissection of the mechanism is not possible with the available system,
this finding provides, as it stands, a simple way of switching neurogenesis to
gliogenesis from a pool of progenitors.
Continued use of FGF2 inhibited the generation of OPCs from hESCs. This is
reminiscent of observations in the past decade that human neural
stem/progenitor cells, following expansion with FGF2, or FGF2 and EGF, rarely
produce oligodendrocytes in vitro (Chandran
et al., 2004; Roy et al.,
1999; Zhang et al.,
2000). Even when the human neural progenitor cultures are enriched
for OPCs, the OPCs quickly disappear after culturing in the presence of FGF2
(Grever et al., 1999). Our
present finding that co-expression of OLIG2 and NKX2.2 is suppressed by FGF2
in the pre-OPCs explains the phenomenon. The inhibitory effect appears
specific to FGF2, as EGF did not affect the co-expression of OLIG2 and NKX2.2
and subsequent generation of OPCs (Fig.
4). FGF2 induces OPC generation from mouse neural stem/progenitor
cells by activating endogenous SHH signaling
(Gabay et al., 2003) or by as
yet unknown SHH-independent pathways
(Chandran et al., 2003;
Kessaris et al., 2004). In our
human cell differentiation system, FGF2 inhibits endogenous SHH expression and
significantly increases the level of GLI2 and GLI3, which are downstream
repressors of SHH signaling (Ruiz i Altaba
et al., 2007), thus disrupting co-expression of OLIG2 and NKX2.2
in pre-OPCs. This results in the loss of pre-OPCs and the conversion to
progenitors expressing OLIG2 (at a low level) or NKX2.2, which generates
astrocytes or neurons (Fig.
5B,D). Nevertheless, we do not exclude the possibility that FGF
does this through other oligodendroglial transcription factors, such as SOX10
and ASCL1 (Liu et al., 2007;
Sugimori et al., 2008), or by
selectively promoting the survival/expansion of neuronal progenitors in our
long-term cultures.
In the present study, we developed an effective strategy for reproducibly
directing hESCs to an enriched population of OPCs with an unequivocal
oligodendrocyte identity and myelination potential. In the previous reports of
OPC differentiation from hESCs, SHH was not applied and the expression of
OLIG2 in neural progenitors was not examined by single-cell-resolution
immunocytochemistry, but instead by PCR on bulk cultures
(Izrael et al., 2007;
Kang et al., 2007). Based on
our findings, we believe that the OPCs described in those reports are
differentiated from OLIG2 progenitors spontaneously induced by endogenous or
alternatively activated SHH signaling. The near pure population of `OPCs'
generated from hESCs without application of SHH
(Nistor et al., 2005) cannot
be explained by our model, nor has the result been replicated by us or any
other reports. The identity of the OPCs in that report has not been
unequivocally verified either.
The SHH-dependent transcriptional network underlying human OPC
specification and the time course of OPC generation, consistent with that in
human embryo development, revealed in the present study suggest that the hESC
differentiation system is a useful tool for understanding the biology of human
cells. The divergent responses to common growth factors such as FGF2 between
human and other vertebrate cells might be related to the in vitro system, but
may also reflect the nature of normal human development. Similar to our
present finding, the maintenance of human and mouse ESCs depends on the same
transcriptional network (Boyer et al.,
2005). However, the common extrinsic factor, BMP, maintains the
self-renewal of mESCs but induces cell differentiation of hESCs
(Xu et al., 2002;
Ying et al., 2003). This
seemingly `trivial' deviation has slowed down the translation of findings from
mESCs to the establishment of hESCs (Evans
and Kaufman, 1981; Thomson et
al., 1998). Similarly, over the past decade, many laboratories
have stumbled in trying to replicate the finding in rodents, so as to
differentiate human neural stem/progenitors to OPCs. Thus, the confirmation of
conserved principles and the revelation of `nuances' using the hESC
differentiation system might bear significant consequences.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/dev.029447/DC1
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Footnotes |
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* These authors contributed equally to this work 
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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