1 MRC Laboratory for Molecular Cell Biology and Cell Biology Unit and the
Biology Department, University College London, London WC1E 6BT, UK
2 Centre for Genome Research, University of Edinburgh, King's Buildings, West
Mains Road, Edinburgh EH9 3JQ, UK
* Author for correspondence (e-mail: n.billon{at}ucl.ac.uk)
Accepted 7 July 2002
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Summary |
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Key words: Oligodendrocyte, Development, ES cells, Genetic selection
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Introduction |
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The development of OPCs from neuroepithelial cells has mostly been studied
in the rodent neural tube. In the mouse spinal cord, for example, the first
OPCs expressing the platelet-derived growth factor receptor
(PDGFR
) are detected as a narrow band in the ventral
neuroepithelium around E12.5-13, well after neuronal development begins at E9
(Hardy, 1997
;
Pringle et al., 1998
). Some
proteins that are characteristic of oligodendrocytes and their precursors are
expressed earlier than PDGFR
, however, and may identify the
earliest stages of oligodendrocyte lineage specification. These include the
Olig1 and Olig2 basic helix-loop-helix gene regulatory
proteins, which are expressed in the same region of the spinal cord as
PDGFR
at E13, but are expressed as early as E9
(Lu et al., 2000
;
Zhou et al., 2000
). The
earliest Olig2-expressing cells in the ventral spinal cord, however,
may develop into neurons rather than OPCs
(Marquardt and Pfaff, 2001
;
Mizuguchi et al., 2001
;
Novitch et al., 2001
;
Sun et al., 2001
;
Zhou et al., 2001
): if
Olig2 is inactivated, neither motor neurons nor OPCs develop in the
spinal cord (Lu et al., 2002
;
Zhou and Anderson, 2002
). By
E11.5-12, however, Olig expression is restricted to the same region,
where PDGFR
will be expressed and OPCs emerge; it is likely,
therefore, that this Olig expression marks the earliest stages of
oligodendrocyte specification, 1-1.5 days before PDGFR
expression is detectable (Lu et al.,
2000
; Zhou et al.,
2000
). By E13, Olig1, Olig2 and PDGFR
, as
well as another OPC marker, the NG2 proteoglycan
(Levine and Nishiyama, 1996
),
are all expressed in the same small region of the ventral spinal cord that
generates OPCs (Lu et al.,
2000
; Pringle et al.,
1996
; Woodruff et al.,
2001
; Zhou et al.,
2000
). OPCs then migrate throughout the CNS, where they
proliferate, largely in response to PDGF
(Fruttiger et al., 1999
) and
terminally differentiate into oligodendrocytes. The first oligodendrocytes
that express galactocerebroside (GC) appear in the mouse CNS around E17
(Calver et al., 1998
).
The differentiation of OPCs into oligodendrocytes is better understood than
the initial commitment of neuroepithelial cells to the oligodendrocyte
lineage. The normal timing of OPC differentiation can be reconstituted in
cultures of dissociated embryonic rat optic nerves, as long as the OPCs are
stimulated to proliferate by PDGF (Raff et
al., 1988), and hydrophobic signals such as thyroid hormone (TH)
or retinoic acid (RA) are present (Ahlgren
et al., 1997
; Barres et al.,
1994
; Gao et al.,
1998
). Without PDGF, the cells rapidly stop dividing and
differentiate (Temple and Raff,
1985
). In the presence of PDGF, but in the absence of TH or RA,
most of the OPCs tend to keep dividing and fail to differentiate
(Barres et al., 1994
). There is
abundant evidence that TH plays an important part in timing oligodendrocyte
development in vivo (Rodriguez-Pena,
1999
). TH may also promote the commitment of CNS stem cells to the
oligodendrocyte lineage (Johe et al.,
1996
).
So far, OPCs have been purified only from the rat optic nerve. Although
studies of these purified cells have provided important insights into the
control of OPC differentiation, the cells cannot be purified in large enough
numbers for conventional biochemical analyses. Moreover, the neural progenitor
cells that give rise to OPCs have not been purified, making it difficult to
study the earliest stages of OPC specification. Mouse embryonic stem (ES)
cells are proliferating, pluripotent stem cells that have been isolated from
the epiblast of blastocyst-stage mouse embryos
(Brook and Gardner, 1997;
Evans and Kaufman, 1981
;
Martin, 1981
). They can be
propagated indefinitely in culture in the presence of leukemia inhibitory
factor (LIF) (Smith et al.,
1988
; Williams et al.,
1988
). When transplanted into a mouse blastocyst, ES cells
integrate into the embryo and contribute to all cell lineages, including germ
cells (Bradley et al., 1984
).
If ES cells are cultured without LIF on a non-adherent surface, they aggregate
to form embryoid bodies (EBs), in which the cells form ectodermal, mesodermal
and endodermal derivatives (Keller,
1995
). ES cells can be produced in large numbers, can be easily
genetically modified and can be induced to differentiate into various CNS cell
types in vitro (Bain et al.,
1995
; Brustle et al.,
1999
; Fraichard et al.,
1995
; Okabe et al.,
1996
). They should therefore provide a powerful system for
studying the early events of neural development. In the present study, we
devised a strategy for using ES cells to study OPC and oligodendrocyte
development.
We first modified the genome of mouse ES cells to allow us both to select
positively for neuroepithelial cells (Li
et al., 1998) and to select negatively against residual
undifferentiated ES cells. We then studied various combinations of
extracellular signal molecules to find optimal conditions for the development
of OPCs from these doubly selected neuroepithelial cells. Using a variety of
markers to follow the fate of the selected cells, we show that oligodendrocyte
lineage cells can be efficiently produced in this way. Most important, we show
that, when exposed to appropriate signal molecules, OPCs and oligodendrocytes
develop on a predictable schedule that is similar to that observed in vivo,
suggesting that the ES-cell-derived neuroepithelial cells follow a normal
pathway of oligodendrocyte development. This model system should thus be
useful for studying the early events of OPC specification and differentiation.
This strategy may also be useful for producing OPCs and oligodendrocytes from
human ES cells for cell therapy and drug screening.
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Materials and Methods |
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Genetic engineering of ES cells
Two independently derived parental ES cell lines were used, CGR8
(Mountford et al., 1994) and
E14Tg2a (Hooper et al., 1987
).
Both lines were subjected to sequential gene targeting in order to integrate
ßgeo into the Sox2 locus
(Li et al., 1998
) and
hygromycin-thymidine kinase
(Lupton et al., 1991
) into the
Oct4 (Pou5f) locus (Mountford et
al., 1994
). Linearised constructs were introduced into ES cells by
electroporation, and stable transfectants were isolated by selection in G418
or hygromycin. Homologous recombinants were then identified by Southern
hybridisation of genomic DNA samples using probes external to the homology
regions. The Sox2-targeting construct and flanking probes were
generously provided by Silvia Nicolis and Robin Lovell-Badge and will be
described in detail elsewhere (S. Avilion and N.B., unpublished). The
Oct4 construct was generated by replacing ßgeo in the
Oct4ßgeo-targeting vector
(Mountford et al., 1994
) with
the hygromycin-thymidine kinase fusion gene. CGR8-derived OSG cells also carry
a puromycin-selectable GFP transgene driven by the ubiquitous CAG expression
unit (Pratt et al., 2000
).
Exposure of undifferentiated OSG or OS25 (E14Tg2a derivative) cells to
gancyclovir eliminated all cells within 4 days. Following in vitro
differentiation, cells became gancyclovir resistant. Neuronal differentiation
and oligodendrocyte development occurred similarly in the two clones.
Differentiation protocol
Experiments with the engineered ES cell lines were performed using the
culture protocol outlined in Fig.
1. On day 0, exponentially growing ES cells were trypsinised and
7x106 cells were plated on 10 cm bacterial dishes without
LIF. In these conditions, ES cells form embryoid bodies (EBs). RA
(10-6 M) was added on day 4 to promote neural development
(Bain et al., 1995). After 2
days, the medium was replaced by a 50:50 mixture of DMEM-F12 containing N2
supplement and Neurobasal medium containing B27 supplement (Gibco). The N2
supplement contains insulin (25 µg/ml), transferrin (100 µg/ml),
progesterone (6 ng/ml), putrescine (16 µg/ml), sodium selenite (30 nM) and
BSA (50 µg/ml). In most experiments with genetically engineered ES cells,
the medium was also supplemented with G-418 (100 µg/ml) and Ganciclovir
(2.5 µM) to select for neuroepithelial cells and against undifferentiated
ES cells, respectively. At day 8 (2 days of selection), EBs were dissociated
with trypsin, and 2x106 cells were replated on poly-D-lysine
(PDL; 10 µg/ml; Sigma) and laminin-coated (10 µg/ml; Sigma) tissue
culture flasks (Falcon, T25) in the same medium, except that FGF-2 (Prepotech)
was added at 20 ng/ml. Fresh G-418 and Ganciclovir were also added.
|
After 2 more days (day 10; 4 days of selection), the medium was changed to
a modified Bottenstein-Sato medium
(Bottenstein et al., 1979) to
promote development along the oligodendrocyte pathway. It contained FGF-2 (20
ng/ml), NT3 (5 ng/ml), insulin (10 µg/ml), human transferin (100 µg/ml),
BSA (100 µg/ml), progesterone (60 ng/ml), sodium selenite (40 ng/ml),
N-acetyl-cysteine (60 µg/ml), putrescine (16 µg/ml), forskolin (5
µM), biotin (10 ng/ml), penicillin, streptomycin and L-glutamine. G418 and
Ganciclovir were omitted, and, in some experiments, recombinant myristylated
Shh-N (gift from Biogen, 300 ng/ml) was added.
In some experiments, 5 days after selection (when the first
PDGFR-positive OPCs were detected), FGF-2 was removed, and the cells
were grown in the presence of PDGF-AA (10 ng/ml) and TH (triodothyronine,
Sigma, 40 ng/ml) to promote the differentiation of OPCs into oligodendrocytes
(Barres et al., 1994
).
In situ hybridisation
Riboprobes were synthesised using a digoxigenin (DIG) RNA-labelling Kit
(SP6/T7; Roche) according to the supplier's instructions. Probes used in this
study included: Olig1, a 735 bp insert derived from the whole coding
region of the mouse olig1 cDNA; Olig2, a 630 bp insert
derived from the 5' region of the mouse Olig2 cDNA;
PDGFR, a 1.6 Kb insert derived from the extracellular domain
of the mouse PDGFR
cDNA. Sense RNA probes were prepared as
controls for each Riboprobe.
In situ hybridisation with DIG-labelled Riboprobes was performed using a procedure developed by D. Anderson's laboratory (http://kclab.webprovider.com/protocols/alish.html). Briefly, following fixation with paraformaldehyde, acetylation with acetic anhydride and permeabilisation with 0.2 N HCl, cells were incubated in hybridisation buffer (50% formamide, 5x SSC, 4.8 units/ml yeast tRNA, 17 units/ml heparin, 1x Denhardt's solution, 0.1% Tween-20, 1.6 mM CHAPS, 5 mM EDTA) for 3-4 hours at room temperature. Hybridisation was carried out for 16 hours at 65°C, using 1-2 µg/ml of probe. After stringent washing with 0.2x saline-sodium citrate buffer (SSC; 3 mM sodium citrate pH 7.0, 30 mM NaCl) and blocking with 20% normal sheep serum, the signal was detected by overnight incubation at 4°C with a sheep anti-DIG antibody conjugated with alkaline phosphatase (Roche), followed by 24 hours at room temperature in a solution containing 100 mM Tris pH 9.5, 50 mM MgCl2, 100 mM NaCl, 0.1% Tween 20, 5 mM Levamisole, 0.4 mM of 5-bromo-4-chloro-3-indolyl phosphate and 46 mM nitroblue tetrazolium.
RT-PCR analysis
Cells were harvested with trypsin and processed immediately for RT-PCR
analysis. Poly (A)+ mRNA was prepared using a QuickPrep Micro mRNA
purification kit (Pharmacia Biotech). cDNAs were synthesised using AMV-Reverse
Transcriptase (Promega) according to the supplier's instructions and were used
as templates for the PCR reaction.
The following oligonucleotide primers were synthesised. For glyceraldehyde-3-phosphate dehydrogenase (G3PDH), the 5' primer was 5'-ACC ACA GTC CAT GCC ATC AC-3', and the 3' primer was 5'-TCC ACC ACC CTG TTG CTG TA-3'; for Oct4, the 5' primer was 5'-CTG CTG AAG CAG AAG AGG ATC AC-3', and the 3' primer was 5'-TGG TTC TGT AAC CGG CGC CAG AAG-3'; for Sox2, the 5' primer was 5'-AAC ATG ATG GAG ACG GAG CTG AAG C-3', and the 3' primer was 5'-TAC GCG CAC ATG AAC GGC TGG AG-3'; for Sox1, the 5' primer was 5'-TTA CTT CCC GCC AGC TCT TC-3', and the 3' primer was 5'-TGA TGC ATT TTG GGG GTA TCT CTC-3'; for myelin basic protein (MBP), the 5' primer was 5'-AAGTACTTGGCCACAGCAAG-3', and the 3' primer was 5'-CAGAGCGGCTGTCTCTTC-3'.
The RT-PCR reactions were carried out as follows: 25 µl of reaction
mixture contained 200 pg of template cDNA, 300 nM of 5' and 3' PCR
primers, 0.2 mM dNTP, 1.25 mM MgCl2, 10 mM Tris-HCl (pH 9.0), 50 mM
KCl, 0.1% Triton X-100, and 1.25 units of Taq DNA polymerase (Promega). The
reaction mixture was denatured for 1 minute at 94°C. The PCR parameters
were 94°C for 10 seconds for the denaturing step, 53°C (MBP),
62°C (Sox2 and Oct4) or 60°C (Olig1, Olig2,
PDGFR and GAPDH) for 30 seconds for the annealing step,
and 72°C for 1 minute for the elongation step. The PCR products were
electrophoresed in a 1.2% agarose gel and stained with EtBr. The number of PCR
cycles was 35 for Sox-2 and Oct4, 26 for MPB and 25 for
G3PDH and Sox-1.
Immunocytochemistry
Cells were cultured on PDL- and laminin-coated 13 mm glass coverslips or
Nunclon multidishes and fixed in 4% paraformaldehyde in PBS for 5 minutes at
room temperature. After washing with PBS, cells were incubated for 30 minutes
in 10% normal goat serum to block non-specific staining. They were then
incubated for 1 hour in the first antibody, washed in PBS and incubated for 1
hour in FITC-coupled goat anti-mouse Ig or goat anti-rabbit Ig antibodies
(diluted 1/100; Jackson Immuno Research Laboratories) and bisbenzamide (5
ng/ml; Hoescht No. 33342; Sigma). Coverslips were mounted in Citifluor
mounting medium (CitiFluor, UK) and examined with a Zeiss Axioplan 2
fluorescence microscope. Nunclon multidishes were examined with a Leica DMIRB
inverted fluorescence microscope.
The following antibodies were used: monoclonal anti-galactocerebroside (GC)
antibody (supernatant, diluted 1/5)
(Ranscht et al., 1982);
monoclonal SSEA-1 antibody (supernatant, diluted 1/5; Developmental Studies
Hybridoma Bank); monoclonal anti-rat nestin antibody (diluted 1/100,
Pharmingen); HA 1297 cocktail of affinity-purified rabbit antibodies that
together recognize all three neurofilament proteins (diluted 1/200; Affiniti
Research Products Limited); rabbit anti-NG2 chondroitin sulfate proteoglycan
antibodies (diluted 1/50; Chemicon International); and O4 monoclonal antibody
(supernatant, diluted 1/20) (Sommer and
Schachner, 1981
).
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Results |
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We therefore genetically engineered ES cells so that we could select
against residual ES cells and select for neuroepithelial cells. To select
against residual ES cells, we introduced a
hygromycin-thymidine-kinase (tk) fusion gene into the
Oct4 locus by homologous recombination. As Oct4 is expressed
in undifferentiated ES cells (Scholer et
al., 1989), we should be able to eliminate such engineered ES
cells by treatment with Gancyclovir. To select for neuroepithelial cells, we
introduced a ßgeo gene into the Sox2 locus
(Li et al., 1998
). As
Sox2 is specifically expressed in neuroepithelial cells
(Zappone et al., 2000
), these
cells should selectively survive treatment with G418 (undifferentiated ES
cells also express sox2 and would be expected, therefore, to survive
G418, but they should be eliminated by Ganciclovir). Thus, by treating the
doubly targeted ES cells with both Ganciclovir and G418, undifferentiated ES
cells could be selected against, and neuroepithelial cells could be selected
for.
When we selected 6-day-old EBs derived from these doubly targeted ES cells
for 4 days in G418 and Ganciclovir, no cells expressing SSEA-1 antigen
(Fig. 3A) or Oct4 mRNA
(Fig. 3B) could be detected,
suggesting that no residual ES cells were present in the culture. By contrast,
85% of the cells expressed nestin protein
(Fig. 3A), and both
Sox1 and Sox2 mRNAs were readily detected
(Fig. 3B), indicating a
significant enrichment in neuroepithelial cells
(Brustle and McKay, 1995).
Thus, combined negative and positive selection strategies proved to be
extremely efficient in generating highly enriched populations of
neuroepithelial cells. It is not clear why some nestin-negative cells survived
the selection, although at least some of these cells are likely to be newly
differentiated neurons.
|
Effect of sonic hedgehog (Shh) on the production of OPCs from
ES-cell-derived neuroepithelial cells
As Shh promotes the development of OPCs from neuroepithelial cells in vitro
and in vivo (Orentas et al.,
1999; Pringle et al.,
1996
; Roelink et al.,
1995
; Tekki-Kessaris et al.,
2001
), we tested its effect on the production of OPCs from our
selected, ES-cell-derived neuroepithelial cells. After drug selection as
before, we cultured the cells in a serum-free, modified Bottenstein-Sato
medium, which is permissive for oligodendrocyte development in vitro
(Bottenstein et al., 1979
). We
added FGF-2, which promotes the proliferation of CNS stem cells in vitro
(Murphy et al., 1990
) and in
vivo (Wagner et al., 1999
),
and, in some cultures, we added Shh to promote OPC development.
We used a variety of markers to follow the fate of the neuroepithelial
cells and to help identify OPCs. We used RT-PCR analysis and in situ
hybridisation to detect Olig1, Olig2 and PDGFR mRNAs,
and we used immunohistochemistry to detect NG2 proteoglycan. We scored cells
as positive in immunohistochemical assays only if they also had the
characteristic morphology of OPCs (Temple
and Raff, 1986
).
As shown in Fig. 4, after 5
days in the presence of FGF-2 alone, less than 12% of the cells expressed
Olig1, Olig2 or PDGFR mRNAs or stained for NG2. By
contrast, after 5 days in FGF-2 and Shh, the percentage of cells expressing
each individual OPC marker ranged between 10 and 50%, depending on the marker
(Fig. 4B); two days later, this
percentage increased to 40-85% (Fig.
6A). Thus, as in the developing mouse neural tube, Shh promoted
the development of OPCs from ES-cell-derived neuroepithelial cells.
|
|
Differentiation of ES-cell-derived OPCs into oligodendrocytes
To promote the differentiation of ES-cell-derived OPCs into
oligodendrocytes, we first cultured selected neuroepithelial cells in FGF-2
and Shh for 5 days to enhance OPC production. We then switched the cells into
PDGF-AA and TH for a further 3 to 9 days. As shown in
Fig. 5A, oligodendrocytes were
identified by their characteristic morphology
(Temple and Raff, 1986) and by
immunostaining for GC (Raff et al.,
1978
). About 40-50% of the cells developed into GC-positive
oligodendrocytes (Fig. 6B).
Interestingly, many of the oligodendrocyte lineage cells accumulated around,
and extended processes to, the axons of neurons that developed in the cultures
(Fig. 5B), just as they do in
vivo.
|
Normal timing of oligodendrocyte development in ES-cell-derived
cultures
Neuronal, OPC and oligodendrocyte development occur in a predictable
sequence in the developing mouse neural tube, with the first neurons appearing
at around E9, the first OPCs around E12 and the first oligodendrocytes at
around E17 (Calver et al.,
1998; Hardy, 1997
;
Pringle et al., 1998
).
To determine if a similar sequence occurred in our cultures, we
drug-selected neuroepithelial cells for 4 days and treated them as above to
optimize oligodendrocyte production (Fig.
1). We then examined the time of first appearance of various
neuronal, OPC and oligodendrocyte markers. As shown in
Fig. 6,
neurofilament-containing neurons were present at all times tested. Their
numbers peaked at 3 days post-selection
(Fig. 6A) and fell rapidly
thereafter (Fig. 6B). The first
OPCs, as determined by morphology, staining for NG2, and PDGFR
mRNA expression detected by in situ hybridisation, appeared at 5 days
post-selection and increased to more than 40% by 8 days post-selection
(Fig. 6A). As in vivo,
Olig2 mRNA-expressing cells appeared 2 days before
PDGFR
- and NG2-expressing cells
(Fig. 6A). Moreover, the first
GC-positive oligodendrocytes appeared 10 days post-selection, about 5 days
after the first PDGFR
-expressing OPCs, just as in vivo
(Fig. 6B). Finally,
MBP mRNA was expressed from 10 days post-selection and increased
thereafter. Thus, the sequence of neural cell development and the timing of
oligodendrocyte differentiation in our ES-cell-derived cultures, closely
resembled that in normal neural development.
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Discussion |
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Timing of oligodendrocyte development
The different cell types in the CNS develop in a predictable sequence. It
is gratifying that a similar sequence occurs in our ES-cell-derived cultures.
Neurons appear first, followed by OPCs and then oligodendrocytes. More
remarkably, the timing of the first appearance of various neural markers
closely resembles that reported in the developing mouse neural tube. As in the
developing ventral spinal cord, Olig2 is expressed in our cultures
before PDGFR and NG2 are first detected. This early expression
of Olig2 might reflect the earliest stages of oligodendrocyte
specification in these ES-cell-derived cultures. It could also reflect
Olig2 expression in motor neuron precursors
(Lu et al., 2002
;
Zhou and Anderson, 2002
).
Again, as in vivo, PDGFR
expression and NG2 immunostaining
precede the appearance of GC immunostaining by about 5 days
(Calver et al., 1998
;
Lu et al., 2000
;
Zhou et al., 2000
). The
emergence of OPCs in the developing spinal cord depends on Shh produced by the
notochord and the floor plate (Echelard et
al., 1993
; Placzek et al.,
1993
; Roelink et al.,
1994
), and we find that the treatment of our ES cell cultures with
Shh increases the expression of OPC markers. It seems likely that in the
absence of added Shh, endogenous Shh helps promote OPC development in these
cultures.
Perhaps the most impressive and convincing normal timing that occurs in our
cultures is the interval between the first appearance of OPCs, identified by
their morphology and expression of Olig2, PDGFR mRNAs and NG2
proteoglycan, and the first appearance of oligodendrocytes, identified by
their morphology and GC staining. It is surprising that this interval so
closely resembles that reported in the ventral mouse spinal cord, as the
controls on the differentiation of OPCs into oligodendrocytes are thought to
be complex, involving a cell-intrinsic timer
(Durand and Raff, 2000
), PDGF
(Raff et al., 1998
) and TH
(Barres et al., 1994
).
Moreover, we treat our cultures with a high concentration of PDGF and TH,
whereas PDGF (and probably TH) is limiting in the developing mouse spinal cord
(Calver et al., 1998
). On the
other hand, it was shown previously that the time of first appearance of
GC-positive oligodendrocyte is remarkably similar in the developing rat brain
and in cultures of dissociated E10 rat brain cells
(Abney et al., 1983
). Our
present finding that the sequence and timing of neural development in our
cultures is similar to that observed in vivo suggests that ES-cell-derived
neuroepithelial cells may follow the same pathways followed by normally
developing neuroepithelial cells.
Our findings are reminiscent of those previously published for the timing
of erythropoieisis in ES-cell-derived cultures, which closely resemble in vivo
timing (Keller, 1995). By
contrast, a recent report suggested that the development of pancreatic ß
cells in cultures of mouse ES cells may have followed an abnormal pathway -
from neuroepithelial cells rather than from endodermal cells - although it was
not shown that ß cells arose from neuroepithelial cells in these
ES-cell-derived cultures (Lumelsky et al.,
2001
).
Our culture system may be especially useful for studying the specification
of neuroepithelial cells toward the oligodendrocyte lineage. During spinal
cord development, OPCs are generated adjacent to the floor plate, just ventral
to where motor neurons develop (Noll and
Miller, 1993; Pringle and
Richardson, 1993
; Richardson
et al., 2000
; Sun et al.,
1998
; Yu et al.,
1994
). This is where Olig2 is first expressed, presumably
under the influence of Shh produced by the notochord and floorplate
(Lu et al., 2000
;
Zhou et al., 2000
). Olig2 is
sequentially expressed in motor neuron progenitors
(Mizuguchi et al., 2001
;
Novitch et al., 2001
;
Takebayashi et al., 2000
) and
OPCs (Lu et al., 2000
;
Zhou et al., 2000
), and it is
required for the development of both cell types in the spinal cord
(Lu et al., 2002
;
Zhou and Anderson, 2002
).
Olig2 apparently acts together with the homeodomain gene regulatory
protein Nkx2.2 to promote OPC specification
(Sun et al., 2001
;
Zhou et al., 2001
), whereas it
acts with other gene regulatory proteins to promote motor neuron specification
(Mizuguchi et al., 2001
;
Novitch et al., 2001
).
Remarkably, most of the known gene regulatory proteins that act in the
specification of neural cells in the ventral spinal cord, including Olig2
protein (Mizuguchi et al.,
2001
; Novitch et al.,
2001
; Sun et al.,
2001
; Zhou et al.,
2001
), seem to act as gene repressors, shutting off genes required
for alternative pathways of development
(Briscoe and Ericson, 2001
;
Marquardt and Pfaff, 2001
).
Our ES cells may be useful for analysing such specification mechanisms.
Prospects for human cell therapy
Human ES cells offer a potential source of cells for therapy and drug
screening. For human ES cells to be useful in cell therapy, it will be
necessary to induce them to develop along particular pathways, to select for
the cell type of interest and to select against any residual ES cells, which
are very efficient at forming tumours in the recipient
(Brustle et al., 1997). The
strategy we describe here should be applicable to human ES cells and may be
useful in these respects, especially for producing large numbers of OPCs and
oligodendrocyte for the treatment of demyelinating diseases such as multiple
sclerosis. Althought it remains to be demonstrated that our ES-cell-derived
oligodendrocytes are capable of myelinating axons in vivo, OPCs derived from
mouse ES cells have already been shown by others to remyelinate axons in
rodent models of demyelinating diseases
(Brustle et al., 1999
;
Liu et al., 2000
). The
availability of human ES cells and the possibility of producing autologous
human ES cells by nuclear transfer offers exciting possibilities for the
treatment of such diseases in human.
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Acknowledgments |
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References |
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