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Distinctions between fetal and adult human platelet-derived growth factorЦresponsive neural precursors.

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ORIGINAL ARTICLES
Distinctions between Fetal and Adult
Human Platelet-Derived Growth Factor–
Responsive Neural Precursors
Andrew Chojnacki, PhD,1 John J. P. Kelly, MD,1 Walter Hader, MD,2 and Samuel Weiss, PhD1
Objective: Platelet-derived growth factor (PDGF)–responsive neural precursors (PRPs; also known as oligodendrocyte progenitor
cells) are one of the best characterized precursor cell populations of the rodent central nervous system. Yet little is known about
the biology of human PRPs because of an apparent inability to culture and expand them in large numbers. This study was
designed to establish an approach that allows direct comparisons between the biology of fetal and adult human PRPs, as a means
to address potential differences in intrinsic myelin-production capabilities.
Methods: We used the neurosphere culture system, under low plating density, to isolate, culture, and compare the properties of
fetal and adult human PRPs.
Results: PDGF stimulated fetal human PRPs to generate neurospheres that differentiated primarily into oligodendrocytes, which
acquired myelin basic protein expression, as well as neurons and a small number of astrocytes. Together with PDGF, fibroblast
growth factor 2 promoted fetal human PRP expansion. In contrast, adult human PRPs isolated from the corpus callosum
required twice the culture period to generate neurospheres, which contained oligodendrocytes, as well as astrocytes, but not
neurons. Strikingly, fibroblast growth factor 2 did not promote adult human PRP self-renewal.
Interpretation: Differences in the intrinsic proliferation, phenotype, and self-renewal properties of fetal and adult human PRPs
suggest they are distinct populations, which may result in distinct myelin-production capabilities.
Ann Neurol 2008;64:127–142
Multiple sclerosis (MS) is thought to be an autoimmune disease of the central nervous system that is characterized by inflammatory cell infiltration, astrogliosis,
and demyelination.1 Limited remyelination has been
observed in chronic silent and active lesions,2 as well as
in acute MS lesions.3– 6 Remyelination of the central
nervous system in rodents has been demonstrated to
require newly generated oligodendrocytes.7 Therefore,
an in-depth knowledge of human oligodendrocyte progenitor cell (OPC) biology is crucial for understanding
why remyelination in MS ultimately fails.
OPCs are one of the best-characterized precursor cell
populations of the rodent central nervous system,8 and
rodent models of MS, such as experimental autoimmune encephalomyelitis, have been used for the development of drugs to treat the human disease. However,
this has sometimes led to the development of strategies
that were efficacious in experimental autoimmune encephalomyelitis but were either ineffective or exacer-
bated (Lenercept) MS in humans,9 and this may be
due, at least in part, to species-specific differences. Little is known about how closely the biology of human
OPCs mirrors that of rodent OPCs largely because of
difficulties in culturing and expanding either fetal10,11
or adult human OPCs.12,13 Furthermore, the requirement of unique culture conditions for human OPCs
makes it difficult to make direct comparisons of their
biology with that of rodent OPCs. Differences in the
biology of rodent and human cells have been previously demonstrated in the remyelinating capacity of
Schwann cells,14,15 the growth factors required for the
self-renewal of embryonic stem cells,16,17 and the capacity of fetal11 and adult18 neural stem cells (NSCs)
to generate oligodendrocytes. Wilson and colleagues19
have found that fetal human OPCs were largely similar
in growth factor responsiveness to their rodent counterparts with the exception of FGF-2 and neurotrophin
3 (NT-3) actions. Fetal and adult OPCs have also been
From the Departments of 1Cell Biology and Anatomy and 2Clinical
Neurosciences, Hotchkiss Brain Institute, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada.
Published online in Wiley InterScience (www.interscience.wiley.com).
DOI: 10.1002/ana.21421
Received Jun 4, 2007, and in revised form Feb 28, 2008. Accepted
for publication Apr 4, 2008.
This article includes supplementary materials available via the Internet at http://www.interscience.wiley.com/jpages/0364-5134/suppmat
Address correspondence to Dr Weiss, Department of Cell Biology
and Anatomy, 3330 Hospital Drive, NW, University of Calgary,
Faculty of Medicine, Calgary, Alberta, Canada T2N 4N1.
E-mail: weiss@ucalgary.ca
© 2008 American Neurological Association
Published by Wiley-Liss, Inc., through Wiley Subscription Services
127
successfully isolated from human brain tissue using
fluorescence-activated cell sorting for the OPC marker
A2B5.20,21 Importantly, these studies demonstrated
differences in the biology of fetal and adult human
OPCs that suggest they may have distinct origins. The
discovery of two separate OPC populations in the rodent spinal cord22,23 and three separate populations in
the forebrain24 supports the contention that fetal and
adult human OPCs may be distinct populations. A
better understanding of the similarities and differences
between rodent and human OPCs, as well as fetal and
adult human OPCs, will contribute essential information for understanding remyelination and developing
repair strategies for MS.
Standardizing cell culture systems used to expand orthologous precursor populations will increase the likelihood that observed differences are meaningful. Using
the neurosphere culture system,25 we previously reported the isolation of fetal mouse OPCs that we
termed platelet-derived growth factor (PDGF)–responsive
precursors (PRPs), because in addition to a robust oligodendroglial differentiation capacity, these precursors
also generated neurons.26 We reasoned that if human
and rodent PRPs possess similarities, then the same
culture protocol utilized for expansion of mouse PRPs
should be sufficient to expand PRPs isolated from human brain tissue. Therefore, the neurosphere culture
system was used without modification to grow fetal
and adult human PRPs. We report that fetal human
brain and adult human corpus callosum white matter
contain PRPs that generate neurospheres when cultured in the presence of PDGF. Fetal human PRPs
demonstrate remarkable conservation of their proliferation, self-renewal, and differentiation properties, when
compared with fetal rodent OPCs/PRPs. However, we
observed differences in the proliferation and selfrenewal properties of fetal versus adult human PRPs,
which may explain, in part, the exhaustion of remyelinating capacity in the adult human brain.
Materials and Methods
Cell Culture
Thirty-two fetal human brain tissue samples, aged 11 to 23
gestational weeks were obtained at therapeutic abortions.
The tissue was rinsed three times with phosphate-buffered
saline (PBS), with the meninges and other nonneural tissue
removed. To assess the ability of different growth factors to
induce proliferation using the neurosphere culture system,25
we mechanically dissociated fetal brain tissue with a P1000
pipetter in enzyme-free media hormone mix (MHM) whose
composition was described previously.25 The dissociated cell
suspension was then cultured at 20,000 to 40,000 cells/ml
for 3 weeks, with half the medium being replaced once every
7 days. The formation of neurospheres in epidermal growth
factor (EGF; human recombinant; 20ng/ml), FGF-2 (human
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recombinant; 20ng/ml; includes 2␮g/ml heparan sulfate),
PDGF-AA (human recombinant; 100ng/ml), EGF ⫹ FGF-2,
EGF ⫹ PDGF-AA, FGF-2 ⫹ PDGF, and EGF ⫹ FGF-2 ⫹
PDGF-AA (all from Peprotech, Rocky Hill, NJ, except for
FGF-2, which was from R&D Systems, Minneapolis, MN)
was assessed. MHM used to generate neurospheres also contained 2% B27 (Invitrogen, La Jolla, CA). The characteristics of the generated neurospheres were then examined as detailed later.
The phenotype potential of the generated neurospheres
was examined by washing the neurospheres three times with
MHM, and plating them as whole neurospheres or as singlecell suspensions on poly-L-ornithine– and/or laminin-coated
glass coverslips. Whole neurospheres were differentiated for 3
days in MHM or in the presence of 20ng/ml leukemia inhibitor factor (LIF; human recombinant; Chemicon, Temecula, CA), 20ng/ml T3 (Sigma, St. Louis, MO), or LIF ⫹
T3. Next, the differentiated neurospheres were assessed for
their phenotype potential using indirect immunohistochemistry as detailed later. In addition, neurospheres dissociated
to a single-cell suspension were plated at 100,000 cells/ml
onto poly-L-ornithine–coated coverslips, differentiated for 3
and 5 days, and assessed for their phenotype potential as detailed later. Self-renewal capacity was assessed by dissociating
generated neurospheres and culturing them at 20,000
cells/ml in the factor used for their derivation and various
other growth factor combinations. The total numbers of cells
generated after 3 weeks of culture were counted.
Adult human brain samples taken during temporal lobe
resections (6 samples ranging in age from 29 – 40 years; average, 36 years) or corpus callosotomies (5 samples, ranging
in age from 8 – 41 years, average, 21 years) were obtained
from the Foothills Hospital (Calgary, Alberta, Canada). The
tissue was rinsed with PBS, minced with a scalpel into 1mm
cubes, and then digested in MHM containing kynurenic acid
(0.2mg/ml), DNASE 1 (1mg/ml; Sigma), and purified collagenase (200U/ml; Worthington) for 1.5 hours at 37°C,
constantly stirring. The digest was then mechanically dissociated with a P1000 pipetter set at 800␮l, and rinsed three
times with MHM to clear the remaining enzymes. Subsequently, the cell suspension (including the myelin debris)
was plated at low (9 cells/mm2) or high (27 cells/mm2) densities into MHM-containing flasks. After 2 hours, the majority of the live cells had attached to the flask bottom, and
therefore all the media was removed and replaced to rid the
culture of the myelin debris. Cultures were maintained for
up to 9 weeks with half the medium being replaced with
fresh medium every 7 days.
Immunofluorescence
Differentiated whole neurospheres or single-cell suspensions
of the neurospheres were fixed for 20 minutes in ice-cold 4%
paraformaldehyde after the appropriate differentiation period. Coverslips were then processed for immunocytochemistry against mouse IgM anti-O4 (1:50; Chemicon, Temecula, CA), mouse anti-␤-III-tubulin (1:1000; Sigma),
rabbit anti–glial fibrillary acidic protein (GFAP; 1:300; BTI,
Stoughton, MA), rabbit anti-calretinin (1:200; Swant, Bellinzona, Switzerland), rabbit anti-GABA (1:500; Sigma), goat
anti-parvalbumin (1:200: Swant), rabbit anti–human myelin
basic protein (MBP; 1:200; Dako, Mississauga, Ontario,
Canada), goat anti–human PDGFR␣ (1:10; R&D Systems),
biotin-conjugated goat anti–human PDGFR␣ (1:5; R&D
Systems), sheep anti–mouse epidermal growth factor receptor
(EGFR; 1:50; Biodesign International, Kennebunk, ME),
and/or rabbit anti–mouse fibroblast growth factor receptor-2
(FGFR2; 1:50; Santa Cruz Biotechnology). The appropriate
biotin-conjugated antibodies were used followed by incubation with streptavidin-Cy3 (1:2,000) for the detection of the
mouse IgM anti-O4 and rabbit anti–mouse FGFR2 antibodies. Biotin-conjugated goat anti–human PDGFR␣ was detected by incubation with streptavidin-Cy3 (1:2,000). All
other antibodies were detected by incubation with the appropriate rhodamine- or fluorescein isothiocyanate–conjugated
donkey secondary antibodies. In addition, Hoechst 33258
(1:100 –1:1,000; Sigma) was used to label nuclei. Fetal human brain tissue was fixed in 4% paraformaldehyde followed
by incubation in 10 and 25% sucrose before embedding in
Tissue Tek OCT (Sakura Finetek, Torrance, CA). The tissue
was then sectioned at 8␮m and allowed to adhere to the
slides overnight. Immunostaining for PDGFR␣ (1:20),
EGFR, and FGFR2 was performed as described earlier for
the cultured cells. Fetal and adult human brain sections were
also processed for staining against PDGFR␣ (1:20) together
with rabbit anti-OLIG2 (1:200; gift from Dr Masato Nakafuku), rabbit anti-NG2 (1:20; gift from Dr Bill Stallcup),
rabbit anti-S100␤ (1:50; RD, Concord, MA), or mouse antiS100␤ (1:50; Sigma). Slides stained for OLIG2 were pretreated with a biotin blocking kit according to the manufacturer’s instructions (Vectastain, Burlingame, CA). Sections
were incubated with the primary antibodies overnight at
room temperature in PBS containing 0.3% Triton X-100
(Sigma), followed by three washes with PBS, and a 1-hour
block in PBS containing 10% normal donkey serum. For
OLIG2 staining, a donkey anti–rabbit biotin-conjugated secondary antibody (1:200) was applied for 1 hour, followed by
incubation with streptavidin-fluorescein isothiocyanate
(1:500). NG2, both S100␤ antibodies, and PDGFR␣ were
detected using the appropriate rhodamine- or fluorescein isothiocyanate–conjugated donkey secondary antibodies. All
secondary antibodies were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA). All slides were
mounted with Fluorsave (Calbiochem, San Diego, CA). Images were captured on an Axiocam camera (Zeiss, Thornwood, NY) mounted on a Zeiss Axioplan2 microscope
(Zeiss) using Axiovision software, and figures were composed
in Adobe Photoshop 6.0 (Adobe Systems, Mountain View,
CA). Confocal images where captured on a Olympus Optical
(Tokyo, Japan) Fluoview BX-50 laser scanning confocal microscope using Fluoview software.
Analysis of Self-Renewal Capacity
An example is provided demonstrating how the numbers for
Figure 4A were generated. For primary culture, the cells were
plated at 20,000 cells/ml in 10ml media containing PDGF.
Therefore, a total of 200,000 cells were plated. Only about
0.25% of the plated cells generate neurospheres after 3 weeks
in culture, whereas the remainder of the cells remained ad-
hered to the flask bottom and differentiated or died. Only
the neurospheres, which are suspended in the media, were
collected. The neurospheres were dissociated, and a cell
count was performed. On average, 200,000 cells were generated from these primary PDGF neurospheres. Thus, in addition to the cells that remained adhered to the flask bottom,
which either died or differentiated, 200,000 newly generated
live cells were produced in the presence of PDGF. This does
not include the cells that died during the mechanical dissociation of the neurosphere and is most likely an underestimate. Thus, 200,000 cells were plated, and 200,000 new
cells were generated, which results in 1 cell plated generating
1 cell.
Statistical Analysis
Statistical analysis was conducted using Prism statistical software by GraphPad. Values are mean ⫾ standard error of the
mean. For multiple-group comparisons, a one-way analysis of
variance was performed followed by a Tukey’s multiplecomparison test. A Student’s t test was performed for comparisons between two groups. p ⬍ 0.05 was considered significant.
Results
Fetal Human Brain Tissue Contains Platelet-Derived
Growth Factor–Responsive Neural Precursors That
Are Distinct from Epidermal Growth Factor– and
Fibroblast Growth Factor–Responsive Precursors
To establish whether fetal human brain tissue contained OPCs similar to those found in rodents, we first
examined fetal human brain tissue using immunohistochemistry. We previously demonstrated that fetal
mouse PRPs express PDGFR␣ and nuclear-localized
FGFR2 in vivo.26 We found that in sections of a
whole human brain aged 18 gestational weeks, obtained from the same tissue later used for cell culture
experiments, all PDGFR␣-expressing cells colabeled for
nuclear FGFR2 (Figs 1A–C). We also found cells that
expressed FGFR2 only (presumably FGF-responsive
precursors) but not PDGFR␣ (Fig 1B, arrowhead). Examination of PDGFR␣ and FGFR2 expression in dissociated primary cells plated for 24 hours in the absence of growth factors demonstrated that PDGFR␣expressing cells continued to coexpress FGFR2, which
was localized either to the nucleus or to the cell membrane (see Figs 1D–G). We found that in contrast with
the exclusive nuclear localization of FGFR2 in
PDGFR␣-expressing cells in vivo, the majority of the
FGFR2 staining was localized to the cell membrane in
vitro, which supports previous findings that ligand
binding is associated with translocation of FGF receptors to the nucleus.27,28 These data suggested that fetal
human brain tissue does contain PRPs, and that they
are distinct from FGF-responsive precursors.
We have also previously reported that E14 mouse
PRPs are distinct from EGF-responsive mouse NSCs.26
Chojnacki et al: Fetal and Adult Human PRPs
129
Fig 1. Fetal human platelet-derived growth factor (PDGF)–responsive precursors are distinct from epidermal growth factor receptor
(EGFR)– and fibroblast growth factor receptor 2 (FGFR2)–expressing neural stem cells (NSCs). (A–C) Tissue sections of an 18week-old fetal brain sample immunolabeled for PDGF receptor ␣ (PDGFR␣) (A) and FGFR2 (B). Note that PDGFR␣-positive
cells express FGFR2 (arrow) that is localized to the nucleus (C), but not all FGFR2-labeled cells express PDGFR␣ (arrowhead)
(B). In fetal brain tissue that had been dissociated, plated in the absence of growth factors, and fixed after 24 hours in culture,
some PDGFR␣-positive cells (D) continue to express nuclear-localized FGFR2 (E), but the majority of FGFR2 expression is restricted to the cell membrane (F, G). In fetal brain tissue sections, PDGFR␣ (H) and EGFR (I) labeling were found to be mutually exclusive (J), but PDGFR␣ (K) and EGFR (L) colocalized in culture (K); however, cells expressing only PDGFR␣ (arrowhead)
were also observed (M, N). Scale bars ⫽ 10␮m (A–N); 5␮m (insets).
Examination of fetal human brain sections aged 18 gestational weeks demonstrated that EGFR and PDGFR␣
labeled mutually exclusive populations (see Figs 1H–J).
In vitro we found that some cells brightly labeled for
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PDGFR␣ started to express EGFR, and vice versa (see
Figs 1K–N). Nevertheless, we still observed PDGFR␣expressing cells that did not label for EGFR (Fig 1M,
arrowhead). In addition, sections of a fetal human
brain aged 21 gestational weeks also showed that
PDGFR␣-expressing cells colabeled for OLIG2, NG2,
and S100␤ (see Supplementary Fig 1), all of which
have been found to be expressed by OPCs.29 –32 Thus,
in vivo, fetal human PRPs represent a distinct population of precursor cells, and this distinction is largely
maintained in vitro.
Fetal Human Platelet-Derived Growth Factor–
Responsive Neural Precursors Generate Neurospheres
That Differentiate into Neurons, Oligodendrocytes,
and Astrocytes
The observation that fetal human brain tissue contained PRPs (see Fig 1) similar to those we found in
the fetal mouse brain led us to ask whether we could
utilize the neurosphere culture system, previously used
to expand fetal mouse PRPs,26 to expand fetal human
PRPs. We cultured the dissociated fetal human brain
tissue at a low cell density of 20,000 cells/ml because
we had previously determined that 95% of the neurospheres generated in PDGF from a 1:1 mix of green
fluorescent protein– and non–green fluorescent protein–expressing fetal mouse brain tissue were clonally
derived. Fetal human brain tissue, which ranged in age
from 11 to 24 gestational weeks, generated neurospheres (50 –200␮m in size) in PDGF-containing medium after 3 weeks of culture (Fig 2A). PDGF neurospheres were also generated at a density of 10 cells/well
for both primary and passaged neurospheres, and also
at 1 cell/well for passaged neurospheres (data not
shown), indicating that these were, in fact, genuine
neurospheres and not the result of cell clumping. Furthermore, neutralizing antibodies directed against
PDGFR␣ greatly diminished the generation of both
primary and passaged neurospheres in the presence of
PDGF (data not shown). PDGF generated more neurospheres compared with medium containing no
growth factors ( p ⬍ 0.05; n ⫽ 8), but significantly
fewer neurospheres ( p ⬍ 0.001; n ⫽ 8) than all the
other conditions tested (see Figs 2B–F). When differentiated for 3 days, PDGF-generated neurospheres differentiated primarily into oligodendrocytes (O4) and
neurons (␤-III-tubulin), with only the occasional
GFAP-positive astrocyte being observed (see Fig 2G).
In contrast with fetal mouse PRPs that generated
GABAergic parvalbumin-positive neurons,26 fetal human PRPs generated neurons that were GABAergic
and labeled for calretinin but not parvalbumin (data
not shown). A larger proportion of EGF-generated
progeny differentiated into neurons (13 ⫾ 1%) in
comparison with PDGF-generated neurospheres (9 ⫾
1%; n ⫽ 5; p ⬍ 0.05; see Figs 2G–H). In contrast, PDGF-generated neurospheres contained, on average, at least fivefold as many oligodendrocytes compared with the next best condition (FGF-2 ⫹ PDGF;
Figs 3A–F). Furthermore, we found that PDGF stim-
Fig 2. Fetal human brain tissue contains precursor cells responsive to platelet-derived growth factor (PDGF) signaling.
(A–F) Examples of the neurospheres generated in the presence
of the growth factors indicated in the panels from a 21 gestational weeks brain tissue sample after 3 weeks of culture. (G,
H) Both PDGF and epidermal growth factor (EGF) neurospheres differentiated for 3 days generated neurons (␤-III-tubulin; green), oligodendrocytes (O4; red), and astrocytes (glial
fibrillary acidic protein [GFAP]; blue), but the majority of
EGF-generated progeny differentiated into neurons and astrocytes, whereas PDGF-derived progeny differentiated primarily
into oligodendrocytes and neurons. Scale bars ⫽ 200␮m (E);
50␮m (H). FGF ⫽ fibroblast growth factor; MHM ⫽ media
hormone mix.
ulation did not enhance the generation of oligodendrocytes by primary EGF ⫹ FGF-2–responsive NSCs (see
Fig 3F).
It is possible that the inability of PDGF to enhance
the generation of oligodendrocytes by primary EGF ⫹
FGF-2–generated neurospheres reflected a limitation of
the differentiation conditions used. It has previously
Chojnacki et al: Fetal and Adult Human PRPs
131
Fig 3. Fetal human neurospheres generated in the presence of platelet-derived growth factor (PDGF), but in the absence of epidermal growth factor (EGF) or fibroblast growth factor 2 (FGF-2), have the greatest capacity for oligodendroglial differentiation.
(A–E) Three-week-old neurospheres generated in various growth factor conditions were differentiated for 3 days in vitro and then
stained for the oligodendrocyte-specific antigen O4 (red) and the nuclear stain Hoechst 33258 (blue). (F) A significantly larger proportion of oligodendrocytes (arrows) differentiated from neurospheres cultured in the presence of PDGF, but in the absence of EGF
or FGF-2 (A), compared with neurospheres generated in EGF- or FGF-2–containing medium (B–E). *p ⬍ 0.05, PDGF significantly different against all other conditions. Scale bar ⫽ 50␮m (E).
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been reported that laminin is required for oligodendroglial differentiation from adult human NSCs.18 However, we found that PDGF-generated neurospheres
continued to generate significantly more oligodendrocytes than neurospheres generated in the other conditions when differentiated onto coverslips that were
coated with laminin together with poly-L-ornithine (see
Supplementary Fig 2A). In addition, we tested the capacity of thyroid hormone (T3) and LIF to promote
oligodendroglial generation because these factors are
known to support oligodendrocyte differentiation33
and survival,34 respectively. We observed no change in
the proportion of O4-labeled cells in PDGF neurospheres treated with (see Supplementary Fig 2B) or
without T3 (see Fig 3). Almost no oligodendroglial differentiation was observed in LIF-treated neurospheres
(see Supplementary Fig 2C). Similarly, oligodendroglial
differentiation was almost completely suppressed in
neurospheres differentiated in the presence of both LIF
and T3 (see Supplementary Fig 2D). Together, the
data suggest that PRPs present in the fetal human
brain proliferate and generate neurospheres in response
to PDGF and have the greatest capacity for oligodendrocyte generation.
Fibroblast Growth Factor 2 Promotes the Expansion
of Fetal Human Platelet-Derived Growth Factor–
Responsive Neural Precursors
Although it is well known that the continued expansion of rodent OPCs/PRPs requires the presence of
FGF-2 in addition to PDGF,26,35,36 it is unclear
whether FGF-2 also promotes the expansion of human
OPCs.11,19,20 Thus, we examined the self-renewal
properties of human fetal PRPs. In contrast with our
findings in the mouse, dissociated primary PDGFgenerated neurospheres could form secondary (pass1)
neurospheres in response to PDGF alone (Fig 4A; refer
to Materials and Methods for an example of how the
numbers were generated). However, the PRP population did not undergo expansion because the number of
cells generated equaled the number of cells plated, although this may be an underestimate because some
cells are lost during the mechanical dissociation of the
neurospheres (see Fig 4C). Fetal mouse PRP expansion
and self-renewal,26 as well as the expansion of rodent
OPCs, requires the presence of FGF-2 in conjunction
with PDGF.35,36 Addition of 20ng/ml FGF-2 to the
culture medium increased the size of the secondary
neurospheres and prevented their differentiation onto
the flask bottom (see Fig 4C). In agreement with the
increase in neurosphere size observed, FGF-2 more
than doubled the number of cells generated per cells
plated (see Fig 4C; p ⬍ 0.05, n ⫽ 5, t test), suggesting
that it can promote the expansion of fetal human
PRPs.
Primary EGF neurospheres derived from the mouse
E14 ventral forebrain can generate neurospheres when
passaged into PDGF, and these neurospheres are indistinguishable from primary PDGF neurospheres.26
Thus, we tested the potential of EGF ⫹ FGF-2–generated fetal human neurospheres to serve as an alternative source of PDGF neurospheres. We found that primary EGF ⫹ FGF-2 neurospheres could generate
pass1 PDGF neurospheres. However, these pass1
PDGF neurospheres (see Fig 4E) derived from primary
EGF ⫹ FGF-2 or EGF ⫹ FGF-2 ⫹ PDGF neurospheres generated significantly fewer ( p ⬍ 0.001) oligodendrocytes than primary PDGF neurospheres (see
Fig 4D) or pass1 PDGF neurospheres (see Fig 4E) derived from primary PDGF neurospheres. Thus, regardless of the passage, PDGF neurospheres always generated the greatest proportion of oligodendrocytes.
It has previously been reported that the capacity of
human NSCs to generate oligodendrocytes decreases
through subsequent passages.11 In agreement, we
found that the capacity for oligodendroglial differentiation decreased with increasing passage, regardless of
the growth factors that were used to generate neurospheres (see Fig 4F). Interestingly, by binning the data
into early gestational age brain tissue (average, 13
weeks; range, 11–14 weeks) and late gestational age tissue (average, 20 weeks; range, 17–23 weeks), we found
that neurospheres generated from younger brain tissue
generated significantly more oligodendrocytes per neurosphere (see Fig 4G; p ⬍ 0.001, n ⫽ 7 each condition, t test). Together, the data suggest that although
human PRPs can be passaged, their capacity for oligodendroglial differentiation declines with passage, at
least in vitro.
Fetal Human Platelet-Derived Growth Factor–
Responsive Neural Precursors Generate Myelin Basic
Protein–Expressing Oligodendrocytes
We had previously observed that fetal mouse PRPs
generated cells that could express immature neuronal
and oligodendroglial antigens simultaneously.26 Therefore, to ensure we were, in fact, identifying oligodendrocytes, we examined the progeny of fetal human
PDGF-generated neurospheres for expression of the
mature oligodendroglial marker MBP. In addition, we
were concerned that oligodendroglial differentiation in
larger neurospheres might not be observed because of
the limited migration out of the core of neurospheres
in some of the conditions. Hence, we cultured neurospheres in PDGF, EGF ⫹ FGF-2, or EGF ⫹ FGF2 ⫹ PDGF for 3 weeks, and then dissociated the neurospheres, plated the suspension at 100,000 cells/ml,
and differentiated the cells for 3 or 5 days in vitro.
After the appropriate differentiation period, the cultures were dual-labeled for O4 and MBP. Again, we
Chojnacki et al: Fetal and Adult Human PRPs
133
Fig 4. Fetal human platelet-derived growth factor (PDGF)–responsive neural precursors generate the greatest proportion of oligodendrocytes over multiple passages. Digital images of 2-week-old secondary neurospheres generated in PDGF (A) or PDGF ⫹ fibroblast
growth factor 2 (FGF-2) (B) derived from primary 3-week-old PDGF neurospheres. (C) The capacity of primary cells to proliferate
in response to PDGF was compared with progeny passaged into PDGF or PDGF ⫹ FGF-2. PDGF-responsive precursors exhibit
significantly greater expansion when passaged into PDGF ⫹ FGF-2–containing medium ( p ⬍ 0.05, t test; n ⫽ 5), in comparison
with medium containing PDGF alone. Comparisons of the capacity of primary (D) and pass1 (E) neurospheres to generate oligodendrocytes demonstrate that only neurospheres originally derived in PDGF retain their capacity to generate a large proportion of
oligodendrocytes (minimum p ⬍ 0.05, Tukey’s Honest Significant Difference). (F) The capacity to generate oligodendrocytes declines
with each subsequent passage regardless of the growth factors used to generate the primary neurospheres (minimum p ⬍ 0.05,
Tukey’s Honest Significant Difference). This decrease in oligodendroglial capacity is also seen with PDGF-generated neurospheres
derived from older gestational age brain samples (G; *p ⬍ 0.05). Scale bars ⫽ 100␮m (A, B).
found that neurospheres generated in PDGF generated
close to fourfold more O4⫹ oligodendrocytes of the
live cells plated in comparison with the other conditions, regardless of whether the cells were differentiated
for 3 (Figs 5A, B; see Supplementary Fig 3; p ⬍ 0.001;
n ⫽ 7) or 5 days in vitro (see Figs 5A, C; see Supplementary Fig 3; p ⬍ 0.001; n ⫽ 5 for P; n ⫽ 6 for E ⫹
F and E ⫹ F ⫹ P). Morphologically, the O4-positive
oligodendrocytes generated from PDGF neurospheres
after 3 days in vitro were predominantly bipolar,
whereas O4-positive cells differentiating from EGF ⫹
FGF-2 ⫹ PDGF neurospheres of the same age were
predominantly multipolar and more reminiscent of
mature oligodendrocytes. Indeed, a larger proportion
of MBP-positive oligodendrocytes (1.4 ⫾ 0.1%; p ⬍
0.05, n ⫽ 7, Tukey’s HSD) differentiated from
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EGF ⫹ FGF-2 ⫹ PDGF neurospheres after 3 days in
vitro than either EGF ⫹ FGF-2– (0.1 ⫾ 0.02) or
PDGF-generated (0.7 ⫾ 0.18) progeny (see Fig 5A).
However, after 5 days of differentiation, the proportion
of MBP-expressing oligodendrocytes was at least 3.5fold greater in PDGF-derived cultures (6.71 ⫾ 0.99%)
compared with EGF ⫹ FGF-2 (0.8 ⫾ 0.02%) or
EGF ⫹ FGF-2 ⫹ PDGF (1.87 ⫾ 0.21%) cultures
( p ⬍ 0.001; n ⫽ 6 for P; n ⫽ 7 for E⫹F and
E⫹F⫹P). Even so, we believe that the numbers of
MBP and O4-positive oligodendrocytes we observed in
the differentiated dissociates of PDGF-generated neurospheres is an underestimate of their true potential for
oligodendroglial differentiation; we observed a large
number of oligodendrocytes in this condition without
nuclei or with fragmented nuclei that were not included
Fig 5. Fetal human platelet-derived growth factor (PDGF)–responsive precursors generate large numbers of myelin basic protein
(MBP)–expressing oligodendrocytes. (A) Primary 3-week-old neurospheres generated in epidermal growth factor (EGF) ⫹ fibroblast
growth factor receptor 2 (FGF-2), EGF ⫹ FGF-2 ⫹ PDGF, or PDGF were dissociated and differentiated at 100,000 cells/ml for
3 or 5 days in vitro (DIV), and then immunostained for O4 (red), MBP (green), and the nuclear stain Hoechst 33258 (blue). At
3 DIV, the majority of the O4-expressing cells derived from PDGF neurospheres possessed a bipolar morphology reminiscent of immature oligodendrocytes, and few of these cells expressed MBP (B). However, even though EGF ⫹ FGF-2 ⫹ PDGF-generated neurospheres generated significantly less O4⫹ (black bars) oligodendrocytes, a larger proportion of these cells expressed MBP (white
bars), and morphologically these cells resembled more mature oligodendrocytes. At 5 DIV, the majority of the O4⫹ cells from
PDGF-generated neurospheres had a complex multidendritic morphology, and a significantly larger proportion of these cells coexpressed MBP expression compared with EGF ⫹ FGF-2- or EGF ⫹ FGF-2 ⫹ PDGF-generated neurospheres (C). Scale bar ⫽
50␮m
Chojnacki et al: Fetal and Adult Human PRPs
135
in the counts (data not shown). Together, the data suggest that fetal human PRPs have the greatest capacity
for generating MBP-expressing oligodendrocytes.
Platelet-Derived Growth Factor and Neurotrophin 3
Induce the Generation of Neurospheres from the
Adult Human Corpus Callosum
Remyelination within MS patients has been demonstrated to occur in cases of MS.2,4 – 6 Currently, however, there is little evidence for proliferating OPCs
within the adult human brain, and attempts to culture
adult human OPCs have been largely unsuccessful. Because we had established the presence of PRPs within
fetal brains aged 11 to 24 gestational weeks, which possessed a robust potential for oligodendroglial generation, we asked whether adult human white matter also
contained such PRPs, and how similar they were to
their fetal counterparts. We obtained samples of white
matter from four corpus callosotomies, which included
the anterior body of the corpus callosum. For two of
the specimens, samples were reserved for immunohistochemistry. We stained 20- and 16-year-old samples
for PDGFR␣ and FGFR2 to establish the presence of
PRPs in the tissue obtained. We found that 3.6 ⫾
0.3% (36,713 cells examined, 29,203 from the 20year-old corpus callosum and 7,510 from the 16-yearold corpus callosum) of the cells expressed PDGFR␣
(Fig 6A), and all PDGFR␣ ⫹ cells labeled for nuclearlocalized FGFR2 (see Figs 6A, B), as we had found for
fetal human PRPs. However, not all FGFR2-labeled
cells expressed PDGFR␣. In addition, we stained the
tissue for Ki67, a marker associated with cell proliferation, and PDGFR␣ (see Fig 6C) and found that approximately 0.5% of PDGFR␣-expressing cells were
proliferating. We also found that PDGFR␣-expressing
cells colabeled for NG2, OLIG2, and S100␤, all of
which have been found to be expressed by OPCs29 –32
(see Supplementary Fig 4). Thus, adult human corpus
callosum contains PRPs that may contribute to remyelination.
Next, we examined whether neurospheres could be
generated from the white matter of the corpus callosum. We included NT-3 in the culture medium in
conjunction with PDGF, because NT-3 has been reported previously to increase fetal human OPC proliferation.19 Neurospheres that ranged in size from 50 to
200␮m were generated after 7 weeks in culture in the
presence of PDGF ⫹ NT-3 (see Fig 6D). When differentiated for 3 days in vitro, extensive migration was
observed away from the center of the neurospheres (see
Fig 6E). The cells migrating from the neurospheres
had a bipolar morphology with a few smaller processes
radiating away from the cell soma (see Fig 6F), characteristic of immature oligodendrocytes and OPCs.
Surprisingly, we found that neurospheres were gener-
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ated at only low-density conditions for all samples (see
Supplementary Fig 5A), whereas no neurospheres were
generated at high cell densities (see Supplementary Fig
5B). We also examined whether we could generate
neurospheres in the presence of PDGF and NT-3 from
the white matter obtained from temporal lobe resections. Despite the fact that we observed PDGFR␣positive cells in samples of temporal lobe myelin (data
not shown), none of the samples generated neurospheres even after 9 weeks in culture (n ⫽ 6). The
addition of FGF-2 in combination with PDGF and
NT-3 to some of the cultures (n ⫽ 4) also failed to
stimulate neurosphere formation. Furthermore, lowdensity cultures of temporal lobe white matter did not
promote the generation of neurospheres by PDGF and
NT-3. The data suggest that adult human corpus callosum contains PRPs that can be cultured as neurospheres in the presence of PDGF and NT-3.
Restricted Phenotypes and Limited Self-Renewal of
Adult Human Corpus Callosum–Derived PlateletDerived Growth Factor–Responsive Neural Precursors
Rodent postnatal and adult OPCs differ in their
proliferation and differentiation characteristics.37 We
sought to determine whether these differences existed
between fetal and adult human PRPs. Therefore, we
first examined the phenotype potential of neurospheres
generated in PDGF ⫹ NT-3 from the adult corpus
callosum. We found O4-expressing oligodendrocytes
and GFAP-expressing astrocytes migrating away from
the neurospheres (see Figs 6G, H). After 3 days of differentiation, 33 ⫾ 3% of the cells adopted O4 expression, whereas 15 ⫾ 3% of the cells expressed GFAP
(n ⫽ 3). However, in contrast with the fetal human
PRPs examined, the majority of the neurospheres did
not contain ␤-III-tubulin–expressing neurons (only
one neuron observed of all the neurospheres examined;
see Figs 6G, H). Furthermore, we occasionally found
MBP-expressing cells within the neurospheres after 5
days in vitro (see Fig 6H), suggesting that the progeny
of the adult human PRPs have the potential to generate the constituents of myelin.
Lastly, we examined the capacity of adult human
PRPs derived from the corpus callosum to self-renew.
Approximately 10 neurospheres, which were at least
150␮m in size, were dissociated per 1ml medium. The
dissociate was then plated in the presence of PDGF
and NT-3 or PDGF, NT-3, and FGF-2. We did not
observe the generation of secondary neurospheres even
after 12 weeks of culture, regardless of the presence of
FGF-2 (n ⫽ 3, data not shown). We did find some
evidence of limited proliferation in the form of adherent colonies of cells comprising 10 to 20 cells, and
these cells had an astroglial morphology. These data
suggest that in contrast with fetal human PRPs, adult
Fig 6. Platelet-derived growth factor–responsive neural precursors (PRPs) isolated from adult human corpus callosum–generated neurospheres in response to platelet-derived growth factor (PDGF) ⫹ neurotrophin 3 (NT-3) that differentiated into oligodendrocytes
and astrocytes. (A) Section of a 20-year-old human corpus callosum sample stained for PDGF receptor ␣ (PDGFR␣) (red) and
fibroblast growth factor receptor 2 (FGFR2) (green) demonstrates that they label the same cell. (B) Confocal microscopy confirms
that FGFR2 labels the nucleus of PDGFR␣-expressing cells. Approximately 0.5% of PDGFR␣-expressing cells also labeled for the
nuclear antigen Ki67 (C), which is associated with cell proliferation. (D) Examples of the neurospheres generated in PDGF ⫹
NT-3 after 7 weeks and 5 days of culture. (E) Large numbers of cells migrated away from the center of the neurosphere after 3
days of differentiation, and these cells had a bipolar or a polydendritic morphology (F). (G, H) PDGF-generated neurospheres differentiated into oligodendrocytes and astrocytes after 3 days, but neurons were rarely observed. (I) MBP-positive cells were also observed after 5 days of differentiation. Scale bars ⫽ 12.5␮m (A), 5␮m (B, C), 100␮m (D), 25␮m (E, F), 50␮m (G), 20␮m
(H), 10␮m (I). GFAP ⫽ glial fibrillary acidic protein.
human corpus callosum PRPs are limited in their phenotype and self-renewal potentials.
Discussion
Utilizing the same culture system we previously used to
examine the properties of fetal mouse forebrain
PRPs,26 we have cultured and examined the properties
of fetal and adult human PRPs. We found that fetal
human PRPs, like fetal mouse PRPs, were distinct
from EGF- and FGF-responsive NSCs, and generated
the greatest numbers of oligodendrocytes. Fetal human
PRPs self-renewed poorly in PDGF, but expanded in
the presence of FGF signaling, and continued to generate the greatest proportion of oligodendrocytes after
passaging. In addition, we found that adult human
PRPs isolated from the corpus callosum, but not the
temporal lobe, could generate neurospheres in response
to PDGF and NT-3, and had a robust capacity for
oligodendroglial generation. However, adult human
PRPs were distinct from fetal PRPs by the prolonged
Chojnacki et al: Fetal and Adult Human PRPs
137
culture period required for the generation of neurospheres, the inability of FGF-2 to promote their selfrenewal, and their inability to generate neurons or a
large number of MBP-expressing oligodendrocytes.
Properties of Mouse and Human Platelet-Derived
Growth Factor–Responsive Neural Precursors/
Oligodendrocyte Progenitor Cells Are Remarkably
Similar
One of the aims of this study was to determine the
degree to which the biology of rodent OPCs is a predictor of human OPC biology (Table). Use of the
same in vitro system to culture both mouse and human
precursor populations enabled the pursuit of this aim.
Many studies have reported that PDGF failed to stimulate the proliferation of human OPCs11–13,19 despite
its well-known capacity to stimulate rodent PRP/OPC
proliferation.26,38,39 We observed that PDGF induced
proliferation and the generation of neurospheres by fetal human PRPs, just as it did for fetal mouse forebrain
PRPs.26 It is possible that the differences observed in
PDGF responsiveness simply stems from the different
culture systems used. Dissociating the tissue mechanically, which we found to be quite effective, without
utilizing enzymes may have increased responsiveness of
our cell preparations to growth factors as we have
found that enzymatic digestion can adversely affect cell
proliferation in response to mitogens (unpublished observations). Nevertheless, Wilson and colleagues19 have
also found that PDGF can stimulate the proliferation
of fetal human OPCs, albeit those derived from the
spinal cord. We did, however, find some differences
with respect to the effect of PDGF on the formation of
neurospheres by fetal human PRPs. We found that it
took three times the culture period for fetal human
PRPs to generate neurospheres of the same size compared with fetal mouse PRPs. We also found that in
contrast with mouse PRPs, human PRPs passaged in
the presence of PDGF. Whether these discrepancies
are a result of an increased cell cycle time or differences
in survival/apoptosis requires further investigation.
Thus, although both fetal rodent and human PRPs/
OPCs proliferate in response to PDGF, subtle differences in PDGF responsiveness do exist between the
two populations.
In addition to similarities in growth factor responsiveness, we found that mouse and fetal human PRPs
were similar in their phenotype potentials (see the Table). Both generate a large proportion of oligodendrocytes that can differentiate into a significant number of
MBP-expressing oligodendrocytes, and both have the
capacity to generate neurons. However, fetal human
PRPs generated calretinin-positive interneurons in contrast with the parvalbumin-positive interneurons generated by fetal mouse PRPs.26 Both precursor populations also have the capacity to generate astrocytes.
Nonetheless, fetal human PRPs appear to generate astrocytes more readily because they were observed even
in control medium, whereas the generation of astrocytes by fetal mouse PRPs required the presence of
bone morphogenetic protein-2 and ciliary neurotrophic
factor.26 We are uncertain of the significance of these
Table. Characteristics of Mouse and Human Platelet-Derived Growth Factor–Responsive Neural Precursors
Characteristics
Embryonic PRPs
Mouse
PDGF stimulates proliferation
Time to neurosphere formation, wk
Self-renewing in factor of derivation
PDGFR␣-expressing cells express nuclear
FGFR2
FGF-2 promotes expansion
Generate large numbers of oligodendrocytes
Generate large numbers of MBP-positive
oligodendrocytes
Generate neurons
Neuronal phenotype
Capacity to generate astrocytes
Adult PRPs
Human
Human
Yes
1
No
Yes
Yes
3
Yes
Yes
Yesa
6-7
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
Yes
No
Yes
GABAergic parvalbuminpositive
Yesb
Yes
GABAergic calretininpositive
Yes
No
N/A
a
Yes
With neurotrophin 3.
Requires bone morphogenetic protein-2 and ciliary neurotrophic factor stimulation.
PRP ⫽ platelet-derived growth factor–responsive neural precursors; PDGF ⫽ platelet-derived growth factor; PDGFR␣ ⫽ plateletderived growth factor receptor ␣; FGFR2 ⫽ fibroblast growth factor receptor 2; FGF-2 ⫽ fibroblast growth factor 2; MBP ⫽ myelin
basic protein.
b
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subtle differences, but they highlight the import of verifying results obtained from rodent studies with human
studies.
FGF-2 is known to promote the self-renewal of rodent optic nerve–derived OPCs35,36,40; however, previous studies of human OPCs have failed to demonstrate
any actions of FGF-2 on self-renewal.11,19 We found
that both fetal mouse26 and human PRPs express
nuclear-localized FGFR2 (see Figs 1A–C), indicative of
ligand activation.27,28,41 This suggests that FGFR2 signaling is active in PRPs, and that it may function in
promoting their self-renewal. In contrast, Bansal and
colleagues42 have found that only mature oligodendrocytes express FGFR2. It is unclear why this discrepancy
in FGFR2 expression is observed, but others43,44 have
found that FGFR2 messenger RNA is expressed in
white matter tracts, which are known to be populated
by OPCs in addition to mature oligodendrocytes.37,45
Nevertheless, we observed that FGF-2 promoted the
expansion of fetal human PRPs (see Figs 4A–C), just as
it did for fetal mouse PRPs. This dissimilarity in the
actions of FGF signaling observed by different groups
may indicate regional distinctions in OPC growth factor requirements for proliferation/self-renewal, given
that both Wilson and colleagues19 and Chandran and
colleagues11 cultured dissociated fetal human spinal
cord, whereas we utilized fetal human brain.
Using indirect immunohistochemistry, we also demonstrated that fetal human PRPs are distinct from both
FGF- and EGF-responsive fetal human NSCs/precursors (see Fig 1). In agreement with previous findings,11,46,47 we found that fetal human NSCs were
severely restricted in their capacity to generate oligodendrocytes (less than 4% of the cells generated) regardless of the differentiation condition (see Figs 3, 4,
5, and Supplementary Fig 2). Also, we could not generate PDGF neurospheres with the same oligedendroglial capacity from EGF ⫹ FGF-2 expanded human
NSCs, despite the fact that fetal mouse NSCs possess
this capability.26 It is possible that some additional factors may be required for the generation of PRPs and/or
oligodendrocytes from fetal human NSCs. In summary, although they differ in some respects, the biology of fetal mouse PRPs is, in principle, predictive of
fetal human PRP biology.
Oligodendroglial Differentiation Capacity of Fetal
Human Platelet-Derived Growth Factor–Responsive
Neural Precursors Declines with Passage and
Gestational Age
Our results demonstrate that the capacity of both fetal
human NSCs and PRPs to generate oligodendrocytes
decreases through subsequent passages. This is in agreement with the findings of Chandran and colleagues,11
who observed that neurospheres generated in FGF-2
from fetal human spinal cord had a limited capacity for
generating GalC-positive oligodendrocytes, and that
later passage neurospheres generated even fewer oligodendrocytes. Bone morphogenetic proteins, previously
shown to inhibit oligodendroglial differentiation in rodents,48 –51 may contribute to the regulation of oligodendrocyte differentiation by fetal human PRPs. Indeed, adult human OPCs, purified using magnetic
activated cell sorting for A2B5, express bone morphogenetic protein-2 and -7.52 The receptor tyrosine phosphatase ␤/␨ may also participate in regulating the differentiation of oligodendrocytes generated by fetal
human PRPs because Sim and colleagues52 found that
it regulates the differentiation of adult human OPCs.
Importantly, we found that the potential of PRPs to
generate oligodendrocytes decreased with an increase in
the age of the fetal brain tissue (see Fig 4G), suggesting
that this restriction also occurs in vivo. Whether adult
human PRPs also demonstrate a similar decrease in the
capacity to generate oligodendrocytes through subsequent divisions needs to be tested because this may also
contribute to the limited remyelination observed in MS.
Fetal and Adult Human Platelet-Derived Growth
Factor–Responsive Neural Precursors Are Distinct
Populations
Similar to fetal human PRPs, adult human corpus callosum–derived PRPs proliferated and generated neurospheres in the presence of PDGF, and generated large
numbers of oligodendrocytes. However, we observed
several differences between fetal and adult human
PRPs. Seven weeks, compared with 3 weeks for fetal
human PRPs, were required for the generation of neurospheres of comparable size from adult human brain
tissue. Also, we rarely observed MBP-expressing cells
after 5 days in vitro from adult human PRPs, whereas
upward of 7% of fetal human PRP progeny expressed
MBP after 5 days in vitro. These findings are similar to
the increased cell-cycle time of adult rodent OPCs, and
the lengthened period required for oligodendroglial
maturation of adult compared with postnatal rodent
OPCs.37 However, the adult brain tissue was obtained
from epilepsy patients, and how this may have influenced the biology of adult PRPs is unknown. Also, in
contrast with the neurogenic capacity of fetal human
PRPs, adult human corpus callosum PRPs essentially
failed to generate neurons, which is similar to our findings for PRPs obtained from the adult mouse corpus
callosum53 and adult O-2A progenitor cells.45 The differences that fetal and postnatal PRPs/OPCs have with
their adult counterparts, whether rodent or human,
serve to highlight the similarities between adult rodent
and human PRPs/OPCs.
We did, however, find an important difference in
the self-renewal/expansion capacity of adult human
Chojnacki et al: Fetal and Adult Human PRPs
139
PRPs compared with adult rodent OPCs, as well as
fetal human PRPs. This was despite the fact that fetal
PRPs were cultured in the presence of PDGF to generate neurospheres, whereas adult PRPs were cultured
in the presence of PDGF ⫹ NT-3, which could not
account for the differences we observed because fetal
PRPs cultured in PDGF ⫹ NT-3 maintain their capacity to self-renew and their phenotype potential (data
not shown). In contrast with its actions on fetal human
PRPs and adult rodent OPCs,54 we found that FGF-2
did not promote the expansion/proliferation of adult
PRPs isolated from the corpus callosum (see the Table). Previous studies have demonstrated that unknown
factors secreted by cortical astrocytes can abrogate the
effects of FGF signaling on adult rodent optic nerve
OPCs.55 However, it is unlikely that autocrine/paracrine effects reduced the ability of FGF-2 to promote
self-renewal of adult human PRPs in our cultures,
given the low cell culture density used. It is possible
that other factors may contribute to the maintenance
of adult human PRP proliferation. Moreover, it is
tempting to speculate that the inability of FGF signaling to promote the expansion of adult human PRPs
may be a factor contributing to the limited remyelination observed in human demyelinating diseases.
Whether adult OPCs continue to display limited selfrenewal in the presence of inflammation, denuded axons, or myelin inhibitory factors remains to be determined. Taken together, the results strongly suggest that
fetal and adult human PRPs are distinct populations of
precursors. In support of such a conclusion, it has been
reported that adult human OPCs/PRPs ensheathed
more host axons per donor cell and generated oligodendrocytes more efficiently than their fetal counterparts when transplanted into shiverer mice.21 Furthermore, evidence from rodent studies has demonstrated
at least three different OPC populations in the mouse
forebrain, in which the earliest population is largely replaced by the later two.24
Finally, several lines of evidence also suggest that
adult human corpus callosum–derived PRPs and temporal lobe subcortical white matter OPCs may be distinct precursor populations. We were unable to generate neurospheres from white matter obtained from
human temporal lobe resections. This is in contrast
with the findings of Nunes and colleagues,56 because
they were able to generate neurospheres from dissociates of the human temporal lobe tissue that had been
sorted for the A2B5 antigen. They also found that only
3 weeks was required for the generation of neurospheres from the adult temporal lobe, in contrast with
the 7 weeks we found are needed for the adult human
corpus callosum. Moreover, the observations that corpus callosum–derived PRPs, in contrast with temporal
lobe OPCs,20,56 do not readily generate neurons or
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self-renew in the presence of FGF-2 further supports
the conclusion that the two populations are distinct.
Again, whether this is due to culture system differences
or distinct precursor populations is unknown. However, the observations that focal MS lesions found in
the human brain predominantly localize to the periventricular white matter of the frontal and parietal
lobes,57–59 whereas only 30% of MS patients have focal lesions in the corpus callosum,60 may be indicative
of underlying differences in oligodendrocyte populations and/or their precursors in specific regions of the
adult human brain.
This work was supported by the Neuroscience Canada Foundation,
the Multiple Sclerosis Research Foundation of Canada, and the Alberta Heritage Foundation for Medical Research (J.J.P.K., S.W.).
We thank Drs M. Noble, M. Raff, and V. W. Yong for reviewing a
previous version of this manuscript. We also thank R. Hassam and
D. Livingstone for excellent technical assistance.
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