1 Medical Research Centre, Polish Academy of Sciences, 5 Pawinskiego St. 02-106
Warsaw, Poland
2 Institute of Oncology, 5 Roentgena St. 02-781 Warsaw, Poland
* Author for correspondence (e-mail: kd-j{at}cmdik.pan.pl )
Accepted 20 February 2002
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Summary |
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Key words: Human stem cells, Cord blood, Neural differentiation, Neural progenitors, Transdifferentiation
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Introduction |
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We selected umbilical cord blood (CB) cells for our study. These cells are
easily available and preserved, and they could potentially serve as a routine
starting material for isolation and expansion of cells for allogenic as well
as authologous transplantations. The preliminary results of this study have
already been presented
(Bua
ska et al.,
2001a
; Machaj et al.,
2001
). Here, using the method of CB cell subfractionation and
their subsequent culturing in the presence of defined media and growth
factors, we were able to generate a self-renewing, clonogenic cell population
with neural-type precursor characteristics.
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Materials and Methods |
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Flow cytometry analysis
A Becton-Dickinson FACS Scan and commercial antibodies (HPCA-2 anti-CD34
phycoerithrine-conjugated and anti-CD45 fluoresceine-conjugated) were used for
FACS examination.
Cell culture expansion and clone formation
For cell culture expansion, trypsin-removed cells were plated in plastic 25
cm2 culture flasks at a density of 5x104
cells/cm2 in DMEM (Gibco) supplemented with 10% FCS, EGF (epidermal
growth factor, Sigma) at 10 ng/ml and antibiotic-antimycotic solution (AAS,
Sigma, 1:100). The cells were grown for 7 days to obtain a monolayer. Some
confluent cultures were re-seeded after trypsinisation, whereas some were kept
for 5 days longer in order to obtain free floating, non-adherent cells. Both
kinds of cell, when transferred to separate flasks or multi-well plates at a
density of about 10 cells/cm2 in the presence of EGF, started to
grow clones within the next 7 days in culture. The clones were observed to
grown in size during the following 14 days. As the cells proliferated, some of
them detached from the plastic and remained floating in suspension; however,
they stayed viable and could give rise to new clones. After reseeding, these
cells can be maintained as an adherent, undifferentiated, clonogenic
population in the presence of EGF and FCS during the six, already tested,
passages.
In additional experiments, these cells were cultured in the commercially available clonal cell culture system (Methocult H 4330, Stem Cell Technologies), which support the growth of both erythroid and myeloid precursors. The cells were analysed for the possible appearance of erythropoietic (BFU-E), granulo/macrophagopoietic (GM-CFC) and mixed (CFU-GEMM) colonies after 14 days of culture at 37°C in 5% CO2 in a fully humidified atmosphere.
Differentiation of nestin-expressing cells derived from cord
blood
In culture media
Clones that had been grown for 14 days in the conditions described above
were treated directly with Neurobasal Media supplemented with 10% FCS and 0.5
µM all-trans-retinoic acid (RA, Sigma) for the following 4 days.
In separate experiments, clone-growing cells were collected by trypsinisation and plated on poly-L-lysine 24-well tissue culture plates at a density 5x104 cells/cm2. The media used for promoting cellular differentiation was as follow: (1) Neurobasal Medium (Gibco) supplemented with 10% FCS (Gibco); (2) Neurobasal Medium supplemented with 10% FCS and 0.5 µM RA; (3) Neurobasal Medium supplemented with 10% FCS, 0.5 µM RA and BDNF (Sigma) at a concentration of 10 ng/ml.
In each case, cells were incubated at 37°C in 5% CO2 in a fully humidified atmosphere for 4 days and fixed for immunocytochemical detection of neural-specific antigens.
In the presence of the cortical primary culture
Mixed primary cultures were prepared from the brain cortex of 18-19 day-old
rat embryos (Wistar) under sterile conditions. Dissected tissue was placed in
Ca2+- and Mg2+-free HBSS (Gibco), dispersed mechanically
(10-12 pipette strokes) and then enzymatically by a 15 minute incubation in
0.2% trypsin (Gibco). After centrifugation at 200 g for 3
minutes the pellets were resuspended in Dulbecco's modified Eagle's medium
(DMEM, Gibco) supplemented with 10% FCS (Gibco) under antibiotic-antimycotic
protection (AAS, Sigma 1:100). After triturating, the debris was removed by
filtration through Millipore cell strainers (45 µm in diameter). Viable
cells were plated at a density of 5x104 cells/cm2
on poly-L-lysine 24-well tissue culture plates in 500 µl DMEM supplemented
with 10% FCS and AAS (1:100). The cells were maintained in a humidified
atmosphere with 5% CO2 at 37°C and allowed to grow for 7 days
before CB-derived cells were added. Undifferentiated cells from
nestin-positive clones were collected by trypsinisation and prelabelled with
green `cell tracker' (5-chloromethyl-fluorescein-diacetate, Molecular Probes
Inc), according to the manufacturer's recommendation. CB-derived cells were
seeded on the monolayer of rat brain cells at a density of
5x104 cells/cm2. Cells were allowed to grow in
such co-culture conditions for between 4 and 8 days and were then fixed for
immunocytochemistry.
Western blotting
The cultures were harvested in PBS, counted and lysed in the Laemmli
(Laemmli, 1970) gel loading
buffer in the proportion of 3.4x105 cells per 100 µl. The
equal-volume samples were separated by SDS-PAGE on a 10% polyacrylamide gel
and transferred onto Hybon-C-Extra. Immunodetection was performed using the
monoclonal anti-ß-tubulin III (Sigma), polyclonal anti-GFAP (DAKO) and
polyclonal anti-PLP/DM-20 (gift from J.-M. Matthieu). The immunoblots were
incubated with horseradish-peroxidase-conjugated secondary antibodies,
anti-rabbit for GFAP and PLP/DM-20 antigens and anti-mouse for ß-tubulin
III detections, then developed by ECL (Amersham).
Immunocytochemistry
Cells were fixed with 4% paraformaldehyde diluted in PBS for 20 minutes,
then washed with PBS and blocked in PBS containing 50% sheep serum and 10% FCS
(60 minutes). First antibodies were applied overnight at 4°C. Anti-human
nestin, a rabbit polyclonal antibody (gift of U. Lendahl, Karolinska
Institute, Stockholm) was applied at a concentration 1:1000, according to
Grigelioniene et al. (Grigelioniene et
al., 1996). The three following primary antibodies, mouse
monoclonal TUJ1 (Easter et al.,
1993
) directed against the ß-tubulin isoform III (gift of A.
Frankfurter, University of Virginia, Charlottesville, VA), mouse monoclonal
anti-MAP-2 (microtubule associated protein 2, Sigma) and rabbit polyclonal
anti-GFAP (glial fibrillary acidic protein, purchased from Dakopatts), were
diluted 1:2000, 1:100 and 1:200, respectively, in PBS/gelatine containing 0.2%
Triton X-100. The mouse monoclonal anti-GalC (galactosylceramide) antibody
(Ranchst et al., 1982
), a
culture supernatant obtained from R-mAb hybridoma cells (gift of B. Zalc,
INSERM U-495, Paris) was used at a dilution of 1:50 in DMEM with 10% FCS.
Secondary antibodies, anti-mouse IgG FITC for MAP2 (Sigma), anti-mouse
IgG2a-TxR for TUJ1, anti-mouse IgG3-TxR for GalC or anti-rabbit IgG-TxR for
GFAP (all from Pharmingen), were diluted 1:100 in the same solution as the
first antibody and applied for 1 hour at room temperature. As a control for
immunocytochemistry (in order to exclude non-specific background staining),
first antibodies were omitted during the procedure. To visualize the nuclei,
the cultures were then incubated with 5 µM Hoechst 33258 (Sigma) (20
minutes at room temperature) before being mounted in Fluoromount-G (Southern
Biotechnology Associate Inc., USA) either directly on the bottom of 24-well
plates or on glass slides of poly-L-lysine-coated cover slips.
Microscopy and quantification
The live growing cells or prefixed immunocytochemically labelled cultures
were observed either in the phase contrast or in the UV light under
fluorescence microscopes using Axiovert 25 or Axioscope 2 (Carl Zeiss),
respectively. Images were captured by the Videotronic CCD-4230 camera coupled
with the microscope and processed using the computer-based programmable image
analyser KS300 (Carl Zeiss).
The formation of clones by mitogen-expanded cells, which were selected from three independent cord blood preparations, was followed for at least four weeks for three or more randomly chosen clones. Differentiation towards a particular cell phenotype was quantified as a percentage of the total number of CB-derived cells growing in defined conditions. Cells from three culture plates (at least 600 cells each time) were counted in parallel for every cord blood preparation using the computer-assisted image analysis system described above.
PCR
Total RNA was isolated from cells using TRIzol Reagent (Life Technologies)
and quantified spectrophotometrically. Then 5 µg samples were reverse
transcribed using Superscript II and oligo (dT)12-18 primers
(Gibco). Each sample was amplified in duplicate, with and without reverse
transcriptase, to control the amplification of genomic DNA.
An equal volume of each sample was amplified by PCR using the following primers: for the human nestin gene 5'-GAGGACCAGGACTCTCTATC-3' and 5'-AGCGAGGAGGATGAGCTCGG-3' and for the GAPDH gene 5'-CATGTGGGCCATGAGGTCCACCAC-3' and 5'-TGAAGGTCGGAGTCAACGGATTTGGT-3'. Following 30 cycles of amplification (1 minute at 94°C, 1 minute at 58°C and 1 minute at 72°C using the MJ Research Thermal Cycler PTC-100), the PCR products were resolved on a 1% agarose gel. The appearance of 998 bp nestin bands was photographed under UV light.
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Results |
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Differentiation of nestin-positive CB-derived cells into
neural-specific cell phenotypes
Differentiation of nestin-positive cells was achieved either by direct
treatment of growing clones with differentiation-promoting media
(Fig. 3A,B) or by plating
clone-forming cells onto poly-L-lysine-coated coverslips in the presence of
neurobasal/10%FCS medium. Cell differentiation was supported by addition of
retinoic acid (RA) alone (Fig.
3A,B,C) or in combination with brain-derived neurotrophic factor
(BDNF) (Fig. 4) as recommended
previously by Sanchez-Ramos et al.
(Sanchez-Ramos et al., 2000).
Under these conditions the CB-derived cells start to differentiate along the
three major CNS lines, which can be identified by their immunochemical
properties. Cell-type-specific antigens were recognised by a TUJ1 monoclonal
antibody directed against a neuron-specific class of III ß-tubulin
(Fig. 3A,B, Fig. 4A,B), by GFAP polyclonal
antibody against an astrocyte-specific fibrillary acidic protein
(Fig. 3A,
Fig. 4C,D) and by a GalC
monoclonal antibody against the oligodendrocyte-specific galactosylceramide
(Fig. 3B, Fig. 4E,F) (for details see the
Materials and Methods). Moreover, as is shown in
Fig. 3A,B, cells belonging to
the same clone can express neuronal/astrocytic or neuronal/oligodendrocytic
markers, confirming directly their dual differentiation potential. The
appearance of the neural marker proteins upon CB cell differentiation was
additionally proved by western blotting
(Fig. 3C). A low level
expression of the neuronal marker ß-tubulin III can be found even in the
initial, non-differentiated clonogenic cultures, whereas two other, astrocytic
(GFAP) and oligodendrocytic (PLP/DM-20) markers, are detected only after
growing the cells in differentiation-promoting conditions. For western blots,
we have used, instead of a classic oligodendrocyte immunomarker, GalC, a
proteolipid protein (PLP) and its splicing variant DM-20 expression. An early
appearance of DM-20, which is known to overtake expression of PLP as well as
GalC in the oligodendrocyte lineage, is clearly visible on the blot
(arrow).
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The degree of differentiation of CB-derived cells depends on supplementation of the medium (Fig. 6). Spontaneous differentiation after plating of the clone-growing cells on poly-L-lysine substratum in 10%-FCS-supplemented neurobasal medium was minimal for neurons and astrocytes (less than 5% of the whole cell population). Addition of RA into the medium promotes differentiation of neurons and, to a lesser extent, astrocytes. Supplementation of the medium with BDNF does not increase the number of neurons in comparison with RA alone, whereas it significantly promotes the development of astrocytes and suppresses that of oligodendrocytes. This may indicate that at this stage of differentiation of CB-derived cells (4 days after poly-L-lysine plating), neurons are not able to produce BDNF at a concentration that is optimal for its physiological paracrine effect on the neighbouring cells.
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Differentiation by reseeding of nestin-expressing CB-derived cells on
a monolayer of rat brain primary culture
A similar or even higher differentiating effect was achieved after plating
CB-derived cells on cover slips with already growing rat primary cortical
culture in the presence of 10% FCS in DMEM medium. The phenotype-specific
markers for neurons, astrocytes and oligodendrocytes (red in
Fig. 5B,F,J) co-stained
numerous CB-derived cells that were pre-labelled with
5-chloromethyl-fluorescein-diacetate (green in
Fig. 5A,E,I). The close
vicinity of rat-brain-differentiated cells appears to promote CB-derived cell
differentiation. It seems that under these conditions the CB-derived neural
precursors get an optimal paracrine neurotrophic support that promotes the
appearance of all three types of neural progeny. After 4 days in co-culture,
almost 40% of cells that were previously marked by green `cell tracker'
differentiate into neurons, and for the other cells types 30% differentiate
into astrocytes and 11% into oligodendrocytes
(Fig. 6).
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Examples of phenotypic diversity among differentiating cells
Fig. 7 presents the
characteristic future of cells belonging to all three types of neural lineage.
Some cells display typical neuron-like morphology, with long neurite
projections. These cells express, in addition to TUJ1-labelled ß-tubulin
III, MAP-2 protein, which is characteristic of later steps of neuronal
development. Overlaying images of cells double-labelled for these two neuronal
proteins are shown in Fig. 7A.
In Fig. 7B, distinct
populations of anti-GFAP-reactive astrocytes (red) and anti-MAP2-stained
neurons (green-labelled cytoskeletal structures) are shown to grow in
proximity. Occasionally cells co-expressing both markers (GFAP and MAP-2) were
observed; however this was seen in less than 2% of all the GFAP-labelled
cells. Cells showing a typical morphology of matured, myelin-forming
oligodendrocytes, with irregular, branched projections that stain with an
anti-GalC antibody (owing to overlaying with the green `cell tracker' they are
yellow in Fig. 7C) were often
found in differentiating CB-derived cell cultures (Figs
4 and
5).
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Discussion |
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Interestingly, neural cells obtained from CB show a relatively high
spontaneous differentiation into oligodendrocytes, which amounts to about 11%
of the cells in co-culturing conditions, a higher percentage than previously
reported for CNS stem cells (Palm et al.,
2000; Zhang et al.,
2000
).
Attempts are under way to test the responses of CB-derived cells to defined
trophic or genetic signals, which are known to be effective in promoting
differentiation of oligodendrocytes and neurons in vitro
(Cameron et al., 1998;
Josephson et al., 1998
;
Bu
a
ska et al.,
2001b
) and to recruit them to the damaged brain in vivo
(Fricker et al., 1999
;
Bjorklund and Lindvall, 2000
;
Rosser et al., 2000
). Our data
utilising a co-culture system as an alternative to in vivo injection studies
indicate that brain tissue itself can provide optimal trophic support for
neural progenitor cell differentiation.
The other challenge is to elucidate further the origin of the CB-derived
neural precursors described here. We have already shown that this selected
cell population, which is able to differentiate towards neural phenotypes, is
practically devoid of cells expressing CD34 and CD45 antigens
(Fig. 1B), which are
characteristic of angiogenic or blood-forming stem cells
(Kim et al., 1999). In this
respect, their antigenic properties are similar to those described in the
foetal human CNS stem cell subpopulation
(Uchida et al., 2000
). In
contrast, the neural precursors examined for this study originate from a
plastic adherent mononuclear fraction, which may suggest a mesenchymal origin.
In spite of this, at the final stage of in vitro propagation and selection,
which directly precedes nestin-expressing clone formation, these cells are
totally unable to produce any hematopoietic colonies in vitro. This result
corresponds with the antigenic properties estimated by FACS analysis in this
paper (Fig. 1B). A similar
fraction of plastic-adherent mouse bone marrow stromal cells was reported to
transdifferentiate into a neural lineage by Kopen et al.
(Kopen et al., 1999
). As we
have already shown, the CB-derived precursor cells can produce
nestin-expressing clones that are able to differentiate toward
neuronal/astrocytic or neuronal/oligodendrocytic phenotypes
(Fig. 3A,B), thus displaying a
bipotentiality. The question of whether these clones can differentiate
simultaneously into all three types of neural progeny, a rigorous demand of a
neural stem cells, must be answered in further experiments. It is also
conceivable that CB-derived neural cells may originate from even more
`primitive' pluripotent stem cells residing in cord blood and resembling those
discovered recently in mouse bone marrow
(Krause et al., 2001
). These
ancestor cells can differentiate in vivo toward a variety of cell types,
including epithelial cells of the lung, gastrointestinal tract, liver, brain
and skin. This striking potential for transdifferentiation of adult stem cells
from various tissues into a neural fate as well as into cells of others organs
(Kopen et al., 1999
;
Peterson et al., 1999
) is a
matter of increasing interest and discussion
(Morrison, 2001
). Thus, it
will be scientifically and practically important to understand by which
mechanisms the cells from cord blood give rise to developmentally unrelated
CNS tissue and to further purify and characterise these cells.
In conclusion, this study has provided evidence, to our knowledge for the first time*, that each of the three cell types of human brain neurons, astrocytes and oligodendrocytes can be propagated in vitro from CB cells. These results raise the possibility that cord blood may provide an efficient source of cells differentiating into the neural lineage, with a potential to be employed in the therapy of human CNS diseases.
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Acknowledgments |
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Footnotes |
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