C.N. S6endsen et al. / Journal of Neuroscience Methods 85 (1998) 141–152
Expansion of human neural precursor cells using conventional passaging methods
Age (weeks) Region Days Total expansion
14 21 28 42 56 63
12 CTX 2.8 2.6 1.7 1 12.36
10 SC 1.7 1.7 1.4 4.06
9 SC 0.9 1.4 1 0.4 0.50
10 MES 2.5 1 2 1.1 5.50
10 MES 1.9 0.4 1.1 1.2 1.00
21 2.0 2.5 1.2 6.00
8 1.8 2 1.4 1.3 6.55
Numbers represent expansion ratios: i.e. number of cells at end of passage over number of cells seeded into ﬂasks. Last number indicates end of
growth for the culture. Cultures were maintained in EGF and FGF-2 for the ﬁrst 28–35 days and then switched to FGF-2 alone.
SC, spinal cord; CTX, cortex; MES, mesencephalon.
neously stop dividing and differentiate. Stem cells have When attached cells or free ﬂoating aggregates reach
been most extensively studied in haemopoetic, epider- the end of a growth cycle, they must be mechanically
mal and intestinal tissues which require frequent cell broken up or ‘passaged’, often using digestion enzymes,
replacement throughout life (Hall and Watt, 1989). to avoid contact mediated growth arrest or lack of
Recent studies have shown that speciﬁc regions of both nutrient diffusion. We postulated that these standard
the developing and adult rodent brain harbour cells passaging techniques may lead to cellular trauma, strip
which divide in response to mitogens, while retaining receptors, deprive cells of contact mediated factors and
the capacity to differentiate into neurons and glia, and remove vital tight junctions known to hold tissues
as such may represent neural stem cells (Weiss et al., together. This may lead to either the terminal differenti-
1996; McKay, 1997; Palmer et al., 1997), although stem ation of precursor cells, or a lack of response to mito-
cell status is often debated and the term neural precur- gens for the rat and human cells. We therefore
sor may better describe cells within these heterogeneous attempted to adapt the passaging technique such that
cultures. Neural precursor cells from the rodent re- enzymatic or mechanical disturbance to the cells was
spond to both epidermal growth factor (EGF) and minimised and then assess the ability of the human
ﬁbroblast growth factor (FGF-2) (for reviews see Gage neural precursor cells to continuously renew over time.
et al., 1995; McKay, 1997) and can be grown as either
monolayer cultures or as free ﬂoating spherical aggre-
gates termed ‘neurospheres’ (Reynolds et al., 1992). The
short term growth ( B60 days) of similar human CNS
precursors has recently been reported (Buc-Caron,
2.1. Tissue collection
1995; Svendsen et al., 1996; Chalmers-Redman et al.,
1997; Murray and Dubois-Dalcq, 1997) and in some
Human fetal tissue (between 7 and 21 weeks post
cases these can survive, migrate, differentiate and re-
conception) was collected from two different sources:
store function following transplantation into rat models
via the Uniform Anatomical Gift Act of the United
of Parkinson’s disease (Svendsen et al., 1997a). How-
States or from a local hospital. The methods of collec-
ever, we and others have also shown that human neuro-
tion conform with the arrangements recommended by
spheres are difﬁcult to expand in vitro over long
the Polkinghorne Committee for the collection of such
periods of time (Svendsen et al., 1997a; Quinn et al.,
tissues and the guidelines set out by the Department of
1997). Furthermore, we have also shown that rat and
Health in the United Kingdom.
mouse neurospheres, grown using identical methods,
have very different long term expansion potentials with
2.2. Cell culture
the rat cells entering senescence within 3 – 4 weeks of
expansion (Svendsen et al., 1997b). Thus, there may be
Tissues collected locally (see Table 1 for details) were
a signiﬁcant species difference when developing meth-
dissected in chilled sterile phosphate buffered saline
ods for the growth and differentiation of these cells.
(PBS, pH 7.4) with 0.6% glucose. Identiﬁed pieces were
Clearly, if neural precursor cells are to become a source
incubated in 0.1% trypsin (Worthington) with 0.04%
of tissue for basic neuroscience and clinical pro-
DNAase (Sigma type II) for 20 min at 37°C. Following
grammes it would be a major advantage if they could
be expanded for long periods of time. three washes in 0.04% DNAase the tissue was triturated
C.N. S6endsen et al. / Journal of Neuroscience Methods 85 (1998) 141–152 143
in the same solution using a ﬁne polished Pasteur above. Following thawing (Clon-3582) or from primary
pipette. Cell counts showed greater than 65% viable (BRC-44), the cells were grown as spheres for 35 days
cells in all cases. Cells were seeded at a concentration of (Clon-3582) or 43 days (BRC-44) in EGF and FGF-2
200000 per ml into substrate free tissue culture ﬂasks. during which time they showed approximately a 5-fold
The growth medium consisted of DMEM/HAMS F12 increase in cell number (see Table 1). Passaging of these
(3:1), penicillin G, streptomycin sulphate, amphotericin cultures consisted of a gentle trituration with a ﬁne
B (1:100; Gibco), B27 (1:50; Gibco), human recombi- polished Pasteur pipette every 14 days in order to break
nant FGF-2 and EGF (both at 20 ng/ml; R&D Sys- up the growing spheres. At day 36 or 44, single spheres
tems) and heparin (5 vg/ml; Sigma). Passaging was were measured (using a lens grid under a dissecting
carried out at the time points shown in Table 1 and microscope). Those which were 0.5 mm or greater in
consisted of a gentle mechanical dissociation using a radius were sectioned into quarters (using two c23
ﬁne polished Pasteur pipette, after which the mixture of Swann-Moston surgical blades without handles in a
intact spheres and single cells were re-seeded into fresh Petri dish with 10 ml of growth medium) and then
medium, as above but with N2 (1:100; Gibco) replacing transferred to a single un-coated well of a 24-well plate
B27, at 200000 cells per ml. This switch from N2 to with 0.5 ml of FGF-2 and heparin supplemented
B27 was due to the fact that although B27 is vital for growth medium. After the ﬁrst sectioning it was impor-
maximum growth of neural precursors from primary tant to wait until each sphere quarter had grown to at
cultures, it is no better than the less expensive supple- least 0.35 mm in radius again before re-sectioning (be-
ment, N2, once neurosphere cultures are established as tween 14 and 21 days growth). All subsequent sections
we have reported previously for rat cultures (Svendsen were performed every 14 days regardless of sphere size
et al., 1995). Therefore in all neurosphere cultures to establish average growth rates over time. Spheres too
described in this study, other than primary cultures, N2 small to section at the end of 14 days were discarded
was used as the medium supplement. At 28 – 35 days in and accounted for in the results (see Table 2). Bulk
vitro all cultures were switched to FGF-2 alone as we cultures were grown at a density of 50 spheres per T75
found no synergistic effect of combining EGF and ﬂask in 20 ml of growth medium and quartered every
FGF-2 on growth after this period as we have reported 14 days. 24 h following sectioning the quarters would
previously for rat cultures (Svendsen et al., 1997b). To occasionally attach to the surface of the well or ﬂask
estimate total cell number per ﬂask, a 1-ml aliquot of but could, in most cases, be shaken off gently at this
spheres (taken from a 20-ml ﬂask of cells which was time. All cultures were fed by replacing 50% of the
shaken to randomly distribute the spheres) was re- medium every 4–5 days.
moved prior to passaging and a single cell suspension
2.4. Automated tissue chopping
achieved using trypsin digestion followed by mechanical
dissociation. Live cells were then counted in a haemocy-
Spheres at the end of a growth cycle ( 0.35 mm
tometer using trypan blue exclusion to exclude dead
cells. radius) were transferred to the lid of a 16-mm Petri dish
Tissue collected via the US Uniform Anatomical Gift and the majority of medium removed using a Pasteur
Act (Clon-5382) consisted of a 21 week post conception pipette. The lid with the spheres was attached to the
fetus. Cortical cells were isolated and passed through a stage of the McIlwain tissue chopper (Mickle Engineer-
190-mesh cell strainer before running through a 30% ing, Gomshall, Surrey, UK) using thin strips of adhe-
percoll column for 20 min. Cells were seeded into sive putty (Blue Tack or equivalent). A sterile razor
growth medium (described above) and grown as blade was inserted into the arm of the tissue chopper.
spheres in EGF and FGF-2 for 7 days before cryopre- Sections were then automatically taken through the
spheres using a distance between chops of 350 vm. The
serving in liquid nitrogen using DMEM with 20% fetal
calf serum with 10% DMSO. Frozen cells were rapidly stage was rotated through 90° and the process repeated
warmed to 37°C, washed three times in DMEM and to generate ‘cubes’ of tissue with a mean width of
approximately 350 vm. These were then carefully
then re-seeded into fresh growth medium. This freezing
process could be used successfully at any stage of washed in fresh growth medium and re-seeded at ap-
sphere growth. proximately 200 spheres per T75 ﬂask containing 20 ml
of growth medium.
2.3. The sectioning method and systematic assessment
2.5. Thymidine incorporation
of growth rates
[3H]Thymidine (Amersham; 0.5 vCi/ml) was added
Two cultures were used to assess exact growth rates
using the sectioning method. BRC-44 was generated to individual spheres for a period of 24 h. At the end of
from an 8-week post conception fetus but was not the incubation period the spheres were washed three
times in DMEM and incorporated [3H]thymidine was
cryopreserved at any stage and Clon-3582 is described
C.N. S6endsen et al. / Journal of Neuroscience Methods 85 (1998) 141–152
Cumulative growth data for human neural stem cell cultures
14 28 42 56 70 84 98 112
0.449 0.02 0.439 0.02 0.37 90.03 0.4290.03 0.39 90.03 0.32 90.03 0.32 90.03
Radius of mother disc NA
0.349 0.01 0.279 0.03 0.34 90.02 0.31 90.03 0.28 9 0.03 0.339 0.02 0.37 9 0.04
Radius of daughter NA
% of mother disc 77 63 92 74 72 103 116
Number of surviving NA 19/24 20/24 22/24 22/24 22/24 22/24 19/24
% of total 79 83 91 92 92 92 79
2.95 9 0.24
Expansion ratio NA 2.43 2.09 3.35 2.72 2.63 3.79 3.67
0.31 9 0.02 0.389 0.03 0.379 0.04 0.29 90.03 0.4690.05 0.49 90.04 0.4690.03 0.5290.01
Radius of mother disc
0.349 0.03 0.309 0.04 0.319 0.03 0.33 90.02 0.32 9 0.08 0.42 90.04 0.42 90.04 0.51 90.02
Radius of daughter
% of mother disc 111 84 84 116 71 86 104 97
Number of surviving 23/24 18/24 19/24 19/24 17/24 23/24 20/24 24/24
% of total 96 75 79 79 71 95 83 100
3.19 9 0.28
Expansion ratio 4.25 2.37 2.66 3.67 2.01 3.27 3.45 3.88
Mother disc radius from six individual discs was measured (mm) before sectioning into quarters. Each quarter was then measured again at the
end of the growth period. If all four quarters had reached the size of the mother disc this would represent a 4-fold increase in cell number. Thus
the ﬁnal expansion ratio = expected number of discs (4)×% of mother disc size×% of total discs surviving. NA, data not available.
Over a 14 day period. Not signiﬁcantly different between the cultures (p0.05; Student’s t-test).
then solubilised using 600 vl NaOH (0.4 M) for 1 h at Sigma) combined with GFAP (polyclonal; Bohringer;
37°C. This solution was then added to 4 ml of scintilla- 1:1000) in 0.1 M PBS/0.1% Triton/3% goat serum.
tion cocktail and counted in a scintillation Others were incubated with antibodies to nestin (poly-
spectrometer. clonal; 1:50; kindly donated by R.D.G. McKay); GAL-
C (monoclonal; kindly donated by B. Ranscht; 1:4; no
2.6. Karyotyping Triton was used with this surface marker) or MAP-2ab
(1:500; Sigma). Goat anti-mouse biotin or ﬂuoroscein
Chromosome number and size was scored using conjugated goat anti rabbit antibodies were used to
Giemsa-stained metaphase spreads by the Department label the primaries, followed by a streptavadin–rho-
of Cytogenetics, Addenbrooke’s Hospital, Cambridge. damine conjugate Hoescht was added to the ﬁnal incu-
bation step (Sigma, 1:10000 in 0.1 M PBS) to visualise
2.7. Immunocytochemistry nuclei. For cell counts at least ﬁve random ﬁelds (at
× 40) were analysed from the monolayer of cells
Whole free ﬂoating spheres were ﬁxed in 4% around the plated spheres. Each ﬁeld contained be-
paraformaldehyde for 20 min, washed in PBS and tween 50 and 100 cells.
dehydrated though 70, 95 and 100% alcohol (20 min Sections were also viewed under a Biorad confocal
each). Following clearing overnight in xylene, spheres scanning microscope at excitation wavelengths of 488
and 564 nm. Optical sections were taken at 20 vm
were embedded in parafﬁn and sectioned on a micro-
tome at 5 vm. For differentiation studies, whole intervals and then merged.
spheres were allowed to attach to a poly-L-lysine
(Sigma) coated coverslips in 24-well plates in the pres-
ence of 0.5 ml of DMEM/B27 with 1% serum for 24 h. 3. Results
Following this period the medium was exchanged for
3.1. Expansion of human precursors
DMEM/B27 alone. Cultures were fed by replacing 50%
of the medium every 4– 5 days. At 14 days, the cultures
were ﬁxed for 30 min in 4% paraformaldehyde. Wax Following seeding into growth medium, aggregates
sections and coverslips were incubated with primary of dividing cells formed into spheres which grew in size
antibodies to beta tubulin III (TuJl; monoclonal; 1:500; over time in response to the mitogens EGF and FGF-2.
C.N. S6endsen et al. / Journal of Neuroscience Methods 85 (1998) 141–152 145
Fig. 1. Cells isolated from the developing cortex and grown in EGF and FGF-2 formed mainly spheres at early passages (A1) but some discs could
also be seen at later passages when grown in FGF-2, possibly as a result of transient attachment to the culture dish (A2). (B), (C), (D) and (E)
show four quarters from a single sphere (A1) 1 h after sectioning. (F) Cumulative growth curves for Clon-5382 and BRC-44 based on expansion
data in Table 2 and subsequent results not shown in Table 2. Note the steady and consistent exponential increase in total sphere number over
time. Scale bar = 0.2 mm.
Between 14 and 21 days of growth the spheres could be showed only modest expansion over this 14 day period
gently dissociated to a mixed suspension of single cells as shown in Table 1. By 40 days of growth using the
and sphere remnants before re-plating into growth sectioning method, some disc shaped clusters could be
medium. Using this technique we have previously re- seen in addition to spheres (Fig. 1A1 and A2). The
ported a 3-4-fold increase in cell number over the ﬁrst discs mainly developed due to temporary attachment of
few weeks in vitro, after which the absolute number of the newly sectioned spheres to the surface of the ﬂask,
cells harvested at each passage declined (Svendsen et and were often concave on one surface. This led to
al., 1997b). In this study, seven separate experiments some disc/sphere aggregates which appeared hollow
using either brain stem, spinal cord or cortical human when sectioned (approximately 30% at 100 days of
fetal tissue showed similar results, with variable growth growth, data not shown). When individual spheres at
for the ﬁrst 4–6 weeks, after which their was very little 100 days growth were dissociated and the number of
further growth with the maximum expansion of total cells counted, those with a size of between 0.35 and 0.45
cells only reaching 12-fold (Table 1). A variety of mm diameter contained an average of 61166+ 3498
modiﬁcations to the culture medium, growth factor viable cells (n= 25 spheres from CTX-44) with less than
combinations and passaging strategies have been at- 5% non viable cells. Very similar results with regard to
tempted. None of these had any effect on the slow cell number and sphere size were found at 50 and 150
growth rate and eventual senescence of these precur- days of growth (data not shown). There was a positive
sors, although some cultures could be kept in a mitotic, correlation between sphere size and number of cells
but non expanding, state (due to concomitant cell (Fig. 2A), although some variation existed due to the
death) for up to 6 months (data not shown). technical difﬁculties of dissociating small single spheres
A novel, non traumatic, passaging approach was (cells attaching to the side of the pipette, incomplete
employed on several cultures. Data for two of these dissociation). We next assessed whether the size of the
isolated from the cortex, which differed in both fetal sphere sections inﬂuenced their ability to grow back to
age and method of cell isolation, are described in detail the size of the mother sphere. Regardless of the mother
here. Instead of mechanical dissociation, individual sphere size, all quarters grew back to the same size in
spheres were sectioned into quarters under a dissecting relation to the mother showing that the expansion is
microscope using a scalpel blade (Fig. 1). The resulting not dependent on either large or small sphere sizes (Fig.
2B). [3H]Thymidine added to the spheres over the last
four quarters were placed into fresh growth medium
containing FGF-2 and over the next 24 h rounded to 24 h of growth at all stages in culture showed that there
form new spheres which grew close to the size of the was a signiﬁcant amount of uptake ( 5000 counts per
mother sphere by 14 days. Using this method there was min/per sphere at each passage) indicating that active
a steady and exponential increase in total sphere num- cell division was occurring. The total amount of expan-
ber which did not decrease with time (Fig. 1; Table 2). sion achieved over the growth period (including a 5-
Parallel cultures which were also switched to FGF-2 fold increase prior to the start of the sectioning
alone but were dissociated rather than chopped, method) was greater than 1.5 million-fold for Clon-
C.N. S6endsen et al. / Journal of Neuroscience Methods 85 (1998) 141–152
Fig. 2. Graphs showing (A) correlation between volume of sphere and number of cells per sphere. (B) Lack of any relationship between size of
sectioned quarter and its subsequent growth relative to its own mother sphere.
migrating out. Between 2 and 7 days, radiating pro-
5382 and both cultures showed a population doubling
cesses often developed which sketched from the edge of
time of approximately 4 days. Metaphase spreads
the sphere onto the substrate (Fig. 3C). Along these
showed that at 150 days of growth the human cells
radiating strands, immature TuJ1 + neurons could be
remained karyotypically normal with regard to chro-
seen which often trailed processes in parallel lines and
mosome number and appearance. Removal of FGF-2
may have been migrating out from the core of the
from the growth medium at any stage resulted in
sphere (Fig. 3D). Using confocal imaging the exact
senescence and eventual death of cultures over a period
anatomy of the sphere following 14 days of differentia-
of 14 days. However, switching FGF-2 responsive
tion could be seen and radial processes emanating from
spheres to a medium containing EGF resulted in the
the core were found to be GFAP positive (Fig. 4).
growth of an EGF responsive spheres with very similar
Migrating cells eventually formed a monolayer
characteristics following plating and which also ex-
around the plated sphere and labelled for either TuJ1
panded exponentially using this sectioning method.
or GFAP but never with both markers at 14 days of
However, the EGF responsive spheres were less prone
differentiation (Fig. 5A, B and C). Detailed cell counts
to attaching to the culture dish and subsequently
within the monolayer around the sphere revealed that
formed less discs. We are also systematically assessing
the majority of the cells were either TuJ1 or GFAP
the effects of sectioning on the exact growth rates of
positive at both early and late passages and that no
neural precursor cells isolated from other brain regions
galactocerbroside (GAL-C) positive oligodendrocytes
mentioned in Table 1, although bulk cultures spinal
could be seen under these plating conditions (Fig. 5D).
cord and brain stem do expand rapidly using this
Although not analysed in detail, every whole sphere
method (data not shown).
plated (over 300) gave rise to both neurons and astro-
cytes, strongly suggesting the presence of a common
3.2. Differentiation of human neural precursors
precursor. At the very periphery of the migrating cells,
lone GFAP+ astrocytes could often be found, onto
Continual growth of the human neural spheres sug-
which a number neurons had selectively migrated, the
gested self renewal was occurring, but did not deter-
processes from which were entirely conﬁned to the
mine the phenotypic potential of these cells. To assess
astrocyte surface (Fig. 6A–C). This suggests that these
this, whole spheres or differentiating spheres (at 100
precursor cell derived astrocytes provide an attractive
days growth) were processed for indirect immunocyto-
surface for the migrating neurons. Some cells also
chemistry. The majority of cells (95%) within the
expressed markers only found in mature neurons such
growing spheres were found to be positive for nestin,
as microtuble associated protein 2ab which is located
(Fig. 3B) a marker for undifferentiated neuroepithelial
mainly in dendrites (MAP-2ab; Fig. 6D) (Riederer and
stem cells (Lendahl et al., 1990), but did not stain for
Matus, 1985). Interestingly, following trituration to a
TuJ1 (a speciﬁc early neuronal marker) (Menezes and
single cell suspension and plating, very few TuJ1 +
Luskin, 1997) or glial acidic ﬁbriallary protein (GFAP;
neurons were found while the majority of cells stained
an astrocyte marker). When whole spheres were ex-
for GFAP (data not shown), indicating that either the
posed to a substrate, they rapidly attached and within
temporal sequence of events following plating of whole
hours cells with a glial morphology could be seen
spheres, or the lack of physical trauma caused by
trituration, may be required for neuronal survival and
C.N. S6endsen et al. / Journal of Neuroscience Methods 85 (1998) 141–152 147
Fig. 3. Staining whole growing spheres at 100 days growth for Hoescht revealed that viable cell nuclei could be seen throughout in most cases
(A). Scale bar: 100 vm. Immunocytochemical staining showed that the majority of cells within the growing spheres were nestin + and
undifferentiated (B). Scale bar: 25 vm. Some spheres were plated onto poly-L-lysine coated coverslips in DMEM/B27 and 1% serum for 24 h to
induce differentiation. Phase (C) and TuJ1 (D) staining of the same ﬁeld from a sphere 4 days following plating. Note the radial ﬁbre outgrowths
along which TuJl + cells with extensive ﬁbres could be seen. Arrow heads represent glial ﬁbres with no TuJl + ﬁbres, arrows represent TuJl + ﬁbres
along processes, double arrowhead shows single neuronal cell body with extended TuJl + processes. Scale bar: 25 vm.
differentiation of cells arising from long term human 4. Discussion
neural precursor cell cultures.
The present study has demonstrated a new method
3.3. Automation of the sectioning technique for the long-term exponential expansion of non immor-
talised or transformed human neural precursor cells,
Through the detailed assessment of manually section- which maintained the capacity to generate a high per-
ing individual spheres we have shown that sustained centage of neurons (see Fig. 7 for a schematic of the
exponential growth of human neural precursor cells can technique).
be achieved (Table 2). However, it is obviously not There are two main methods in the literature com-
practical to section large numbers of spheres using this monly used to generate populations of neural precursor
manual method. We therefore developed an automated cells. The ﬁrst uses FGF-2 and a substrate to expand
procedure using the McIlwain tissue chopper originally colonies of cells which grow attached to the culture
designed to slice fresh brain tissue. Using this device up ﬂask while the second uses EGF to expand aggregates
to 1000 spheres can be automatically sectioned within a of cells (neurospheres) although it is now clear that
few minutes. Each section rapidly rounds up after these growth factors are often interchangeable in their
seeding into fresh medium and forms a new growing effects (for recent review, see Svendsen (1997)). We
sphere as described previously using the manual tech- have not attempted to grow human cells attached to a
nique, although obviously the spheres differ in size substrate in the presence of FGF-2 in this study, but
depending on exactly where they were sectioned. As have focused instead on the neurosphere culture
sphere size is not a critical determinant of cell expan- method. The advantage of the aggregate culture
sion (see Fig. 2) we feel that this method should provide method is that large numbers of cells can be expanded
an automated technique for growing large numbers of in a small volume of medium. There are no published
human neural precursor cells. reports on the long term growth of human neural
C.N. S6endsen et al. / Journal of Neuroscience Methods 85 (1998) 141–152
Fig. 4. Confocal image (Biorad Inc, UK) of a sphere plated for 14 days showing both TuJ1 positive neurons (red) and GFAP positive astrocytes
(green) which had migrated from the core of a sphere (left) and formed a monolayer culture. Note the many radial ﬁbres which stain for GFAP
(arrowheads). Scale bar: 100 vm. Insert is a high power view of a plated region around the sphere ( × 4 magniﬁcation compared to rest of plate)
showing how neuronal processes pass both under and over the astrocytes.
precursor cells, although preliminary data suggests that published observations). Furthermore, we have found
EGF can drive a human precursor for extended periods the expansion of human neurospheres to be very slow,
of time (Vescovi et al., 1997). This cell may be similar and although division continued (based on incorpora-
to the EGF responsive cell isolated from the developing tion of mitotic labels) real expansion stopped after 35
mouse striatum which can grown for long periods of days of growth (Svendsen et al., 1997a). Using the
time in vitro as spheroid neurospheres and may repre- sectioning method presented here, human neurospheres
sent a population of stem cells (Reynolds et al., 1992; continue to expand and give rise to high numbers of
Reynolds and Weiss, 1996) although even as early as neurons even at late passages. We are currently identi-
passage two (using conventional passaging methods) fying the phenotype of neurons generated from these
these mouse neurospheres spontaneously gave rise to long term human cultures.
virtually no neurons but rather glial cells (Arsenijevic What is the exact nature of the dividing cells within
and Weiss, 1998). We have previously found that in the sectioned spheres?. Their growth rate was remark-
contrast to mouse neurospheres, rat neurospheres enter ably stable and relatively slow, with a cell cycle time of
a programmed senescence between 28 and 35 days of approximately 4 days throughout the culture period.
growth using routine passaging methods (Svendsen et They were karyotypically normal on gross inspection
al., 1997b) and also give rise to high numbers of which suggests they had not transformed but are main-
astrocytes at later passages (Rosser and Svendsen, un- taining a normal cellular phenotype with a slow cell
C.N. S6endsen et al. / Journal of Neuroscience Methods 85 (1998) 141–152 149
Fig. 5. Photomicrograph showing phase (A), TuJ1 (B) and GFAP (C) staining of an identical ﬁeld of cells around the periphery of a sphere plated
for 14 days. Many TuJI + and GFAP + cells could be found but no cells labelled with both markers. Arrows show neurons and arrowheads show
astrocytes. Graph shows the numbers of labelled cells around the sphere as a percentage of total cells (n =12 spheres at each time) at either 50
or 150 days of growth (D). There was no signiﬁcant difference between the numbers of neurons and astrocytes generated at early or late passages.
cycle time consistent with a slowly dividing stem cell passages, but only neurons and astrocytes at late pas-
population. Clonal analysis has classically been re- sages using the plating conditions in this study. To
quired to prove pluri-potency, but is difﬁcult to per- investigate this further we are currently assessing the
form with this culture system since we have shown that effects of other growth factors and substrates on the
groups of cells must maintain contact in order to grow differentiation of these long term human precursor
for long periods. However, the observation that every cells, which have previously been shown to inﬂuence the
sphere we have plated produced both neurons and fate rat hippocampal precursors and immortalised hu-
astrocytes, but never only one phenotype, argues man neural precursors (Joh et al., 1996; Sah et al.,
strongly in favour of a common self renewing stem cell. 1997). It was of interest that the cultures generated
It also remains possible that two uni-potent stem cells from widely differing fetal ages (8 and 21 weeks) gave
with similar division rates and the capacity to produce rise to similar numbers of neurons and astrocytes at
either neurons or astrocytes are dividing alongside each late passages. This would further suggest that a com-
other. Oligodendrocytes were never seen to arise from mon cell is being driven in these cultures following an
the late passage sectioned spheres, but have been seen initial period of instability where progenitors with a
to develop from early passage spheres (Murray and more limited mitotic life span are ﬁltered out. In sum-
Dubois-Dalcq, 1997; Svendsen et al., unpublished ob- mary, the cell which is dividing in these cultures is
servations). This suggests that either, (i) an oligoden- maybe best described as a precursor cell until we know
drocyte precursor may be capable of dividing for a more about its exact phenotypic potential under a
certain period of time and then be lost from the cul- variety of circumstances.
tures during the extended period of growth which then There is currently some confusion regarding the dif-
only consists of cells capable of giving rise to neurons ferential effects of EGF and FGF-2 on neural precursor
and astrocytes or, (ii) a common precursor is able to cells. Based on clonal analysis, EGF was only found to
make oligodendrocytes, neurons and astrocytes at early stimulate a glial progenitor from the mouse cortex late
C.N. S6endsen et al. / Journal of Neuroscience Methods 85 (1998) 141–152
Fig. 6. Photomicrographs showing cells which had migrated out from the sphere and established colonies at the limit of the migration wave.
Isolated epitheloid glial cells could be seen with many smaller cells on top which conformed exactly to the shape of the underlying cell suggesting
a strong attraction of neurons to the developing astrocytes (A). The smaller cells were TuJI + neurons (B) and the epitheloid cells were GFAP +
astrocytes (C) (C is the same ﬁeld as A and B). MAP-2ab was found to label the dentritic processes of more mature neurons arising from the
spheres (D). Scale bar: 15 vm.
in development and had no effect on earlier cortical increase the mitotic effects of FGF-2 on embryonic
precursors, in contrast to FGF-2 which was able to precursor cells (Caldwell and Svendsen, 1998) and it
induce the division of a multipotent cell at both early was used throughout the current study. Interestingly,
and late developmental ages using the same model we found that changing the growing human precursors
(Kilpatrik and Bartlett, 1995). Furthermore, EGF ap- from FGF-2 to EGF had no obvious effect on the
pears to stimulate glial division in adult subventricular ability to generate neurons following plating, although
zones and may in fact repress neuronal development in this is the subject of a more detailed comparison cur-
vivo (Kuhn et al., 1997). We have recently shown that rently in preparation. Clearly there is much more exper-
following priming with FGF-2, the same cell responds imental work required to resolve these issues.
to both EGF and FGF-2 in primary E14 mouse striatal The reason why the sectioning method is so effective
tissue (Ciccolini and Svendsen, 1998). Perhaps in early in maintaining stable and rapid growth may be in part
and late adulthood there are more restricted precursors due to the fact that there is no disruption of cell–cell
which respond separately to these factors, whereas dur- contact within the intact regions of the spheres in
ing development a common precursor exists. However, contrast to standard process of mechanical dissociation.
the mixture of in vivo and in vitro data across different Membrane associated factors are known to be impor-
species and culture conditions makes it impossible to tant for the division of neural precursor cells (Temple
draw conclusions at present. It is of interest that the and Davis, 1994) and a ‘niche’ hypothesis has been
adult mouse subventricular zone has recently been proposed which suggests stem cells will only retain their
shown to contain FGF-2 responsive cells (Gritti et al., pluripotency within an appropriate environment
1996) which appear almost identical to the EGF re- (Schoﬁeld, 1978), both of which may be sustained using
sponsive cells described originally by Reynolds and this sectioning method. Equally important may be the
Weiss (1992) who claimed that FGF-2 was unable to reduction in cellular trauma that results from sectioning
stimulate division of the same cells. This discrepancy rather than dissociating intact spheres. It is clear that
may be due to the fact that heparin was added to the partial dissociation may also lead to intact remnants
medium with FGF-2 in the later study. We have re- remaining which have cell–cell contact. Indeed, our
cently shown that this proteoglycan can signiﬁcantly normal passaging methods often result in non-complete
C.N. S6endsen et al. / Journal of Neuroscience Methods 85 (1998) 141–152 151
Fig. 7. Schematic summarising the overall method. Conventional passaging techniques resulted in slow growth or senescence. By sectioning
growing spheres there was less trauma to the cells and the quarters grew close to the size of the mother over a period of 14 days. This pattern
continued for extensive periods of time in culture and allowed the exponential growth of human neural precursor cells.
dissociation. The major disadvantage of this is that provide a valuable source of normal human neural
tissue for both testing novel neuroactive compounds in
spheres are generally stripped of cells leaving only the
vitro and clinical neural transplantation programmes
cores. Many of the stripped cells die but the cores may
which are currently dependent on the collection of fresh
continue to grow. However, this does not result in such
human fetal tissues (Bjorklund, 1993; Svendsen, 1997).
a rapid growth rate as simply sectioning the spheres
and is far more inconsistent as it is difﬁcult to control
exactly how much dissociation is performed in one
culture to the next.
It is tempting to speculate that the stages of sphere
We thank S.B. Dunnett for his continual support,
attachment, formation of radial processes and apparent
Biorad Inc. for preparation of the confocal images and
neuronal migration may recapitulate the normal pro-
Ziggy Zhang and Irena Sarel of Blowhittiker Inc. for
cess of primate development where neuronal precursors
providing human neurospheres. The authors would also
divide within ventricular zones before migrating to the
like to thank Dr Scott Whittemore for critically ap-
pial surface along radial glia (Rakic, 1985). However,
praising an early version of this manuscript. This re-
this needs to be substantiated with further studies
search was funded by a Wellcome Fellowship to CNS
which are the focus of ongoing work. Genetic manipu-
and by the MRC.
lations to these dividing cells should be possible, as
achieved previously with both human and rat neural
precursors (Sabate et al., 1995; Svendsen et al., 1996),
to allow the expression of speciﬁc proteins following
grafting, thereby facilitating ex vivo CNS gene therapy
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