Keywords: Stem cell; Progenitor cell; Proliferation; Differentiation; Expansion; Human
1. Introduction
There is at present no source of non-transformedhuman neurons other than primary foetal tissue. Thishas posed major limitations to both research and indus-try with regard to studying the basic biology and drugresponsiveness of human neurons, and has limited clin-ical neural transplantation programmes to very smallnumbers of patients. The only strategy currently avail-able for obtaining large amounts of well characterisedhuman neurons is the use of cell lines. There have beentwo such human lines described previously, one derivedfrom a tetratocarcinoma and the other oncogenically
transformed (Pleasure and Lee, 1993; Sah et al., 1997).Although of great interest, transformed cell lines maynot share the exact features of primary human neuraltissue, and their oncogenic status makes them lessattractive as a source of tissue for clinical transplanta-tion. An alternative approach to producing largeamounts of neural tissue is to isolate and expand neuralprecursor cells from the CNS.
The ideal human precursor cell to expand would be aneural stem cell. A stem cell can be most simply definedas any cell which is capable of self renewal for extendedperiods of time, the progeny from which are capable offorming the components of a defined tissue. Asymmet-ric division allows stem cells to generate a progenitorcell, in addition to another stem cell, which have alimited potential for self renewal and often sponta-
Numbers represent expansion ratios: i.e. number of cells at end of passage over number of cells seeded into flasks. Last number indicates end ofgrowth 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.a Represents CLON-5382.b Represents BRC-44.
neously stop dividing and differentiate. Stem cells havebeen most extensively studied in haemopoetic, epider-mal and intestinal tissues which require frequent cellreplacement throughout life (Hall and Watt, 1989).Recent studies have shown that specific regions of boththe developing and adult rodent brain harbour cellswhich divide in response to mitogens, while retainingthe capacity to differentiate into neurons and glia, andas such may represent neural stem cells (Weiss et al.,1996; McKay, 1997; Palmer et al., 1997), although stemcell status is often debated and the term neural precur-sor may better describe cells within these heterogeneouscultures. Neural precursor cells from the rodent re-spond to both epidermal growth factor (EGF) andfibroblast growth factor (FGF-2) (for reviews see Gageet al., 1995; McKay, 1997) and can be grown as eithermonolayer cultures or as free floating spherical aggre-gates termed ‘neurospheres’ (Reynolds et al., 1992). Theshort term growth (B60 days) of similar human CNSprecursors has recently been reported (Buc-Caron,1995; Svendsen et al., 1996; Chalmers-Redman et al.,1997; Murray and Dubois-Dalcq, 1997) and in somecases these can survive, migrate, differentiate and re-store function following transplantation into rat modelsof Parkinson’s disease (Svendsen et al., 1997a). How-ever, we and others have also shown that human neuro-spheres are difficult to expand in vitro over longperiods of time (Svendsen et al., 1997a; Quinn et al.,1997). Furthermore, we have also shown that rat andmouse neurospheres, grown using identical methods,have very different long term expansion potentials withthe rat cells entering senescence within 3–4 weeks ofexpansion (Svendsen et al., 1997b). Thus, there may bea significant species difference when developing meth-ods for the growth and differentiation of these cells.Clearly, if neural precursor cells are to become a sourceof tissue for basic neuroscience and clinical pro-grammes it would be a major advantage if they couldbe expanded for long periods of time.
When attached cells or free floating aggregates reachthe end of a growth cycle, they must be mechanicallybroken up or ‘passaged’, often using digestion enzymes,to avoid contact mediated growth arrest or lack ofnutrient diffusion. We postulated that these standardpassaging techniques may lead to cellular trauma, stripreceptors, deprive cells of contact mediated factors andremove vital tight junctions known to hold tissuestogether. This may lead to either the terminal differenti-ation of precursor cells, or a lack of response to mito-gens for the rat and human cells. We thereforeattempted to adapt the passaging technique such thatenzymatic or mechanical disturbance to the cells wasminimised and then assess the ability of the humanneural precursor cells to continuously renew over time.
2. Methods
2.1. Tissue collection
Human fetal tissue (between 7 and 21 weeks postconception) was collected from two different sources:via the Uniform Anatomical Gift Act of the UnitedStates or from a local hospital. The methods of collec-tion conform with the arrangements recommended bythe Polkinghorne Committee for the collection of suchtissues and the guidelines set out by the Department ofHealth in the United Kingdom.
2.2. Cell culture
Tissues collected locally (see Table 1 for details) weredissected in chilled sterile phosphate buffered saline(PBS, pH 7.4) with 0.6% glucose. Identified pieces wereincubated in 0.1% trypsin (Worthington) with 0.04%DNAase (Sigma type II) for 20 min at 37°C. Followingthree washes in 0.04% DNAase the tissue was triturated
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in the same solution using a fine polished Pasteurpipette. Cell counts showed greater than 65% viablecells in all cases. Cells were seeded at a concentration of200000 per ml into substrate free tissue culture flasks.The growth medium consisted of DMEM/HAMS F12(3:1), penicillin G, streptomycin sulphate, amphotericinB (1:100; Gibco), B27 (1:50; Gibco), human recombi-nant FGF-2 and EGF (both at 20 ng/ml; R&D Sys-tems) and heparin (5 mg/ml; Sigma). Passaging wascarried out at the time points shown in Table 1 andconsisted of a gentle mechanical dissociation using afine polished Pasteur pipette, after which the mixture ofintact spheres and single cells were re-seeded into freshmedium, as above but with N2 (1:100; Gibco) replacingB27, at 200000 cells per ml. This switch from N2 toB27 was due to the fact that although B27 is vital formaximum growth of neural precursors from primarycultures, it is no better than the less expensive supple-ment, N2, once neurosphere cultures are established aswe have reported previously for rat cultures (Svendsenet al., 1995). Therefore in all neurosphere culturesdescribed in this study, other than primary cultures, N2was used as the medium supplement. At 28–35 days invitro all cultures were switched to FGF-2 alone as wefound no synergistic effect of combining EGF andFGF-2 on growth after this period as we have reportedpreviously for rat cultures (Svendsen et al., 1997b). Toestimate total cell number per flask, a 1-ml aliquot ofspheres (taken from a 20-ml flask of cells which wasshaken to randomly distribute the spheres) was re-moved prior to passaging and a single cell suspensionachieved using trypsin digestion followed by mechanicaldissociation. Live cells were then counted in a haemocy-tometer using trypan blue exclusion to exclude deadcells.
Tissue collected via the US Uniform Anatomical GiftAct (Clon-5382) consisted of a 21 week post conceptionfetus. Cortical cells were isolated and passed through a190-mesh cell strainer before running through a 30%percoll column for 20 min. Cells were seeded intogrowth medium (described above) and grown asspheres in EGF and FGF-2 for 7 days before cryopre-serving in liquid nitrogen using DMEM with 20% fetalcalf serum with 10% DMSO. Frozen cells were rapidlywarmed to 37°C, washed three times in DMEM andthen re-seeded into fresh growth medium. This freezingprocess could be used successfully at any stage ofsphere growth.
2.3. The sectioning method and systematic assessmentof growth rates
Two cultures were used to assess exact growth ratesusing the sectioning method. BRC-44 was generatedfrom an 8-week post conception fetus but was notcryopreserved at any stage and Clon-3582 is described
above. Following thawing (Clon-3582) or from primary(BRC-44), the cells were grown as spheres for 35 days(Clon-3582) or 43 days (BRC-44) in EGF and FGF-2during which time they showed approximately a 5-foldincrease in cell number (see Table 1). Passaging of thesecultures consisted of a gentle trituration with a finepolished Pasteur pipette every 14 days in order to breakup the growing spheres. At day 36 or 44, single sphereswere measured (using a lens grid under a dissectingmicroscope). Those which were 0.5 mm or greater inradius were sectioned into quarters (using two c23Swann-Moston surgical blades without handles in aPetri dish with 10 ml of growth medium) and thentransferred to a single un-coated well of a 24-well platewith 0.5 ml of FGF-2 and heparin supplementedgrowth medium. After the first sectioning it was impor-tant to wait until each sphere quarter had grown to atleast 0.35 mm in radius again before re-sectioning (be-tween 14 and 21 days growth). All subsequent sectionswere performed every 14 days regardless of sphere sizeto establish average growth rates over time. Spheres toosmall to section at the end of 14 days were discardedand accounted for in the results (see Table 2). Bulkcultures were grown at a density of 50 spheres per T75flask in 20 ml of growth medium and quartered every14 days. 24 h following sectioning the quarters wouldoccasionally attach to the surface of the well or flaskbut could, in most cases, be shaken off gently at thistime. All cultures were fed by replacing 50% of themedium every 4–5 days.
2.4. Automated tissue chopping
Spheres at the end of a growth cycle (\0.35 mmradius) were transferred to the lid of a 16-mm Petri dishand the majority of medium removed using a Pasteurpipette. The lid with the spheres was attached to thestage of the McIlwain tissue chopper (Mickle Engineer-ing, Gomshall, Surrey, UK) using thin strips of adhe-sive putty (Blue Tack or equivalent). A sterile razorblade was inserted into the arm of the tissue chopper.Sections were then automatically taken through thespheres using a distance between chops of 350 mm. Thestage was rotated through 90° and the process repeatedto generate ‘cubes’ of tissue with a mean width ofapproximately 350 mm. These were then carefullywashed in fresh growth medium and re-seeded at ap-proximately 200 spheres per T75 flask containing 20 mlof growth medium.
2.5. Thymidine incorporation
[3H]Thymidine (Amersham; 0.5 mCi/ml) was addedto individual spheres for a period of 24 h. At the end ofthe incubation period the spheres were washed threetimes in DMEM and incorporated [3H]thymidine was
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Table 2Cumulative growth data for human neural stem cell cultures
Days Averageexpansiona
84 98 11214 28 42 56 70
BRC-440.3290.030.3290.03Radius of mother disc NA 0.3990.030.4490.02 0.4390.02 0.3790.03 0.4290.03
0.3190.03 0.2890.03 0.3390.02Radius of daughter 0.3790.04NA 0.3490.01 0.2790.03 0.3490.02discs
103 116% of mother disc 77 63 92 74 7222/24Number of surviving 22/24 19/24NA 22/2419/24 20/24 22/24
discs92 92 79% of total 79 83 91 92
3.79 3.67Expansion ratio NA 2.43 2.09 3.35 2.72 2.63 2.9590.24
0.5190.020.4290.04Radius of daughter 0.3290.080.3490.03 0.4290.040.3090.04 0.3190.03 0.3390.02discs
104 97% of mother disc 111 84 84 116 71 8623/24 20/24Number of surviving 24/2423/24 18/24 19/24 19/24 17/24
discs83% of total 10096 9575 79 79 71
3.1990.283.88Expansion ratio 4.25 2.37 2.66 3.67 2.01 3.27 3.45
Mother disc radius from six individual discs was measured (mm) before sectioning into quarters. Each quarter was then measured again at theend 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. Thusthe final expansion ratio=expected number of discs (4)×% of mother disc size×% of total discs surviving. NA, data not available.a Over a 14 day period. Not significantly different between the cultures (p\0.05; Student’s t-test).
then solubilised using 600 m l NaOH (0.4 M) for 1 h at37°C. This solution was then added to 4 ml of scintilla-tion cocktail and counted in a scintillationspectrometer.
2.6. Karyotyping
Chromosome number and size was scored usingGiemsa-stained metaphase spreads by the Departmentof Cytogenetics, Addenbrooke’s Hospital, Cambridge.
2.7. Immunocytochemistry
Whole free floating spheres were fixed in 4%paraformaldehyde for 20 min, washed in PBS anddehydrated though 70, 95 and 100% alcohol (20 mineach). Following clearing overnight in xylene, sphereswere embedded in paraffin and sectioned on a micro-tome at 5 mm. For differentiation studies, wholespheres 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.Following this period the medium was exchanged forDMEM/B27 alone. Cultures were fed by replacing 50%of the medium every 4–5 days. At 14 days, the cultureswere fixed for 30 min in 4% paraformaldehyde. Waxsections and coverslips were incubated with primaryantibodies to beta tubulin III (TuJl; monoclonal; 1:500;
Sigma) combined with GFAP (polyclonal; Bohringer;1:1000) in 0.1 M PBS/0.1% Triton/3% goat serum.Others were incubated with antibodies to nestin (poly-clonal; 1:50; kindly donated by R.D.G. McKay); GAL-C (monoclonal; kindly donated by B. Ranscht; 1:4; noTriton was used with this surface marker) or MAP-2ab(1:500; Sigma). Goat anti-mouse biotin or fluorosceinconjugated goat anti rabbit antibodies were used tolabel the primaries, followed by a streptavadin–rho-damine conjugate Hoescht was added to the final incu-bation step (Sigma, 1:10000 in 0.1 M PBS) to visualisenuclei. For cell counts at least five random fields (at×40) were analysed from the monolayer of cellsaround the plated spheres. Each field contained be-tween 50 and 100 cells.
Sections were also viewed under a Biorad confocalscanning microscope at excitation wavelengths of 488and 564 nm. Optical sections were taken at 20 mmintervals and then merged.
3. Results
3.1. Expansion of human precursors
Following seeding into growth medium, aggregatesof dividing cells formed into spheres which grew in sizeover time in response to the mitogens EGF and FGF-2.
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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 couldalso 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 expansiondata in Table 2 and subsequent results not shown in Table 2. Note the steady and consistent exponential increase in total sphere number overtime. Scale bar=0.2 mm.
Between 14 and 21 days of growth the spheres could begently dissociated to a mixed suspension of single cellsand sphere remnants before re-plating into growthmedium. Using this technique we have previously re-ported a 3-4-fold increase in cell number over the firstfew weeks in vitro, after which the absolute number ofcells harvested at each passage declined (Svendsen etal., 1997b). In this study, seven separate experimentsusing either brain stem, spinal cord or cortical humanfetal tissue showed similar results, with variable growthfor the first 4–6 weeks, after which their was very littlefurther growth with the maximum expansion of totalcells only reaching 12-fold (Table 1). A variety ofmodifications to the culture medium, growth factorcombinations and passaging strategies have been at-tempted. None of these had any effect on the slowgrowth rate and eventual senescence of these precur-sors, although some cultures could be kept in a mitotic,but non expanding, state (due to concomitant celldeath) for up to 6 months (data not shown).
A novel, non traumatic, passaging approach wasemployed on several cultures. Data for two of theseisolated from the cortex, which differed in both fetalage and method of cell isolation, are described in detailhere. Instead of mechanical dissociation, individualspheres were sectioned into quarters under a dissectingmicroscope using a scalpel blade (Fig. 1). The resultingfour quarters were placed into fresh growth mediumcontaining FGF-2 and over the next 24 h rounded toform new spheres which grew close to the size of themother sphere by 14 days. Using this method there wasa steady and exponential increase in total sphere num-ber which did not decrease with time (Fig. 1; Table 2).Parallel cultures which were also switched to FGF-2alone but were dissociated rather than chopped,
showed only modest expansion over this 14 day periodas shown in Table 1. By 40 days of growth using thesectioning method, some disc shaped clusters could beseen in addition to spheres (Fig. 1A1 and A2). Thediscs mainly developed due to temporary attachment ofthe newly sectioned spheres to the surface of the flask,and were often concave on one surface. This led tosome disc/sphere aggregates which appeared hollowwhen sectioned (approximately 30% at 100 days ofgrowth, data not shown). When individual spheres at100 days growth were dissociated and the number ofcells counted, those with a size of between 0.35 and 0.45mm diameter contained an average of 61166+3498viable cells (n=25 spheres from CTX-44) with less than5% non viable cells. Very similar results with regard tocell number and sphere size were found at 50 and 150days of growth (data not shown). There was a positivecorrelation between sphere size and number of cells(Fig. 2A), although some variation existed due to thetechnical difficulties of dissociating small single spheres(cells attaching to the side of the pipette, incompletedissociation). We next assessed whether the size of thesphere sections influenced their ability to grow back tothe size of the mother sphere. Regardless of the mothersphere size, all quarters grew back to the same size inrelation to the mother showing that the expansion isnot dependent on either large or small sphere sizes (Fig.2B). [3H]Thymidine added to the spheres over the last24 h of growth at all stages in culture showed that therewas a significant amount of uptake (\5000 counts permin/per sphere at each passage) indicating that activecell division was occurring. The total amount of expan-sion achieved over the growth period (including a 5-fold increase prior to the start of the sectioningmethod) was greater than 1.5 million-fold for Clon-
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Fig. 2. Graphs showing (A) correlation between volume of sphere and number of cells per sphere. (B) Lack of any relationship between size ofsectioned quarter and its subsequent growth relative to its own mother sphere.
migrating out. Between 2 and 7 days, radiating pro-cesses often developed which sketched from the edge ofthe sphere onto the substrate (Fig. 3C). Along theseradiating strands, immature TuJ1+ neurons could beseen which often trailed processes in parallel lines andmay have been migrating out from the core of thesphere (Fig. 3D). Using confocal imaging the exactanatomy of the sphere following 14 days of differentia-tion could be seen and radial processes emanating fromthe core were found to be GFAP positive (Fig. 4).
Migrating cells eventually formed a monolayeraround the plated sphere and labelled for either TuJ1or GFAP but never with both markers at 14 days ofdifferentiation (Fig. 5A, B and C). Detailed cell countswithin the monolayer around the sphere revealed thatthe majority of the cells were either TuJ1 or GFAPpositive at both early and late passages and that nogalactocerbroside (GAL-C) positive oligodendrocytescould be seen under these plating conditions (Fig. 5D).Although not analysed in detail, every whole sphereplated (over 300) gave rise to both neurons and astro-cytes, strongly suggesting the presence of a commonprecursor. At the very periphery of the migrating cells,lone GFAP+astrocytes could often be found, ontowhich a number neurons had selectively migrated, theprocesses from which were entirely confined to theastrocyte surface (Fig. 6A–C). This suggests that theseprecursor cell derived astrocytes provide an attractivesurface for the migrating neurons. Some cells alsoexpressed markers only found in mature neurons suchas microtuble associated protein 2ab which is locatedmainly in dendrites (MAP-2ab; Fig. 6D) (Riederer andMatus, 1985). Interestingly, following trituration to asingle cell suspension and plating, very few TuJ1+
neurons were found while the majority of cells stainedfor GFAP (data not shown), indicating that either thetemporal sequence of events following plating of wholespheres, or the lack of physical trauma caused bytrituration, may be required for neuronal survival and
5382 and both cultures showed a population doublingtime of approximately 4 days. Metaphase spreadsshowed that at 150 days of growth the human cellsremained karyotypically normal with regard to chro-mosome number and appearance. Removal of FGF-2from the growth medium at any stage resulted insenescence and eventual death of cultures over a periodof 14 days. However, switching FGF-2 responsivespheres to a medium containing EGF resulted in thegrowth of an EGF responsive spheres with very similarcharacteristics following plating and which also ex-panded exponentially using this sectioning method.However, the EGF responsive spheres were less proneto attaching to the culture dish and subsequentlyformed less discs. We are also systematically assessingthe effects of sectioning on the exact growth rates ofneural precursor cells isolated from other brain regionsmentioned in Table 1, although bulk cultures spinalcord and brain stem do expand rapidly using thismethod (data not shown).
3.2. Differentiation of human neural precursors
Continual growth of the human neural spheres sug-gested self renewal was occurring, but did not deter-mine the phenotypic potential of these cells. To assessthis, whole spheres or differentiating spheres (at \100days growth) were processed for indirect immunocyto-chemistry. The majority of cells (\95%) within thegrowing spheres were found to be positive for nestin,(Fig. 3B) a marker for undifferentiated neuroepithelialstem cells (Lendahl et al., 1990), but did not stain forTuJ1 (a specific early neuronal marker) (Menezes andLuskin, 1997) or glial acidic fibriallary protein (GFAP;an astrocyte marker). When whole spheres were ex-posed to a substrate, they rapidly attached and withinhours cells with a glial morphology could be seen
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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 mm. Immunocytochemical staining showed that the majority of cells within the growing spheres were nestin+ andundifferentiated (B). Scale bar: 25 mm. Some spheres were plated onto poly-L-lysine coated coverslips in DMEM/B27 and 1% serum for 24 h toinduce differentiation. Phase (C) and TuJ1 (D) staining of the same field from a sphere 4 days following plating. Note the radial fibre outgrowthsalong which TuJl+ cells with extensive fibres could be seen. Arrow heads represent glial fibres with no TuJl+ fibres, arrows represent TuJl+ fibresalong processes, double arrowhead shows single neuronal cell body with extended TuJl+ processes. Scale bar: 25 mm.
differentiation of cells arising from long term humanneural precursor cell cultures.
3.3. Automation of the sectioning technique
Through the detailed assessment of manually section-ing individual spheres we have shown that sustainedexponential growth of human neural precursor cells canbe achieved (Table 2). However, it is obviously notpractical to section large numbers of spheres using thismanual method. We therefore developed an automatedprocedure using the McIlwain tissue chopper originallydesigned to slice fresh brain tissue. Using this device upto 1000 spheres can be automatically sectioned within afew minutes. Each section rapidly rounds up afterseeding into fresh medium and forms a new growingsphere as described previously using the manual tech-nique, although obviously the spheres differ in sizedepending on exactly where they were sectioned. Assphere size is not a critical determinant of cell expan-sion (see Fig. 2) we feel that this method should providean automated technique for growing large numbers ofhuman neural precursor cells.
4. Discussion
The present study has demonstrated a new methodfor the long-term exponential expansion of non immor-talised or transformed human neural precursor cells,which maintained the capacity to generate a high per-centage of neurons (see Fig. 7 for a schematic of thetechnique).
There are two main methods in the literature com-monly used to generate populations of neural precursorcells. The first uses FGF-2 and a substrate to expandcolonies of cells which grow attached to the cultureflask while the second uses EGF to expand aggregatesof cells (neurospheres) although it is now clear thatthese growth factors are often interchangeable in theireffects (for recent review, see Svendsen (1997)). Wehave not attempted to grow human cells attached to asubstrate in the presence of FGF-2 in this study, buthave focused instead on the neurosphere culturemethod. The advantage of the aggregate culturemethod is that large numbers of cells can be expandedin a small volume of medium. There are no publishedreports on the long term growth of human neural
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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 mm. 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 thatEGF can drive a human precursor for extended periodsof time (Vescovi et al., 1997). This cell may be similarto the EGF responsive cell isolated from the developingmouse striatum which can grown for long periods oftime in vitro as spheroid neurospheres and may repre-sent a population of stem cells (Reynolds et al., 1992;Reynolds and Weiss, 1996) although even as early aspassage two (using conventional passaging methods)these mouse neurospheres spontaneously gave rise tovirtually no neurons but rather glial cells (Arsenijevicand Weiss, 1998). We have previously found that incontrast to mouse neurospheres, rat neurospheres entera programmed senescence between 28 and 35 days ofgrowth using routine passaging methods (Svendsen etal., 1997b) and also give rise to high numbers ofastrocytes at later passages (Rosser and Svendsen, un-
published observations). Furthermore, we have foundthe expansion of human neurospheres to be very slow,and although division continued (based on incorpora-tion of mitotic labels) real expansion stopped after 35days of growth (Svendsen et al., 1997a). Using thesectioning method presented here, human neurospherescontinue to expand and give rise to high numbers ofneurons even at late passages. We are currently identi-fying the phenotype of neurons generated from theselong term human cultures.
What is the exact nature of the dividing cells withinthe sectioned spheres?. Their growth rate was remark-ably stable and relatively slow, with a cell cycle time ofapproximately 4 days throughout the culture period.They were karyotypically normal on gross inspectionwhich suggests they had not transformed but are main-taining a normal cellular phenotype with a slow cell
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Fig. 5. Photomicrograph showing phase (A), TuJ1 (B) and GFAP (C) staining of an identical field of cells around the periphery of a sphere platedfor 14 days. Many TuJI+ and GFAP+ cells could be found but no cells labelled with both markers. Arrows show neurons and arrowheads showastrocytes. Graph shows the numbers of labelled cells around the sphere as a percentage of total cells (n=12 spheres at each time) at either 50or 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 cellpopulation. Clonal analysis has classically been re-quired to prove pluri-potency, but is difficult to per-form with this culture system since we have shown thatgroups of cells must maintain contact in order to growfor long periods. However, the observation that everysphere we have plated produced both neurons andastrocytes, but never only one phenotype, arguesstrongly in favour of a common self renewing stem cell.It also remains possible that two uni-potent stem cellswith similar division rates and the capacity to produceeither neurons or astrocytes are dividing alongside eachother. Oligodendrocytes were never seen to arise fromthe late passage sectioned spheres, but have been seento develop from early passage spheres (Murray andDubois-Dalcq, 1997; Svendsen et al., unpublished ob-servations). This suggests that either, (i) an oligoden-drocyte precursor may be capable of dividing for acertain period of time and then be lost from the cul-tures during the extended period of growth which thenonly consists of cells capable of giving rise to neuronsand astrocytes or, (ii) a common precursor is able tomake oligodendrocytes, neurons and astrocytes at early
passages, but only neurons and astrocytes at late pas-sages using the plating conditions in this study. Toinvestigate this further we are currently assessing theeffects of other growth factors and substrates on thedifferentiation of these long term human precursorcells, which have previously been shown to influence thefate rat hippocampal precursors and immortalised hu-man neural precursors (Joh et al., 1996; Sah et al.,1997). It was of interest that the cultures generatedfrom widely differing fetal ages (8 and 21 weeks) gaverise to similar numbers of neurons and astrocytes atlate passages. This would further suggest that a com-mon cell is being driven in these cultures following aninitial period of instability where progenitors with amore limited mitotic life span are filtered out. In sum-mary, the cell which is dividing in these cultures ismaybe best described as a precursor cell until we knowmore about its exact phenotypic potential under avariety of circumstances.
There is currently some confusion regarding the dif-ferential effects of EGF and FGF-2 on neural precursorcells. Based on clonal analysis, EGF was only found tostimulate a glial progenitor from the mouse cortex late
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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 suggestinga 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 thespheres (D). Scale bar: 15 mm.
in development and had no effect on earlier corticalprecursors, in contrast to FGF-2 which was able toinduce the division of a multipotent cell at both earlyand late developmental ages using the same model(Kilpatrik and Bartlett, 1995). Furthermore, EGF ap-pears to stimulate glial division in adult subventricularzones and may in fact repress neuronal development invivo (Kuhn et al., 1997). We have recently shown thatfollowing priming with FGF-2, the same cell respondsto both EGF and FGF-2 in primary E14 mouse striataltissue (Ciccolini and Svendsen, 1998). Perhaps in earlyand late adulthood there are more restricted precursorswhich respond separately to these factors, whereas dur-ing development a common precursor exists. However,the mixture of in vivo and in vitro data across differentspecies and culture conditions makes it impossible todraw conclusions at present. It is of interest that theadult mouse subventricular zone has recently beenshown to contain FGF-2 responsive cells (Gritti et al.,1996) which appear almost identical to the EGF re-sponsive cells described originally by Reynolds andWeiss (1992) who claimed that FGF-2 was unable tostimulate division of the same cells. This discrepancymay be due to the fact that heparin was added to themedium with FGF-2 in the later study. We have re-cently shown that this proteoglycan can significantly
increase the mitotic effects of FGF-2 on embryonicprecursor cells (Caldwell and Svendsen, 1998) and itwas used throughout the current study. Interestingly,we found that changing the growing human precursorsfrom FGF-2 to EGF had no obvious effect on theability to generate neurons following plating, althoughthis is the subject of a more detailed comparison cur-rently in preparation. Clearly there is much more exper-imental work required to resolve these issues.
The reason why the sectioning method is so effectivein maintaining stable and rapid growth may be in partdue to the fact that there is no disruption of cell–cellcontact within the intact regions of the spheres incontrast to standard process of mechanical dissociation.Membrane associated factors are known to be impor-tant for the division of neural precursor cells (Templeand Davis, 1994) and a ‘niche’ hypothesis has beenproposed which suggests stem cells will only retain theirpluripotency within an appropriate environment(Schofield, 1978), both of which may be sustained usingthis sectioning method. Equally important may be thereduction in cellular trauma that results from sectioningrather than dissociating intact spheres. It is clear thatpartial dissociation may also lead to intact remnantsremaining which have cell–cell contact. Indeed, ournormal 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 sectioninggrowing 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 patterncontinued for extensive periods of time in culture and allowed the exponential growth of human neural precursor cells.
dissociation. The major disadvantage of this is thatspheres are generally stripped of cells leaving only thecores. Many of the stripped cells die but the cores maycontinue to grow. However, this does not result in sucha rapid growth rate as simply sectioning the spheresand is far more inconsistent as it is difficult to controlexactly how much dissociation is performed in oneculture to the next.
It is tempting to speculate that the stages of sphereattachment, formation of radial processes and apparentneuronal migration may recapitulate the normal pro-cess of primate development where neuronal precursorsdivide within ventricular zones before migrating to thepial surface along radial glia (Rakic, 1985). However,this needs to be substantiated with further studieswhich are the focus of ongoing work. Genetic manipu-lations to these dividing cells should be possible, asachieved previously with both human and rat neuralprecursors (Sabate et al., 1995; Svendsen et al., 1996),to allow the expression of specific proteins followinggrafting, thereby facilitating ex vivo CNS gene therapy(Friedmann, 1994). The spheres can be frozen andstored which facilitates shipping and banking of suchtissue. Finally, as these sectioned neurospheres produceconsistently large numbers of neurons, they may
provide a valuable source of normal human neuraltissue for both testing novel neuroactive compounds invitro and clinical neural transplantation programmeswhich are currently dependent on the collection of freshhuman fetal tissues (Bjorklund, 1993; Svendsen, 1997).
Acknowledgements
We thank S.B. Dunnett for his continual support,Biorad Inc. for preparation of the confocal images andZiggy Zhang and Irena Sarel of Blowhittiker Inc. forproviding human neurospheres. The authors would alsolike to thank Dr Scott Whittemore for critically ap-praising an early version of this manuscript. This re-search was funded by a Wellcome Fellowship to CNSand by the MRC.
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