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Stem Cells 2004;22:344-354 www.StemCells.com
© 2004 AlphaMed Press

Enhancement of Oligodendrocyte Differentiation from Murine Embryonic Stem Cells by an Activator of gp130 Signaling

Peilin Zhang, Judith Chebath, Peter Lonai, Michel Revel

Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel

Key Words. Embryonic stem cells • Glial differentiation • gp130 • IL-6R/IL-6 fusion protein • In vitro differentiation • Oligodendrocytes

Michel Revel, M.D., Ph.D., Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, 76100, Israel. Telephone: 972-50-419763; Fax: 972-89-346106; e-mail: michel.revel{at}weizmann.ac.il


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Embryonic stem (ES) cells derived from the inner cell mass of blastocyst-stage embryos are a potential large scale source of oligodendrocytes and of their progenitors for transplantation into the central nervous system for the repair of demyelinating lesions. We found previously that interleukin-6 (IL-6) fused to its soluble receptor (IL-6R), a potent activator of the gp130 receptor, induces myelin gene expression in Schwann cells of embryonic dorsal root ganglia. Like leukemia inhibitory factor, IL-6R/IL-6 inhibits the differentiation of murine ES cells into embryoid bodies. In the present study, we show that this recombinant cytokine may be efficiently used to stimulate the differentiation of oligodendrocytes if added to ES cell-derived neural precursors. IL-6R/IL-6 leads to an increase in early chondroitin sulfate proteoglycan positive and late O4 positive progenitors and to a stimulation of maturation into O1 and myelin basic protein expressing oligodendrocytes. Expression of the genes for transcription factor genes Olig-1 and Sox10, which appear early in the oligodendrocyte lineage, was stimulated by IL-6R/IL-6 addition. We conclude that this cytokine can significantly enhance the derivation of oligodendrocytes from ES cells.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Oligodendrocytes, which make the myelin sheaths in the central nervous system (CNS), evolve from multipotent neural stem cells (NSCs) through a series of developmental stages [1, 2]. The principal stages are: A) round pre-progenitors that express nestin as well as polysialylated neural cell adhesion molecule (PSA-NCAM) and which share with NSCs the property to grow as floating spheres when cultured in presence of growth factors [3, 4]; B) early bipolar progenitors or oligodendrocyte-type-2 astrocyte (O-2A) [5] staining with anti-ganglioside A2B5, and later forms becoming multipolar and positive for chondroitin sulfate proteoglycan (NG2); C) multipolar or arborizing late progenitors expressing O4 sulfatide glycolipids [6]; D) arborized premyelinating oligodendrocytes positive for galactocerebroside (GalC) and O1, and E) mature oligodendrocytes synthesizing the myelin membrane with its structural components such as myelin basic protein (MBP).

Embryonic stem (ES) cell lines derived from the inner cell mass of blastocysts are a potential large scale source of oligodendrocytes and of their progenitors, which have been used for transplantion into myelin deficient CNS [7–9]. Several culture conditions have been defined under which murine ES cells differentiating into embryoid bodies (EB) may be directed toward neural lineages, neurons, astrocytes, and oligodendrocytes. One approach is selection in serum-free defined medium in which neural precursor cells survive, proliferate in response to basic fibroblast growth factor (FGF-2), and differentiate when plated on adherent substrates after withdrawing the growth factor [10]. Some O4+ oligodendrocytes can then be derived provided tri-iodothyronine (T3) is added [10] in analogy to the effect of T3 on brain cell cultures [11]. Platelet derived growth factor (PDGF) promotes proliferation of brain glial precursors, in cooperation with epidermal growth factor (EGF) [12]. Similarly, on EB cells, combinations of FGF-2 with EGF and PDGF-AA provide more efficient growth of A2B5+ O-2A progenitors [8]. Yet another approach uses retinoic acid (RA) to induce neural precursors in mouse EB cultures [13, 14].

Brain NSCs can be further enriched by selecting non-adherent cells forming floating spheres in defined medium with FGF-2 or EGF and then by expanding them as multipotent neurospheres or as oligodendrocyte progenitor-enriched oligospheres [3, 15]. Similarly, oligospheres were obtained from RA-induced mouse EB cell cultures [14]. Floating neurospheres were also derived with FGF-2 from human ES cell lines and transplanted in vivo or plated in vitro on polycationic substrates yielding neurons, astrocytes, and oligodendrocytes [16, 17]. In these procedures, the growth factors used to obtain progenitors actually inhibit their differentiation [12]. A factor that stimulates oligodendrocyte differentiation from ES cells would therefore be of importance.

The cytokine ciliary neurotrophic factor (CNTF) has been reported to contribute to the proliferation of oligodendrocyte precursor cells from optic nerve [18]. However, the effect of CNTF on the differentiation of glial progenitors remains unclear as in several studies CNTF mainly induced astrocytes expressing glial fibrillary acidic protein (GFAP) with little effect on O4+ oligodendrocytes [5, 19–23], whereas in others it increased astrocytes and also survival and proportion of GalC+, O1+ and MBP+ cells in the cultures [24–26]. CNTF belongs to the interleukin-6 (IL-6) family of cytokines that signal via gp130 either as a heterodimer with the receptor for leukemia inhibitory factor ([LIFR] for CNTF, LIF, Oncostatin M[OSM]) or as a homodimer (for IL-6, IL-11) [27]. Differentiation of murine ES cells into EB is inhibited by LIF [28], CNTF [29], OSM [30] as well as by combinations or fusions of IL-6 with the extracellular portion of the soluble IL-6 receptor (sIL-6R) [31, 32]. Due to this inhibitory effect, the gp130 ligands were not investigated as differentiation agents for ES cells.

In previous studies we found that the IL-6R/IL-6 protein, a potent gp130 ligand in which IL-6 is fused to sIL-6R [33], is an efficient inducer of myelin gene expression in embryonic Schwann cells [34, 35] and an activator of myelin gene promoters [36]. Here we report that in cultures derived from murine ES cells, IL-6R/IL-6 strongly enhances the differentiation of early and late oligodendrocyte progenitors and their maturation into MBP+ cells.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
ES Cell Cultures and Neurosphere Production
The murine ES cell line ROSA 11 [37] was maintained as before [38]. The cells were removed from the feeder layer with 0.05% trypsin and transferred without LIF to tissue culture plates in ES1 (Dulbecco’s modified Eagle’s medium [DMEM/F12] with 15% fetal calf serum [FCS], 1% glutamine, 0.1 mM ß-mercaptoethanol, 2 µg/ml heparin) with 4 ng/ml of FGF-2. Media from (GIBCO/Invitrogen; Carlsbad, CA; http://www.invitrogen.com) were replenished daily. After 2 days the culture was treated with 0.2% dispase (GIBCO/Invitrogen) for 15 min at 37°C, and the clumps were reseeded into 9 cm tissue culture dishes in ES1 medium lacking FGF. After 4 days, clumps of differentiating EB cells were seen loosely bound to the dish by the intermediate of a few attached cells. The clumps, easily harvested using a needle, were transferred to new tissue culture dishes and cultured for another day in ES1 medium to facilitate attachment. Afterwards, selection for survival and growth of neural precursors [10] was achieved using EB-defined medium (DMEM/F12, 25 µg/ml insulin, 100 µg/ml transferrin, 60 µM putrescine, 30 nM sodium selenite, 2 µg/ml heparin, 20 nM progesterone) with 20 ng/ml of FGF-2. Medium was changed every 2 days. After 8–10 days, the cores of spherical aggregates containing neural precursors, as identified by surrounding radiating axons, were picked up with a needle. These spherical aggregates were then transferred into bacterial culture plates (Sterilin; Staffordshire, UK; http://www.bibby-sterilin.com) with the same EB-defined medium with 20 ng/ml FGF-2 and kept in suspension for at least 8 days. During the suspension culture, many cells detached from the aggregates and the latter acquired a regular shape typical of neurospheres [3]. Spheres expanding to more than 0.5 mm diameter were cut into two before reseeding in suspension for longer cultures. The composition of the floating neurospheres was examined after complete dissociation with 0.25% trypsin-EDTA by plating 25,000 cells on coverslips for immunostaining (as below).

Cell Differentiation Assay
Four neurospheres from the suspension cultures were deposited on each glass coverslip precoated with a solution of 20 µg/ml poly-D-lysine, 250 µg/ml fibronectin (PDL-FN), which were then placed into wells of 12-well plates in differentiation N2 medium (DMEM/F12 with 5 µg/ml insulin, 100 µg/ml transferrin, 16.1 µg/ml putrescine, 5.2 ng/ml selenite, and 6.3 ng/ml progesterone—all added as 1% N2 supplement (from GIBCO). Laminin (2.5 µg/ml) and FGF-2, (5 ng/ml) were added for the first 4 days to facilitate attachment and then removed (laminin was omitted in some experiments without altering the results). Half of the wells were supplemented with IL-6R/IL-6 chimera, 100 or 200 ng/ml, produced in Chinese hamster ovary cells and purified as before [33]. The medium was replaced every 3 days, and in prolonged cultures 50 µg/ml ascorbic acid were added to the medium starting at day 21. At indicated times after plating the neurospheres, the cultures were fixed in 4% paraformaldehyde and kept in phosphate buffered saline/solution (PBS) at 4°C. After blocking with 5% normal goat serum (NGS), fixed cells were stained for early progenitors or oligodendroblasts with rabbit polyclonal anti-NG2 (chondroitin sulfate proteoglycan; Chemicon International; Temecula, CA; http://www.chemicon.com; 1:200) for 1 h at room temp (RT) and then Alexa Fluor 566-conjugated goat anti-rabbit antibody (Ab; Molecular Probes; Eugene, OR; http://www.probes.com; 1:250). Staining for late progenitors or pre-oligodendrocytes was with anti-sulfatide O4 mouse monoclonal (Mc) immunoglobulin M (IgM) antibodies (McAb 345 Chemicon; 1:75) for 1h at RT and fluorescein-conjugated goat anti-mouse IgM (Chemicon; 1:50). Immunostaining for GFAP, ßIII-tubulin, nestin, and MBP was after permeabilization with 0.5% Triton-X100 and blocking with 10% NGS. Staining with mouse Mc anti-GFAP conjugated with the fluorescent Cy3 tag (Sigma; St Louis, MO; http://www.sigmaaldrich.com; 1:400) was for 1 h at RT. The mouse Mc immunoglobulin G (IgG) Tuj-1 anti-tubulin-ßIII (Covance; Berkeley, CA; http://www.covance.com; 1:400) was used with goat anti-mouse IgG conjugated with Alexa Fluor 488 (Molecular Probes; 1:250). Staining for myelinating cells was with mouse Mc IgG anti-MBP (McAb 386; Chemicon; 1:400) and Cy3-conjugated affinity purified goat anti-mouse IgG, F(ab')2 fragment specific (Jackson ImmunoResearch Laboratories; West Grove, PA; http://www.jacksonimmuno.com; 1:400). Staining for neural precursors was with Mc IgG1 anti-nestin (Rat-401, Developmental Studies Hybridoma Bank at University of Iowa; Iowa City, IA; http://www.uiowa.edu/~dshbwww; 1:100) followed by the same Cy3-conjugated IgG as above.

Live cells were stained for O1. After blocking with 5% FCS, anti-O1 mouse IgM Mc antibodies (McAb 344, Chemicon; 1:75) and fluorescein-conjugated goat anti-mouse IgM (Chemicon; 1:50) were used for 1h at 37°C in humidified atmosphere, followed by fixation with 5% acetic acid in methanol. In all cases, the nuclear fluorescent dye DAPI (Sigma; 0.05 µg/ml) was added last. Coverslips were mounted in Mowiol (Calbiochem; La Jolla, CA; http://www.calbiochem.com), viewed in an Olympus IX-70 FLA microscope with a DVC-1310C digital camera (DVC; Austin, TX; http://www.dvcco.com) and images processed by Photoshop. Double-stained preparations are shown as overlayed images. A manual count program in the AlphaEase software (Alpha Innotech; San Leandro, CA; http://www.alphainnotech.com) was used to measure sizes and enumerate NG2, O4 and GFAP stained cells, as well as total cell nuclei visualized by DAPI.

Gene Expression Assays
Procedures for RNA extraction and reverse transcriptase-polymerase chain reaction (RT-PCR) for measuring levels of Sox10, GFAP, MBP and glyceraldehyde 3'-phosphodehydrogenase (G3PDH) gene transcripts were as described in detail previously, including the number of cycles and the primers used [36]. For the Olig-1 gene (accession NM_016968 [GenBank] ), the primers were: forward, 5'-TGCGCGCGAGAAGGCCGAAG and reverse, 5'-CCCAGCCAGCCCTCACTTG. Conditions for PCR amplification: 94°C, 2 minutes then 30 cycles at 94°C, 30 seconds; 56°C, 30 seconds; 72°C, 1 minute. The PCR buffer [36] was supplemented with 10% DMSO. After gel electrophoresis, the amplified DNA bands were photographed under UV-light, scanned, and their intensity was quantified using the AlphaEase spot density software. Band intensity was verified to be in the linear range by varying the amount of PCR reaction loaded on the gels.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Addition Of IL-6R/IL-6 To EB Cell-Derived Neurospheres Enhances Oligodendrocyte Progenitor Differentiation
Like LIF and other members of the IL-6 cytokine family, IL-6R/IL-6 inhibited mouse EB formation (not shown). We, therefore, studied the effects of IL-6R/IL-6 when added to neurosphere cells derived from already preformed EBs. To produce neurospheres [16, 17], murine ROSA 11 ES cells, removed from the feeder layer, were induced to form EBs which were then subjected to selection for neural precursors [10] in serum-free medium supplemented with 20 ng/ml FGF-2. Under these conditions one observes the formation of spherical cell aggregates surrounded by outgrowing axons. The cores of these aggregates were dislodged and transferred to suspension culture, in which the floating spheres were maintained in the same selection medium containing FGF-2 for 8 or more days.

Dissociation of the floating spheres with trypsin and plating on glass coverslips confirmed that they are mainly composed of small round or elongated bipolar cells, 90% of which are positive for nestin, the intermediate filament protein found in neural precursors (Fig. 1AGo). Very few differentiating cells were seen at this stage (not shown), less than 1% staining for GFAP (astrocytes) or ßIII-tubulin (neurons), and none staining for the O4 sulfatide marking the progenitors differentiating into oligodendrocytes. We also examined cells staining for NG2, which was thought to be a specific marker of perinatal early oligodendrocyte progenitors but is now known to be also present in earlier neural multipotent precursors [39]. Up to 10% NG2+ cells were found, but all had a bipolar morphology resembling the other nestin-positive neural precursors present in the neurosphere and not oligodendrocyte progenitors (see below).



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  		Figure 1. Neurosphere cells and effect of IL-6R/IL-6 on the outgrowth of glial cells. A) Mouse ES cell-derived floating neurospheres, after 19 days in suspension cultures, were dissociated with trypsin, plated, and stained for nestin (red) as marker of neural precursors and by DAPI (blue) to visualize nuclei. B) Outgrowth from neurospheres plated on PDL-FN adherent substratum double stained after the first 4 days for O4 (green) and GFAP (red). Note absence of O4+ cells. C) After 7 days of culture on PDL-FN (FGF removed at day 4), the outgrowth surrounding the neurospheres was double stained as in B and is seen to contain mainly GFAP+ astrocytes with few O4+ oligodendrocytes. D) Addition of 100 ng/ml IL-6R/IL-6 during the 7 days of culture on PDL-FN produces a large increase in O4+ oligodendrocyte progenitors (green). A series of enlarged microphotographs were used to quantify the results (Table 1Go). E) Culture after 15 days, under control conditions. F) After 15 days with IL-6R/IL-6, a large network of O4+ cells has formed. Size bars: 100 µm in all the panels.

 
To investigate the effect of the gp130 activator IL-6R/IL-6 on differentiation, the floating spheres were placed on glass coverslips coated with PDL-FN, an adherent substratum that favors glial cell development [17], and the coverslips were incubated in defined N2 medium with or without IL-6R/IL-6 addition. To promote cell outgrowth, FGF-2 (5 ng/ml) and laminin (2.5 µg/ml) were added during the first 4 days, after which the cultures were continued without these additions. After the first 4 days, the outgrowth had formed a monolayer of GFAP+ cells but no O4+ cells were observed (Fig. 1BGo). On day 7 (i.e., 3 days after removal of FGF), O4+ oligodendrocyte progenitors became apparent in the control cultures (Fig. 1CGo), but their number and their size was much larger in the presence of IL-6R/IL-6 (Fig. 1DGo). In comparison, the GFAP+ astrocytes surrounding the neurosphere appeared similar in both conditions. Quantitative analysis of the stained cells versus the total cells labeled by DAPI showed that in the absence of IL-6R/IL-6, more than half the cells in the outgrowth were GFAP+ and about 2% were O4+ oligodendrocytes (Table 1Go). The data show that in the presence of IL-6R/IL-6 the proportion of GFAP+ cells was actually reduced whereas the percentage of O4+ cells became 6.4-fold higher than in the control conditions. IL-6R/IL-6 not only increased the proportion of O4+ progenitors, but accelerated their differentiation. Thus, at 15 days (Fig. 1FGo), the IL-6R/IL-6-treated cultures showed a marked expansion and branching of the O4+ oligodendrocyte progenitors, which formed a network over the astrocyte layer. Without IL-6R/IL-6, this differentiation was not apparent in the 15-day cultures (Fig. 1EGo).


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  		Table 1. Effect of IL-6R/IL-6 on oligodendrocyte number and morphology
 
In another experiment, the outgrowing cells were double stained for NG2 and O4 to compare the effect on early and late progenitors, respectively. To facilitate quantitative analysis in these 19-day cultures, the cells were observed at higher magnification (Figs. 2AGo–2BGo) which allowed clear visualization of the increase both in number and in size of the O4+ progenitors produced by IL-6R/IL-6 treatment (Fig. 2BGo). The percentage of O4+ cells rose from 0.8% to 7.3%, or 9.1-fold in response to the cytokine (Table 1Go). The percentage of NG2+ cells was also increased in response to IL-6R/IL-6 but only by 2.7-fold (Table 1Go). The higher increment of O4+ cells suggests that the main effect of IL-6R/IL-6 may be on the transition from NG2+ early progenitors to more differentiated O4+ cells. When examined at 7 days (Figs. 2CGo–2DGo), the increase in NG2+ cells was similar (percentage increase of 2.5-fold). Although NG2 may also be present in multipotent neural progenitors [39], the NG2+ cells in the outgrowth were mostly multipolar or branched (Figs. 2CGo–2DGo arrows) as typical for oligodendrocyte progenitors. Moreover, the size of the multipolar and branched NG2+ cells was larger in the IL-6R/IL-6-treated cultures (Fig. 2DGo arrows). Hence, although less pronounced than for O4, the enhancement of NG2+ early progenitors was reproducibly observed. On the other hand, when the cultures were stained for tubulin-ßIII we did not observe significant changes in the density of the neuron axonal network in the outgrowth surrounding the neurospheres (Figs. 2EGo–2FGo). This makes it unlikely that the effect of IL-6R/IL-6 on oligodendrocyte differentiation would be a secondary effect resulting from an increase in the axon network.



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  		Figure 2. IL-6R/IL-6 increases differentiation of oligodendrocyte progenitors without affecting the axonal network. A, B) Outgrowth from ES cell-derived neurospheres after 19 days on PDL-FN adherent substratum, fixed and double stained for NG2 (red, early oligodendrocyte progenitors) and for O4 (green, arborized late progenitors). The neurospheres are in the lower left corner. A) control culture, B) culture with 100 ng/ml IL-6R/IL-6, showing increase in stained cell number and size. A quantitative analysis appears in Table 1Go. C, D) similar cultures at 7 days, stained for NG2 (red) and by DAPI (blue) to visualize nuclei. C) control culture, D) culture with 100 ng/ml IL-6R/IL-6, showing increase in NG2+ cell size and proportion (6.7% in control versus 16.9% with IL-6R/IL-6). E, F) Similar cultures for 21 days, fixed and stained to visualize the axonal network with anti-ßIII-tubulin. E) control culture, F) culture with 200 ng/ml IL-6R/IL-6 (E, F same magnification). Size bars: 50 µm.

 
IL-6R/IL-6 Promotes Maturation of EB Cell Derived Oligodendrocytes
In long-term cultures (6 weeks), the control cultures developed small arborized O4+ oligodendrocyte progenitors (Fig. 3AGo), which were spread among the underlying layer of cells outgrowing from the neurosphere (Fig. 3CGo). In contrast, cultures treated with IL-6R/IL-6 formed a dense network of O4+ cells with considerably more arborization, which formed the majority of the cells in certain areas of the outgrowth and surrounded thickened nerve fibers (Figs. 3BGo, 3DGo). The O4+ oligodendrocytes in the IL-6R/IL-6 treated cultures reached much larger size than in the control cultures (Fig. 4Go, panels A, B, and E for untreated versus C, D, and F for IL-6R/IL-6-treated cultures). Measuring the length of the branches indicated a significant fourfold increase in the mean length as compared to control cultures (Table 1Go). Hence, while oligodendrocyte progenitors survived and increased in number in these long-term cultures under control conditions, the addition of IL-6R/IL-6 produced a notable expansion of the oligodendrocytes, both in number, in size, and in arborization.



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  		Figure 3. IL-6R/IL-6 enhances oligodendrocyte differentiation in long-term cultures. ES cell-derived neurospheres plated on PDL-FN and cultured for 6 weeks; outgrowing cells fixed and stained for O4. A) control culture, B) culture with IL-6R/IL-6, 200 ng/ml. The same fields are shown under light phase contrast. C) control culture, D) with IL-6R/IL-6. Under control conditions (A and C), a number of multipolar O4+ cells are seen within a monolayer of unstained cells. With IL-6R/IL-6 (B and D), a network of larger O4+ cells with long branches has developed, which represents the majority of the cells seen between thickened neuronal fibers. All panels at same magnification. Size bars: 100 µm.

 


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  		Figure 4. Comparative morphology of O4+ oligodendrocytes in ES cell-derived neurosphere cultures. A, B, E) Representative cells from control cultures; C, D, F) cells from IL-6R/IL-6-treated cultures (as in Fig. 3Go). With IL-6R/IL-6, the size and thickness of the oligodendrocyte branches increased markedly and myelin membranes were formed (see in panels C, D, and F). Quantitative results on branch length appear in Table 1Go. Panels A, B, and C, D are at the same 160x magnification. Size bars: 50 µm.

 
Besides promoting oligodendrocyte differentiation, IL-6R/IL-6 also promoted their maturation. This is denoted first by the development of the cell processes into flattened myelin-like membrane sheaths that were visible in many cells from IL-6R/IL-6-treated cultures (Figs. 4CGo–4DGo, 4FGo). Such large membranes were not seen in untreated cultures (Figs. 4AGo–4BGo, 4EGo). Second, IL-6R/IL-6 enhanced the development of mature O1+ oligodendrocytes, whose size was considerably increased as compared to the control cultures (Fig. 5Go, panel A for control versus panels 5B-5C for IL-6R/IL-6-treated). In other experiments, we also found that the presence of IL-6R/IL-6 during the first 7 days was enough to produce the increase in O1+ cells at the end of the 6-week culture. Furthermore, the stimulating effect of IL-6R/IL-6 was similar in media supplemented with T3 and thyroxine (0.4 ng/ml each; not shown), indicating that the effect of IL-6R/IL-6 is in addition to that of these hormones.



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  		Figure 5. Increased oligodendrocyte maturation in response to IL-6R/IL-6. A-C) ES cell-derived neurospheres cultured for 6 weeks on PDL-FN and outgrowing cells stained live for O1. A) control culture; B, C) culture with IL-6R/IL-6, 200 ng/ml, which induces much larger branched O1+ oligodendrocytes. D, E) fixed cells stained for MBP. D) control culture shows weak MBP stain. E) culture with IL-6R/IL-6 200 ng/ml shows extensive accumulation of MBP. Panels A-C as well as D, E are at the same magnification. Size bars: 50 µm.

 
In line with the morphological development of myelin membranes, immunostaining for MBP was much higher in the oligodendrocyte network of IL-6R/IL-6-treated cultures at 6 weeks (Fig. 5EGo), than in the control cultures where only weakly labeled and small size cells were seen (Fig. 5DGo). Enhancement in MBP+ cells by IL-6R/IL-6 was already observed at 14 days (not shown). The gp130 activator, therefore, not only stimulated differentiation of ES-cell derived oligodendrocyte progenitors but also their maturation toward the myelinating phenotype.

IL-6R/IL-6 Enhances Oligodendrocyte Lineage-Specific Gene Expression
Olig-1 is a transcription factor of the bHLH group with a restricted expression seen in the oligodendrocyte lineage but not in astrocytes or other glial cells [40, 41]. Olig-1 is expressed early and appears specifically required for the development and maturation of oligodendrocytes [42]. Sox10 is also expressed early in the oligodendrocyte lineage [41] and is a transcription factor acting on the promoters of myelin genes [36, 43]. The expression of these oligodendrocyte marker genes and of the astrocyte marker GFAP was examined by RT-PCR. We first analyzed RNA extracted from the spherical aggregates formed in the EB cultures after selection in serum-free medium with 20 ng/ml FGF-2 for 12 days. Little expression of Olig-1, Sox10, or GFAP RNA was detected in these spherical aggregates even when treated with IL-6R/IL-6 (Fig. 6Go, lanes 1–2). When RNA was extracted from outgrowing neurospheres cultured under the differentiation conditions on PDL-FN (4 days with 5 ng/ml FGF-2 and 2.5 µg/ml laminin and then four more days without these additions), expression of the three marker genes was observed (Fig. 6Go, lane 3). Addition of IL-6R/IL-6 for the last 4 days of this differentiation culture produced a marked increase in Olig-1 and Sox-10, whereas GFAP was unaffected (Fig. 6Go, lane 4). Furthermore, an induction of MBP RNA was observed in response to IL-6R/IL-6 (Fig. 6Go, lanes 5–6). Photometric scanning indicated increases of up to 20-fold for Olig-1 and 7.6-fold for Sox-10 in response to IL-6R/IL-6. In these short-term cultures, MBP was increased threefold. These gene expression profiles support the conclusion that the gp130 activator exerts enhancing effects on early phases of cell differentiation along the oligodendrocyte lineage (as denoted by Sox-10 and Olig-1 expression), as well as on the maturation toward myelinating MBP-expressing oligodendrocytes.



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  		Figure 6. IL-6R/IL-6 enhances expression of oligodendrocyte lineage specific gene. Lanes 1, 2: gene expression in spherical aggregates formed in EB cultures after selection for 12 days in defined medium with 20 ng/ml FGF-2. Lanes 3–6: outgrowing neurospheres on PDL-FN for 8 days (FGF removed at day 4). Where indicated, IL-6R/IL-6 was added for the last 4 days before extracting RNA. For lane 2, IL-6R/IL-6 at 200 ng/ml and for lanes 4 and 6, IL-6R/IL-6 at 100 ng/ml. Expression levels measured by RT-PCR are shown for GFAP (astrocyte lineage), for Olig-1 and Sox10 (early oligodendrocyte progenitors), and for MBP (oligodendrocyte maturation), versus the housekeeping gene G3PDH as control for RNA loading.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The IL-6R/IL-6 recombinant protein is a prototype of the IL-6 cytokine family. IL-6R/IL-6 has a high affinity for the gp130 receptor and can act on cells which do not respond to IL-6 itself [33, 44]. This results from the fact that the extracellular (soluble) portion of the gp 80 IL-6 binding receptor (sIL-6R) has the unusual property of being an agonist of IL-6 [45], so that in cells having only gp130 the IL-6/sIL-6R combination allows gp130 dimerization, thereby mediating signal transduction and activation of transcription factors of the STAT family [46].

This study demonstrates that a cytokine acting through the IL-6 family gp130 receptor may be effective in stimulating the differentiation of ES cell derivatives when added after the formation of EB. The differentiation-promoting activity of IL-6R/IL-6 was examined on EB-cell derived neurospheres consisting mainly of nestin positive neural precursor cells. Addition of IL-6R/IL-6 to neurospheres plated on an adherent substratum resulted in a marked increase in the total number and percentage of outgrowing O4+ late oligodendrocyte progenitors. This effect of IL-6R/IL-6 was observed already after 7 days, and in longer term cultures IL-6R/IL-6 enhanced the differentiation of highly branched large O4+ oligodendrocytes and their maturation into O1+ and MBP+ cells.

The steps leading to the development of the three main neural lineages from multipotent NSCs are still incompletely understood, in particular whether astrocytes and oligodendrocytes derive from a common glial SC or whether both neurons and oligodendrocytes derive from a common pathway distinct from that leading to astrocytes [42]. In the present experimental system, the proportion of GFAP+ astrocytes among the cells outgrowing from the neurospheres was rather reduced by IL-6R/IL-6, and the density of the axons coming out from the neurospheres appeared unchanged (although we did not study the number of neuronal bodies inside the neurosphere). We focused on the action of IL-6R/IL-6 on differentiation of the oligodendrocyte lineage and examined whether IL-6R/IL-6 has an effect on early oligodendrocyte progenitors. Our experiments show that there is a moderate increase in NG2+ early multipolar progenitors, although smaller than the increase in development of O4+ late progenitors. Since no effect was seen on earlier bipotential O-2A A2B5+ cells (not shown), the first target of IL-6R/IL-6 may be the NG2+ committed progenitors. Further evidence for an early effect is seen in the expression of Sox10 and Olig-1 transcription factor genes, which are early markers of the oligodendrocyte lineage [40–42]. These genes started to be expressed when the neurospheres were plated on adherent substratum under conditions of outgrowth and differentiation following removal of FGF-2. However, supplementing these cultures with IL-6R/IL-6 caused within 4 days a marked increase in the expression of Sox10 and Olig-1 genes, but not in the astrocyte GFAP marker gene. This suggests that IL-6R/IL-6 has some lineage-specific effects, such as the commitment or early differentiation of specific oligodendrocyte progenitors. This does not exclude that additional actions on cell growth and survival may be involved at this or later stages in the process of oligodendrocyte differentiation. In the control cultures, small arborized oligodendrocytes developed in the long-term cultures indicating that progenitors do survive. However, the mechanism(s) by which IL-6R/IL-6 caused the formation of much denser networks of larger long-branched oligodendrocytes and increased accumulation of MBP may be complex, and involve both enhancements of differentiation and of survival.

We previously showed that IL-6R/IL-6 induces myelin gene expression in Schwann cells from dorsal root ganglia of day-14 rat embryos [34]. Molecular effects of the gp130 ligand included a profound downregulation of transcription factor Pax3 [35], a step required for the onset of MBP gene expression [47]. Similarly, IL-6R/IL-6 activated the promoter transcriptional activities of the MBP and peripheral myelin Po genes in a transdifferentiating melanoma cell line, effects mediated in part by the downregulation of the Pax-3 repressor and by the increased expression of activators such as Sox10 [36]. The in vivo importance of gp130 signaling for Schwann cells has been shown by postnatal gp130 gene deletion which led, among others, to loss of Schwann cells and of peripheral myelin sheaths [48].

Effects of IL-6R/IL-6 could be observed also on neurospheres derived from post-natal mouse brain striatum, resulting in increased MBP mRNA (Ben-Hur, Chebath and Revel, unpublished observations). Increase of number of cells expressing a transgenic MBP promoter reporter gene in mouse brain cell cultures was recently reported in response to CNTF, LIF, OSM or the combination of IL-6 with sIL-6R [49]. In a previous study of the IL-6R/IL-6 protein using rat brain cortical cell cultures, a very marked increase in highly arborized oligodendrocytes was noted [50]. With IL-6R/IL-6 the number and length of MBP-stained branches was 2.3-fold larger than with CNTF, at their respective optimal doses [50]. In brain-derived cells, CNTF and LIF have been associated to astrocyte differentiation, with more variable effects on oligodendrocytes depending on the in vitro assays and type of cells examined [5, 19–26]. On the other hand, addition of CNTF to EGF-grown brain neurospheres was also reported to maintain NSC multipotency and reduce their transition to glial fate [23]. More recently, LIF was found to enhance formation of neurospheres, containing multipotent neural precursors, in sparse mouse ES cell cultures [51]. The differences in conditions and cells used in these studies make it difficult to compare the effects of CNTF or LIF to the action of IL-6R/IL-6 reported here, namely an enhancement of oligodendrocyte differentiation with no significant effect on astrocytes. This is the first study on ES cell-derived neurospheres showing that an IL-6 type cytokine stimulates the production of oligodendrocyte progenitors and their differentiation toward mature myelinating cells.

The potential of ES cell lines for expansion together with the availability of agents promoting their directed differentiation into various cell types holds much promise for the repair of injured tissues. Transplantations of murine ES cell-derived EBs and neurosphere cells have demonstrated remyelination, including examples of functional recovery [8, 9]. Recently, brain-derived neurospheres injected intravenously could induce recovery in a mouse model of multiple sclerosis [52]. Human ES cell lines have been used to prepare neuroglial cells transplantable into mouse brain [16, 17]. These strategies may lead to medical applications. IL-6R/IL-6 might have applications in the ex vivo preparation of myelinating cell transplants, or could be co-injected in vivo to stimulate differentiation of the myelinating cells in the CNS, as done previously to promote remyelination in rats subjected to sciatic nerve transections [35]. The improved derivation of oligodendrocytes from ES cells will also help in studying the molecular mechanisms and the control of their differentiation.


   ACKNOWLEDGMENT
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We thank Dr. Tamir Ben-Hur, Neurology Department, Hadassah Medical School, Jerusalem, for many helpful discussions. The assistance of Dr. Shalom Haggiag, Dr. Li Li, Dr. Dalia Gurari, Mrs. Rosalie Kaufmann, Lia Chazin and Raya Zwang are gratefully acknowledged. Work supported by InterPharm, Ares-Serono Group (Nes-Ziona, Israel and Geneva, Switzerland).


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received August 3, 2003; accepted for publication November 24, 2003.



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