Enhancement of Oligodendrocyte Differentiation from Murine Embryonic Stem Cells by an Activator of gp130 Signaling
http://www.100md.com
《干细胞学杂志》
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@weizmann.ac.il
ABSTRACT
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 . 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 ; B) early bipolar progenitors or oligodendrocyte-type-2 astrocyte (O-2A) 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 ; 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 . 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 . Some O4+ oligodendrocytes can then be derived provided tri-iodothyronine (T3) is added in analogy to the effect of T3 on brain cell cultures . Platelet derived growth factor (PDGF) promotes proliferation of brain glial precursors, in cooperation with epidermal growth factor (EGF) . Similarly, on EB cells, combinations of FGF-2 with EGF and PDGF-AA provide more efficient growth of A2B5+ O-2A progenitors . Yet another approach uses retinoic acid (RA) to induce neural precursors in mouse EB cultures .
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 . Similarly, oligospheres were obtained from RA-induced mouse EB cell cultures . 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 . In these procedures, the growth factors used to obtain progenitors actually inhibit their differentiation . 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 . 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 , whereas in others it increased astrocytes and also survival and proportion of GalC+, O1+ and MBP+ cells in the cultures . 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 ( for CNTF, LIF, Oncostatin M) or as a homodimer (for IL-6, IL-11) . Differentiation of murine ES cells into EB is inhibited by LIF , CNTF , OSM as well as by combinations or fusions of IL-6 with the extracellular portion of the soluble IL-6 receptor (sIL-6R) . 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 , is an efficient inducer of myelin gene expression in embryonic Schwann cells and an activator of myelin gene promoters . 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
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 , murine ROSA 11 ES cells, removed from the feeder layer, were induced to form EBs which were then subjected to selection for neural precursors 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. 1A). Very few differentiating cells were seen at this stage (not shown), less than 1% staining for GFAP (astrocytes) or ?III-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 . 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).
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 1). 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 , 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. 1B). On day 7 (i.e., 3 days after removal of FGF), O4+ oligodendrocyte progenitors became apparent in the control cultures (Fig. 1C), but their number and their size was much larger in the presence of IL-6R/IL-6 (Fig. 1D). 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 1). 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. 1F), 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. 1E).
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. 2A–2B) 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. 2B). The percentage of O4+ cells rose from 0.8% to 7.3%, or 9.1-fold in response to the cytokine (Table 1). The percentage of NG2+ cells was also increased in response to IL-6R/IL-6 but only by 2.7-fold (Table 1). 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. 2C–2D), the increase in NG2+ cells was similar (percentage increase of 2.5-fold). Although NG2 may also be present in multipotent neural progenitors , the NG2+ cells in the outgrowth were mostly multipolar or branched (Figs. 2C–2D 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. 2D 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-?III we did not observe significant changes in the density of the neuron axonal network in the outgrowth surrounding the neurospheres (Figs. 2E–2F). 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.
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 1. 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-?III-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. 3A), which were spread among the underlying layer of cells outgrowing from the neurosphere (Fig. 3C). 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. 3B, 3D). The O4+ oligodendrocytes in the IL-6R/IL-6 treated cultures reached much larger size than in the control cultures (Fig. 4, 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 1). 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.
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.
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. 3). 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 1. 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. 4C–4D, 4F). Such large membranes were not seen in untreated cultures (Figs. 4A–4B, 4E). Second, IL-6R/IL-6 enhanced the development of mature O1+ oligodendrocytes, whose size was considerably increased as compared to the control cultures (Fig. 5, 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.
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. 5E), than in the control cultures where only weakly labeled and small size cells were seen (Fig. 5D). 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 . Olig-1 is expressed early and appears specifically required for the development and maturation of oligodendrocytes . Sox10 is also expressed early in the oligodendrocyte lineage and is a transcription factor acting on the promoters of myelin genes . 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. 6, 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. 6, 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. 6, lane 4). Furthermore, an induction of MBP RNA was observed in response to IL-6R/IL-6 (Fig. 6, 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.
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
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).
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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@weizmann.ac.il
ABSTRACT
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 . 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 ; B) early bipolar progenitors or oligodendrocyte-type-2 astrocyte (O-2A) 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 ; 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 . 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 . Some O4+ oligodendrocytes can then be derived provided tri-iodothyronine (T3) is added in analogy to the effect of T3 on brain cell cultures . Platelet derived growth factor (PDGF) promotes proliferation of brain glial precursors, in cooperation with epidermal growth factor (EGF) . Similarly, on EB cells, combinations of FGF-2 with EGF and PDGF-AA provide more efficient growth of A2B5+ O-2A progenitors . Yet another approach uses retinoic acid (RA) to induce neural precursors in mouse EB cultures .
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 . Similarly, oligospheres were obtained from RA-induced mouse EB cell cultures . 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 . In these procedures, the growth factors used to obtain progenitors actually inhibit their differentiation . 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 . 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 , whereas in others it increased astrocytes and also survival and proportion of GalC+, O1+ and MBP+ cells in the cultures . 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 ( for CNTF, LIF, Oncostatin M) or as a homodimer (for IL-6, IL-11) . Differentiation of murine ES cells into EB is inhibited by LIF , CNTF , OSM as well as by combinations or fusions of IL-6 with the extracellular portion of the soluble IL-6 receptor (sIL-6R) . 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 , is an efficient inducer of myelin gene expression in embryonic Schwann cells and an activator of myelin gene promoters . 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
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 , murine ROSA 11 ES cells, removed from the feeder layer, were induced to form EBs which were then subjected to selection for neural precursors 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. 1A). Very few differentiating cells were seen at this stage (not shown), less than 1% staining for GFAP (astrocytes) or ?III-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 . 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).
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 1). 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 , 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. 1B). On day 7 (i.e., 3 days after removal of FGF), O4+ oligodendrocyte progenitors became apparent in the control cultures (Fig. 1C), but their number and their size was much larger in the presence of IL-6R/IL-6 (Fig. 1D). 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 1). 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. 1F), 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. 1E).
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. 2A–2B) 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. 2B). The percentage of O4+ cells rose from 0.8% to 7.3%, or 9.1-fold in response to the cytokine (Table 1). The percentage of NG2+ cells was also increased in response to IL-6R/IL-6 but only by 2.7-fold (Table 1). 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. 2C–2D), the increase in NG2+ cells was similar (percentage increase of 2.5-fold). Although NG2 may also be present in multipotent neural progenitors , the NG2+ cells in the outgrowth were mostly multipolar or branched (Figs. 2C–2D 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. 2D 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-?III we did not observe significant changes in the density of the neuron axonal network in the outgrowth surrounding the neurospheres (Figs. 2E–2F). 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.
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 1. 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-?III-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. 3A), which were spread among the underlying layer of cells outgrowing from the neurosphere (Fig. 3C). 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. 3B, 3D). The O4+ oligodendrocytes in the IL-6R/IL-6 treated cultures reached much larger size than in the control cultures (Fig. 4, 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 1). 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.
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.
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. 3). 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 1. 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. 4C–4D, 4F). Such large membranes were not seen in untreated cultures (Figs. 4A–4B, 4E). Second, IL-6R/IL-6 enhanced the development of mature O1+ oligodendrocytes, whose size was considerably increased as compared to the control cultures (Fig. 5, 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.
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. 5E), than in the control cultures where only weakly labeled and small size cells were seen (Fig. 5D). 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 . Olig-1 is expressed early and appears specifically required for the development and maturation of oligodendrocytes . Sox10 is also expressed early in the oligodendrocyte lineage and is a transcription factor acting on the promoters of myelin genes . 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. 6, 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. 6, 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. 6, lane 4). Furthermore, an induction of MBP RNA was observed in response to IL-6R/IL-6 (Fig. 6, 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.
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
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).
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