Regulation of Rat Schwann Cell Po Expression and DNA Synthesis by Insulin-like Growth Factors In Vitro

Myelination by Schwann cells is likely to be regulated in Vitro by positive and negative epigenetic factors. In Vitro , the positive regulation of myelin differentiation, in particular expression of the major myelin protein P_o, can be mimicked by cAMP elevating agents, while serum, transforming growth factor (TGF)βs, and fibroblast growth factor (FGF)2 have been shown to exert a negative effect on this differentiation. Growth factors which promote P_o induction have not, however, been identified previously. Using a forskolin concentration (0.4 μM) which alone produces little P_o mRNA or protein induction, we show that insulin-like growth factor (IGF)-I, IGF-II and high concentrations of insulin promote high levels of P_o induction, although in the absence of forskolin they have no effect. Another event related to Schwann cell differentiation, induction of galactocerebroside expression in response to cAMP analogues, is also potentiated by IGFs. In a different context, IGFs regulate Schwann cell DMA synthesis. We find that in defined medium forskolin plus FGF2, TGFβ or platelet-derived growth factor (PDGF) BB causes minimal DNA synthesis in the absence of IGFs and that IGFs act as potent mitogens under these conditions. IGFs also potentiate DNA synthesis induced by β isoforms of neu-differentiation factors (NDFs), although in this case considerable DNA synthesis occurs even in the absence of IGF. These results show that IGFs can act as powerful stimulators of both proliferation and differentiation in Schwann cells, and that the total growth factor input determines which of these pathways IGFs will promote.     

Fibroblast Growth Factors and Insulin Growth Factors Combine to Promote Survival of Rat Schwann Cell Precursors Without Induction of DNA Synthesis

In embryonic rat nerves, we recently identified an early cell in the Schwann cell lineage, the Schwann cell precursor. We found that when these cells were removed from contact with axons they underwent rapid apoptotic death, and that in a proportion of the cells this death could be prevented by basic fibroblast growth factor (bFGF, FGF-2). We now report that 100% of Schwann cell precursors isolated from peripheral nerves of 14-day-old-rat embryos can be rescued by a combination of insulin-like growth factor (IGF) 1 or 2 in combination with either acidic FGF (aFGF, FGF-1), bFGF or Kaposi's sarcoma FGF (K-FGF; FGF-4). The precursors display an absolute requirement for both an IGF and an FGF to achieve maximal survival. Elevation of intracellular levels of cAMP by forskolin does not result in a significant shift in the IGF/FGF dose-response curves. In contrast, the percentage of precursors rescued by FGF in the presence of insulin is dramatically increased by elevation of cAMP. These growth factor combinations did not stimulate DNA synthesis significantly in Schwann cell precursors. These findings show that cooperation between growth factors is required to suppress cell death in Schwann cell precursors, and suggest that survival and DNA synthesis are regulated by distinct growth factor combinations in these cells. The observations are consistent with the idea that survival regulation by FGFs and IGFs plays an important role in the development of glial cells in early embryonic nerves.     

Response of Schwann cells to mitogens in vitro is determined by pre-exposure to serum,time in vitro,and developmental age

We compared the mitogenic response of Schwann cells freshly isolated from adult, neonatal, and embryonic nerves, and compared these cells with cells that had been cultured in serum for 5 days. DNA synthesis in response to growth factors was measured using bromodeoxyuridine and immunocytochemistry. Freshly isolated adult Schwann cells were unresponsive to growth factors with or without forskolin to elevate intracellular cAMP levels. After 5 days of culture in serum, or alternatively in defined medium containing fibroblast growth factor 2 plus forskolin, or neu-differentiation factor β2, adult cells were responsive to mitogens, whereas cells cultured in defined medium alone remained unresponsive. Serum also increased expression of type 1 fibroblast growth factor receptor. Freshly isolated embryonic and neonatal Schwann cells in contrast responded to growth factors even in the absence of forskolin. This responsiveness changed with time in culture. Neonatal cells cultured for 5 days in defined medium in the presence or absence of serum no longer responded to FGF alone, but required forskolin for a mitogenic response. Thus, the response of freshly isolated cells to mitogens is developmentally regulated; extrinsic signals are required to render adult cells responsive to mitogens; and with time in culture, neonatal cells develop a requirement for cAMP elevation for mitogenic response. GLIA 20:219–230, 1997. © 1997 Wiley-Liss, Inc.     

Growth factors for human glial cells in culture

Using a new double-labeling immunofluorescence technique, we assessed various growth factors on their ability to promote proliferation of cultured human glial cells. Cells studied were fetal astrocytes, fetal Schwann cells, adult astrocytes, and adult oligodendrocytes. Effective agents for fetal astrocytes were glial growth factor from the bovine pituitary, platelet-derived growth factor, fibroblast growth factor, and 4 beta-phorbol 12,13-dibutyrate. For fetal Schwann cells, mitogens were glial growth factor from the bovine pituitary, platelet-derived growth factor, nerve growth factor, and 4 beta-phorbol 12,13-dibutyrate. Adult astrocytes and oligodendrocytes did not normally divide in culture, and none of the agents tested were effective in inducing their proliferation. The report that interleukin-2 was a mitogen for oligodendrocytes could not be replicated in the present study on any of the glial cell types.     

Mitogn induced proliferation of isolated adult mouse schwann cells

The proliferation of neonatal Schwann cells (SCs) in response to mitogenic agents has been well analyzed in vitro, but a limited range of mitogens have been defined. We investigated whether three identified neonatal SC mitogens [glial growth factor (GGF), platelet-derived growth factor BB (PDGF-BB), and basic fibroblast growth factor (bFGF)] are required to stimulate mitosis of adult SCs. Adult SCs were isolated from mouse sciatic nerves by mechanical and chemical dissociation, following three experimental steps: (1) culturing the dissociated cells for 24 hr in 10% FCS-F12 medium, (2) culturing these cells in serum-free medium for the next 48 hr, and (3) purifying adult SCs by differential adhesion. We describe a new method for preparation of SCs from peripheral nerves of adult mouse that provides 99.5% pure SCs populations at cell yields of greater than 3 × 10³ cells/mg of starting nerve wet weight within 5 culture days. Although mitosis of SCs in culture in response to mitogens requires the presence of serum, the complex nature of serum renders difficult a complete analysis of mitogens required for SCs DNA synthesis, so we examined the proliferating response of adult SCs to GGF, PDGF-BB, and bFGF in serum-free medium. GGF alone had mitogenicity for adult SCs in a dose-dependent manner, and synergistic activation coupling with forskolin was not observed. Neither PDGF-BB nor bFGF was mitogenic for adult SCs when used alone or with forskolin. Measurement of intracellular cyclic AMP levels in SCs cultured with these growth factors showed that cAMP levels were not changed by GGF and bFGF, and PDGF-BB reduced the cAMP level to half of basal one. This study demonstrated that adult SCs could proliferate without serum factors and forskolin in response to GGF, and adult SCs were activated by GGF through a signal transduction pathway separate from the cyclic AMP-dependent system. In addition, PDGF-BB or bFGF has no mitogenic effect on adult SCs under serum-free conditions. © 1995 Wiley-Liss, Inc.     

Platelet-derived growth factor and regulation of Schwann cell proliferation in vivo.

To examine the role of platelet-derived growth factor (PDGF) in the in vivo regulation of Schwann cell proliferation, steady-state levels of mRNAs encoding PDGF A and B chains, and PDGF alpha and beta receptors were measured in immature and adult rat sciatic nerves and in cultured rat Schwann cells. PDGF B chain and PDGF beta receptor mRNAs are present in immature rat sciatic nerves and to a lesser extent in adult rat nerves. Short-term cultures of neonatal rat Schwann cells express PDGF beta receptor mRNA, but not PDGF B chain mRNA, and are stimulated to synthesize DNA by addition of PDGF BB to the medium. These data indicate that PDGF BB is a developmentally regulated paracrine growth factor for rat Schwann cells. Very long-term cultures of rat Schwann cells, which have lost normal dependence on exogenous growth factors, express PDGF B chain mRNA as well as mRNAs encoding the PDGF alpha and beta receptors, suggesting that, under these circumstances, PDGF BB also act as an autocrine growth factor. PDGF A chain mRNA is present in both immature and adult rat sciatic nerves and is expressed by primary and secondary cultures of rat Schwann cells as well. However, because the abundance of PDGF alpha receptor mRNA is very low in rat Schwann cells, PDGF AA is not likely to be a significant autocrine growth factor for rat Schwann cells.     

Mitogenic effects of platelet-derived growth factor,fibroblast growth factor,transforming growth factor-β, and heparin-binding serum factor for adult mouse Schwann cells

Mitogenic effects of fetal calf serum (FCS), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), transforming growth factor-β (TGF-β), and forskolin to adult mouse Schwann cells were examined by bromodeoxyuridine (BrdU) incorporation and double immunofluorescence for S100 and BrdU. PDGF-BB, basic FGF, and TGF-β1 and β2 were all mitogenic for Schwann cells in media containing FCS. Forskolin suppressed the mitogenic activity of these factors. In serum-free media, PDGF-BB and bFGF were also mitogenic, but TGF-β1 and β2 were not. Heparin-binding fractions of FCS obtained by heparin-Sepharose chromatography synergized with TGF-β1 and β2 to produce a mitogenic response. Since PDGF-BB, acidic FGF, and basic FGF were not detected in these fractions by immunoabsorption and immunoblot assays, the presence of unidentified heparin-binding molecules in FCS bioactive for adult mouse Schwann cells is suggested. © 1994 Wiley-Liss, Inc.     

Transforming growth factor-beta and gamma-interferon have dual effects on growth of peripheral glia

The influence of transforming growth factor-beta (TGF-beta) and gamma-interferon on DNA synthesis in Schwann cells and enteric glia in culture has been studied. TGF-beta stimulated the DNA synthesis of short-term (less than 2 weeks in culture) Schwann cells, whereas gamma-interferon was ineffective. The stimulatory effect of TGF-beta was additive to the stimulation of DNA synthesis due to axonal membrane fragments. In contrast to their effect on short-term Schwann cells, both TGF-beta and gamma-interferon inhibited DNA synthesis in enteric glial cells and in long-term (over 3 months in culture) Schwann cells. When short-term Schwann cells were stimulated to divide by axolemma or glial growth factor, gamma-interferon did not inhibit this enhanced DNA synthesis although it suppressed DNA synthesis induced by cAMP analogues. These results raise the possibility that TGF-beta and gamma-interferon might have a role in controlling glial proliferation during development and/or regeneration of the peripheral nervous system.     

Differentiation of rat adipose tissue-derived stem cells into Schwann-like cells in vitro

In this study, we explored the competence of adipose-derived stem cells to differentiate into Schwann cells in vitro. Rat adipose-derived stem cells were sequentially treated with various factors beta-mercaptoethanol, all-trans-retinoic acid, followed by a mixture of forskolin, basic fibroblast growth factor, platelet-derived growth factor and heregulin. We found that differentiated adipose-derived stem cells displayed the morphology of Schwann cells. Western blotting and dual immunofluorescence staining confirmed that they produced proteins characteristic for Schwann cells, including S100 and glial fibrillary acidic protein. Furthermore, differentiated adipose-derived stem cells could enhance neurite outgrowth in coculture with sensory neurons. These results demonstrate that adipose-derived stem cells can differentiate into Schwann-like cells with morphological, phenotypic, and functional characteristics of Schwann cells.     

体外诱导获得雪旺细胞的可靠性研究

目的 研究骨髓基质细胞(BMSCs)在体外条件下向周围神经雪旰细胞(SCs)分化的可靠性. 方法 分离提取SD大鼠股骨和胫骨部位BMSCs.利用其贴壁生长的特性.培养纯化,传代扩增.用复合诱导因子(β.巯基乙醇+全反式维甲酸+血小板凝集抑制剂+m小板源性生长因子+碱性成纤维细胞生长因子)在体外诱导BMSCs分化.免疫细胞化学方法 检测P75、S-100及胶质原纤维酸性蛋白(GFAP)的表达,荧光实时定量PCR检测P75、S100及CD104的表达. 结果诱导后的BMSCs形态类似SCs,免疫荧光染色鉴定其具有SCs性质,表达SCs的表面标志物(GFAP、S100和P75).荧光实时定量PCR结果显示诱导后BMSCs S-100、CD104的表达量达到了SCs的表达量水平,但P75的表达量与SCs的表达量水平还有较大差距. 结论 体外诱导BMSCs可部分获得SCs的特征,传代后恢复至未诱导状态,这种预诱导加复合因子诱导的方法 尚待完善.     

Schwann Cells Secrete a PDGF-like Factor: Evidence for an Autocrine Growth Mechanism involving PDGF

We have investigated the influence of platelet-derived growth factor (PDGF) in peripheral nervous system gliogenesis using two types of Schwann cell cultures. Short-term Schwann cell cultures grow very slowly, but when maintained in culture for several months the division rate of some cells increases, and cell lines can be established. We show that Schwann cells in both short- and long-term culture possess PDGF receptors and synthesize DNA in response to PDGF. Competitive binding experiments show that Schwann cells express mainly PDGF beta-receptors and respond better to PDGF-BB than to PDGF-AA. Conditioned media from short- and long-term Schwann cell cultures contain PDGF-like mitogenic activity, and anti-PDGF immunoglobin partially inhibits DNA synthesis in long-term Schwann cell cultures. Antibody neutralization experiments and Northern blot analyses both indicate that the predominant PDGF isoform in these cultures is PDGF-BB. PDGF-like activity is also detected in extracts of rat sciatic nerve. Taken together, these results suggest that PDGF-BB may stimulate Schwann cell proliferation in an autocrine manner during normal development.     

cAMP-dependent protein kinase A is required for Schwann cell growth: Interactions between the cAMP and neuregulin/tyrosine kinase pathways

Schwann cell proliferation is stimulated by contact with neurons or exposure to growth factor ligands for tyrosine kinase receptors, effects of which are potentiated by cAMP. Here we show that treatment of rat Schwann cells with recombinant human glial growth factor 2 (rhGGF2), but not with other mitogenic factors, transiently increases intracellular cyclic AMP (cAMP), with maximal elevation at the G0/G1 boundary. The cAMP-dependent protein kinase (PKA) inhibitor H-89 strongly antagonized GGF- and neuron-induced Schwann cell proliferation, with maximum inhibition observed at G0/G1. H-89 also inhibited Schwann cell proliferation induced by growth factors that did not increase intracellular cAMP. Stimulation of Schwann cells with rhGGF2 resulted in 70-fold activation of MAP kinase; forskolin treatment resulted in a 50% decrease in MAP kinase activity but did not alter Raf-1 phosphorylation on Ser-43. These results demonstrate that the MAP kinase cascade represents an intersection between receptor tyrosine kinase and cAMP signaling pathways in Schwann cells and that PKA plays a critical role in Schwann cell cycle progression. J. Neurosci. Res. 49:236–247, 1997. © 1997 Wiley-Liss, Inc.     

Apoptosis occurs in the oligodendroglial lineage,and is prevented by basic fibroblast growth factor

During the perinatal period, oligodendroglial precursor cells proliferate rapidly, then cease dividing and differentiate into oligodendroglia. Many of these newly formed oligodendroglia are destined to die. We now demonstrate that oligodendroglia generated in passaged cultures of rat forebrain oligodendroglial precursor cells after removal of basic fibroblast growth factor (basic FGF) from the medium often undergo internucleosomal DNA nicking and nuclear fragmentation, features characteristic of apoptosis. These alterations are rare in cultures maintained continuously in basic FGF. As in many other cellular lineages susceptible to apoptosis, these degenerative changes can be prevented by treatment with the endonuclease antagonist, aurintricarboxylic acid, or by inhibiting de novo RNA or protein synthesis. Supplementation of the basic FGF-free medium with insulin, insulin-like growth factor-1, platelet-derived growth factor, or ciliary neuronotrophic growth factor also diminishes DNA nicking. Both oligodendroglial differentiation and DNA nicking are induced in basic FGF-treated cultures by inhibiting DNA synthesis with aphidicholin or excess thymidine, thus suggesting a close linkage between the anti-apoptotic, anti-differentiation, and mitogenic effects of basic FGF on the oligodendroglial lineage. © 1995 Wiley-Liss, Inc.     

Mitogenic response of rat Schwann cells to fibroblast growth factors is potentiated by increased intracellular cyclic AMP levels.

Rat sciatic nerve Schwann cells either do not proliferate, or proliferate very slowly, in medium containing 10% fetal bovine serum (FBS). They were previously shown to respond only to a limited number of mitogens associated with cells of central and peripheral nervous systems, which appeared to be distinct from FGFs and PDGF, and to agents that raise intracellular cAMP levels. In a basal medium consisting of 75% DMEM, 25% Ham's F-12, 5 nM sodium selenite, 50 microM 2-amino ethanol, and 2 mM histidine, supplemented with 5% FBS, we showed that aFGF, bFGF, and PDGF were all capable of stimulating Schwann cell growth and the stimulation was greatly potentiated by forskolin and dibutyryl-cAMP. In addition, pretreating culture surface with purified matrix proteins such as laminin, fibronectin, or type 1 collagen, was necessary for obtaining a better cellular response to the mitogenesis of these growth factors even in 10% FBS. Our results clearly indicated that providing a suitable medium and substratum, aFGF, bFGF and PDGF are mitogens for rat sciatic nerve Schwann cells in medium with and without forskolin or dibutyryl-cAMP.     

Interferon-gamma, tumor necrosis factor-alpha, and transforming growth factor-beta inhibit cyclic AMP-induced Schwann cell differentiation.

Schwann cells differentiate in vivo in response to contact with axons, and cAMP simulates some of these aspects of differentiation in vitro, particularly morphologic changes and expression of certain phenotypic molecules. Unfractionated inflammatory cytokines inhibit cAMP-induced Schwann cell expression of galactolipids (Gal). We sought to identify which cytokines were responsible for this inhibition and to determine whether other phenotypic indicators of Schwann cell differentiation were also affected. Neonatal rat Schwann cells were incubated in vitro with 1 mM 8 Bromo cAMP (8 Br cAMP) with or without the addition of interleukin-1 alpha (IL-1 alpha), IL-1 beta, IL-2, IL-6, tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), or transforming growth factor-beta (TGF-beta). Cells were then examined for morphologic changes and for expression of surface Gal and low-affinity nerve growth factor receptor (NGFRp75), employing indirect immunofluorescence. 8 Br cAMP induced Schwann cell upregulation of Gal, downregulation of NGFRp75, and the cells became enlarged and somewhat amorphous and irregular in appearance. Cells treated with IFN-gamma or TNF-alpha alone were more bipolar and more evenly distributed on coverslips than were control cells, whereas TGF-beta alone induced elongated cells often in a swirling pattern. None of the cytokines alone induced upregulation of Gal or downregulation of NGFRp75. TNF-alpha, IFN-gamma, and TGF-beta inhibited the 8 Br cAMP-induced morphologic changes, as well as the upregulation of Gal and downregulation of NGFRp75. The other cytokines had no effects on Gal or NGFRp75 expression. Thus, these three cytokines, which are present in inflammatory lesions in the peripheral nervous system, are capable of inhibiting Schwann cell differentiation.     

Effect of dibutyryl cAMP and several reagents on apoptosis in PC12 cells induced by a sialoglycopeptide from bovine brain

The sialoglycopeptide (SGP) prepared from bovine brain induces apoptosis in rat pheochromocytoma (PC12) cells. This apoptosis accompanies a decrease in cell growth, DNA fragmentation to oligonucleotide repeats, and morphological changes involving cell shrinkage. Although the growth of PC12 cells was maintained by nerve growth factor (NGF) or basic fibroblast growth factor (bFGF), the apoptosis induced by SGP occurred in the presence of these reagents. The addition of macromolecule synthesis inhibitors or depolarization of membrane potential by extracellular K⁺ did not prevent apoptosis. Apoptosis was prevented only by a cAMP analog, dibutyryl cAMP, or high concentrations of serum.     

Influence of basic fibroblast growth factor on carbonic anhydrase expression by rat glial cells in primary culture

Modifications of the morphology, the proliferation and the synthesis of carbonic anhydrase of glial cells in primary cultures maintained in defined medium have been investigated under the action of basic fibroblast growth factor. Cultures contained essentially three cell types: astrocytes which expressed glial fibrillary acidic protein, oligodendrocytes which were characterized by the presence of carbonic anhydrase and precursor cells in which these two proteins were detected by immunocytochemistry. In the presence of basic fibroblast growth factor astrocytes and oligodendrocytes underwent morphological changes, characterized by a fibrous aspect; astroglial cells acquired essentially several long processes and oligodendroglial cells formed generally two long processes. The factor increased the proliferation of these two cell types. The quantity of carbonic anhydrase per oligodendrocyte was enhanced in treated cultures. The double-stained precursor cells were present between days 7 and 11 of culture in defined medium, while in the presence of fibroblast growth factor these cells were more numerous and were still present after 14 days. The basic fibroblast growth factor stimulated the proliferation of these young glial cells and modified their morphology. But the differentiation of precursor cells towards one glial cell type appeared to be delayed.     

Contrasting effects of mitogenic growth factors on oligodendrocyte precursor cell migration

We have examined the effects of the mitogenic growth factors platelet derived growth factor (PDGF), basic fibroblast growth factor (bFGF) and glial growth factor-2 (GGF-2) on oligodendrocyte precursor migration. In an agarose drop migration assay PDGF and bFGF stimulated migration while GGF-2 had no effect. The migration-enhancing effect of bFGF cannot be blocked by neutralising antibodies against PDGF, confirming that this effect is direct and not mediated via upregulation of PDGF receptors. Based on our results, we propose a model in which the differing effects of PDGF and GGF-2 ensure appropriate numbers of oligodendrocyte precursor cells in the vicinity of axons to be myelinated during development. GLIA 19:85–90, 1997. © 1997 Wiley-Liss, Inc.