Enlarged lateral ventricles and aberrant behavior in mice overexpressing PDGF-B in embryonic neural stem cells

Platelet-derived growth factor (PDGF) is important in central nervous system (CNS) development, and aberrant expression of PDGF and its receptors has been linked to developmental defects and brain tumorigenesis. We previously found that neural stem and progenitor cells in culture produce PDGF and respond to it by autocrine and/or paracrine signaling. We therefore aimed to examine CNS development after PDGF overexpression in neural stem cells in vivo.Transgenic mice were generated with PDGF-B under control of a minimal nestin enhancer element, which is specific for embryonic expression and will not drive adult expression in mice.The resulting mouse showed increased apoptosis in the developing striatum, which suggests a disturbed regulation of progenitor cells. Later in neurodevelopment, in early postnatal life, mice displayed enlarged lateral ventricles. This enlargement remained into adulthood and it was more pronounced in male mice than in transgenic female mice. Nevertheless, there was an overall normal composition of cell types and numbers in the brain and the transgenic mice were viable and fertile. Adult transgenic males, however, showed behavioral aberrations and locomotor dysfunction. Thus, a tightly regulated expression of PDGF during embryogenesis is required for normal brain development and function in mice.     

PDGF function in diverse neural crest cell populations

Activation of platelet derived growth factor (PDGF) receptors causes context-dependent cellular responses, including proliferation and migration, and studies in model organisms have demonstrated that this receptor family (PDGFRα and PDGFRβ) is required in many mesenchymal and migratory cell populations during embryonic development. One of these migratory cell populations is the neural crest, which forms cranial bone and mesenchyme, sympathetic neurons and ganglia, melanocytes and smooth muscle. Mice with disruption of PDGF signaling exhibit defects in some of these neural crest derivatives including the palate, aortic arch, salivary gland and thymus. Although many of these neural crest defects were identified many years ago, the mechanism of action of PDGF in neural crest remains controversial. In this review, we examine the current knowledge of PDGF function during neural crest cell (NCC) development, focusing on its role in the formation of different neural crest-derived tissues and the implications for PDGF receptors in NCC-related human birth defects.Key words: neural crest, aortic arch, PDGF, melanocytes, thymus, cleft palate, ventricular septal defects     

Effect of neurotrophic factors on neuronal stem cell death

Neural cell survival is an essential concern in the aging brain and many diseases of the central nervous system. Neural transplantation of the stem cells are already applied to clinical trials for many degenerative neurological diseases, including Huntington\'s disease, Parkinson\'s disease, and strokes. A critical problem of the neural transplantation is how to reduce their apoptosis and improve cell survival. Neurotrophic factors generally contribute as extrinsic cues to promote cell survival of specific neurons in the developing mammalian brains, but the survival factor for neural stem cell is poorly defined. To understand the mechanism controlling stem cell death and improve cell survival of the transplanted stem cells, we investigated the effect of plausible neurotrophic factors on stem cell survival. The neural stem cell, HiB5, when treated with PDGF prior to transplantation, survived better than cells without PDGF. The resulting survival rate was two fold for four weeks and up to three fold for twelve weeks. When transplanted into dorsal hippocampus, they migrated along hippocampal alveus and integrated into pyramidal cell layers and dentate granule cell layers in an inside out sequence, which is perhaps the endogenous pathway that is similar to that in embryonic neurogenesis. Promotion of the long term-survival and differentiation of the transplanted neural precursors by PDGF may facilitate regeneration in the aging adult brain and probably in the injury sites of the brain.     

Essential role for PDGF signaling in ophthalmic trigeminal placode induction

Much of the peripheral nervous system of the head is derived from ectodermal thickenings, called placodes, that delaminate or invaginate to form cranial ganglia and sense organs. The trigeminal ganglion, which arises lateral to the midbrain, forms via interactions between the neural tube and adjacent ectoderm. This induction triggers expression of Pax3, ingression of placode cells and their differentiation into neurons. However, the molecular nature of the underlying signals remains unknown. Here, we investigate the role of PDGF signaling in ophthalmic trigeminal placode induction. By in situ hybridization, PDGF receptor beta is expressed in the cranial ectoderm at the time of trigeminal placode formation, with the ligand PDGFD expressed in the midbrain neural folds. Blocking PDGF signaling in vitro results in a dose-dependent abrogation of Pax3 expression in recombinants of quail ectoderm with chick neural tube that recapitulate placode induction. In ovo microinjection of PDGF inhibitor causes a similar loss of Pax3 as well as the later placodal marker, CD151, and failure of neuronal differentiation. Conversely, microinjection of exogenous PDGFD increases the number of Pax3+ cells in the trigeminal placode and neurons in the condensing ganglia. Our results provide the first evidence for a signaling pathway involved in ophthalmic (opV) trigeminal placode induction.     

Mitogen and substrate differentially affect the lineage restriction of adult rat subventricular zone neural precursor cell populations.

The effects of specific mitogens and substrates on the proliferative capacity and the differentiated phenotypic plasticity of neural precursor cell populations isolated from the adult rat subventricular zone (SVZ) were examined. SVZ cells were grown on uncoated tissue culture plastic, extracellular matrix, or poly-D-ornithine with either laminin or fibronectin. SVZ neural precursor cells could not be generated with platelet-derived growth factor (PDGF), granulocyte macrophage colony stimulating factor, stem cell factor, heparin-binding epidermal growth factor (HB-EGF), granulocyte colony stimulating factor, or ciliary neurotrophic factor (CNTF), but could be with EGF, fibroblast growth factor 2 (FGF2), and FGF2 plus heparin. Varying combinations of substrate and mitogen resulted in very different expansion rates and/or lineage potential. Neurons, oligodendrocytes, and astrocytes differentiated from all cultures, but EGF-generated neural precursor cells were more restricted to an astrocytic lineage and FGF2-generated neural precursor cells had a greater capacity for neuronal differentiation. In both EGF- and FGF2-generated cell populations, CNTF increased the number of differentiated astrocytes, triiodothyronine oligodendrocytes, PDGF neurons, and brain-derived neurotrophic factor neurons only from EGF cells. Electrophysiological analysis of differentiated cells showed three distinct phenotypes, glial, neuronal, and presumed precursor cells, although the neuronal properties were immature. Collectively, these data indicate that CNS neural precursor cell populations isolated with different mitogens and substrates are intrinsically different and their characteristics cannot be directly compared.     

Platelet-derived growth factor receptor regulates salivary gland morphogenesis via fibroblast growth factor expression

A coordinated reciprocal interaction between epithelium and mesenchyme is involved in salivary gland morphogenesis. The submandibular glands (SMGs) of Wnt1-Cre/R26R mice have been shown positive for mesenchyme, whereas the epithelium is beta-galactosidase-negative, indicating that most mesenchymal cells are derived from cranial neural crest cells. Platelet-derived growth factor (PDGF) receptor alpha is one of the markers of neural crest-derived cells. In this study, we analyzed the roles of PDGFs and their receptors in the morphogenesis of mouse SMGs. PDGF-A was shown to be expressed in SMG epithelium, whereas PDGF-B, PDGFRalpha, and PDGFRbeta were expressed in mesenchyme. Exogenous PDGF-AA and -BB in SMG organ cultures demonstrated increased levels of branching and epithelial proliferation, although their receptors were found to be expressed in mesenchyme. In contrast, short interfering RNA for Pdgfa and -b as well as neutralizing antibodies for PDGF-AB and -BB showed decreased branching. PDGF-AA induced the expression of the fibroblast growth factor genes Fgf3 and -7, and PDGF-BB induced the expression of Fgf1, -3, -7, and -10, whereas short interfering RNA for Pdgfa and Pdgfb inhibited the expression of Fgf3, -7, and -10, indicating that PDGFs regulate Fgf gene expression in SMG mesenchyme. The PDGF receptor inhibitor AG-17 inhibited PDGF-induced branching, whereas exogenous FGF7 and -10 fully recovered. Together, these results indicate that fibroblast growth factors function downstream of PDGF signaling, which regulates Fgf expression in neural crest-derived mesenchymal cells and SMG branching morphogenesis. Thus, PDGF signaling is a possible mechanism involved in the interaction between epithelial and neural crest-derived mesenchyme.     

PDGF function in diverse neural crest cell populations

Activation of platelet derived growth factor (PDGF) receptors causes context-dependent cellular responses, including proliferation and migration, and studies in model organisms have demonstrated that this receptor family (PDGFRα and PDGFRβ) is required in many mesenchymal and migratory cell populations during embryonic development. One of these migratory cell populations is the neural crest, which forms cranial bone and mesenchyme, sympathetic neurons and ganglia, melanocytes, and smooth muscle. Mice with disruption of PDGF signaling exhibit defects in some of these neural crest derivatives including the palate, aortic arch, salivary gland, and thymus. Although many of these neural crest defects were identified many years ago, the mechanism of action of PDGF in neural crest remains controversial. In this review, we examine the current knowledge of PDGF function during neural crest cell (NCC) development, focusing on its role in the formation of different neural crest-derived tissues and the implications for PDGF receptors in NCC-related human birth defects.     

PDGFR alpha-positive B cells are neural stem cells in the adult SVZ that form glioma-like growths in response to increased PDGF signaling

Neurons and oligodendrocytes are produced in the adult brain subventricular zone (SVZ) from neural stem cells (B cells), which express GFAP and have morphological properties of astrocytes. We report here on the identification B cells expressing the PDGFRalpha in the adult SVZ. Specifically labeled PDGFRalpha expressing B cells in vivo generate neurons and oligodendrocytes. Conditional ablation of PDGFRalpha in a subpopulation of postnatal stem cells showed that this receptor is required for oligodendrogenesis, but not neurogenesis. Infusion of PDGF alone was sufficient to arrest neuroblast production and induce SVZ B cell proliferation contributing to the generation of large hyperplasias with some features of gliomas. The work demonstrates that PDGFRalpha signaling occurs early in the adult stem cell lineage and may help regulate the balance between oligodendrocyte and neuron production. Excessive PDGF activation in the SVZ in stem cells is sufficient to induce hallmarks associated with early stages of tumor formation.     

The JAK3 inhibitor WHI-P154 prevents PDGF-evoked process outgrowth in human neural precursor cells

The prospect of manipulating endogenous neural stem cells to replace damaged tissue and correct functional deficits offers a novel mechanism for treating a variety of CNS disorders. The aim of this study was to investigate pathways controlling neurite outgrowth in human neural precursor cells, in particular in response to platelet-derived growth factor (PDGF). PDGF-AA, -AB and -BB were found to initiate calcium signalling and produce robust increases in neurite outgrowth. PDGF-induced outgrowth of Tuj1-positive precursors was abolished by the addition of EGTA, suggesting that calcium entry is a critical part of the signalling pathway. Wortmannin and PD098059 failed to inhibit PDGF-induced outgrowth. Clostridium Toxin B increased the amount of PDGF-induced neurite branching but had no effect on basal levels. In contrast, WHI-P154, an inhibitor of Janus protein tyrosine kinase (JAK3), Hck and Syk, prevented PDGF-induced neurite outgrowth. PDGF activates multiple signalling pathways with considerable potential for cross-talk. This study has highlighted the complexity of the pathways leading to neurite outgrowth in human neural precursors, and provided initial evidence to suggest that calcium entry is critical in producing the morphological changes observed.     

The effect of controlled growth factor delivery on embryonic stem cell differentiation inside fibrin scaffolds

The goal of this project was to develop 3-D biomaterial scaffolds that present cues to direct the differentiation of embryonic stem (ES) cell-derived neural progenitor cells, seeded inside the scaffolds, into mature neural phenotypes, specifically neurons and oligodendrocytes. Release studies were performed to determine the appropriate conditions for retention of neurotrophin-3 (NT-3), sonic hedgehog, and platelet-derived growth factor (PDGF) by an affinity-based delivery system incorporated into fibrin scaffolds. Embryoid bodies containing neural progenitors were formed from mouse ES cells, using a 4−/4+ retinoic acid treatment protocol, and then seeded inside fibrin scaffolds containing the drug delivery system. This delivery system was used to deliver various growth factor doses and combinations to the cells seeded inside the scaffolds. Controlled delivery of NT-3 and PDGF simultaneously increased the fraction of neural progenitors, neurons, and oligodendrocytes while decreasing the fraction of astrocytes obtained compared to control cultures seeded inside unmodified fibrin scaffolds with no growth factors present in the medium. These results demonstrate that such a strategy can be used to generate an engineered tissue for the potential treatment of spinal cord injury and could be extended to the study of differentiation in other tissues.     

A Pdgf‐cCreERT2 knock‐in mouse model for tracing PDGF‐C cell lineages during development

PDGF‐C, a member of the platelet‐derived growth factor (PDGF) family, plays important roles in the development of craniofacial structures, the neural system, the vascular system, and tumors. PDGF‐C could also be required for the regulation of certain types of stem or progenitor cells as suggested by its expression in the regions where these cells are located. To further characterize the role of PDGF‐C in development, we generated a Pdgf‐c^CreERT2 mouse strain, in which a tamoxifen‐inducible Cre (CreERT2) cDNA was specifically targeted into the Pdgf‐c genomic locus and controlled by the endogenous Pdgf‐c regulatory elements. We also showed that Cre activity in this mouse strain could be specifically induced by tamoxifen, which allowed the fate of PDGF‐C‐expressing cells to be traced at various stages of development. Using this model system, we demonstrated for the first time that PDGF‐C‐expressing cells could be multipotent, generating multiple cell lineages required for the formation of the cerebellum. Therefore, the Pdgf‐c^CreERT2 mouse strain generated in this study will be a valuable transgenic tool for exploring the function of PDGF‐C in development and stem cell biology.     

DNA synthesis in U-2 OS human osteosarcoma cells is independent of PDGF binding to functional cell surface receptors

Previous studies have shown that suramin reveals specific PDGF binding sites on U-2 OS human osteosarcoma cells. Studies presented here indicate that U-2 OS cells pretreated with suramin internalize and degrade 125I-PDGF and respond to PDGF by increased tyrosine kinase activity and amino acid transport. However, DNA synthesis in these cells is not reduced by incubation with the PDGF blocking agent suramin and is not stimulated by exogenous PDGF. These data indicate that U-2 OS cells possess functional PDGF receptors but that high levels of DNA synthesis in these cells is unrelated to the binding of secreted PDGF to these cell surface receptors. Thus, it is unlikely that the PDGF mitogen produced by U-2 OS cells stimulates proliferation through an autocrine mechanism involving secretion and subsequent binding to PDGF receptors.     

Control of progenitor cell number by mitogen supply and demand

BACKGROUND: Much is known about how cell proliferation is controlled at the single cell level, but much less about the control of cell numbers in developing populations. Cell number might be determined by an intracellular division limiter or, alternatively, by the availability of mitogens or other factors outside the cell. We investigated the relative importance of intracellular and extracellular controls for one well-defined population of neural precursor cells, namely the glial progenitors that give rise to oligodendrocytes in the mouse spinal cord. RESULTS: We found by cumulative BrdU labeling in vivo that the progenitor cell division cycle slows down markedly as their numbers increase during embryogenesis. When cultured in saturating PDGF, the main mitogen for these cells, their cell cycle accelerated and was independent of their prior rate of division in vivo. This shows that mitogens are limiting in vivo, and suggests that division normally slows down because the PDGF concentration declines. In PDGF-transgenic mice, cell number was proportional to the PDGF supply and apparently unsaturable; at ten times the normal rate of supply, cell number was still increasing but the animals were no longer viable. CONCLUSIONS: Progenitor cell proliferation in the embryo is limited by environmental factors, not a cell-intrinsic mechanism. The linear relationship between PDGF supply and final cell number strongly suggests that cells deplete the mitogenic activity in their environment at a rate proportional to the total number of cells. The cells might simply consume the available PDGF or they might secrete autocrine inhibitors, or both.     

PDGF beta-receptor stimulates tyrosine phosphorylation of GAP and association of GAP with a signaling complex

Platelet-derived growth factor (PDGF) stimulated the tyrosine phosphorylation of the GTPase activating protein (GAP) in 3T3 cells and in CHO cells expressing wild-type PDGF receptors, but not in several CHO cell lines expressing mutant receptors defective in transmitting mitogenic signals. Following PDGF treatment of cells, GAP physically associated with the PDGF receptor and with Raf-1, phospholipase c-gamma, and PI-3 kinase, suggesting that PDGF induced the formation of complexes of signaling molecules. The association of GAP with the PDGF receptor and the phosphorylation of GAP with the PDGF receptor and the phosphorylation of GAP were reconstituted in vitro using purified protein and in insect cells expressing murine PDGF receptor and human GAP. However, in cells transformed by activated c-Ha-ras, which are defective in certain responses to PDGF, GAP failed to associate with the PDGF receptor or increase its phosphotyrosine content in response to PDGF. The association of GAP with ligand-activated PDGF receptors may directly link PDGF and ras signaling pathways.     

多瘤病毒癌基因产物对PDGF刺激应答的影响

多瘤病毒癌基因产物对ＰＤＧＦ刺激应答的影响于爱鸣冈野幸雄＊于秉治（中国医科大学基础医学院生化教研室,沈阳１１０００１）＊（日本岐阜大学医学部生化学教室,日本岐阜５００）多瘤病毒癌基因主要表达三种转化蛋白．其中,中等分子肿瘤抗原（ｍｉｄｄｌｅｓｉｚｅｄ...     

Effect of platelet-derived growth factor on DNA synthesis and gene expression in bone marrow stromal cells derived from adult and old rats

The effects of platelet-derived growth factor (PDGF) on DNA synthesis and mRNA expression of osteoblast markers in marrow stromal cells derived from adult (6 months) and old (24 months) rats were examined. Treatment of stromal cells from adult rats with dexamethasone induced the appearance of osteoblast-like cells. PDGF partially also inhibited the differentiation of stromal cells induced by dexamethasone. In cultures of serum-starved stromal cells, PDGF stimulated [³H]-thymidine incorporation into DNA in a dose-dependent manner with a maximum stimulation of 15-fold at 500 ng/ml. By comparison, insulin-like growth factor (IGF-I) has a small effect on [³H] -thymidine incorporation. The effect of PDGF and IGF-I on DNA synthesis was additive. Treatment of the confluent stromal cells from adult rats with PDGF increased the mRNA level of osteopontin fourfold without any significant effect on alkaline phosphatase and type I collagen mRNAs. In contrast, dexamethasone stimulated the mRNA expression of alkaline phosphatase, type I collagen, and osteopontin 2.1-, 2.3-, and 14-fold, respectively. Addition of PDGF to dexamethasone-treated cells failed to induce any further increase in osteopontin expression whereas the expression of alkaline phosphatase and type I collagen was partially reduced. The expression of osteocalcin mRNA was negligible in stromal cells but stimulated several fold by dexamethasone and 1,25(OH)₂D₃. PDGF inhibited drastically the elevation of osteocalcin mRNA. In contrast, IGF-I stimulated type I collagen expression 100% without any appreciable effect on the expression of osteopontin and alkaline phosphatase. The stimulatory effect of PDGF on osteopontin expression was augmented by IGF-I. Furthermore, PDGF attenuated the stimulatory effect of IGF-I on type I collagen expression. The responses of cultured cells from old rats to growth factors were also examined. PDGF or PDGF plus IGF-I increased [³H]-thymidine incorporation in stromal cells from old rats but to a lesser extent. However, PDGF was equally effective in stimulating osteopontin expression in cells from both adult and old rats. We concluded that PDGF is a potent mitogen but that the response of stromal cells from old rats is impaired. In addition, PDGF stimulates osteopontin expression in stromal cells and this effect is not age dependent. © 1995 Wiley-Liss, Inc.     

Glucose reduces PDGF production and cell proliferation of cultured vascular endothelial cells

The effect of glucose on PDGF production and cell proliferation was studied on cultured bovine aortic endothelial cells. PDGF levels were measured using an enzyme-linked immunosorbent assay technique newly developed in our laboratory. The cell proliferation rate was determine on the basis of 3H-thymidine incorporation into cellular DNA. PDGF levels in culture medium were below the detection limit of the assay. However, PDGF levels were measurable in cultured endothelial cells at confluence. Both PDGF production and thymidine incorporation were significantly reduced in the endothelial cells cultured with high concentrations of glucose. These results suggest that reduced PDGF production and cell proliferation may be involved in altered vascular endothelial function in diabetics.     

Passaging human neural stem cells

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro provides a means to investigate their utility as cell transplants for therapeutic purposes as well as to explore many fundamental processes of human neural development and pathology. This protocol presents a simple method of culturing and passaging hNSPCs in hopes of standardizing this technique and increasing reproducibility of human stem cell research. The hNSPCs we use were isolated from cadaveric postnatal brain cortices by the National Human Neural Stem Cell Resource and grown as adherent cultures on flasks coated with fibronectin (Palmer et al., 2001; Schwartz et al., 2003). We culture our hNSPCs in a DMEM:F12 serum-free media supplemented with EGF, FGF, and PDGF and passage them 1:2 approximately every seven days. Using these conditions, the majority of the cells in the culture maintain a bipolar morphology and express markers of undifferentiated neural stem cells (such as nestin and sox2).