Epidermal Growth Factor (EGF) Promotes the In Vitro Differentiation of Neural Crest Cells to Neurons and Melanocytes

Proliferation of neural crest (NC) stem cells and their subsequent differentiation into different neural cell types are key early events in the development of the peripheral nervous system. Soluble growth factors present at the sites where NC cells migrate are critical to the development of NC derivatives in each part of the body. In the present study, we further investigate the effect of microenvironmental factors on quail trunk NC development. We show for the first time that EGF induces differentiation of NC to the neuronal and melanocytic phenotypes, while fibroblast growth factor 2 (FGF2) promotes NC differentiation to Schwann cells. In the presence of both EGF and FGF2, the neuronal differentiation predominates. Our results suggest that FGF2 stimulates gliogenesis, while EGF promotes melanogenesis and neurogenesis. The combination of both growth factors stimulates neurogenesis. These findings suggest that these two growth factors may play an important role in the fate decision of NC progenitors and in the development of the peripheral nervous system.     

Fate of stem cell transplants in peripheral nerves

While damaged peripheral nerves demonstrate some potential to regenerate, complete functional recovery remains infrequent, owing to a functional loss of supportive Schwann cells distal to the injury. An emerging solution to improve upon this intrinsic regenerative capacity is to supplement injured nerves with stem cells derived from various tissues. While many of these strategies have proven successful in animal models, few studies have examined the behavior of transplanted stem cells in vivo, including whether they survive and differentiate. In previous work, we demonstrated that cells derived from neonatal rodent dermis (skin-derived precursor cells, or SKPs) could improve regenerative parameters when transplanted distal to both acute and chronic nerve injuries in Lewis rats. The aim of this work was to track the fate of these cells in various nerve injury paradigms and determine the response of these cells to a known glial growth factor. Here, we report that SKPs survive, respond to local cues, differentiate into myelinating Schwann cells, and avoid complete clearance by the host's immune defenses for a minimum of 10 weeks. Moreover, the ultimate fate of SKPs in vivo depends on the nerve environment into which they are injected and can be modified by inclusion of heregulin-1β.     

Expression of EGF receptor and FGF receptor isoforms during neuroepithelial stem cell differentiation

To characterize the role of epidermal growth factor (EGF) and fibroblast growth factor (FGF) in regulating neuroepithelial stem cells differentiation, we have examined the expression of FGF, EGF, and their receptors by neuroepithelial (NEP) cells and their derivatives. Our results indicate that undifferentiated NEP cells express a subset of FGF receptor (FGFR) isoforms, but do not express platelet-derived growth factor receptors (PDGFRs) or epidermal growth factor receptor (EGFR). The FGFR pattern of expression by differentiated neuron and glial cells differs from that found on NEP stem cells. FGFR-4 is uniquely expressed on NEP cells, while FGFR-1 is expressed by both NEP cells and neurons, and FGFR-2 is down-regulated during neuronal differentiation. FGFRs present on astrocytes and oligodendrocytes also represent a subset of those present on NEP cells. Expression of FGF and EGF by NEP cells and their progeny was also examined. NEP cells synthesize detectable levels of both FGF-1 and FGF-2, and EGF. FGF-1 and FGF-2 synthesis is likely to be biologically relevant, as cells grown at high density do not require exogenous FGF for their survival and cells grown in the presence of neutralizing antibodies to FGF show a reduction in cell survival and division. Thus, neuroepithelial cells synthesize and respond to FGF, but not to EGF, and are therefore distinct from other neural stem cells (neurospheres). The unique pattern of expression of FGF isoforms may serve to distinguish NEP cells from their more differentiated progeny.     

EGF responsiveness and receptor regulation in normal and differentiation-defective mouse myoblasts

The interrelationship between cell proliferation and terminal myogenic differentiation has been analyzed by studying a differentiation-defective subclone (DD-1) of the permanent mouse myoblast line MM14. Parental MM14 myoblasts withdraw irreversibly from the cell cycle and initiate terminal differentiation when they are deprived of certain mitogens. In contrast, DD-1 cells become quiescent in a mitogen-depleted environment and less than 0.4% of the cells differentiate. When refed with mitogen-rich medium quiescent DD-1 cells resume proliferation. Expression of this differentiation-defective phenotype is apparently coupled to an alteration in mitogen sensitivity: MM14 myoblasts require horse serum plus either chick embryo extract or fibroblast growth factor (FGF) to sustain cell growth: DD-1 variants are responsive to FGF, but also proliferate in response to serum alone or to reduced serum plus epidermal growth factor (EGF). Interestingly, EGF also appears to retard DD-1 cell differentiation in a manner similar to the FGF repression of differentiation in normal myoblasts. Normal and differentiation-defective myoblasts which have been maintained under growth-promoting conditions exhibit similar EGF binding, internalization, and degradation. However, whereas the EGF binding capacity of MM14 myoblasts declines to less than 5% of its initial level within 24 hr of FGF removal, DD-1 variants exhibit an increase in EGF binding when switched to an FGF-depleted medium. The relationship of altered EGF receptor regulation to changes in mitogen sensitivity and differentiation capacity of the DD-1 variant is discussed, and implications for general in vivo processes governing cell proliferation and differentiation are considered.     

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.     

Combined effects of somatomedin-like growth factors with fibroblast growth factor or epidermal growth factor in DNA synthesis in rabbit chondrocytes

Summary The somatomedin-like growth factors cartilage-derived factor (CDF) and multiplication-stimulating activity (MSA) stimulate DNA synthesis and proliferation of rabbit costal chondrocytes under serum-free conditions. Previously, we suggeted that CDF and MSA act on chondrocytes in an early G₁ phase to stimulate DNA synthesis. CDF and MSA have synergistic effects with epidermal growth factor (EGF) or fibroblast growth factor (FGF) in stimulating DNA synthesis of the cells. The mode of combined action of CDF or MSA with EGF or FGF in chondrocytes was studied by sequential treatments with these agents. EGF or FGF had synergistic effects with CDF or MSA in stimulating DNA synthesis, even when added 10 h after the latter. Synergism was also observed in cells pretreated with CDF or MSA; That is, the cultures were treated for 5 h with CDF or MSA and then washed, and treated with FGF or EGF. However, when CDF or MSA was added more than 5 h after EGF or FGF, no synergism of effects was observed. These findings suggest that the cultured chondrocytes become activated to interact with FGF or EGF for commitment to DNA synthesis when they are exposed to somatomedin-like growth factors at an early stage in the G₁ phase. Thus chondrocytes are under a different mechanism of growth control from fibroblastic cells.Abbreviations CDF cartilage-derived factor - MSA multiplication-stimulating activity - EGF epidermal growth factor - FGF fibroblast growth factor     

Regulation of rat ovarian cell growth and steroid secretion

A cultured rat ovarian cell line (31 A-F(2)) was used to study the effect of growth factors (epidermal growth factor [EGF] and fibroblast growth factor [FGF]), a survival factor (ovarian growth factor [OGF]), a hormone (insulin), and an iron-binding protein (transferring) on cell proliferation and steroid production under defined culture conditions. EGF and insulin were shown to be mitogenic (half-maximal response at 0.12 nM and 0.11 muM, respectively) for 31A-F(2) cells incubated in serum-free medium. EGF induced up to three doublings in the cell population, whereas insulin induced an average of one cell population doubling. FGF, OGF, and transferrin were found not to have any prominent effect on cell division when incubated individually with 31A-F(2) cells in serum-free medium. However, a combination of EGF, OGF, insulin, and transferrin stimulated cell division to the same approximate extent as cells incubated in the presence of 5 percent fetal calf serum. EGF or insulin did not significantly affect total cell cholesterol levels (relative to cells incubated in serum-free medium) when incubated individually with 31A-F(2) cells. However, cell cholesterol levels were increased by the addition of OGF (250 percent), FGF (370 percent), or a combination of insulin and EGF (320 percent). Progesterone secretion from 31A-F(2) cells was enhanced by EGF (25 percent), FGF (80 percent), and insulin (115 percent). However, the addition of a mitogenic mixture of EGF, OGF, insulin, and transferrin suppressed progesterone secretion 150 percent) below that of control cultures. These studies have permitted us to determine that EGF and insulin are mitogenic factors that are required for the growth of 31A-F(2) cells and that OGF and transferrin are positive cofactors that enhance growth. Also, additional data suggest that cholesterol and progesterone production in 31A-F(2) cells can be regulated by peptide growth factors and the hormone insulin.     

Plant-derived human recombinant growth factors and serum albumin maintain stemness of human-induced pluripotent stem cells

Stem cells are an important therapeutic source for recovery and regeneration, as their ability of self-renewal and differentiation offers an unlimited supply of highly specialized cells for therapeutic transplantation. Growth factors and serum are essential for maintaining the characteristics of stem cells in culture and for inducing differentiation. Because growth factors are produced mainly in bacterial (Escherichia coli) or animal cells, the use of such growth factors raises safety concerns that need to be resolved for the commercialization of stem cell therapeutics. To overcome this problem, studies on proteins produced in plants have been conducted. Here, we describe the functions of plant-derived fibroblast growth factor 2 (FGF2) and human serum albumin in the maintenance and differentiation of human-induced pluripotent stem cells (hiPSCs). Plant-derived FGF2 and human epidermal growth factor EGF were able to differentiate hiPSCs into neural stem cells (NSCs). These NSCs could differentiate into neuronal and glial cells. Our results imply that culturing stem cells in animal-free culture medium, which is composed of plant-derived proteins, would facilitate stem cell application research, for example, for cell therapy, by reducing contamination risk.     

In vitro regulation of pig Sertoli cell growth and function: effects of fibroblast growth factor and somatomedin-C

The effects of insulin, somatomedin-C (Sm-C), epidermal growth factor (EGF), fibroblast growth factor (FGF), vitamin E, and retinoic acid on growth and function of immature cultured pig Sertoli cells were investigated. All these factors, except vitamin E, stimulated Sertoli cell DNA synthesis and proliferation. The mitogenic effects of insulin observed only at micromolar concentrations were similar to those induced by nanomolar concentrations of Sm-C or EGF, but significantly less than those induced by FGF. The effects of EGF and Sm-C were almost additive, whereas those of Sm-C and FGF were synergistic. After a 6-day treatment, FGF and retinoic acid induced a significant increase in the number of follicle-stimulating hormone (FSH) receptors per cell, and in FSH-induced cyclic adenosine 3',5'-monophosphate (cAMP) production. Sm-C, which alone had no effect on these two parameters, potentiated FGF action. Basal plasminogen activator activity was enhanced after the 6-day treatment with EGF plus insulin and, particularly, with FGF plus insulin. Similarly, the response of the latter group to FSH was significantly higher than in any other group of cells. FGF was also able to stimulate cell multiplication and enhanced the FSH receptor number of Sertoli cells isolated from 15- and 26-day-old rats. Thus, FGF is the most potent known mitogenic factor for cultured Sertoli cells, and it stimulates the phenotypic expression of these cells.     

Superinduction by cycloheximide of mitogen-induced secreted proteins produced by Balb/c 3T3 cells

We describe here some of the characteristics of the regulation of a group of secretory proteins whose secreted levels rise within 2-4 h of adding fibroblast growth factor (FGF), epidermal growth factor (EGF), or serum to quiescent Balb/c 3T3 cells. The levels of these secretory proteins are regulated similarly to the interferons. When cycloheximide is present during the induction period, the amounts of [35S]methionine incorporated into five of these proteins that we have called "superinducible proteins" (SIPs) is increased 2-5-fold. Superinduction of the SIPs is seen also in response to polyribol-polyriboC, the classical inducer of interferons. None of the SIPs, however, are immuno-precipitated by anti-beta-interferon antibody. Induction and superinduction of the SIPs is inhibited by actinomycin D. Superinduction occurs at concentrations of cycloheximide that inhibit protein synthesis by at least 85%. The SIPs are not major intracellular proteins; they are barely detectable in cellular fractions. Their induction is, however, correlated with the ability of the polypeptide growth factor to stimulate DNA synthesis; EGF, FGF, and serum induce the SIPs, whereas insulin does not, and insulin alone weakly stimulates DNA synthesis in these cells. Because FGF, EGF, and serum cause the SIPs to be produced at concentrations of cycloheximide that inhibit 85% of bulk protein and DNA synthesis, it follows that the SIPs are produced directly from the action of the growth factor and not as a consequence of increased growth. Although probably not interferons, in analogy to the lymphokines, the SIPs could be a set of autocrine or paracrine factors that rapidly convey the growth or differentiation signal between cells.     

Separated human breast epithelial and myoepithelial cells have different growth factor requirements in vitro but can reconstitute normal breast lobuloalveolar structure

In order to investigate the specific factors controlling the growth of normal breast cell types, purified populations of human breast epithelial and myoepithelial cells from reduction mammoplasties were grown in primary culture in three defined media and their response to foetal calf serum (FCS), epidermal growth factor (EGF) and basic fibroblast growth factor (FGF2) measured using MTT growth assays. Epithelial and myoepithelial cells differed markedly in their growth requirements. Whereas epithelial cell survival was dependent on the presence of FCS, myoepithelial cell growth was dramatically inhibited by serum. EGF and FGF2 were mitogenic for epithelial cells but not myoepithelial cells, the addition of insulin being the only essential supplement required for myoepithelial cell growth. Heparin inhibited FGF2-stimulated epithelial cell growth but also basal myoepithelial cell proliferation and this inhibition could be overcome by the addition of EGF. Neutralizing antibodies to EGF also inhibited basal myoepithelial cell growth. This suggests the possibility of an autocrine role for a heparin-binding member of the EGF family in the growth of myoepithelial cells. Purified cells combined to form lobuloalveolar structures when incubated in a reconstituted basement membrane matrix (Matrigel) in the presence of EGF and FGF2. J. Cell. Physiol. 171:11–19, 1997. © 1997 Wiley-Liss, Inc.     

Generation of an environmental niche for neural stem cell development by the extracellular matrix molecule tenascin C

Stem cells in the embryonic mammalian CNS are initially responsive to fibroblast growth factor 2 (FGF2). They then undergo a developmental programme in which they acquire epidermal growth factor (EGF) responsiveness, switch from the production of neuronal to glial precursors and become localized in specialized germinal zones such as the subventricular zone (SVZ). Here we show that extracellular matrix molecules act as regulators of this programme. Tenascin C is highly expressed in the SVZ, and transgenic mice lacking tenascin C show delayed acquisition of the EGF receptor. This results from alterations in the response of the stem cells to the growth factors FGF2 and bone morphogenic protein 4 (BMP4), which normally promote and inhibit acquisition of the EGF receptor, respectively. Tenascin C-deficient mice also have altered numbers of CNS stem cells and these stem cells have an increased probability of generating neurones when grown in cell culture. We conclude that tenascin C contributes to the generation of a stem cell 'niche' within the SVZ, acting to orchestrate growth factor signalling so as to accelerate neural stem cell development.     

hSef potentiates EGF-mediated MAPK signaling through affecting EGFR trafficking and degradation

Sef (similar expression to fgf genes) was identified as an effective antagonist of fibroblast growth factor (FGF) in vertebrates. Previous reports have demonstrated that Sef interacts with FGF receptors (FGFRs) and inhibits FGF signaling, however, its role in regulating epidermal growth factor receptor (EGFR) signaling remains unclear. In this report, we found that hSef localizes to the plasma membrane (PM) and is subjected to rapid internalization and well localizes in early/recycling endosomes while poorly in late endosomes/lysosomes. We observed that hSef interacts and functionally colocalizes with EGFR in early endosomes in response to EGF stimulation. Importantly, we demonstrated that overexpression of hSef attenuates EGFR degradation and potentiates EGF-mediated mitogen-activated protein kinase (MAPK) signaling by interfering EGFR trafficking. Finally, our data showed that, with overexpression of hSef, elevated levels of Erk phosphorylation and differentiation of rat pheochromocytoma (PC12) cells occur in response to EGF stimulation. Taken together, these data suggest that hSef plays a positive role in the EGFR-mediated MAPK signaling pathway. This report, for the first time, reveals opposite roles for Sef in EGF and FGF signalings.     

Basic fibroblast growth factor and epidermal growth factor downmodulate the growth of hematopoietic cells in long-term stromal cultures

The bone marrow microenvironment consists of stromal cells and extracellular matrix components which act in concert to regulate the growth and differentiation of hematopoietic stem cells. There is little understanding of the mechanisms which modulate the regulatory role of stromal cells. This study examined the hypothesis that mesenchymal growth factors such as basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) modulate stromal cell activities and thereby influence the course of hematopoiesis. Both bFGF and EGF were potent mitogens for marrow stroma. However, both factors proved to be inhibitory to hematopoiesis in primary log-term marrow cultures. Inhibition was also observed when hematopoietic cells and bFGF or EGF were added to subconfluent irradiated stromal layers, demonstrating that the decline of hematopoiesis was not due to overgrowth of the stromal layer. Loss of hematopoietic support in bFGF and EGF was dose-dependent. Removal of bFGF and EGF permitted stromal layers to regain their normal capacity to support hematopoiesis. In stroma-free long-term cultures, neither factor affected CFU-GM expansion. Basic FGF slightly enhanced granulocyte-macrophage colony forming unit (CFU-GM) cloning efficiency in short-term agarose culture. Basic FGF did not reduce the levels of interleukin-6 (IL-6), GM-CSF, or G-CSF released by steady state or IL-1-stimulated stroma. Similarly, the constitutive levels of steel factor (SF) mRNA and protein were not affected by bFGF. Basic FGF did not alter the level of TGF-β1 in stromal cultures. We conclude that bFGF and EGF can act as indirect negative modulators of hematopoietic growth in stromal cultures. The actual mediators of regulation, whether bound or soluble, remain to be identified. © 1995 Wiley-Liss, Inc.     

Cell surface fibroblast growth factor and epidermal growth factor receptors are permanently lost during skeletal muscle terminal differentiation in culture

One characteristic of skeletal muscle differentiation is the conversion of proliferating cells to a population that is irreversibly postmitotic. This developmental change can be induced in vitro by depriving the cultures of specific mitogens such as fibroblast growth factor (FGF). Analysis of cell surface FGF receptor (FGFR) in several adult mouse muscle cell lines and epidermal growth factor receptor (EGFR) in mouse MM14 cells reveals a correlation between receptor loss and the acquisition of a postmitotic phenotype. Quiescent MM14 cells, mitogen-depleted, differentiation-defective MM14 cells, and differentiated BC3H1 muscle cells (a line that fails to become postmitotic upon differentiation) retained their cell surface FGFR. These results indicate that FGFR loss is not associated with either reversible cessation of muscle cell proliferation or biochemical differentiation and thus, further support a correlation between receptor loss and acquisition of a postmitotic phenotype. Comparison of the kinetics for growth factor receptor loss and for commitment of MM14 cells to a postmitotic phenotype reveals that FGFR rises transiently from approximately 700 receptors/cell to a maximum of approximately 2,000 receptors/cell 12 h after FGF removal, when at the same time, greater than 95% of the cells are postmitotic. FGFR levels then decline to undetectable levels by 24 h after FGF removal. During the interval in which FGFR increases and then disappears there is no change in its affinity for FGF. The transient increase in growth factor receptors appears to be due to a decrease in ligand-mediated internalization because EGFR, which undergoes an immediate decline when cultures are deprived of FGF (Lim, R. W., and S. D. Hauschka. 1984. J. Cell Biol. 98:739-747), exhibits a similar transient rise when cultures are grown in media containing both EGF and FGF before switching the cells to media without these added factors. These results indicate that the loss of certain growth factor receptors is a specific phenotype acquired during skeletal muscle differentiation, but they do not resolve whether regulation of FGFR number is causal for initiation of the postmitotic phenotype. A general model is presented in the discussion.