Human melanoblasts in culture: Expression of BRN2 and synergistic regulation by fibroblast growth factor-2, stem cell factor, and endothelin-3

The BRN2 transcription factor (POU3F2, N-Oct-3) has been implicated in development of the melanocytic lineage and in melanoma. Using a low calcium medium supplemented with stem cell factor, fibroblast growth factor-2, endothelin-3 and cholera toxin, we have established and partially characterised human melanocyte precursor cells, which are unpigmented, contain immature melanosomes and lack L-dihydroxyphenylalanine reactivity. Melanoblast cultures expressed high levels of BRN2 compared to melanocytes, which decreased to a level similar to that of melanocytes when cultured in medium that contained phorbol ester but lacked endothelin-3, stem cell factor and fibroblast growth factor-2. This decrease in BRN2 accompanied a positive L-dihydroxyphenylalanine reaction and induction of melanosome maturation consistent with melanoblast differentiation seen during development. Culture of primary melanocytes in low calcium medium supplemented with stem cell factor, fibroblast growth factor-2 and endothelin-3 caused an increase in BRN2 protein levels with a concomitant change to a melanoblast-like morphology. Synergism between any two of these growth factors was required for BRN2 protein induction, whereas all three factors were required to alter melanocyte morphology and for maximal BRN2 protein expression. These finding implicate BRN2 as an early marker of melanoblasts that may contribute to the hierarchy of melanocytic gene control.

Regulation of stem cell factor expression in inflammation and asthma

Stem cell factor (SCF) is a major mast cell growth factor, which could be involved in the local increase of mast cell number in the asthmatic airways. In vivo, SCF expression increases in asthmatic patients and this is reversed after treatment with glucocorticoids. In vitro in human lung fibroblasts in culture, IL-1beta, a pro-inflammatory cytokine, confirms this increased SCF mRNA and protein expression implying the MAP kinases p38 and ERK1/2 very early post-treatment, and glucocorticoids confirm this decrease. Surprisingly, glucocorticoids potentiate the IL-1beta-enhanced SCF expression at short term treatment, implying increased SCF mRNA stability and SCF gene transcription rate. This potentiation involves p38 and ERK1/2. Transfection experiments with the SCF promoter including intron1 also confirm this increase and decrease of SCF expression by IL-1beta and glucocorticoids, and the potentiation by glucocorticoids of the IL-1beta-induced SCF expression. Deletion of the GRE or kappaB sites abolishes this potentiation, and the effect of IL-1beta or glucocorticoids alone. DNA binding of GR and NF-kappaB are also demonstrated for these effects. In conclusion, this review concerns new mechanisms of regulation of SCF expression in inflammation that could lead to potential therapeutic strategy allowing to control mast cell number in the asthmatic airways.

Stem cell factor protects against neuronal apoptosis by activating AKT/ERK in diabetic mice

Neuronal apoptosis occurs in the diabetic brain due to insulin deficiency or insulin resistance, both of which reduce the expression of stem cell factor (SCF). We investigated the possible involvement of the activation of the MAPK/ERK and/or AKT pathways in neuroprotection by SCF in diabetes. Male C57/B6 mice (20-25 g) were randomly divided into four groups of 10 animals each. The morphology of the diabetic brain in mice treated or not with insulin or SCF was evaluated by H&E staining and TUNEL. SCF, ERK1/2 and AKT were measured by Western blotting. In diabetic mice treated with insulin or SCF, there was fewer structural change and apoptosis in the cortex compared to untreated mice. The apoptosis rate of the normal group, the diabetic group receiving vehicle, the diabetic group treated with insulin, and the diabetic group treated with SCF was 0.54 ± 0.077%, 2.83 ± 0.156%, 1.86 ± 0.094%, and 1.78 ± 0.095% (mean ± SEM), respectively. SCF expression was lower in the diabetic cortex than in the normal cortex; however, insulin increased the expression of SCF in the diabetic cortex. Furthermore, expression of phosphorylated ERK1/2 and AKT was decreased in the diabetic cortex compared to the normal cortex. However, insulin or SCF could activate the phosphorylation of ERK1/2 and AKT in the diabetic cortex. The results suggest that SCF may protect the brain from apoptosis in diabetes and that the mechanism of this protection may, at least in part, involve activation of the ERK1/2 and AKT pathways. These results provide insight into the mechanisms by which SCF and insulin exert their neuroprotective effects in the diabetic brain.

Lipopolysaccharide-induced Stem Cell Factor Messenger RNA Expression and Production in Odontoblast-like Cells

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Stem cell factor estimula células da musculatura lisa da traqueia a produzir TGF-β, FGF-2 E CCL3/MIP-1α

Pós-graduação em Ciências Fisiológicas - FOA

Stem cell factor: a hemopoietic cytokine with important targets in asthma

We review evidence that Stem Cell Factor (SCF) plays an important role in the pathophysiology of asthma. SCF is produced by a wide variety of cells present in asthmatic lung, including mast cells and eosinophils. Its receptor, c-kit, is broadly expressed on mature mast cells and eosinophils. SCF promotes recruitment of mast cell progenitors into tissues, as well as their local maturation and activation. It also promotes eosinophil survival, maturation and functional activation. SCF enhances IgE-dependent release of mediators from mast cells, including histamine, leukotrienes, cytokines (TNF-alpha, IL-5, GM-CSF) and chemokines (RANTES/CCL5, MCP-1/CCL2, TARC/CCL17 e MDC/CCL22); it is required for IL-4 production in mast cells. SCF, acting in concert with IgE, also upregulates the expression and function of CC chemokine receptors in mast cells. Structural and resident airway cells express increased levels of SCF in the bronchus of asthmatic patients. In a murine model of asthma, allergen exposure increased production of SCF by epithelial cells and alveolar macrophages, which was transient and paralleled by histamine release. SCF induced long-lived airway hyperreactivity, which was prevented by local neutralization of SCF, as well as by inhibitors of the production or activity of cysteinyl-leukotrienes. Together, these observations suggest that SCF has an important role in asthma.

Chymase cleavage of stem cell factor yields a bioactive, soluble product

Stem cell factor (SCF) is produced by stromal cells as a membrane-bound molecule, which may be proteolytically cleaved at a site close to the membrane to produce a soluble bioactive form. The proteases producing this cleavage are unknown. In this study, we demonstrate that human mast cell chymase, a chymotrypsin-like protease, cleaves SCF at a novel site. Cleavage is at the peptide bond between Phe-158 and Met-159, which are encoded by exon 6 of the SCF gene. This cleavage results in a soluble bioactive product that is 7 amino acids shorter at the C terminus than previously identified soluble SCF. This research shows the identification of a physiologically relevant enzyme that specifically cleaves SCF. Because mast cells express the KIT protein, the receptor for SCF, and respond to SCF by proliferation and degranulation, this observation identifies a possible feedback loop in which chymase released from mast cell secretory granules may solubilize SCF bound to the membrane of surrounding stromal cells. The liberated soluble SCF may in turn stimulate mast cell proliferation and differentiated functions; this loop could contribute to abnormal accumulations of mast cells in the skin and hyperpigmentation at sites of chronic cutaneous inflammation.

Improved retroviral gene transfer into murine and Rhesus peripheral blood or bone marrow repopulating cells primed in vivo with stem cell factor and granulocyte colony-stimulating factor.

In previous studies we showed that 5 days of treatment with granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF) mobilized murine repopulating cells to the peripheral blood (PB) and that these cells could be efficiently transduced with retroviral vectors. We also found that, 7-14 days after cytokine treatment, the repopulating ability of murine bone marrow (BM) increased 10-fold. In this study we examined the efficiency of gene transfer into cytokine-primed murine BM cells and extended our observations to a nonhuman primate autologous transplantation model. G-CSF/SCF-primed murine BM cells collected 7-14 days after cytokine treatment were equivalent to post-5-fluorouracil BM or G-CSF/SCF-mobilized PB cells as targets for retroviral gene transfer. In nonhuman primates, CD34-enriched PB cells collected after 5 days of G-CSF/SCF treatment and CD34-enriched BM cells collected 14 days later were superior targets for retroviral gene transfer. When a clinically approved supernatant infection protocol with low-titer vector preparations was used, monkeys had up to 5% of circulating cells containing the vector for up to a year after transplantation. This relatively high level of gene transfer was confirmed by Southern blot analysis. Engraftment after transplantation using primed BM cells was more rapid than that using steady-state bone marrow, and the fraction of BM cells saving the most primitive CD34+/CD38- or CD34+/CD38dim phenotype increased 3-fold. We conclude that cytokine priming with G-CSF/SCF may allow collection of increased numbers of primitive cells from both the PB and BM that have improved susceptibility to retroviral transduction, with many potential applications in hematopoietic stem cell-directed gene therapy.

Paracrine effects of oocyte secreted factors and stem cell factor on porcine granulosa and theca cells in vitro

Oocyte control of granulosa and theca cell function may be mediated by several growth factors via a local feedback loop(s) between these cell types. This study examined both the role of oocyte-secreted factors on granulosa and thecal cells, cultured independently and in co-culture, and the effect of stem cell factor (SCF); a granulosa cell derived peptide that appears to have multiple roles in follicle development. Granulosa and theca cells were isolated from 2-6 mm healthy follicles of mature porcine ovaries and cultured under serum-free conditions, supplemented with: 100 ng/ml LR3 IGF-1, 10 ng/ml insulin, 100 ng/ml testosterone, 0-10 ng/ml SCF, 1 ng/ml FSH (granulosa), 0.01 ng/ml LH (theca) or 1 ng/ml FSH and 0.01 ng/ml LH (co-culture) and with/without oocyte conditioned medium (OCM) or 5 oocytes. Cells were cultured in 96 well plates for 144 h, after which viable cell numbers were determined. Medium was replaced every 48 h and spent medium analysed for steroids.Oocyte secreted factors were shown to stimulate both granulosa cell proliferation (P < 0.001) and oestradiol production (P < 0.001) by granulosa cells throughout culture. In contrast, oocyte secreted factors suppressed granulosa cell progesterone production after both 48 and 144 hours (P < 0.001). Thecal cell numbers were increased by oocyte secreted factors (P = 0.02), together with a suppression in progesterone and androstenedione synthesis after 48 hours (P < 0.001) and after 144 hours (P = 0.02), respectively. Oocyte secreted factors also increased viable cell numbers (P < 0.001) in co-cultures together with suppression of progesterone (P < 0.001) and oestradiol (P < 0.001). In granulosa cell only cultures, SCF increased progesterone production in a dose dependent manner (P < 0.001), whereas progesterone synthesis by theca cells was reduced in a dose dependent manner (P = 0.002). Co-cultured cells demonstrated an increase in progesterone production with increasing SCF dose (P < 0.001) and an increase in oestradiol synthesis at the highest dose of SCF (100 ng/ml). In summary, these findings demonstrate the presence of a co-ordinated paracrine interaction between somatic cells and germ cells, whereby oocyte derived signals interact locally to mediate granulosa and theca cell function. SCF has a role in modulating this local interaction. In conclusion, the oocyte is an effective modulator of granulosa-theca interactions, one role being the inhibition of luteinization

Stem cell populations derived from heart and pancreas show a with a unique phenotype and give rise to functional cardiomyocytes and insulin-producing cells, respectively

Introduction: Recently, mesenchymal stem cells (MSC) of perivascular origin have been identified in several organs not including the heart. Using a novel cell isolation protocol, we have isolated cells sharing common characteristics from mouse hearts and pancreas. The aim of the present study was to characterize these cells in vitro.Methods: Cells were isolated from neonatal and adult mouse hearts and pancreas and cultured for more than 6 months. Surface marker expression was analyzed by flow cytometry and immunocytochemistry. Cell differentiation was tested using multiple differentiation media. Insulin production by pancreas-derived cells was tested by dithizone staining.Results: Cells showing a similar, distinctive morphology were obtained from the heart and pancreas after 4-8 weeks of culture. Cells from the two organs also showed a very similar immunophenotype, characterized by expression of c-kit (stem cell factor receptor), CD44, the common leukocyte marker CD45, and the monocytic markers CD11b and CD14. A significant proportion of cardiac and pancreatic cells expressed NG2, a marker for pericytes and other vascular cells. A significant proportion of cardiac, but not of pancreatic cells expressed stem cell antigen-1 (Sca-1). However, cells did not express T, B or dendritic cell markers. Cells of both cardiac and pancreatic origin spontaneously formed "spheres" (spherical cell aggregates similar to "neurospheres" formed by neural stem cells) in vitro. Cardiosphere formation was enhanced by TNF-alpha. Several cardiospheres (but no "pancreatospheres") derived from neonatal (but not adult) cells showed spontaneous rhythmic contractions, thus demonstrating cardiac differentiation (this was confirmed by immunostaining for alpha-sarcomeric actinin). Beating activity was enhanced by low serum conditions. Cells from both organs formed adipocytes, osteocytes and osteocytes under appropriate conditions, the typical differentiation pattern of MSCs. Pancreas-derived cells also formed dithizonepositive insulin-producing cells.Conclusions: We have defined cardiac and pancreatic cell populations that share a common morphology, growth characteristics, and a unique immunophenotype. Expression of perivascular and monocytic markers, along with stem/priogenitor cell markers by these cells suggests a relationship with pericytes-mesoangioblasts and so-called multipotent monocytes. Cells show MSC-typical growth and differentiation patterns, together with tissue-specific differentiation potential: cardiomyocytes for cardiac-derived cells and insulinproducing cells for pancreas-derived cells.

Cytokine expression patterns and mesenchymal stem cell karyotypes from the bone marrow microenvironment of patients with myelodysplastic syndromes

The purpose of this study was to explore cytokine expression patterns and cytogenetic abnormalities of mesenchymal stem cells (MSCs) from the bone marrow microenvironment of Chinese patients with myelodysplastic syndromes (MDS). Bone marrow samples were obtained from 30 cases of MDS (MDS group) and 30 healthy donors (control group). The expression pattern of cytokines was detected by customized protein array. The karyotypes of MSCs were analyzed using fluorescence in situ hybridization. Compared with the control group, leukemia inhibitory factor, stem cell factor (SCF), stromal cell-derived factor (SDF-1), bone morphogenetic protein 4, hematopoietic stem cell (HSC) stimulating factor, and transforming growth factor-β in the MDS group were significantly downregulated (P<0.05), while interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and programmed death ligand (B7-H1) were significantly upregulated (P<0.05). For chromosome abnormality analysis, the detection rate of abnormal karyotypes (+8, -8, -20, 20q-, -Y, -7, 5q-) was 30% in the MDS group and 0% in the control group. In conclusion, the up- and downregulated expression of these cytokines might play a key role in the pathogenesis of MDS. Among them, SCF and SDF-1 may play roles in the apoptosis of HSCs in MDS; and IFN-γ, TNF-α, and B7-H1 may be associated with apoptosis of bone marrow cells in MDS. In addition, the abnormal karyotypes might be actively involved in the pathogenesis of MDS. Further studies are required to determine the role of abnormal karyotypes in the occurrence and development of MDS.

Plerixafor and granulocyte colony-stimulating factor for first-line steady-state autologous peripheral blood stem cell mobilization in lymphoma and multiple myeloma: results of the prospective PREDICT trial.

In Europe, the combination of plerixafor + granulocyte colony-stimulating factor is approved for the mobilization of hematopoietic stem cells for autologous transplantation in patients with lymphoma and myeloma whose cells mobilize poorly. The purpose of this study was to further assess the safety and efficacy of plerixafor + granulocyte colony-stimulating factor for front-line mobilization in European patients with lymphoma or myeloma. In this multicenter, open label, single-arm study, patients received granulocyte colony-stimulating factor (10 μg/kg/day) subcutaneously for 4 days; on the evening of day 4 they were given plerixafor (0.24 mg/kg) subcutaneously. Patients underwent apheresis on day 5 after a morning dose of granulocyte colony-stimulating factor. The primary study objective was to confirm the safety of mobilization with plerixafor. Secondary objectives included assessment of efficacy (apheresis yield, time to engraftment). The combination of plerixafor + granulocyte colony-stimulating factor was used to mobilize hematopoietic stem cells in 118 patients (90 with myeloma, 25 with non-Hodgkin's lymphoma, 3 with Hodgkin's disease). Treatment-emergent plerixafor-related adverse events were reported in 24 patients. Most adverse events occurred within 1 hour after injection, were grade 1 or 2 in severity and included gastrointestinal disorders or injection-site reactions. The minimum cell yield (≥ 2 × 10(6) CD34(+) cells/kg) was harvested in 98% of patients with myeloma and in 80% of those with non-Hodgkin's lymphoma in a median of one apheresis. The optimum cell dose (≥ 5 × 10(6) CD34(+) cells/kg for non-Hodgkin's lymphoma or ≥ 6 × 10(6) CD34(+) cells/kg for myeloma) was harvested in 89% of myeloma patients and 48% of non-Hodgkin's lymphoma patients. In this prospective, multicenter European study, mobilization with plerixafor + granulocyte colony-stimulating factor allowed the majority of patients with myeloma or non-Hodgkin's lymphoma to undergo transplantation with minimal toxicity, providing further data supporting the safety and efficacy of plerixafor + granulocyte colony-stimulating factor for front-line mobilization of hematopoietic stem cells in patients with non-Hodgkin's lymphoma or myeloma.

Stem cell-related "self-renewal" signature and high epidermal growth factor receptor expression associated with resistance to concomitant chemoradiotherapy in glioblastoma.

PURPOSE: Glioblastomas are notorious for resistance to therapy, which has been attributed to DNA-repair proficiency, a multitude of deregulated molecular pathways, and, more recently, to the particular biologic behavior of tumor stem-like cells. Here, we aimed to identify molecular profiles specific for treatment resistance to the current standard of care of concomitant chemoradiotherapy with the alkylating agent temozolomide. PATIENTS AND METHODS: Gene expression profiles of 80 glioblastomas were interrogated for associations with resistance to therapy. Patients were treated within clinical trials testing the addition of concomitant and adjuvant temozolomide to radiotherapy. RESULTS: An expression signature dominated by HOX genes, which comprises Prominin-1 (CD133), emerged as a predictor for poor survival in patients treated with concomitant chemoradiotherapy (n = 42; hazard ratio = 2.69; 95% CI, 1.38 to 5.26; P = .004). This association could be validated in an independent data set. Provocatively, the HOX cluster was reminiscent of a "self-renewal" signature (P = .008; Gene Set Enrichment Analysis) recently characterized in a mouse leukemia model. The HOX signature and EGFR expression were independent prognostic factors in multivariate analysis, adjusted for the O-6-methylguanine-DNA methyltransferase (MGMT) methylation status, a known predictive factor for benefit from temozolomide, and age. Better outcome was associated with gene clusters characterizing features of tumor-host interaction including tumor vascularization and cell adhesion, and innate immune response. CONCLUSION: This study provides first clinical evidence for the implication of a "glioma stem cell" or "self-renewal" phenotype in treatment resistance of glioblastoma. Biologic mechanisms identified here to be relevant for resistance will guide future targeted therapies and respective marker development for individualized treatment and patient selection.

Recent advances in the epidermal growth factor receptor/ligand system biology on skin homeostasis and keratinocyte stem cell regulation.

The epidermal growth factor (EGF) receptor/ligand system stimulates multiple pathways of signal transduction, and is activated by various extracellular stimuli and inter-receptor crosstalk signaling. Aberrant activation of EGF receptor (EGFR) signaling is found in many tumor cells, and humanized neutralizing antibodies and synthetic small compounds against EGFR are in clinical use today. However, these drugs are known to cause a variety of skin toxicities such as inflammatory rash, skin dryness, and hair abnormalities. These side effects demonstrate the multiple EGFR-dependent homeostatic functions in human skin. The epidermis and hair follicles are self-renewing tissues, and keratinocyte stem cells are crucial for maintaining these homeostasis. A variety of molecules associated with the EGF receptor/ligand system are involved in epidermal homeostasis and hair follicle development, and the modulation of EGFR signaling impacts the behavior of keratinocyte stem cells. Understanding the roles of the EGF receptor/ligand system in skin homeostasis is an emerging issue in dermatology to improve the current therapy for skin disorders, and the EGFR inhibitor-associated skin toxicities. Besides, controlling of keratinocyte stem cells by modulating the EGF receptor/ligand system assures advances in regenerative medicine of the skin. We present an overview of the recent progress in the field of the EGF receptor/ligand system on skin homeostasis and regulation of keratinocyte stem cells.

Involvement of Eukaryotic Translation Initiation Factor 5A (eIF5A) in Skeletal Muscle Stem Cell Differentiation

The eukaryotic translation initiation factor 5A (eIF5A) contains a special amino acid residue named hypusine that is required for its activity, being produced by a post-translational modification using spermidine as substrate. Stem cells from rat skeletal muscles (satellite cells) were submitted to differentiation and an increase of eIF5A gene expression was observed. Higher content of eIF5A protein was found in satellite cells on differentiation in comparison to non-differentiated satellite cells and skeletal muscle. The treatment with NI-guanyl- 1,7-diaminoheptane (GC7), a hypusination inhibitor, reversibly abolished the differentiation process. In association with the differentiation blockage, an increase of glucose consumption and lactate production and a decrease of glucose and palmitic acid oxidation were observed. A reduction in cell proliferation and protein synthesis was also observed. L-Arginine, a spermidine precursor and partial suppressor of muscle dystrophic phenotype, partially abolished the GC7 inhibitory effect on satellite cell differentiation. These results reveal a new physiological role for eIF5A and contribute to elucidate the molecular mechanisms involved in muscle regeneration.