Basic fibroblast growth factor and transforming growth factor beta-1: Synergistic mediators of angiogenesis in vitro

We investigated the relative roles of basic fibroblast growth factor (bFGF) and transforming growth factor beta-1 (TGF-b) on bovine aortic endothelial cell mitogenesis and morphogenesis using two-dimensional Petri dish cultures and a threedimensional hydrated collagen gel. bFGF alone stimulated endothelial cell proliferation with an EC₅₀ of 0.5 ng/ml. At bFGF levels greater than 2.5 ng/ml, morphologic alterations in confluent monolayers predominated; cells changed from a cobblestone morphology to an elongated cell pattern and showed enhanced migration into a denuded area of a Petri dish. In the three-dimensional model, exposure of endothelial cell monolayers to high bFGF levels stimulated minor cell migration directly under the monolayer but no invasion into the gel matrix. In combination with bFGF, heparin potentiated morphogenic changes, but not mitogenesis. bFGF, modification of the antiproliferative effect of TGF-b in confluent cultures was evidenced by induction of endothelial cell sprouting in response to 0.5 ng/ml TGF-b and 10–20 ng/ml bFGF in two-dimensional cultures. On collagen gels, endothelial cells migrated into the deep layers of the gel in a dose-dependent manner: invasion was maximal at 0.3–0.7 ng/ml TGF-b with decreased invasion at higher concentrations. The optimal collagen concentration that supported cell invasion was 0.075% collagen with the number of invading cells decreasing with increasing collagen gel density. By scanning electron microscopy, invading endothelial cells assumed a fibroblast-like appearance with slender cell extensions. We concluded that bFGF and TGF-b had independent effects on endothelial cell morphology and mitogenesis in culture. In combination at specific doses, these agents stimulated sprouting in the two-dimensional model and cell invasion in a collagen gel model. Morphogenic changes may be the primary event in determining angiogenesis. © 1993 Wiley-Liss, Inc.     

Dosage and Cell Line Dependent Inhibitory Effect of bFGF Supplement in Human Pluripotent Stem Cell Culture on Inactivated Human Mesenchymal Stem Cells

Many different culture systems have been developed for expanding human pluripotent stem cells (hESCs and hiPSCs). In general, 4–10 ng/ml of bFGF is supplemented in culture media in feeder-dependent systems regardless of feeder cell types, whereas in feeder-free systems, up to 100 ng/ml of bFGF is required for maintaining long-term culture on various substrates. The amount of bFGF required in native hESCs growth niche is unclear. Here we report using inactivated adipose-derived human mesenchymal stem cells as feeder cells to examine long-term parallel cultures of two hESCs lines (H1 and H9) and one hiPSCs line (DF19-9-7T) in media supplemented with 0, 0.4 or 4 ng/ml of bFGF for up to 23 passages, as well as parallel cultures of H9 and DF19 in media supplemented with 4, 20 or 100 ng/ml bFGF for up to 13 passages for comparison. Across all cell lines tested, bFGF supplement demonstrated inhibitory effect over growth expansion, single cell colonization and recovery from freezing in a dosage dependent manner. In addition, bFGF exerted differential effects on different cell lines, inducing H1 and DF19 differentiation at 4 ng/ml or higher, while permitting long-term culture of H9 at the same concentrations with no apparent dosage effect. Pluripotency was confirmed for all cell lines cultured in 0, 0.4 or 4 ng/ml bFGF excluding H1-4 ng, as well as H9 cultured in 4, 20 and 100 ng/ml bFGF. However, DF19 demonstrated similar karyotypic abnormality in both 0 and 4 ng/ml bFGF media while H1 and H9 were karyotypically normal in 0 ng/ml bFGF after long-term culture. Our results indicate that exogenous bFGF exerts dosage and cell line dependent effect on human pluripotent stem cells cultured on mesenchymal stem cells, and implies optimal use of bFGF in hESCs/hiPSCs culture should be based on specific cell line and its culture system.     

Basic fibroblast growth factor stimulates hepatocyte growth factor/scatter factor secretion by human mesenchymal cells

Basic fibroblast growth factor (bFGF) together with other pleiotropic factors plays an important role in many complex physiological processes such as embryonic development, angiogenesis, and wound repair. Among these factors, hepatocyte growth factor/scatter factor (HGF/SF) which is secreted by cells of mesodermal origin exerts its mito- and motogenic activities on cells of epithelial and endothelial origin. Knowledge of the regulatory mechanisms of HGF/SF may contribute to the understanding of its role in physio-pathological processes. We observed that the secretion of HGF/SF by MRC-5 cells and by other fibroblast-derived cell cultures in conditioned media was enhanced by exposure to bFGF. HGF/SF was measured by the scatter assay, a bioassay for cell motility, and was further characterized by Western blot analysis with anti-HGF/SF antibodies. Exposure of MRC-5 cultures to 10 ng/ml of bFGF resulted already 6 h posttreatment in a threefold higher amount of scatter factor secreted into the medium as compared to untreated cultures. HGF/SF secretion was sustained after bFGF treatment for the following 72 h when increased amounts of HGF/SF were detected both in conditioned media as well as associated to the extracellular matrix. The secretion of HGF/SF in cell supernatants increased dose dependently upon treatment with bFGF starting from basal levels of 6 U/ml and reaching 27 U/ml at 30 ng/ml bFGF, plateauing thereafter. Upregulation of HGF/SF by IL-1, already described by others, was confirmed in this study. Based on our findings an articulated interaction can be speculated for bFGF, HGF/SF, and IL-1, e.g., in tissue regeneration during inflammatory processes or in wound healing. © 1996 Wiley-Liss, Inc.     

Stimulation of Cultured Melanocytes in Medium Containing a Serum Substitute: Ultroser-G

Melanocyte cultures were established and maintained routinely in Ham's F-10 medium containing 12-O-tetradecanoyl-phorbol-13-acetate (TPA), isobutylmethylxanthine (IBMX), cholera toxin (CT) and fetal calf serum (FCS). Three serum substitutes (Ultroser-G, Nutridoma-Hu and Nutricyte-H) were tested in order to obtain a medium without FCS having a more constant composition. Melanocyte proliferation was examined in long-term culture experiments by in situ cell counts at different periods of time. Only with Ultroser-G (1-2%) was the proliferation of melanocytes maintained without both FCS and CT, whereas the addition of the other two serum substitutes resulted in stabilization of melanocyte densities in the cultures up to 28 days. In the medium containing 1% Ultroser-G and IBMX without TPA minimal or no increases in melanocyte density were found. Addition of basic fibroblast growth factor (bFGF, 1 ng/ml) to the medium without TPA resulted in a partial restimulation of growth in different experiments. In this system with 1% Ultroser-G and 1 ng/ml bFGF, IBMX could also be replaced by other factors (dbcAMP LTC4 and a purified form of α-melanocyte stimulating hormone). The culture medium with 1% Ultroser-G containing TPA and IBMX is now used for routine melanocyte culture. In this medium TPN/IBMX can easily be replaced by bFGF/dbcAMP with optimal growth stimulation. The combination bFGF/α-MSH and other more physiological stimulators offers an alternative to study responses of melanocytes in culture with respect to proliferation, metabolism, and phenotype.     

Isolation of thecal cells: an assessment of purity and steroidogenic potential

In the present investigation, the responsiveness of rat thecal cells, prepared by means of an optimised discontinuous Percoll density gradient centrifugation procedure and cultured under serum-free cell culture conditions, to different concentrations of follitropin (FSH), basic fibroblast growth factor (FGF-2 or bFGF), and lutropin (LH) has been examined. The estradiol (E(2)) and progesterone (P(4)) contents of the cell culture medium were simultaneously determined with aliquots collected after different times of exposure to these regulatory proteins, either individually or in combination. The results confirm that no E(2) could be detected in the cell culture medium of the rat thecal cells prepared and cultured in this manner following all of these different treatments, and hence no contamination of the thecal cell preparations by granulosa cells was evident. The effects of FGF-2 and LH on the steroidogenic and cytodifferentiational properties of these rat thecal cells under serum-free cell culture procedures were also examined. The production of P(4) in the Percoll-purified rat thecal cell cultures receiving different treatments of FSH, and/or FGF-2 did not differ from the basal cell cultures, and no E(2) was detected from the same culture media. In contrast, LH (20 or 50 ng/ml) was found to enhance the production of P(4) (P<0.05) in the serum-free cell culture media. The stimulation of P(4) production was greater at higher LH concentration (50 ng/ml) (P<0.05). Concurrent treatment of LH (20 or 50 ng/ml) and FGF-2 (1-100 ng/ml) showed that FGF-2 inhibited the production of P(4) by LH-stimulated thecal cell cultures (P<0.05). The inhibition by FGF-2 was greater when LH was at a lower concentration (EC(50)<1 ng/ml at LH-20 ng/ml vs. EC(50)>1 ng/ml at LH-50 ng/ml). The results of the present study thus indicate that rat thecal cells isolated by this optimised Percoll density centrifugation procedure maintain a very high steroidogenic potential and specificity. Consistent with the absence of contaminating granulosa cells, these rat theca cell preparations do not respond to FSH treatment in terms of E(2) production. However, these rat theca cell preparations can be stimulated by LH to express their differentiated status in serum-free medium and respond to growth factors such as FGF-2.     

Growth factor profile of irradiated human dermal fibroblasts using a serum-free method

Radiation therapy for cancer permanently damages tissue in the line of treatment. This study sought to establish a serum-free protocol to evaluate the growth of irradiated fibroblasts and to analyze the levels of basic fibroblast growth factor (bFGF) and transforming growth factor-beta (TGF-beta) compared with normal fibroblasts. One irradiated cell line of human dermal fibroblasts was established from an intraoperative specimen obtained from a patient who had undergone radiation therapy for head and neck cancer. Irradiated and normal fibroblasts were then plated in UltraCULTURE (serum and growth factor free), modified Webber's medium (bFGF 50 ng/ml, insulin-like growth factor 100 ng/ml), and Dulbecco's Modified Eagle Medium with 10% fetal bovine serum (serum with undefined basal growth factors). Irradiated cells were also seeded in UltraCULTURE with 50 and 100 ng/ml of bFGF. Cell counts were performed at 0, 1, 3, 5, and 7 days, and cell supernatants were assayed for bFGF and TGF-beta. Irradiated and normal fibroblasts exhibited stronger growth in modified Webber's medium than in Dulbecco's Modified Eagle Medium with 10% fetal bovine serum. Growth of irradiated fibroblasts under bFGF modulation was similar to their growth in Webber's medium. Furthermore, irradiated fibroblasts remained viable in a serum-free and growth factor-free environment for at least 7 days; however, their growth and autocrine growth factor production was less than that of normal cells. This confirms the results of previous studies suggesting that cells from irradiated tissue undergo cellular changes. This study provides an effective model for the first-line evaluation of agents to improve wound healing, and it helps to establish standard levels of bFGF and TGF-beta production for irradiated fibroblasts.     

Characterization of the receptor for insulin-like growth factor II in bone cells

We have previously shown that insulin-like growth factor II (IGF-II) is produced by bone cells and that IGF-II stimulates cell proliferation and collagen synthesis in bone cells. We now extend these in vitro findings by demonstrating specific IGF-II binding to bone cells derived from newborn mouse calvaria and embryonic chick calvaria. The kinetics of [125I] IGF-II binding in embryonic chick calvaria cells showed time and temperature dependence. Scatchard analysis of [125I]IGF-II binding to chick calvaria cells showed an apparent Kd of 1.4 x 10(-10) M, with a calculated receptor site concentration of 40,000/cell. The specificity characteristics showed that IGF-II was significantly more potent than IGF-I or insulin in displacing IGF-II tracer. Competition for binding of [125I]IGF-II by unlabeled IGF-II showed a dose-dependent displacement between 0.5 and 25 ng/ml. Fifty percent displacement of [125I]IGF-II binding to chick and mouse calvarial cells was achieved at 1-2 ng/ml; 90% of specific binding of [125I]IGF-II was displaceable in the presence of 125 ng/ml of unlabeled IGF-II. IGF-I showed less than 5% cross reactivity for displacement of [125I]IGF-II binding to chick and mouse bone cells. Type II receptor inhibitory antibodies, R-II-PAB1 inhibited the binding of [125I]IGF-II to mouse bone cells and H-35 rat hepatoma cells (which contain type II but not type I receptors) in a dose-dependent manner. R-II-PAB1 also inhibited basal cell proliferation as well as IGF-II-, IGF-I-, and fibroblast growth factor (FGF)-induced cell proliferation in mouse bone cells. In chick calvaria bone cells and TE89 human osteosarcoma cells, R-II-PABI inhibited neither binding of [125I]IGF-II nor IGF-II-induced cell proliferation. These results together with our findings that IGF-II increased chick bone cell proliferation in the presence of maximal doses of IGF-I suggest that at least part of the mitogenic action of IGF-II is mediated through type II rather than type I receptors in bone cells.     

Regulation of cerebroside sulfotransferase activity in cultured oligodendrocytes: effect of growth factors and insulin

Cerebroside sulfotransferase (EC 2.8.2.11, CST) specific activity has been determined in oligodendrocyte (OL)-enriched glial cell cultures from newborn rat brain grown in serum supplemented medium. This activity is detectable at 5 days in vitro (DIV) and reaches its maximum value at 12 DIV. This period corresponds to that of oligodendrocyte precursor proliferation in these cultures. The activity decreases thereafter and remains nearly constant after 24 DIV. The developmental curve of CST activity is parallel in pure oligodendrocyte subcultures but twice higher than in primary cultures. These data confirm that CST is highly enriched in OL. Basic fibroblast growth factor (bFGF) (15 ng/ml) and platelet derived growth factor (PDGF) (0.75 U/ml) both enhance CST activity by 90% and 72%, respectively. This increase is in the same range than that of DNA content in treated cultures, whereas protein increase is smaller (50% and 22%, respectively). In contrast, transforming growth factor beta 1 (TGF beta 1, 0.5 and 5 ng/ml) does not significantly enhance CST activity nor DNA content of OL cultures. Insulin at high concentrations (5 micrograms/ml) also enhances CST activity but has no effect at physiological concentrations (20 ng/ml). These results show that CST activity can be controlled by growth factors. They suggest that CST activity is more closely related to OL and OL precursor proliferation than to myelination itself since its maximal activity preceeds myelination in vitro.     

Basic FGF and TGF-beta 1 influence commitment to melanogenesis in neural crest-derived cells of avian embryos

In previous studies, we showed that neural crest (NC)-derived cells from embryonic quail dorsal root ganglia (DRG) and peripheral nerve (PN), which do not normally give rise to melanocytes, become committed to melanogenesis following treatment in culture with the phorbol ester drug 12-O-tetradecanoyl phorbol-13-acetate (TPA). These and other observations support the notion that melanocytes and Schwann cells are derived from a common bipotent intermediate in the neural crest lineage--the melanocyte/Schwann cell progenitor. In this study, we test the possibility that peptide growth factors found in the embryonic environment might act similarly to TPA to influence the fates of these cells. DRG and PN explants were cultured in medium supplemented with a variety of growth factors, and then the cultures were examined for the presence of pigment cells. We found that basic fibroblast growth factor (bFGF), but not various other growth factors, induced pigmentation in about 20% of these cultures. When low concentrations of TPA were included in the culture medium, bFGF augmented the TPA-induced pigmentation, significantly increasing the proportion of pigmented cultures. These effects of bFGF were age-dependent, and could be blocked by addition of a bFGF-neutralizing antibody to the culture medium. In contrast to these stimulatory effects of bFGF, transforming growth factor-beta 1 (TGF-beta 1) was found to inhibit the TPA- or bFGF-induced pigmentation of DRG cultures. These data suggest, therefore, that at least some NC-derived cells are responsive to bFGF and TGF-beta 1, and that these growth factors may play an important role in the control of NC cell fate.     

Effect of the association of IGF-I, IGF-II, bFGF, TGF-β1, GM-CSF, and LIF on the development of bovine embryos produced in vitro

This study examined the influence of the following growth factors and cytokines on early embryonic development: insulin-like growth factors I and II (IGF-I, IGF-II), basic fibroblast growth factor (bFGF), transforming growth factor (TGF-β), granulocyte-macrophage colony-stimulating factor (GM-CSF), and leukemia inhibitory factor (LIF). Synthetic oviduct fluid (SOF) was used as the culture medium. We studied the development of bovine embryos produced in vitro and cultured until Day 9 after fertilization. TGF-β1, bFGF, GM-CSF, and LIF used on their own significantly improved the yield of hatched blastocysts. IGF-I, bFGF, TGF-β1, GM-CSF, and LIF significantly accelerated embryonic development, especially the change from the expanded blastocyst to hatched blastocyst stages. Use of a combination of these growth factors and cytokines (GF-CYK) in SOF medium produced higher percentages of blastocysts and hatched blastocysts than did use of SOF alone (45% and 22% vs. 24% and 12%; P < 0.05) on Day 8 after in vitro fertilization and similar results to use of SOF + 10% fetal calf serum (38% and 16%, at the same stages, respectively). The averages of total cells, inner cell mass cells, and trophectoderm cells of exclusively in vitro Day-8 blastocysts for pooled GF-CYK treatments were higher than those for SOF and similar to those for fetal calf serum. The presence of these growth factors and cytokines in the embryo culture medium therefore has a combined stimulatory action on embryonic development; in particular through an increase in hatching rate and in the number of cells of both the inner cell mass and trophoblast. These results are the first to demonstrate that use of a combination of recombinant growth factors and cytokine, as IGF-I, IGF-II, bFGF, TGF-β1, LIF, and GM-CSF, produces similar results to 10% fetal calf serum for the development of in vitro-produced bovine embryos. This entirely synthetic method of embryo culture has undeniable advantages for the biosecurity of embryo transfer.     

Production of stem cells with embryonic characteristics from human umbilical cord blood

When will embryonic stem cells reach the clinic? The answer is simple -- not soon! To produce large quantities of homogeneous tissue for transplantation, without feeder layers, and with the appropriate recipient's immunological phenotype, is a significant scientific hindrance, although adult stem (ADS) cells provide an alternative, more ethically acceptable, source. The annual global 100 million human birth rate proposes umbilical cord blood (UCB) as the largest untouched stem cell source, with advantages of naive immune status and relatively unshortened telomere length. Here, we report the world's first reproducible production of cells expressing embryonic stem cell markers, - cord-blood-derived embryonic-like stem cells (CBEs). UCB, after elective birth by Caesarean section, has been separated by sequential immunomagnetic removal of nucleate granulocytes, erythrocytes and haemopoietic myeloid/lymphoid progenitors. After 7 days of high density culture in microflasks, (10(5) cells/ml, IMDM, FCS 10%, thrombopoietin 10 ng/ml, flt3-ligand 50 ng/ml, c-kit ligand 20 ng/ml). CBE colonies formed adherent to the substrata; these were maintained for 6 weeks, then were subcultured and continued for a minimum 13 weeks. CBEs were positive for TRA-1-60, TRA-1-81, SSEA-4, SSEA-3 and Oct-4, but not SSEA-1, indicative of restriction in the human stem cell compartment. The CBEs were also microgravity--bioreactor cultured with hepatocyte growth medium (IMDM, FCS 10%, HGF 20 ng/ml, bFGF 10 ng/ml, EGF 10 ng/ml, c-kit ligand 10 ng/ml). After 4 weeks the cells were found to express characteristic hepatic markers, cytokeratin-18, alpha-foetoprotein and albumin. Thus, such CBEs are a viable human alternative from embryonic stem cells for stem cell research, without ethical constraint and with potential for clinical applications.     

Human recombinant insulin-like growth factor I. I. Development of a serum-free medium for clonal density assay of growth factors using balb/c 3T3 mouse embryo fibroblasts

Summary A serum-free clonal density growth assay was developed for the quantification of the biological activity of human recombinant insulin-like growth factor I (IGF-I). The assay measures IGF-I stimulated growth of Balb/c 3T3 cells cultured over 4 d on poly-d-lysine-coated plastic surfaces in a serum-free medium formulation composed of a 1∶1 (vol/vol) mixture of Ham's F12 and Dulbecco's modified Eagle's media, supplemented with 3.0 ng/ml bovine basic fibroblast growth factor (bFGF), 10 μg/ml human transferrin, 100 μg/ml ovalbumin, and 1.0 μM dexamethanose. Low-temperature trypsinization of serum-supplemented stock cultures combined with the use of poly-d-lysine-coated plates made it unnecessary to use serum or fibronectin to promote cell attachment and survival. Serum-free growth conditions were optimized with respect to the concentrations of the supplements. Addition of IGF-I resulted in 3.5-fold more cells than control cultures without IGF-I after 4 d. Deletion of bFGF resulted in no IGF-I stimulation of growth. The concentrations of various preparations of IGF-I required to achieve one-half maximal stimulation of cell number (ED₅₀), ranged between 1.25 and 4.7 ng/ml. In parallel assays, IGF-I was 6.6 times more potent than human recombinant insulin-like growth factor II and 32 times more potent than insulin. When cells were seeded into medium containing IGF-I, transferrin, ovalbumin, and dexamethasone but no bFGF, growth was minimal. Dose-response addition of bFGF showed an ED₅₀, of 0.9 ng/ml. The methods reported are useful to monitor the biological potency of recombinant and natural-source growth factors as well as providing a new means of studying the multiple growth factor requirements of Balb/c 3T3 cells in cultures. This work was supported by a contract from IMCERA Bioproducts, Inc.     

Effects of acidic and basic fibroblast growth factors (aFGF, bFGF) on glial precursor cell proliferation: age dependency and brain region specificity.

Acidic fibroblast growth factor (aFGF) and basic fibroblast growth factor (bFGF) are present in high levels in most areas of the embryonic rodent brain. To begin to understand the role of these growth factors in brain development, the effects of aFGF and bFGF on dissociated cell cultures prepared from embryonic and neonatal rat brain were studied. Addition of aFGF and heparin or bFGF alone to serum-free cultures of the dissociated Embryonic Day (E) 14.5 mesencephalon stimulates cell proliferation, as judged by [3H]thymidine autoradiography, leading to a maximal 75-fold increase in the total number of cells. This effect is dose-dependent with half-maximal increases at concentrations of about 5-6 ng/ml of aFGF or bFGF and is inhibited by the FGF antagonist HBGF-1U. The effect of aFGF on cell proliferation in cultures prepared from E14.5 mesencephalon is similar to that in cultures prepared from E14.5 cortex. However, in cultures prepared from E14.5 rhombencephalon or diencephalon, the proliferative effect of aFGF is much reduced. In all brain areas studied, the proliferative effect of aFGF declines with increasing age. Immunocytochemical analysis of E14.5 mesencephalic cultures demonstrated that the aFGF-induced increase in cell number is due to the proliferation of A2B5-immunoreactive (IR) glial precursor cells, but not of neuronal precursors, fibroblasts, or microglial cells. Moreover, differentiated glial fibrillary acidic protein-IR astrocytes and 2',3'-cyclic nucleotide 3'-phosphohydrolase-IR oligodendrocytes were not observed in cultures continuously treated with aFGF or bFGF, but were observed in high numbers after removal of the growth factors. These results suggest (1) that aFGF and bFGF are potent mitogens for glial precursor cells in all embryonic brain regions, (2) that the magnitude of the effects of aFGF depends on embryonic age and brain region, and (3) that both growth factors inhibit the differentiation of astrocyte or oligodendrocyte precursors. These observations made in vitro strongly support the hypothesis that FGF plays a critical role in gliogenesis and the timing of glial differentiation in the brain.     

Regulation of Astrocytic Tenascin by Basic Fibroblast Growth Factor

Extracellular matrix (ECM) molecules have been implicated in the regulation of neuronal adhesion and neurite outgrowth both during development and after injury. It has been demonstrated in our laboratory that astrocytes are heterogenous in expression of the ECM molecule tenascin. High-tenascin astrocytes have a reduced ability to support neurite outgrowth. In addition, astrocytes treated with exogenous basic fibroblast growth factor (bFGF) supported reduced neuronal growth and adhesion. In the current study, the hypothesis was tested that bFGF could increase the expression of tenascin by these cells. Basic FGF was added to cultures of rat cerebral cortical astrocytes at concentrations of up to 30 ng/ml, concentrations shown to have a significant effect on neuronal adhesion. Tenascin levels were evaluated by Western blot analysis of both cell extracts and conditioned media and also by immunocytochemistry techniques. Tenascin levels began to increase after 24-48 hr and continued to increase throughout 8 days in culture. The increase in tenascin was concentration-dependent, with the largest increase seen at 5 ng/ml bFGF. Tenascin production was increased approximately 5.5-fold in serum-containing medium but only about 2-fold in serum-free medium. When heparin (10 μg/ml) was included along with bFGF in serum-free medium, tenascin production was further enhanced. The bFGF treatment was discontinued after 8 days, and the cells were maintained for an additional 8 days in culture. Tenascin levels returned to control values, demonstrating that the bFGF effect is transient. It is our hypothesis that the action of bFGF during injury may evoke the induction of tenascin on astrocytes, thereby reducing regeneration in the central nervous system.     

The combination of basic fibroblast growth factor and kit ligand promotes the proliferation,activity and steroidogenesis of granulosa cells during human ovarian cortical culture

Different factors, such as basic fibroblast growth factor (bFGF) and kit ligand (KL), are used in ovarian cortical culture to promote activation of primordial follicles. In the present study, the effects of bFGF and KL, alone and in combination, were evaluated on human follicular activation and growth during in-situ cortical culture. Slow frozen-thawed human ovarian cortical tissues (n = 6) were cultured in 4 different groups: 1) control (base medium), 2) KL (base medium; BM + 100 ng/ml KL), 3) bFGF (BM + 100 ng/ml bFGF) and 4) bFGF + KL (BM + 100 ng/ml KL + 100 ng/ml bFGF) for a week. The proportion of morphologically normal and degenerated follicles at different developmental stages, secreted hormonal levels and specific gene expressions were compared. Although the proportion of growing follicles was higher than primordial counterpart in all cultured groups, no significant differences were observed among the cultured groups. In all cultured groups, anti-Müllerian hormone (AMH), progesterone and estradiol hormones levels increased after 7 days of culture; however, this increase was only significant for estradiol in the bFGF + KL group. The expression of Ki67 gene indicated an increase in ovarian cell proliferation in the three experimental groups compared to the control group, however this increment was only significant for the bFGF + KL group. It can be concluded that KL and bFGF factors individually have no beneficial effects on in-situ follicular growth, but their combination positively influences steroidogenesis of granulosa cells without significantly increasing the number of growing follicles.     

In vitro neurite extension by granule neurons is dependent upon astroglial-derived fibroblast growth factor

When grown in the absence of astroglial cells, purified mouse cerebellar granule neurons survive less than 36 hr and do not extend neurites. Here we report that low concentrations of basic fibroblast growth factor (bFGF, 1-25 ng/ml) maintained the viability and promoted the differentiation of purified granule neurons. The effect of bFGF on granule cell neurite outgrowth was dose dependent. Neurite outgrowth was stimulated markedly in the presence of 1-25 ng/ml bFGF, but effects were not seen below 1 ng/ml or above 50 ng/ml. When affinity-purified antibodies against bFGF (1-5 micrograms/ml) were added either to purified granule cells or to co-cultures of neurons and astroglial cells, process extension by granule neurons was severely impaired. The inhibition of neurite outgrowth in the presence of anti-bFGF antibodies was reversed by the addition of 25 ng/ml of exogenous bFGF. In addition to neuronotrophic effects, bFGF influenced the rate of growth of the astroglial cells. This result depended on whether the astroglia were grown in isolation from neurons, where low doses of bFGF (10-25 ng) stimulated glial growth, or in coculture with neurons, where much higher doses of bFGF (100-250 ng/ml) were needed for glial mitogenesis. Immunoprecipitation of lysates from 35S-labeled cerebellar astroglial cells with anti-bFGF antibodies revealed a single band after SDS-PAGE at 18,000 Da, the molecular weight of bFGF. These results indicate that glial cells synthesize bFGF and are possibly an endogenous source of bFGF in cerebellar cultures. Thus, astroglial cells synthesize soluble factors needed for neuronal differentiation.     

FSH and growth factors affect the growth and endocrine function in vitro of granulosa cells of bovine preantral follicles

The hypothesis was tested that bovine preantral follicles can be stimulated to grow in vitro by FSH and by the mitogens, epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF), but not by transforming growth factor-beta (TGFbeta), which generally inhibits EGF and bFGF action. Preantral follicles, 60 to 179 mum in diameter, were isolated from fetal ovaries by treatment with collagenase and DNase and cultured for 6 d in serum-free medium, with or without FSH and growth factors. Basic FGF (50 ng/ml), and to a lesser extent FSH (100 ng/ml) and EGF (50 ng/ml), stimulated thymidine incorporation by granulosa cells in bovine preantral follicles compared to control cultures (8-, 4- and 2.5-fold the labeling index of the controls; P < 0.05). Alone TGFbeta (10 ng/ml) had no effect on (3)H-thymidine incorporation, but it completely inhibited the bFGF- but not the FSH-stimulated increase in the labeling index and mean follicular diameter of preantral follicles (P < 0.05). By the end of the culture period oocytes in most treatments had degenerated, and the few surviving oocytes were in preantral follicles cultured with FSH or bFGF. Progesterone accumulation was greater (P < 0.05) in the presence of FSH (100 ng/ml) or EGF (50 ng/ml) than with bFGF, TGFbeta or control medium. Basic FGF strongly inhibited the effect of FSH on progesterone secretion (P < 0.05). Only FSH stimulated the conversion of exogenous testosterone to estradiol and both bFGF and TGFbeta markedly inhibited FSH-stimulated estradiol accumulation. These results indicate that proliferation of granulosa cells of bovine preantral follicles can be stimulated by bFGF, FSH and EGF, whereas TGFbeta inhibits growth, and that they are steroidogenically active in culture. Basic FGF and TGFbeta antagonize FSH-stimulated steroid production by granulosa cells of cultured bovine preantral follicles.     

Effect of various growth-promoting factors on preimplantation bovine embryo development in vitro

One-cell bovine embryos produced by in vitro oocyte maturation (IVM) and in vitro fertilization (IVF) were cultured in chemically defined medium (CDM) to which the following growth-promoting factors were added separately: transferrin (Tf, 10 ug/ml), epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha), transforming growth factor-betal (TGF-beta1), insulin-like growth factor-one (IGF-I), insulin-like growth factor-two (IGF-II), platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), and nerve growth factor (NGF), all at 10 ng/ml. The embryos were cultured for a total of 10 days and were assessed. The culture medium was changed every 2 days. There were no beneficial effects of growth factors on embryo development to morula or hatched blastocyst stages of development. However, supplementation with EGF approached significance (P<0.08) when compared with that of CDM alone at the blastocyst stage of development. The results suggest that supplementation with growth factors does not improve bovine IVM-IVF embryo development when cultured under chemically-defined conditions.     

人神经干细胞的体外生物学特性

本实验利用有丝分裂因子,体外诱导生成人神 经干细胞(NSCs),观察其生长特性并进行鉴定。取胎龄10-22周的大脑半球,分散细胞后种于添加表皮生长因子(EGF,20ng/ml)和/或碱性成纤维生长因子(bFGF,20ng/ml)的培养基中。利用免疫组织化学方法鉴定分化后的细胞类型。同时,进行细胞克隆分析、传代培养及端粒酶活性检测。结果显示:NSCs呈悬浮生长的干细胞球,其特异性抗原nestin阳性。NSCs具有增殖能力,可连续传代而不丢失其增殖和多分化潜能的干细胞特性。撤除EGF和bFGF的作用,细胞停止分裂,并分化为神经元、星形胶质细胞和少突胶质细胞。克隆分析显示NSCs生长呈密度依赖性。人NSCs表达较低的端粒酶水平,并随培养时间延长而下调。研究表明,利用有丝分裂因子,可在体外成功诱导生成人NSCs,其生长,分化受内外源因素的调节,相关的机制还有待阐明。