Presence Of Fibroblast Growth Factor Research Articles

Trophoblast Stem Cell Maintenance by Fibroblast Growth Factor 4 Requires MEKK4 Activation of Jun N-Terminal Kinase

Trophoblast differentiation during placentation involves an epithelial-mesenchymal transition (EMT) with loss of E-cadherin and gain of trophoblast invasiveness. Mice harboring a point mutation that renders inactive the mitogen-activated protein kinase kinase kinase MEKK4 exhibit dysregulated placental development with increased trophoblast invasion. Isolated MEKK4 kinase-inactive trophoblast stem (TS) cells cultured under undifferentiating, self-renewing conditions in the presence of fibroblast growth factor 4 (FGF4) display increased expression of Slug, Twist, and matrix metalloproteinase 2 (MMP2), loss of E-cadherin, and hyperinvasion of extracellular matrix, each a hallmark of EMT. MEKK4 kinase-inactive TS cells show a preferential differentiation to Tpbp alpha- and Gcm1-positive trophoblasts, which are indicative of spongiotrophoblast and syncytiotrophoblast differentiation, respectively. FGF4-stimulated Jun N-terminal kinase (JNK) and p38 activity is markedly reduced in MEKK4 kinase-inactive TS cells. Chemical inhibition of JNK in wild-type TS cells induced a similar EMT response as loss of MEKK4 kinase activity, including inhibition of E-cadherin expression and increased expression of Slug, MMP2, Tpbp alpha, and Gcm1. Chromatin immunoprecipitation analyses revealed changes in AP-1 composition with increased Fra-2 and decreased Fra-1 and JunB binding to the regulatory regions of Gcm1 and MMP2 genes in MEKK4 kinase-inactive TS cells. Our results define MEKK4 as a signaling hub for FGF4 activation of JNK that is required for maintenance of TS cells in an undifferentiated state.

FGF signalling modulates transcriptional repression by Xenopus groucho‐related‐4

Developmental cell signals co-operate in the processes of cell specification and tissue patterning during embryogenesis. Interactions between the FGF (fibroblast growth factor) and Wnt signalling pathways have been demonstrated in a number of developmental processes, including mesoderm formation in amphibian embryos. However, the mechanism underlying the interactions between these key signalling pathways remains unclear. In the present study, we find that the ability of TLE4/Xgrg4 (transducin-like enhancer of split 4/Xenopus groucho-related gene 4) to inhibit a transcriptional target of canonical Wnt signalling is reduced in the presence of FGF and that this is partially dependent on a consensus site for MAPK (mitogen-activated protein kinase) phosphorylation in TLE4/Xgrg4. These data suggest to us a novel molecular mechanism where FGF and Wnt signalling pathways interact at the level of the co-repressor TLE4/Xgrg4: the weakening of TLE4/Xgrg4 repression by FGF signalling, combined with the stabilization of beta-catenin by Wnt signals, enhances expression of Wnt target genes.

Inhibition of glycogen synthase kinase-3 enhances the expression of alkaline phosphatase and insulin-like growth factor-1 in human primary dermal papilla cell culture and maintains mouse hair bulbs in organ culture

Dermal papilla (DP) at the hair follicle base is important for hair growth. Recent studies demonstrated that mouse vibrissa DP cells can be cultured in the presence of fibroblast growth factor-2 (FGF-2), but lose expression of versican and their follicle-inducing activity during the culture, and that activation of the Wnt signal, which is inhibited by glycogen synthase kinase-3 (GSK-3), in the DP cells promotes hair growth activity. We therefore investigated the influence of a GSK-3 inhibitor, (2'Z,3'E)-6-bromoindirubin-3'-oxime (BIO), on the growth of human DP cells and mouse vibrissa follicles in culture. We first demonstrated that, similarly to mouse DP cells, human DP cells were able to be cultured up to 15 passages in the presence of FGF-2, and lost the expression of alkaline phosphatase (ALP). When human DP cells later than ten passages were treated with BIO, the expression of ALP as well as insulin-like growth factor-1 (IGF-1), another DP marker, was significantly elevated. Nuclear and perinuclear translocation of beta-catenin was also observed. We then cultured mouse vibrissa follicles. In the presence of BIO, the follicles could be maintained for at least 3 days without detectable regression of the hair bulbs. The morphology and ALP expression were well preserved. BIO successfully retrieved the expression of DP marker molecules, such as ALP and IGF-1 in cultured human DP cells, and maintained mouse hair bulbs. Thus, treatment with BIO may be useful to prepare DP cells with hair follicle-inducing activity.

Impaired differentiation potential of human trabecular bone mesenchymal stromal cells from elderly patients

Advances in bone tissue engineering with mesenchymal stromal cells (MSC) as an alternative to conventional orthopedic procedures has opened new horizons for the treatment of large bone defects. Bone marrow (BM) and trabecular bone are both sources of MSC. Regarding clinical use, we tested the potency of MSC from different sources. We obtained MSC from 17 donors (mean age 64.6 years) by extensive washing of trabecular bone from the femoral head and trochanter, as well as BM aspirates of the iliac crest and trochanter. The starting material was evaluated by histologic analysis and assessment of colony-forming unit-fibroblasts (CFU-F). The MSC populations were compared for proliferation and differentiation potential, at RNA and morphologic levels. MSC proliferation potential and immunophenotype (expression of CD49a, CD73, CD90, CD105, CD146 and Stro-1) were similar whatever the starting material. However, the differentiation potential of MSC obtained by bone washing was impaired compared with aspiration; culture-amplified cells showed few Oil Red O-positive adipocytes and few mineralized areas and formed inconsistent Alcian blue-positive high-density micropellets after growth under adipogenic, osteogenic and chondrogenic conditions, respectively. MSC cultured with 1 ng/mL fibroblast growth factor 2 (FGF-2) showed better differentiation potential. Trabecular bone MSC from elderly patients is not good starting material for use in cell therapy for bone repair and regeneration, unless cultured in the presence of FGF-2.

Activated Notch1 is a stronger astrocytic stimulus than leukemia inhibitory factor for rat neural stem cells

Neural stem cells (NSC) self-renew and generate specialized cell types. There are reports indicating that Notch and Leukemia Inhibitory Factor (LIF) signaling are involved in cell determination of NSC, either preventing differentiation or promoting astrocytic fate. In this work, we aimed to compare the astrocytogenic effect of activated Notch with that induced by LIF. To this end, rat cerebral cortex neural progenitors/NSC were transduced with retroviral vectors in order to express green fluorescent protein (GFP), or a fusion protein of GFP with the active Notch1 intracellular domain (NICD). In parallel, other cultures were treated with increasing concentrations of LIF. We confirmed, in proliferating NSC, that LIF activated intracellular effectors by measuring STAT3 phosphorylation and Socs3 transcription. In NICD-expressing cells, Hes5 mRNA was induced, an effect not present in GFP-transduced NSC. We quantified the proportion of cells expressing Nestin in the presence of Fibroblast Growth Factor-2 (FGF-2) with LIF or NICD treatments. LIF significantly increased the proportion of cells co-expresssing Nestin and Glial Fibrillary Acidic Protein (GFAP), an effect absent in cells with activated Notch. After FGF2 withdrawal to promote differentiation, Nestin was markedly down-regulated, and neuronal and glial markers appeared in control cultures. LIF treatment caused a significant increase in the proportion of GFAP-positive cells, but cells expressing NICD showed a significantly higher percentage of astrocytes than control and LIF-treated cultures. These experiments show that cells stimulated with NICD differentiate more readily to astrocytes than LIF-treated NSC.

An FGF‐responsive astrocyte precursor isolated from the neonatal forebrain

Gliogenesis in the mammalian CNS continues after birth, with astrocytes being generated well into the first two postnatal weeks. In this study, we have isolated an A2B5(+) astrocyte precursor (APC) from the postnatal rat forebrain, which is capable of differentiating into mature astrocytes in serum-free medium without further trophic support. Exposure to basic fibroblast growth factor (bFGF) selectively induces the APCs to proliferate, forming clusters of vimentin(+) cells, which, within 2 weeks, differentiate into GFAP(+) astrocytes. While bFGF functions as a potent mitogen, neither is it necessary to induce or maintain astrocyte differentiation, nor is it capable of maintaining the precursors in an immature, proliferative state. APCs exit the cell cycle and differentiate, even in the continued presence of fibroblast growth factor alone or in combination with other mitogenic factors such as platelet-derived growth factor. Under the culture conditions used, it was not possible to cause the astrocytes to re-enter cell cycle. After transplantation into the neonatal forebrain, APCs differentiated exclusively into astrocytes, regardless of brain region. Initially distributed widely within the forebrain, the precursors are most greatly concentrated within the subventricular zone (SVZ) and subcortical white matter, where they are maintained throughout postnatal development. APCs can be isolated from the SVZ and white matter of animals as late as 4 weeks after birth.

Cleavage of the Wnt Receptor Ryk Regulates Neuronal Differentiation during Cortical Neurogenesis

Ryk is a transmembrane receptor tyrosine kinase (RTK). It functions as a receptor of Wnt proteins required for cell-fate determination, axon guidance, and neurite outgrowth in different organisms; however, the molecular mechanism of Ryk signaling is unknown. Here, we show that Ryk is cleaved, permitting the intracellular C-terminal fragment of Ryk to translocate to the nucleus in response to Wnt3 stimulation. We also show that the cleaved intracellular domain of Ryk is required for Wnt3-induced neuronal differentiation in vitro and in vivo. These results demonstrate an unexpected mechanism of signal transduction for Ryk as a Wnt receptor, in which the intracellular domain itself functions as the transducing molecule to bring extracellular signals from the cell surface into the nucleus, to regulate neural progenitor cell differentiation.

Fibroblast Growth Factors Decrease Inducible Nitric Oxide Synthase mRNA Accumulation in Bovine Retinal Pigmented Epithelial Cells

Bovine retinal pigmented epithelial cells (RPE cells), after activation with interferon gamma (IFN gamma) and lipopolysaccharide (LPS), express an inducible nitric oxide (NO) synthase. This activity can be inhibited by the fibroblast growth factors (FGF), FGF-1 (acidic FGF) and FGF-2 (basic FGF). We have attempted to elucidate the molecular mechanisms involved in the FGF inhibition of NO synthase activity. Analysis by immunocytochemistry and Western blot using a polyclonal antibody against the inducible NO synthase of rat liver reveals that RPE cells co-stimulated with FGF-2 and LPS/IFN gamma accumulate lower levels of NO synthase than in the absence of FGF-2. Northern blot analysis by cross-species hybridization with a mouse macrophage NO synthase cDNA probe shows that a 4.4-kb mRNA accumulates when RPE cells are activated with LPS/IFN gamma. The level of inducible NO synthase mRNA in LPS/IFN gamma-activated RPE cells is markedly reduced by FGF-1 or FGF-2 treatment. Message stability studies revealed that the presence of FGF did not accelerate mRNA degradation, implying that FGF did not act on inducible NO synthase mRNA stability, but more probably on its expression. Furthermore an effect of FGF on IFN gamma receptors was excluded, since IFN gamma binding was not altered by FGF. Since NO acts as a cytostatic compound, FGF, by preventing NO synthase expression in RPE cells, may protect the retina from endotoxin and cytokine-mediated tissue damage.

Generation of dopamine neurons from embryonic stem cells in the presence of the neuralizing activity of bone marrow stromal cells derived from adult mice

Stromal cell lines such as PA6 and MS5 have been employed for generating dopamine (DA) neurons from embryonic stem (ES) cells. The present study was designed to test whether bone marrow stromal cells (BMSC) derived from adult mice might be available as a feeder layer to produce DA cells efficiently from ES cells. When ES cells were grown on BMSC in the presence of fibroblast growth factor 8 (FGF8) and sonic hedgehog (SHH), about 40% of TuJ1-positive neurons expressed tyrosine hydroxylase (TH). Because these cells labeled with TH were negative for dopamine-beta-hydroxylasae (DBH), the marker for noradrenergic and adrenergic neurons, the TH-positive cells were most likely DA neurons. They indeed expressed midbrain DA neuron markers such as Nurr 1, Ptx-3, and c-ret and were capable of synthesizing and releasing DA in vitro. Furthermore, DA neurons differentiated from ES cells in this differentiation protocol survived transplantation in rats with 6-hydroxydopamine lesions and reversed the lesion-induced circling behavior. The data indicate that BMSC can facilitate an efficient induction of DA neurons from ES cells and that the generated DA neurons are biologically functional both in vitro and in vivo. Insofar as BMSC have recently been employed in autologous cell therapy for ischemic heart and arteriosclerotic limb diseases, the present study raises the possibility that autologous BMSC can be applied in future cell transplantation therapy in Parkinson's disease.

Inhibition of adipocyte differentiation by phytoestrogen genistein through a potential downregulation of extracellular signal‐regulated kinases 1/2 activity

In the current study, we investigated the effects of genistein on adipogenic differentiation of mouse bone marrow-derived mesenchymal stem cell (BMSC) cultures and its potential signaling pathway. The terminal adipogenic differentiation was assessed by western-blotting analysis of adipogenic-specific proteins such as PPARgamma, C/EBPalpha, and aP2 and the formation of adipocytes. Treatment of mouse BMSC cultures with adipogenic cocktail resulted in sustained activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), which are members of the mitogen-activated protein kinase (MAPK) family, at the early phase of adipogenesis (from days 3 to 9). Inhibition of ERK1/2 activation by PD98059, a specific MEK inhibitor, reversed the induced adipogenic differentiation. Genistein dose-dependently decreased the phosphorylation of ERK1/2 in mouse BMSC cultures. Genistein incubation for the entire culture period, as well as that applied during the early phase of the culture period, significantly inhibited the adipogenic differentiation of mouse BMSC cultures. While genistein was incubated at the late stage (after day 9), no inhibitory effect on adipogenic differentiation was observed. BMSC cultures treated with genistein in the presence of fibroblast growth factor-2 (FGF-2), an activator of the ERK1/2 signaling pathway, expressed normal levels of ERK1/2 activity, and, in so doing, are capable of undergoing adipogenesis. Our results suggest that activation of the ERK1/2 signaling pathway during the early phase of adipogenesis (from days 3 to 9) is essential to adipogenic differentiation of BMSC cultures, and that genistein inhibits the adipogenic differentiation through a potential downregulation of ERK1/2 activity at this early phase of adipogenesis.

Growth factors, stem cells, and stroke

Postischemic neurogenesis has been identified as a compensatory mechanism to repair the damaged brain after stroke. Several factors are released by the ischemic tissue that are responsible for proliferation, differentiation, and migration of neural stem cells. An understanding of their roles may allow future therapies based on treatment with such factors. Although damaged cells release a variety of factors, some of them are stimulatory whereas some are inhibitory for neurogenesis. It is interesting to note that factors like insulin-like growth factor-I can induce proliferation in the presence of fibroblast growth factor-2 (FGF-2), and promote differentiation in the absence of FGF-2. Meanwhile, factors like transforming growth factor-beta can induce the differentiation of neurons while inhibiting the proliferation of neural stem cells. Therefore, understanding the role of each factor in the process of neurogenesis will help physicians to enhance the endogenous response and improve the clinical outcome after stroke. In this article the authors discuss the role of growth factors and stem cells following stroke.

Effect of leukemia inhibitory factor on long-term propagation of precursor cells derived from rat forebrain subventricular zone and ventral mesencephalon

Tissue blocks containing neural precursor cells were isolated from the rat forebrain subventricular zone (SVZ) and ventral mesencephalon (VM) and propagated as neural tissue-spheres (NTS). In the presence of fibroblast growth factor-2 (FGF2) and epidermal growth factor (EGF), SVZ-derived NTS were propagated and maintained for more than 6 months with a cell population doubling time of 21.5 days. The replacement of EGF by leukemia inhibitory factor (LIF) resulted in a cell population doubling time of 19.8 days, corresponding to a 10-fold increase in estimated cell numbers over a period of 70 days, at which point these NTS ceased to grow. In the presence of FGF2 and LIF, VM-derived NTS displayed a cell population doubling time of 24.6 days, which was maintained over a period of more than 200 days. However, when LIF was replaced by EGF, the cell numbers only increased 1.2 fold over 50 days. Using different immunohistochemical markers, we observed a distinct compartmentalization of cells within the spheres. In SVZ-derived NTS an outer compartment of proliferating (nestin +/Ki67 +), preferentially neurogenic (β-tubulin III +/MAP2 +) cells, surrounded by an inner compartment of glial (GFAP +/CNPase +) cells. The inner compartment of long-term propagated VM-derived NTS contained GFAP + cells as well as cells immunoreactive for the precursor cell marker nestin, even where minimal cell proliferation was observed. Our results demonstrate that tissues from rat SVZ and VM can be propagated as NTS. However, the cellular organization of the NTS and the need for mitogens to maintain long-term proliferative capacity differ with the origin of the tissue.

Molecular mechanisms of FGF-2 inhibitory activity in the osteogenic context of mouse adipose-derived stem cells (mASCs)

Adipose-derived adult stem cells (ASCs), like their bone-marrow derived counterparts, possess the ability to differentiate down osteogenic, chondrogenic, adipogenic, and myogenic pathways. For bone differentiation of mouse ASCs (mASCs), retinoic-acid mediated upregulation of BMPR-IB has been found to be necessary. Interestingly, our previous work has also shown Fibroblast Growth Factor-2 (FGF-2) to strongly inhibit this osteogenic differentiation, even in the presence of retinoic acid. In this report, we investigated the molecular mechanisms underlying FGF-2 mediated osteogenic inhibition, demonstrating that addition of exogenous FGF-2 to mASCs antagonizes upregulation of BMPR-IB gene expression in response to retinoic acid. In addition, constitutive expression of BMPR-IB, but not BMPR-IA or BMPR-II, was found to counteract the inhibitory effects of FGF-2. Finally, p53 −/− mASCs and human ASCs, both of which express high levels of endogenous BMPR-IB, underwent normal osteogenic differentiation even in the presence of FGF-2. Collectively, our data therefore indicate that FGF-2 antagonizes the response of mASCs to retinoic acid and also suggest that threshold levels of BMPR-IB may play a crucial role both in counteracting the inhibitory role of FGF-2 and in promoting osteogenic differentiation of ASCs in the absence of retinoic acid. Moreover, the present study also indicates that differences exist between mouse and human ASCs in relationship to FGF-2 activity in the osteogenic context.

IN VITRO HEPATIC DIFFERENTIATION OF HUMAN UMBILICAL CORD BLOOD AND BONE MARROW CELLS

The purpose of the present study was to investigate whether human umbilical cord blood (UCB) as well as bone marrow (BM) can generate hepatocyte lineage cells in a simple culture condition. Mononuclear cells (MNCs) separated from UCB and BM were cultured in the presence of fibroblast growth factor (FGF)-1, FGF-2, stem cell factor (SCF), and hepatocyte growth factor (HGF). The cultured cells were analyzed for morphology and for the expression of mRNAs and/or proteins of hepatocyte lineage markers. Both the UCB and BM MNCs grown in the given culture condition yielded large, round cells that were adherent to the culture dishes. RT-PCR analysis showed that mRNAs of albumin (ALB), cytokeratin (CK)-18, and alpha-fetoprotein were expressed from day 7 in both the UCB- and BM-derived cells. Immunofluorescent staining showed that the large, round cells expressed not only ALB and CK-19 but also proliferating cell nuclear antigen, implying the proliferative potential of hepatocyte lineage cells. Therefore, UCB as well as BM can give rise to hepatocyte lineage cells in the simple culture condition with HGF, SCF, FGF-1, and FGF-2. These cells may be one of the potential candidates of cell sources for the cytotherapy of hepatic disease, although it remains to be determined if the hepatocyte lineage cells are derived from plastic hematopoietic stem cells or from liver stem cells that reside in UCB or BM.

FGF-2 increases osteogenic and chondrogenic differentiation potentials of human mesenchymal stem cells by inactivation of TGF-β signaling

Human mesenchymal stem cells (hMSCs) are able to self-replicate and differentiate into a variety of cell types including osteoblasts, chondrocytes, adipocytes, endothelial cells, and muscle cells. It was reported that fibroblast growth factor-2 (FGF-2) increased the growth rate and multidifferentiation potentials of hMSCs. In this study, we investigated the genes involved in the promotion of osteogenic and chondrogenic differentiation potentials of hMSCs in the presence of FGF-2. hMSCs were maintained in the medium with FGF-2. hMSCs were harvested for the study of osteogenic or chondrogenic differentiation potential after 15 days' culture. To investigate osteogenic differentiation, the protein levels of alkaline phosphatase (ALP) and the mRNA expression levels of osteocalcin were measured after the induction of osteogenic differentiation. Moreover, the investigation for chondrogenic differentiation was performed by measuring the mRNA expression levels of type II and type X collagens after the induction of chondrogenic differentiation. The expression levels of ALP, type II collagen, and type X collagen of hMSCs cultured with FGF-2 were significantly higher than control. These results suggested that FGF-2 increased osteogenic and chondrogenic differentiation potentials of hMSCs. Furthermore, microarray analysis was performed after 15 days' culture in the medium with FGF-2. We found that the overall insulin-like growth factor-I (IGF-I) and transforming growth factor-beta (TGF-beta) signaling pathways were inactivated by FGF-2. These results suggested that the inactivation of IGF-I and TGF-beta signaling promotes osteogenic and chondrogenic differentiation potential of hMSCs in the presence of FGF-2.

Expansion and characterization of ventral mesencephalic precursor cells: Effect of mitogens and investigation of FA1 as a potential dopaminergic marker

Methods for identification and in vitro expansion of ventral mesencephalic dopaminergic precursor cells are of interest in the search for transplantable neurons for cell therapy in Parkinson's disease (PD). We investigated the potential use of fibroblast growth factor 2 (FGF2) and fibroblast growth factor 8 (FGF8) for expansion of such dopaminergic precursor cells, and fetal antigen-1 (FA1), a secreted neuronal protein of unknown function, as a non-invasive dopaminergic marker. Tissue from embryonic day (ED) 12 rat ventral mesencephalon was dissociated mechanically and cultured for 4 days in the presence of FGF2, FGF8, or without mitogens (control). After mitogen withdrawal and addition of 0.5% bovine serum, cells were differentiated for 6 days. Before differentiation, significantly more cells incorporated BrdU in cultures exposed to FGF2 (19-fold; P < 0.001) and FGF8 (3-fold; P < 0.05) compared to controls. After differentiation, biochemical analyses showed significantly more dopamine and FA1 in conditioned medium from both FGF2 and FGF8 expanded cultures than in controls. Correspondingly, numbers of tyrosine hydroxylase (TH)- and FA1-immunoreactive cells had increased 16-fold (P < 0.001) and 2.1-fold (P < 0.001), respectively in the FGF2 group and 10-fold (P < 0.001) and 1.8-fold (P < 0.05), respectively in the FGF8 group. In conclusion, the present procedure allows efficient expansion and differentiation of dopaminergic precursor cells and provides novel evidence of FGF8 as a mitogen for these cells. Furthermore, FA1 was identified as a potential supplementary non-invasive marker of cultured dopaminergic neurons.

Tripotential Differentiation of Adherently Expandable Neural Stem (NS) Cells

BackgroundA recent study has shown that pure neural stem cells can be derived from embryonic stem (ES) cells and primary brain tissue. In the presence of fibroblast growth factor 2 (FGF2) and epidermal growth factor (EGF), this population can be continuously expanded in adherent conditions. In analogy to continuously self-renewing ES cells, these cells were termed ‘NS’ cells (Conti et al., PLoS Biol 3: e283, 2005). While NS cells have been shown to readily generate neurons and astrocytes, their differentiation into oligodendrocytes has remained enigmatic, raising concerns as to whether they truly represent tripotential neural stem cells.Methodology/Principal FindingsHere we provide evidence that NS cells are indeed tripotent. Upon proliferation with FGF2, platelet-derived growth factor (PDGF) and forskolin, followed by differentiation in the presence of thyroid hormone (T3) and ascorbic acid NS cells efficiently generate oligodendrocytes (∼20%) alongside astrocytes (∼40%) and neurons (∼10%). Mature oligodendroglial differentiation was confirmed by transplantation data showing that NS cell-derived oligodendrocytes ensheath host axons in the brain of myelin-deficient rats.Conclusions/SignificanceIn addition to delineating NS cells as a potential donor source for myelin repair, our data strongly support the view that these adherently expandable cells represent bona fide tripotential neural stem cells.

Immunohistochemical demonstration of growth factors at the tendon–bone interface in anterior cruciate ligament reconstruction using a rabbit model

BackgroundThe success of anterior cruciate ligament (ACL) reconstruction using tendon grafts depends on biological integration between the tendon and bone. Growth factors play a significant role in this integration process, but few studies have defined the regulating mechanisms of these growth factors during tendon–bone healing. The aim of the present study was to clarify the relationship between the histological changes and the expression of endogenous growth factors at the tendon–bone interface. MethodsUsing intra-articular tendon transfer in rabbits to stimulate ACL reconstruction, the presence of fibroblast growth factor-2 (FGF-2), vascular endothelial growth factor (VEGF), bone morphogenetic protein-2 (BMP-2), and BMP-7 at the interface between the tendon and bone was evaluated immunohistochemically. Histological and immunohistochemical investigations were performed at 1, 3, 6, and 12 weeks after surgery. ResultsFibrous integration of the tendon graft to the bone was observed immediately after tendon transfer and followed remodeling of the bone tunnel. Fibroblast and vascular growth factors were found in abundance at the tendon–bone interface in the first 3 weeks of graft incorporation, but were absent in the 12-week specimens. BMPs were found throughout the 12-week study period and were observed at high concentrations near the bone. ConclusionsThese results indicate that FGF-2 and VEGF contribute to fibrous integration between the tendon and bone during the early postoperative stage, and that BMP-2 and BMP-7 are specifically involved in bone remodeling leading to osseous integration. The early stages of tendon–bone healing might be important in controlling the integration process of the interface in ACL reconstruction surgery as seen in this rabbit model.

Neural conversion of human embryonic stem cell colonies in the presence of fibroblast growth factor-2

Pluripotency and the capability for unlimited self-renewal make human embryonic stem cells a promising tool for studying development and new cell replacement strategies. Here, we present a simple differentiation protocol, which permits the direct conversion of human embryonic stem cells into neurogenic precursors without formation of embryoid bodies or coculture with other cell types. In this protocol, human embryonic stem cells propagated as adherent cultures are induced to differentiate into the neural lineage in media containing fibroblast growth factor-2. The adherent cells are proliferated to form detaching neurospheres. Upon plating, these neurospheres give rise to a homogenous population of neural precursors capable of generating neurons, astrocytes and oligodendrocytes. Our findings suggest that fibroblast growth factor-2 exposure alone suffices to promote neural conversion of adherently growing human embryonic stem cell cultures.

Growth factor-dependent actions of PACAP on oligodendrocyte progenitor proliferation

We previously reported that rat oligodendrocyte progenitors (OLP) express receptors for the pituitary adenylyl cyclase-activating peptide (PACAP) in vivo and in vitro. Addition of PACAP to cultured OLP triggered a potent elevation in intracellular cAMP contents, a dose-dependent stimulation of proliferation, and a delay in myelinogenesis (Lee M, Lelievre V, Zhao P, Torres M, Rodriguez W, Byun JY, Doshi S, Ioffe Y, Gupta G, de los Monteros AE, de Vellis J, Waschek J. Pituitary adenylyl cyclase-activating polypeptide stimulates DNA synthesis but delays maturation of oligodendrocyte progenitors. J Neurosci. 2001 21:3849–59.). In an attempt to understand how PACAP might interact with growth factors known to stimulate OLP proliferation, we investigated PACAP actions on OLP proliferation in the presence of Fibroblast Growth Factor-2 (FGF-2) and PDGF. Multiple PACAP receptor subtype mRNAs and splice variants were detected in these cultures. PACAP by itself potently stimulated OLP proliferation and enhanced the ability of FGF-2 to stimulate DNA synthesis. In contrast, this peptide strongly antagonized the mitogenic effects of PDGF in association with a reduction of PDGFα receptor gene expression. Additionally, we investigated the interaction of PACAP with the morphogenetic factor sonic hedgehog (Shh), which recently was shown to be crucial for oligodendrocyte generation. OLP cultures were found to express mRNAs for both ptc1 (Shh receptor) and gli1 (Shh target gene) and responded to Shh treatment with an increase in proliferation. PACAP antagonized the ability of Shh to stimulate OLP proliferation. Moreover, transcriptional targets of Shh signaling were also reduced by this treatment, suggesting that PACAP directly antagonized Shh signaling. These studies reveal complex in vitro interactions of PACAP with other factors involved in OLP development.