Combination Of Fibroblast Growth Factor Research Articles

In vitro trans-differentiation of human umbilical cord derived hematopoietic stem cells into hepatocyte like cells using combination of growth factors for cell based therapy.

The aim of the study was to develop a new strategy for the differentiation of hematopoietic stem cell (HSC) derived from UCB into hepatocyte like cells and also to estimate the effects of combination of fibroblast growth factor 4 (FGF 4) and hepatocyte growth factor (HGF) on hematopoietic stem cell differentiation. HSCs were isolated and purified by magnetic activated cell sorting. HSCs were induced to hepatocyte like cells under a 2-step protocol with combination of growth factors. Reverse transcription polymerase chain reaction was performed to detect multiple genes related to hepatocyte like cells development and function. Hepatocyte like morphology was illustrated by inverted repeat microscope and the secretion of albumin and α- fetoprotein by these cells was confirmed by enzyme linked immunosorbent assay. Hepatocyte like cells was observed at the end of the protocol (days 14). These differentiated cells were observed to show high expression of genes related to hepatocytes (tryptophan 2, 3-dioxygenase [TO], glucose 6-phosphate [G6P], cytokeratin 18 [CK 18], albumin and α- fetoprotein [AFP]). The quantities of albumin and AFP at day 0 were low and upon differentiation the cells were able to produce albumin and AFP at high levels. Our results show a new strategy for differentiation in a short duration, using a combination of growth factors for the differentiation of umbilical cord blood derived HSC into hepatocyte like cells under certain in vitro conditions. After further studies this approach has the potency, for widespread cell replacement therapy for liver diseases.

Transplantation of microencapsulated umbilical-cord-blood-derived hepatic-like cells for treatment of hepatic failure

To investigate intraperitoneal transplantation of microencapsulated hepatic-like cells from human umbilical cord blood for treatment of hepatic failure in rats. CD34+ cells in umbilical cord blood cells were isolated by magnetic cell sorting. In the in vitro experiment, sorted CD34+ cells were amplified and induced into hepatic-like cells by culturing with a combination of fibroblast growth factor 4 and hepatocyte growth factor. Cultures without growth factor addition served as controls. mRNA and protein levels for hepatic-like cells were analyzed by reverse transcription-polymerase chain reaction, immunohistochemistry and immunofluorescence. In the in vivo experiment, the hepatic-like cells were encapsulated and transplanted into the abdominal cavity of acute hepatic failure (AHF) rats at 48 h after D-galactosamine induction of acute hepatic failure. Transplantation with PBS and unencapsulated hepatic-like cells served as controls. The mortality rate, hepatic pathological changes and serum biochemical indexes were determined. The morphology and structure of microcapsules in the greater omentum were observed. Human albumin, alpha-fetoprotein and GATA-4 mRNA and albumin protein positive cells were found among cultured cells after 16 d. Albumin level in culture medium was significantly increased after culturing with growth factors in comparison with culturing without growth factor addition (P < 0.01). Compared with the unencapsulated group, the mortality rate of the encapsulated hepatic-like cell-transplanted group was significantly lower (P < 0.05). Serum biochemical parameters, alanine aminotransferase, aspartate aminotransferase and total bilirubin in the encapsulated group were significantly improvement compared with the PBS control group (P < 0.01). Pathological staining further supported these findings. At 1-2 wk post-transplantation, free microcapsules with a round clear structure and a smooth surface were observed in peritoneal lavage fluid, surviving cells inside microcapsules were found by trypan blue staining, but some fibrous tissue around microcapsules was also detected in the greater omentum of encapsulated group by hematoxylin and eosin staining. Transplantation of microencapsulated hepatic-like cells derived from umbilical cord blood cells could preliminarily alleviate the symptoms of AHF rats.

Pluripotency of bank vole embryonic cells depends on FGF2 and activin A signaling pathways

The objective of this study was to investigate the capability of bank vole (Myodes glareolus) embryonic cells to sustain their pluripotent character during in vitro culture, and to determine the optimal conditions for derivation of embryonic stem (ES) cells. We compared the presence of specific pluripotency (Oct4, Ssea1) and differentiation markers (Gata4 - primitive endoderm marker; Cdx2 - trophectoderm marker) in blastocysts and inner cell mass (ICM) outgrowths obtained from blastocysts of bank vole, and two mouse hybrids F1(C57Bl/6xCBA/H) and F1(C57Bl/6x129/Sv), which differ in the permissiveness of giving rise to ES cells. We found that, in contrast to mouse, the expression of pluripotency markers in the cells of bank vole ICM outgrowths is progressively downregulated and rapidly lost by the 4th day of culture. This correlates with the appearance of cells expressing Gata4 and Cdx2, indicating differentiation towards primitive endoderm and derivatives of trophectoderm, respectively. We have also shown that heterologous cytokine leukaemia inhibitory factor (LIF) in conjunction with either homologous or heterologous feeder layer is unable to delay differentiation and preserve pluripotency of bank vole embryonic cells. Thus, the conditions optimised for mouse do not support the maintenance of bank vole embryonic cells in the undifferentiated state and do not allow for the isolation of the ES cells. Instead, combination of fibroblast growth factor 2 and activin A allows retention of Oct4 expression in bank vole blastocyst outgrowths during 4-day culture, indicating that signaling pathways operating in human, rather than mouse ES cells, might be involved in the process of self-renewal of bank vole embryonic cells.

D012 Development of novel therapeutic strategies to stimulate revascularization in heart failure: biopolymeric delivery of a synergistic combination of angiogenic growth factors

Therapeutic angiogenesis is a promising approach to re-establish cardiac perfusion following ischemia or in heart failure. Recently, it has been proposed that a combination of growth factors may be key to the generation of stable vascular networks. However, it is currently unknown what growth factor combinations are optimal for therapeutic angiogenesis and what mode of administration is most adapted to achieve functional revascularization of the heart. The aim of this study was to investigate the effects of novel growth factor combinations and to evaluate their angiogenic effects in vivo following delivery with a new biodegradable polymer system. First, we found that FGF-2 (Fibroblast Growth Factor-2) and HGF (Hepatocyte Growth Factor) display synergistic stimulatory effects on the migration and proliferation of murine endothelial cells in vitro. Furthermore, we show that whereas HGF potentiates the proliferative effect of FGF-2 in smooth muscle cells, the HGFinduced mobility response in these cells is enhanced by FGF-2. The molecular mechanisms of the reciprocal stimulatory effects involve upregulation in endothelial cells of the HGF receptor, c-Met, by FGF-2, and vice versa upregulation of the FGFR-1 by HGF stimulation. Next, we determined the release potential of a new polymeric delivery system based on biodegradable alginate microparticules adapted for cardiac injection. We found that FGF-2 is liberated during less than 1 month with a strong initial level of release during the first ten days. In contrast, HGF is continuously liberated during 40 days with a maximal release around day eighth. Our in vivo data, from a Matrigel plug assay in mice, indicate a significantly increased efficacy of angiogenic therapy using growth factors delivered by alginate polymers. Moreover, we found that a combination of HGF and FGF-2 displays a potent synergistic angiogenic effect in vivo. In conclusion, we demonstrate the potential of the specific combination of FGF-2 and HGF in obtaining a synergistic stimulatory effect in vitro and in vivo. Further, our new alginate-based injectable polymeric system seems well adapted for delivery of growth factors for therapeutic angiogenic approaches to ameliorate cardiac perfusion.

Inhibition of histone deacetylases antagonized FGF2 and IL-1β effects on MMP expression in human articular chondrocytes

Fibroblast growth factor-2 (FGF2) and interleukin-1β (IL-1β) stimulate the expression of matrix metalloproteinases (MMPs) in articular chondrocytes, which may contribute to cartilage degradation and development of osteoarthritis. Histone deacetylases (HDACs) have recently been implicated in the regulation of MMP gene expression. To investigate the functional involvement of HDACs in the signaling pathway of FGF2 and IL-1β, we examined the effects of HDAC inhibition on activities of FGF2 or IL-1β on gene expression of MMP-1, MMP-3, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs-5 (ADAMTS5), collagen type II, and aggrecan. Human articular chondrocyte cultures were treated with FGF2 or IL-1β in the presence or absence of HDAC inhibitor (trichostatin A, TSA). Gene expression levels after treatments were assessed using quantitative real time PCR. Results showed that FGF2 and IL-1β both increased MMP-1 and -13 expression, while IL-1β also increased MMP-3 mRNA levels. These effects were attenuated in the presence of TSA in a dose dependent manner. In contrast to the effects on MMPs, FGF2 decreased mRNA levels of ADAMTS-5, which was not affected by HDAC inhibition. FGF2, IL-1β, and TSA inhibited expression of aggrecan, while TSA also decreased mRNA levels of collagen type II. These findings showed that HDAC inhibition antagonized FGF2 and IL-1β induced MMP expression. Combination of FGF2 and the HDAC inhibitor decreases both anabolic and catabolic genes, which may slow the cartilage turnover and be beneficial for maintaining cartilage integrity.

Three Steps to Commitment

Mouse embryonic stem (ES) cells that differentiate in serum-supplemented media give rise to hematopoietic cells such as erythrocytes and macrophages. Along the way, they pass through three intermediate stages, epiblast-like, mesodermal, and hemangioblast, which are distinguishable by gene expression profile, developmental potential, and morphology. Pearson et al . describe a serum-free culture method that recapitulates this differentiation pathway, and they used it to identify the stage-specific requirements for each cytokine. Cell identities were determined by quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis of marker gene expression and by developmental potential as assayed by cell morphology in colonies after replating. A cocktail of bone morphogenetic protein 4 (BMP4), fibroblast growth factor 2 (FGF2), activin A, and vascular endothelial growth factor (VEGF) mimicked serum in directing ES cell fate. ES cells treated with BMP4 passed through an epiblast-like stage before expressing mesoderm-specific genes such as Mesp2 and Tbx6 . Without further treatment, mesodermal precursor cells gave rise to hemangioblasts and other mesodermal derivatives that were not observed in serum-directed differentiation, indicating that they retained pluripotency. However, continuing the treatment of these cells with a combination of FGF2 and activin A induced only the formation of hemangioblasts. Addition of VEGF caused the cells to differentiate into hematopoietic cells. The authors have identified a handful of factors that can direct the differentiation of ES cells to a particular subset of fates in vitro, a necessary prelude to understanding how lineage restriction may by regulated in stem cells in vivo and how it may be manipulated in vitro. S. Pearson, P. Sroczynska, G. Lacaud, V. Kouskoff, The stepwise specification of embryonic stem cells to hematopoietic fate is driven by sequential exposure to Bmp4, activin A, bFGF and VEGF. Development 135, 1525-1535 (2008). [Abstract] [Full Text]

Survival, synaptogenesis, and regeneration of adult mouse spiral ganglion neurons in vitro

The inner ear spiral ganglion is populated by bipolar neurons connecting the peripheral sensory receptors, the hair cells, with central neurons in auditory brain stem nuclei. Hearing impairment is often a consequence of hair cell death, e.g., from acoustic trauma. When deprived of their peripheral targets, the spiral ganglion neurons (SGNs) progressively degenerate. For effective clinical treatment using cochlear prostheses, it is essential to maintain the SGN population. To investigate their survival dependence, synaptogenesis, and regenerative capacity, adult mouse SGNs were separated from hair cells and studied in vitro in the presence of various neurotrophins and growth factors. Coadministration of fibroblast growth factor 2 (FGF-2) and glial cell line-derived neurotrophic factor (GDNF) provided support for long-term survival, while FGF-2 alone could strongly promote neurite regeneration. Fibroblast growth factor receptor FGFR-3-IIIc was found to upregulate and translocate to the nucleus in surviving SGNs. Surviving SGNs formed contacts with other SGNs after they were deprived of the signals from the hair cells. In coculture experiments, neurites extending from SGNs projected toward hair cells. Interestingly, adult mouse spiral ganglion cells could carry out both symmetric and asymmetric cell division and give rise to new neurons. The authors propose that a combination of FGF-2 and GDNF could be an efficient route for clinical intervention of secondary degeneration of SGNs. The authors also demonstrate that the adult mammalian inner ear retains progenitor cells, which could commit neurogenesis.

BMP-2-dependent integration of adult mouse subventricular stem cells into the neural crest of chick and quail embryos

Central nervous system (CNS) stem cells isolated from the subventricular zone (SVZ) show a remarkable differentiation potential into neural derivatives. Surprisingly adult SVZ cells can also be induced in vitro to differentiate into neural crest cell fates. This fate switch is dependent on the combination of fibroblast growth factor 2 (FGF2) and bone morphogenetic proteins (BMPs). Here we transplanted adult SVZ stem cells from GFP mice as neurospheres into the trunk neural tube of chick and quail embryos. Only neurospheres pre-exposed to BMP-2 and FGF2 formed close contacts with the dorsal neuroepithelium corresponding to the neural crest area. GFP-positive cells emigrated from the neurosphere and were identified in the roof plate, the dorsal neuroepithelium and among emigrating neural crest cells adjacent to the neural tube. Neurospheres not treated with BMP-2 did not integrate into the neuroepithelium. Our data demonstrate that adult CNS stem cells can be efficiently prepared in vitro for integration into the embryonic neural crest. BMP-2 treatment conveys the necessary morphogenetic capabilities to adult stem cells for future clinical transplantation strategies.

Cell Sourcing and Culture Conditions for Fibrin-Based Valve Constructs

Cell sourcing for tissue-engineered heart valves remains a critical issue. In this work, human dermal fibroblasts (HDF) or porcine valve interstitial cells (PVIC) were entrapped in adherent fibrin disk constructs and harvested at 3 and 5 weeks. We compared the fibrin remodeling abilities of each cell type in Dulbecco's Modified Eagle's Medium (DMEM) and DMEM/F12 supplemented with transforming growth factor beta (TGF), and the response of PVIC to DMEM/F12 supplemented with fibroblast growth factor (FGF), a combination of FGF and TGF, and TGF with varying ascorbic acid (AA) concentrations. Culture media were supplemented with serum, insulin, AA, a fibrinolysis inhibitor, and antibiotics. DMEM maximized collagen and elastin deposition by HDF, while DMEM/F12 with FGF yielded the highest fibrin remodeling response by PVIC. HDF degraded fibrin slower than PVIC, and PVIC constructs had higher cellularity than HDF constructs in DMEM and DMEM/F12 at 3 weeks. FGF addition increased collagen content, collagen deposited per cell, and collagen as percentage of total protein compared to medium supplemented with TGF or TGF and FGF. AA addition increased collagen deposition by PVIC, but there was no dose dependence between 50 and 150 microg/mL AA. These results collectively show that PVIC are able to remodel fibrin faster and exhibit greater mechanical stiffening compared to HDF. Conditions for increased collagen deposition are also identified toward the engineering of valve constructs.

Niche-Independent Symmetrical Self-Renewal of a Mammalian Tissue Stem Cell

Pluripotent mouse embryonic stem (ES) cells multiply in simple monoculture by symmetrical divisions. In vivo, however, stem cells are generally thought to depend on specialised cellular microenvironments and to undergo predominantly asymmetric divisions. Ex vivo expansion of pure populations of tissue stem cells has proven elusive. Neural progenitor cells are propagated in combination with differentiating progeny in floating clusters called neurospheres. The proportion of stem cells in neurospheres is low, however, and they cannot be directly observed or interrogated. Here we demonstrate that the complex neurosphere environment is dispensable for stem cell maintenance, and that the combination of fibroblast growth factor 2 (FGF-2) and epidermal growth factor (EGF) is sufficient for derivation and continuous expansion by symmetrical division of pure cultures of neural stem (NS) cells. NS cells were derived first from mouse ES cells. Neural lineage induction was followed by growth factor addition in basal culture media. In the presence of only EGF and FGF-2, resulting NS cells proliferate continuously, are diploid, and clonogenic. After prolonged expansion, they remain able to differentiate efficiently into neurons and astrocytes in vitro and upon transplantation into the adult brain. Colonies generated from single NS cells all produce neurons upon growth factor withdrawal. NS cells uniformly express morphological, cell biological, and molecular features of radial glia, developmental precursors of neurons and glia. Consistent with this profile, adherent NS cell lines can readily be established from foetal mouse brain. Similar NS cells can be generated from human ES cells and human foetal brain. The extrinsic factors EGF plus FGF-2 are sufficient to sustain pure symmetrical self-renewing divisions of NS cells. The resultant cultures constitute the first known example of tissue-specific stem cells that can be propagated without accompanying differentiation. These homogenous cultures will enable delineation of molecular mechanisms that define a tissue-specific stem cell and allow direct comparison with pluripotent ES cells.

Heparin structures in FGF‐2–dependent morphological transformation of astrocytes

Fibroblast growth factor-2 (FGF-2) participates in the morphological transformation of astrocytes (stellation) during the formation of glial scars in injured brains. In the current study, we used quantitative morphometric analysis to investigate the structural requirements for heparin's enhancement of FGF-2-induced stellation of cultured cortical astrocytes. Native heparin significantly promoted FGF-2-dependent astrocytic stellation, whereas heparin hexasaccharide inhibited FGF-2-dependent stellation. Furthermore, 2-O-, 6-O-, and N-desulfated heparins were unable to promote FGF-2-dependent stellation. The stellation induced by FGF-2 or by a combination of FGF-2 and native heparin was inhibited by SU5402, an FGF receptor inhibitor. These results demonstrate that the length and sulfated position of heparin are important for its enhancement of FGF-2-dependent astrocyte stellation. In addition, our findings show that heparin oligosaccharides are useful for regulating the FGF-2-dependent astrocytic transformation.

Fibroblast Growth Factor-2 Stimulates Interleukin-6 Secretion in Human Pancreatic Periacinar Myofibroblasts

Fibroblast growth factor-2 (FGF-2) plays an important role in the pathophysiology of acute and chronic pancreatitis. In the present study, to evaluate the proinflammatory nature of FGF-2, we investigated the effects of FGF-2 on IL-6 secretion in human pancreatic periacinar myofibroblasts. IL-6 supernatant levels were determined by enzyme-linked immunosorbent assays (ELISA). IL-6 mRNA expression were determined by Northern blots and quantitative PCRs. Activated protein (AP)-1 DNA-binding activities were evaluated by electrophoretic gel mobility shift assays (EMSA). FGF-2 induced IL-6 release in a dose- and time-dependent manner. FGF-2 activity for IL-6 induction was the same as that of IL-17. The combination of FGF-2 and IL-17 exerted additive effects at mRNA and protein levels. FGF-2 induced AP-1 DNA-binding activity, but blockage of AP-1 signaling by adenovirus-mediated transfer of a dominant negative c-Jun gene did not affect FGF-2-induced IL-6 mRNA expression. FGF-2 rapidly induced activation of ERK1/2 and p38 MAP kinases, and specific inhibitors for these enzymes significantly reduced FGF-2-induced IL-6 release. In the pancreas, FGF-2 may not only play a role as a growth factor in tissue injury repair processes but also as an inducer of acute-phase response via stimulation of IL-6 release.

Hypoxia in combination with FGF-2 induces tube formation by human microvascular endothelial cells in a fibrin matrix: involvement of at least two signal transduction pathways.

Hypoxia in combination with a growth factor is a strong inducer of angiogenesis. Among several effects, hypoxia can activate endothelial cells directly, but the mechanism by which it acts is not fully elucidated. In vitro, human microvascular endothelial cells (hMVEC) form capillary-like tubules in fibrin solely after stimulation with a combination of fibroblast growth factor (FGF)-2 or vascular endothelial growth factor (VEGF) and the cytokine tumour necrosis factor (TNF)alpha. We show in this paper that in hypoxic conditions, FGF-2-stimulated hMVEC form tube-like structures in a fibrin matrix in the absence of TNFalpha. Hypoxia/FGF-2-stimulated cells express more urokinase-type plasminogen activator (u-PA) receptor than normoxia/FGF-2-stimulated cells and display a slightly higher turnover of u-PA. This small increase in u-PA activation probably cannot fully explain the hypoxia/FGF-2-induced tube formation. Hypoxia activated at least two signal pathways that may contribute to the enhanced angiogenic response. In hypoxia/FGF-2-stimulated hMVEC the transcription factor p65 was activated and translocated to the nucleus, whereas in normoxia/FGF-2-stimulated cells p65 remained inactive. Furthermore, in hypoxic conditions, the amounts of phosphorylated mitogen-activated protein kinases ERK1/2 were increased compared to normoxic conditions. We conclude that hypoxia is able to activate different signal pathways in FGF-2-stimulated human endothelial cells, which may be involved in hypoxia-induced angiogenesis.

Growth factor synergism and antagonism in early neural crest development

This review article focuses on data that reveal the importance of synergistic and antagonistic effects in growth factor action during the early phases of neural crest development. Growth factors act in concert in different cell lineages and in several aspects of neural crest cell development, including survival, proliferation, and differentiation. Stem cell factor (SCF) is a survival factor for the neural crest stem cell. Its action is neutralized by neurotrophins, such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) through apoptotic cell death. In contrast, SCF alone does not support the survival of melanogenic cells (pigment cell precursors). They require the additional presence of a neurotrophin (NGF, BDNF, or NT-3). Fibroblast growth factor-2 (FGF-2) is an important promoter of proliferation in neuronal progenitor cells. In neural crest cells, fibroblast growth factor treatment alone does not lead to cell expansion but also requires the presence of a neurotrophin. The proliferative stimulus of the fibroblast growth factor - neurotrophin combination is antagonized by transforming growth factor beta-1 (TGFbeta-1). Moreover, TGFbeta-1 promotes the concomitant expression of neuronal markers from two cell lineages, sympathetic neurons and primary sensory neurons, indicating that it acts on a pluripotent neuronal progenitor cell. Moreover, the combination of FGF-2 and NT3, but not other neurotrophins, promotes expression or activation of one of the earliest markers expressed by presumptive sympathetic neuroblasts, the norepinephrine transporter. Taken together, these data emphasize the importance of the concerted action of growth factors in neural crest development at different levels and in several cell lineages. The underlying mechanisms involve growth-factor-induced dependence of the cells on other factors and susceptibility to growth-factor-mediated apoptosis.

Inhibition of angiogenesis on glycated collagen lattices.

Advanced glycation endproduct (AGE) accumulation in extracellular matrix proteins has been demonstrated in diabetic patients with a significant correlation with the severity of diabetic complications. AGE accumulation induces matrix protein cross-link formation, resulting in an increased stiffness of matrix fibres and the reduction of the susceptibility of matrix proteins to proteolytic degradation. We examined whether glycation-induced collagen cross-linking may affect vascular endothelial cell behaviours such as invasion, proliferation and differentiation, using the in vitro angiogenesis model of capillary-like structure formation in three-dimensional matrices of collagen type I. Endothelial cells cultured on collagen gel with angiogenic factors (the combination of fibroblast growth factor-2 and vascular endothelial growth factor) invaded the underlying collagen matrix, and organized capillary-like cord structures in the gel. We found that endothelial cell invasion into glycated collagen gel was significantly attenuated without any effect on proteinase activity including cell-associated plasminogen activator and matrix metalloproteinase in the conditioned medium. In addition, subsequent capillary-like cord formation was also inhibited in glycated collagen gel. In contrast, endothelial cell proliferation was enhanced on glycated collagen gel with or without angiogenic factors compared with control collagen gel. These results suggest that the structural alterations of extracellular matrix proteins through the glycation-induced cross-link formation affect the interaction between endothelial cell and extracellular matrix, resulting in the impairment of an adequate neovascularization in diabetic patients.

The effect of growth factors on the proliferation of human endometrial stromal cells in culture

Development of ectopic implants of endometriosis is associated with both an inflammatory response by macrophages and endometrial stromal cell proliferation. Macrophages are capable of releasing a variety of inflammatory mediators, including growth factors. To assess the impact of such factors on endometrial tissue, we have studied the effects of recombinant growth factors, fibroblast growth factor, epidermal growth factor, transforming growth factor-alpha, and inflammation mediators transforming growth factor-beta, and tumor necrosis factor-alpha on human endometrial stromal cell proliferation. Increasing concentrations of these compounds were added to cultures of primary, secondary, and long-term stromal cells and the cells were harvested at 24, 48, and 72 hours. Epidermal growth factor, transforming growth factor-alpha, transforming growth factor-beta, and fibroblast growth factor induced a statistically significant, dose-dependent increase in stromal cell thymidine uptake of 1.5- to fivefold. The cytokine tumor necrosis factor had no effect alone, but the combination of fibroblast growth factor and tumor necrosis factor had a synergistic effect, increasing cell proliferation 25% to 84% over fibroblast growth factor alone. The stromal cell response to a wide range of cell growth effectors and the potential of mediators like tumor necrosis factor-alpha to synergize suggest that such macrophage-secretory products may contribute to proliferation of endometrial implants in vivo.

Competence and progression growth factors stimulate different tRNA lys modification reactions in Balb/C 3T3 cells

Sparse cultures of Balb/C 3T3 cells were growth arrested in a medium containing 1% calf plasma. This treatment caused a decrease in tRNA4lys, a tRNA species related to cell division, and a corresponding increase in tRNA5lys. A restoration of tRNA4lys levels and an increase in cell number was obtained by the addition of either 10% calf serum or a combination of fibroblast growth factor and melanocyte stimulating activity. Neither growth factor alone evoked a proliferative response. The progression factors, insulin and multiplication stimulating activity stimulated the rapid conversion of tRNA5lys to tRNA2lys, whereas the competence factors, fibroblast growth factor and platelet-derived growth factor stimulated a slight conversion of tRNA2lys to tRNA4lys.

Stimulation of amniotic fluid cells by fibroblast growth factor

Amniotic fluid cells were grown in modified Dulbecco's medium with and without the following additives in different combinations: fibroblast growth factor (FGF), dexamethasone (DME), insulin and cyclic GMP under controlled conditions. Experiments were also conducted with varying amounts of foetal calf serum and FGF to establish optimum levels. Comparative growth, determined by measuring 3H-thymidine uptake, showed that the combination of FGF, DME and insulin in medium with 20% foetal calf serum produced the highest growth rate. These experiments indicate that the culture time of amniotic fluid cells for chromosome studies could be reduced by 25%. FGF did not have any deleterious effect on the chromosomes.

Uptake of α‐aminoisobutyric acid and phosphate by membrane vesicles derived from growing and quiescent fibroblasts

Membrane vesicles derived principally from the plasma membrane and endoplasmic reticulum of mouse 3T3 cells transformed by Simian virus 40 take up alpha-aminoisobutyric acid (AIB) and phosphate (Pi). When NaCl is added simultaneously with AIB or Pi, uptake rises two- to three-times above the equilibrium to accumulate AIB or Pi over the control value, in the presence of a Na+ gradient, is almost lost in membrane vesicles derived from benzpyrene-transformed 3T3 cells (BP3T3) arrested in the G1 phase of the cell cycle by serum starvation. When added to the membranes with NaCl and the uptake substrate, a combination of fibroblast growth factor (FGF) and epidermal growth factor EGF restores the ability of the membranes to accumulate AIB and Pi over the control value.