Fibroblast growth factor signaling in embryonic and cancer stem cells

Cancer stem cells are cancer cells that originate from the transformation of normal stem cells. The most important property of any stem cell is the ability to self-renew. Through this property, there are striking parallels between normal stem cells and cancer stem cells. Both cell types share various markers of “stemness”. In particular, normal stem cells and cancer stem cells utilize similar molecular mechanisms to drive self-renewal, and similar signaling pathways may induce their differentiation.The fibroblast growth factor 2 (FGF-2) pathway is one of the most significant regulators of human embryonic stem cell (hESC) self-renewal and cancer cell tumorigenesis. Here we summarize recent data on the effects of FGF-2 and its receptors on hESCs and leukemic stem/progenitor cells. Also, we discuss the similarities of these findings with stem cell renewal and differentiation phenotypes.     

Inhibition of invasiveness and expression of epidermal growth factor receptor in human colorectal carcinoma cells induced by retinoic acid

INTRODUCTIONAsaderivativeofvitaminA,RAcaninhibittheproliferati0nofmanymalignantcel1sallde1icitdifferentiationinsometumorce1lsI1-5].RecentstudieshaveshownthatRAmodu1atessynthesisofover4Oproteinsthroughitsnucleicrecept0r[6].Forinstance,RAinducesthesynthesis0ffibronectin(FN)incertaintumorcellsandactivatesthegenecodingfOrB1sllbunitoflaminin(LN)causingitsexpressi0n[7,8].FNandLN'areknownt0bethemostimportantcomponentsofnon-c0llageng1ycoproteinsintheextracellu1armatrixandareclose1yrelatedt…     

Mesenchymal to embryonic incomplete transition of human cells by chimeric OCT4/3 (POU5F1) with physiological co-activator EWS

POU5F1 (more commonly known as OCT4/3) is one of the stem cell markers, and affects direction of differentiation in embryonic stem cells. To investigate whether cells of mesenchymal origin acquire embryonic phenotypes, we generated human cells of mesodermal origin with overexpression of the chimeric OCT4/3 gene with physiological co-activator EWS (product of the EWSR1 gene), which is driven by the potent EWS promoter by translocation. The cells expressed embryonic stem cell genes such as NANOG, lost mesenchymal phenotypes, and exhibited embryonal stem cell-like alveolar structures when implanted into the subcutaneous tissue of immunodeficient mice. Hierarchical analysis by microchip analysis and cell surface analysis revealed that the cells are subcategorized into the group of human embryonic stem cells and embryonal carcinoma cells. These results imply that cells of mesenchymal origin can be traced back to cells of embryonic phenotype by the OCT4/3 gene in collaboration with the potent cis-regulatory element and the fused co-activator. The cells generated in this study with overexpression of chimeric OCT4/3 provide us with insight into cell plasticity involving OCT4/3 that is essential for embryonic cell maintenance, and the complexity required for changing cellular identity.     

Transcriptional profiling of initial differentiation events in human embryonic stem cells

Currently, there are no differentiation strategies for human embryonic stem cells (hESCs) that efficiently produce one specific cell type, possibly because of lack of understanding of the genes that control signaling events prior to overt differentiation. sed HepG2 cell conditioned medium (MEDII), which induces early differentiation in mouse ES cells while retaining pluripotent markers, to query gene expression in hESCs. Treatment of adherent hESCs with 50% MEDII medium effected differentiation to a cell type with gene expression similar to primitive streak stage cells of mouse embryos. MEDII treatment up-regulates TDGF1 (Cripto), a gene essential for anterior-posterior axis and mesoderm formation in mouse embryos and a key component of the TGFB1/NODAL signaling pathway. LEFTYA, an antagonist of NODAL/TDGF1 signaling expressed in anterior visceral endoderm, is down-regulated with MEDII treatment, as is FST, an inhibitor of mesoderm induction via the related INHBE1 pathway. In summary, the TGFB1/NODAL pathway is important for primitive-streak and mesoderm formation and in using MEDII, we present a means for generating an in vitro cell population that maintains pluripotent gene expression (POU5F1, NANOG) and SSEA-4 markers while regulating genes in the TGFB1/NODAL pathway, which may lead to more uniform formation of mesoderm in vitro.     

The expression of insulin-like growth factor binding proteins in human hepatocellular carcinoma

Insulin-like growth factors (IGF), IGF receptors and IGF binding proteins (IGFBPs) play an important role in cell growth and differentiation. The liver is the major source of IGF-1 and at least two IGFBPs (IGFBP-1 and IGFBP-3). IGFBPs most often serve to attenuate the effects of IGF at the receptor level and thereby limit IGF-induced cell growth and differentiation. Although changes in IGFBP expression have been described during controlled liver growth such as hepatic regeneration following partial hepatectomy, there is limited knowledge of IGFBPs gene expression in uncontrolled growth or hepatocellular carcinoma. In the present study, we employed Northern blotting techniques to document the expression of IGFBP-1, 3 and 4 in normal human livers, cirrhotic and hepatocellular carcinoma tissues. The results revealed no differences in IGFBP-1, 3 and 4 mRNA levels between normal and cirrhotic tissues. However, the expression of all three IGFBPs mRNA were significantly down regulated in hepatocellular carcinoma tissues. These findings are in keeping with IGFBPs playing an important inhibitory role in the development and/or growth of hepatocellular carcinoma in humans.     

Endoderm-secreted factor stimulates growth of embryonal carcinoma stem cells

Summary Stem cells of the embryonal carcinoma cell line called H6 can be induced to differnetiate to endoderm-like cells by retinoic acid (3×10⁻⁶ M). We have detected a diffusible and stable factor which is secreted by H6 endoderm-like cells and stimulates the growth of H6 stem cells. The stimulation by the endoderm-like cells is considereably greater than that by mouse fibroblasts or H6 stem cells themselves. No reciprocal stimulation of endoderm-like cells by stem cells occurs. Part but not all of the stimulation might be due to extracellular matrix proteins or to insulin-like growth factor type 2, each of which also stimulates the growth of H6 stem cells. Insulin causes no such stimulation. This work was supported by research rant no. CA-16754 from the National Cancer Institute to J. W. L. E. L. G. was supported by an American Heart Association Medical Student Research Award. Editor's Statement This paper presents a good example of cooperativity between undifferentiated teratoma stem cells and differentiated parietal endoderm-derived countrparts in terms of growth support. It raises the interesting question of the relationship between factors produced by paprietal and visceral endoderm cells. Gordon H. Sato     

Overexpression of hepatocyte growth factor receptor in renal carcinoma cells indirectly stimulates tumor growth in vivo

We examined the role of increased expression of HGFR kinase in in vivo growth of renal carcinoma. Human renal carcinoma cell line, ACHN cells, was transfected with plasmid encoding wild-type HGFR gene to generate cell lines with increased HGFR protein. ACHN cells with elevated HGFR expression, denoted clones 8 and 10, respectively, showed higher basal kinase activities of HGFR and PI3-kinase than those of empty-vector (mock)-transfected cells. Clone 8 and 10 cells grew similar to mock cells in culture. In mice, tumors of these clones grew more rapidly than those of mock cells. Microvessel density of clone 8 or 10 tumors was higher than that of mock tumors. Clone 8 and 10 cells secreted vascular endothelial growth factor-A (VEGF-A) more than mock cells and the secretion was PI3-kinase inhibitor, LY294002-sensitive. Anti-VEGF-A neutralizing antibody significantly inhibited tumor growth of clones 8 and 10 in mice. These results indicate for the first time that overexpression of HGFR tyrosine kinase in renal carcinoma cells participates in rapid tumor growth in vivo.     

Betacellulin and nicotinamide sustain PDX1 expression and induce pancreatic beta-cell differentiation in human embryonic stem cells

The major obstacle in cell therapy of diabetes mellitus is the limited source of insulin-producing β cells. Very recently, it was shown that a five-stage protocol recapitulating in vivo pancreatic organogenesis induced pancreatic β cells in vitro; however, this protocol is specific to certain cell lines and shows much line-to-line variation in differentiation efficacy. Here, we modified the five-stage protocol for the human embryonic stem cell line SNUhES3 by the addition of betacellulin and nicotinamide. We reproduced in vivo pancreatic islet differentiation by directing the cells through stages that resembled in vivo pancreatic organogenesis. The addition of betacellulin and nicotinamide sustained PDX1 expression and induced β-cell differentiation. C-peptide—a genuine marker of de novo insulin production—was identified in the differentiated cells, although the insulin mRNA content was very low. Further studies are necessary to develop more efficient and universal protocols for β-cell differentiation.     

Spontaneous differentiation of mouse embryonic stem cells in vitro: characterization by global gene expression profiles

We characterized the temporal gene expression changes during four weeks of spontaneous differentiation of mouse ES cells in a monolayer culture in order to obtain better insight into the differentiation process. The overall gene expression pattern was changed dramatically during the first two weeks of spontaneous differentiation, but stabilized after the second week. Most of the genes regulated within the first two weeks of spontaneous differentiation were genes related to development including morphogenesis, cell differentiation, embryonic development, pattern specification, mesoderm development, post-embryonic development, and blastocyst development. While most of the ectoderm lineage related genes were down-regulated, genes related to the mesoderm or endoderm lineage were up-regulated through the first week and second week, respectively. This study revealed that the development of ectoderm lineage is a recessive process during the spontaneous differentiation of mouse ES cells in monolayer culture. Our time-course characterization might provide a useful time line for directed differentiation of mouse ES cells.     

Growth and differentiation in cultured human thyroid cells: Effects of epidermal growth factor and thyrotropin

Summary Human thyroid cells were grown and subcultured in vitro to examine their responses to known hormones and growth factors, and to serum. The cells were obtained from surgical specimens and were either neoplastic or nonneoplastic. The effects of culture conditions on cell growth were measured by changes in cell numbers and by stimulation of [³H]thymidine incorporation. The results showed that serum (0.5%) was essential for cell proliferation, and that a mixture of insulin (10 μg/ml), transferrin (5 μg/ml), hydrocortisone (10 μg/ml), somatostatin (10 ng/ml), and glycyl-histidyl-lysine (10 ng/ml) enhanced the effect of serum. Maximum growth of the cells was obtained when epidermal growth factor was present at 10⁻⁹ M. Differentiation was measured by production of thyroglobulin, which was found to be stimulated by thyrotropin. This system provides a means to study the hormonal control of growth and differentiation in human thyroid cells. This work was supported by grants from the Medical Research Council of Canada; the Department of Medicine, University of Toronto; and the National Cancer Institute of Canada. J. E. E. is a C.H. Best Foundation and Department of Medicine postdoctoral fellow.     

Neurogenic effect of vascular endothelial growth factor during germ layer formation of human embryonic stem cells

Vascular endothelial growth factor (VEGF), a potent mitogen for vascular endothelial cells, has been suggested as a modulator that is involved in neurogenesis as well as angiogenesis. Here, we directly examined the effect of VEGF on neuroectodermal differentiation using human embryonic stem cells (hESCs). VEGF treatment upregulated the expression of neuroectodermal genes (Sox1 and Nestin) during germ layer formation in embryoid bodies (EBs) and efficiently increased the number of neural rosettes expressing both Pax6 and Nestin. The neural progenitors generated from VEGF-treated EBs further differentiated into cells that showed a similar pattern of gene expression observed in the development of dopaminergic neurons upon terminal differentiation. These results support the neurogenic effect of VEGF on hESC differentiation.