Production of cytokines by immature erythroid cells derived from human embryonic liver

It is well known that regulatory interactions between hematopoietic and lymphoid cells are mediated by different mediators. The cells of erythroid lineage are not an exception and have a regulatory effect on hemato- and immunopoiesis that can be mediated through the production of cytokines i.e. by soluble factors - a universal mechanism for cell regulation in hematopoietic and immune systems. It has been previously shown that erythroid progenitor cells from mice express mRNA of cytokines such as IL-1 alpha and beta, IL-4, IL-6, IFN-gamma, GM-CSF and TGF-beta. In this report we present the results of the production of the main immunoregulatory cytokines by erythroid cells derived from human embryonic liver. It was revealed that the cell population enriched with erythroid progenitors, isolated from human fetal liver, can produce IL-1 beta, IL-2, IL-4, IL-6. The levels of production of cytokines by immature erythroid progenitor cells is compared to the levels of corresponding cytokines produced by mitogen-stimulated peripheral blood mononuclear cells. The production of these cytokines changed quantitatively under the effect of erythropoietin, and are correlated with the expression of differentiation markers of erythroid cells such as AG-EB and Glycophorin A. The role of cytokine production by erythroid cells in hemato- and immunopoiesis and the mechanisms of self-regulation of proliferation and differentiation of erythroid progenitor cells is discussed.     

Epidermal growth factor and transforming growth factor-alpha decrease gamma interferon receptors and induction of intercellular adhesion molecule (ICAM-1) on cultured keratinocytes.

The link between the epidermal keratinocytes of the skin and the activated T lymphocytes of the immune system is mediated by a variety of cytokines, including gamma interferon (IFN-gamma). We studied the influence of keratinocyte mitogens such as transforming growth factor-alpha (TGF-alpha), epidermal growth factor (EGF), and somatomedin-C (SM-C) on the ligand binding of 32P-labeled IFN-gamma to cultured keratinocytes derived from normal appearing adult human skin. Keratinocytes placed in a medium devoid of mitogens become growth arrested, and these quiescent cells expressed 2.4 times (28,900 versus 12,200 sites/cell) as many high affinity IFN-gamma receptors (Kd = 0.22 nM) compared to keratinocytes which were actively growing in medium containing TGF-alpha (25 ng/ml) or EGF (10 ng/ml). The reduction in IFN-gamma receptor sites by TGF-alpha/EGF was mitogen specific, as adding SM-C (500 ng/ml) did not have any effect on ligand binding, although it similarly stimulated keratinocyte growth. The reduction in IFN-gamma receptors was time dependent, occurring primarily after 24-48 hours of change in tissue culture conditions. The reduction in the number of high affinity IFN-gamma receptors by TGF-alpha/EGF had immunobiological consequences, because quiescent keratinocytes in basal medium had an increased expression of HLA-DR and intercellular adhesion molecule-1 (ICAM-1) induced by IFN-gamma, compared to actively growing TGF-alpha/EGF treated keratinocytes. These results suggest that rapidly proliferating keratinocytes exposed to TGF-alpha/EGF but not SM-C are capable of altering their response to IFN-gamma by decreasing their number of cell surface high affinity receptors for IFN-gamma.     

Signalling pathways involved in the process of mesenchymal stem cells differentiating into hepatocytes

End‐stage liver disease can be the termination of acute or chronic liver diseases, with manifestations of liver failure; transplantation is currently an effective treatment for these. However, transplantation is severely limited due to the serious lack of donors, expense, graft rejection and requirement of long‐term immunosuppression. Mesenchymal stem cells (MSCs) have attracted considerable attention as therapeutic tools as they can be obtained with relative ease and expanded in culture, along with features of self‐renewal and multidirectional differentiation. Many scientific groups have sought to use MSCs differentiating into functional hepatocytes to be used in cell transplantation with liver tissue engineering to repair diseased organs. In most of the literature, hepatocyte differentiation refers to use of various additional growth factors and cytokines, such as hepatocyte growth factor (HGF), fibroblast growth factor (FGF), epidermal growth factor (EGF), oncostatin M (OSM) and more, and most are involved in signalling pathway regulation and cell–cell/cell–matrix interactions. Signalling pathways have been shown to play critical roles in embryonic development, tumourigenesis, tumour progression, apoptosis and cell‐fate determination. However, mechanisms of MSCs differentiating into hepatocytes, particularly signalling pathways involved, have not as yet been completely illustrated. In this review, we have focused on progress of signalling pathways associated with mesenchymal stem cells differentiating into hepatocytes along with the stepwise differentiation procedure.     

EGF responsiveness and receptor regulation in normal and differentiation-defective mouse myoblasts

The interrelationship between cell proliferation and terminal myogenic differentiation has been analyzed by studying a differentiation-defective subclone (DD-1) of the permanent mouse myoblast line MM14. Parental MM14 myoblasts withdraw irreversibly from the cell cycle and initiate terminal differentiation when they are deprived of certain mitogens. In contrast, DD-1 cells become quiescent in a mitogen-depleted environment and less than 0.4% of the cells differentiate. When refed with mitogen-rich medium quiescent DD-1 cells resume proliferation. Expression of this differentiation-defective phenotype is apparently coupled to an alteration in mitogen sensitivity: MM14 myoblasts require horse serum plus either chick embryo extract or fibroblast growth factor (FGF) to sustain cell growth: DD-1 variants are responsive to FGF, but also proliferate in response to serum alone or to reduced serum plus epidermal growth factor (EGF). Interestingly, EGF also appears to retard DD-1 cell differentiation in a manner similar to the FGF repression of differentiation in normal myoblasts. Normal and differentiation-defective myoblasts which have been maintained under growth-promoting conditions exhibit similar EGF binding, internalization, and degradation. However, whereas the EGF binding capacity of MM14 myoblasts declines to less than 5% of its initial level within 24 hr of FGF removal, DD-1 variants exhibit an increase in EGF binding when switched to an FGF-depleted medium. The relationship of altered EGF receptor regulation to changes in mitogen sensitivity and differentiation capacity of the DD-1 variant is discussed, and implications for general in vivo processes governing cell proliferation and differentiation are considered.     

Cell-cell and cell-ECM interactions in epithelial apoptosis and cell renewal during frog intestinal development

Amphibian intestinal remodeling during metamorphosis is a developmental system that is entirely controlled by thyroid hormone. It transforms a simple tubular organ into a complex multiply folded frog intestine similar to that in higher vertebrates. This process involves the degeneration of the larval epithelium through programmed cell death (apoptosis) and concurrent proliferation and differentiation of adult cell types. Earlier morphological and cellular studies have provided strong evidence implicating the importance of cell-cell and cell-ECM (extracellular matrix) interactions in this process. The recent molecular characterization of the genes that are regulated by thyroid hormone has begun to reveal some molecular clues underlying such interactions. In particular, theXenopus putative morphogen hedgehog appears to be involved in regulating/mediating cell-cell interactions during adult epithelial proliferation, differentiation, and/or intestinal morphogenesis. On the other hand, several matrix metalloproteinases (MMPs) may be involved in remodeling the ECM. Of special interest is stromelysin-3, whose spatial and temporal expression profile during intestinal metamorphosis implicates a role in ECM remodeling, which in turn facilitates cell fate determination, i.e., apoptosis vs proliferation and differentiation. Understanding the mechanisms of action for those extracellular molecules will present a future challenge in developmental research.     

Cell survival: differences and differentiation

The regulatory mechanisms of cell survival and apoptosis are very complex in nature, implicating numerous players and signaling pathways not only in the decision-making process of surviving (or dying), but as well as in the execution of apoptosis itself. The same complex nature applies with regards to anoikis, a form of apoptosis that is largely regulated by integrin-mediated, cell-extracellular matrix interactions. However, cell survival, apoptosis and anoikis also happen to implicate further mechanistic distinctions according to the specific tissue and/or cell type concerned. Incidentally, recent studies in a particular tissue, the human intestinal epithelium, have unearthed yet another layer of complexity in the regulation of these three cellular processes, namely the implication of differentiation state-specific mechanisms. Although our understanding of the molecular underpinnings of this new concept of differentiation state-distinct regulation of cell survival, apoptosis and/or anoikis is in its infancy, there is already evidence that such principle applies as well to cell types other than intestinal epithelial cells. Further studies on the differentiation state-specific regulation of these three cellular processes, either under normal or physiopathological situations, should prove crucial in increasing our understanding of pathologies which implicate a dysregulation of apoptosis and/or anoikis - such as cancer.     

Real-time studies of the interactions between epidermal growth factor and its receptor during endocytic trafficking.

The interactions of growth factors with cell surface receptors regulate fundamental cell processes, such as growth, differentiation and transformation. Understanding the nature of these interactions at the molecular level is of fundamental importance in cell biology. This is not only from the point of view of basic science, but also because of the repercussions such knowledge might have in understanding the mode of action of drugs in cells. Receptor mediated endocytosis has been implicated in the downregulation of the mitogenic signal. However, no data are thus far available on how growth factor/receptor interactions might control endocytic trafficking. Here we show that information on modes of binding and receptor conformational changes can be obtained using time-resolved fluorescence methods. We have found that fluorescent probes bound to epidermal growth factor (EGF) show dynamic fluorescence quenching when EGF is bound to internalising EGF receptors (EGFR). We propose that this dynamic quenching takes place because EGF-bound probes interact with tryptophan residues in the extracellular domain of the EGF-EGFR complex. Real-time accumulation of fluorescent decays has also allowed us to follow the time course of a conformational change in EGFR occurring during endocytosis, and correlate this information with endosomal trafficking and EGFR recycling.     

Regulation of intestinal stem cell fate specification

The remarkable ability of rapid self-renewal makes the intestinal epithelium an ideal model for the study of adult stem cells. The intestinal epithelium is organized into villus and crypt, and a group of intestinal stem cells located at the base of crypt are responsible for this constant self-renewal throughout the life. Identification of the intestinal stem cell marker Lgr5, isolation and in vitro culture of Lgr5+ intestinal stem cells and the use of transgenic mouse models have significantly facilitated the studies of intestinal stem cell homeostasis and differentiation, therefore greatly expanding our knowledge of the regulatory mechanisms underlying the intestinal stem cell fate determination. In this review, we summarize the current understanding of how signals of Wnt, BMP, Notch and EGF in the stem cell niche modulate the intestinal stem cell fate.     

Role of polypeptide growth factors in the regulation of epidermal keratinocyte proliferation

The growth factors, cytokines, adhesive molecules and extracellular matrix components play the leading role in the processes of intercellular interactions. Literary data on structure and mechanisms of functioning of the growth factors and their receptors are summarised in the present review. Some aspects of regulatory functions of such growth factors as EGF, TGFalpha, TGFbeta, FGF, KGF, AR, and HGF in these processes in epidermis keratinocytes both in vivo and in vitro as example were also considered.     

EGF-responsive rat neural stem cells: molecular follow-up of neuron and astrocyte differentiation in vitro

Neural stem cells (NSCs) could be very useful for the "cell therapy" treatment of neurological disorders. For this reason basic studies aiming to well characterize the biology of NSCs are of great interest. We carried out a molecular and immunocytochemical analysis of EGF-responsive NSCs obtained from rat pups. After the initial growth of NSCs as floating neurospheres in EGF-containing medium, cells were plated on poly-L-ornithine-coated dishes either in the presence or absence of EGF. We followed cell differentiation and apoptosis for 21 days in vitro and analyzed the expression levels of some genes having a major role in these processes, such as pRB, pRB2/p130, p27, and p53. We observed that EGF impairs neuronal differentiation. Furthermore, in the absence of mitogens, apoptosis, which appeared to proceed through the "p53 network," was significantly lower than in the presence of EGF. The cyclin kinase inhibitor p27, while important for cell cycle exit, seemed dispensable for cell survival and differentiation.     

Inhibition of T cell differentiation into effectors by NKT cells requires cell contacts

NKT cells are potent regulatory T cells that prevent the development of several autoimmune diseases. Analysis of NKT cell regulatory function in the NOD mouse has revealed that NKT cells inhibit the development of type 1 diabetes by impairing the differentiation of anti-islet T cells into Th1 effector cells. In the present study, we have performed in vitro and in vivo experiments to determine the respective role of cytokines and cell contacts in the blockade of T cell differentiation by NKT cells. These experiments reveal that cytokines such as IL-4, IL-10, IL-13, and TGF-beta, that have been involved in other functions of NKT cells, play only a minor role if any in the blockade of T cell differentiation by NKT cells. Diabetes is still prevented by NKT cells in the absence of functional IL-4, IL-10, IL-13, and TGF-beta. In contrast, we show for the first time that cell contacts are crucial for the immunoregulatory function of NKT cells.     

Analysis of proteomic changes induced upon cellular differentiation of the human intestinal cell line Caco-2

The human intestinal cell line Caco-2 is a well-established model system to study cellular differentiation of human enterocytes of intestinal origin, because these cells have the capability to differentiate spontaneously into polarized cells with morphological and biochemical features of small intestinal enterocytes. Therefore, the cells are widely used as an in vitro model for the human intestinal barrier. In this study, a proteomic approach was used to identify the molecular marker of intestinal cellular differentiation. The proteome of proliferating Caco-2 cells was compared with that of fully differentiated cells. Two-dimensional gel analysis yielded 53 proteins that were differently regulated during the differentiation process. Pathway analysis conducted with those 34 proteins that were identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analysis revealed subsets of proteins with common molecular and cellular function. It was shown that proteins involved in xenobiotic and drug metabolism as well as in lipid metabolism were upregulated upon cellular differentiation. In parallel, proteins associated with proliferation, cell growth and cancer were downregulated, reflecting the loss of the tumorigenic phenotype of the cells. Thus, the proteomic approach in combination with a literature-based pathway analysis yielded valuable information about the differentiation process of Caco-2 cells on the molecular level that contributes to the understanding of the development of colon cancer or inflammatory diseases such as ulcerative colitis--diseases associated with an imbalanced differentiation process of intestinal cells.     

Experimental basis of cancer combination chemotherapy with retinoids,cytokines, 1,25-dihydroxyvitamin D3, and analogs

Retinoids, cytokines as well as 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] and analogs possess properties known to contribute potentially to cancer chemopreventive and chemotherapeutic effects. They induce cell differentiation, inhibit cell prolifeation, suppress expression of viral oncogenes, and inhibit angiogenesis nexessary for tumor growth. Since clinical combination chemotherapy of cytotoxic agents has proven superior to monotherapy, This moddality might also be useful for other classes of antitumor drugs. A series of retinoids, such as all-trans-, 13-cis-, 9-cis retinoic acid, and acitreti, cytokines, 1,25(OH)₂D₃, and analogs have been investigated in mocdel systems of defferentiation, proliferation, viral oncogenes, and angiogenesis. The three classes of compounds have common effects but nevertheless show a variance depending on the particular representative of each class. Combination of compounds of the different classes led in the various models to a hegher efficacy compared with the compounds given alone. Cytokines such as IFNα, IFNγ, G-CSF, TNFα, IL-1, and IL-4 markedly potentiate the differentiation-inducing effect of retinoids. Cytokines as well as retinoids combined with 1,25(OH)₂D₃ and analogs synergistically enhanced differentiation induction in human transformed hemopoietic cell lines. On a series of human transformed epithelial cell lines a panel of cytokies, such as IFNα, IFNβ, TNFα, TGFβ, and EGF acted synergistically with retinoies on inhibition of proliferation. This was also observed by combining retinoids with 1,25(OH)₂D₃ and analogs. Retinoids as well as interferons α and γ have the capacity to suppress the capacity to supperess the oncogene expression of human papilloma viruses which are involved in induction and growth of certain malignancies such as cervical cancer. All-trans-, 13-cis-, 9-cis retinoic acid, and acitretin as well as IFNα inhinited the formation of newly formed blood vessels induced by injection of HPV harboring, tumorigenic cell lines into Balb/C mice. The combination of retinoids with IFNα was more efficacious in inhibition angiogenesis than a retinoid or IFNα given alone. Since there is evidence that cell differentiationl, cell proliferation, viral oncogene expression as well as angiogenesis play a role in tumor induction and/or progression of tumor growth, retinoids, cytokines, as well as 1,25(OH)₂D₃ and analogs may be useful in chemoprevention and chemotherapy of neoplasms. based on the superior effect of combinations compared with administration of single compounds of these three classes under experimental conditions there is hope that also clinical application of combination treatment might bring as a step forward in chemopreventio and chemotherapy of cancer. This has already been proven in a very limited number of very limited numberr of clinical trials.     

小肠干细胞的命运调控

小肠上皮具有快速更新的能力,是研究成体干细胞的理想系统.小肠上皮由绒毛和隐窝两部分组成,而位于小肠隐窝底部的小肠干细胞是其持续更新的源泉.近年来,以Lgr5为代表的小肠干细胞标记物的发现、Lgr5+小肠干细胞的分离培养和多种转基因小鼠模型的出现,极大地促进了对小肠干细胞自我更新和分化调控的研究,使得人们可以更加深入地认识小肠干细胞命运决定的分子机制.本文简要综述了近年来人们对Wnt,BMP,Notch和EGF等信号如何在小肠干细胞命运调控中发挥作用的认识.     

In vitro expanded stem cells from the developing retina fail to generate photoreceptors but differentiate into myelinating oligodendrocytes

Cell transplantation to treat retinal degenerative diseases represents an option for the replacement of lost photoreceptor cells. In vitro expandable cells isolated from the developing mammalian retina have been suggested as a potential source for the generation of high numbers of donor photoreceptors. In this study we used standardized culture conditions based on the presence of the mitogens FGF-2 and EGF to generate high numbers of cells in vitro from the developing mouse retina. These presumptive 'retinal stem cells' ('RSCs') can be propagated as monolayer cultures over multiple passages, express markers of undifferentiated neural cells, and generate neuronal and glial cell types upon withdrawal of mitogens in vitro or following transplantation into the adult mouse retina. The proportion of neuronal differentiation can be significantly increased by stepwise removal of mitogens and inhibition of the notch signaling pathway. However, 'RSCs', by contrast to their primary counterparts in vivo, i.e. retinal progenitor cells, loose the expression of retina-specific progenitor markers like Rax and Chx10 after passaging and fail to differentiate into photoreceptors both in vitro or after intraretinal transplantation. Notably, 'RSCs' can be induced to differentiate into myelinating oligodendrocytes, a cell type not generated by primary retinal progenitor cells. Based on these findings we conclude that 'RSCs' expanded in high concentrations of FGF-2 and EGF loose their retinal identity and acquire features of in vitro expandable neural stem-like cells making them an inappropriate cell source for strategies aimed at replacing photoreceptor cells in the degenerated retina.     

Stem cells: a view from the roots

In both plants and animals, regeneration requires the activation of stem cells. This is possibly related to the origin and requirements of multicellularity. Although long diverged from a common ancestry, plant and animal models such as Arabidopsis, Drosophila and mouse share considerable similarities in stem cell regulation. This includes stem cell niche organisation, epigenetic modification of DNA and histones, and the role of small RNA machinery in differentiation and pluripotency states. Dysregulation of any of these can lead to premature ageing, patterning and specification defects, as well as cancers. Moreover, emerging basal animal and plant systems are beginning to provide important clues concerning the diversity and evolutionary history of stem cell regulatory mechanisms in eukaryotes. This review provides a comparative framework, highlighting both the commonalities and differences among groups, which should promote the intelligent design of artificial stem cell systems, and thereby fuel the field of biomaterials science.