Induction of cytokeratin expression in human mesenchymal cells

We studied the phenotypic features of some typical human mesenchymal cells, including decidual stromal cells and adult and fetal fibroblasts under different cell culture conditions by using antibodies to intermediate filament proteins and desmoplakins. In cell culture, the decidual stromal cells rapidly acquired typical fibroblastoid appearance with abundant arrays of vimentin filaments while the cytokeratin-positive epithelial cells, occasionally found in typical epithelioid colonies, lacked vimentin positivity and showed desmoplakin positivity. Within a few days, many of the stromal cells started to present cytokeratin positivity when cultured either in Condimed or in Chang medium. The cytokeratin positivity was first detected in small, scattered cytoplasmic dotted fibrils or in perinuclear dotlike aggregates with fibrillar projections. Later, denser cytokeratin-positive fibrillar arrays could also be seen in stromal cells, which lacked desmoplakin positivity as judged by two monoclonal antibodies. Decidual stromal cells were also cloned and in five out of ten clones some of the cells acquired a similar cytokeratin positivity when transferred into Chang or Condimed medium. Immunoblotting results indicated that cytokeratins 8, 18, and 19 can be found in these cultures. Similar cytokeratin positivity could also be seen in the same culture conditions in cultured fetal fibroblasts from skin, chorionic villi, and lung but not in young or adult skin fibroblast cultures. The present results suggest that decidual stromal cells as well as some embryonal mesenchymal cells can acquire epithelial differentiation in vitro as judged by the emergence of cytokeratin proteins. This ability appears to be lost in the corresponding adult cell. The results furthermore suggest that cytokeratin fibrils can be organized in the cytoplasm without an apparent organization center and that neither the appearance of desmoplakins nor the formation of cell-to-cell contacts are required for cytokeratin filament assembly.     

Distribution of vimentin, cytokeratins, and desmosomal-plaque proteins in human nephroblastoma as revealed by specific antibodies: co-existence of cell groups of different degrees of epithelial differentiation

The distribution of cytokeratins, desmosomal-plaque proteins (desmoplakins), and vimentin in nephroblastoma tissue was studied by immunofluorescence microscopy using specific antibodies. In undifferentiated blastema cells, desmosomes, as revealed by antibodies to desmoplakins, preceded the advent of significant amounts of cytokeratins, indicating that desmosomes are early and sensitive markers of epithelial differentiation. Cytokeratin-positive tumor cells were seen in the following distribution patterns: groups of loosely arranged and scattered cells containing only scant cytokeratin fibrils surrounded by negative stroma cells; focal accumulation of cytokeratin-positive cells with cytokeratin-specific cytoplasmic fibril meshwork staining; rosettes of cytokeratin-positive cells without formation of distinct lumina, showing concentration of cytokeratin staining in the center; tubules with distinct lumina made up of cytokeratin-positive cells, with cytokeratin staining concentrated in the subapical cell portions. In cytokeratin-positive cells, the numbers of desmoplakin-positive dots were generally increased; in well-formed tubules, enrichment of desmoplakin-positive spots, corresponding to the subapical skeletal disks, was most conspicuous. Vimentin was demonstrated in stromal areas, but also in blastema cells showing coexpression of desmosomes and vimentin filaments. Moreover, in certain blastema cells, an overlap of cytokeratin and vimentin immunostaining was observed. Epithelial cells of nephroblastoma tubules did not react with vimentin antibodies. Our results show that the appearance of desmosomal plaques, as demonstrated by antibodies to desmoplakins, may be a very early feature of epithelial differentiation, and they also emphasize the value of antibodies to desmoplakins in tumor cell typing and diagnosis.     

Transient change of organization of vimentin filaments during mitosis as demonstrated by a monoclonal antibody

A monoclonal antibody specific for vimentin is described which, by immunofluorescence and immunoelectron microscopy, decorates fibrillar and/or granular structures in mitotic and early postmitotic cells but does not react with vimentin filaments of interphase stages of various cultured cells (rat vascular smooth muscle-derived cell line RVF-SM; SV40-transformed human fibroblasts; bovine kidney epithelial cells of line MDBK). These observations indicate that the organization of vimentin filaments varies during the cell cycle, undergoing a perimitotic change of filament organization. These changes of vimentin filaments are described in relation to those reported for cytokeratin filaments of various epithelial and carcinoma cells. The possible functional implications of filament protein rearrangements both during the cell cycle and in cell differentiation processes are discussed.     

Control of kidney differentiation by soluble factors secreted by the embryonic liver and the yolk sac

Since transferrin is necessary for the differentiation of the embryonic kidney in organ culture, we have suggested that the component is a growth factor for in vivo development as well. In the present study we demonstrate that transferrin is present in the serum of 11-day-old mouse embryos, at the time when kidney differentiation starts. We have also tested whether various embryonic tissues can replace transferrin as stimulators of the differentiation and proliferation of the metanephric mesenchyme. We used a transfilter model system where nephrogenic mesenchymes are cultured with spinal cord, a known inductor of kidney tubules. The embryonic liver could not replace the spinal cord as an inducer of tubular differentiation. However, when the kidney mesenchymes were cultured together with both the spinal cord and the liver, the mesenchymes proliferated and differentiated also in the absence of exogenous transferrin. In such cocultures the spinal cord had to be in close contact with the mesenchyme while the embryonic liver could be located several cell layers apart. The liver-mediated stimulation of proliferation of the induced mesenchyme could be inhibited by anti-transferrin antibodies. Immunoprecipitation and immunoblotting with these antibodies of the liver-conditioned medium demonstrated that the 11-day mouse liver produces transferrin. Other potential mitogens produced by liver cells, alpha-fetoprotein, or multiplication stimulating activity, did not in any way stimulate the proliferation of induced mesenchymes. These studies suggest that the mitogen in the liver medium is transferrin. This is supported by data which show that another embryonic transferring producer, the visceral yolk sac, can replace the effect of the liver, whereas a tissue not producing transferrin, the salivary mesenchyme, cannot. In conclusion, an essential function of the inducer is to make the mesenchyme responsive to transferrin. The liver and the yolk sac stimulate early kidney differentiation by producing the soluble factor, transferrin, but they are ineffective as inductors of the transferrin responsiveness.     

TGF beta 2, LIF and FGF2 cooperate to induce nephrogenesis

The metanephric kidney develops from interactions between the epithelial ureteric bud and adjacent metanephric mesenchyme, which is induced by the bud to form the epithelia of the nephron. We have found that leukemia inhibitory factor (LIF) and transforming growth factor beta 2 (TGF beta 2) are secreted by inductive rat bud cells and cooperate to enhance and accelerate renal tubule formation in uninduced rat metanephric mesenchymal explants. LIF alone or TGF beta 2 with fibroblast growth factor 2 induced numerous tubules in isolated mesenchymes over an 8 day period, while (in combination) all three caused abundant tubule formation in 72 hours. Furthermore, neutralization of Wnt ligands with antagonist-secreted Frizzled-related protein 1 abrogated these responses and combinatorial cytokine/growth factor stimulation of explants augmented nuclear activation of Tcf1/Lef1, suggesting that LIF and TGF beta 2/FGF2 cooperate to regulate nephrogenesis through a common Wnt-dependent mechanism.     

Differentiation and vascularization of the metanephric kidney grafted on the chorioallantoic membrane

The origin and development of mouse kidney vasculature were examined in chorioallantoic grafts of early kidney rudiments and of experimentally induced explants of separated metanephric mesenchymes. Whole kidney rudiments developed into advanced stages, expressed the segment-specific antigenic markers of tubules and the polyanionic coat of the glomeruli. In contrast to development in vitro, these grafts regularly showed glomeruli with an endothelial component and a basement membrane expressing type IV collagen and laminin. The glomerular endothelial cells in these grafts were shown to carry the nuclear structure of the host. This confirms the outside origin of these cells and the true hybrid nature of the glomeruli. When in vitro induced mesenchymes were grafted on chorioallantoic membranes, abundant vascular invasion was regularly found but properly vascularized glomeruli were exceptional. Uninduced, similarly grafted mesenchymal explants remained avascular as did the undifferentiated portions of partially induced mesenchymal blastemas. It is concluded that the stimulation of the host endothelial cells to invade into the differentiating mesenchyme requires the morphogenetic tissue interaction between the ureter bud and the mesenchyme. The induced metanephric cells presumably start to produce chemoattractants for endothelial cells at an early stage of differentiation. Kidney development thus seems to require an orderly, synchronized development of the three cell lineages: the branching ureter, the induced, tubule-forming mesenchyme, and the invading endothelial cells of outside origin.     

Immunohistochemical characterization of cloned lamb nephropathy.

Kidneys from lambs derived by nuclear transfer are frequently abnormal and are characterized by an enlarged pelvis and narrow medulla, consistent with lower urinary tract obstruction and development of variable hydronephrosis. The precise pathogenesis of this entity is unknown. Immunohistochemical staining for intermediate filaments was used to further characterize the lesions seen in this condition and was compared with age-matched control tissue. Major findings were upregulation of cytokeratin on damaged tubules, desmin and vimentin in undifferentiated mesenchyme, and smooth muscle actin in mesenchyme and on smooth muscle "collars" around dilated tubules. In addition, some cases showed reexpression of vimentin and desmin on proximal tubular epithelial cells. Taken together, these findings provide a valuable database for tracking the expression of intermediate filaments throughout renal development in sheep and have further characterized the nature of the response to injury by the developing kidney, a response that is characterized by proliferation of mesenchyme and both reexpression and upregulation of intermediate filaments within renal cells. In addition, the study has confirmed that the changes in cloned lamb nephropathy are established by day 85 of development.     

Rearrangement of the vimentin cytoskeleton during adipose conversion: formation of an intermediate filament cage around lipid globules

During adipose conversion of murine 3T3-L1 cells, the arrangement of vimentin intermediate filaments (IFs) changes from an extended fibrillar state to a complex cage formation tightly associated with the forming lipid globules. The fully developed cage complex surrounding the lipid globule consists of a monolayer of groups of regularly spaced vimentin IFs that in turn is closely ensheathed by a special endoplasmic reticulum cisterna. The same IF cage is also seen in other adipocytes in culture and in tissues. The specificity of the association of lipid globules with vimentin IFs during adipose conversion is discussed as a special form of compartmentalization supporting adipogenesis and is taken as an example of a possible IF function in relation to a cell differentiation process.     

Modulations of the epithelial phenotype and functional activity of cultured bovine tracheal gland cells: dependence on the culture medium and passage number.

Bovine tracheal gland (BTG) cells in culture show an epithelial-fibroblastoid transition after several passages. To investigate these BTG cell phenotype changes, we studied the effects of both the culture medium and passage number on the expression of epithelial cytoskeletal proteins and glandular serous cell markers. We also analyzed the intracellular cAMP level in the basal state and after adrenergic stimulation. Three culture media were used: 1) serum-free defined medium (SFDM); 2) medium supplemented with 2% Ultroser G; and 3) medium supplemented with 10% fetal calf serum (FCS). Using immunofluorescence microscopy, we showed that, in the first 4 passages whatever the culture conditions, BTG cells expressed immunoreactivities to cytokeratin filaments and desmoplakins I and II, whereas vimentin filaments were not detected. After four passages, BTG cells cultured in 10% FCS or 2% Ultroser G became progressively fibroblastoid and showed immunoreactivities to both vimentin and cytokeratin intermediate filaments. No immunoreactivity to vimentin filaments was observed on BTG cells cultured in a SFDM. Using biochemical analysis, we showed that basal levels of cAMP in cultured BTG cells and lysozyme secretion by these cells vary according to the culture medium and passage number. It was higher in BTG cells cultured in a SFDM compared to that recovered from cells cultured in medium supplemented with Ultroser G or FCS. Whatever the culture medium, BTG cells responded to stimulation by isoproterenol. However, the results of stimulation in a SFDM were higher than in Ultroser G or FCS supplemented medium. We conclude that the BTG epithelial cell organization and the regulation of biosynthesis of secretory proteins by these cells in culture depend on both the culture medium and passage number.(ABSTRACT TRUNCATED AT 250 WORDS)     

Connections of intermediate filaments with the nuclear lamina and the cell periphery

We investigated the relationship between intermediate filaments (IFs) and other detergent- and nuclease-resistant filamentous structures of cultured liver epithelial cells (T51B cell line) using whole mount unembedded preparations which were sequentially extracted with Triton X-100 and nucleases. Immunogold labelling and stereoscopic observation facilitated the examination of each filamentous structure and their three-dimensional relationships to each other. After solubilizing phospholipid, nucleic acid and soluble cellular protein, the resulting cytoskeleton preparation consisted of a network of cytokeratin and vimentin IFs linked by 3 nm filaments. The IFs were anchored to and determined the position of the nuclear lamina filaments (NLF) network and the centrioles. The NLF was composed of the nuclear lamina filaments measuring 3-6 nm in diameter which radiated from and anchored to the skeleton of the nuclear pores. The IFs located in the nuclear region appeared to be interwoven with the NLF. At the cell surface, the IFs seemed to be attached to the putative actin filament network. They formed a focally interrupted plexus-like structure at the cell periphery. Fragments of vimentin filaments were found among the filamentous network located at the cell surface, and some filaments terminated blindly there.     

In vitro segregation of the metanephric nephron

In vitro segregation of the metanephric nephron was examined using three probes for the main segments: fluorochrome-conjugated wheat germ agglutinin (WGA) binding to the glomerular epithelial surface, an antiserum against the brush-border antigens (BB) of the proximal tubules, and an antiserum against the Tamm-Horsfall glycoprotein (TH) of the distal tubules. In vivo, these markers appeared sequentially on Days 13 to 15. The same sequence was obtained in experimental recombinants of the metanephric mesenchyme and its inductor. When the inductor was removed after a 24 hr initial transfilter contact with the mesenchyme, segregation was similarly observed after subculture of the isolated mesenchyme. Hence, the sequential, multiphase differentiation of the nephron is initiated during a short induction period.     

Midkine Promotes Selective Expansion of the Nephrogenic Mesenchyme during Kidney Organogenesis

During kidney development, the growth and development of the stromal and nephrogenic mesenchyme cell populations and the ureteric bud epithelium is tightly coupled through intricate reciprocal signaling mechanisms between these three tissue compartments. Midkine, a target gene activated by retinoid signaling in the metanephros, encodes a secreted polypeptide with mitogenic and anti-apoptotic activities in a wide variety of cell types. Using immmunohistochemical methods we demonstrated that Midkine is found in the uninduced mesenchyme at the earliest stages of metanephric kidney development and only subsequently concentrated in the ureteric bud epithelium and basement membrane. The biological effects of purified recombinant Midkine were analyzed in metanephric organ culture experiments carried out in serum-free defined media. These studies revealed that Midkine selectively promoted the overgrowth of the Pax-2 and N-CAM positive nephrogenic mesenchymal cells, failed to stimulate expansion of the stromal compartment and suppressed branching morphogenesis of the ureteric bud. Midkine suppressed apoptosis and stimulated cellular proliferation of the nephrogenic mesenchymal cells, and was capable of maintaining the viability of isolated mesenchymes cultured in the absence of the ureteric bud. These results suggest that Midkine may regulate the balance of epithelial and stromal progenitor cell populations of the metanephric mesenchyme during renal organogenesis.     

Cytoskeletal markers and specific protein production in cells cultured from human first and third trimester placentae

Summary In primary, short-term cultures derived from first and third trimester placentae, 60 to 90 and 70 to 95%, respectively, of the total cell population positively stain for cytokeratin intermediate filaments, typical of epithelial, i.e. trophoblastic cells. The rest of the cells express only vimentin intermediate filaments and thus are of mesenchymal origin. Only the cytokeratin-positive cells express human chorionic gonadotropin (hCG), whereas both the epithelial and the mesenchymal cells stain positively for pregnancy-specific beta-1-glycoprtein (SP₁). Cytokeratin-negative and vimentin-positive cell overgrowth is observed in cultures derived from first and early third trimester placentae. The cells constituting the monolayer thus formed are of fetal origin as evidenced by the expression of Y-body in over 80% of them. The cultured cells synthesize and secrete hCG and SP₁. The activity of these trophoblast-specific functions is inversely proportional to the gestational age of the placenta. Production of specific proteins and expression of intermediate filaments are proposed as criteria for defining the nature and origin of placental cells in primary, short-term cultures.     

Both Humoral Mesenchymal Factors and the Close Association between the Hepatic Endoderm and Mesenchyme can be Involved in Liver Formation of Mouse Embryos

Previous studies with tissue recombination experiments demonstrated that the splanchnic mesenchymes, including hepatic, pulmonary and stomach mesenchymes can support hepatocyte differentiation from the hepatic endoderm in 9.5-day mouse embryos. This phenomenon corresponds to the second hepatic induction. The present study was undertaken to determine whether direct cell-cell contacts between the hepatic endoderm and mesenchyme are required for hepatocyte differentiation, using transfilter experiments in which membrane filters with various pore sizes were inserted between the endoderm and the hepatocyte-inducing mesenchyme (the chick lung mesenchyme). Hepatocyte differentiation occurred even when the direct cell-cell contacts between the hepatic endoderm and the mesenchyme were absent, suggesting that humoral factors may work in this interaction. However, growth of hepatocytes was most prominent in the transfilter experiments with filters having pore sizes of 0.2 and 0.8 mum, which permitted mesenchymal cells or their cell processes to penetrate to the side of the endoderm. These results suggest that two types of tissue interactions, including humoral mesenchymal factors and very local tissue interactions such as direct cell-cell contacts, may be involved in the second step of hepatic induction.     

Wnt9b plays a central role in the regulation of mesenchymal to epithelial transitions underlying organogenesis of the mammalian urogenital system

The vertebrate urogenital system forms due to inductive interactions between the Wolffian duct, its derivative the ureteric bud, and their adjacent mesenchymes. These establish epithelial primordia within the mesonephric (embryonic) and metanephric (adult) kidneys and the Müllerian duct, the anlage of much of the female reproductive tract. We show that Wnt9b is expressed in the inductive epithelia and is essential for the development of mesonephric and metanephric tubules and caudal extension of the Müllerian duct. Wnt9b is required for the earliest inductive response in metanephric mesenchyme. Further, Wnt9b-expressing cells can functionally substitute for the ureteric bud in these interactions. Wnt9b acts upstream of another Wnt, Wnt4, in this process, and our data implicate canonical Wnt signaling as one of the major pathways in the organization of the mammalian urogenital system. Together these findings suggest that Wnt9b is a common organizing signal regulating diverse components of the mammalian urogenital system.     

On the relation between distinct components of the cytoskeleton: an epitope shared by intermediate filaments, microfilaments and cytoplasmic foci.

Monoclonal antibodies were generated against detergent-insoluble cytoskeletal proteins isolated from low-density membrane fractions of rat liver. By immunofluorescence, one of the antibodies stains three distinct structures in cultured rat fibroblast and hepatocyte lines as well as the PtK2 rat-kangaroo kidney epithelial line. These structures are: i) many tangled filaments similar to intermediate filaments (IFs), ii) fewer and variable numbers of straight filaments, and iii) punctate cytoplasmic foci, often most intense around the nucleus. All three of these structures are resistant to extraction by non-ionic detergent. Close examination reveals that the tangled and straight filaments are not stained uniformly, but as a series of bright patches. In cells treated with nocodazole, the antibody reacts strongly with a perinuclear filamentous cage. Very few tangled filaments are detected in these cells, however, the straight filaments and punctate cytoplasmic staining are resistant to nocodazole treatment. Double-label immunofluorescence shows that, even though tangled filament distribution and nocodazole sensitivity are similar to the behavior of vimentin IFs, there is only partial coincidence of staining with either vimentin or cytokeratin IFs. The straight filaments coincide with some actin stress fibers, but the punctate cytoplasmic staining is not related to IFs, actin, or tubulin. Thus, this monoclonal antibody stains a novel group of three seemingly unrelated cytoskeletal structures, including a previously undescribed insoluble nonfilamentous pool. Taken as a whole, two hypotheses are consistent with these data. i) The antigen recognized may be a protein which has a large insoluble cytoplasmic pool and binds both IFs and actin, but only binds to a subset of each class of filaments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression and distribution of vimentin and keratin filaments in heterokaryons of human fibroblasts and amnion epithelial cells

The expression of intermediate filaments of the keratin- and the vimentin-type was studied in heterokaryons of human fibroblasts and amnion epithelial cells by immunofluorescence microscopy. Fibroblasts and their homokaryons showed a fibrillar, vimentin-specific fluorescence throughout the cytoplasm but were negative when stained for keratin. Amnion epithelial cells and their homokaryons, on the other hand, showed a keratin-specific fibrillar staining, and only some of them contained also detectable vimentin. When suspended epithelial cells were fused with adherent fibroblasts, keratin fibrils spread within 3 h into the fibroblasts, intermixing with the vimentin fibrils. 1-3 d after fusion, both vimentin and keratin filaments were expressed as typical fibrillar cytoplasmic arrays, and the distribution of keratin in heterokaryons resembled closely that of vimentin. A typical cell-to-cell arrangement of keratin fibrils, seen in cultures of amnion epithelial cells, could also be found between heterokaryons. Treatment of the cultures with vinblastine sulphate induced coiling of the vimentin filaments in both homo- and heterokaryons, whereas the keratin organization was only slightly affected. Our results show that both vimentin and keratin filaments are incorporated into the cytoskeleton of heterokaryons formed between fibroblasts and epithelial cells, and that they behave in the same way as in their parental cells. Both epithelial and fibroblastic characteristics thus appear to the coexpressed in such heterokaryons.     

Midkine Promotes Selective Expansion of the Nephrogenic Mesenchyme During Kidney Organogenesis

During kidney development, the growth and development of the stromal and nephrogenic mesenchyme cell populations and the ureteric bud epithelium is tightly coupled through intricate reciprocal signaling mechanisms between these three tissue compartments. Midkine, a target gene activated by retinoid signaling in the metanephros, encodes a secreted polypeptide with mitogenic and anti-apoptotic activities in a wide variety of cell types. Using immmunohistochemical methods we demonstrated that Midkine is found in the uninduced mesenchyme at the earliest stages of metanephric kidney development and only subsequently concentrated in the ureteric bud epithelium and basement membrane. The biological effects of purified recombinant Midkine were analyzed in metanephric organ culture experiments carried out in serum-free defined media. These studies revealed that Midkine selectively promoted the overgrowth of the Pax-2 and N-CAM positive nephrogenic mesenchymal cells, failed to stimulate expansion of the stromal compartment and suppressed branching morphogenesis of the ureteric bud. Midkine suppressed apoptosis and stimulated cellular proliferation of the nephrogenic mesenchymal cells, and was capable of maintaining the viability of isolated mesenchymes cultured in the absence of the ureteric bud. These results suggest that Midkine may regulate the balance of epithelial and stromal progenitor cell populations of the metanephric mesenchyme during renal organogenesis.Key Words: growth factor, proliferation, apoptosis, ureteric bud, branching morphogenesis, epithelial progenitor, development, signaling