Two different laminin domains mediate the differentiation of human endothelial cells into capillary-like structures in vitro

Endothelial cells, both microvascular as well as large vessel, undergo differentiation slowly in culture under most conditions. When endothelial cells are cultured on Matrigel, a solid gel of basement membrane proteins, they rapidly align and form hollow tube-like structures. We show here that tube formation is a multi-step process induced by laminin. An RGD-containing sequence in the A chain of laminin through an integrin receptor on the endothelial cell induces their attachment to the protein while a YIGSR site in the B1 chain induces cell-cell interactions and the resulting tube formation. We also show that the laminin-derived synthetic peptide YIGSR contains sufficient information to induce single endothelial cells to form ring-like structures surrounding a hollow lumen, the basic putative unit in the formation of capillaries.     

Role of laminin terminal globular domains in basement membrane assembly

Laminins contribute to basement membrane assembly through interactions of their N- and C-terminal globular domains. To further analyze this process, recombinant laminin-111 heterotrimers with deletions and point mutations were generated by recombinant expression and evaluated for their ability to self-assemble, interact with nidogen-1 and type IV collagen, and form extracellular matrices on cultured Schwann cells by immunofluorescence and electron microscopy. Wild-type laminin and laminin without LG domains polymerized in contrast to laminins with deleted alpha1-, beta1-, or gamma1-LN domains or with duplicated beta1- or alpha1-LN domains. Laminins with a full complement of LN and LG domains accumulated on cell surfaces substantially above those lacking either LN or LG domains and formed a lamina densa. Accumulation of type IV collagen onto the cell surface was found to require laminin with separate contributions arising from the presence of laminin LN domains, nidogen-1, and the nidogen-binding site in laminin. Collectively, the data support the hypothesis that basement membrane assembly depends on laminin self-assembly through formation of alpha-, beta-, and gamma-LN domain complexes and LG-mediated cell surface anchorage. Furthermore, type IV collagen recruitment into the laminin extracellular matrices appears to be mediated through a nidogen bridge with a lesser contribution arising from a direct interaction with laminin.     

Mitogen-induced disorganization of capillary-like structures formed by human large vessel endothelial cells in vitro

Endothelial cells (EC) from human aorta, umbilical vein and pulmonary artery were grown in Medium 199 supplemented with 20% human serum (HS), endothelial cell growth factor (ECGF) from bovine and human brain (200 micrograms/ml) and heparin (100 micrograms/ml) in gelatin-coated flasks. Under these conditions cells rapidly proliferated and survived 15-25 passages (40-60 cumulative population doublings). When cells were cultured on plastic substrate and without growth factors a capillary-like network appeared after 3-4 weeks of growth. According to TEM, this network consisted of tubes with the lumen encircled by one or several cells. The reduction of serum concentration in the medium or the replacement of plasma-derived serum (PDS) for HS reduced the time of network formation to 3-5 days. S-180 conditioned medium mitogenic for EC induced a rapid spreading of the cells and a partial reversion to a two-dimensional monolayer structure. Trypsin inhibitor did not abolish the effect of tumour conditioned medium. Other EC mitogens, e.g. ECGF and fibroblast growth factor (FGF), also disorganized the capillary-like network. In a day or two the network was completely restored. In contrast, culturing EC on gelatin-coated substrate is a sufficient condition for monolayer formation from tubes and long-term maintenance. We suggest that mitogens can influence the EC morphology but that it is the nature of the substrate that determines the stage of large vessel EC differentiation.     

Astrocytes induce neural microvascular endothelial cells to form capillary-like structures in vitro

Astrocytes maintain a unique association with the central nervous system microvasculature and are thought to play a role in neural microvessel formation and differentiation. We investigated the influence of astroglial cells on neural microvascular endothelial differentiation in vitro. Using an astroglial-endothelial coculture system, rat brain astrocytes and C6 cells of astroglial lineage are shown to induce bovine retinal microvascular endothelial (BRE) cells to form capillary-like structures. Light microscopic evidence for endothelial reorganization began within 48 hours and was complete 72-96 hours following the addition of BRE cells to 1-day-old astroglial cultures. The extent of BRE reorganization was quantitated by computer-assisted analysis and shown to be dependent upon the density of both the BRE and C6 cells within the cocultures. Coculture conditions in which BRE cells were separated from C6 cells by porous membranes failed to generate this endothelial cell change. Likewise, C6-conditioned media and C6-endothelial coculture conditioned media did not induce BRE cell reorganization. Extracellular laminin within the C6-endothelial cocultures, identified by indirect immunofluorescence, was concentrated at the endothelial-astroglial interface of capillary-like structures consistent with incipient basement membrane formation. Astroglial cells accumulated adjacent to capillary-like structures suggesting the presence of bidirectional influences between the reorganized endothelial cells and astroglia. This is the first demonstration of astroglial induction of angiogenesis in vitro and these findings support a functional role for perivascular astrocytes in the vascularization of neural tissue such as retina and brain.     

Type IV collagen synthesis by cultured human microvascular endothelial cells and its deposition into the subendothelial basement membrane

Cultured microvascular endothelial cells isolated from human dermis were examined for the synthesis of basement membrane specific (type IV) collagen and its deposition in subendothelial matrix. Biosynthetically radiolabeled proteins secreted into the culture medium were analyzed by sodium dodecyl sulfate gel electrophoresis after reduction, revealing a single collagenous component with an approximate Mr of 180 000 that could be resolved into two closely migrating polypeptide chains. Prior to reduction, the 180 000 bands migrated as a high molecular weight complex, indicating the presence of intermolecular disulfide bonding. The 180 000 material was identified as type IV procollagen on the basis of its selective degradation by purified bacterial collagenase, moderate sensitivity to pepsin digestion, immunoprecipitation with antibodies to human type IV collagen, and comigration with type IV procollagen purified from human and murine sources. In the basement membrane like matrix elaborated by the microvascular endothelial cells at their basal surface, type IV procollagen was the predominant constituent. This matrix-associated type IV procollagen was present as a highly cross-linked and insoluble complex that was solubilized only after denaturation and reduction of disulfide bonds. In addition, there was evidence of nonreducible dimers and higher molecular weight aggregates of type IV procollagen. These findings support the suggestion that the presence of intermolecular disulfide bonds and other covalent interactions stabilizes the incorporation of the type IV procollagen into the basement membrane matrix. Cultured microvascular endothelial cells therefore appear to deposit a basal lamina-like structure that is biochemically similar to that formed in vivo, providing a unique model system that should be useful for understanding microvascular basement membrane metabolism, especially as it relates to wound healing, tissue remodeling, and disease processes.     

Identification of a subpopulation of human renal microvascular endothelial cells with capacity to form capillary-like cord and tube structures

Summary Endothelial specialization is a prominent feature within distinct capillary beds of organs such as mammalian kidney, yet immunological markers for functionally distinct subpopulations of cultured endothelial cells from tissue sources such as kidney have not been available. We developed a simple and reproducible isolation and culture procedure to recover human renal microvascular endothelial cells (HRMEC) from the cortex of unused donor kidneys. This procedure yields highly purified preparations of cells that display endothelial markers that include Factor VIII antigen, acetyl-LDL receptors, and determinants that bind Ulex europaeus lectin. HRMEC assemble into capillary-like cord and tube structures when plated on the surface of basement membrane-like matrix (BMM) in media containing phorbol myristate acetate. To further define subpopulations of HRMEC, we generated a panel of monoclonal antibodies and screened for those recognizing cell surface determinants. One monoclonal antibody recovered from this screen recognized a cell surface protein expressed on a subpopulation of HRMEC that we have designated PEC-1 (pioneer endothelial cell antigen-1). Cells expressing PEC-1 extended long, interconnecting filopodial processes in response to phorbol myristate acetate and assembled into capillary-like structures when plated on BMM. Anti-PEC-1 immunoprecipitated proteins of 25 and 27 kDa. Magnetic bead separation of PEC-1 (+) cells selected cells that assemble into capillary-like cord and tube structures. The remaining PEC-1 (−) HRMEC population formed matrix adherent patches. In the kidney, the PEC-1 determinant is expressed on a small subpopulation of microvascular glomerular cells and is prominently expressed on the apical membrane of proximal tubule cells. The PEC-1 determinant discriminates among subpopulations of HRMEC, identifying a subpopulation that contributes to assembly of capillary-like structures.     

Efficient differentiation of embryonic stem cells into hepatic cells in vitro using a feeder-free basement membrane substratum

The endoderm-inducing effect of the mesoderm-derived supportive cell line M15 on embryonic stem (ES) cells is partly mediated through the extracellular matrix, of which laminin α5 is a crucial component. Mouse ES or induced pluripotent stem cells cultured on a synthesized basement membrane (sBM) substratum, using an HEK293 cell line (rLN10-293 cell) stably expressing laminin-511, could differentiate into definitive endoderm and subsequently into pancreatic lineages. In this study, we investigated the differentiation on sBM of mouse and human ES cells into hepatic lineages. The results indicated that the BM components played an important role in supporting the regional-specific differentiation of ES cells into hepatic endoderm. We show here that knockdown of integrin β1 (Itgb1) in ES cells reduced their differentiation into hepatic lineages and that this is mediated through Akt signaling activation. Moreover, under optimal conditions, human ES cells differentiated to express mature hepatocyte markers and secreted high levels of albumin. This novel procedure for inducing hepatic differentiation will be useful for elucidating the molecular mechanisms controlling lineage-specific fates during gut regionalization. It could also represent an attractive approach to providing a surrogate cell source, not only for regenerative medicine, but also for pharmaceutical and toxicologic studies.     

In vitro growth and differentiation of human kidney tubular cells on a basement membrane substrate

Summary Kidney cortical tubular cells, mainly proximal tubular cells, isolated from human kidney and grown either on a basement membrane substrate in chemically defined medium or on plastic in serum-supplemented medium, had substantial proliferative potential and could be propagated for more than 10 generations or 8 passages before senescence. Basement membrane produced on a plastic substrate by the HR-9 endodermal cell line could replace serum supplementation in promoting tubular cell growth. Tubular cells grown on an HR-9 basement membrane substrate exhibited stable epithelial morphology over an extended period of time; in the presence of 5% serum they differentiated into organized structures such as hemicysts and cell cords. Cells grown on plastic failed to differentiate and gradually degenerated. Tubular cells on HR-9 basement membrane were characterized by densely packed microvilli, abundant rough endoplasmic reticulum and free polysomes, basal cell membrane interdigitations, a well-developed endocytotic apparatus, and conspicuous junctional complexes—all features of the proximal tubular cell. Compared with cells on plastic substrate, there were higher levels of the brush border enzymes γ-glutamyl transpeptidase,l-leucine aminopeptidase, and alkaline phosphatase in cells maintained on an HR-9 basement membrane substrate, further supporting the conclusion that a basement membrane substrate promoted differentiation of tubular cells. These data and morphological observations indicate that a basement membrane substrate can promote growth and both functional and morphologic differentiation of human kidney tubular cells. This work was supported by the Veterans Administration.     

Enhanced synthesis of basement membrane proteins during the differentiation of rat mammary tumour epithelial cells into myoepithelial-like cells in vitro

Rama 25, an epithelial cell line obtained from a dimethylbenzanthracene-induced rat mammary tumour differentiates spontaneously in culture forming elongated myoepithelial-like cells. The elongated cells form multilayered ridge structures from which cultures of elongated cells, relatively uncontaminated by epithelial cells, can be obtained. By using immunofluorescence techniques, both the elongated cells and the cells in ridges, but not undifferentiated Rama 25 cells, have been demonstrated to synthesize three basement membrane proteins, laminin, type IV collagen, and fibronectin. The identity of these basement membrane proteins has been confirmed by immunoprecipitation. These proteins appear to be located in a fibrillar extracellular matrix. We suggest that the ability to synthesize basement membrane proteins by mammary epithelial cells in vitro on plastic is a characteristic of myoepithelial-like cells.     

Inhibition of laminin alpha 1-chain expression leads to alteration of basement membrane assembly and cell differentiation

The expression of the constituent alpha 1 chain of laminin-1, a major component of basement membranes, is markedly regulated during development and differentiation. We have designed an antisense RNA strategy to analyze the direct involvement of the alpha 1 chain in laminin assembly, basement membrane formation, and cell differentiation. We report that the absence of alpha 1-chain expression, resulting from the stable transfection of the human colonic cancer Caco2 cells with an eukaryotic expression vector comprising a cDNA fragment of the alpha 1 chain inserted in an antisense orientation, led to (a) an incorrect secretion of the two other constituent chains of laminin-1, the beta 1/gamma 1 chains, (b) the lack of basement membrane assembly when Caco2-deficient cells were cultured on top of fibroblasts, assessed by the absence of collagen IV and nidogen deposition, and (c) changes in the structural polarity of cells accompanied by the inhibition of an apical digestive enzyme, sucrase-isomaltase. The results demonstrate that the alpha 1 chain is required for secretion of laminin-1 and for the assembly of basement membrane network. Furthermore, expression of the laminin alpha 1-chain gene may be a regulatory element in determining cell differentiation.     

Synergism of biochemical and mechanical stimuli in the differentiation of human placenta-derived multipotent cells into endothelial cells

There have been intensive studies on the differentiation of endothelial progenitor cells (EPCs) into endothelial cells. We investigated the endothelial differentiation of placenta-derived multipotent cells (PDMCs), a population of CD34(-)/CD133(-)/Flk-1(-) cells. PDMCs were cultured in basal media or media containing endothelial growth factors (EGM), including vascular endothelial growth factor (VEGF), for 3 days and then subjected to shear stress of 6 or 12dyn/cm(2) for 24h. Culture of PDMCs in EGM under static conditions resulted in significant increases in VEGF receptor-1 (Flt-1) and receptor-2 (Flk-1) expression. Application of shear stress at 12dyn/cm(2) to these cells led to significant increases in their expression of von Willebrand Factor and platelet-endothelial cell adhesion molecule-1 at both the gene and protein levels. Shear stress at 6dyn/cm(2) had lesser effects. Uptakes of acetylated low-density lipoproteins as well as formation of tube-like structures on Matrigel were significantly increased after subjecting to shear stress of 12dyn/cm(2) for 24h. Our findings suggest that the combined use of endothelial growth factors and high shear stress is synergistic for the endothelial differentiation of PDMCs.     

Role of gangliosides in the differentiation of human mesenchymal-derived stem cells into osteoblasts and neuronal cells

Gangliosides are complex glycosphingolipids that are the major component of cytoplasmic cell membranes, and play a role in the control of biological processes. Human mesenchymal stem cells (hMSCs) have received considerable attention as alternative sources of adult stem cells because of their potential to differentiate into multiple cell lineages. In this study, we focus on various functional roles of gangliosides in the differentiation of hMSCs into osteoblasts or neuronal cells. A relationship between gangliosides and epidermal growth factor receptor (EGFR) activation during osteoblastic differentiation of hMSCs was observed, and the gangliosides may play a major role in the regulation of the differentiation. The roles of gangliosides in osteoblast differentiation are dependent on the origin of hMSCs. The reduction of ganglioside biosynthesis inhibited the neuronal differentiation of hMSCs during an early stage of the differentiation process, and the ganglioside expression can be used as a marker for the identification of neuronal differentiation from hMSCs. [BMB Reports 2013; 46(11): 527-532]     

Ultrastructural orientation of laminin 5 in the epidermal basement membrane: an updated model for basement membrane organization.

Laminin 5 is a trimeric glycoprotein involved in cell adhesion in the epidermal basement membrane. To determine the precise orientation of laminin 5 in adult human skin, we used plural epitope-specific monoclonal antibodies, a polyclonal antiserum, and postembedding immunogold electron microscopy (IEM). Immunogold labeling distances from the basal keratinocyte plasma membrane (PM) were measured for each gold particle (>200 particles) and the mean distance (nm) calculated. Antibodies included BM165 (recognizing the alpha 3-chain first globular domain) that was measured at 35.40 +/- 2.20 nm from the keratinocyte PM, K140 (recognizing a region adjacent to the beta 3-chain globular domain IV) that measured 45.20 +/- 3.60 nm from the PM, and an anti-laminin 5 polyclonal antiserum that was 43.43 +/- 6.28 nm from the PM. The laminin 5 gamma 2-chain short arm hinge domain was previously localized to the lower lamina densa (LD) at approximately 56.30 +/- 1.65 nm from the keratinocyte PM. Taken together with previous gamma 2-chain data and the distribution of the polyclonal antisera, we estimate that the long axis of laminin 5 is oriented at an angle of approximately 27 degrees from the horizontal lamina lucida (LL)/LD border and propose that the gamma 2-chain lies farthest from the PM. This novel orientation, with the majority of the laminin 5 molecule lying obliquely along the LL/LD border and not perpendicularly, as was first thought, sheds new light on the organization of the basement membrane and likely molecular interactions.     

Demonstration of laminin,a basement membrane glycoprotein,in routinely processed formalin-fixed human tissues

Summary Laminin was demonstrated by immunoperoxidase and immunofluorescence staining in sections of normal human tissues fixed in formalin and routinely processed in paraffin. Exposure of the sections to a solution of pepsin (Burns et al. (1980) Histochemistry 6773–78) revealed the antigenicity of this basement membrane glycoprotein. Sections from paraffin blocks stored for years at room temperature could be stained with this procedure. Normal human tissues, developing fetal tissues and tumors could be stained with this method. The staining patterns were similar to those seen in unfixed frozen sections. It thus appears that basement membrane components can be detected by immunohistological means from routinely processed histological samples, once the sections are pretreated with proteases. Staining for laminin could be used in embryonic studies and in histopathology to study the relation of cells to basement membranes and for the visualization of normal and abnormal vascularization.To whom offprint requests should be sent     

Demonstration of laminin, a basement membrane glycoprotein, in routinely processed formalin-fixed human tissues

Laminin was demonstrated by immunoperoxidase and immunofluorescence staining in sections of normal human tissues fixed in formalin and routinely processed in paraffin. Exposure of the sections to a solution of pepsin (Burns et al. (1980) Histochemistry 67:73-78) revealed the antigenicity of this basement membrane glycoprotein. Sections from paraffin blocks stored for years at room temperature could be stained with this procedure. Normal human tissues, developing fetal tissues and tumors could be stained with this method. The staining patterns were similar to those seen in unfixed frozen sections. It thus appears that basement membrane components can be detected by immunohistological means from routinely processed histological samples, once the sections are pretreated with proteases. Staining for laminin could be used in embryonic studies and in histopathology to study the relation of cells to basement membranes and for the visualization of normal and abnormal vascularization.     

Staurosporine-induced morphological differentiation of human neuroblastoma cells

SH-SY-5Y human neuroblastoma cells rapidly elaborated an extensive network of neuritic processes following treatment with staurosporine, an inhibitor of protein kinase C. These neurites were retracted within 24hr following removal of inhibitor. Another inhibitor of protein kinase C, H7 [1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride], also induced rapid, reversible neurite outgrowth. However, neurites induced by these two inhibitors were morphologically distinct: staurosporine-treated cells elaborated a branching neuritic network adjacent to cell bodies, with some longer, unbranching neurites extending out of this network, while H7-treated cells elaborated only long, unbranching neurites. HA-1004 [N-(2-guanidinoethyl)-5-isoquinolinesulfonamide], which inhibits of cAMP- and cGMP-dependent protein kinases but not protein kinase C, did not induce neuritogenesis. Staurosporine-induced neurite outgrowth did not require protein synthesis but did require microtubule assembly, suggesting that cells contained the necessary components for neuritogenesis, and that alterations in protein phosphorylation alone was sufficient to initiate neurite outgrowth by rearrangement of existing structures or cytoskeletal precursors. These results implicate phosphorylation in the regulation of neuronal differentiation and neuritogenesis.     

Fibronectin and laminin promote differentiation of human mesenchymal stem cells into insulin producing cells through activating Akt and ERK

Background  

Islet transplantation provides a promising cure for Type 1 diabetes; however it is limited by a shortage of pancreas donors. Bone marrow-derived multipotent mesenchymal stem cells (MSCs) offer renewable cells for generating insulin-producing cells (IPCs).     

In vitro and in vivo differentiation of human umbilical cord derived stem cells into endothelial cells

The successful use of tissue-engineered transplants is hampered by the need for vascularization. Recent advances have made possible the using of stem cells as cell sources for therapeutic angiogenesis, including the vascularization of engineered tissue grafts. The goal of this study was to examine the endothelial potential of human umbilical cord-derived stem (UCDS) cells. UCDS cells were initially characterized and differentiated in an endothelial differentiation medium containing VEGF and bFGF. Differentiation into endothelial cells was determined by acetylated low-density lipoprotein incorporation and expression of endothelial-specific proteins, such as PECAM and CD34. In vivo, the transplanted UCDS cells were sprouting from local injection and differentiated into endothelial cells in a hindlimb ischemia mouse model. These findings indicate the presence of a cell population within the human umbilical cord that exhibits characteristics of endothelial progenitor cells. Therefore, human umbilical cord might represent a source of stem cells useful for therapeutic angiogenesis and re-endothelialization of engineered tissue grafts.