Gerd Klein

Expression of laminin α1, α5 and β2 chains during embryogenesis of the kidney and vasculature

Abstract Laminins, found predominantly in basement membranes, are large glycoproteins consisting of different subsets of α, β and γ chain subunits. To resolve conflicting data in the literature concerning coexpression of α1 and β2 chains, expression of α1 chain was studied with two different antisera against the E3 fragment of laminin α1 chain. Expression of the α1 chain was seen in several types of epithelial basement membranes throughout development, but its expression in rat glomerular basement membranes and some other types of epithelial basement membranes occurred only during early stages of development. By contrast, β2 chains were detected by immunofluorescence only during advanced stages of glomerulogenesis and vascular development. By Northern and Western blots, β2 chains were detected somewhat earlier, but in situ hybridization revealed that β chain was also confined to vasculature during the earlier stages. It thus seems that, in the tissues studied here, the expression of α1 and β2 chains was mutually exclusive. To explore whether the newly described α5 chain is expressed in locations lacking α1 chain, expression of α5 chain was studied by Northern blots and in situ hybridization. The α5 chain was not uniformly expressed in all embryonic epithelial cell types but was present mainly in epithelial sheets which produce very little α1 chain. There also appeared to be a developmental trend, with α1 chain appearing early and α5 later, in maturing epithelial sheets. The α5 chain could be a major α chain of the adult glomerular basement membrane.

Perlecan in human bone marrow: A growth-factor-presenting, but anti-adhesive, extracellular matrix component for hematopoietic cells

Human perlecan is a heparan sulfate proteoglycan with a large core protein of 467 kDa to which three glycosaminoglycan side chains are attached. It belongs to the heparan sulfate proteoglycan family which has been implicated in strong interactions between developing hematopoietic cells and their microenvironment in the bone marrow. Here we report that perlecan is highly expressed in the human bone marrow, as well as in long-term bone marrow cultures which are thought to mimic hematopoiesis in vitro. Expression of perlecan in this tissue was shown by Northern blotting of the 14-kb mRNA of the core protein and by immunofluorescence stainings. Functionally, perlecan shows a strong anti-adhesive effect on unfractionated bone marrow cells and on various hematopoietic cell lines, repelling the cells from the perlecan-coated area. In contrast, perlecan is adhesive for fibroblasts and endothelial cells. It is suggested that the anti-adhesive site is located within the core protein of perlecan since heparitinase-treated perlecan still shows the repellent effect. Although anti-adhesive for hematopoietic cells, perlecan is able to bind growth factors like granulocyte/macrophage-colony stimulating factor and present them to hematopoietic progenitor cells in a semi-solid colony assay. The functional role of a growth-factor-binding extracellular matrix component in the bone marrow microenvironment with anti-adhesive properties is uncertain but may be related to compartmentalization.

Characterization of a conduit system containing laminin-5 in the human thymus: a potential transport system for small molecules

T cells develop in the thymus in a highly specialized cellular and extracellular microenvironment. The basement membrane molecule, laminin-5 (LN-5), is predominantly found in the medulla of the human thymic lobules. Using high-resolution light microscopy, we show here that LN-5 is localized in a bi-membranous conduit-like structure, together with other typical basement membrane components including collagen type IV, nidogen and perlecan. Other interstitial matrix components, such as fibrillin-1 or -2, tenascin-C or fibrillar collagen types, were also associated with these structures. Three-dimensional (3D) confocal microscopy suggested a tubular structure, whereas immunoelectron and transmission electron microscopy showed that the core of these tubes contained fibrillar collagens enwrapped by the LN-5-containing membrane. These medullary conduits are surrounded by thymic epithelial cells, which in vitro were found to bind LN-5, but also fibrillin and tenascin-C. Dendritic cells were also detected in close vicinity to the conduits. Both of these stromal cell types express major histocompatibility complex (MHC) class II molecules capable of antigen presentation. The conduits are connected to blood vessels but, with an average diameter of 2 μm, they are too small to transport cells. However, evidence is provided that smaller molecules such as a 10 kDa dextran, but not large molecules (>500 kDa), can be transported in the conduits. These results clearly demonstrate that a conduit system, which is also known from secondary lymphatic organs such as lymph nodes and spleen, is present in the medulla of the human thymus, and that it might serve to transport small blood-borne molecules or chemokines to defined locations within the medulla.

The significance of integrin ligand nanopatterning on lipid raft clustering in hematopoietic stem cells.

Hematopoietic stem cells (HSCs) are the vital, life-long source of all blood cell types. They are found in stem cell niches, specific anatomic locations that offer all the factors and signals necessary for the maintenance of the stem cell potential of HSCs. Much attention has been paid to the biochemical composition of the niches, but only little is known about the influence of physical parameters, such as ligand nanopatterns, on HSCs. To investigate the impact of nanometer-scale spacing between cell ligands on HSC adhesion, integrin distribution and signal transduction, we employed geometrically defined, nanostructured, bio-functionalized surfaces. HSCs proved to be sensitive to the lateral distance between the presented ligands with regard to adhesion and lipid raft clustering, the latter being a prerequisite for the formation of signaling complexes. Furthermore, an extensive redistribution of stem cell markers, integrins and phosphorylated proteins in HSCs was observed. In conclusion, integrin-mediated adhesion and signaling of HSCs proved to depend on the nanostructured presentation of ligands in their environment. In this work, we show that the nanostructure of the matrix is an important parameter influencing HSC behavior that should be integrated into biomaterial-based approaches aiming at HSC multiplication or differentiation.

The integrin α9β1 on hematopoietic stem and progenitor cells: involvement in cell adhesion, proliferation and differentiation

Integrins have been shown to play a major role in the interaction of hematopoietic stem cells with their supportive microenvironment. In this article, the authors show that integrin α9β1, in addition to the previously implicated integrin α4β1, contributes to the interaction of human CD34+ cells with primary osteoblasts, and that selective inhibition of its function by inhibitory antibodies affects the proliferation and differentiation of CD34+ cells. See related perspective article on page 1477. Background Hematopoietic stem and progenitor cells can interact with their microenvironment via integrins which are adhesion receptors consisting of α and β subunits. Current knowledge suggests that the integrin subunits α4 and α6 expressed on hematopoietic stem and progenitor cells have distinct roles in retaining stem cells in the bone marrow. The aim of our study was to gain insight into the expression and functions of the integrin subunits α7-α11 within the endosteal stem cell niche. Design and Methods Human osteoblasts isolated from trabecular bone and hematopoietic stem and progenitor cells purified from umbilical cord blood or bone marrow aspirates were analyzed for the expression of integrin α7-α11 chains by reverse transcriptase polymerase chain reaction. The involvement of the integrin α9β1 in hematopoietic stem and progenitor cell adhesion, proliferation and differentiation was analyzed in functional assays. Results Transcripts for all investigated integrin chains were found in primary osteoblasts. Highly purified hematopoietic stem and progenitor cells, however, expressed only transcripts encoding integrin subunits α7 and α9. Flow cytometric analysis verified extracellular expression of the integrin α9β1 on hematopoietic stem and progenitor cells. Cell-cell adhesion assays with osteoblasts and dye-labeled CD34+ hematopoietic stem and progenitor cells in the presence of function-blocking antibodies revealed a role of integrin α9 in hematopoietic stem and progenitor cell adhesion to osteoblasts. Furthermore, the addition of anti-integrin α9 antibodies significantly inhibited proliferation and in vitro differentiation of CD34+ hematopoietic stem and progenitor cells. Conclusions The integrin α9β1 has been identified as a new member of the integrin β1-subfamily expressed on human hematopoietic stem and progenitor cells. The functional studies strongly suggest that integrin α9β1 contributes to adhesion and differentiation of hematopoietic stem and progenitor cells in the endosteal stem cell niche.

Laminin γ2 chain as a stromal cell marker of the human bone marrow microenvironment

Summary. Laminins are large heterotrimeric molecules consisting of α, β and γ chains. At present, five α chains, three β chains and three γ chains have been characterized. Laminin‐5 (α3β3γ2) is the only isoform known to date which contains a γ2 chain. In human bone marrow, non‐haematopoietic stromal cells expressed the laminin γ2 chain, whereas bone marrow mononuclear cells did not. Co‐localization of the γ2 chain was detected with the laminin α4 and α5 chains, and co‐immunoprecipitation studies revealed a new isoform consisting of α5, β2 and γ2 chains. The laminin γ2 chain was also co‐localized with α‐sm‐actin in bone marrow, but it was not expressed in endothelial cells or megakaryocytes, indicating that the γ2 chain is exclusively expressed in vivo in bone marrow stromal cells. The laminin γ2 chain containing isoform LN‐5 was shown to be an adhesive substrate for a small subpopulation of bone marrow mononuclear cells and also for peripheral blood platelets. Taken together, these results indicate that (I) laminin isoforms containing the γ2 chain can act as adhesive substrates for human haematopoietic cells, and (II) the laminin γ2 chain can be used as a specific marker molecule for human bone‐marrow‐derived stromal cells.

Cell binding properties of collagen type XIV for human hematopoietic cells

Collagen XIV, which belongs to the subclass of fibril-associated collagens with interrupted triple helices (FACITs), is a homotrimeric molecule consisting of three alpha 1 (XIV) chains. Collagen type XIV is strongly expressed in the native human bone marrow, as shown by immunofluorescence staining and immunoblotting with an affinity-purified antibody. Hematopoietic cell lines of myeloid (KG1a, U937, K562) and lymphoid (U266, IM-9) origin were able to attach firmly to purified human collagen XIV preparations. Attachment of these cells was shown to be concentration-dependent. However, other hematopoietic cell lines tested were unable to adhere to collagen XIV, indicating restriction of this cellular interaction. The cellular receptors involved in cell binding to collagen type XIV are probably membrane-bound heparansulfate proteoglycans, since only the the addition of heparin inhibited attachment of the hematopoietic cells to collagen XIV in a concentration-dependent manner. Antibodies against the beta 1-integrin subunit could not interfere with binding to collagen type XIV. Using purified fragments of collagen XIV, it could be demonstrated that at least two different heparin-sensitive adhesion sites are present in the N-terminal globular domain and in the triple-helical domain. These data indicate that collagen XIV represents another collagen type expressed in human bone marrow with strong cell binding properties for defined populations of hematopoietic cells.

TGF-β Enhances the Integrin α2β1-Mediated Attachment of Mesenchymal Stem Cells to Type I Collagen

The heterodimeric integrins are important receptors for the attachment of cells to their extracellular matrix. Here, we studied the attachment of human mesenchymal stem cells (MSCs) to type I collagen (col-1), which is part of the extracellular matrix in bone, skin, and connective tissues. Furthermore, we examined how TGF-beta influences the integrin expression and attachment of MSC. Using flow cytometry, immunoblot, and RT-PCR, we report that MSC express several integrin subunits, including the alpha(2)beta(1) integrin (VLA-2, CD49b/CD29). TGF-beta increases the expression of integrin subunits alpha(2), alpha(6), and beta(1) in MSC, thereby enhancing the attachment of MSC to col-1. The TGF-beta-mediated up-regulation of the expression of the integrin subunits alpha(2) and alpha(6) is mainly mediated in MSC by Smad2.

Processing of Laminin α Chains Generates Peptides Involved in Wound Healing and Host Defense

Laminins play a fundamental role in basement membrane architecture and function in human skin. The C-terminal laminin G domain-like (LG) modules of laminin α chains are modified by proteolysis to generate LG1-3 and secreted LG4-5 tandem modules. In this study, we provide evidence that skin-derived cells process and secrete biologically active peptides from the LG4-5 module of the laminin α3, α4 and α5 chain in vitro and in vivo. We show enhanced expression and processing of the LG4-5 module of laminin α3 in keratinocytes after infection and in chronic wounds in which the level of expression and further processing of the LG4-5 module correlated with the speed of wound healing. Furthermore, bacterial or host-derived proteases promote processing of laminin α3 LG4-5. On a functional level, we show that LG4-5-derived peptides play a role in wound healing. Moreover, we demonstrate that LG4-derived peptides from the α3, α4 and α5 chains have broad antimicrobial activity and possess strong chemotactic activity to mononuclear cells. Thus, the data strongly suggest a novel multifunctional role for laminin LG4-5-derived peptides in human skin and its involvement in physiological processes and pathological conditions such as inflammation, chronic wounds and skin infection.

Human mesenchymal stromal cells from different sources diverge in their expression of cell surface proteins and display distinct differentiation patterns

When germ-free cell cultures became a laboratory routine, hopes were high for using this novel technology for treatment of diseases or replacement of cells in patients suffering from injury, inflammation, or cancer or even refreshing cells in the elderly. Today, more than 50 years after the first successful bone marrow transplantation, clinical application of hematopoietic stem cells is a routine procedure, saving the lives of many every day. However, transplanting other than hematopoietic stem and progenitor cells is still limited to a few applications, and it mainly applies to mesenchymal stromal cells (MSCs) isolated from bone marrow. But research progressed and different trials explore the clinical potential of human MSCs isolated from bone marrow but also from other tissues including adipose tissue. Recently, MSCs isolated from bone marrow (bmMSCs) were shown to be a blend of distinct cells and MSCs isolated from different tissues show besides some common features also some significant differences. This includes the expression of distinct antigens on subsets of MSCs, which was utilized recently to define and separate functionally different subsets from bulk MSCs. We therefore briefly discuss differences found in subsets of human bmMSCs and in MSCs isolated from some other sources and touch upon how this could be utilized for cell-based therapies.