Yes / The individual molecular pathways downstream of Cdc42, Rac, and Rho GTPases are well documented, but we know surprisingly little about how these pathways are coordinated when cells move in a complex environment in vivo. In the developing embryo, melanoblasts originating from the neural crest must traverse the dermis to reach the epidermis of the skin and hair follicles. We previously established that Rac1 signals via Scar/WAVE and Arp2/3 to effect pseudopod extension and migration of melanoblasts in skin. Here we show that RhoA is redundant in the melanocyte lineage but that Cdc42 coordinates multiple motility systems independent of Rac1. Similar to Rac1 knockouts, Cdc42 null mice displayed a severe loss of pigmentation, and melanoblasts showed cell-cycle progression, migration, and cytokinesis defects. However, unlike Rac1 knockouts, Cdc42 null melanoblasts were elongated and displayed large, bulky pseudopods with dynamic actin bursts. Despite assuming an elongated shape usually associated with fast mesenchymal motility, Cdc42 knockout melanoblasts migrated slowly and inefficiently in the epidermis, with nearly static pseudopods. Although much of the basic actin machinery was intact, Cdc42 null cells lacked the ability to polarize their Golgi and coordinate motility systems for efficient movement. Loss of Cdc42 de-coupled three main systems: actin assembly via the formin FMNL2 and Arp2/3, active myosin-II localization, and integrin-based adhesion dynamics. / Cancer Research UK (to L.M.M. [A17196], R.H.I. [A19257], and S.W.G.T.) and NIH grants P01-GM103723 and P41-EB002025 (to K.M.H.). N.R.P. is supported by a Pancreatic Cancer Research Fund grant (to L.M.M.). Funding to Prof. Rottner by the Deutsche Forschungsgemeinschaft (grant RO2414/3-2).

Microsporidia are a distant fungal pathogen that have severe clinical consequences for the immunocompromised. Previous work identified a microsporidian pathogen protein termed Microsporidian ADAM or MADAM. This protein has close sequence homology to other ADAM proteins (A Disintegrin and Metalloproteinase) in two microsporidian species, Encephalitozoon intestinalis and E. cuniculi. ADAM proteins have a wide range of functions, including binding to integrins and host signaling. It is known that many pathogens manipulate integrins to invade host cells, and it is predicted that microsporidia are also exploiting this host target. Previous work with the MADAM protein demonstrated that this protein has a role in adherence to host cells. Separate work showed integrin inhibitors can also decrease spore adherence to cells. Experiments in this project complement previous research and further characterize the binding of microsporidia to host integrins and the intracellular consequences of that binding. This work found the integrin binding sequence of MADAM (MADAM peptide) is important for spore binding to host cells. Separate work shows that the host β1 integrin is also involved in spore adherence. Additional work demonstrated that spores and the MADAM peptide elicited an increase in host integrin signaling in Western blotting experiments. And finally, preliminary acellular interferometry experiments suggest the MADAM protein binds specifically to α5β1 and α6β4 integrins. Together, these results suggest microsporidia spores rely, in part, on host integrins to bind to host cells before infection.

Cell adhesion to extracellular matrices (ECM) is critical to many cellular processes including differentiation, proliferation, migration, and apoptosis. Alterations in adhesive mechanisms are central to the behavior of cells in pathological conditions including cancer, atherosclerosis, and defects in wound healing. Although significant progress has been made in identifying molecules involved in adhesion, the mechanisms that dictate the generation of strong adhesive forces remain poorly understood. Specifically, the role of nanoscale geometry of the adhesive interface in integrin recruitment and adhesion forces remains elusive due to limitations in the techniques available for engineering cell adhesion environments. The objective of this project was to analyze the role of nanoscale geometry in cell adhesion strengthening to ECM. Our central hypothesis was that adhesive interactions are regulated by integrin clusters whose recruitment is determined by the nanoscale geometry of the adhesive interface and whose heterogeneity in size, spacing, and orientation modulates adhesion strength. The objective of this project was accomplished by 1) developing an experimental technique capable of producing nanoscale patterns of proteins on surfaces for cell adhesion arrays, 2) assessing the regulation of integrin recruitment by geometry of the adhesive interface, and 3) determining the functional implications of adhesive interface geometry by systematically analyzing the adhesion strengthening response to nanoscale patterns of proteins. A printing technique was developed that patterns proteins into features as small as 90nm with high contrast and high reproducibility. Cell adhesion arrays were produced by directly immobilizing proteins into patterns on mixed-SAMs surfaces with a protein-resistant background. Colocalization analysis of integrin recruitment to FN patterns demonstrated a concentrating effect of bound integrins at pattern sizes with areas equivalent to small nascent focal adhesions. At adhesion areas below 333 × 333 nm2, the frequency of integrin recruitment events decreased significantly indicating a threshold size for integrin clustering. Functionally, pattern sizes below the threshold were unable to participate in generation of adhesion strength. In contrast, patterns between the threshold and micron sizes showed a relationship between adhesion strength and area of individual adhesion points, independent of the total available adhesion area. These studies introduce a robust platform for producing nanoscale patterns of proteins in biologically relevant geometries. Results obtained using this approach yielded new insights on the role of nanoscale organization of the adhesive interface in modulating adhesion strength and integrin recruitment.

No description available.

No description available.

Artificial lipid bilayers formed on solid surface supports are widespread model systems to study physical, chemical, as well as biological aspects of cell membranes and fundamental interfacial interactions. The approach to use a thin polymer film representing a cushion for lipid bilayers prevents incorporated membrane proteins from pinning to the support and mimics the native environment of a lipid bilayer in certain aspects of the extracellular matrix and intracellular structures. A key component for cell anchorage to extracellular fibronectin is the transmembrane adhesion receptor alpha(5)beta(1) integrin. Its transport dynamics and clustering behavior plays a major role in the assembly of focal adhesions, which mediate mechanical forces and biochemical signals of cells with their surrounding. The system investigated herein is envisioned to use extrinsically controlled stimuli-responsive polymer cushions to tune the frictional drag between polymer cushion and mobile membranes with incorporated integrins to actively regulate lipid membrane characteristics. To attain this goal, a temperature- and pH-responsive polymer based on poly(N-isopropylacrylamide) copolymers containing varying amounts of carboxyl-group-terminated comonomers at different aliphatic spacer lengths (PNIPAAm-co-carboxyAAM) was surface-grafted to a poly(glycidyl methacrylate) anchorage layer. The swelling transitions were characterized using atomic force microscopy, ellipsometry and quartz crystal microbalance with dissipation monitoring (QCM-D) and found to be tunable over a wide range of temperature and pH. In agreement with the behavior of the polymers in solution, longer alkyl spacers decreased the phase transition temperature T(P) and higher contents of carboxylic acid terminated comonomers increased T(P) at alkaline conditions and decreased T(P) at acidic conditions. Remarkably, the point where the degree of carboxyl group deprotonation balances the T(P)-lowering effect of the alkyl spacer was distinctive for each alkyl spacer length. These findings illustrate how the local and global balance of hydrophilic and hydrophobic interactions along the copolymer chain allows to adjust the swelling transition to temperatures below, comparable, or above those observed for PNIPAAm homopolymers. Additionally, it could be shown that surface-grafting leads to a decrease in T(P) for PNIPAAm homopolymers (7°C) and copolymers (5°C - 10°C). The main reason is the increase in local polymer concentration of the swollen film constrained by dense surface anchorage in comparison to the behavior of dilute free chains in solution. In accordance with the Flory-Huggins theory, T(P) decreases with increasing concentration up to the critical concentration. Biological functionalization of the PNIPAAm-co-carboxyAAm thin films was demonstrated for the cell adhesion ligand peptide cRGD via carbodiimide chemistry to mimic extracellular binding sites for the cell adhesion receptors integrin. The outcome of QCM-D measurements of cRGD-functionalized surfaces showed a maintained stimuli-responsiveness with slight reduction in T(P). A drying/rehydration procedure of a 9:1 lipid mixture of the cationic lipid dioleoyl-trimethylammoniumpropane (DOTAP) and the zwitterionic dioleoyl-phosphatidylcholine (DOPC) was utilized to form lipid bilayer membranes on PNIPAAm-co-carboxyAAM cushions. Fluorescence recovery after photobleaching (FRAP) revealed that lipid mobility was distinctively higher (6.3 - 9.6) µm2 s-1 in comparison to solid glass support ((3.0 - 5.9) µm2 s-1). In contradiction to the initial expectations, modulation of temperature and pH led to poor variations in lipid mobility that did not correlate with the PNIPAAm cushion swelling state. The results suggested a weak coupling of the lipid bilayer with PNIPAAm polymer cushions that can be slightly tuned by electrostatic interactions. The transmembrane adhesion receptor alpha(5)beta(1) integrin was reconstituted into liposomes consisting of DOPC/sphingomyelin/cholesterol 2:2:1 for the formation of polymer cushioned bilayers. PNIPAAm- co-carboxyAAM and maleic acid (MA) copolymers were used as cushions, both with the option for cRGD functionalization. On the MA copolymer cushions, fusion of proteoliposomes resulted in supported bilayers with mobile lipids as confirmed by FRAP. However, incorporated integrins were immobile. In an attempt to explain this observation, the medium-sized cytoplasmic integrin domain was accounted to hamper the movement by steric interactions with the underlying polymer chains in conjunction with electrostatic interactions of the cationic cytoplasmic domain with the oppositely charged MA copolymer. On the PNIPAAm-co-carboxyAAM cushion only a drying/rehydration procedure lead to bilayer formation. However, again the integrins were immobile, presumably due to the harsh treatment during preparation. Nevertheless, the results of the investigated set of PNIPAAm copolymer films suggest their application as temperature- and pH-responsive switchable layers to control interfacial phenomena in bio-systems at different physiological conditions. The PNIPAAm-co-carboxyAAm cushioned bilayer system represents a promising step towards extrinsically controlled membrane – substrate interactions.

Thesis (Ph.D. in Pharmacology) -- University of Colorado Denver, 2007. / Typescript. Non-Latin script record Includes bibliographical references (leaves 112-128). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;

As graft source for lymphoma or leukemia treatment, hematopoietic stem and progenitor cells (HSPCs) have been the focus of translational medicine for decades. HSPCs are defined by their self-renewing capacity and their ability to give rise to all mature blood cells. They are found anchored to a specialized microenvironment in the bone marrow (BM) called the hematopoietic niche. HSPCs can be enriched by sorting them based on the presence of the surface antigen CD34 before clinical or tissue engineering use. As these cells represent a minority in most graft sources and the amount of applicable cells is limited, ex vivo expansion-cultures were established using cytokine cocktails or small molecules. However, in vitro culture of HSPCs as suspension-cultures result in heterogeneous cell populations with undefined cellular identities. In the BM niche, HSPCs are not exclusively maintained by cytokines but also by cell-matrix adhesions mediated by integrins (ITGs). Thus, β1 and β2 ITGs were found to promote initial contact of HSPCs with mesenchymal stromal cells (MSCs) and ITGβ3 expression was shown to be a marker for long-term repopulating HSPCs in vivo. Consequently, ex vivo remodeling of the BM niche using co-cultures of HSPCs and niche cells like MSCs came into spotlight and was proven to be a promising tool for stem cell expansion. However, in clinical and research applications, direct contact of two cell populations necessitates HSPC post-culture purification. To address these problems, we established a novel culture method for remodeling the BM extra cellular stroma in vitro wherein we used decellularized extracellular matrix (ECM) scaffolds derived from immortalized mesenchymal stromal cells (SCP-1). Such scaffolds were found to be highly reproducible and served as in vitro niche for HSPCs by being more effective for the expansion of CD34+ cells, compared to classical suspension cultures. ECMs were shown to consist of multiple proteins including fibronectins, collagens, and a major niche chemokine responsible for BM homing and retention of HSPCs in vivo, namely, stromal derived factor 1 (SDF-1). SDF-1 is known to be secreted by MSCs and is anchored to matrix proteins. This reveals that ECM scaffolds produced by SCP-1 cells are a naïve reconstructed microenvironment. When CD34+ cells were seeded, only around 20% of the cells adhered to the provided ECM scaffold. These cells recognized SDF-1 via C-X-C chemokine receptor type 4 (CXCR-4), as shown by laser scanning confocal microscopy. Thus, adhesive sides as they are present in the BM niche are provided. However, CD34+ cells isolated from G-CSF mobilized peripheral blood of healthy donors were found to be heterogenous with respect to adhesion capacity. Nonetheless, it was similar to HSPC co-cultures with SCP-1 cells as feeder layer. Therefore, we separated and analyzed two cell fractions, the adherent (AT-cells) and the non- adherent supernatant (SN-cells) cells. Other signals provided by the BM extracellular stroma to HSPCs are physical cues that control HSPC fate. HSPCs sense these physical features through focal contacts and accordingly remodel their morphological and biomechanical properties. Using real-time deformability cytometry (RT-DC) to uncover biomechanical phenotypes of freshly isolated HSPCs, SN-cells, AT-cells, and classical suspension cultured HSPCs in plastic culture dishes (PCD) were analyzed. We found freshly isolated cells to be less deformable and small. AT-cells displayed actin polymerization to stress fibers, and exhibited a stiffer mechanical phenotype compared to PCD-cultured or SN-cells. This might constitute the first hint of functional adaptation. Integrins are known to establish mechanosensing focal contacts. Thus, we analyzed ITG surface expression and identified ITGαIIb, ITGαV, and ITGβ3 to be enriched on AT-cells compared to freshly isolated cells or SN-cells. Active integrins need to form heterodimers consisting of one α- and one β subunit. Interestingly, the identified ITGs exclusively interact with each other to form RGD peptide receptors. RGD is a tripeptide consisting of the amino acids arginine, glycine, and aspartic acid and was identified as an adhesion sequence within fibronectin and other extracellular proteins. Consequently, we could confirm an important role for ITGαVβ3 in HSPC- ECM interaction with respect to adhesion and migration. However, we also identified ITGβ3 expression on a subset of CD34+ cells either freshly isolated or ECM cultured cells, as a marker for erythrocyte differentiation. These findings demonstrate that, in vitro, the ECM compartment acts as a regulator of HSPC fate and portray mechanical recognition as a potent driver of differentiation. In this context, targeted modulation of ECM scaffolds could enhance cell-ECM interactions and accelerate stem cell expansion or differentiation. These modulations could also provide further insights into HSPC-niche regulation. We demonstrate that ECMs derived from osteogenic differentiated SCP-1 cells increase HSPC expansion but do not lead to increased cell adhesion. As ECM adhesion preliminary alters HSPC function, we aimed at developing ECM scaffolds with increased adhesion capacity. Using lentiviral transduction, we generated a stable knock down of fibulin-1 in SCP-1 cells. Fibulin-1 is an ECM protein known to form anti-adhesion sites with fibronectin. However, we failed to increase adherent cell numbers or enhance HSPC expansion in the fibulin-1 knock down ECMs. Taken together, SCP-1 cell-derived ECM protein scaffolds provide an in vitro niche for HSPCs capable of stem cell expansion. Integrin mediated signaling altered the biomechanical and functional properties of HSPCs and hints at suspension cultures as being inappropriate to study the physiological aspects of HSPCs. Targeted modulation of ECM scaffolds could theoretically generate suitable ex vivo environments with the capacity to gain detailed insight into HSPC regulation within their niche. This will enhance the functionality of new biomaterials and will lead to improved regenerative therapies like BM transplantation.

Die strahleninduzierte Mucositis enoralis ist eine der bedeutendsten und häufig dosislimitierenden frühen Nebenwirkungen der Strahlentherapie fortgeschrittener Kopf Hals Tumoren. Bis heute hat sich noch kein allgemein gültiges Konzept zur Therapie und Prophylaxe der Mundschleimhautentzündung durchsetzen können. Ein Ansatz zur selektiven, auf der Tumorbiologie beruhenden Beeinflussung der Strahlenempfindlichkeit von Tumoren ist die Blockade des epidermalen Wachstumsfaktor-Rezeptors (EGFR). In Kombination mit Strahlentherapie sollen so die lokale Tumorkontrolle und die Heilungschancen verbessert werden. Die Wirkung der Tyrosinkinase-Inhibitoren BIBX1382BF und Erlotinib auf histomorphologische Parameter in der Mundschleimhaut sowie auf die Expression der als Stammzellmarker diskutierten Proteine p63, Integrin β1 und CD44 wurde in der vorliegenden Arbeit im Vergleich zur alleinigen fraktionierten Bestrahlung untersucht. Für die histologischen Studien erfolgte die zweiwöchige fraktionierte Bestrahlung der Schnauzen von Mäusen des Inzuchtstammes C3H/Neu mit zehn Fraktionen zu je 3 Gy (Tag 0-4, Tag 7-11). Die Versuche gliederten sich in vier Gruppen: • I/A (54 Tiere) und II/A (40 Tiere): fraktionierte Bestrahlung, keine weitere Behandlung • I/B (51 Tiere): fraktionierte Bestrahlung, zusätzlich orale Gabe von BIBX1382BF, 50 mg/kg KG per os, von Tag 0-14 je 30 min nach der Bestrahlung • II/B (35 Tiere): fraktionierte Bestrahlung, zusätzlich orale Gabe von Erlotinib, 50 mg/kg KG per os, von Tag 0-11 je 30 min nach der Bestrahlung. Die Entnahme der Zungen erfolgte im Versuch I bei jeweils drei Tieren pro Tag von Tag 0 bis Tag 17. Im Versuch II wurden an den Tagen 0, 2, 4, 6, 8, 10, 12 und 14 jeweils die Zungen von fünf Tieren entnommen. Anschließend folgten die Fixierung der Zungen in Formalin, die Einbettung in Paraffin und die Anfertigung 3 µm dicker Gewebeschnitte. Die Zungenpräparate wurden für die histologischen Untersuchungen mit Hämatoxylin-Eosin gefärbt. Für die immunhistochemischen Färbungen wurde die ABC-Methode eingesetzt. Das Epithel der Zungenunterseite wurde lichtmikroskopisch hinsichtlich Zellzahl, Schichtdicke und Expression der potentiellen Stammzellmarker p63, Integrin β1 und CD44 ausgewertet. Aufgrund der geringen Gruppengröße (Versuch I: drei Tiere pro Datenpunkt; Versuch II: fünf Tiere pro Datenpunkt) wurde auf eine eingehende statistische Testung verzichtet. Die vorliegende Arbeit beschränkt sich auf eine beschreibende Darstellung des Verlaufs der Einzelparameter über den Gesamtzeitraum. Die Zellzahlen verringerten sich während der ersten Bestrahlungswoche auf 60-70 % der Ausgangswerte, stagnierten in der zweiten Woche und stiegen schließlich bis zum Ende der Nachbeobachtung wieder an. Zwischen den nur bestrahlten und den zusätzlich mit BIBX1382BF behandelten Tieren war kein Unterschied feststellbar. Ein gleichsinniger Verlauf war auch in Versuch II zu beobachten, wobei die Zellzahlen der mit Erlotinib behandelten Tiere in der Funktionsschicht durchgängig höher ausfielen als in Versuchsreihe A. Die Dicke des Gesamtepithels bzw. der einzelnen Epithelschichten zeigte im Versuch I unter Bestrahlung große individuelle Schwankungen. Unter zusätzlicher BIBX1382BF-Gabe wurden oft niedrigere Werte gemessen. Im Versuch II blieb die Dicke des Gesamtepithels unter Fraktionierung konstant. Von Tag 0-12 wurden bei zusätzlicher Erlotinib-Applikation geringere Werte der Gesamtdicke gemessen als unter alleiniger Bestrahlung, ansonsten fielen die Veränderungen der Epitheldicke unabhängig von der Erlotinib-Gabe gering aus. Der kurzzeitigen, mit dem allgemeinen Zellverlust einhergehenden Verringerung der p63-Expression zu Beginn der Bestrahlung folgt bis zum Ende des Beobachtungszeitraumes die Normalisierung der p63-positiven Zellen. Mit EGFR-Blockade sind gegenüber der alleinigen Bestrahlung keine Unterschiede in der p63-Expression festzustellen. Die Integrin β1-Expression nahm im Verlauf der Bestrahlung ab. Unter EGFR-Blockade mit BIBX1382BF zeigte sich an den Tagen 2-9 und 12-16 ein schwächeres Färbesignal als im fraktioniert bestrahlten Epithel, was für eine mögliche Interaktion des EGF-Rezeptors mit Integrin β1 spricht. Im Versuch II waren unabhängig von der Erlotinib-Gabe keine Unterschiede in der Expression von Integrin β1 feststellbar. Die CD44-Expression im Epithel wurde durch Bestrahlung gefördert. Übereinstimmend konnte in der vorliegenden Arbeit in beiden Versuchen eine Steigerung der CD44-Färbeintensität über den jeweiligen Referenzbereich festgestellt werden. Eine Blockade der EGFR-Aktivität durch Erlotinib reduzierte die Expression von CD44, wie in Versuch II/B im initialen Abfall der CD44-Färbeintensität deutlich wurde. Doch schon ab Tag 4 wurden im Versuch II/A und II/B gleich starke Färbesignale für CD44 erfasst. Insgesamt ergaben sich unter EGFR-Inhibition mittels BIBX1382BF oder Erlotinib keine Hinweise auf Veränderungen der untersuchten Parameter während einer zweiwöchigen fraktionierten Bestrahlung. Ob diese Ergebnisse auch auf andere Tyrosinkinase-Inhibitoren bzw. unterschiedliche Wirkstoffklassen (z. B. Anti-EGFR-Antikörper) übertragbar sind, muss in weiteren Studien untersucht werden. / Radiation-induced oral mucositis is one of the most important and often dose limiting early side effects of radiotherapy of advanced tumours in the head-and-neck region. To this day, no general concept for therapy and prophylaxis of the oral mucositis has been established. The inhibition of the epidermal growth factor receptor (EGFR) is one approach to a selective increase of the radiosensitivity of tumours based on the tumour biology. In combination with radiotherapy, application of EGFR-inhibitors is supposed to increase the local tumour control and the chances of cure. The aim of the present study was to investigate the effect of the tyrosine kinase inhibitors BIBX1382BF and Erlotinib on the radiation response of the oral mucosa and on the expression of different proteins that are discussed to be markers of epithelial stem cells. For the histological studies, the snouts of C3H/Neu mice were irradiated with ten daily fractions of 3 Gy over two weeks (on days 0-4, 7-11). The experiments comprised four treatment groups: • I/A (54 animals) and II/A (40 animals): fractionated irradiation, no further treatment • I/B (51 animals): fractionated irradiation, administration of BIBX1382BF, 50 mg/kg per os, once daily (days 0-14) 30 min after the radiation treatment • II/B (35 animals): fractionated irradiation, administration of Erlotinib, 50 mg/kg per os, once daily (days 0-11) 30 min after the radiation treatment. Between day 0 and 17, three animals of the groups I/A and I/B were euthanised per day. In the experimental arms II/A and II/B five mice were killed on day 0, 2, 4, 6, 8, 10, 12 and 14, respectively. The tongues were excised, fixed in formalin, embedded in paraffin and 3 µm thick sections were prepared. Subsequently, the tongue sections were stained with haematoxylin and eosin or with an ABC kit to visualise proteins of interest. The epithelium of the lower tongue was examined by light microscopy regarding the following parameters: cell numbers, thickness of epithelial layers and expression of the potential stem cell markers p63, integrin β1 and CD44. Due to the limited number of animals per data point (experiment I: three mice per data point; experiment II: five mice per data point), a detailed statistical analysis was not performed. The present study is determined to describe the parameter variations over the observation period. Cell numbers decreased to 60-70 % of the pre-treatment control values within the first week of irradiation alone, remained constant in the second week, and then slowly increased until the end of the observation period. There was no difference between radiotherapy alone or combined treatment with BIBX1382BF. In experiment II similar observations were made with higher cell numbers in the functional layer of the epithelium of the Erlotinib treated animals than in the irradiated group. The thickness of the epithelium and its individual layers showed high inter individual differences in experiment I. In treatment group I/B, lower values of thickness were often detected in comparison to group I/A. In experiment II the thickness of the epithelium remained constant under fractionated irradiation. Between day 0 and 12 the Erlotinib treatment slightly decreased the thickness of the whole epithelium in comparison to the irradiated group. Besides, there were only minor changes in the thickness of the different layers. Associated with the general loss of cells, radiation treatment led to a transient decrease in the expression of p63. The number of p63-positive cells recovered until the end of the observation period. A similar expression pattern of p63-positivity was found independent of EGFR inhibition. The expression of integrin β1 decreased during fractionated irradiation. On days 2-9 and 12-16, the changes were more pronounced in combination with BIBX1382BF treatment which indicates a potential interaction of the EGF receptor with integrin β1. In experiment II, no differences between the exclusively irradiated group and the combined treatment with Erlotinib were found for the expression patterns of integrin β1. Irradiation alone resulted in a higher epithelial expression of CD44. Accordingly, a general increase of CD44 staining intensity was observed in both experiments exceeding control values. Due to the EGFR inhibition with Erlotinib, the expression of CD44 initially decreased. However, by day 4 no persisting differences in staining intensity could be observed independent of EGFR inhibition. In summary, EGFR inhibition via BIBX1382BF or Erlotinib did not result in alterations of the analysed parameters during two weeks of fractionated irradiation. Further studies are required to demonstrate if the present findings are transferable to other tyrosine kinase inhibitors or different substance classes (e.g. inhibiting receptor antibodies).

As graft source for lymphoma or leukemia treatment, hematopoietic stem and progenitor cells (HSPCs) have been the focus of translational medicine for decades. HSPCs are defined by their self-renewing capacity and their ability to give rise to all mature blood cells. They are found anchored to a specialized microenvironment in the bone marrow (BM) called the hematopoietic niche. HSPCs can be enriched by sorting them based on the presence of the surface antigen CD34 before clinical or tissue engineering use. As these cells represent a minority in most graft sources and the amount of applicable cells is limited, ex vivo expansion-cultures were established using cytokine cocktails or small molecules. However, in vitro culture of HSPCs as suspension-cultures result in heterogeneous cell populations with undefined cellular identities. In the BM niche, HSPCs are not exclusively maintained by cytokines but also by cell-matrix adhesions mediated by integrins (ITGs). Thus, β1 and β2 ITGs were found to promote initial contact of HSPCs with mesenchymal stromal cells (MSCs) and ITGβ3 expression was shown to be a marker for long-term repopulating HSPCs in vivo. Consequently, ex vivo remodeling of the BM niche using co-cultures of HSPCs and niche cells like MSCs came into spotlight and was proven to be a promising tool for stem cell expansion. However, in clinical and research applications, direct contact of two cell populations necessitates HSPC post-culture purification. To address these problems, we established a novel culture method for remodeling the BM extra cellular stroma in vitro wherein we used decellularized extracellular matrix (ECM) scaffolds derived from immortalized mesenchymal stromal cells (SCP-1). Such scaffolds were found to be highly reproducible and served as in vitro niche for HSPCs by being more effective for the expansion of CD34+ cells, compared to classical suspension cultures. ECMs were shown to consist of multiple proteins including fibronectins, collagens, and a major niche chemokine responsible for BM homing and retention of HSPCs in vivo, namely, stromal derived factor 1 (SDF-1). SDF-1 is known to be secreted by MSCs and is anchored to matrix proteins. This reveals that ECM scaffolds produced by SCP-1 cells are a naïve reconstructed microenvironment. When CD34+ cells were seeded, only around 20% of the cells adhered to the provided ECM scaffold. These cells recognized SDF-1 via C-X-C chemokine receptor type 4 (CXCR-4), as shown by laser scanning confocal microscopy. Thus, adhesive sides as they are present in the BM niche are provided. However, CD34+ cells isolated from G-CSF mobilized peripheral blood of healthy donors were found to be heterogenous with respect to adhesion capacity. Nonetheless, it was similar to HSPC co-cultures with SCP-1 cells as feeder layer. Therefore, we separated and analyzed two cell fractions, the adherent (AT-cells) and the non- adherent supernatant (SN-cells) cells. Other signals provided by the BM extracellular stroma to HSPCs are physical cues that control HSPC fate. HSPCs sense these physical features through focal contacts and accordingly remodel their morphological and biomechanical properties. Using real-time deformability cytometry (RT-DC) to uncover biomechanical phenotypes of freshly isolated HSPCs, SN-cells, AT-cells, and classical suspension cultured HSPCs in plastic culture dishes (PCD) were analyzed. We found freshly isolated cells to be less deformable and small. AT-cells displayed actin polymerization to stress fibers, and exhibited a stiffer mechanical phenotype compared to PCD-cultured or SN-cells. This might constitute the first hint of functional adaptation. Integrins are known to establish mechanosensing focal contacts. Thus, we analyzed ITG surface expression and identified ITGαIIb, ITGαV, and ITGβ3 to be enriched on AT-cells compared to freshly isolated cells or SN-cells. Active integrins need to form heterodimers consisting of one α- and one β subunit. Interestingly, the identified ITGs exclusively interact with each other to form RGD peptide receptors. RGD is a tripeptide consisting of the amino acids arginine, glycine, and aspartic acid and was identified as an adhesion sequence within fibronectin and other extracellular proteins. Consequently, we could confirm an important role for ITGαVβ3 in HSPC- ECM interaction with respect to adhesion and migration. However, we also identified ITGβ3 expression on a subset of CD34+ cells either freshly isolated or ECM cultured cells, as a marker for erythrocyte differentiation. These findings demonstrate that, in vitro, the ECM compartment acts as a regulator of HSPC fate and portray mechanical recognition as a potent driver of differentiation. In this context, targeted modulation of ECM scaffolds could enhance cell-ECM interactions and accelerate stem cell expansion or differentiation. These modulations could also provide further insights into HSPC-niche regulation. We demonstrate that ECMs derived from osteogenic differentiated SCP-1 cells increase HSPC expansion but do not lead to increased cell adhesion. As ECM adhesion preliminary alters HSPC function, we aimed at developing ECM scaffolds with increased adhesion capacity. Using lentiviral transduction, we generated a stable knock down of fibulin-1 in SCP-1 cells. Fibulin-1 is an ECM protein known to form anti-adhesion sites with fibronectin. However, we failed to increase adherent cell numbers or enhance HSPC expansion in the fibulin-1 knock down ECMs. Taken together, SCP-1 cell-derived ECM protein scaffolds provide an in vitro niche for HSPCs capable of stem cell expansion. Integrin mediated signaling altered the biomechanical and functional properties of HSPCs and hints at suspension cultures as being inappropriate to study the physiological aspects of HSPCs. Targeted modulation of ECM scaffolds could theoretically generate suitable ex vivo environments with the capacity to gain detailed insight into HSPC regulation within their niche. This will enhance the functionality of new biomaterials and will lead to improved regenerative therapies like BM transplantation.:List of contents I List of figures IV List of tables VI Abbreviations VII 1 Introduction 1 1.1 The stem cell microenvironment 3 1.1.1 The cellular endosteal bone marrow microenvironment 6 1.1.1.1 Mesenchymal stem/stromal cells 7 1.1.1.2 Hematopoietic stem and progenitor cells 8 1.1.2 Extracellular bone marrow microenvironment 10 1.1.2.1 Extracellular matrix 11 Chemokines and Cytokines 12 Cell adhesion to ECM 13 1.2 Native ex vivo ECM scaffolds 16 2 Aim of the study 19 3 Materials and methods 21 3.1 Materials 21 3.1.1 Chemicals and reagents 21 3.1.2 Kits 23 3.1.3 Media 24 3.1.4 Antibodies 24 3.1.5 Primers, sh-RNA sequences, and vectors 25 3.1.6 Equipment 26 3.1.7 Software 27 3.2 Methods 27 3.2.1 Cell preparation and culture 27 3.2.1.1 Mesenchymal stromal cells 27 3.2.1.2 Hematopoietic stem cells 28 3.2.1.3 Single cell picked clone 1 (SCP-1) cells 28 3.2.2 Generation of surface immobilized ECM preparations 29 3.2.2.1 Surface functionalization 29 3.2.2.2 ECM preparation 29 3.2.3 Flow cytometry and fluorescent activated cell sorting 30 3.2.4 Cell cycle analyses 30 3.2.5 Proliferation analyses 31 3.2.6 Colony forming unit cell assay (CFU-GEMM) 31 3.2.7 Migration assays 31 3.2.7.1 Transwell migration 31 3.2.7.2 Live cell migration 32 3.2.8 Confocal laser scanning microscopy 32 3.2.9 Real-time deformability cytometry (RT-DC) 32 3.2.10 Molecular biological methods 33 3.2.10.1 RNA isolation, reverse transcription, and PCR 33 3.2.10.2 Lentiviral shRNA transduction 34 3.2.10.3 Western blot 35 3.2.10.4 ELISA 36 3.2.11 Statistical analysis 37 4 Results 38 4.1 Extracellular matrix scaffolds for HSPCs 38 4.1.1 ECM properties 39 4.1.2 HSPC survival in ECM and PCD cultures 40 4.1.3 HSPC expansion in ECM and PCD cultures 41 4.2 HSPC morphological and mechanical adaptation to ECM 44 4.2.1 Actin polymerization and polarization 45 4.2.2 Biomechanical phenotype 46 4.3 Bioactive SDF-1 is incorporated in ECM scaffolds 49 4.3.1 CXCR4 polarization towards ECM 50 4.4 HSPC integrin expression and migration 52 4.4.1 Integrin surface expression on HSPC subsets 52 4.4.2 Focal contact formation 53 4.4.3 Integrin activation via ECM adhesion 55 4.4.4 Clonogenicity of ECM cultured HSPCs 57 4.4.5 HSPC migration when attached to ECM scaffolds 60 4.4.5.1 Reduced migratory behavior via ITGαVβ3 inhibition 61 4.4.5.2 SDF-1 induces migration but not adhesion 64 4.5 Targeted modulation of ECM scaffolds 65 4.5.1 Fibulin-1 knock down in SCP-1 cells 66 4.5.2 HSPC support of fibulin-1 reduced ECM scaffolds 70 5 Discussion 73 5.1 SCP-1 cells as a source for ECM scaffold production 74 5.2 Cell adhesion and focal contact formation 75 5.3 HSPC multilineage potential 78 5.4 ECM scaffold modulation 79 6 Summary 83 7 Zusammenfassung 86 Bibliography 89 Danksagung 108 Anlagen 110 Erklärung zur Eröffnung des Promotionsverfahrens [Formblatt 1.2.1] 110 Erklärung zur Einhaltung rechtlicher Vorschriften [Formblatt 1.1] 110