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Despite intense research efforts, the repair of large bone defects is still not satisfactory and remains a major challenge in Orthopaedic Surgery. In this context bone tissue engineering has emerged as a promising strategy. However, one of the fundamental principles underlying tissue engineering approaches is that newly formed tissue must maintain sufficient vascularization to support its growth. Thus an active blood vessel network is an essential pre-requisite for scaffold constructs to integrate within existing host tissue. Currently, great efforts are made to address this problem employing transplantation of vascular cells and loading of appropriate biological factors. Endothelial progenitor cells (EPCs) are a heterogeneous subpopulation of bone marrow mononuclear progenitor cells with potential for differentiation to the endothelial lineage and thus vasculogenic capacity. However, clinical studies reported that with the increase of age, increased susceptibility to apoptosis and accelerated senescence may contribute to the numerical and functional impairments observed in EPCs, which may lead to a reduced angiogenic capacity and an increased risk of vascular disease. Hence attention has increasingly been paid to enhance mobilization and differentiation of EPCs for therapeutic purposes. A large body of evidence indicates that in Traditional Chinese Medicine (TCM) a plethora of herbs and herbal extracts are effective in the treatment of vascular diseases such as chronic wounds, diabetic retinopathy and rheumatoid arthritis. Thus, it seems rational to explore these medicinal plants as potential sources of novel angiomodulatory factors. In this thesis we demonstrated that treatment with TCM herbal extracts promote cell growth, cell migration, cell-matrix and capillary-like tube formation of BM-EPCs. Among these TCM extracts, Salidroside (SAL) and Icariin (ICAR) incubation increased VEGF and nitric oxide secretion, which in turn mediated the enhancement of angiogenic differentiation of BM-EPCs. A mechanic evaluation provided evidence that SAL stimulates the phosphorylation of Akt, mammalian target of rapamycin (mTOR) and ribosomal protein S6 kinase (p70S6K), as well as phosphorylated ERK1/2, which is associated with the cell migration and tube formation. Furthermore, a pilot in vivo study showed that SAL has the potential to enhance bone formation in a murine femoral critical-size bone defects model. Another new finding of the present study is that hydrogen peroxide (H2O2)-induced cytotoxicity is counteracted by TCM extracts. We found that SAL, Salvianolic acid B (SalB) and ICAR significantly abrogated H2O2-induced cell apoptosis, reduced the intracellular level of reactive oxygen species (ROS) and nicotinamide adenine dinucleotide phosphate-oxidase (NADPH) expression, and restored the mitochondrial membrane potential of BM-EPCs. Our data suggest that this protective effect of SalB is mediated by the activation of mTOR, p70S6K, 4EBP1, and by the suppression of MKK3/6-p38 MAPK-ATF2 and ERK1/2 signaling pathways after H2O2 stress. In addition, the investigation also demonstrates that ICAR owns the ability to inhibit apoptotic and autophagic programmed cell death via restoring the loss of mTOR and attenuation of ATF2 activity upon oxidative stress. Based on the outcomes of the present work, we propose SAL, SalB and ICAR as novel proanigiogenic and cytoprotective therapeutic agents with potential applications in the fields of systemic and site-specific tissue regeneration including ischaemic disease and extended musculoskeletal tissue defects.

Despite intense research efforts, the repair of large bone defects is still not satisfactory and remains a major challenge in Orthopaedic Surgery. In this context bone tissue engineering has emerged as a promising strategy. However, one of the fundamental principles underlying tissue engineering approaches is that newly formed tissue must maintain sufficient vascularization to support its growth. Thus an active blood vessel network is an essential pre-requisite for scaffold constructs to integrate within existing host tissue. Currently, great efforts are made to address this problem employing transplantation of vascular cells and loading of appropriate biological factors. Endothelial progenitor cells (EPCs) are a heterogeneous subpopulation of bone marrow mononuclear progenitor cells with potential for differentiation to the endothelial lineage and thus vasculogenic capacity. However, clinical studies reported that with the increase of age, increased susceptibility to apoptosis and accelerated senescence may contribute to the numerical and functional impairments observed in EPCs, which may lead to a reduced angiogenic capacity and an increased risk of vascular disease. Hence attention has increasingly been paid to enhance mobilization and differentiation of EPCs for therapeutic purposes. A large body of evidence indicates that in Traditional Chinese Medicine (TCM) a plethora of herbs and herbal extracts are effective in the treatment of vascular diseases such as chronic wounds, diabetic retinopathy and rheumatoid arthritis. Thus, it seems rational to explore these medicinal plants as potential sources of novel angiomodulatory factors. In this thesis we demonstrated that treatment with TCM herbal extracts promote cell growth, cell migration, cell-matrix and capillary-like tube formation of BM-EPCs. Among these TCM extracts, Salidroside (SAL) and Icariin (ICAR) incubation increased VEGF and nitric oxide secretion, which in turn mediated the enhancement of angiogenic differentiation of BM-EPCs. A mechanic evaluation provided evidence that SAL stimulates the phosphorylation of Akt, mammalian target of rapamycin (mTOR) and ribosomal protein S6 kinase (p70S6K), as well as phosphorylated ERK1/2, which is associated with the cell migration and tube formation. Furthermore, a pilot in vivo study showed that SAL has the potential to enhance bone formation in a murine femoral critical-size bone defects model. Another new finding of the present study is that hydrogen peroxide (H2O2)-induced cytotoxicity is counteracted by TCM extracts. We found that SAL, Salvianolic acid B (SalB) and ICAR significantly abrogated H2O2-induced cell apoptosis, reduced the intracellular level of reactive oxygen species (ROS) and nicotinamide adenine dinucleotide phosphate-oxidase (NADPH) expression, and restored the mitochondrial membrane potential of BM-EPCs. Our data suggest that this protective effect of SalB is mediated by the activation of mTOR, p70S6K, 4EBP1, and by the suppression of MKK3/6-p38 MAPK-ATF2 and ERK1/2 signaling pathways after H2O2 stress. In addition, the investigation also demonstrates that ICAR owns the ability to inhibit apoptotic and autophagic programmed cell death via restoring the loss of mTOR and attenuation of ATF2 activity upon oxidative stress. Based on the outcomes of the present work, we propose SAL, SalB and ICAR as novel proanigiogenic and cytoprotective therapeutic agents with potential applications in the fields of systemic and site-specific tissue regeneration including ischaemic disease and extended musculoskeletal tissue defects.

There is convincing evidence in vivo that the vascular homing of endothelial progenitor cells (EPCs) contributes to rapid endothelial regeneration, which could prevent thrombosis and restenosis of cardiovascular devices. To enhance the EPC homing on cardiovascular devices, immobilization of an EPC capture agent (e.g. an anti-CD34 antibody) on the surface of cardiovascular devices is critical. We describe a way of immobilizing anti-CD34 Ab on 316L Stainless Steel (316L SS). For this, surface modification of 316L SS was performed via self-polymerization of dopamine (DA) and covalent grafting of staphylococcal protein A (SPA). On this coating the anti-CD34 Abs were oriented immobilized through their Fc constant region with SPA. In this process, the results of quartz crystal microbalance, X-ray photoelectron spectroscopy and water contact angle studies indicate that DA, SPA and anti-CD34 Ab were successfully immobilized onto the surface step by step. In vitro blood-compatibility tests confirmed that the modified surface induced less pro-coagulant fibrinogen denaturation, less platelet adhesion and lower activation of the adherent platelets. The affinity of EPCs for the modified surface has been demonstrated under flow conditions. This study provides potential applications for cardiovascular implant materials.

Efficient immobilization of the antibody to the substrate is of crucial importance in the development of anti-CD34-based endothelial progenitor cells capturing cardiovascular devices. This should go along with precise control of the antibody orientation by appropriate immobilization technology for retaining antibody activity, like in immunosensors. Recently, great attention was paid to immobilization of anti-CD34 antibody onto substrates by covalent binding, but at random orientation. Here, to investigate the biological effect of antibody orientation, we have prepared two kinds of anti-CD34 antibody coated surfaces, with random immobilization and oriented immobilization. The immunological binding activity (IBA) of the antibody at oriented immobilization was 3.48 times higher than at random immobilization, indicating that the two different surfaces were successfully prepared. The endothelial progenitor cell-capturing capability of oriented antibody-immobilized surface was 1.35 and 1.64 times higher than for the random immobilized surface after seeding for 2 and 12 h under flow condition, respectively. The endothelial progenitor cell-capturing efficiency per antibody by oriented immobilization was 5.16 and 6.26 times higher than for the random after seeding for 2 and 12 h under flow condition, respectively. In addition, the oriented antibody-immobilized surface possessed better blood-compatibility. These results clearly revealed the significance of antibody orientation which could retain its biological effect and may revolutionize the antibody-immobilization protocols used in cardiovascular and other bloodcontacting biomedical devices.

The principle of trained immunity represents innate immune memory due to sustained, mainly epigenetic, changes triggered by endogenous or exogenous stimuli in bone marrow (BM) progenitors (central trained immunity) and their innate immune cell progeny, thereby triggering elevated responsiveness against secondary stimuli. BM progenitors can respond to microbial and sterile signals, thereby possibly acquiring trained immunity-mediated long-lasting alterations that may shape the fate and function of their progeny, for example, neutrophils. Neutrophils, the most abundant innate immune cell population, are produced in the BM from committed progenitor cells in a process designated granulopoiesis. Neutrophils are the first responders against infectious or inflammatory challenges and have versatile functions in immunity. Together with other innate immune cells, neutrophils are effectors of peripheral trained immunity. However, given the short lifetime of neutrophils, their ability to acquire immunological memory may lie in the central training of their BM progenitors resulting in generation of reprogrammed, that is, “trained”, neutrophils. Although trained immunity may have beneficial effects in infection or cancer, it may also mediate detrimental outcomes in chronic inflammation. Here, we review the emerging research area of trained immunity with a particular emphasis on the role of neutrophils and granulopoiesis.

Endothelzell-basierte Therapien vermitteln regenerative Effekte hinsichtlich der Revaskularisierung von ischämischen Geweben. Doch ist die Verfügbarkeit von autologen Endothelzellen aufgrund einer krankheitsbedingt reduzierten Frequenz im peripheren Blut oder einer verminderten Integrität der endogenen Endothelzell-Populationen eingeschränkt. Hingegen ist es möglich, allogene endotheliale Vorläuferzellen aus der Nabelschnur in zelltherapeutisch relevanten Mengen zu isolieren. In der vorliegenden Arbeit wurden zunächst die Eigenschaften allogener humaner Nabelschnur (NS)-abgeleiteter sog. Endothelial Colony-Forming Cells (ECFCs) mit denen von venösen NS-abgeleiteten Endothelzellen verglichen. Aufgrund der nachgewiesenen Immunogenität von allogenen ECFCs wurde eine weiterführende Strategie zur Reduktion dieser immunogenen Eigenschaften durch die Koapplikation mit Mesenchymalen Vorläuferzellen (MSCs) verfolgt. Humane ECFCs wurden mit MSCs desselben Spenders kombiniert und in funktionellen in vitro- und in vivo-Assays untersucht. Dadurch konnte nachgewiesen werden, dass IFNγ-stimulierte ECFC/MSC-Kokulturen eine reduzierte Expression von HLA-Molekülen zeigen. Entsprechend induzierten spezifische CD8+ T-Zellen eine reduzierte Lyse der kokultivierten ECFCs und MSCs. Die Kokultur von ECFCs und MSCs mit allogenen Immunzellen führte zu einer nahezu vollständigen Inhibition der T-Zell-Proliferation. Um die reduzierte Immunogenität von ECFC und MSC in vivo zu verifizieren, wurden die Zellen in immundefiziente Mäuse injiziert, welche nachfolgend mit humanen PBMCs rekonstituiert wurden. So konnte nachgewiesen werden, dass die Koapplikation von ECFCs und MSCs nicht nur die Entstehung von stabilen Gefäßnetzwerken begünstigt, sondern zudem in den Transplantaten zu einer verringerten Immunzell-Infiltration führte. Die Koapplikation von ECFCs mit MSCs könnte daher eine klinische Nutzung dieser allogenen Quelle für die therapeutische Unterstützung der Vaskularisierung ermöglichen. / Endothelial cell-based therapies promote tissue regeneration and vascularization after ischemic damage. The availability of autologous endothelial progenitor cells is restricted in diseased patients, however therapeutically relevant numbers of allogeneic Endothelial Progenitor Cells can be isolated from an umbilical cord (UC). In the present study, the immunogenic properties of these Endothelial Colony Forming Cells (ECFCs) were first compared to human umbilical vein endothelial cells (HUVECs). Both cytokine-treated endothelial cells induced CD4+ and CD8+ T cell proliferation after coculture with allogeneic immune cells. So far, the potential interactions between ECFCs and Mesenchymal Stem/Progenitor Cells (MSCs) concerning their immunological features is poorly understood, but we hypothesize that MSCs might improve the immune compatibility and vessel building characteristics of ECFCs. Therefore, human UC-derived ECFC and MSC cocultures from the same donor were analyzed using various functional in vitro and in vivo assays. Stimulation of these cocultures with IFNγ caused strongly reduced expression levels of HLA-molecules compared to ECFC monocultures. The decreased molecular density on the cocultured ECFCs resulted in reduced cytotoxic CD8+ T cell-mediated lysis. Further, during IFNγ stimulation, the combination of ECFCs with MSCs prevented initiation of allogeneic T cell proliferation. To verify this concept in vivo, ECFCs and MSCs were co-transplanted in a humanized allograft mouse model in immunodeficient mice in order to effectively induce stable microvessels. These experiments demonstrate that when MSCs are co-applied with ECFCs, they not only support the formation of stable blood vessels, but also lead to fewer HLA-DR+ human vascular structures and fewer infiltrating human leukocytes. The data presented indicate that crosstalk between UC-derived ECFCs and MSCs might lower the risk of allogeneic ECFC rejection.

L’allogreffe des cellules souches hématopoïétiques (CSH) dans les situations d’incompatibilité HLA partielle représente une option thérapeutique irremplaçable pour des patients nécessitant une greffe de cellules souches hématopoïétiques, en absence d’un donneur HLA-identique. Toutefois, le retard de la restauration du système immunitaire en particulier dans du compartiment lymphocytaire après greffe est l'une des complications majeures. Une nouvelle stratégie pour promouvoir la reprise de la thymopoïèse à partir des CSH provenant du donneur et d'accélérer la reconstitution cellulaire T chez des patients après greffe de CSH consiste en le transfert adoptif des progéniteurs T générés in vitro. L’identification de Notch1 comme le régulateur-clé du développement lymphocytaire T a permis l’établissement de systèmes de culture à base de ligands de Notch, qui permettent la génération efficace de progéniteurs lymphoïdes T in vitro. L'efficacité des progeniteurs T-lymphoïdes murins pour promouvoir la reconstitution des lymphocytes T a été bien démontrée dans des modèles de greffe chez la souris. De même, des progéniteurs T-lymphopoïétiques humains générés in vitro et greffés aux souris humanisées favorisent la reprise de la thymopoïèse. Pourtant, aucune donnée n’a encore démontré leur capacité à donner naissance à un compartiment lymphocytaire T périphérique. De plus, les systèmes de co-culture à base de ligand de Notch actuellement utilisés consistent en des lignées stromales murines génétiquement modifiées. Afin d'établir un système cliniquement applicable, il est donc indispensable d’établir des systèmes de culture qui soutiennent la génération de progéniteurs T en absence d’un support des cellules nourricières. Au cours de mon projet de thèse, j'ai développé un nouveau système de culture pour la génération des progéniteurs T-lymphopoïétiques humains T basé sur l’immobilisation du ligand de Notch Delta-like-4 (DL-4) sous sa forme protéique. La culture des progéniteurs hématopoïétiques CD34+ issue de sang en présence de DL-4 immobilisé permet la génération d’un grand nombre de cellules ayant un phénotype de progéniteurs thymiques précoces (early thymic progenitor: ETP) et de prothymocytes (proT). Les cellules ETP et ProT ainsi générées expriment à des niveaux élevés des gènes impliqués dans le développement lymphocytaire précoce (i.e. pTa, Rag1, IL7Ra et BCL11b). Elles montrent des signes de réarrangement du récepteur des cellules T (TCR) similaires à leurs homologues thymiques. Par des expériences de dilution limite sur une co-culture OP9/DL-1 secondaire, j’ai pu montrer que les progéniteurs générés sur DL-4 possédaient un potentiel lymphoïde T très augmenté, qui pourrait être entièrement attribué aux sous populations ETP et ProT. Suite à leur transfert dans des souris NOD/SCID/γc-/-, les progéniteurs lymphoïde T générés par exposition a DL-4 sont capable de migrer dans le thymus, d’y poursuivre des étapes ultérieures de leur développement et d’accélérer la différentiation T intra thymique ainsi que l’émergence des lymphocytes T mature, polyclonaux et fonctionnels en périphérie. Dans une approche de co-transplantation, qui se rapproche des conditions cliniques envisagées, j’ai simultanément injecté dans le même récipient des progéniteurs générées sur DL-4 et des cellules CD34+ non traitées (d’un 2èm donneur HLA-incompatible). Cette procédure a permis une reconstitution des lymphocytes T encore plus rapide et plus. Etant donné que les progéniteurs T générées sur DL-4 et les cellules CD34+ non-traitées étaient issue de deux donneurs avec un HLA différent, cette expérience a permis de montrer que les progéniteurs préalablement exposés à DL-4 reconstituaient spécifiquement les compartiments lymphoïdes T alors que les autres lignées hématopoïétiques provenaient des progéniteurs CD34+ non-traités... / Human leukocyte antigen (HLA)-mismatched haematopoietic stem cell transplantation (HSCT) represents an important therapeutic option for patients lacking suitable donors. Delayed posttransplant immune recovery constitutes one of its major complications and is most pronounced in the T cellular compartment. A novel strategy to promote de novo thymopoiesis from donor derived HSCs and to accelerate T cellular reconstitution in patients after HSCT consists in the adoptive transfer of in vitro generated T cell progenitor cells. Identification of Notch1 as the key regulator of early T-lineage development has allowed the generation of Notch ligand-based culture systems, which provide a powerful tool to generate T-lymphoid progenitors in vitro. The efficacy of murine T-lymphoid progenitors to promote T cell reconstitution has been well demonstrated in conventional mouse models. In consistency, in vitro-generated human T cell progenitors were demonstrated to promote thymic recovery in humanized mice. Yet, positive effects of in vitro generated human T cell precursors on peripheral T cell reconstitution have not been demonstrated. Moreover currently used Notch-based co-culture systems consist of genetically modified murine cell lines. With view to establishing a clinically applicable system, feeder-cell-free Notch-ligand culture systems for the generation of T-lymphopoietic progenitors are warranted. During my PhD project I developed a new culture system based on the immobilized Notch ligand Delta-like-4 (DL-4). Exposure of human CD34+ cord blood cells to immobilized DL-4 enabled the in vitro generation of high number of T cell progenitors, which harboured the phenotype of immature early thymic progenitor cells (ETP) and prothymocytes (proT). ETP and proT cell generated during DL-4 culture upregulated essential genes involved in early T-lymphoid development (i.e. IL7Rα, PTα, RAG1 and BCL11b) and had undergone stage-specific recombination of the T cell receptor (TCR) locus in a similar way as in native human thymopoiesis. In limiting dilution analysis after secondary OP9/DL-1 co-culture, DL-4 progenitors displayed a highly increased T-lymphoid potential, which could be entirely attributed to the ETP and proT subset. When transferred into NOD/SCID/γc-/- mice, DL-4 primed T cell progenitors migrated to the thymus and accelerated intrathymic T cell differentiation and emergence of functional, mature and polyclonal αβ T cells in the periphery. In a co-transplantation approach, which more closely mimics a clinical setting, DL-4 progenitors and untreated CD34+ cells from HLA-disparate donors were simultaneously injected in the same recipient. This procedure allowed even more rapid and more robust T cell reconstitution. HLA-tracking of the distinct graft sources further showed, that DL-4 progenitors specifically reconstituted the T-lymphoid compartments. This work provides further evidence for the ability of in vitro-generated human T cell progenitors to promote de novo thymopoiesis and shows for the first time, that these cells accelerate peripheral T cell reconstitution in humanized mice. The availability of the efficient feeder-cell-free DL-4 culture technique represents an important step towards the future clinical exploitation translation of in vitro generated T-lymphoid progenitor cells to improve posttransplant immune reconstitution / Die Wiederherstellung der T-lymphozytären Immunität nach T-Zell depletierter hämatopoetischer Stammzelltransplantation (HSZT) ist ein langwieriger Prozess. Eine potentielle Strategie zur Beschleunigung der Neubildung von T-Zellen aus den transplantierten Stammzellen besteht in der Gabe von T-lymphozytären Vorläuferzellen. Die Entdeckung von Notch1 als wichtigster Regulator der frühen T-Zell-Entwicklung hat zur Etablierung Notchligand-basierter Zellkulturen geführt, mit deren Hilfe T-lymphoide Vorläuferzellen aus hämatopoetischen Stammzellen in vitro gebildet werden können. Das therapeutische Potential dieses Zelltyps wurde eindrucksvoll in konventionellen, syngenen und allogenen Maustransplantationsmodellen belegt, in denen nach Injektion in vitro generierter, muriner T-Vorläuferzellen eine Verbesserung der Neubesiedlung des Thymus sowie eine beschleunigte Wiederherstellung der T-zellulären Immunität erreicht werden konnte. Notchbasierte Co-Kultursysteme wurden ebenfalls für die invitro Herstellung humaner T-lymphoider Vorläuferzellen verwendet. Das in-vivo Potential humaner T Vorläuferzellen ist bislang jedoch nur lückenhaft charakterisiert: Zwar konnte gezeigt werden, dass humane T-Vorläuferzellen den hypoplastischen Thymus von immundefizienten NOD/SCID/γc-/- Mäusen besiedeln können. Ihre Wirksamkeit, die Wiederherstellung eines funktionellen, peripheren T-Zellkompartiments zu beschleunigen, gelang bislang jedoch nicht. Darüber hinaus werden Notchliganden in derzeit verwendeten Kultursystemen von genetisch modifizierten, murinen Stromazellen präsentiert. Die Entwicklung stromazellfreier, proteinbasierter Notchligand-Kultursysteme ist daher von grosser Bedeutung für eine mögliche therapeutische Nutzung in vitro generierter T-Vorläuferzellen. Durch Immobilisierung von Notchligand Delta-like 4 (DL-4) habe ich im Rahmen meines PhD Projekts ein stromazellfreies Kultursystem zur Züchtung T-zellulärer Vorläuferzellen aus humanen CD34+ Nabelschnurblutzellen etabliert. In DL-4 Kultur generierte Zellen besitzen phänotypische und molekulare Eigenschaften von frühen thymischen Vorläuferzellen (ETP) und Prothymocyten (proT). ETP und proT Zellen aus DL-4 Kulturen exprimieren wesentliche Geneder frühen T-Zellentstehung (z.B. IL7Ra, PTa, RAG1 und BCL11b). Die entwicklungsstadiumspezifischen TCR-Rekombinationsprozesse in DL-4 Zellen erfolgen nach dem gleichen Muster wie in der nativen Thymusentstehung. Die in DL4 Kultur generierten T-Vorläuferzellen können sich in reife T-Zellen weiterentwickeln und durchlaufen die weitere T-Zelldifferenzierung innerhalb kürzerer Zeit als native CD34+ hämatopoetische Vorläuferzellen. 13 Darüber hinaus können DL-4 generierte T-Vorläuferzellen nach Xenotransplantation den hypoplastischen Thymus von immundefizienten NOD/SCID/γc-/- Mäusen besiedeln, intrathymische T-Zellentwicklung begünstigen und die Neubildung reifer und funktionaler TZellen in der Peripherie beschleunigen. Zur Simulation einer klinischen Anwendung führte ich weiterhin Co-Transplantationen mit DL-4 Vorläuferzellen und unbehandelten CD34+ Zellen in gleiche Empfänger durch und konnte hiermit eine weitere Verbesserung der Immunrekonstitution erzielen. Durch Verwendung HLA-divergenter Spender in diesen Versuchen konnte ich zeigen, dass DL-4 Zellen sich vornehmlich in T-Zellen weiterentwickelten, während die restlichen Blutzellreihen von unbehandelten CD34-poitiven Zellen gebildet wurden. Im Rahmen dieses Projekts konnte ich mit einem für die klinische Anwendung geeigneten Kulturmodell wichtige präklinische Belege für das therapeutische Potential in vitro generierter TVorläuferzellen erbringen. Diese Arbeit bildet somit eine wichtige Grundlage für eine zukünftige klinische Anwendung von T-Vorläuferzellen zur Verbesserung der T-Zell-Immunität nach HSZT