Establishment and maintenance of the blood system relies on the cellular and spatial organization of bone marrow. In the BM niche, sinusoidal endothelial cells (SECs) are mainly located in the trabecular zone of the metaphysis area of long bones. SECs present a fenestrated structure characterized by high permeability, low shear rates, and oxygen pressure. The hypoxic environment surrounding SECs is needed for the movement and engraftment of hematopoietic cells, but it might also facilitate the homing of malignant cells. SECs’ adaptive response to hypoxia depends on the Hypoxia Inducible Factors (HIFs) promoting angiogenesis, hematopoiesis, and other processes. The upstream regulator of HIFs, the prolyl hydroxylase domain-2 (PHD2) is considered the key cellular oxygen sensor. Our group has shown the crucial regulatory effects that PHD2 has on different physiological and pathological settings. During steady state, PHD2 modulates proliferation and mobilization of hematopoietic progenitor cells (HSCPs), as well as bone metabolism. On the other hand, PHD2 is crucial for neutrophil motility affecting their extravasation during arthritis. We have also demonstrated that loss of PHD2 in different humans and mouse tumor cell lines, as well as in myeloid cells and T-lymphocytes where it impairs tumor development. Others have shown that heterozygous deletion of PHD2 in tumor ECs can reduce distant metastasis. Due to the intricate interplay between the different players of the PHD2/HIFs axis, as well as their effect on other signaling pathways, the positive or negative impact of the hypoxia pathways has been shown to be cell type and context dependent. In the present study, we analyzed the role of hypoxia pathway proteins, mainly PHD2, in ECs in the bone/BM niche, as well as its consequent influence on the environment during physiological and pathological scenarios. We have demonstrated that endothelial PHD2 has a profound intrinsic effect on vessel morphology and functionality, affecting the crucial communication between ECs and the bone/BM niche. Further, using different transgenic mouse lines, we have identified the BM endothelial cells (BM-ECs) PHD2/HIF-2α axis directly increasing leukocytosis via vascular cell adhesion protein 1 (VCAM-1) protein downregulation. Moreover, during steady state conditions we have discovered a novel regulatory effect that PHD2 exerts on VCAM-1 by increasing miR-126-3p transcription, a well-known inhibitor of VCAM-1 expression. Lastly, the PHD2/HIF-2/miR-126-3p/VCAM-1 axis not only influenced the myeloid cell intravasation towards circulation but also increased the extravasation and homing of metastatic breast carcinoma cells into the bone/BM tissue, increasing tumor burden throughout the bone. BM-ECs PHD2 offers a protective role against tumor homing cells in the bone/BM while serving as an important regulator in the communication between endothelium and BM niche. Concluding Remarks:  Loss of PHD2 in ECs generates profound changes in vessel function and in the hematopoietic compartment leading to increase myelopoiesis resulting in leukocytosis. The above-mentioned effect occurs in a HIF-2α-dependent manner.  PHD2/HIF-2α also transcribed in an increase of miR-126-3p expression leading to VCAM-1 downregulation. Process that resulted in increased leukocytes in circulation due to hematopoiesis dysregulation. The novel PHD2/HIF-2/miR-126-3p/VCAM-1 axis enhanced extravasation and homing of metastatic breast carcinoma cells into the bone/BM tissue, increasing tumor burden throughout the bone.:Introduction 8 The Endothelium 9 Endothelial barrier. 9 Leukocyte transendothelial migration. 11 Intracellular mechanism for activation of adhesion molecules. 12 Cellular migration in pathological conditions. 13 Tumor Dissemination: Metastasis. 14 Metastatic cascade. 15 The bone is a preferential site for malignant cells arrival. 16 Bone/BM physiology influences metastasis. 17 The endosteal niche. 18 The perivascular niche. 19 Hypoxia pathway proteins impact on metastasis. 20 Thesis Aims 23 Aim 1: Characterize the role of the hypoxia pathway proteins in bone vasculature and the impact on the niche. 24 Aim 2: Analyze the genetic changes that resulted from PHD2 deletion in BM-ECs and their impact on the cross-talk communication with the different BM-niches. 24 Aim 3: Investigate the impact of PHD2 and downstream regulators (HIF-1/2α) on vessel functionality. 25 Materials and Methods 26 Mice. 27 Histology: tissue processing and immunofluorescence staining 28 Bone tissue processing. 28 Staining of Bone cryosections. 28 Induced skin inflammatory model. 29 Staining of ears treated with PMA. 29 Vascular morphology quantification. 29 Microscopy 31 Antibodies used for immunofluorescence. 31 Bone analysis. 31 Bone µCT measurements and analysis 31 Tartrate-resistant acid phosphatase (TRAP) staining 32 Blood and BM analysis. 32 Sysmex. 32 Flow cytometry. 32 BM cell extraction from bones. 32 BM cells staining. 33 Meso Scale Discovery (MSD) 35 Evans Blue assay. 35 BM soup ELISA. 35 RNAseq of CD31+ EMCN+ BM-ECs 36 BM-EC cell sorting for RNAseq. 36 RNA extraction and qPCRs 36 Tumor model. 36 Tumor breast carcinoma cells. 36 Tumor homing model. 37 Statistical analyses. 37 Results 38 PHD2 Conditional Knockout from Endothelial Cell Compartment. 39 Further P2EC mice characterization. 41 Transgenic deletion of PHD2 showed slight developmental retardation. 41 Spleen size showed not to be affected by deletion endothelial PHD2. 41 P2EC mice displayed increased vessel leakiness. 42 Endothelial PHD2 deletion does not affect lung endothelial cells. 43 BM-ECs PHD2-HIF-2α axis modulates leukocytosis and vessel morphology. 44 HIF-2α modulates P2EC leukocytosis and thrombocytopenia. 44 BM-ECs PHD2 deficient mice hinder vessel morphology in a HIF-2α dependent manner. 44 Endothelial PHD2-deficient mice exhibit perturbed hematopoiesis. 45 P2EC mice early progenitor displayed reduced total cell number, but frequency remained unchanged. 46 P2EC mice favor differentiation of committed progenitors with a myeloid bias. 48 P2EC mice significantly reduced the numbers and frequency of megakaryocyte/erythroid progenitor’s linage. 48 PHD2-HIF-2α deletion restored normal hematopoiesis. 50 P2EC vascular functionality during pathological conditions. 53 Endothelial PHD2 modulates leukocyte migration during localized inflammation. 53 Endothelial PHD2 shapes bone/BM tumor homing. 55 Tumor homing in the bone: generation of an early metastatic model. 56 Early metastasis limitation. 58 Endothelial PHD2 modulates tumor colonization to the bone/BM. 59 Simultaneous deletion of PHD2 and HIF-1 in BM-ECs worsen tumor metastasis to bone. 61 Simultaneous deletion of PHD2 and HIF-2 in BM-ECs showed no differences in tumor homing. 62 Deep sequencing of PHD2 deficient BM-ECs. 63 BM EC from P2EC mice display enriched leukocyte migration gene signatures. 63 P2EC mice presented genetic dysfunction in the integrin-binding system. 64 P2EC steady-state VCAM-1 expression is HIF-2α dependent. 66 BM-ECs VCAM-1 + is regulated by PHD2 through HIF-2α. 66 PHD2-dependent downregulation of VCAM-1 does not affect VE-cadherin expression. 68 BM pro-inflammatory cytokines do not contribute to VCAM-1 lower expression. 69 During steady-state, loss of VCAM-1 increased frequency BM resident mature cells. 69 BM-ECs VCAM-1 deficient mice 71 VCAM1EC mice developed leukocytosis. 71 VCAM1EC does not exhibit significant changes in hematopoiesis. 73 P2EC vessel morphology is independent of downregulation of VCAM-1 74 VCAMEC mice showed increased tumor homing in the diaphysis. 75 PHD2-HIF-2 regulatory effect on VCAM-1 is modulated by mir-126-3p. 76 HIF-2α regulates mir-126 expression in PHD2 deficient BM-ECs. 77 BM-ECs PHD2 influence bone homeostasis. 78 Loss of BM-ECs PHD2 lead to increase Osteoclast numbers and activity. 79 Osteoclast differentiation and activity could be independent of OBs. 79 Loss of PHD2 in BM-ECs leads to osteoclastogenesis. 80 BM resident Tcell CD8+ could be Increasing Osteoclast Activation. 82 Discussion. 83 Mouse Model: Conditional Deletion of Endothelial PHD2. 85 Endothelial PHD2 Modulates Myelopoiesis. 86 BM-EC PHD2 regulates vessel morphology and functionality under steady-state independent of VCAM-1. 88 Endothelial VCAM-1 downregulation does not impaired neutrophil migration during inflammation. 88 BM-ECs PHD2 is a Gatekeeper of Tumor Homing in the Bone. 89 HIF-2α dependent Mir-126 activation leads to VCAM-1 downregulation 91 Endothelial PHD2 controls Osteoclastogenesis independent of BM RANKL. 94 References 96 List of Abbreviations 107 Summary 109 Zusammenfassung 111 Acknowledgements 113 Deklaration 114 Appendix 118 List of Figures. 118 List of tables 119 / Die Organisation und Aufrechterhaltung des Blutsystems hängt von der zellulären und räumlichen Organisation des Knochenmarks ab. In der BM-Nische befinden sich, hauptsächlich in der Trabekelzone des Metaphysenbereichs langer Knochen, die sinusoidale Endothelzellen (SECs). SECs weisen eine gefensterte Struktur auf, die von hoher Durchlässigkeit, geringer Scherrate und Sauerstoffdruck geprägt ist. Das hypoxische Milieu der SECs ist notwendig für Bewegung und Einwanderung der hämatopoetischen Zellen und könnte dies ebenso für bösartige Zellen begünstigen. Die Anpassung der SECs an Hypoxie hängt von den Hypoxia Inducible Factors (HIFs) ab. HIFs fördern die Angiogenese, die Hämatopoese und andere Prozesse. Die prolyl hydorxylase domain-2 (PHD2) ist der vorgeschaltete Regulator der HIFs und gilt als wichtigster zellulärer Sauerstoffsensor. Unsere Gruppe konnte zeigen welche zentralen regulatorischen Effekte die PHD2 auf verschiedene physiologische und pathophysiologische Mechanismen ausübt. Im Gleichgewichtszustand moduliert PHD2 Proliferation und Mobilisation der hämatopoetischen Vorläuferzellen (HSCPs) sowie den Knochenmetabolismus. Auf der einen Seite spielt PHD2 eine entscheidende Rolle bei der Motilität der Neutrophilen und beeinflusst daher die Extravasation bei Arthritis. Wir konnten außerdem zeigen, dass der Verlust von PHD2 in verschiedenen humanen und murinen Tumorzelllinien, sowie in myeloischen Zellen und T-Lymphozyten die Tumorentwicklung beeinträchtigt. In anderen Arbeiten wurde gezeigt, dass eine heterozygote PHD2-Deletion in Tumor-ECs eine Fernmetastasierung reduziert. In Anbetracht der komplizierten Wechselwirkung zwischen den verschiedenen Komponenten der PHD2/HIF-Signalkaskade sowie deren Effekte auf andere Signalkaskaden, übt sich in Abhängigkeit des Zelltyps und des Kontexts ein positiver oder negativer Einfluss auf die Hypoxie-Signalwege aus. In der vorliegenden Studie haben wir die Rolle von Proteinen des Hypoxiewegs, hauptsächlich PHD2, in den ECs der Knochen-/BM-Nische sowie deren daraus resultierenden Einfluss auf die Umwelt in physiologischen und pathologischen Szenarien analysiert. Wir konnten zeigen, dass das endotheliale PHD2 einen tiefgreifenden intrinsischen Effekt auf die Gefäßmorphologie und Funktionalität besitzt und damit entscheidend die Kommunikation zwischen ECs und der Knochen-/BM-Nische beeinflusst. Weiterhin konnte unter Nutzung verschiedener transgener Muslinien identifiziert werden, dass die Knochenmarksendothelzellen (BM-ECs) PHD2/HIF-2α-Achse direkt die Myelopoese, durch eine Herabsetzung der vascular cell adhesion protein 1 (VCAM-1) -Expression, steigert. Darüber hinaus haben wir einen neuartigen regulatorischen Effekt der PHD2 auf das VCAM-1 entdeckt. Hierbei wird die Expression des VCAM-1 Inhibitors miR-126-3p gesteigert. Des Weiteren beeinflusst die PHD2/HIF-2/miR-126-3p/VCAM-1 Achse nicht nur die Intravasation der Myloidzellen in Richtung des Kreislaufs, sondern auch eine Steigerung der Extravastion und Einwanderung von metastasierenden Brustkarzinomzellen in die Knochen/BM-Gewebe und steigert somit die Tumorlast in den Knochen. In Anbetracht klinischer Versuche der Krebsbehandlung mit PHD2-Inhibitoren, bietet BM-ECs PHD2 einen schützenden Effekt gegen Tumorzelleinwanderung in die Knochen/BM, während es gleichzeitig als ein wichtiger Regulator in der Kommunikation zwischen Endothelium und der BM-Nische dient. Abschließende Bemerkungen: Der Verlust von PHD2 in ECs führt zu tiefgreifenden Veränderungen in der Gefäßfunktion und im hämatopoetischen Kompartiment, was zu einer verstärkten Myelopoese und damit zu Leukozytose führt. Die oben erwähnte Wirkung tritt in einer HIF-2α-abhängigen Weise auf. PHD2/HIF-2α führte auch zu einem Anstieg der miR-126-3p-Expression, was zu einer Herunterregulierung von VCAM-1 führte. Dieser Prozess führte zu einer erhöhten Anzahl von Leukozyten im Blutkreislauf aufgrund einer Dysregulation der Hämatopoese. Die neuartige PHD2/HIF-2/miR-126-3p/VCAM-1-Achse förderte die Extravasation und Ansiedlung von metastatischen Brustkrebszellen im Knochen/BM-Gewebe, wodurch die Tumorlast im gesamten Knochen erhöht wurde.:Introduction 8 The Endothelium 9 Endothelial barrier. 9 Leukocyte transendothelial migration. 11 Intracellular mechanism for activation of adhesion molecules. 12 Cellular migration in pathological conditions. 13 Tumor Dissemination: Metastasis. 14 Metastatic cascade. 15 The bone is a preferential site for malignant cells arrival. 16 Bone/BM physiology influences metastasis. 17 The endosteal niche. 18 The perivascular niche. 19 Hypoxia pathway proteins impact on metastasis. 20 Thesis Aims 23 Aim 1: Characterize the role of the hypoxia pathway proteins in bone vasculature and the impact on the niche. 24 Aim 2: Analyze the genetic changes that resulted from PHD2 deletion in BM-ECs and their impact on the cross-talk communication with the different BM-niches. 24 Aim 3: Investigate the impact of PHD2 and downstream regulators (HIF-1/2α) on vessel functionality. 25 Materials and Methods 26 Mice. 27 Histology: tissue processing and immunofluorescence staining 28 Bone tissue processing. 28 Staining of Bone cryosections. 28 Induced skin inflammatory model. 29 Staining of ears treated with PMA. 29 Vascular morphology quantification. 29 Microscopy 31 Antibodies used for immunofluorescence. 31 Bone analysis. 31 Bone µCT measurements and analysis 31 Tartrate-resistant acid phosphatase (TRAP) staining 32 Blood and BM analysis. 32 Sysmex. 32 Flow cytometry. 32 BM cell extraction from bones. 32 BM cells staining. 33 Meso Scale Discovery (MSD) 35 Evans Blue assay. 35 BM soup ELISA. 35 RNAseq of CD31+ EMCN+ BM-ECs 36 BM-EC cell sorting for RNAseq. 36 RNA extraction and qPCRs 36 Tumor model. 36 Tumor breast carcinoma cells. 36 Tumor homing model. 37 Statistical analyses. 37 Results 38 PHD2 Conditional Knockout from Endothelial Cell Compartment. 39 Further P2EC mice characterization. 41 Transgenic deletion of PHD2 showed slight developmental retardation. 41 Spleen size showed not to be affected by deletion endothelial PHD2. 41 P2EC mice displayed increased vessel leakiness. 42 Endothelial PHD2 deletion does not affect lung endothelial cells. 43 BM-ECs PHD2-HIF-2α axis modulates leukocytosis and vessel morphology. 44 HIF-2α modulates P2EC leukocytosis and thrombocytopenia. 44 BM-ECs PHD2 deficient mice hinder vessel morphology in a HIF-2α dependent manner. 44 Endothelial PHD2-deficient mice exhibit perturbed hematopoiesis. 45 P2EC mice early progenitor displayed reduced total cell number, but frequency remained unchanged. 46 P2EC mice favor differentiation of committed progenitors with a myeloid bias. 48 P2EC mice significantly reduced the numbers and frequency of megakaryocyte/erythroid progenitor’s linage. 48 PHD2-HIF-2α deletion restored normal hematopoiesis. 50 P2EC vascular functionality during pathological conditions. 53 Endothelial PHD2 modulates leukocyte migration during localized inflammation. 53 Endothelial PHD2 shapes bone/BM tumor homing. 55 Tumor homing in the bone: generation of an early metastatic model. 56 Early metastasis limitation. 58 Endothelial PHD2 modulates tumor colonization to the bone/BM. 59 Simultaneous deletion of PHD2 and HIF-1 in BM-ECs worsen tumor metastasis to bone. 61 Simultaneous deletion of PHD2 and HIF-2 in BM-ECs showed no differences in tumor homing. 62 Deep sequencing of PHD2 deficient BM-ECs. 63 BM EC from P2EC mice display enriched leukocyte migration gene signatures. 63 P2EC mice presented genetic dysfunction in the integrin-binding system. 64 P2EC steady-state VCAM-1 expression is HIF-2α dependent. 66 BM-ECs VCAM-1 + is regulated by PHD2 through HIF-2α. 66 PHD2-dependent downregulation of VCAM-1 does not affect VE-cadherin expression. 68 BM pro-inflammatory cytokines do not contribute to VCAM-1 lower expression. 69 During steady-state, loss of VCAM-1 increased frequency BM resident mature cells. 69 BM-ECs VCAM-1 deficient mice 71 VCAM1EC mice developed leukocytosis. 71 VCAM1EC does not exhibit significant changes in hematopoiesis. 73 P2EC vessel morphology is independent of downregulation of VCAM-1 74 VCAMEC mice showed increased tumor homing in the diaphysis. 75 PHD2-HIF-2 regulatory effect on VCAM-1 is modulated by mir-126-3p. 76 HIF-2α regulates mir-126 expression in PHD2 deficient BM-ECs. 77 BM-ECs PHD2 influence bone homeostasis. 78 Loss of BM-ECs PHD2 lead to increase Osteoclast numbers and activity. 79 Osteoclast differentiation and activity could be independent of OBs. 79 Loss of PHD2 in BM-ECs leads to osteoclastogenesis. 80 BM resident Tcell CD8+ could be Increasing Osteoclast Activation. 82 Discussion. 83 Mouse Model: Conditional Deletion of Endothelial PHD2. 85 Endothelial PHD2 Modulates Myelopoiesis. 86 BM-EC PHD2 regulates vessel morphology and functionality under steady-state independent of VCAM-1. 88 Endothelial VCAM-1 downregulation does not impaired neutrophil migration during inflammation. 88 BM-ECs PHD2 is a Gatekeeper of Tumor Homing in the Bone. 89 HIF-2α dependent Mir-126 activation leads to VCAM-1 downregulation 91 Endothelial PHD2 controls Osteoclastogenesis independent of BM RANKL. 94 References 96 List of Abbreviations 107 Summary 109 Zusammenfassung 111 Acknowledgements 113 Deklaration 114 Appendix 118 List of Figures. 118 List of tables 119

Das Knochenmark (BM) beherbergt wesentliche Komponenten des adaptiven Immunsystems, die einen langfristigen Schutz gegen wiederkehrende Pathogene vermitteln können, sodass es sich als Reservoir für ein immunologisches Gedächtnis qualifiziert. Neben langlebiger Antikörper-produzierender Plasmazellen bleiben auch Antigen (Ag)-spezifische CD8+ und CD4+ T-Gedächtniszellen dauerhaft im Knochenmark erhalten, auch wenn sie in den sekundären lymphoiden Organen (SLOs) und im Blut abwesend sind. Es wird angenommen, dass diese T-Gedächtniszellen bei erneutem Kontakt mit den gleichen systemischen Pathogenen schnell reagieren können. Allerdings sind die biologischen Mechanismen für ihre langfristige Aufrechterhaltung immer noch umstritten und demnach ungeklärt. Unklar ist auch, wie die T-Gedächtniszellen des Knochenmarks bei erneuter Konfrontation mit demselben Antigen reagieren. Hier wird dieser Frage begegnet, indem durch klassiche Immunisierung mit definieren Antigenen eine stabile Population Ag-spezifischer CD8+ und CD4+ T-Gedächtniszellen im Knochenmark erzeugt wird. / The bone marrow (BM) harbors critical components of the adaptive immune system being able to provide long-lasting protection against previously encountered pathogens, thus qualifying as a reservoir of immunological memory. In addition to long-lived antibody producing plasma cells, antigen (Ag)-specific CD8+ and CD4+ memory T lymphocytes are maintained long-term in the BM even when they are absent from secondary lymphoid organs (SLOs) and blood. Those memory T cells are thought to respond fast upon re-encounter of systemic pathogens. However, the biological mechanisms behind their long-term maintenance in the BM are still a matter of debate and thus remain unclear. Similarly, it is also unclear how the memory T cells of the BM react to antigenic re-challenge. Here we address these issues by generating a stable pool of Ag-specific CD8+ and CD4+ memory T lymphocytes in the BM by classical immunizations with defined antigens.

Background: Multipotent mesenchymal stromal cells (MSC) can be recovered from a variety of tissues in the body. Yet, their functional properties were shown to vary depending on tissue origin. While MSC have emerged as a favoured cell type for tendon regenerative therapies, very little is known about the influence of the MSC source on their properties relevant to tendon regeneration. The aim of this study was to assess and compare the expression of tendon extracellular matrix proteins and tendon differentiation markers in MSC derived from different sources as well as in native tendon tissue. MSC isolated from equine bone marrow, adipose tissue, umbilical cord tissue, umbilical cord blood and tendon tissue were characterized and then subjected to mRNA analysis by real-time polymerase chain reaction. Results: MSC derived from adipose tissue displayed the highest expression of collagen 1A2, collagen 3A1 and decorin compared to MSC from all other sources and native tendon tissue (p < 0.01). Tenascin-C and scleraxis expressions were highest in MSC derived from cord blood compared to MSC derived from other sources, though both tenascin-C and scleraxis were expressed at significantly lower levels in all MSC compared to native tendon tissue (p < 0.01). Conclusions: These findings demonstrate that the MSC source impacts the cell properties relevant to tendon regeneration. Adipose derived MSC might be superior regarding their potential to positively influence tendon matrix reorganization.

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.

This data article contains data related to the research article entitled, 'in vitro characterization of bone marrow stromal cells from osteoarthritic donors' [1]. Osteoarthritis (OA) represents the main indication for total joint arthroplasty and is one of the most frequent degenerative joint disorders. However, the exact etiology of OA remains unknown. Bone marrow stromal cells (BMSCs) can be easily isolated from bone marrow aspirates and provide an excellent source of progenitor cells. The data shows the identification of pivotal genes and pathways involved in osteoarthritis by comparing gene expression patterns of BMSCs from osteoarthritic versus healthy donors using an array-based approach.

Knochenmarks-Stromazellen sind in den letzten Jahren in den Fokus der Forschung gerückt. Es konnte gezeigt werden, dass sie durch Bereitstellung von Überlebenssignalen essenziell für die Erhaltung hämatopoetischer Nischen sind. Stromales Interleukin-7 (IL-7) konnte dabei für T Zellen als Überlebenssignal identifiziert werden. Gemeinsam ist allen Stromazellen die Expression des Oberflächenmarkers CD106/VCAM-1. Ein effizientes Protokoll erlaubte die qualitative wie quantitative Isolation von Stromazellen aus dem murinen Knochenmark mit anschließender ex vivo Microarray-Analyse. Die auf diese Weise ermittelten Kandidaten-Marker wurden auf Proteinebene via Histologie und (Hochdurchsatz-) Durchflusszytometrie validier. Dazu gehören z.B. die Marker CD1d, gas6 oder ANXA2R. CD1d wurde als guter Interimsmarker für VCAM-1+PECAM-1- Stromazellen identifiziert, wohingegen die IL-7-Produzenten in der Population von CD200int/BP 1+/CD73+/CD105- angereichert sind. Gleiches gilt für den Transkriptionsfaktor Prrx1. CD55, BP-1 and Cadherin-11 zeigten eine Expressionsmuster in Abhängigkeit des verwendeten IL-7-Reportermaus-Haplotyps. Für BP-1 und Cadherin 11 konnte die Abwesenheit von reifen Lymphozyten als Ursache des Feedbacks ausgeschlossen werden. Die Haplotypen der Reportermaus legten auch eine monoallele Expression des IL-7 nahe. Die Ergebnisse dieser Arbeit zeigen VCAM-1+ (IL-7+/-) Stromazellen als heterogene Population, wenn es nach der Vielzahl der möglichen exprimierten Marker geht. Zwischen vielen dieser Marker gibt es aber wiederum auf Zelloberflächenebene einen großen Überlapp. Die funktionelle Relevanz dieser Oberflächenmarker-Diversität wird in weiteren Arbeiten zu klären sein, gibt aber den Stromazellen ein breites Repertoire vor, um Interaktionen mit Lymphozyten zu initiieren, modulieren und inhibieren. Abschließend ist zu erwarten, dass diese Erkenntnisse in die klinische Behandlung der Stroma-Nischen in Autoimmun-Fragestellungen einfließen. / Bone marrow stromal cells receive increasing amounts of attention lately. They have been shown to support survival of hematopoietic stem cells as well as memory lymphocytes which is of great importance when targeting the perseverance of autoimmune diseases. CD4+ memory T lymphocytes reside in the proximity of VCAM-1 expressing stromal cells which provide them with survival signals such as Interleukin-7. Herein, a protocol was developed to quantitatively obtain VCAM-1+ and VCAM-1+ IL-7+/- stromal cells via enzymatic/mechanic digestion and cytoskeleton-inhibition. Ex vivo gene expression analysis was performed from sorted, pure cells with good recovery. Candidate genes/markers were validated in (high-throughput) flow cytometry and histological analysis including subsequent semi-automated colocalization was performed. CD1d was found to be good surrogate marker for VCAM-1+PECAM-1- non-endothelial stroma while the population of CD200int/BP-1+/CD73+/CD105- stromal cells is greatly enriched in IL-7 producers which was equally true for the stromal transcription factor Prrx1. CD55, BP-1 and Cadherin-11 were found to be differentially expressed in differing IL-7 reporter mice haplotypes. The reporter mice haplotypes revealed monoallelic expression features of IL-7. All methodologies suggest that VCAM-1+ as well as IL-7+/- stromal cells are heterogeneous by marker expression yet don’t cluster extensively in flow cytometry co-stains. The functional relevance of the marker diversity described in this thesis remains to be tested but insinuates a broad repertoire for bone marrow stroma cells for new interaction pathways with lymphocyte subsets. Ultimately, this knowledge will hopefully feedback to clinical questions of autoimmunity for targeted treatment of stromal niches.

Background: The beneficial impact of mesenchymal stem cells (MSC) on both acute and chronic liver diseases has been confirmed, although the molecular mechanisms behind it remain elusive. We aim to identify factors secreted by undifferentiated and hepatocytic differentiated MSC in vitro in order to delineate liver repair pathways potentially targeted by MSC. Methods: Secreted factors were determined by protein arrays and related pathways identified by biomathematical analyses. Results: MSC from adipose tissue and bone marrow expressed a similar pattern of surface markers. After hepatocytic differentiation, CD54 (intercellular adhesion molecule 1, ICAM-1) increased and CD166 (activated leukocyte cell adhesion molecule, ALCAM) decreased. MSC secreted different factors before and after differentiation. These comprised cytokines involved in innate immunity and growth factors regulating liver regeneration. Pathway analysis revealed cytokine-cytokine receptor interactions, chemokine signalling pathways, the complement and coagulation cascades as well as the Januskinase-signal transducers and activators of transcription (JAK-STAT) and nucleotide-binding oligomerization domain-like receptor (NOD-like receptor) signalling pathways as relevant networks. Relationships to transforming growth factor beta(TGF-beta) and hypoxia-inducible factor 1-alpha (HIF1-alpha) signalling seemed also relevant. Conclusion: MSC secreted proteins, which differed depending on cell source and degree of differentiation. The factors might address inflammatory and growth factor pathways as well as chemo-attraction and innate immunity. Since these are prone to dysregulation in most liver diseases, MSC release hepatotropic factors, potentially supporting liver regeneration.

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

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.

Functional impairment of the bone marrow (BM) niche has been suggested as a major reason for prolonged cytopenia and secondary graft failure after allogeneic hematopoietic cell transplantation (alloHCT). Because mesenchymal stromal cells (MSCs) serve as multipotent progenitors for several niche components in the BM, they might play a key role in this process. We used collagenase digested trephine biopsies to directly quantify MSCs in 73 patients before (n = 18) and/or after alloHCT (n = 65). For the first time, we demonstrate that acute graft-versus-host disease (aGvHD, n = 39) is associated with a significant decrease in MSC numbers. MSC reduction can be observed even before the clinical onset of aGvHD (n = 10). Assessing MSCs instantly after biopsy collection revealed phenotypic and functional differences depending on the occurrence of aGvHD. These differences vanished during ex vivo expansion. The MSC endotypes observed revealed an enhanced population of donor-derived classical dendritic cells type 1 and alloreactive T cells as the causing agent for compartmental inflammation and MSC damage before clinical onset of aGvHD was ascertained. In conclusion, MSCs endotypes may constitute a predisposing conductor of alloreactivity after alloHCT preceding the clinical diagnosis of aGvHD.