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.

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