Bone marrow niche-mimetics modulate hematopoietic stem cell function via adhesion signaling in vitro

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] 11

Immune Cell Deformability in Depressive Disorders: Longitudinal Associations Between Depression, Glucocorticoids and Cell Deformability

Background Cell deformability of all major blood cell types is increased in depressive disorders (DD). Furthermore, impaired glucocorticoid secretion is causally related to DD. Nevertheless, there are no longitudinal studies examining changes in glucocorticoid output and depressive symptoms regarding cell deformability in DD. Aim To investigate, whether changes in depressive symptoms or hair glucocorticoids predict cell deformability in DD. Methods In 136 individuals, depressive symptoms (PHQ-9) and hair glucocorticoids (cortisol and cortisone) were measured at timepoint one (T1), while one year later (T2) depressive symptoms and hair glucocorticoids were remeasured and additionally cell deformability of peripheral blood cells was assessed and DD status was determined by clinical interview. Results Depression severity at T1 predicted higher cell deformability in monocytes and lymphocytes over the entire sample. Subjects with continuously high depressive symptoms at T1 and T2 showed elevated monocyte deformability as compared to subjects with low depressive symptoms. Depression severity at T1 of subjects with a lifetime persistent depressive disorder (PDD) was associated with elevated monocyte, neutrophil, and granulo-monocyte deformability. Depression severity at T1 of subjects with a 12-month PDD was positively associated with monocyte deformability. Furthermore, increases in glucocorticoid concentrations from T1 to T2 tended to be associated with higher immune cell deformability, while strongest associations emerged for the increase in cortisone with elevated neutrophil and granulo-monocyte deformability in the 12-month PDD group. Conclusion Continuously elevated depressive symptomatology as well as an increase in glucocorticoid levels over one year are associated with higher immune cell deformability, particularly in PDD. These findings suggest, that persistent depressive symptomatology associated with increased glucocorticoid secretion may lead to increased immune cell deformability thereby compromising immune cell function and likely contributing to the perpetuation of PDD

Depressive disorders are associated with increased peripheral blood cell deformability: a cross-sectional case-control study (Mood-Morph)

Pathophysiological landmarks of depressive disorders are chronic low-grade inflammation and elevated glucocorticoid output. Both can potentially interfere with cytoskeleton organization, cell membrane bending and cell function, suggesting altered cell morpho-rheological properties like cell deformability and other cell mechanical features in depressive disorders. We performed a cross-sectional case-control study using the image-based morpho-rheological characterization of unmanipulated blood samples facilitating real-time deformability cytometry (RT-DC). Sixty-nine pre-screened individuals at high risk for depressive disorders and 70 matched healthy controls were included and clinically evaluated by Composite International Diagnostic Interview leading to lifetime and 12-month diagnoses. Facilitating deep learning on blood cell images, major blood cell types were classified and morpho-rheological parameters such as cell size and cell deformability of every individual cell was quantified. We found peripheral blood cells to be more deformable in patients with depressive disorders compared to controls, while cell size was not affected. Lifetime persistent depressive disorder was associated with increased cell deformability in monocytes and neutrophils, while in 12-month persistent depressive disorder erythrocytes deformed more. Lymphocytes were more deformable in 12-month major depressive disorder, while for lifetime major depressive disorder no differences could be identified. After correction for multiple testing, only associations for lifetime persistent depressive disorder remained significant. This is the first study analyzing morpho-rheological properties of entire blood cells and highlighting depressive disorders and in particular persistent depressive disorders to be associated with increased blood cell deformability. While all major blood cells tend to be more deformable, lymphocytes, monocytes, and neutrophils are mostly affected. This indicates that immune cell mechanical changes occur in depressive disorders, which might be predictive of persistent immune response

Bone marrow niche-mimetics modulate hematopoietic stem cell function via adhesion signaling in vitro

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] 11

Bone marrow niche-mimetics modulate hematopoietic stem cell function via adhesion signaling in vitro

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] 11

Mechanical phenotyping of primary human skeletal stem cells in heterogeneous populations by real-time deformability cytometry.

Skeletal stem cells (SSCs) are a sub-population of mesenchymal stromal cells (MSCs) present in bone marrow with multipotent differentiation potential. A current unmet challenge hampering their clinical translation remains the isolation of homogeneous populations of SSCs, in vitro, with consistent regeneration and differentiation capacities. Cell stiffness has been shown to play an important role in cell separation using microfluidic techniques such as inertial focusing or deterministic lateral displacement. Here we report that the mechanical properties of SSCs, and of a surrogate human osteosarcoma cell line (MG-63), differ significantly from other cell populations found in the bone marrow. Using real-time deformability cytometry, a recently introduced method for cell mechanical characterization, we demonstrate that both MG-63 and SSCs are stiffer than the three primary leukocyte lineages (lymphocytes, monocytes and granulocytes) and also stiffer than HL-60, a human leukemic progenitor cell line. In addition, we show that SSCs form a mechanically distinct sub-population of MSCs. These results represent an important step towards finding the bio-physical fingerprint of human SSCs that will allow their label-free separation from bone marrow with significant physiological and therapeutic implications

Alterations in Cell Mechanics by Actin Cytoskeletal Changes Correlate with Strain-Specific Rubella Virus Phenotypes for Cell Migration and Induction of Apoptosis

The cellular cytoskeleton is central for key cellular functions, and as such is a marker for diseased and infected cell states. Here we analyzed infection with rubella virus (RV) strains with respect to phenotypes in cellular mechanical properties, cell movement, and viral cytopathogenicity. Real-time deformability cytometry (RT-DC), as a high-throughput platform for the assessment of cell mechanics, revealed a correlation of an increase in cortical filamentous-actin (F-actin) with a higher cellular stiffness. The additional reduction of stress fibers noted for only some RV strains as the most severe actin rearrangement lowered cell stiffness. Furthermore, a reduced collective and single cell migration speed in a wound healing assay was detected in addition to severe changes in cell morphology. The latter was followed by activation of caspase 3/7 as a sign for induction of apoptosis. Our study emphasizes RT-DC technology as a sensitive means to characterize viral cell populations and to implicate alterations of cell mechanical properties with cell functions. These interdependent events are not only promising options to elucidate viral spread and to understand viral pathologies within the infected host. They also contribute to any diseased cell state, as exemplified by RV as a representative agent for cytoskeletal alterations involved in a cytopathological outcome

AIDeveloper: Deep Learning Image Classification in Life Science and Beyond

Abstract Artificial intelligence (AI)‐based image analysis has increased drastically in recent years. However, all applications use individual solutions, highly specialized for a particular task. Here, an easy‐to‐use, adaptable, and open source software, called AIDeveloper (AID) to train neural nets (NN) for image classification without the need for programming is presented. AID provides a variety of NN‐architectures, allowing to apply trained models on new data, obtain performance metrics, and export final models to different formats. AID is benchmarked on large image datasets (CIFAR‐10 and Fashion‐MNIST). Furthermore, models are trained to distinguish areas of differentiated stem cells in images of cell culture. A conventional blood cell count and a blood count obtained using an NN are compared, trained on >1.2 million images, and demonstrated how AID can be used for label‐free classification of B‐ and T‐cells. All models are generated by non‐programmers on generic computers, allowing for an interdisciplinary use

Machine learning assisted real‑time deformability cytometry of CD34+ cells allows to identify patients with myelodysplastic syndromes

Diagnosis of myelodysplastic syndrome (MDS) mainly relies on a manual assessment of the peripheral blood and bone marrow cell morphology. The WHO guidelines suggest a visual screening of 200 to 500 cells which inevitably turns the assessor blind to rare cell populations and leads to low reproducibility. Moreover, the human eye is not suited to detect shifts of cellular properties of entire populations. Hence, quantitative image analysis could improve the accuracy and reproducibility of MDS diagnosis. We used real-time deformability cytometry (RT-DC) to measure bone marrow biopsy samples of MDS patients and age-matched healthy individuals. RT-DC is a high-throughput (1000 cells/s) imaging flow cytometer capable of recording morphological and mechanical properties of single cells. Properties of single cells were quantified using automated image analysis, and machine learning was employed to discover morpho-mechanical patterns in thousands of individual cells that allow to distinguish healthy vs. MDS samples. We found that distribution properties of cell sizes differ between healthy and MDS, with MDS showing a narrower distribution of cell sizes. Furthermore, we found a strong correlation between the mechanical properties of cells and the number of disease-determining mutations, inaccessible with current diagnostic approaches. Hence, machine-learning assisted RT-DC could be a promising tool to automate sample analysis to assist experts during diagnosis or provide a scalable solution for MDS diagnosis to regions lacking sufficient medical experts