Here we describe some of the crucial steps to generate induced pluripotent stemcells (iPSCs) usingmRNA transfection. Our approach uses a V. virus-derived capping enzyme instead of a cap-analog, ensuring 100% proper cap orientation for in vitro transcribedmRNA. V. virus\'' 2′-O-Methyltransferase enzymecreates a cap1 structure found in higher eukaryotes and has higher translation efficiency compared to other methods. Use of the polymeric transfection reagent polyethylenimine proved superior to other transfection methods. The mRNA created via this method did not trigger an intracellular immune response via human IFN-gamma (hIFN-γ) or alpha (hIFN-α) release, thus circumventing the use of suppressors. Resulting mRNA and protein were expressed at high levels for over 48 h, thus obviating daily transfections. Using this method, we demonstrated swift activation of pluripotency associated genes in human fibroblasts. Low oxygen conditions further facilitated colony formation. Differentiation into different germ layers was confirmed via teratoma assay. Reprogramming with non-synthetic mRNA holds great promise for safe generation of iPSCs of human origin. Using the protocols described herein we hope to make this method more accessible to other groups as a fast, inexpensive, and non-viral reprogramming approach.

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.

No description available.

Homeostasis of hematopoietic stem cells (HSC) in the mammalian bone marrow stem cell niche is regulated by signals of the local microenvironment. Besides juxtacrine, endocrine and metabolic cues, paracrine and autocrine signals are involved in controlling quiescence, proliferation and differentiation of HSC with strong implications on expansion and differentiation ex vivo as well as in vivo transplantation. Towards this aim, a cell culture analysis on a polymer microcavity carrier platform was combined with a partial least square analysis of a mechanistic model of cell proliferation. We could demonstrate the discrimination of specific autocrine and paracrine signals from soluble factors as stimulating and inhibitory effectors in hematopoietic stem and progenitor cell culture. From that we hypothesize autocrine signals to be predominantly involved in maintaining the quiescent state of HSC in single-cell niches and advocate our analysis platform as an unprecedented option for untangling convoluted signaling mechanisms in complex cell systems being it of juxtacrine, paracrine or autocrine origin.

Hematopoiesis in the adult organism is maintained by a complex, hierarchically organized cascade of differentiating cells that ultimately originate from adult hematopoietic stem cells (HSCs). HSCs have been extensively studied in perturbative settings (e.g transplantation assays), which are well suited for exploring cell potential but tell very little about cell behaviour in a physiological state. Novel \textit{in situ} techniques have made it possible to observe hematopoiesis in its natural environment, yet many questions about native HSC behaviour remain controversial. It is yet unclear whether and to what extent HSCs contribute to steady-state blood production, and whether they represent a reserve population that can be activated upon emergency. As, to date, common definitions of HSCs comprise cells of heterogeneous function, incoherent results are likely linked by another fundamental question: what is the identity of the HSC population residing on the apex of the hematopoietic hierarchy? To answer these questions is essential to gain a deeper understanding of fatal human diseases like leukemia or aplastic anemia. We describe an integrative approach combining non-invasive experimental methods with mathematical inference to uncover underlying pathways and differentiation patterns of steady state hematopoiesis. By this, we identify a population of Sca-1\textsuperscript{hi} CD201(EPCR)\textsuperscript{hi} HSCs as the hithertho elusive apex population, and show that they contribute marginally, but continuously, to native steady-state hematopoiesis. Further, we clarify the architecture of a shortcut route of thrombopoiesis, which emanates directly from HSCs and links them trough a previously undefined CD48\textsuperscript{-/low} megakaryocyte progenitor population directly to platelets. Finally, we provide an extensive analysis of the dynamic label propagation and proliferation behaviour of hematopoietic stem and progenitor cells in prototypical stress situations mimicking blood loss, infection and inflammation. We find that apex stem cells do not directly contribute to emergency hematopoiesis and are only activated upon severe myeloablation and chronic inflammation. Further, innate immune training did not influence HSC contribution in response to reoccurring stimulation. In sum, we demonstrate that primitive HSCs do neither represent a major contributor to steady-state blood production, nor a reservoir for emergency supply. We thus question current dogma of hematopoiesis, and argue that the primary function of HSCs in the adult organism remains yet to be discovered.:Contents Abstract iv Zusammenfassung vii List of Figures xii List of Tables xiii List of Abbreviations xv 1 Introduction 1 1.1 Hematopoiesis and the hematopoietic system 1 1.1.1 Developmental origins 1 1.1.2 The hematopoietic hierarchy 3 1.2 Hematopoietic stem cells 5 1.2.1 Characteristics of hematopoietic stem cells 7 1.2.2 Identification of hematopoietic stem and progenitor cells 7 1.2.3 HSC heterogeneity 11 1.3 Novel genetic tools to analyse native hematopoiesis 12 1.3.1 Conditional gene targeting 12 1.4 Hematopoietic stem cell behaviour in adult steady-state hematopoiesis 15 1.4.1 Models of native Hematopoiesis 16 1.4.2 Models of HSC quiescence 23 1.4.3 Aged hematopoiesis 25 1.5 Hematopoietic stem cell behaviour in stress response 26 1.5.1 Inflammation and Infection 27 1.5.2 Myeloablation 30 1.5.3 Innate immune training 32 2 Aim of Thesis 35 3 Material and Methods 37 3.1 Materials 37 3.1.1 Antibodies 37 3.1.2 Buffer and Solutions 38 3.1.3 Chemicals and Reagents 39 3.1.4 Cultivation Media 40 3.1.5 Kit Systems 41 3.1.6 Software 41 3.2 Mice 42 3.2.1 Animal housing and husbandry 42 3.2.2 Mouse strains 42 3.3 Genotyping of mice 43 3.4 Treatments of mice 46 3.4.1 Reporter induction 46 3.4.2 Hematopoietic stress induction 46 3.5 Hemograms 48 3.6 Transplantation and in vivo repopulation assays 49 3.6.1 Preconditioning and transplantation of recipients 49 3.6.2 Competitive repopulation assay 50 3.6.3 Limiting dilution assay 50 3.6.4 Transplantation into sublethally irradiated hosts 51 3.7 Cell preparation for transplantation and flow cytometry 51 3.7.1 Bone marrow 51 3.7.2 Splenocytes 51 3.7.3 Peripheral blood 51 3.7.4 Peritoneal cells 52 3.7.5 Cell depletion by magnetic activated cell sorting (MACS) 52 3.8 Flow Cytometry 53 3.8.1 Cell staining 54 3.8.2 Cell counting 55 3.8.3 Cell identification and gating 55 3.8.4 Chimerism analysis 57 3.8.5 Cell sorting 58 3.9 Cytokine detection assay 58 3.10 in vitro single cell expansion assay 59 3.11 Sequencing 61 3.11.1 Single cell RNA sequencing 61 3.11.2 Bulk RNA sequencing 62 3.11.3 Single cell and bulk transcriptome analysis 62 3.12 Mathematical modelling 62 3.13 Data Normalization 63 3.14 Statistical Analysis 64 4 Results 67 4.1 Identification of an apex HSC population 67 4.1.1 Fgd5 Cre-System preferentially labels primitive HSCs 68 4.1.2 ES HSCs reside at the top of the hierarchy 71 4.2 A combination of proliferation and differentiation modelling uncovers lineage trajectories 76 4.2.1 Iterative modelling reveals subtle, but continuous contribution of HSC to Steady state hematopoiesis 77 4.2.2 The myeloid lineage diverges within phenotypic HSCs 80 4.3 Fate mapping uncovers an alternative pathway of thrombopoiesis 82 4.3.1 CD201-/lo Sca-1lo HSCs feed thrombopoiesis via CD48-/lo MkPs 82 4.3.2 CD48-stratified MkP subsets display variable thrombopoietic potential 88 4.3.3 Thrombopoietin signalling enhances platelet production via the direct pathway 92 4.4 Ageing modulates the dynamics of fate mapping 95 4.5 HSCs in times of crisis: Contribution to stress recovery 99 4.5.1 Faithful reporters are necessary to study HSPC behaviour during stress hematopoiesis 100 4.5.2 Severe myeloablation provokes HSC activation 105 4.5.3 Inflammation-induced emergency hematopoiesis only mildly amplifies HSC activity 107 4.5.4 Innate immune training does not alter HSC proliferation and differentiation 113 4.5.5 Compensation of blood cell loss is achieved without HSC contribution 118 4.5.6 Emergency hematopoiesis proceeds without activation of primitive Hematopoietic stem cells (HSCs) 120 5 Discussion 123 5.1 Labels matter: What is a true HSC? 123 5.1.1 Fgd5ZsGreen:CreERT2R26LSL-tdRFP fate mapping preferentially labels primitive HSCs 124 5.1.2 ES HSCs reside at the top of the hierarchy 126 5.1.3 Subtle, but continuous contribution of HSCs to steady state hematopoiesis 127 5.1.4 The myeloid lineage diverges within phenotypic HSCs 130 5.2 Fate mapping uncovers an alternative pathway of thrombopoiesis 132 5.2.1 CD201-/lo Sca-1lo HSCs feed thrombopoiesis via CD48-/lo MkPs 132 5.2.2 Thrombopoietin signalling enhances platelet production via the direct pathway 135 5.3 Fundamental dynamics of label propagation are preserved in aged mice 136 5.4 HSCs in times of crisis: are HSC a reserve population for emergency response? 138 5.4.1 Faithful reporters: Caveats and merits of using in situ models to study HSC activation 139 5.4.2 Severe myeloablation provokes HSC activation 143 5.4.3 Inflammation-induced emergency hematopoiesis only mildly amplifies HSC activity 145 5.4.4 Compensation of blood cell loss is achieved without HSC contribution 149 5.4.5 Innate immune training does not alter HSC proliferation and differentiation 150 6 Conclusion 153 References 155 Acknowledgements 185 Appendices 187 Anlage 1: Erklärungen zur Eröffnung des Promotionsverfahrens 189 Anlage 2: Erklärung über die Einhaltung der gesetzlichen Vorgaben 190 / Die Blutbildung in erwachsenen Organismen wird durch eine komplexe, hierarchisch organisierte Kaskade von sich fortwährend differenzierenden Zellen aufrechterhalten, und hat ihren Ursprung in adulten h\'amatopoietischen Stammzellen (HSZs). HSZs wurden ausgiebig unter unphysiologischen Extrembedingungen (z.B. Transplantation) untersucht, in welchen sich viel über das maximale potential einer Zelle, aber wenig über ihr Verhalten in ungestörtem Zustand sagen lässt. Obwohl durch moderne \textit-Techniken mittlerweile h\'amatopoietische Differenzierung in ihrer ursprünglichen Umgebung beobachtet werden kann, bleiben etliche Fragen zum nativen Verhalten von HSZs offen. So ist es noch unklar, ob und in welchem Ausmaß HSZs überhaupt zur stationären Blutbildung beitragen, oder ob sie eine Reservepopulation darstellen, die im Notfall aktiviert werden kann. Da bis dato gebr\'auchliche Definitionen von HSZs verschiedene Sub-Populationen mit heterogener Funktionalität umfassen, sind die inkohärenten Ergebnisse wahrscheinlich mit einer anderen grundlegenden Frage verknüpft: Was ist die wahre Identit\'at der HSZ-Population, die an der Spitze der h\'amatopoetischen Hierarchie steht? Die Beantwortung dieser Fragen ist essenziell für ein besseres Verst\'andnis von fatalen humanen Krankheiten, wie beispielsweise Leuk\'amie oder aplastische An\'amie. Wir beschreiben einen integrativen Ansatz, der nicht-invasive experimentelle Methoden mit mathematischer Modellierung vereint, um die zugrundeliegenden Pfade und Differenzierungsmuster der station\'arer H\'amatopoese zu ergründen. Auf diese Weise können wir eine Population von Sca-1\textsuperscript{hi} und CD201(EPCR)\textsuperscript{hi} HSZs als die bisher unbekannte Apex-Population identifizieren, und zeigen, dass diese Zellen geringf\'ugig, aber kontinuierlich, zur nativen H\'amatopoese beitragen. Darüber hinaus klären wir die Architektur eines direkten thrombozytischen Differenzierungspfades, welcher unmittelbar von HSZs abzweigt und diese über eine neu definierte CD48\textsuperscript{-/lo} Megakaryozyten-Vorläuferpopulation direkt mit Thrombozyten ver- bindet. Schließlich liefern wir eine umfassende Analyse des dynamischen Differenzierungs- und Proliferationsverhaltens von hämatopoetischen Stamm- und Vorläuferzellen in prototypischen Stresssituationen, die Blutverlust, Infektion und Entzündung nachahmen. Wir zeigen, dass primitive Stammzellen nicht direkt zur Notfall-Hämatopoese beitragen und nur bei schwerer Myeloablation und chronischer Entzündung aktiviert werden. Ebenfalls hat das Training des angeborenen Immunsystems keinen Einfluss auf die Aktivität von HSZs bei wiederholter Stimulation. Unsere Ergebnisse belegen, dass primitive HSZs weder einen wesentlichen Beitrag zur stationären Blutbildung leisten, noch ein Reservoir für die Notfallversorgung darstellen. Wir stellen somit das derzeitige Dogma der Hämatopoese in Frage und argumentieren, dass die primäre Funktion der HSZ im erwachsenen Organismus noch zu entdecken ist.:Contents Abstract iv Zusammenfassung vii List of Figures xii List of Tables xiii List of Abbreviations xv 1 Introduction 1 1.1 Hematopoiesis and the hematopoietic system 1 1.1.1 Developmental origins 1 1.1.2 The hematopoietic hierarchy 3 1.2 Hematopoietic stem cells 5 1.2.1 Characteristics of hematopoietic stem cells 7 1.2.2 Identification of hematopoietic stem and progenitor cells 7 1.2.3 HSC heterogeneity 11 1.3 Novel genetic tools to analyse native hematopoiesis 12 1.3.1 Conditional gene targeting 12 1.4 Hematopoietic stem cell behaviour in adult steady-state hematopoiesis 15 1.4.1 Models of native Hematopoiesis 16 1.4.2 Models of HSC quiescence 23 1.4.3 Aged hematopoiesis 25 1.5 Hematopoietic stem cell behaviour in stress response 26 1.5.1 Inflammation and Infection 27 1.5.2 Myeloablation 30 1.5.3 Innate immune training 32 2 Aim of Thesis 35 3 Material and Methods 37 3.1 Materials 37 3.1.1 Antibodies 37 3.1.2 Buffer and Solutions 38 3.1.3 Chemicals and Reagents 39 3.1.4 Cultivation Media 40 3.1.5 Kit Systems 41 3.1.6 Software 41 3.2 Mice 42 3.2.1 Animal housing and husbandry 42 3.2.2 Mouse strains 42 3.3 Genotyping of mice 43 3.4 Treatments of mice 46 3.4.1 Reporter induction 46 3.4.2 Hematopoietic stress induction 46 3.5 Hemograms 48 3.6 Transplantation and in vivo repopulation assays 49 3.6.1 Preconditioning and transplantation of recipients 49 3.6.2 Competitive repopulation assay 50 3.6.3 Limiting dilution assay 50 3.6.4 Transplantation into sublethally irradiated hosts 51 3.7 Cell preparation for transplantation and flow cytometry 51 3.7.1 Bone marrow 51 3.7.2 Splenocytes 51 3.7.3 Peripheral blood 51 3.7.4 Peritoneal cells 52 3.7.5 Cell depletion by magnetic activated cell sorting (MACS) 52 3.8 Flow Cytometry 53 3.8.1 Cell staining 54 3.8.2 Cell counting 55 3.8.3 Cell identification and gating 55 3.8.4 Chimerism analysis 57 3.8.5 Cell sorting 58 3.9 Cytokine detection assay 58 3.10 in vitro single cell expansion assay 59 3.11 Sequencing 61 3.11.1 Single cell RNA sequencing 61 3.11.2 Bulk RNA sequencing 62 3.11.3 Single cell and bulk transcriptome analysis 62 3.12 Mathematical modelling 62 3.13 Data Normalization 63 3.14 Statistical Analysis 64 4 Results 67 4.1 Identification of an apex HSC population 67 4.1.1 Fgd5 Cre-System preferentially labels primitive HSCs 68 4.1.2 ES HSCs reside at the top of the hierarchy 71 4.2 A combination of proliferation and differentiation modelling uncovers lineage trajectories 76 4.2.1 Iterative modelling reveals subtle, but continuous contribution of HSC to Steady state hematopoiesis 77 4.2.2 The myeloid lineage diverges within phenotypic HSCs 80 4.3 Fate mapping uncovers an alternative pathway of thrombopoiesis 82 4.3.1 CD201-/lo Sca-1lo HSCs feed thrombopoiesis via CD48-/lo MkPs 82 4.3.2 CD48-stratified MkP subsets display variable thrombopoietic potential 88 4.3.3 Thrombopoietin signalling enhances platelet production via the direct pathway 92 4.4 Ageing modulates the dynamics of fate mapping 95 4.5 HSCs in times of crisis: Contribution to stress recovery 99 4.5.1 Faithful reporters are necessary to study HSPC behaviour during stress hematopoiesis 100 4.5.2 Severe myeloablation provokes HSC activation 105 4.5.3 Inflammation-induced emergency hematopoiesis only mildly amplifies HSC activity 107 4.5.4 Innate immune training does not alter HSC proliferation and differentiation 113 4.5.5 Compensation of blood cell loss is achieved without HSC contribution 118 4.5.6 Emergency hematopoiesis proceeds without activation of primitive Hematopoietic stem cells (HSCs) 120 5 Discussion 123 5.1 Labels matter: What is a true HSC? 123 5.1.1 Fgd5ZsGreen:CreERT2R26LSL-tdRFP fate mapping preferentially labels primitive HSCs 124 5.1.2 ES HSCs reside at the top of the hierarchy 126 5.1.3 Subtle, but continuous contribution of HSCs to steady state hematopoiesis 127 5.1.4 The myeloid lineage diverges within phenotypic HSCs 130 5.2 Fate mapping uncovers an alternative pathway of thrombopoiesis 132 5.2.1 CD201-/lo Sca-1lo HSCs feed thrombopoiesis via CD48-/lo MkPs 132 5.2.2 Thrombopoietin signalling enhances platelet production via the direct pathway 135 5.3 Fundamental dynamics of label propagation are preserved in aged mice 136 5.4 HSCs in times of crisis: are HSC a reserve population for emergency response? 138 5.4.1 Faithful reporters: Caveats and merits of using in situ models to study HSC activation 139 5.4.2 Severe myeloablation provokes HSC activation 143 5.4.3 Inflammation-induced emergency hematopoiesis only mildly amplifies HSC activity 145 5.4.4 Compensation of blood cell loss is achieved without HSC contribution 149 5.4.5 Innate immune training does not alter HSC proliferation and differentiation 150 6 Conclusion 153 References 155 Acknowledgements 185 Appendices 187 Anlage 1: Erklärungen zur Eröffnung des Promotionsverfahrens 189 Anlage 2: Erklärung über die Einhaltung der gesetzlichen Vorgaben 190

Mutations in TREX1, encoding three prime repair exonuclease 1, cause Aicardi-Goutières syndrome (AGS) 1, an autoinflammatory disease characterized by neurodegeneration and constitutive activation of the antiviral cytokine type I interferon. Here, we report the generation and characterization of induced pluripotent stem cells (iPSCs) derived from fibroblasts from two AGS patients with biallelic TREX1 mutations. These cell lines offer a unique resource to investigate disease processes in a cell-type specific manner.

Mammals have two orthologs, Mll and Trx2, for the Drososphila protein Trithorax (TRX), which is the founding member of the trithorax group (TrxG) of epigenetic regulators. TrxG proteins are characterized by an evolutionary conserved SET domain. A major function of all SET domain- containing proteins is to modulate gene activity, but the underlying mechanisms are poorly understood. Apparently TRX, Mll and Trx2 are histone H3 lysine 4 specific methyltransferases. So far all evidence points to roles in expression of specific target genes. However, target genes and function of the epigenetic regulator Trx2 were still unknown. Homozygous trx2 mutant embryos arrest in development because of severe and widespread defects {Glaser, 2005 #296}. Thus mouse embryonic stem (ES) cells carrying a null mutation of trx2 were used as an alternative model system to address the implication of Trx2 in differentiation. This study showed that Trx2 is redundant for ES cell self-renewal. Homozygous trx2 knockout ES cells did not exhibit cell cycle defects. However, loss of Trx2 resulted in reduced proliferation and increased apoptosis rates in trx2-/- ES cells. Due to the fact that differentiation requires an appropriate rate of population growth, trx2-/- cells were affected adversely upon in vitro differentiation. Neurogeneic differentiation of trx2 mutant cells generated fewer mature neurons than wild type cells. Moreover a temporal delay in the developmental progression to differentiation became apparent. Cardiac differentiation of trx2-/- cells confirmed the developmental defect and temporal delay. Notably differentiation of trx2-/- cells was merely delayed or impaired but it was not absent, implying that Trx2 is not required for gene expression programs specific for neurons or cardiac myocytes. We propose that differentiation of trx2-/- ES cells is impaired because apoptosis is disturbing differentiation. Apart from analyzing the phenotype of trx2 mutant cells, this work was focused on the identification of Trx2 target genes. Oligonucleotide expression arrays were used to identify genes whose expression levels were affected by the absence of Trx2. In general, loss of Trx2 function resulted in more genes with decreased than increased expression levels. This is consistent with the hypothesis that Trx2 functions as a transcriptional activator. Comparison of gene expression profiles for constitutive and conditional trx2 mutant cells enabled a distinction between direct and indirect target genes for Trx2. As a result Magoh2 was identified as the key candidate target gene for Trx2. Interaction between Trx2 and Magoh2 suggested a potential regulatory role for Trx2 in alternative splicing. Furthermore this work provided evidence that Trx2 could be involved in the maintenance of CpG island promoter gene expression, thus providing a potent regulatory mechanism for ubiquitously expressed genes.

No description available.

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.