Indiana University-Purdue University Indianapolis (IUPUI) / Internal tandem duplications in FMS-like receptor tyrosine kinase (FLT3-ITDs) are seen in approximately 25% of all acute myeloid leukemia (AML) patients. FLT3-ITDs induce FLT3 ligand (FL)-independent cellular hyperproliferation, promiscuous and aberrant activation of STAT5, and confer a poor prognosis in patients; however, the molecular mechanisms contributing to FLT3-ITD-induced malignancy remain largely unknown. The protein tyrosine phosphatase, Shp2, is important for normal hematopoiesis as well as hematopoietic stem cell (HSC) differentiation, engraftment, and self-renewal. Furthermore, FLT3-ITD- or constitutive active STAT5-expressing CD34+ cells demonstrate enhanced hematopoietic stem cell self-renewal. Together with the previous findings that Shp2 is critical for normal hematopoiesis, that dysregulated Shp2 function contributes to myeloid malignancies, and that Shp2 has been shown to interact with WT-FLT3 tyrosine 599, which is commonly duplicated in FLT3-ITDs, a positive role for Shp2 in FLT3-ITD-induced signaling and leukemogenesis is implied. I demonstrated that Shp2 is constitutively associated with the reported FLT3-ITDs, N51-FLT3 and N73-FLT3, compared to WT-FLT3; therefore, I hypothesized that increased Shp2 recruitment to N51-FLT3 or N73-FLT3 contributes to hyperproliferation and hyperactivation of STAT5. I also hypothesized that Shp2 cooperates with STAT5 to activate STAT5 transcriptional targets contributing to the up-regulation of pro-leukemic proteins. Finally, I hypothesized that reduction of Shp2 would result in diminished N51-FLT3-induced hyperproliferation and activation of STAT5 in vitro, and prevent FLT3-ITD-induced malignancy in vivo. I found that genetic disruption of Ptpn11, the gene encoding Shp2, or pharmacologic inhibition of Shp2 with the novel Shp2 inhibitor, II-B08, resulted in significantly reduced FLT3-ITD-induced hematopoietic cell hyperproliferation and STAT5 hyperphosphorylation. I also demonstrated a novel role of Shp2 in the nucleus of FLT3-ITD-expressing hematopoietic cells where Shp2 and STAT5 co-localized at the promoter region of STAT5-transcriptional target and pro-survival protein, Bcl-XL. Furthermore, using a Shp2flox/flox;Mx1Cre+ mouse model, I demonstrated that reduced Shp2 expression in hematopoietic cells resulted in an increased latency to and reduced severity of FLT3-ITD-induced malignancy. Collectively, these findings demonstrate that Shp2 plays an integral role in FLT3-ITD-induced malignancy and suggest that targeting Shp2 may be a future therapeutic option for treating FLT3-ITD-positive AML patients.

Myelodysplastic syndromes (MDS) comprise a heterogeneous group of hematologic malignancies characterized by clonal hematopoiesis, one or more cytopenias such as anemia, neutropenia, or thrombocytopenia, abnormal cellular maturation, and a high risk of progression to acute myeloid leukemia. The bone marrow microenvironment (BMME) in general and mesenchymal stromal cells (MSCs) in particular contribute to both the initiation and progression of MDS. However, little is known about the role of MSC-derived extracellular matrix (ECM) in this context. Therefore, we performed a comparative analysis of in vitro deposited MSC-derived ECM of different MDS subtypes and healthy controls. Atomic force microscopy analyses demonstrated that MDS ECM was significantly thicker and more compliant than those from healthy MSCs. Scanning electron microscopy showed a dense meshwork of fibrillar bundles connected by numerous smaller structures that span the distance between fibers in MDS ECM. Glycosaminoglycan (GAG) structures were detectable at high abundance in MDS ECM as white, sponge-like arrays on top of the fibrillar network. Quantification by Blyscan assay confirmed these observations, with higher concentrations of sulfated GAGs in MDS ECM. Fluorescent lectin staining with wheat germ agglutinin and peanut agglutinin demonstrated increased deposition of N-acetyl-glucosamine GAGs (hyaluronan (HA) and heparan sulfate) in low risk (LR) MDS ECM. Differential expression of N-acetyl-galactosamine GAGs (chondroitin sulfate, dermatan sulfate) was observed between LR- and high risk (HR)-MDS. Moreover, increased amounts of HA in the matrix of MSCs from LR-MDS patients were found to correlate with enhanced HA synthase 1 mRNA expression in these cells. Stimulation of mononuclear cells from healthy donors with low molecular weight HA resulted in an increased expression of various pro-inflammatory cytokines suggesting a contribution of the ECM to the inflammatory BMME typical of LR-MDS. CD34+ hematopoietic stem and progenitor cells (HSPCs) displayed an impaired differentiation potential after cultivation on MDS ECM and modified morphology accompanied by decreased integrin expression which mediate cell-matrix interaction. In summary, we provide evidence for structural alterations of the MSC-derived ECM in both LR- and HR-MDS. GAGs may play an important role in this remodeling processes during the malignant transformation which leads to the observed disturbance in the support of normal hematopoiesis.

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

High transduction rates of viral vectors in gene therapies (GT) and experimental hematopoiesis ensure a high frequency of gene delivery, although multiple integration events can occur in the same cell. Therefore, tracing of integration sites (IS) leads to mis-quantification of the true clonal spectrum and limits safety considerations in GT. Hence, we use correlations between repeated measurements of IS abundances to estimate their mutual similarity and identify clusters of co-occurring IS, for which we assume a clonal origin. We evaluate the performance, robustness and specificity of our methodology using clonal simulations. The reconstruction methods, implemented and provided as an R-package, are further applied to experimental clonal mixes and preclinical models of hematopoietic GT. Our results demonstrate that clonal reconstruction from IS data allows to overcome systematic biases in the clonal quantification as an essential prerequisite for the assessment of safety and long-term efficacy of GT involving integrative vectors.

Indiana University-Purdue University Indianapolis (IUPUI) / The hematopoietic system is highly proliferative, making hematopoietic stem and progenitor cells (HSPC) sensitive to radiation damage. Total body irradiation and chemotherapy, as well as the risk of radiation accident, create a need for countermeasures that promote recovery of hematopoiesis. Substantive damage to the bone marrow from radiation exposure results in the hematopoietic syndrome of the acute radiation syndrome (HS-ARS), which includes life-threatening neutropenia, lymphocytopenia, thrombocytopenia, and possible death due to infection and/or hemorrhage. Given adequate time to recover, expand, and appropriately differentiate, bone marrow HSPC may overcome HS-ARS and restore homeostasis of the hematopoietic system. Prostaglandin E2 (PGE2) is known to have pleiotropic effects on hematopoiesis, inhibiting apoptosis and promoting self-renewal of hematopoietic stem cells (HSC), while inhibiting hematopoietic progenitor cell (HPC) proliferation. We assessed the radiomitigation potential of modulating PGE2 signaling in a mouse model of HS-ARS. Treatment with the PGE2 analog 16,16 dimethyl PGE2 (dmPGE2) at 24 hours post-irradiation resulted in increased survival of irradiated mice compared to vehicle control, with greater recovery in HPC number and colony-forming potential measured at 30 days post-irradiation. In a sublethal mouse model of irradiation, dmPGE2-treatment at 24 hours post-irradiation is associated with enhanced recovery of HSPC populations compared to vehicle-treated mice. Furthermore, dmPGE2-treatment may also act to promote recovery of the HSC niche through enhancement of osteoblast-supporting megakaryocyte (MK) migration to the endosteal surface of bone. A 2-fold increase in MKs within 40 um of the endosteum of cortical bone was seen at 48 hours post-irradiation in mice treated with dmPGE2 compared to mice treated with vehicle control. Treatment with the non-steroidal anti-inflammatory drug (NSAID) meloxicam abrogated this effect, suggesting an important role for PGE2 signaling in MK migration. In vitro assays support this data, showing that treatment with dmPGE2 increases MK expression of the chemokine receptor CXCR4 and enhances migration to its ligand SDF-1, which is produced by osteoblasts. Our results demonstrate the ability of dmPGE2 to act as an effective radiomitigative agent, promoting recovery of HSPC number and enhancing migration of MKs to the endosteum where they play a valuable role in niche restoration.

Indiana University-Purdue University Indianapolis (IUPUI). / Tumor necrosis factor-induced protein 6 (TSG-6) has been shown to mitigate inflammation. Its presence in the secretome of adipose stem / stromal cells (ASC) and its role in activities of ASC have been overlooked. This thesis described for the first time the release of TSG-6 from ASC, and its modulation by endothelial cells. It also revealed that protection of endothelial barrier function was a novel mechanism underlying the anti-inflammatory activity of both ASC and TSG-6. Moreover, TSG-6 was found to inhibit mitogen-activated lymphocyte proliferation, extending the understanding of its pleiotropic effects on major cell populations involved in inflammation. Next, enzyme-linked immunosorbent assays (ELISA) were established to quantify secretion of TSG-6 from human and murine ASC. To study the importance of TSG-6 to specific activities of ASC, TSG-6 was knocked down in human ASC by siRNA. Murine ASC from TSG-6-/- mice were isolated and the down-regulation of TSG-6 was verified by ELISA. The subsequent attempt to determine the efficacy of ASC in ameliorating ischemic limb necrosis and the role of TSG-6, however, was hampered by the highly variable ischemic tissue necrosis in the BALB/c mouse strain. Afterwards in a mouse model of cigarette smoking (CS), in which inflammation also plays an important role, it was observed, for the first time, that 3-day CS exposure caused an acute functional exhaustion and cell cycle arrest of hematopoietic progenitor cells; and that 7-week CS exposure led to marked depletion of phenotypic bone marrow stem and progenitor cells (HSPC). Moreover, a dynamic crosstalk between human ASC and murine host inflammatory signals was described, and specifically TSG-6 was identified as a necessary and sufficient mediator accounting for the activity of the ASC secretome to ameliorate CS-induced myelotoxicity. These results implicate TSG-6 as a key mediator for activities of ASC in mitigation of inflammation and protection of HSPC from the myelotoxicity of cigarette smoke. They also prompt the notion that ASC and TSG-6 might potentially play therapeutic roles in other scenarios involving myelotoxicity.

No description available.

Dogs are useful preclinical models for the translation of cell transplantation therapies from the bench to the bedside. In order for canine models to be utilized for stem cell transplantation research, it is necessary to advance discoveries in the fields of canine stem cell biology and transplantation. The use of side population hematopoietic stem cells (HSCs) has garnered much interest for the purification of mouse HSCs and has been translated to several other species, including human. In order to assess if this method of purification of HSCs could be useful for stem cell therapies in humans, safety and efficacy studies in a large animal model, such as the dog would be required. With this objective in mind, we isolated canine bone marrow-derived side population (SP) stem cells and assessed their multilineage differentiation in vitro and engraftment potential in vivo. Utilizing a pregating strategy to enrich for small, agranular SP cells we were able to enrich for blast cells, expressing the ABCG2 transmembrane pump known to be associated with murine and human SP cells. Canine SP cells were also enriched for C-KIT positive cells and lacked expression of CD34 as identified in other species. The small, agranular SP fraction had high CFU potential after long-term culture with canine bone marrow stromal cells and cytokine supplementation. Yet, canine SP cells demonstrated low-level engraftment within the NOD/SCID-β2m-/- xenotransplantation model as compared to unfractionated canine bone marrow, which was indicative of suboptimal activation of quiescent canine SP cells within the murine bone marrow niche. A second source of transplantable canine stem cells was examined through the derivation of canine embryonic stem cells (cESCs). The cESC lines described herein were determined to have similar pluripotent stem cell characteristics to human embryonic stem cells, in that they were maintained in an undifferentiated state upon extended passaging as determined by their expression of the human stem cell markers, OCT3/4, NANOG, SOX2, SSEA3, SSEA4, TRA1-60, TRA1-81 and alkaline phosphatase. In addition, cESCs could be induced to differentiate to cells of the three germ layers within in vitro embryoid body cultures and adherent differentiation cultures. Importantly, these cESC lines were the first reported to differentiate in vivo within teratomas. One method of transplanting stem cells to canine recipients involves the delivery of donor cells to the yolk sacs of developing fetuses in utero. Utilizing cells labeled with supraparamagnetic particles conjugated to a Dragon Green fluorophore and the intracellular fluorescent dye, CMTMR, donor cells were tracked from the yolk sac injection site to fetal tissues after transplantation in early (day-25) and mid (day-35) gestation canine fetuses. Labeled cells were localized primarily to the fetal liver and developing bone marrow cavities when examined at gestational day 32, and had been redistributed to not only the fetal liver and bone marrow by day 42, but also to nonhematopoietic tissues, including the lungs and hearts. No labeled cells were detected within the yolk sacs of transplanted fetuses at either time point. These studies demonstrated the efficacy of yolk sac in utero transplantation for the delivery of donor cells to fetal tissues. Collectively, these results indicate that canine stem cells with characteristics similar to human can be isolated and their engraftment, proliferation and differentiation may be assessed in future studies utilizing the canine in utero transplantation model employing yolk sac delivery.

Klasické schéma vývoje hematopoetických buněk předpokládá časné oddělení lymfoidního a myeloidního prekurzoru. V poslední době jsou navrhovány složitější modely, které předpokládají větší flexibilitu hematopoezy a navrhují existenci progenitorů s lymfoidním i myeloidním potenciálem. Akutní hybridní leukémie jsou malignity, které podle různých kritérií nelze jednoznačně zařadit k lymfoidní nebo k myeloidní linii a jejichž chování spíše dává za pravdu novým modelům hematopoezy. Předkládaná práce se zabývala především výzkumem dětských leukémií s přesmykem z lymfoidní do myeloidní linie během indukční léčby. Jedná se o rozsáhlý projekt, v jehož rámci si diplomová práce si kladla za úkol určit liniové zařazení leukemických blastů pomocí detekce přestaveb genů pro imunoglobuliny a T-buněčné receptory (TCR). Potvrdili jsme, že myeloidní buňky derivované v průběhu léčby pochází u všech pacientů z původního lymfoidního klonu. Dále jsme u těchto případů zkoumali expresi vytipovaných genů ve srovnání s běžnými druhy leukémií. Třetí částí práce byl výzkum prognostického významu přítomnosti přestaveb TCR (a tedy příslušnosti k lymfoidní linii) u leukémií z T-lymfoidní řady.

Introduction Adipocytes are part of hematopoietic microenvironment, even though they are generally believed to play no active role during hematopoiesis. We have shown that accumulation of fat cells in femoral bone marrow (BM) coincides with increased expression of neuropilin-1 (NP-1), while it is weakly expressed in hematopoietic iliac crest BM. Starting from this observation, we postulated that adipocytes might exert a negative effect on hematopoiesis mediated through NP-1. Material and Methode To test this hypothesis, we set up BM adipocytes differentiated into fibroblast-like fat cells (FLFC), which share the major characteristics of primitive unilocular fat cells, as an experimental model. Results Morphological and immunophenotypic analysis of FLFCs in co-culture with CD34+ cells revealed that FLFCs constitutively produced M-CSF and induced CD34+ differentiation into macrophages independently of cell-to-cell contact. By contrast, granulopoiesis was hampered by cell-to-cell contact but could be restored in transwell culture conditions, together with G-CSF production. Both functions were also recovered when FLFCs cultured in contact with CD34+ cells were treated with an antibody neutralizing NP-1, which proved its critical implication in contact inhibition. Conclusion Our data provide the first evidence that adipocytes exert regulatory functions during hematopoiesis that might be implicated in some pathological processes.