Hematopoietic stem and progenitor cells (HSPCs) repopulate the blood system upon transplantation. A large-scale genetic approach to understand the factors that participate in successful engraftment has not been undertaken. In this thesis, I present the development of a novel live imaging-based competitive marrow repopulation assay in adult zebrafish, which allows fast and quantitative measurement of HSPC engraftment capability. Using this assay, a transplantation-based chemical screen was performed, which led to the discovery of 10 compounds that can enhance the marrow engraftment capability in zebrafish. Among them, the arachidonic acid-derived epoxyeicosatrienoic acids (EET), had conserved effects on both short- and long-term bone marrow engraftment in mice. Genetic analysis in zebrafish embryos demonstrated that EET acts through a \(G\alpha12/13\)-mediated receptor, which activates PI3K and induces transcription factors of the AP-1 family. This PI3K/AP-1 pathway directly induced the transcription of HSC marker, runx1, in embryos. The activation of PI3K by EET promoted HSPC migration and interactions with niche cells. Our studies define a role for EETs in the development of blood stem cells during embryogenesis, and in engraftment in adult vertebrates. The other compounds discovered in the screen implicate additional novel signaling pathways involved in the HSPC engraftment process, which require further investigation. In summary, this thesis elucidated an important role of bioactive lipids in regulating HSC engraftment in adults and during embryo development. Systematically mapping out the regulatory network will tremendously benefit both the basic understanding of stem cell biology and the clinical manipulation to generate better stem cells for transplantation.

Gain- and loss-of-function studies of the BCL-2 family of proteins have shown that they can impact chemotherapeutic sensitivity. However, cells contain myriad anti-apoptotic and pro-apoptotic BCL-2 family members making it difficult to predict cell fate decisions based on the initial conditions of these proteins. BH3 profiling is a tool that measures mitochondrial priming, the readiness of a cell to die through the intrinsic (or mitochondrial) apoptotic pathway. Priming is due to the cumulative effect of the BCL-2 family of proteins that act as the gate keepers of the mitochondrial apoptotic pathway. Priming is measured by determining the sensitivity of mitochondria to perturbation by peptides derived from the BH3 domains of pro-apoptotic proteins. Using BH3 profiling, we now have a functional readout that can quantify priming and assess its contribution to drug sensitivity. Here we show that priming affects the sensitivity of acute myeloid leukemia (AML) cell lines to various standard chemotherapeutics, especially topoisomerase II inhibitors. Priming predicts clinical response to conventional induction chemotherapy as well as the long term maintenance of remission in AML patients. Interestingly, the priming of normal hematopoietic stem cells (HSCs) sits at the boundary line between the priming of cured and refractory patient AML. This HSC priming likely defines the therapeutic index since AML that are lower primed than HSCs are often refractory and cannot be cured without transplantation. Additionally, our BH3 profiles revealed that AML cells are more sensitive to BCL-2 antagonism than normal HSCs, which are primarily dependent on MCL-1. Indeed, we were able to kill primary refractory AML cells in vitro with the BCL-2 antagonist ABT-737 at doses that left HSCs unharmed. Cumulatively, these findings show that priming is a major mechanistic determinant of AML response in vitro and in the clinic to standard induction chemotherapy. With the ability to predict outcome, BH3 profiling may offer physicians and patients a promising tool for treatment decision-making.

Stem cell biologists creating cells and tissues for therapies, disease modeling, and drug screening have observed that differentiating pluripotent stem cells (PSCs) tend to produce cells at an embryonic stage of development but have difficulty maturing into adult definitive cells. A better understanding of developmental maturation will provide insights into embryogenesis and permit more accurate disease modeling. In the hematopoietic system, primitive and definitive cells are distinguished by functional transplantation assays, well characterized cell surface antigens, and gene expression signatures. We examined the transition in vivo in transplanted murine hematopoietic stem cells (HSCs) and in vitro in human PSC (hPSC) derived red blood cells (RBCs). We found that the hematopoietic microenvironment of the recipient significantly affects the outcome of HSC transplantation. The earliest embryonic HSCs perform better in neonatal recipients, whereas more mature adult-like HSCs perform better in adult recipients. The preference may be related to different active hematopoietic niches in neonates and adults, as we observed adult HSCs homing to different tissues in neonatal and adult recipients. Additionally, we found that proliferation may enhance the neonatal engraftment potential of adult-like HSCs. Our data highlight the importance of the host environment on transplantation outcomes, and point to the neonatal transplant model as a tool to functionally examine the earliest HSCs and primitive derivatives of PSCs.

<p>Stem cells are defined by their ability to make more stem cells, a property known as self-renewal and their ability to generate cells that enter differentiation. One mechanism by which fate decisions can be effectively controlled in stem cells is through asymmetric division and the correct partitioning and inheritance of cell fate determinants. While hematopoietic stem cells have the capacity to divide through asymmetric division, the molecular machinery that regulates this process is unknown and whether its activity is required in vivo remains unclear. Here we show that Lis1, a dynein-binding protein and regulator of asymmetric division, is critically required for blood development and for hematopoietic stem cell renewal in fetal and adult life. In particular, conditional deletion of Lis1 led to a severe bloodless phenotype and embryonic lethality in vivo. In both fetal and adult mice, loss of Lis1 led to a failure of normal self-renewal, which included impaired colony-forming ability in vitro and defects in long-term reconstitution ability following transplantation. As a possible mechanism, we find that the absence of Lis1 in hematopoietic cells, in part, accelerates differentiation linked to the incorrect inheritance of cell fate determinants. Furthermore, using a live cell imaging strategy, we find that the incorrect inheritance of cell fate determinants observed following the loss of Lis1 is due defects in spindle positioning and orientation. Finally, using two animal models of undifferentiated myeloid leukemia, we show that Lis1 is critical for the aberrant cell growth that occurs in cancer. Deletion of Lis1 both at the early and late stages of myeloid leukemia blocked its propagation in vivo and led to a marked improvement in survival. Together, these data identify Lis1 and the directed control of asymmetric division as key regulators of normal and malignant hematopoietic development.</p> / Dissertation

T lymphocytes play a critical role in adaptive immunity by eliciting and regulating specific immune responses against viral and bacterial pathogens. The development of T cells occurs within the highly specialized thymus and follows a defined set of stage-specific differentiation steps. However, the molecular and cellular events occurring at early stages of human T-cell development remain to be fully elucidated. This was in part due to the inability to obtain substantial numbers of T-lineage cells from hybrid/human fetal thymic organ culture (FTOC) and the inability to recapitulate human T-lymphopoiesis using other systems. To address the molecular and cellular events occurring during early human T-lymphopoiesis, human umbilical cord-blood (UCB) hematopoietic stem cells (HSCs) were induced to differentiate to the T-lineage utilizing OP9-DL1 stromal cells. A developmental program involving a sequential and temporally discrete expression of key differentiation markers was revealed. In addition, this Thesis demonstrates that in vitro-generated CD34+CD7++ progenitors effectively engrafted the thymus of immunodeficient mice. In addition, two distinct progenitor subsets, CD34+CD45RA+CD7++CD5-CD1a- (proT1) and CD34+CD45RA+CD7++CD5+CD1a- (proT2), were identified with proT2 cells showing a 3-fold enhanced engrafting capacity than the proT1 subset. As proT2 cells exhibit superior engrafting capacity, these cells were tested for their ability to enhance T cell generation following hematopoietic stem cell transplant (HSCT). We observe that when HSCs are coinjected with proT2 cells, a dramatic improvement in HSC-derived T-lymphopoiesis is observed. This Thesis demonstrates that in vitro-derived proT2 cells reorganize the thymus stromal compartment of the host NOD/SCID/γcnull mouse compared to the highly disorganized cortical and medullary compartments in mice not receiving proT cells. This alteration in thymic architecture likely favours the recruitment of BM derived progenitors. Lastly, we address whether functional CD8 T cells can be generated in vitro using hematopoietic stem cells (HSCs) in coculture with OP9-DL1 cells and indeed these cells were capable of proliferating, and secreting effector molecules typical of cytotoxic T cells. Taken together, the ability to generate proT cells and mature T cells from Notch-ligand cultures offers a new tool to study human T cell development.

T lymphocytes play a critical role in adaptive immunity by eliciting and regulating specific immune responses against viral and bacterial pathogens. The development of T cells occurs within the highly specialized thymus and follows a defined set of stage-specific differentiation steps. However, the molecular and cellular events occurring at early stages of human T-cell development remain to be fully elucidated. This was in part due to the inability to obtain substantial numbers of T-lineage cells from hybrid/human fetal thymic organ culture (FTOC) and the inability to recapitulate human T-lymphopoiesis using other systems. To address the molecular and cellular events occurring during early human T-lymphopoiesis, human umbilical cord-blood (UCB) hematopoietic stem cells (HSCs) were induced to differentiate to the T-lineage utilizing OP9-DL1 stromal cells. A developmental program involving a sequential and temporally discrete expression of key differentiation markers was revealed. In addition, this Thesis demonstrates that in vitro-generated CD34+CD7++ progenitors effectively engrafted the thymus of immunodeficient mice. In addition, two distinct progenitor subsets, CD34+CD45RA+CD7++CD5-CD1a- (proT1) and CD34+CD45RA+CD7++CD5+CD1a- (proT2), were identified with proT2 cells showing a 3-fold enhanced engrafting capacity than the proT1 subset. As proT2 cells exhibit superior engrafting capacity, these cells were tested for their ability to enhance T cell generation following hematopoietic stem cell transplant (HSCT). We observe that when HSCs are coinjected with proT2 cells, a dramatic improvement in HSC-derived T-lymphopoiesis is observed. This Thesis demonstrates that in vitro-derived proT2 cells reorganize the thymus stromal compartment of the host NOD/SCID/γcnull mouse compared to the highly disorganized cortical and medullary compartments in mice not receiving proT cells. This alteration in thymic architecture likely favours the recruitment of BM derived progenitors. Lastly, we address whether functional CD8 T cells can be generated in vitro using hematopoietic stem cells (HSCs) in coculture with OP9-DL1 cells and indeed these cells were capable of proliferating, and secreting effector molecules typical of cytotoxic T cells. Taken together, the ability to generate proT cells and mature T cells from Notch-ligand cultures offers a new tool to study human T cell development.

Moklsiniame darbe tirtas chimerizmas vaikams po alogeninės kraujodaros kamieninių ląstelių transplantacijos. Tyrimas pradėtas Charité klinikose (Humboldto universitetas, Berlynas), kur chimerizmo tyrinėjimams pasirinkti recipientai, sergantys ūmia limfoblastine leukemija (ŪLL), Fanconi anemija (FA) ir adrenoleukodistrofija (ALD). Sergant šiomis ligomis nuoseklus chimerizmo stebėjimas ALP atliktas pirmą kartą. Darbas tęstas Vilniaus universitete, kuomet pirmą kartą buvo atlikti chimerizmo tyrinėjimai Lietuvoje transplantuotiems vaikams. Donoro ir recipiento ląstelėms atskirti naudoti polimorfiniai DNR žymenys, kurie tirti periferinio kraujo leukocituose (PKL) ir atskirose ląstelių populiacijose (ALP): CD3, CD19 ir CD34. Ląstelių frakcijoms atskirti naudotos imunomagnetinės dalelės. Atlikus chimerizmo analizę nutatyta, kad esant visiškam donoro chimerimui PKL, 74,4% ŪLL ir 66,7% ALD recipientų po mieloabliacinio kondicionavimo ląstelių populiacijose išlieka autologinė, aptinkama iki kelerių metų po transplantacijos. Po sumažinto intensyvumo kondicionavimo 45,5% FA recipientų autologinės kraujodaros pėdsakų nerandama nei PKL, nei ALP. FA ligoniams vystosi stabilus ilgalaikis mišrus chimerizmas, kuris susijęs su su ilgesniu išgyvenamumu po transplantacijos. Nustatėme, kad sergantiems FA galimybė išsivystyti mišriam chimerizmui yra 20 kartų didesnė perpylus kaulų čiulpus, nei periferinio kraujo kamienines ląsteles. / The doctoral thesis aimed to evaluate in a comparative way the benefit chimerism assessment in isolated cell populations (ICP) versus conventional monitoring in whole blood cells (WBC). The study was initiated in Pediatric Bone Marrow Transplant Service of Charité Children’s Hospital (Humboldt University, Berlin). Children suffered from: acute lymphoblastic leukemia (ALL), Fanconi anemia (FA) and adrenoleukodystrophy (ALD) were included into the study. Thereafter the doctoral thesis was accomplished at the Vilnius University where evaluation of the own experience of chimerism analysis in pediatric patients was performed. Chimerism was prospectively monitored in WBC and three cell subsets: CD3, CD19 and CD34. Cell populations were extracted from peripheral blood using immunomagnetic beads. Following polymorphisms of short tandem repeats between donor and recipient were compared. The analysis reveled that following myeloablative conditioning in patients with ALL and ALD analysis of ICP revealed persistent autologous hemopoiesis despite stable donor chimerism in WBC. In contrast after reduced intensity conditioning 45.5% of FA recipients had no evidence of autologous signals either in WBC, or in ICP. FA were found to develop stable long-lasting mixed chimerism associated with better overall survival. development of MC was related to the infusion of bone marrow but not to peripheral blood stem cells. Chimerism in ICP did no affected transplant outcome in any disease group.

The doctoral thesis aimed to evaluate in a comparative way the benefit chimerism assessment in isolated cell populations (ICP) versus conventional monitoring in whole blood cells (WBC). The study was initiated in Pediatric Bone Marrow Transplant Service of Charité Children’s Hospital (Humboldt University, Berlin). Children suffered from: acute lymphoblastic leukemia (ALL), Fanconi anemia (FA) and adrenoleukodystrophy (ALD) were included into the study. Thereafter the doctoral thesis was accomplished at the Vilnius University where evaluation of the own experience of chimerism analysis in pediatric patients was performed. Chimerism was prospectively monitored in WBC and three cell subsets: CD3, CD19 and CD34. Cell populations were extracted from peripheral blood using immunomagnetic beads. Following polymorphisms of short tandem repeats between donor and recipient were compared. The analysis reveled that following myeloablative conditioning in patients with ALL and ALD analysis of ICP revealed persistent autologous hemopoiesis despite stable donor chimerism in WBC. In contrast after reduced intensity conditioning 45.5% of FA recipients had no evidence of autologous signals either in WBC, or in ICP. FA were found to develop stable long-lasting mixed chimerism associated with better overall survival. development of MC was related to the infusion of bone marrow but not to peripheral blood stem cells. Chimerism in ICP did no affected transplant outcome in any disease group. / Moklsiniame darbe tirtas chimerizmas vaikams po alogeninės kraujodaros kamieninių ląstelių transplantacijos. Tyrimas pradėtas Charité klinikose (Humboldto universitetas, Berlynas), kur chimerizmo tyrinėjimams pasirinkti recipientai, sergantys ūmia limfoblastine leukemija (ŪLL), Fanconi anemija (FA) ir adrenoleukodistrofija (ALD). Sergant šiomis ligomis nuoseklus chimerizmo stebėjimas ALP atliktas pirmą kartą. Darbas tęstas Vilniaus universitete, kuomet pirmą kartą buvo atlikti chimerizmo tyrinėjimai Lietuvoje transplantuotiems vaikams. Donoro ir recipiento ląstelėms atskirti naudoti polimorfiniai DNR žymenys, kurie tirti periferinio kraujo leukocituose (PKL) ir atskirose ląstelių populiacijose (ALP): CD3, CD19 ir CD34. Ląstelių frakcijoms atskirti naudotos imunomagnetinės dalelės. Atlikus chimerizmo analizę nutatyta, kad esant visiškam donoro chimerimui PKL, 74,4% ŪLL ir 66,7% ALD recipientų po mieloabliacinio kondicionavimo ląstelių populiacijose išlieka autologinė, aptinkama iki kelerių metų po transplantacijos. Po sumažinto intensyvumo kondicionavimo 45,5% FA recipientų autologinės kraujodaros pėdsakų nerandama nei PKL, nei ALP. FA ligoniams vystosi stabilus ilgalaikis mišrus chimerizmas, kuris susijęs su su ilgesniu išgyvenamumu po transplantacijos. Nustatėme, kad sergantiems FA galimybė išsivystyti mišriam chimerizmui yra 20 kartų didesnė perpylus kaulų čiulpus, nei periferinio kraujo kamienines ląsteles.

Chronic myelogenous leukemia (CML) is a clonal hematopoetic stem cell disorder characterized by proliferation of cells expressing BCR-ABL fusion protein. In the BCR-ABL+ leukemia murine model, 12B1, we explored the therapeutic applicability of chaperone-rich cell lysate (CRCL) in the context of syngeneic hematopoietic cell transplantation (HCT) to treat pre-existing leukemia. Our results demonstrate that tumor growth is significantly delayed in mice receiving syngeneic HCT from 12B1 tumor CRCL immunized donors compared to animals receiving HCT from non-immunized donors. CRCL immunization post-immune HCT further hindered tumor growth when compared to immune HCT without post-transplant vaccination. The magnitude of the immune response was consistent with the anti-tumor effects observed in vivo. We also demonstrated that cured mice had developed long-term tumor specific immunity against 12B1 tumor cells. In addition, we documented that both T cells and NK cells contributed to the anti-tumor effect of CRCL vaccination as depletion of either subset hampered tumor growth delay. Thus, our results suggest that CRCL represents a promising vaccine capable of generating specific immune responses. This anti-tumor immunity can be effectively transferred to a host via HCT and further enhanced post-HCT with additional tumor CRCL immunizations.CD4+CD25+ regulatory T cells (Treg) play a central role in the prevention of autoimmunity and in the control of immune responses by down-regulating the function of effector CD4+ or CD8+ T cells. The role of Treg in Mycobacterium tuberculosis infection and persistence is inadequately documented. Therefore, the current study was designed to determine whether CD4+CD25+ FoxP3+ regulatory T cells may modulate immunity against human tuberculosis (TB). Ourresults indicate that the number of CD4+CD25+FoxP3+ Treg increases in the blood or at the site of infection in active TB patients. The frequency of CD4+CD25+FoxP3+ Treg in pleural fluid inversely correlates with local MTB-specific immunity(p<0.002). These CD4+CD25+FoxP3+ T lymphocytes isolated from the blood and pleural fluid are capable of suppressing MTB-specific IFN-γ and IL-10 production in TB patients. Therefore, CD4+CD25+FoxP3+ Treg expanded in TB patients suppress Mycobacterium tuberculosis immunity and may therefore contribute to the pathogenesis of human TB.

<p>Hematopoietic stem cell (HSC) self-renewal, proliferation and differentiation are regulated by signaling through protein tyrosine kinases (PTK) such as c-kit, Flt-3 and Tie2. PTKs work in concert with receptor protein tyrosine phosphatases (PTPs) to maintain cellular equilibrium. The functions of PTPs in counterbalancing PTK signaling in HSCs however remain incompletely understood. Our laboratory has demonstrated that a heparin binding growth factor, Pleiotrophin (PTN), promotes the expansion of murine long-term (LT)-HSCs via binding to a PTP, protein tyrosine phosphatase receptor type Z (PTPRZ). The addition of PTN to murine PTPRZ-/- c-Kit+Sca-1+Lineage- (KSL) cells caused no expansion of HSCs in culture, suggesting that PTPRZ mediates PTN effects on HSC growth. We subsequently screened for the expression of other receptor PTPs in murine HSCs. Among 21 different receptor PTPs, we found that protein tyrosine phosphatase receptor type S (PTPRS) was significantly overexpressed in mouse and human HSCs compared to more mature hematopoietic cells. Ptprs-/- mice displayed no difference in mature blood counts or phenotypic HSC frequency compared to Ptprs+/+ mice. However, competitive transplantation of bone marrow (BM) cells from Ptprs-/- mice resulted in more than 8-fold increased multilineage hematopoietic repopulation in primary and secondary recipient mice compared to mice transplanted with BM cells from Ptprs+/+ mice. While Ptprs-/- mice displayed no differences in cell cycle status, HSC survival or homing capability compared to Ptprs+/+ mice, PTPRS-/- BM cells expressed significantly increased levels of activated Rac1, a RhoGTPase which regulates HSC engraftment capacity, compared to PTPRS+/+ BM cells. PTPRS-/- BM cells displayed significantly increased transendothelial migration capacity and cobblestone area forming cells (CAFC), consistent with increased Rac1 activation. Furthermore, inhibition of Rac1 abrogated the increased transendothelial migration capacity of PTPRS-/- BM cells, suggesting that the augmented engraftment capacity of PTPRS-/- BM cells was mediated via Rac1. Translationally, we demonstrated that negative selection of human cord blood Lin-CD34+CD38-CD45RA- cells for PTPRS expression yielded a 15-fold enrichment for human long term HSCs compared to Lin-CD34+CD38-CD45RA- cells or Lin-CD34+CD38-CD45RA- PTPRS+ cells. These data suggest that PTPRS regulates HSC repopulating capacity via inhibition of Rac1 and selection of human PTPRS - negative HSCs is a translatable strategy to significantly enrich human cord blood HSCs for transplantation.</p> / Dissertation