Browsing by Subject "Hematopoiesis"
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Item Identification of novel signatures of murine definitive hematopoiesis(2014-01) Webber, BeauPluripotent stem cells (PSC) are a tantalizing prospect for a renewable source of patient-specific hematopoietic stem cells (HSC), however efforts to obtain PSC derived HSC capable of long-term engraftment have largely failed. We set out with the primary aim of identifying novel molecular signatures of definitive hematopoiesis, so that these signatures could be applied to improve generation and isolation of HSC in vitro. Toward this end we pursued both discovery and application based strategies centered on Runx1; a transcription factor that is critical for the development of definitive HSC. The discovery arm identified epigenetic modifications at Runx1 cis-regulatory elements that temporally associate with the transition from primitive to definitive hematopoiesis in vivo. We replicated these signatures in vitro by overexpressing HOXB4 in hematopoietic progenitors derived from murine embryonic stem cells (ESC), and found that HOXB4 directly interacts with the definitive-specific distal Runx1 promoter and mediates increased transcription, loss of DNA methylation, and acquisition of active histone modifications at this locus. We next applied our understanding of Runx1 regulation to generate a panel of clonal mESC lines harboring targeted, single-copy fluorescent reporters under the transcriptional control of Runx1 cis-regulatory elements. These lines were used to interrogate the hematopoietic activity of each element independent of copy number and chromosomal position, allowing us to identify combinations that provided optimal activity and fidelity. Building upon this, we established mESC lines harboring synthetic fluorescent and bioluminescent mini genes replicating the structure of the endogenous Runx1 locus and demonstrated that these lines reflect the dynamic promoter switching that occurs at Runx1 during hematogenesis. Sub-fractionation of embryoid body cells based on promoter activity revealed that nearly all colony forming cells (CFC) reside in the distal promoter expressing fraction. With this information we identified specific conditions that could further mature and expand distal positive cells. Collectively, this work identified a previously undescribed molecular signature of definitive hematopoiesis and the mechanism by which it is established. In addition, we applied this knowledge to generate tools with which to interrogate hematopoietic development in vitro, and have demonstrated their utility in optimizing strategies for obtaining definitive hematopoietic progenitors from PSC.Item Mechanisms of early hemato-endothelial development from human pluripotent stem cells(2016-09) Angelos, Mathew GeorgeHemogenic endothelium is a highly specialized population of vascular endothelial cells that produces hematopoietic stem cells (HSCs) during embryonic development. This process, referred to as the endothelial-to-hematopoietic transition (EHT), is critical to establish a functional hematopoietic system that persists throughout adulthood. The underlying genetic and cell signaling mechanisms that regulate human EHT remain poorly defined. Human pluripotent stem cells, including embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) provide a well-defined cellular platform that can be used to study these mechanisms. In this work, functional human hemogenic endothelium was identified and isolated from the earliest hemato-endothelial cells differentiated from hESCs. Analysis of hemogenic endothelial cells at a single-cell resolution found hESC-derived hemogenic endothelium was transcriptionally distinct from vascular endothelial cells lacking hematopoietic potential. Novel genetic markers distinguishing human hemogenic endothelium are also presented. Contributions from the aryl hydrocarbon receptor (AHR), an important cell signaling molecule in HSC biology, were also assessed at the level of human EHT. Small molecule inhibition and gene deletion of AHR significantly improved functional hematopoietic stem and progenitor cell development from hESCs. Importantly, a novel role for AHR in the development of hESC-derived innate lymphoid cells is also presented. Collectively, this dissertation identifies and describes key transcriptional and signaling mechanisms that support human EHT. This information will be useful to optimize the development of HSCs and other hematopoietic lineages that are suitable for future clinical application.Item The role of endoglin during mesoderm specification(2015-05) Baik, JuneEmbryogenesis requires spatiotemporally regulated cellular signals and gene expressions that influence lineage specification, progenitor patterning, and morphogenesis. However the molecular mechanisms that explain how the progenitors commit into different lineages are still poorly understood. Endoglin (ENG) is an ancillary receptor for transforming growth factor-beta (TGF-β) and lack of ENG leads impaired hematopoiesis, cardiac defects and embryonic lethality. Considering the fact that certain mesodermal population can give rise to both cardiac and hematopoietic cells, it indicates that endoglin may play a role in these cell types. Thereby during my predoctoral training, I have aimed to elucidate how endoglin regulates the cell fate choice that results in the specification of early mesodermal precursors into the cardiac or hematopoietic cells. In Chaper 2, I demonstrate that ENG promotes the commitment of early mesodermal progenitors to the hematopoietic lineage at the expense of the cardiac cell fate and ENG-mediated hematopoietic commitment occurs through BMP signaling pathway. In Chapter 3, I illustrate the mechanistic insights how ENG, through activation of BMP and WNT signals, regulates the cell fate decision to secure mesoderm commitment towards the hematopoietic lineage. These studies will uncover a novel role of ENG as a potential mediator between BMP and WNT signaling during mesoderm specification and contribute to broaden our understanding of TGF-β signaling in cell fate decision.Item Tracking hematopoietic development from human pluripotent stem cells(2013-11) Ferrell, Patrick IanHematopoietic stem cells (HSCs) are a powerful resource for both regenerative medicine and the study of human developmental biology. Though much is known about HSC physiology and development in mice, experimental limitations make their characterization a greater challenge in human. As such, human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) are currently the best systems with which to model early human hematopoietic development in vitro, thus providing insight regarding crucial factors delineating HSC emergence, maintenance and subsequent differentiation. However, generation of HSCs from hESCs and iPSCs relies on an intimate understanding of both the in vivo hematopoietic microenvironment as well as HSC phenotype for their prospective isolation. Though current in vitro protocols can readily generate hESC and iPSC-derived cells with hematopoietic progenitor function, none of these populations has exhibited what should be the hallmark of an HSC: robust, long-term, multilineage reconstitution of an immunodeficient recipient upon transplantation. These studies address this issue by using transgenic hESC and iPSC lines which report the expression of genes known to be crucial for early hematopoietic events in mice so that they may help us to understand how they translate to human development in vitro. Furthermore, this effort is complemented by additional studies using hESC-derived stromal populations to provide assays that help assess putative HSC quality, maintenance and the hematopoietic niche.