Browsing by Subject "Pluripotent stem cell"
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Item Derivation of lymphocytes from human induced pluripotent stem cells(2014-09) Ma, ChaoHuman pluripotent stem cells have the potential to produce essentially unlimited numbers of mature and functional blood lineage populations to study human hematopoiesis. Particularly, human induced pluripotent stem cells (hiPSCs) have the advantage to provide a source of autologous transplantable blood cell populations suitable for treatment of patient specific hematological diseases. This research aims to derive human lymphocytes from hiPSCs. There are three projects: The overall generation of human lymphocytes (B cell, T cell and NK cell) from hiPSCs is explored in Project I. In Project II and III, based on the derivation of NK cells, two human immunodeficiency disease models, both caused by specific somatic gene mutation, are established using human pluripotent stem cells. The ultimate goal of this research is to use hiPSCs to study the normal development of human lymphocytes in vitro, as well as model human immunodeficiency diseases by combining with gene therapy methods, thus providing a novel approach for immunotherapy. The hypothesis is that human lymphocytes can be derived from hiPSCs and this will enable the establishment of in vitro models to study human immunodeficiency diseases. Specific aims:1. To generate human lymphocytes from hiPSCs in vitro;2. To establish two human immunodeficiency disease models (X-SCID and WAS) through in vitro derivation of lymphocytes from hESCs/hiPSCs.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.