Retinoic acid (RA) and RA receptors (RARs) are involved in regulating the growth and differentiation of multiple different cell types. To explore the role of RA in hematopoiesis, the investigators have utilized retroviral vectors harboring a dominant negative RAR to interfere with normal endogenous RAR activity in different hematopoietic cells. They have observed in cultures of normal mouse bone marrow that inhibiting endogenous RA receptor activity in the presence of different hematopoietic growth factors results in the establishment of hematopoietic cell lines frozen at specific stages of neutrophil differentiation. The investigators wish to further extend these studies defining the role of RA receptors in hematopoiesis as follows.
Specific Aim 1 : Determine whether the cultured lymphohematopoietic EML cells can function as hematopoietic stem cells in irradiated mice. By infecting normal mouse bone marrow with a retroviral vector harboring a dominant negative RA receptor construct, the investigators have reproducibly derived stem cell factor-dependent cell lines that display characteristics of self-renewing lympho-hematopoietic progenitors (designated EML). The investigators will formally evaluate the stem cell activity of these cultured EML cells by assessing their ability to act as hematopoietic stem cells in vivo in irradiated syngeneic mice.
Specific Aim 2 : Utilize the dominant negative RA receptor retroviral vector to establish hematopoietic growth factor dependent hematopoietic stem cell lines. To further explore the RA receptors in regulating the commitment and differentiation of lineages of hematopoietic stem cells and progenitors, the investigators will culture bone marrow infected with the dominant negative RA receptor retroviral construct with specific hematopoietic growth factors including IL-1, IL-3 IL-4, IL-6, flk2/flt3 ligand and TPO in an attempt to establish other growth factor dependent cell lines frozen at different stages of hematopoietic differentiation.
Specific Aim 3 : Utilize the dominant negative RA receptor retroviral vector to establish growth factor dependent lymphohematopoietic progenitor lines from human bone marrow.
Specific Aim 4 : Determine the effect of RA receptor antagonists in the self renewal and proliferation of cultured mouse and human stem cells. The investigators will use synthetic retinoids exhibiting RA receptor antagonist activity in attempts to optimize in vitro conditions that enhance self-renewal and proliferation of hematopoietic stem cells while blocking their differentiation.
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