HEMATOPOIETIC STEM CELL BIOLOGY

Human Genome Research

Investigators

Linked publications & trials

Abstract

The research of the Hematopoiesis Section is focused on the basic biology of stem cells and the use of stem cells as vehicles for gene therapy. Hematopoietic stem cells (HSC) are a rare population of self renewing cells that give rise to all cells in the peripheral blood, making them ideal vehicles for gene replacement therapy of inherited hematopoietic diseases. In addition, we have shown that cells highly enriched for HSC can generate cardiac myocytes when injected into the healthy tissue surrounding a myocardial infarct. We have previously shown that treatment with G-CSF and SCF mobilizes HSC into the peripheral blood. When mice treated with G-CSF and SCF were given a myocardial infarct, new cardiac myocytes were observed in the area of the infarct. Others have shown that enriched populations of HSC can give rise to vascular, skeletal muscle, hepatic and neuronal cells. Project 1 will examine stem cell plasticity and biology. Project 1: Biology of Hematopoietic Stem Cells Specific Aim 1.1: We hypothesize that the same hematopoietic stem cells that repopulate the bone marrow are responsible for the regeneration of cardiac myocytes after experimental myocardial infraction (MI). To test this hypothesis, we will transplant mice with bone marrow cells marked with a retrovirus vector containing the GFP gene. These mice will be undergo a coronary artery ligation and stem cells will be mobilized with G-CSF and SCF. The origin of the cardiac cells will be established by the presence of GFP, and clonality will be analyzed by comparison of retroviral insertion sites in the heart and blood. Specific Aim 1.2: We hypothesize that injured regions of the heart and liver express particular gene products that attract HSC and induce them to differentiate into cardiac myocytes or hepatocytes. We will analyze DNA microarrays hybridized with RNA extracted from normal heart tissue and post MI tissue, and from normal and necrotic liver to identify differentially expressed genes. We will test whether these genes can direct HSC to differentiate into cardiac myocytes or hepatocytes using an in vitro culture system. Specific Aim 1.3: We hypothesize that specific genes expressed in both HSC and stem cells isolated from skeletal muscle are responsible for maintaining an undifferentiated state. To test this hypothesis, we will isolate the ?side population? stem cells from mouse skeletal muscle cultures. Due to the limiting amounts of RNA obtainable, we will use cDNA subtraction to generate a library of sequences expressed in these cells. The muscle stem cell transcripts will be compared to those we have identified previously in a cDNA subtraction library of HSC. We will select transcripts common to both libraries for analysis in transgenic mice. Gene transfer to HSC has recently been shown to cure Severe Combined Immune Deficiency, demonstrating that HSC gene therapy could be applied to more common diseases. We would like to develop a gene therapy for Sickle Cell Disease. However, current levels of gene transfer to HSC are too low to treat this disease. We have found that one important reason that gene transfer is so low is that the conventional retrovirus receptors on HSC are nearly undetectable. A second problem has been the instability of retrovirus vectors containing globin genes. Project 2 will examine these problems separately. Project 2: Gene therapy for the hemoglobinopathies Specific aim 2.1: We hypothesize that the endogenous high levels of RD114 or FeLV-C retrovirus receptors on hematopoietic stem cells will result in a higher frequency of human HSC transduction. We will test this hypothesis by simultaneously transducing human HSC with a control GALV pseudotyped vector and either RD114 or FeLV-C pseudotyped vectors for transplantation into fetal sheep. Peripheral blood and bone marrow cells from the sheep chimeras will be analyzed for the relative level of the GALV and RD114 or FeLV-C pseudotyped transgenes in human hematopoietic cells. Specific Aim 2.2: We hypothesize that stable retrovirus vectors containing globin genes linked to the promoters of genes expressed in erythroid cells can be generated that will allow expression of globin mRNA at levels adequate to treat Sickle Cell Disease and b-thalassemia. We will continue our evaluation of the relative level of expression of red cell gene promoters using a transgenic mouse assay to identify promoters that direct position independent, uniform, high-level, and copy number dependent expression. The best promoters will be tested for stability in retrovirus vectors. Stable viruses will be evaluated in the mouse b-thalassemia model.