Purification and characterization of hematopoietic stem cells by microarray analysis

Stem cells are defined by their ability to both self-renew and produce other cell lineages. Hematopoietic stem cells (HSCs), are among the most thoroughly characterized somatic stem cells in the body. Despite this, little is known regarding the mechanisms of HSC engraftment, differentiation, or self-renewal. In an effort to increase this understanding, microarray analysis was used to determine the gene expression profile of HSCs relative to total marrow. One gene identified by this method, encoding the endothelial protein C receptor (EPCR) was found to be expressed over 40 fold higher in HSC as compared to MP. This unique expression pattern was utilized to isolate cells that were highly enriched for hematopoietic reconstitution activity in vivo . In an effort to determine the functional significance of EPCR expression in HSCs, a series of lentiviral vectors were utilized to increase or decrease the expression of EPCR in HSCs prior to in vivo transplantation. Alteration of EPCR expression did not affect engraftment of HSCs. Furthermore, EPCR deficient HSCs were capable of normal engraftment and contribution to peripheral blood demonstrating that EPCR expression is not required for normal HSC function. Attempts to identify the transcriptional elements which impart HSC specific expression determined that 500bp of genomic sequence upstream of the EPCR translational start site is sufficient for expression in HSCs with further specificity provided by an additional 4.5kb of upstream sequence. Transgenic mice created using these genomic sequences failed to demonstrate transgene fidelity for unknown reasons. During the course of these studies a novel lentiviral vector system was produced with significantly improved features including modular elements and reduced vector size. Additional modifications to the transfer vector were made to allow for the production of high-titer lentivirus in the absence of all accessory proteins.