Gene Expression Profiling of Ex-vivo expanded Hematopoietic Stem and Progenitor Cells using SCF+TPO+FLT3L and homing factor SDF1

Efficient bone marrow homing is a prerequisite for successful engraftment of transplanted HSPC. This study aims at determining factors important in homing of these cells from the blood to the marrow and their re-engraftment. We have isolated nucleated cells from mobilized peripheral blood stem cell harvests (PBSC). CD34+ hematopoietic stem and progenitor cells (HSPC) were enriched from PBSC harvests by immunomagnetic separation using negative selection method. Enriched cells were cultured in vitro for expansion in presence or absence of cytokine (GF: SCF+TPO+FLT3L) and/or chemokine (homing factor SDF1) mixture for 8 days at 370C in humidified atmosphere of 5% CO2 in air. GF+SDF1 –stimulated HSPC showed significantly increased total nucleated ells as well as CD34+ cells as compared to GF-stimulated HSPC as well s unstimulated cultured HSPC. On transplantation of these cells in vivo in mice model who were previously irradiated at sublethal dose of 375cGy, it was observed that GF+SDF1-stimulated HSPC exhibited significantly better engraftment in terms of presence of human CD45+ cells in mouse blood at 6 week post –transplantation. We have compared gene expression profiles of mobilized PBSC harvest cells, enriched CD34+ HSPC population and HSPC cultured in plain media, in presence with GF and in presence of GF+SDF1 derived from mobilized PBSC harvests with respect to genes involved in homing and engraftment capacities. SDF1 induced gene expression was measured in CD34-enriched HSPC expanded for 8-day in ex-vivo culture. This Gene expression profiles were compared with that of freshly mobilized PBSC harvests, CD34-enriched HSPC and HSPC cultured in absence of any stimulus and in presence of GF mixture. Five independent experiments were performed at each time using different transplantation donors. Agilent one-color experiment,Organism: Human ,Agilent-Custom Whole Genome Human 8x60k designed by Genotypic Technology Pvt. Ltd. (AMADID: 027114) , Labeling kit: Agilent Quick-Amp labeling Kit (p/n5190-0442)