Development of a novel approach to expand umbilical cord blood-derived haematopoietic stem/progenitor cells ex vivo for clinical transplantation

摘要

Umbilical cord blood (UCB) is a well established alternative source of haematopoietic stem/progenitor cells (HSPC) for haematopoietic stem cell transplantation (HSCT). One factor limiting the wider use of UCB for transplantation is the relatively low cell dose from a UCB unit compared to bone marrow (BM) and peripheral blood (PB). To overcome this barrier, many strategies have been tested to expand HSPC numbers ex vivo. The aims of this thesis were to define the phenotypic, functional and transcriptional changes that occur during ex vivo expansion of UCB HSPC and investigate how activation of the Wingless (Wnt) pathway via GSK-3 inhibition can provide a novel approach to improve transplantation outcomes using expanded cells. In vitro and in vivo models of HSPC function were used to evaluate the impact of expansion and GSK-3 inhibition on progenitor function while gene array technology was used to investigate potential molecular mechanisms.We have shown that expansion of UCB HSPC results in a loss of primitive phenotype, an increase in progenitors with reduced function and a shift in transcriptional profile from ‘stem-like quiescent’ to ‘divide and differentiate’. Brief treatment of expansion cultures with a GSK-3 inhibitor resulted in delayed cell cycle progression which translated into improved PB and BM repopulation. Gene expression analysis revealed modulation of Notch and Tie2 pathways, suggesting their potential role in improving stem cell engraftment. In addition, down-regulation of cyclin D1 and upregulation of CDK inhibitor p57 known to be important in maintenance of quiescence was observed.This work is a timely reminder of the medical revolution that E. Donnall Thomas pioneered in the 1950’s, paving the way for successful treatment of those with haematological malignancies and other blood disorders. In addition, the year 2013 marked the 25th anniversary of the first UCB transplant that took place in France in October 1988. Since then over 600,000 UCB units have been banked and more than 30,000 UCB transplants performed worldwide. This thesis contributes to the understanding of HSPC biology and how this can be modulated to improve clinical transplant outcomes in the future.