THE BNX HU XENOGRAFT MODEL OF HUMAN HEMATOPOIESIS TO OPTIMIZE METHODSFOR RETROVIRAL-MEDIATED STEM-CELL TRANSDUCTION (REVIEW)/

The potentiality of primitive human hematopoietic cells can be profoun dly affected by in vitro culture. Due to the growing number of protoco ls proposed for stem cell gene therapy and ex vivo expansion, it is cr ucial to define methods to preserve the generative capacity of human s tem cells in culture while promoting self-renewal divisions. Stem cell division, homing, and subsequent lineage development can only be stud ied definitively by marking of pluripotent cells, followed by tracking and clonal analysis of the progeny in a long-term transplantation sys tem. We have developed a bnx/hu xenograft model, in which transduced h uman hematopoietic cells can be individually tracked into different li neages over the course of one year post-transplantation. The tracking is accomplished by single cell cloning of individual T lymphoid and my eloid progenitors recovered from the marrow of the mice, and clonal in tegration analysis by the sensitive technique of single-colony inverse PCR. All cells derived from a stem cell transduced by a retroviral ve ctor will carry the unique restriction fragment length polymorphism (R FLP) created by the random integration event. We have used the bnx/hu xenograft system coupled with single-colony inverse PCR to determine t hat human stem cells require stromal support, fibronectin support with cytokines, or the presence of Flt3 ligand during a 72-h Ex vivo cultu re to maintain the ability to sustain long-term multilineage hematopoi esis.