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Genetic modification of human hematopoietic stem cells

University of British Columbia

Human hematopoiesis originates in a population of stem cells with transplantable lympho-myeloid reconstituting potential but an in vivo method for quantitating these cells has not been available. In this study an assay was developed that allows human cord blood (CB) cells with in-vivo repopulating potential to be quantitated. It is based on the ability of immunodeficient mice to be engrafted by intravenously injected human hematopoietic cells and uses limiting dilution analysis to measure the frequency of human cells that produce both lymphoid and myeloid cells in the marrow of the recipient mice. The frequency of human competitive repopulating units (CRU) was shown to be ~1 per 6x10[superscript 5] light density CB cells, 1 per 900 CD34+CD38- CB cells and 1 per 18,000 CD34+CD38+ CB cells. In addition, it was demonstrated that under selected culture conditions, a significant expansion of both CRU and Long-Term Culture-Initiating Cells (LTC-IC) could be obtained. The ability to reliably transfer genes into hematopoietic stem cells remains an important but elusive goal. To date the power of recombinant retroviral gene transfer has been severely compromised by the low efficiency of retroviral infection. A series of experiments was undertaken to develop improved, clinically applicable, gene transfer conditions. These focused on the use of retroviral supernatant and the use of fibronectin coated dishes. The applicability of this protocol was tested by infecting CB cells capable of repopulating immunodeficient mice. The gene transfer efficiency as determined by G418-resistance to CRU and LTC-IC was 17 ± 3% and 17 ± 8 respectively. There was a significant correlation between the gene transfer to LTC-IC and CRU, however there was no correlation between gene transfer to CFC and LTC-IC or CFC and CRU. To further optimize the utility of recombinant retroviruses, the murine heat stable antigen (HSA) a cell surface antigen was developed as dominant selectable marker in a retroviral vector to enable the identification and selection of retrovirally marked human hematopoietic cells. Using this strategy, virtually pure populations of transduced hematopoietic cells including LTC-IC could be specifically isolated on the basis of their ability to express the transferred HSA gene. Taken together these studies provide a means to quantitate human in vivo repopulating cells and describe culture conditions that allows their modest expansion. The results indicate the utility of the NOD/SCID model for optimizing gene transfer to human repopulating cells. Additionally these studies have provided procedures which will allow purification of genetically modified cells ex vivo and the subsequent tracking of infected hematopoietic cells following transplantation. [Scientific formulae used in this abstract could not be reproduced.]

Thesis/Dissertation

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2009-05-29

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http://hdl.handle.net/2429/8472

Pathology

University of British Columbia

UBCV

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