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Stem Cells 2004;22:570-579 www.StemCells.com
© 2004 AlphaMed Press

Clonal Analysis of Individual Marrow-Repopulating Cells after Experimental Peripheral Blood Progenitor Cell Transplantation

K. Zsuzsanna Nagya, Stephanie Laufsa, Bernhard Gentnera, Sonja Naundorfb, Klaus Kuehlckeb, Julian Topalyc, Eike C. Bussc, W. Jens Zellera, Stefan Fruehaufc

a German Cancer Research Center, Heidelberg, Germany;
b EUFETSAG, Idar-Oberstein, Germany;
c Department of Internal Medicine V, University of Heidelberg, Heidelberg, Germany

Key Words. Experimental PBPC transplantation • SCID-repopulating cells • Single cell analysis

Correspondence: Stefan Fruehauf, M.D., Department of Internal Medicine V, University of Heidelberg, Im Neuenheimer Feld 410, 69120 Heidelberg, Germany. Telephone: 49-6221-562781; Fax: 49-6221-565722; e-mail: stefan_fruehauf{at}med.uni-hei-delberg.de

Methods to analyze the clonality of an adverse event in preclinical or clinical retroviral stem cell gene therapy protocols are needed. We analyzed the progeny of retrovirally transduced human peripheral blood progenitor cells (PBPCs) after transplantation and engraftment in immune-deficient mice. The integration site of the provirus serves as a unique tag of the individual transduced PBPC. A plasmid library of junctions between proviral and human genomic DNA was generated. We were able to detect individual transduced cell clones that amounted to 0.14%–0.0001% of chimeric bone marrow cells. This is the first report in which the contribution of individual marrow-repopulating cells to human hematopoiesis is directly quantified.




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