Lentivector-mediated clonal tracking reveals intrinsic heterogeneity in the human hematopoietic stem cell compartment and culture-induced stem cell impairment

• Published in: Blood Vol. 103; no. 2; pp. 545 - 552
• Main Authors: Mazurier, Frédéric, Gan, Olga I., McKenzie, Joby L., Doedens, Monica, Dick, John E.
• Format: Journal Article
• Language: English
• Published: Washington, DC Elsevier Inc 15.01.2004; The Americain Society of Hematology
• Subjects: Anesthesia. Intensive care medicine. Transfusions. Cell therapy and gene therapy; Animals; Applied cell therapy and gene therapy; Biological and medical sciences; Cell Culture Techniques - methods; Colony-Forming Units Assay; Fetal Blood - cytology; Flow Cytometry; Genetic Vectors; Hematopoietic Stem Cells - cytology; Hematopoietic Stem Cells - physiology; Humans; Infant, Newborn; Lentivirus - genetics; Medical sciences; Mice; Mice, Inbred NOD; Mice, SCID; Mice, Transgenic; Phosphoglycerate Kinase - genetics; Promoter Regions, Genetic; Stem Cell Transplantation; Stem Cells - cytology; Stem Cells - physiology; Transfection; Transfusions. Complications. Transfusion reactions. Cell and gene therapy; Human; Clonality; Hematopoiesis; Animal; Stem cell; Cell labelling; Hematopoietic cell; Genetic transfer
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood-2003-05-1558

Knowledge of the composition and interrelationship of the various hematopoietic stem cells (HSCs) that comprise the human HSC pool and the consequence of culture on each class is required for effective therapies based on stem cells. Clonal tracking of retrovirally transduced HSCs in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice revealed heterogeneity in the repopulation capacity of SCID-repopulating cells (SRCs). However, it is impossible to establish whether HSC heterogeneity is intrinsic or whether the culture conditions required for retroviral transduction induce qualitative and quantitative alterations to SRCs. Here, we report establishment of a clonal tracking method that uses lentivectors to transduce HSCs with minimal manipulation during overnight culture without cytokine stimulation. By serial bone marrow (BM) sampling of mice receiving transplants, short-term SRCs (ST-SRCs) and long-term SRCs (LT-SRCs) were identified on the basis of repopulation dynamics demonstrating that their existence is not an experimental artifact but reflects the state of the HSC pool. However, 4 days of culture in conditions previously used for SRC retroviral transduction significantly reduced SRC number as assessed by clonal analysis. These studies provide a foundation to understand the molecular and cellular determinants of human HSC development and to develop therapies targeted to specific HSC classes.