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

Abstract

Knowledge of the composition and inter- relationship of the various hematopoietic stem cells (HSCs) that comprise the hu- man HSC pool and the consequence of culture on each class is required for effec- tive therapies based on stem cells. Clonal tracking of retrovirally transduced HSCs in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice re- vealed heterogeneity in the repopulation capacity of SCID-repopulating cells (SRCs). However, it is impossible to estab- lish whether HSC heterogeneity is intrin- sic or whether the culture conditions re- quired for retroviral transduction induce qualitative and quantitative alterations to SRCs. Here, we report establishment of a clonal tracking method that uses lentivec- tors to transduce HSCs with minimal ma- nipulation during overnight culture with- out cytokine stimulation. By serial bone marrow (BM) sampling of mice receiving transplants, short-term SRCs (ST-SRCs) and long-term SRCs (LT-SRCs) were iden- tified on the basis of repopulation dynam- ics 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 sig- nificantly reduced SRC number as as- sessed 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. (Blood. 2004;103:545-552)