Intrinsic potential of human hemopoietic stem cells to self-renewal during EX vivo cultures

Self-renewal of p I uri potential hemopoietic stem cells (HSCs) is a primary requirement for their ex vivo expansion as well as for the long-lasting genetic correction of the hemopoietic system. In our search for culture conditions that will facilitate HSC replication while preserving their primitive properties, we have made use of a multi-parameter FACS assay based on the phenotypic properties of the primitive cells. The HSCs were defined as cells expressing high levels of CD34 but lack the CD38, CD33 and CD? I antigens. Cell replication in this study was determined by PKH26 membrane dye. Loading CD34+ cells before culture with PKH26 enables to monitor the replication history of cells meeting, at the lime of analysis, the characteristics of HSCs. In total 31 samples of bone marrow and umbilical cord blood were included in the study. Isolated CD34+ cells from each sample were submitted to parallel culture conditions including varying hemopoietic growth factor combinations, the presence or absence of pre-established allogeneic stroma and serum supplementation. This experimental set-up enabled us to compare the effects of various stimuli on the replication of the CD34+CD33,38,71- population of individual samples as well as the response pattern of cells from different samples. A most striking observation in this study was the large intra-sample variation in the proliferative response of the CD34+CD33.38.7S- cells. The replication potential of the CD34+CD33,38,71- population did not correspond to that of the other cellular components in the culture. The replication of the culture as a whole was determined clearly by culture stimuli. In general, the samples from the two sources could be divided according to the replication property of the CD34+CD33,38,71- sub-set into either good or poorly replicating. In comparison to this 'intrinsic' potential, the effect of growth stimuli on proliferation was négligeable. If the CD34+-CD33,38,71 sub set divided it was once and only occasionally twice, during a 6-day period. Other cell components replicated up to 8 times. In conclusion, ex vivo replication of CD34++CD33,38,71- HSCs is apparently intrinsically regulated and their replication potential remains limited under many culture conditions. As the determinants for the intrinsic capacity of HSCs to replicate in culture are unsolved, their ex vivo behavior remains unpredictable. This knowledge should be taken in consideration in the practice of ex vivo HSC expansion and gene therapy.