LONG-TERM REPOPULATING ABILITIES OF ENRICHED FETAL LIVER STEM-CELLS MEASURED BY COMPETITIVE REPOPULATION

To characterize hematopoietic cell biology, many investigators have us ed protocols that enrich for primitive hematopoietic stem cells (PHSC) . In this study, we quantified the long-term repopulating ability (LTR A) of enriched and discarded fractions of PHSC from day-14 murine feta l liver using the competitive repopulation assay. We fractionated popu lations of fetal cells using the antigenic markers AA4.1(+), AA4.1(+)/ Sca(+), and AA4.1(+)/Lin(low)/Sca(+). Differentiating and repopulating abilities of each of these populations were directly compared using c ompetitive repopulation. Adult bone marrow was mixed with fetal cell f ractions from congenic donors having genetically distinguishable marke rs, and mixtures were given to irradiated recipients. Differentiating and repopulating abilities of the enriched donor cells were measured b y the proportions of myeloid and lymphoid cells having donor markers t hat repopulated the recipients. LTRA was found primarily in the AA4.1( +) and AA4.1(+)/Sca(+) subpopulations. Further fractionation of the AA 4.1(+) cells to derive an AA4.1(+)/Lin(low)/Sca(+) fraction showed tha t virtually all of the long-term stem cell activity was found in this subpopulation. These cells were 1400- to 1600-fold enriched in long-te rm functional ability compared to fresh marrow. This very high multili neage repopulating ability per cell was directly measured using a long -term functional assay in vivo. Importantly, the measured repopulating ability for AA4.1(+)/Lin(low)/Sca(+) cells was about five-fold less t han expected from the fraction of cells enriched and remained two- to three-fold less even after compensating for repopulating ability in di scarded fractions. This illustrates that long-term functional abilitie s of enriched PHSC cannot be estimated from fractions enriched but sho uld be quantitatively assayed.