ENHANCED DETECTION, MAINTENANCE, AND DIFFERENTIATION OF PRIMITIVE HUMAN HEMATOPOIETIC-CELLS IN CULTURES CONTAINING MURINE FIBROBLASTS ENGINEERED TO PRODUCE HUMAN STEEL FACTOR, INTERLEUKIN-3, AND GRANULOCYTE-COLONY-STIMULATING FACTOR

To determine whether the sensitivity of the human long-term culture-in itiating cell (LTC-IC) assay could be increased, we have evaluated a s pectrum of different fibroblast cell lines for their abilities to infl uence the number of cells detectable as LTC-IC, to influence LTC-IC ma intenance, and/or to influence LTC-IC differentiation into colony-form ing cells (CFC) in cocultures containing various sources of LTC-IC. In a series of initial experiments with highly purified subpopulations o f CD34(+) cells from normal human marrow, no significant difference co uld be found between any of 3 different murine stromal fibroblast cell s in terms of their support of either LTC-IC detection (CFC production ) or maintenance (over a B-week period), and all were equivalent to pr imary human marrow feeders (HMF). On the other hand, murine M2-10B4 fi broblasts engineered to produce high levels of both human granulocyte colony-stimulating factor (G-CSF) and interleukin-3 (IL-3; 190 and 4 n g/mL, respectively), either alone or mixed 1:1 with SI/SI fibroblasts engineered to produce high levels of soluble Steel factor (SF), with o r without production of the transmembrane form of SF (60 and 4 ng/mL, respectively), stimulated the production of up to 20-fold more CFC in LTC of cells from normal human marrow, G-CSF-mobilized blood or cord b lood when compared with parallel cocultures containing HMF. Limiting d ilution analysis of the CFC output from all three sources of LTC-IC sh owed that most of this increase was due to an ability of the engineere d feeders to increase the plating efficiency of the LTC-IC assay (simi lar to 14-fold for marrow LTC-IC and similar to 4-fold for cord blood or mobilized blood LTC-IC). Analysis of the phenotype of these additio nally recruited LTC-IC from marrow showed they had the same primitive CD34(+)CD45RA(-)CD71(-) phenotype as conventionally defined LTC-IC. Th e limiting dilution studies also showed that the average number of CFC produced per LTC-IC was additionally and independently increased to y ield values of 18 CFC per LTC-IC in marrow, 28 for LTC-IC in cord bloo d, and 25 for LTC-IC in G-CSF-mobilized blood. Replating of cells from primary LTC with different feeders into secondary LTC-IC assays conta ining the best combination of engineered feeders showed that LTC-IC ma intenance could be significantly enhanced (up to 7-fold as compared wi th primary cocultures containing HMF). However, this enhancement was s till not sufficient to amplify the number of LTC-IC present after 6 we eks above the input value. Thus, engineering murine fibroblasts to pro duce sufficient SF, G-CSF, and IL-3 can markedly enhance the detection as well as the maintenance in vitro of a very primitive population of human progenitor cells present in normal adult marrow, mobilized bloo d, and cord blood by providing the most sensitive assay conditions thu s far described. The present findings also provide new evidence of bio logic heterogeneity between different cell populations that can be ope rationally identified as LTC-IC, thus re-emphasizing the importance of limiting dilution analyses to distinguish between quantitative and qu alitative effects on these cells. (C) 1996 by The American Society of Hematology.