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Long-term marrow cultures (LTMC) allow the proliferation and differentiation of primitive human hematopoietic progenitor cells to be maintained for many weeks in the absence of exogenously provided hematopoietic growth factors. Previous investigations focused on defining various types of cells that are present in this culture system and on measuring the cycling behavior of the different subpopulations of colony-forming cells maintained within it. These studies suggested that mesenchymal stromal elements derived from the input marrow play a key role in regulating the turnover of the most primitive, high-proliferative potential erythroid and granulopoietic colony-forming cells that are found almost exclusively in the adherent layer of LTMC. In this study we show that the re-entry into S-phase of these primitive hematopoietic progenitors that occurs after each weekly medium change is due to an as yet undefined constituent of horse serum, which is absent from fetal calf serum. However, this effect is not unique to the factor present in horse serum. It is also elicited by the addition to LTMC of several well-defined growth regulatory molecules, ie, platelet-derived growth factor (PDGF), interleukin-1 (IL-1), transforming growth factor α (TGF-α). and IL-2. None of these was able to stimulate hematopoietic colony-forming cells in methylcellulose assays, although all have known actions on mesenchymal cells including, in some cases, the ability to increase production of growth factors that can stimulate primitive high-proliferative potential hematopoietic progenitors in clonogenic assays. Interestingly, a stimulating effect was not obtained after addition of endotoxin to LTMC. TGF-β, a direct-acting negative regulator that acts selectively on primitive hematopoietic progenitor cells if added to LTMC simultaneously with new medium or IL-1, blocked their stimulating activity. These results suggest a model in which indirect, local modulation of both positive and negative regulatory factors via effects on mesenchymal elements determines the rate of turnover of adjacent populations of very primitive hematopoietic cells that are normally maintained in a quiescent state in vivo.

Numerous factors that can influence the proliferation and differentiation in vitro of cells at various stages of he- matopoiesis have been identified, but the mechanisms used by stromal cells to regulate the cycling status of the most primitive human hematopoietic cells are still poorly understood. Previous studies of long-term cultures (LTC) of human marrow have suggested that cytokine-induced variations in stromal cell production of one or more stimulators and inhibitors of hematopoiesis may be important. To identify the specific regulators involved, we performed Northern analyses on RNA extracted from human marrow LTC adherent layers, or stromal cell types derived from or related to those present in the adherent layer. These analyses showed marked increases in interleukin-1β (IL-1β), IL-6, and granulocyte colony-stimulating factor (G-CSF) mRNA levels within 8 hours after treatments that lead to the activation within 2 days of primitive hematopoietic progenitors in such cultures. Increases in granulocyte-macrophage (GM)-CSF and M-CSF mRNA were also sometimes seen. Bioassays using cell lines responsive to G-CSF, GM-CSF, and IL-6 showed significant elevation in growth factor levels 24 hours after IL-1β stimula tion. Neither IL-3 nor IL-4 mRNA was detectable at any time. In contrast, transforming growth factor-β (TGF-β) mRNA and nanogram levels of TGF-β bioactivity in the medium were detected at all times in established LTC, and these levels were not consistently altered by any of the manipulations that stimulated hematopoietic growth factor production and primitive progenitor cycling. We also found that addition of anti-TGF-β antibody could prolong or reactivate primitive progenitor proliferation when added to previously stimulated or quiescent cultures, respectively. Together, these results indicate a dominant negative regulatory role of endog- enously produced TGF-β in unperturbed LTC, with activation of primitive hematopoietic cells being achieved by mechanisms that stimulate stromal cells to produce G-CSF, GM- CSF, and IL-6. Given the similarities between the LTC system and the marrow microenvironment, it seems likely that the control of human stem cell activation in vivo may involve similar variations in the production of these factors by stromal cells.

Erythropoietic progenitors from peripheral blood of normal individuals or patients with polycythemia vera (PV) were cultured in methylcellulose medium containing 2.5 U/ml of erythropoietin (Ep). After 7-9 days, colonies considered to be early stage large bursts were individually removed, resuspended in a small volume of fresh methylcellulose medium, and then divided between 2 dishes. To one of these secondary cultures, sufficient Ep was added to bring the concentration of Ep up to ~3 U/ml. To the other was added an equal volume of medium but no Ep. The final concentration of Ep in these cultures was determined to be <0.01 U/ml. Nine days later, both types of secondary cultures were scored for the presence of colonies containing 8 or more hemoglobinized erythroblasts. Of 90 primary colonies from 3 normal individuals assessed in this way, 59 gave secondary erythroid colonies in the high Ep cultures, while none gave secondary erythroid colonies in the low Ep cultures. Additional control experiments in which primary colonies from normal individuals were divided into duplicate high Ep cultures showed that on average, the procedure used divided primary colonies equally. Of 109 primary colonies from 5 PV patients that yielded secondary erythroid colonies in the high Ep cultures, 21 yielded no secondary erythroid colonies in the low Ep cultures. The other 88 yielded erythroid colonies in both, but the secondary colonies in the low Ep cultures were consistently smaller in size and significantly fewer in number. Similar results were obtained when primary colonies were generated in cultures to which no Ep was added. These findings indicate that primitive BFU-E in patients with PV can be subdivided into 2 populations: a minor population restricted to the production of erythroid colony-forming cells (Ep-dependent progenitors) that require Ep for their detection, and a major population that is not restricted in this way. In addition, these experiments show that most of the primitive BFU-E that generate Ep-independent progenitors also produce significant numbers of cells that are Ep-dependent.