The Role of Transforming Growth Factor-B₁ in Murine Fetal Liver Hematopoiesis

Abstract

The liver and spleen are the major sites of hematopoiesis during mammalian development. Transforming growth factor-β1 (TGF-131) is a naturally produced growth factor known to affect the regulation of adult hematopoietic cells and to be involved in embryogenesis. Receptors for TGF-β1 are ubiquitous and the protein is produced by a variety of cells. Given the above information, the purpose of this analysis was to examine the role of TGF-β1 in murine fetal hematopoiesis through soft agar assays, liquid culture morphological analysis and Northern analysis. The presence of hematopoietic progenitor cells as assayed by soft agar analysis was performed on 16-day fetal and 5-day-old neonatal spleen cells and, also on 16 and 21-day fetal liver cells and 5-day-old neonatal liver cells. This analysis identified a decrease in the number of progenitor cells responding to interleukin-3 (IL-3), macrophagecolony stimulating factor (M-CSF) and granulocyte-macrophage CSF (GMCSF) in both the liver and spleen from 16 days gestation to 5 days postbirth. Also, greater numbers of progenitors responded to the growth factor signals in the spleen than in the liver at corresponding gestational ages. TGF-β1 did not support colony formation in any system tested and inhibited colony formation stimulated by IL-3 and M-CSF. In contrast, TGF-β1 increased the number (5-7-fold) and size of GM-CSF stimulated colonies in all fetal and neonatal tissues examined. Twenty-one-day fetal liver cells incubated in the absence of exogenous growth factors showed no increase in the number of progenitors responding to GM-CSF. However, cells responding to GM-CSF-plus-TGF-β1 displayed a 2-fold increase in responsive progenitor cells after 1 week in culture followed by a decrease upon further incubation. The effects of TGF-β1 on the differentiation of 21-day fetal liver cells in liquid culture were analyzed by morphological examination of the cultured cells. Cells incubated in TGF-P1 for 1 week displayed a high percentage of macrophages (85%) compared to the untreated control, which consisted of approximately equal numbers of granulocytes and monocytes. Cells incubated with GM-CSF were morphologically similar to the control culture but proliferated 33% above the control culture. Cells treated with GM-CSF-plus-TGF-β1 proliferated 167% (above the control) and contained an increased proportion of early myeloid progenitor cells. Cultures incubated with IL-3 contained basophilic granulocytes (70%) and macrophages (25%), while those treated with TGFβ1-plus-IL-3 contained fewer basophilic granulocytes (40%) and more macrophages (40%). These data suggest that TGF-β1 favors the development of the monocyte lineage in liquid cultures of 21-day fetal liver cells. Whether this is due to differentiation or survival of certain cell types cannot be deduced from these data. Also, production of endogenous growth factors by the cultured cells may have an effect on their differentiation. Northern analysis of liver cells from different gestational ages indicated that TGF-β1 mRNA is temporally regulated in the developing fetus. TGF-β1 mRNA decreases in abundance as the fetus matures from the 16th gestational day to the fifth day post-birth, especially with regard to the 2.5 kilobase (Kb) species. These data clearly demonstrate that TGF-β1 regulates murine fetal hematopoiesis in vitro and suggest that TGF-β1 may play a role in regulating murine fetal hematopoietic events in vivo. The results presented here have opened new avenues for research and raised additional questions regarding the role of growth factors, especially TGF-β1, in regulating mammalian fetal hematopoietic events.