Ethanol exhibits specificity in its effects on differentiation of hematopoietic progenitors.

Ethanol is a known teratogen but the mechanisms by which this simple compound affects fetal development remain unresolved. The goal of the current study was to determine the mechanism by which ethanol affects lymphoid differentiation using an in vitro model of ethanol exposure. Primitive hematopoietic oligoclonal-neonatal-progenitor cells (ONP), with the phenotype Lin(-)HSA(lo)CD43(lo)Sca-1(-)c-Kit(+) that are present in neonatal but not adult bone marrow were sorted from the bone marrow of 2-week-old C57BL/6J mice and cultured under conditions that favor either B cell or myeloid cell differentiation with or without addition of ethanol. The overall growth of the ONP cells was not significantly affected by inclusion of up to 100mM ethanol in the culture medium. However, the differentiation of the progenitor cells along the B-cell pathway was significantly impaired by ethanol in a dose-dependent manner. Exposure of ONP cells to 100mM ethanol resulted in greater than 95% inhibition of B cell differentiation. Conversely, ethanol concentrations up to and including 100mM had no significant effect on differentiation along the myeloid pathway. The effect of ethanol on transcription factor expression was consistent with the effects on differentiation. ONP cells grown in 100mM ethanol failed to upregulate Pax5 and EBF, transcriptional regulators that are necessary for B cell development. However, ethanol had no significant effect on the upregulation of PU.1, a transcription factor that, when expressed in high concentration, favors myeloid cell development. Taken together, these results suggest that ethanol has specificity in its effects on differentiation of hematopoietic progenitors.

In utero exposure to alcohol alters cell fate decisions by hematopoietic progenitors in the bone marrow of offspring mice during neonatal development.

Fetal alcohol syndrome and alcohol related birth defects represent a spectrum of disorders that can result from the consumption of alcohol during pregnancy. Previous studies from this laboratory have shown that alcohol exposure in utero adversely affects hematopoietic progenitors in the bone marrow. Neonatal mice that were exposed in utero to alcohol showed a marked delay in B lymphocyte development. Recent studies have focused on an oligopotential progenitor cell, with the phenotype of HSA(lo)CD43(lo)Lin(-), which yields both B cells and myeloid lineage cells at a high frequency when cultured in vitro with stromal cells and the appropriate cytokines. However, these progenitor cells isolated from neonatal offspring of alcohol fed dams showed a significant decrease in the frequency of B cell formation following in vitro culture. In order to understand the mechanism underlying this defect we examined the expression of key transcription factors (early B cell factor, EBF, and Pax5) in this progenitor pool. Here, we report that >95% of HSA(lo)CD43(lo)Lin(-) cells express EBF and 5% express Pax5. Following liquid culture in the presence of IL-7, these progenitor cells respond by up-regulating Pax5 and the surface expression of CD19 indicating that the cells have committed to the B lineage. By contrast 75% of HSA(lo)CD43(lo)Lin(-) cells isolated from the bone marrow of neonatal animals exposed in utero to alcohol expressed EBF but at a level that was less than 25% the level of cells isolated from control animals. Furthermore, these alcohol-exposed progenitor cells failed to up-regulate Pax5 in response to IL-7 indicating a greatly reduced capacity to expand and differentiate to B lineage cells in liquid cultures. However, the HSA(lo)CD43(lo)Lin(-) cells isolated from the alcohol exposed animals retained the capacity to differentiate to myeloid lineage cells. These results suggest that the interference with the sequential expression of transcription factors in early progenitor cells by in utero alcohol exposure is a potential mechanism for the observed decrease in B lymphocytes in neonatal mice.