Although myeloid and lymphoid lineage cells have shown to be derived from different hematopoietic precursors, some previous studies have identified the existence of biphenotypic myeloid/lymphoid cells (Ford et al., 1992
; Graf et al., 1999
; Zeisig et al., 2003
). The data present here demonstrate that ONP cells can differentiate into both myeloid and lymphoid lineages under different culture conditions. In this study, cells with multipotential properties were analyzed phenotypically by anti-mouse B220 and CD11b after culture in a timely and ordered manner. The observations obtained from our experiments illustrated that these cells first differentiated into two cell populations; B220−
precursors, which differentiated into a lymphoid lineage and B220−
precursors, which differentiate into myeloid cells.
Moreover, in vitro
culturing of progenitor B cells requires stromal cell support (Rolink et al., 2000
). Cells without stromal cell support will either undergo apoptosis or failure to further differentiate into a B lineage (Crooks et al., 2000
; Gimble et al., 1993
; Nutt et al., 1999
; Rolink et al., 2000
). Our results showed that ONP cells first differentiated into a stage which contained both B220−
cells. It is the B220−
cells, which function as stromal cells, supporting the B220−
cells differentiation into B cells.
Previous studies showed that alcohol can affect the ONP cell differentiation into a B lineage both in vitro
and in vivo
(Wang et al., 2006
). However, it is important to know; (a) whether alcohol affects cell differentiation at an early stage, late stage or on both stages, (b) does the effect of alcohol on B-cell differentiation temporary or permanent and (c) the mechanism by which alcohol affects the progenitor B-cell differentiation.
To determine whether alcohol affects cell differentiation at an early or late stage, 100 mM of alcohol was added at different time points during the cell culture. The100 mM concentration of alcohol was used in this study, because it has no direct cytotoxicity to ONP cells and will not affect the proliferation of ONP cell cultures in vitro
(Wang et al., 2006
). Results of this experiment showed that alcohol does not affect the early differentiation of ONP cells until the B220−
stage. Alcohol-exposed B220−
cells were not the same as undifferentiated ONP cells since they started to lose the expression of SCF receptor (c-kit) on their surface indicating cells underwent further differentiation. However, our results showed that the exposure of 100 mM alcohol affects the ONP cell differentiation to a B lineage. Significant reduction in B lymphocytes after 12 days indicates that alcohol affects the ONP cell differentiation in the late stage only. The effect of alcohol on the late stage ONP cell differentiation was permanent.
Further investigation on the molecular basis of B lineage commitment was performed by measuring the transcription factor EBF and Pax5 and cytokine receptor IL-7Rα among these ONP cells. EBF and Pax5 are believed to be the important TFs in the development of B lymphocytes (Busslinger et al., 2000
; Medina and Singh, 2005
). Previous studies showed that maturation of B cells was severely blocked in B progenitors of transgenic mice lacking of EBF or Pax5 (Kee and Murre, 1998
; Nutt et al., 1999
). It is still uncertain whether cells will strictly differentiate into B lineage with the Pax5 gene turned on. Current experimental evidence indicates that Pax5 expression within cells does not block the early myeloid lineage, or natural killer cell development (Cotta et al., 2003
). Moreover, Pax5 can be reversibly switched in immature hematopoietic progenitors (Okubo et al., 2002
). Based on the results of these studies, it is possible that the expression of TFs can be dynamically modulated within multipotential progenitors by environmental factors in favor of a specific lineage commitment. Our findings seem to support this hypothesis. Among ONP cells, directly sorted from mouse bone marrow, EBF and Pax5 message RNA were measured by using quantitative real-time RT-PCR. After 3 days, culture with the addition of stem cell reagent including SCF, FL and IL-3, which favors the myeloid lineage in vitro
, both EBF and Pax5 are down-regulated. Compared with their phenotypes, the majority of cultured cells showed CD11b positive as well. With exposure to IL-7, which favors B lineage commitment, EBF and Pax5 were up-regulated rapidly. Meanwhile, without the addition of IL-7, Pax5 was undetectable. This dynamic switch of the expression level of EBF and Pax5 is likely to address, at least partially, the molecular basis of B lineage differentiation in ONP cells. However, the addition of 100 mM of alcohol to an in vitro
culture down-regulates the expression level of TF EBF, causing minimal expression of Pax5. Low Pax5 expression results in the paucity of B-cell commitment.
In conclusion, our data shows that development of myeloid or lymphoid cells from ONP cells diverges through B220−CD11b+/ B220−CD11b− branch points. Expression of transcription factor EBF and Pax5 does not block early myeloid lineage commitment. The microenvironment that favors the specific lineage differentiation seems to play a critical role. With the exposure of IL-7, EBF and Pax5 reversibly switch to a high level and progenitor cells generate a B lineage. However, the exposure to 100 mM of alcohol, at a late stage of ONP cell differentiation in vitro, severely blocks the expression of TFs, resulting in impairment of B-cell differentiation. Taken together, these results may indicate the common developmental pathway and the molecular basis of myeloid/lymphoid divergence among ONP cells.