Molecular Evidence for Hierarchical Transcriptional Lineage Priming in Fetal and Adult Stem Cells and Multipotent Progenitors

Abstract

Recent studies implicated the existence of adult lymphoid-primed multipotent progenitors (LMPPs) with little or no megakaryocyte-erythroid potential, questioning common myeloid and lymphoid progenitors as obligate intermediates in hematopoietic stem cell (HSC) lineage commitment. However, the existence of LMPPs remains contentious. Herein, global and single-cell analyses revealed a hierarchical organization of transcriptional lineage programs, with downregulation of megakaryocyte-erythroid genes from HSCs to LMPPs, sustained granulocyte-monocyte priming, and upregulation of common lymphoid (but not B and T cell-specific) genes. These biological and molecular relationships, implicating almost mutual exclusion of megakaryocyte-erythroid and lymphoid pathways, are established already in fetal hematopoiesis, as evidenced by existence of LMPPs in fetal liver. The identification of LMPPs and hierarchically ordered transcriptional activation and downregulation of distinct lineage programs is compatible with a model for HSC lineage commitment in which the probability for undergoing different lineage commitment fates changes gradually when progressing from HSCs to LMPPs.

Down-regulation of Mpl marks the transition to lymphoid-primed multipotent progenitors with gradual loss of granulocyte-monocyte potential

“…10,12 To investigate whether LSKFlt3 hi cells with CFU-S potential could be separated from LSKFlt3 hi LMPPs without such activity, we analyzed previous Affymetrix array results. 15 Whereas we had shown that most investigated genes affiliated with the Mk and E lineages are no longer expressed in LSKFlt3 hi cells, we did find that Mpl was still expressed in a fraction of LSKFlt3 hi cells, although at reduced levels and frequencies when compared with LSKCD34 Ϫ Flt3 Ϫ and LSKCD34 ϩ Flt3 Ϫ cells. 10,15 Of further note, the fraction of BM LSKFlt3 hi cells expressing Mpl coexpressed genes of the GM lineage but predominantly not of the lymphoid lineage, in contrast to LSKFlt3 hi cells negative for Mpl, which frequently coexpressed lymphoid and GM genes.…”

“…15 Furthermore, a similar pattern of multilineage transcriptional priming could be observed in fetal LSKFlt3 hi cells with the distinction that a larger fraction of LSKFlt3 hi cells from the fetal liver were transcriptionally primed compared with LSKFlt3 hi adult BM cells. 15 Thus, we also compared the multilineage transcriptional priming of LSKFlt3 hi Mpl Ϫ and LSKFlt3 hi Mpl hi fetal liver (E14.5-E15.5) cells. The expression pattern of Mpl in relationship to Flt3 was comparable to that in adult BM, allowing purification of equivalent LSKFlt3 hi Mpl hi and LSKFlt3 hi Mpl Ϫ populations ( Figure 3B).…”

“…3,10,12,14,15 Further, molecular analysis of putative LMPPs show down-regulated transcriptional priming of genes specific for the MkE lineage, and up-regulation of lymphoid-specific genes, not yet expressed in HSCs. 15,16 The identification of a putative LMPP, representing the earliest lineage-restricted lympho-myeloid progenitor identified in adult hematopoiesis, has provided a potential avenue toward uncovering alternative HSC lineage commitment pathways. 2,3,17 However, the existence of the LMPP remains contentious, 2,3,[18][19][20] largely reflecting functional heterogeneity of phenotypically defined candidate LMPPs.…”

“…To establish an optimal time-point for read-out of each cell population investigated, cells were cultured for 4, 6, 8, 10, or 12 days, at which time erythroid potential was evaluated using 2,7-diaminofluorene staining (DAF; Sigma-Aldrich, St Louis, MO). 15,25 DAF-positive cells were identified as cells with intracellular blue granules and LSKFlt3 hi Mpl Ϫ -derived pure GM clones (generated in methylcellulose [M3134; StemCell Technologies] supplemented with cytokines, but not hEPO or hTHPO) were used as a negative control to confirm the specificity of DAF staining.To evaluate the individual GM, T, and B lineage potentials of the different LSKFlt3 hi subpopulations, single cells were seeded by an automated cell deposition unit (ACDU) coupled to a BD FACSAria (providing …”

c-Kit is required for growth and survival of the cells of origin of Brca1-mutation-associated breast cancer

“…Crucially, however, gene expression analysis and clustering demonstrated that both the basal CD49f Low c-Kit þ and basal CD49f High c-Kit þ cells were in fact most closely related to the MaSCs. This suggests that the occasional c-Kit þ basal cells may be variant MaSCs undergoing 'lineage priming', in which stem/progenitor cells express genes associated with their differentiated daughter populations (Huang et al, 2007;Mansson et al, 2007). Such cells are unlikely to comprise a stable population, but will revert to a canonical stem cell state if they do not receive signals that promote their differentiation.…”