Notch signals are required for in vitro but not in vivo maintenance of human hematopoietic stem cells and delay the appearance of multipotent progenitors.

All blood cell lineages start from hematopoietic stem cells (HSCs), which were recently shown to represent a heterogeneous group of cells. In mice, Notch signaling promotes the maintenance of "stemness" as well as the expansion of self-renewing HSCs in vitro. Additionally, human CD34(+) cells were shown to expand in vitro in response to Notch signals. However, it is unclear whether Notch directly affects all HSCs, and whether this role is relevant in vivo. Here, we developed culture conditions that support the maintenance of CD34(+)CD133(+)CD90(low)CD38(-)CD7(-)CD10(-)CD45RA(-) (CD90(low)) cells, phenotypically defined HSCs, as well as 2 early progenitor cells (CD34(+)CD38(-)CD7(-)CD10(-)CD45RA(int) [RA(int)] and CD34(+)CD38(-)CD7(-)CD10(-)CD45RA(hi) [RA(hi)]) that were functionally equivalent to multipotent progenitor-2 and lymphoid-primed multipotent progenitor, respectively, found in cord blood. Using a genetic approach, we show that Notch signals were required for HSC preservation, with cultured HSCs being equal to ex vivo HSC cells in their ability to reconstitute immunodeficient mice; however, dnMaml-transduced HSCs were not maintained in vitro. Interestingly, Notch signaling did not appear to be required for the self-renewal of human HSCs in vivo. Our findings support the notion that Notch signals maintain human HSCs in vitro that have hematopoietic-reconstituting ability in vivo and delay the appearance of 2 newly described early progenitor cells.

HES1 opposes a PTEN-dependent check on survival, differentiation, and proliferation of TCRß-selected mouse thymocytes.

The developmental progression of immature thymocytes requires cooperative input from several pathways, with Notch signals playing an indispensable role at the T-cell receptor (TCR)-ß selection checkpoint. Notch signals affect the activation of the PI3K/Akt pathway, which is required for pTα/TCRß (pre-TCR)-induced survival, differentiation, and proliferation of developing αß-lineage thymocytes. However, the molecular players responsible for the interaction between the Notch and PI3K pathways at this critical developmental stage are unknown. Here, we show that Notch induction of Hes1 is necessary to repress the PI3K/Akt pathway inhibitor, PTEN (phosphatase and tensin homolog), which in turn facilitates pre-TCR-induced differentiation. In support of this mechanism, deletion or down-regulation of Pten overcomes the Notch signaling requirement for survival and differentiation during ß-selection. In addition, c-Myc is a critical target of Notch at this stage, as c-Myc expression overcomes the Notch signaling requirement for proliferation during ß-selection. Collectively, our results point to HES1, via repression of PTEN, and c-Myc as critical mediators of Notch function at the ß-selection checkpoint.

Stage-specific and differential notch dependency at the alphabeta and gammadelta T lineage bifurcation.

Signals transduced by Notch receptors are indispensable for T cell specification and differentiation of alphabeta T lineage cells. However, the role of Notch signals during alphabeta versus gammadelta T lineage decision remains controversial. Here, we addressed this question by employing a clonal analysis of CD4(-)CD8(-) (DN) progenitor potential to position the divergence of alphabeta and gammadelta T cell lineages to the late DN2 to DN3 developmental stages. Accordingly, alphabeta and gammadelta precursor frequencies within these T cell progenitor subsets were determined, both in the presence and absence of Notch signaling through Delta-like 1. Notch signals were found to be critical for the DN to CD4(+)CD8(+) (DP) transition, irrespective of the identity (pTalphabeta or gammadelta) of the inducing T cell receptor complex, whereas gammadelta T cells developed from gammadeltaTCR-expressing T cell progenitors in the absence of further Notch ligand interaction. Collectively, our findings demonstrate a differential, stage-specific requirement for Notch receptor-ligand interactions in the differentiation of alphabeta and gammadelta T cells from T cell progenitors.