Human mesenchymal stem cells license adult CD34+ hemopoietic progenitor cells to differentiate into regulatory dendritic cells through activation of the Notch pathway.

The mechanisms underlying the immunomodulatory functions of mesenchymal stem cells (MSC) on dendritic cells (DC) have been shown to involve soluble factors, such as IL-6 or TGF-beta, or cell-cell contact, or both depending on the report referenced. In this study, we intend to clarify these mechanisms by examining the immunosuppressive effect of human adult MSC on adult DC differentiated from CD34(+) hemopoietic progenitor cells (HPC). MSC have been shown to inhibit interstitial DC differentiation from monocytes and umbilical CD34(+) HPC. In this study, we confirm that MSC not only halt interstitial DC but also Langerhans cell differentiation from adult CD34(+) HPC, as assessed by the decreased expression of CD1a, CD14, CD86, CD80, and CD83 Ags on their cell surface. Accordingly, the functional capacity of CD34(+) HPC-derived DC (CD34-DC) to stimulate alloreactive T cells was impaired. Furthermore, we showed that 1) MSC inhibited commitment of CD34(+) HPC into immature DC, but not maturation of CD34-DC, 2) this inhibitory effect was reversible, and 3) DC generated in coculture with MSC (MSC-DC) induced the generation of alloantigen-specific regulatory T cells following secondary allostimulation. Conditioned medium from MSC cultures showed some inhibitory effect independent of IL-6, M-CSF, and TGF-beta. In comparison, direct coculture of MSC with CD34(+) HPC resulted in much stronger immunosuppressive effect and led to an activation of the Notch pathway as assessed by the overexpression of Hes1 in MSC-DC. Finally, DAPT, a gamma-secretase inhibitor that inhibits Notch signaling, was able to overcome MSC-DC defects. In conclusion, our data suggest that MSC license adult CD34(+) HPC to differentiate into regulatory DC through activation of the Notch pathway.

Distinct effects of the soluble versus membrane-bound forms of the notch ligand delta-4 on human CD34+CD38low cell expansion and differentiation.

Although Notch ligands are considered to activate signaling through direct cell-cell contact, the existence of soluble forms has been demonstrated. However, their roles remain controversial: soluble forms have been reported to mimic the biological activity of membrane-bound form, whereas other studies rather suggested an antagonistic activity toward their full-length counterparts. We previously observed that membrane-bound Delta4-expressing S17 stroma (mbD4/S17) reduced human CD34+CD38(low) cell proliferation and favored self-renewal. Here, we assessed the effects of a soluble form of Delta4 (solD4) by exposing CD34+CD38(low) cells to S17 feeders engineered to express solD4 (solD4/S17). In contrast to mbD4/S17, (a) solD4/S17 increased 10-fold cell production after 2 weeks, through enhanced cell proliferation, and (b) it did not preserve colony-forming cell and long-term culture-initiating cell potential of output CD34+ cells. mbD4 and solD4 appeared to also differ in their signaling. Indeed, mbD4, but not solD4, strongly activated both CSL (the nuclear mediator of Notch signaling) in Hela cells overexpressing Notch1 and transcription of some classic Notch target genes in CD34+CD38(low) cells. Furthermore, both biological effects and CSL activation elicited by mbD4 were strictly dependent upon the gamma-secretase complex, whereas solD4 enhanced cell expansion in a partially gamma-secretase-independent manner. Altogether, these results suggest that part of solD4 activity did not rely upon canonical Notch pathway.

Notch/Delta4 interaction in human embryonic liver CD34+ CD38- cells: positive influence on BFU-E production and LTC-IC potential maintenance.

We investigated whether Notch signaling pathways have a role in human developmental hematopoiesis. In situ histochemistry analysis revealed that Notch1, 2, and 4 and Notch ligand (Delta1-4, and Jagged1) proteins were not expressed in the yolk sac blood islands, the para-aortic splanchnopleure, the hematopoietic aortic clusters, and at the early stages of embryonic liver hematopoiesis. Notch1-2, and Delta4 were eventually detected in the embryonic liver, from 34 until 38 days postconception. Fluorescence-activated cell sorter analysis showed that first-trimester embryonic liver CD34(+)CD38(low) cells expressed both Notch1 and Notch2. When these cells were cultured on S17 stroma stably expressing Delta4, a 2.6-fold increase in BFU-E number was observed at day 7, as compared with cultures with control stroma, and this effect was maintained for 2 weeks. Importantly, exposure of these cells to Delta4 under these conditions maintained the original frequency and quality of long-term culture-initiating cells (LTC-ICs), while control cultures quickly resulted in the extinction of this LTC-IC potential. Furthermore, short-term exposure of embryonic liver adherent cells to erythropoietin resulted in a dose-dependent increase in Delta4 expression, almost doubling the expression observed with untreated stroma. This suggests that Delta4 has a role in the regulation of hematopoiesis after a hypoxic stress in the fetus.