| Microenvironment plays a crucial role in regulating the differentiation and function of immune cells. Following the development of transplantation medicine, more and more researchers focus on the alternatively activated dendritic cells (AADC) induced by anti-inflammatory agents as IL-10, TGF-βand PGE2, and AADC are specially immune tolerant. Dendritic cells (DC) are derived from bone marrow and educated by hematopoietic microenvironment. While mesenchymal stem cells (MSC) have been regarded as progenitors of hematopoietic microenvironment, an important question that remains unanswered is whether MSC medicate AADC development and immune function. Here, we investigated the effect of MSC on the differentiation of CD34+ progenitors into AADC with respect to their phenotypes and functions.Firstly, to address whether MSC impact DC differentiation from CD34+ cells, DC (also as MSC-DC) was separated by coincubation of CD34+ cells with MSC induction system. MSC-DC was then compared with immature or mature DC and AADC induced by IL-10 according to the method documented previously, and studied their morphology, phenotype and function. The results showed that MSC-DC displayed many pseudopodia in morphology, lower expression of CD86, CD80 and HLA-DR, and reduction of MSC-DC mediated proliferation of allogeneic T cells. MSC-DC formed tube-like and network structures in Matrigel in vitro. All these data described strongly indicated that MSC-DC was AADC resembling those from other sources. Furthermore, MSC-DC seemed as the mature DC since it was not capable of phagocyte FITC-Dextran and expression of CCR7 and CXCR4.Secondly, we investigated the effect of MSC on AADC development and its modulation mechanism. As previous reports, Notch pathway plays an important role in the DC development. Using RNA interfere technology, gene Jagged1 in MSC was knocked down, and then explored the interaction between MSC and AADC. Here, Notch receptor and ligand were tested because they are the essential factors in the Notch signaling pathway. After cocultured of MSC and CD34+ cells, Notch receptor and ligand showed a significantly change by real-time PCR assay. Moreover, to knock down Jagged1-MSC (also as Jag1-MSC) was obtained by GFP lentivirus transfection system. Hes1 is one of the main target genes regulated by Notch. Hes1 mRNA expression was significantly decreased after incubation of CD34+ cells with Jag1-MSC (also as Jag1-MSC-DC). As expected, Jag1-MSC-DC increased CD83, CD86, HLA-DR expression and IL-12 for Th1 cytokine secretion, but decreased secretion of IL-10 for Th2 cytokine. Functionally, Jag1-MSC-DC resulted in increased allostimulatory capacity by alloreactive T cells. In a word, these results indicate that Notch signaling pathway play a critical role in the impairment of AADC differentiation and function by MSC. On the other hand, the MAPK pathway plays a key role in the signal transduction of stem cell proliferation and differentiation. Whether Notch pathway deficiency affected the activation of MAPK pathway was examined next. The results showed that the phosphorylation of ERK was significantly increased in Jag1-MSC by coculture induction system, which implicated MAPK signal activation after Notch signal blocked.Finally, we evaluated whether the differentiation of AADC impacts the biological characteristics of MSC. Cocultured with AADC, MSC showed no morphological change, but down-regulated the expression of CD29, CD73, HLA-ABC and CD86. RT-PCR assay demonstrated that MSC could be increased the expression of TGF-β, G-CSF, M-CSF and GM-CSF, but lost potential to differentiate into adipocyte. The results imply that MSC is enhanced its ability of immune modulation by DC stimulation.In conclusion, our data suggested for the first time that the differentiation of CD34+ cells regulated by mesenchymal stem cell through activation of the Notch signal pathway, providing a novel idea and theory instruction for understanding the DC development and clinical therapy.
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