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P-3.14 Directed differentiation of iPS into imDCs under the action of SN can induce immunoreactivity in vitro

Cuixiang Xu, People's Republic of China

Third Affiliated Hospital of Medical College of Xi’an Jiaotong University, Xi’an

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Abstract

Directed differentiation of iPS into imDCs under the action of SN can induce immunoreactivity in vitro

Cuixiang Xu1,2, Zhankui Jin1, Puxun Tian2, Feng Han1, Chengguang Ding2.

1Center of Shaanxi Provincial Clinical Laboratory, Shaanxi Provincial People’s Hospital, Xi’an, People's Republic of China; 2 Kidney Transplantation, First Affiliated Hospital of Medical College of Xi’an Jiaotong University, Xi’an , People's Republic of China

Introduction: The immature dendritic cells (imDCs) do not provide active signals for effector T cells and have immunomodulatory effects because of the lack of expression of co-stimulatory molecules on its surface, which is considered a regulatory target for inducing immune tolerance. However, imDCs have limited resources and are easy to mature in vivo, which has become a bottleneck in their application. The Chinese medicine monomer sinomenine (SN) can inhibit the maturation of imDCs. In this study, induced pluripotent stem cells (iPS) were differentiated into imDCs (SN-iPS-imDCs) under the action of SN, and we observes the immunological function of SN-iPS-imDCs in vitro and the mechanism in inducing immune hyporesponsiveness.
Methods: IPS cells were cultured and expanded in a mixed system of mouse embryonic fibroblasts (MEFs) and leukemia inhibitory factor (LIF), and iPS cells were identified as monoclonal. Optimal concentration of SN and OP9 cells for seeding density were screened. iPS cells were differentiated into imDCs (SN-iPS-imDCs and iPS-imDCs) with three-steps differentiation method with or without SN, respectively. The functions and mechanisms of imDCs inducing immunoreactivity in vitro were analyzed from molecular, cellular and protein levels.
Results: The imDCs (SN-iPS-imDCs and iPS-imDCs) were successfully obtained from iPS-directed differentiation. The process of directed differentiation of iPS is divided into three stages, which took a total of 25 days. The first stage consisted of 6 days; on the 3rd day, the cells appeared the three-germ-layer structure; on the 6th day, the cell's three germ layer structure disappeared. The second stage consisted of 6 days; on the 3rd day, hematopoietic progenitor-like cells appeared, and on the 6th day, bone marrow progenitor-like cells appeared, and the CD45 and CD11b were highly expressed on the surface of the cells. On the 13th day of the third step, the imDCs were successfully obtained with heterogeneous cell morphology and protrusions on its surface, and the CD80, CD86 and MHC-II were low expressed and CD11c was high expressed on its surface; the positive rate of CD11c on the surface of SN-iPS-imDCs was significantly higher than that of iPS-imDCs (P<0.05); imDCs were stimulated by LPS for 48 hours, and the CD80, CD86, and MHC-Ⅱ positive rates on the surface of SN-iPS-DCs cells were significantly lower than those of iPS-DCs (P<0.05); the secretions of IL-2, IL-12, IFN-γ and TNF-α in the culture supernatant of SN-iPS-DCs were lower than that of iPS-DCs (P<0.05), while the secretions of IL-10 and TGF-β were higher than iPS-DCs (P<0.05); in the mixed lymphocyte response, the ability of SN-iPS-DCs to stimulate CD4+T cell proliferation was significantly lower than that of iPS-DCs (P<0.05). The proportion of CD4+CD25+Foxp3+Tregs in the SN-iPS-DCs group was significantly higher than that of iPS-DCs group (P<0.05); the early apoptosis rate of SN-iPS-DCs was significantly higher than that of iPS-DCs (P<0.05); the relative expression levels of Bax and Fas genes, actived-caspase3, actived-caspase9 and actived-PARP proteins in SN-iPS-DCs were higher than iPS-DCs. Conclusions: SN-iPS-imDCs inhibited the proliferation and activation of T lymphocytes by inducing CD4+CD25+Tregs and CD4+CD25+Foxp3+Tregs and by promoting the secretions of IL-10 and TGF-β; the transplant immune hyporesponsiveness induced by SN-iPS-imDCs, which was related to the early apoptosis.

National Nature Science Foundation of China (No. 81900686). National Nature Science Foundation of China (No. 81100179).

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