Immunoregulatory Properties of Apoptotic Human Amniontic Mesenchymal Stromal Cells
Authors: Xu Ying , Gao Ya , Yang Yan chun , Zhu Dongmao , Zhang Yintian , Zhao Ke , Zhang Yu , Liu Qifa , Sun Haitao , Ping Baohong 
Published: October 2019

Abstract
Objective Mesenchymal stromal cells (MSCs) have been used in preventing and treating acute graft-versus-host disease (aGVHD), but the mechanism is not fully understood. Apoptotic bone marrow mesenchymal stromal cells (BMSCs) were showed could induce vivo recipient-mediated immunomodulation in mice GVHD model. We had demonstrated that, similar to BM-MSCs, human amniontic mesenchymal stromal cells (hAMSCs) exhibit potent immunosuppressive and anti-inflammatory activities but possess a higher proliferation activity and clearer stem cell properties in vitro. This study focuses on the immunoregulatory properties of apoptotic human amniontic mesenchymal stromal cells (apo-hAMSCs) in an inflammatory microenvironment.
Methods hAMSCs from human amniotic membrane were cultured with tissue mass cell culture. The cell phenotype of the 3rd passage were detected by flow cytometry. Transwell co-culture experiments and cell-cell contact co-culture experiments were conducted, consisting of hAMSCs and peripheral blood mononuclear cells stimulated with phytohemagglutinin (PHA-PBMCs), as the positive control group. While other groups were PBMCs without PHA and hAMSCs(PBMCs+hAMSCs), PBMCs and PHA (PHA-PBMCs), hAMSCs and PBMCs. For apoptosis evalution, the morphological features of hAMSCs were recorded in different groups, and apo-hAMSCs were analyzed by flow cytometry at 24 hours. The production of Interferon-γ (IFN-γ), transforming growth factor-β1 (TGF-β1), prostaglandin E2 (PGE-2), soluble human leukocyte antigen G (sHLA-G), Tumor necrosis factor-α(TNF-α) and interleukin-17A (IL-17A) in the co-culture supernatant was detected using enzyme-linked immunosorbent assay (ELISA), and kynurenine were dectected by spectrophotometer. CD4+CD25+FOXP3+ regulatory T cells (Tregs) in PBMC were analyaed by flow cytometry.
Result hAMSCs expressed CD105, CD73, CD90, while not CD19, CD34, CD45, CD11b, HLA-DR. In the group of hAMSCs and PHA-PBMCs, the number of hAMSCs reduced. The morphological features were that cells shrinked, turned round, separated from the bottle and suspended in supernatant. However, hAMSCs in the groups of hAMSCs+PBMCs and hAMSCs stayed the same. Apoptosis in hAMSCs cultivated with PHA-PBMCs via transwell or cell-cell contact experiment increased compared with the group of hAMSCs+PBMCs (P<0.05) and hAMSCs (P<0.05). In the two co-culture experments, the secretion level of PGE-2, TGF-β1, sHLA-G, and KYN significantly increased in hAMSCs with PHA-PBMCs compared with hAMSC (P<0.05) and hAMSCs with PBMCs (P<0.05). The level of IFN-γ and TNF-α decreased in hAMSCs with PHA-PBMCs compared with PBMCs with PHA (P<0.05). While the level of IL-17A was significantly increase in hAMSCs with PHA-PBMCs compared with hAMSCs (P<0.05), hAMSCs with PBMCs (P<0.05) and PHA-PBMCs (P>0.05). Evident difference of CD4+CD25+FOXP3+ Tregs was shown between hAMSCs with PHA-PBMCs and PHA-PBMCs (P<0.05).
Conclusion Activated, but not resting, PBMCs induce extensive early apoptosis in hAMSCs. And apoptosis in hAMSCs need inflammatory microenvirenment. Apoptotic hAMSCs still have immunoregulatory effects in cytokines and immune cells.
Funding This work was supported by Natural Science Foundation of China (81701243), Key Sci-Tech Research Projects of Guangdong Province (2014A02021102), Natural Science Foundation of Guangdong Province, China (2014A030310373), the Pearl River S&T Nova Program of Guangzhou (201710010047).

Disclosures
No relevant conflicts of interest to declare.