Study of immune‐tolerized cell lines and extracellular vesicles inductive environment promoting continuous expression and secretion of HLA‐G from semiallograft immune tolerance during pregnancy

Abstract

An immune reaction is a protector of our body but a target to be overcome for all non-self-derived medicine. Extracellular Vesicles (EVs), noted as a primary alternative to cell therapy products that exhibit immune rejection due to mismatching-major histocompatibility complex (MHC), were discovered to have excellent curative effects through the delivery of various biologically active substances. Although EVs are sure to incur immune reaction by immunogenicity due to alloantigens from their parental cells, their immune rejection is rarely known. Hence, to develop cell lines and EVs as medicines with no immune rejection, we noted the immune tolerance where the foetus, as semiallograft, is perfectly protected from the maternal immune system. We designed the ex-vivo culture systems to simulate in-vivo environmental factors inducing extravillous trophoblast (EVT)-specific Human Leukocyte Antigen-G (HLA-G) expression and secretion of HLA-G-bearing EVs at the motherfoetus interface. Using them, we confirmed that immune-tolerized stem cells (itSCs) continuously expressing and secreting HLA-G like EVTs during pregnancy can be induced. Also, EVs secreted from itSCs are verified as immune-tolerized EVs (itSC-EVs) containing HLA-G and not causing immune rejection through various analytical methods. These findings can provide a new perspective on the local and extensive immune tolerance environment where HLA-G is expressed and secreted by pregnancy-related hormones and different biological conditions. Furthermore, they show the new way to develop itSCs-EVs-based therapeutics that are free from time, space, and donor limitation causing immune rejection.

The Different Immune Responses by Age Are due to the Ability of the Fetal Immune System to Secrete Primal Immunoglobulins Responding to Unexperienced Antigens

“…Human amniotic mesenchymal stem cells (hAM-MSCs) and human umbilical cord blood mesenchymal stem cells (UCB-MSCs) were purchased from ScienCell (Carlsbad, CA, USA). Human amniotic fluid-derived mesenchymal stem cells (hAF-MSCs) were established as described in the previous study [22]. These MSCs were maintained in DMEM medium (Welgene, Republic of Korea) supplemented with 100 U/mL of penicillin, 100 μg/mL of streptomycin, and 10% heat-inactivated FBS.…”

“…An in vivo-like culture condition for inducing immune-tolerized cell lines was applied using temperature change, pH, and circulation conditions established in the previous study [22]. In summary, temperature change between 36.0 ℃ and 37.0 ℃ with a five or six-day cycle and an acidic pH of 6.2 to 6.8 by hyaluronic acid (HA)-based matrix were applied to induce the secretion of various pregnancy-related hormones as shown in Figure 2C.…”

“…To increase the efficiency of the subsequent stage filtration and recovery a high yield of EVs, multi-stage filtration method was applied, established in the previous study [22]. In summary, the cell culture supernatant, centrifuged at 1,500 RPM for 5 min, was filtered with a 0.8-μm filter to remove cell debris and apoptotic bodies completely, and the supernatant was filtered with a 0.45-μm filter twice.…”

“…The conditioned medium obtained from serumfree co-cultivation of hAM-MSCs and hAF-MSCs in multi-dish plate, as described in the previous study [22], was centrifuged at 1,500 RPM for 5 min at the room temperature, and the supernatant was filtered with a 0.45-µm filter after multi-stage filtration to obtain AF/AM-MSC CO -EVs.…”

A Brief Analysis of Tissue-Resident NK Cells in Pregnancy and Endometrial Diseases: The Importance of Pharmacologic Modulation

“…Progesterone's effect on different cell types and trophoblast, like the newly described variety of trophoblast expressing HLA-G, is unknown [113]. However, in this case, the critical element modulates inhibitory signals through the KIR receptors, which can be crucial for embryo implantation and successful pregnancy.…”