Poly(ethylene glycol)-maleimide hydrogels modulate trophoblast phenotype immunomodulation capacity

Introduction::
During pregnancy, the semi-allogeneic fetus is protected by the placenta, which is composed of differentiating trophoblasts that lack antigens to stimulate a maternal immune attack yet facilitate a specific fetal tolerance in the absence of nonspecific systemic immunosuppression. Cytotrophoblasts (CT) are the proliferative progenitor to the syncytiotrophoblasts (ST) that line the intervillous space and extravillous trophoblasts (EVT) that remodel and invade into the maternal decidua. Trophoblasts secrete tolerogenic factors to instruct tolerance and prevent immune activation; however, the distinctive phenotypes’ role in fetal tissue tolerance is insufficiently understood. It is currently difficult to study this tolerance due to physiological differences between species, functional differences between early and term trophoblasts, and ethical considerations. We employed a tunable, synthetic, 3D hydrogel platform to support trophoblast viability and function to recapitulate placenta cues that cause tolerogenic factor secretion. Our previous data showed significant effects of varying hydrogel environments on the morphology, viability, and function of three trophoblast-like cell lines compared to 2D culture; however, cancer-derived cell lines are a poor model for healthy pregnancies. Here, we tuned this in vitro culture system for human trophoblastic stem-like cells and expanded this model to study human trophoblast-immune interactions.




Materials and Methods:: CT isolated from human trophoblast stem cells were cultured in 3D hydrogel matrices made of 4-arm poly(ethylene glycol) (PEG)-maleimide macromer with adhesive ligands (RGD, RDG, GFOGER, YIGSR, IKVAV) from extracellular matrix sequences abundant in the placenta and protease-sensitive (VPM, GDQ, GPQ-W) or nondegradable (DTT, PEG-DT) crosslinkers for six days in CT media or chemically differentiated within the matrices to ST or EVT phenotypes using previously published protocols (Bai, 2021). Naturally derived hydrogels, such as Matrigel, and standard 2D culture were used as controls. Cell viability was assessed through confocal imaging (CalceinAM, ethidium homodimer) with image analysis on percent viability and average cell cluster size and metabolism through alamarBlue over six days. Day 6 CT, ST, and EVT supernatants from the various culture conditions were assessed for human chorionic gonadotropin (hCGβ) by ELISA. Day 6 supernatants from PEG-GFOGER-DTT, PEG-GFOGER-VPM, Matrigel, and 2D from each cell phenotype were assessed for chemokines and cytokines by LEGENDplex^TM and added to Jurkat T cell line cultures for 48 hours to determine differences in protein expression (CD3, CD4, CD25, CD38, CD69, CD71, CD154, HLA-DR) by flow cytometry after activation with PMA/ionomycin during the last 24 hours. Ongoing studies are analyzing trophoblast protein expression in varying culture conditions through mass spectrometry and characterizing human T, B, and NK cell protein expression from PBMCs in response to trophoblast supernatants through flow cytometry.


Results, Conclusions, and Discussions:: The metabolic activity of CT and ST with RGD and GFOGER adhesive ligands was significantly higher compared to the others tested, (Fig. 1) which corresponded to a significantly higher percent viability and larger live cluster size for CT. The average metabolic activity of EVT was also higher with RGD and GFOGER adhesive ligands; though this was not significant, EVT cultured in hydrogels with RGD and GFOGER had significantly larger average live cluster sizes. ST cultured with RGD and GFOGER adhesive ligands secreted significantly more hCGβ (Fig. 2), indicative of the ST phenotype, while CT and EVT secreted significantly less hCGβ, suggesting these hydrogels better support phenotype stability. The metabolic activity of CT and EVT cultured in nondegradable PEG-DTT hydrogels was significantly higher compared to Matrigel controls, while the metabolic activity of ST cultured in PEG-DTT and Matrigel was significantly higher than other PEG crosslinked hydrogels. Secretion of hCGβ by ST was higher than CT and EVT in each culture condition and roughly two times higher for phenotypes cultured in degradable versus nondegradable PEG hydrogels, suggesting degradable hydrogels may skew cells to a ST-like phenotype. Significantly higher IL-6 was secreted in 2D cultured CT and EVT, while IL-8 was primarily secreted by 3D cultured CT (Fig. 3), which are both important in successful pregnancies. GROα, RANTES, and I-TAC secretion was significantly higher in all 3D cultured phenotypes, 3D cultured EVT, and 2D cultured ST, respectively, suggesting phenotypic roles in immune cell recruitment to the maternal-fetal interface. Jurkat cells incubated with supernatants from the various culture conditions expressed significantly lower CD69 and CD154 in CT (all), ST (VPM, all, respectively), and EVT (VPM, 2D) groups compared to the media only controls after activation (Fig. 4). Interestingly, CD38, which has higher expression in the peripheral blood of pregnant people, was expressed significantly higher in all phenotype groups of all culture conditions without activation. Synthetic hydrogels support the viability, metabolic activity, and immunomodulatory secretion of the three human trophoblast phenotypes, which provide a platform to study the immunomodulatory powers of trophoblasts that will be applied to allogeneic transplant therapies in the future.


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References (Optional): :
Bai, T., Peng, C. Y., Aneas, I., Sakabe, N., Requena, D. F., Billstrand, C., Nobrega, M., Ober, C., Parast, M., & Kessler, J. A. (2021). Establishment of human induced trophoblast stem-like cells from term villous cytotrophoblasts. Stem cell research, 56, 102507. https://doi.org/10.1016/j.scr.2021.102507