60 - Assessing the Effects of Tunicamycin in Trophoblast Stem Cell Differentiation

Publication Number: 2057.60

Kay Chi Pua, McGovern Medical School at the University of Texas Health Science Center at Houston, Houston, TX, United States; Kadeshia Campbell, Campbell & Co Scientific, Dickinson, TX, United States; Madison Vu, McGovern Medical School at the University of Texas Health Science Center at Houston, Houston, TX, United States; Tina O. Findley, McGovern Medical School at the University of Texas Health Science Center at Houston, Houston, TX, United States; Jacqueline Parchem, McGovern Medical School at the University of Texas Health Science Center at Houston, Houston, TX, United States

Background: Placental dysfunction underlies numerous neonatal conditions, including preeclampsia, fetal growth restriction, preterm birth, and stillbirth. A population of progenitors known as trophoblast stem cells (TSCs) maintain proper function of the placenta through the generation of syncytiotrophoblasts - the cells that form the specialized epithelial barrier of the placenta responsible for nutrient and gas exchange and hormone production. While TSC depletion occurs with normal placental maturation, cellular stress can accelerate differentiation and contribute to premature loss of placental function.

Objective: We hypothesize that endoplasmic reticulum (ER) stress accelerates TSC differentiation and promotes premature placental aging. Our objective was to determine a dosing strategy for tunicamycin, a drug that causes ER stress, to induce differentiation of human TSCs.

Design/Methods: Human TSCs (CT-29) were cultured and exposed to incremental doses of tunicamycin (0.5, 1, 5, and 10 µg/mL) for 24 hours. Untreated cells served as controls. Western blot for BiP and phospho-eIF2α/total eIF2α was used to confirm ER stress and evaluate integrated stress response signaling, respectively. Quantitative RT-PCR was performed to evaluate expression of stress response transcription factors, ATF4 and ATF6. To assess differentiation, immunofluorescence staining was performed for HCG-β produced by syncytiotrophoblasts and qRT-PCR was used to detect markers of syncytiotrophoblast differentiation: HCG-β, SDC-1, and loss of TEAD4.

Results: Tunicamycin induced ER stress in TSCs, as evidenced by increased BiP protein expression (4.05-fold change [FC]) for tunicamycin 10 µg/mL vs. untreated control (Fig. 1A). Activation of integrated stress response downstream of ER stress was also evident based on increased phospho-eIF2α/eIF2α protein expression (3.16 FC vs. control) (Fig. 1B). ER stress transcriptional responses were confirmed by upregulation of ATF4 (1.58 FC) and ATF6 (1.25 FC) mRNA. Differentiation was not observed, as indicated by the absence of HCG-β protein expression on immunofluorescence (Fig. 1C) and by qRT-PCR showing minimal changes in differentiation markers.

Conclusion(s): Our findings confirm that tunicamycin reliably induces ER stress but does not drive differentiation under current culture conditions. Ongoing work will refine the culture environment to study how cellular stress influences trophoblast maturation and the trajectory of placental aging. Ultimately, this model may help uncover how placental stress disrupts trophoblast function, revealing mechanisms of fetal growth restriction and preterm birth.

Figure 1. Tunicamycin (TN) induces ER stress but not differentiation in human trophoblast stem cells in current culture conditions
(A) Western blot showing BiP protein expression in cells treated with tunicamycin (0.5-10 µg/mL) for 24 h compared to untreated control and differentiated positive control.
(B) Western blot showing phospho-eIF2α/total eIF2α expression in cells treated with tunicamycin (0.5-10 µg/mL) for 24 h compared to untreated control and differentiated positive control.
(C) Immunofluorescence staining for HCG-β (white), DAPI (blue), and phalloidin (pink) in cells treated with tunicamycin (10 µg/mL) compared to differentiated positive control.