Bisphenol A Impairs the Developmental Potential of Mouse Totipotent-like Stem Cell-Derived Blastoids through Oxidative Stress : A Stem Cell-Derived Embryo Model Approach to Developmental Toxicology = 줄기세포 유래 배아모델을 이용한 비스페놀 A의 발생독성 평가 연구

다국어 초록 (Multilingual Abstract)

Bisphenol A (BPA) is a pervasive environmental endocrine disruptor known to interfere with early embryonic development and implantation. Conventional reproductive toxicity assessments primarily depend on animal gametes or embryos, posing ethical and technical limitations in studying early developmental processes. To overcome these challenges, this study employed a synthetic embryo model generated from pluripotent stem cells (PSCs) to investigate BPA-induced developmental toxicity without using fertilized embryos. Mouse PSCs were reprogrammed into chemically induced totipotent-like stem cells (ciTotiSCs) through exposure to a defined combination of small molecules. These ciTotiSCs subsequently self-organized into blastoid structures within inverted pyramid-shaped microwells (AggreWell system) and were cultured for five days. Exposure to 5 μM BPA during blastoid formation markedly decreased formation efficiency to approximately 14%, compared with 73% in untreated controls. In Matrigel-based post-implantation culture, BPA-exposed blastoids showed significant reductions in inner cell mass (ICM) and trophectoderm (TE) expansion— approximately 96% and 72% of control values, respectively—indicating impaired implantation potential. BPA exposure also elevated intracellular reactive oxygen species (ROS) levels, suggesting oxidative stress as a major mechanism of toxicity. Co- treatment with the antioxidant glutathione (GSH, 500 μM) restored blastoid formation efficiency (~54%) and normalized ICM and TE areas. Transcriptomic profiling further revealed that BPA triggered endoplasmic reticulum stress, DNA damage responses, and apoptosis, all of which were mitigated by GSH co-treatment. Collectively, these findings suggest that oxidative stress underlies BPA-induced developmental defects and underscore the potential of stem cell-derived blastoid models as an egg-free platform for assessing reproductive toxicity of environmental toxicants.

국문 초록 (Abstract)

비스페놀 A (Bisphenol A, BPA)는 광범위하게 사용되는 환경 내분비계 교란물질로, 초기 배아 발달 및 착상 과정에 악영향을 미치는 것으로 알려져 있다. 기존 생식독성 평가는 주로 동물의 생식세포나 수정란을 이용하기 때문에, 초기 발생 과정을 연구하는 데 윤리적·기술적 한계가 존재한다. 본 연구에서는 이러한 제약을 극복하고 BPA의 발생독성을 규명하기 위해, 다능성 줄기세포 (pluripotent stem cells, PSCs)로부터 합성 배아모델을 구축하였다. 마우스 PSC를 화학 조합 처리를 통해 화학적 유도 전능성 유사 줄기세포 (chemically induced totipotent-like stem cells, ciTotiSCs)로 재프로그래밍한 후, 이 세포들은 역피라미드형 마이크로웰 (AggreWell 시스템)에서 5일간 자가조직화시켜 블라스토이드 (blastoid)를 형성하였다. 5 μM BPA 처리 시 블라스토이드 형성 효율은 약 14% 감소해 대조군 (약 73%)에 비해 뚜렷한 차이를 보였다. 또한 Matrigel 기반의 착상 이후 (in vitro post-implantation) 배양에서 내부세포괴 (inner cell mass, ICM)와 영양외배엽 (trophectoderm, TE)의 확장 면적이 각각 대조군의 약 96%, 72% 수준으로 감소하여 착상 능력이 저하됨을 확인하였다. BPA 노출은 세포 내 활성산소종 (reactive oxygen species, ROS)을 유의하게 증가시켜 산화 스트레스가 주요 독성 기전임을 시사하였다. 항산화제 글루타치온 (glutathione, GSH, 500 μM) 병용 시 블라스토이드 형성 효율을 약 54%까지 회복되고 ICM 및 TE의 구조도 부분적으로 개선되었다. 전사체 분석 결과에서는 BPA가 소포체 스트레스, DNA 손상 반응 및 세포사멸 관련 경로를 활성화하였고, GSH 처리로 이러한 변화가 완화되는 양상이 관찰되었다. 이 결과는 산화 스트레스가 BPA에 의한 발생 결함의 주요 원인임을 보여주며, 줄기세포 유래 블라스토이드 모델이 환경 독성물질의 생식독성 평가에 활용될 수 있는 비수정란 기반의 대체 플랫폼임을 시사한다.

목차 (Table of Contents)

• List of Tables ⅲ
• List of Figures ⅳ
• ABSTRACT ⅴ
• Ⅰ. Introduction 1
• 1.1. Background: Early Embryonic Development and Stem Cell-Derived models 1
• 1.2. Bisphenol A and Developmental Toxicity 3
• Ⅱ. Materials and Methods 7
• 2.1. Culture of mouse PSCs 7
• 2.2. Induction of ciTotiSCs 7
• 2.3. Generation of ciToti-blastoids 8
• 2.4. Collection and morphometric analysis of blastoids 9
• 2.5. Post-implantation culture of ciToti-blastoids 9
• 2.6. Treatment of blastoids with BPA 9
• 2.7. Immunofluorescence staining 10
• 2.8. Quantitative real-rime polymerase chain reaction (qRT-PCR) 10
• 2.9. Assessment of cytotoxicity in ciToti-blastoids 11
• 2.10. Evaluation of ROS levels in ciToti-blastoids treated with BPA 11
• 2.11. Protective effects of GSH against BPA-induced ROS generation 11
• 2.12. Cell viability assay 12
• 2.13. Bulk RNA-sequencing (RNA-seq) and data analysis 12
• 2.14. Statistical analysis 13
• Ⅲ. Results 16
• 3.1. Induction of totipotent-like stem cells from ESCs and iPSCs 16
• 3.2. Assessment of BPA toxicity during blastoid formation 19
• 3.3. BPA-induced oxidative stress impairs blastoid development via ROS accumulation and Dysregulated antioxidant gene expression 22
• 3.4. Antioxidant GSH rescues blastoid formation efficiency from BPA-induced cytotoxicity 26
• 3.5. GSH mitigates oxidative stress and cell death in BPA-treated blastoids 28
• 3.6. Transcriptomic profiling of BPA- and BPA + GSH- treated ciToti-blastoids 30
• 3.7. GSH modulates BPA-induced stress and restores developmental pathways 33
• 3.8. GSH restores the in vitro implantation capacity of BPA-exposed blastoids by mitigating oxidative stress 37
• Ⅳ. Discussion 39
• Ⅴ. Conclusion 44
• References 45
• Abstract (in Korean) 55