Applications of the long-term culture human primordial germ cell-like cells to toxicological assessments and mechanistic studies on chemically caused heritable human health threats

长期培养人类原始生殖细胞样细胞在化学引起的遗传性人类健康威胁的毒理学评估和机制研究中的应用

• 批准号: 10666200
• 负责人: TOSHIHIRO SHIODA
• 金额: $ 25.05万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10666200
• 关键词: Adult; Affect; Apoptosis; Apoptotic; BAK1 gene; BAX gene; Benzo(a)pyrene; Biological Assay; Cell Culture Techniques; Cell Death Induction; Cell Separation; Cells; ChIP-seq; Characteristics; Chemical Actions; Chemical Exposure; Chemicals; Chemotherapy-Oncologic Procedure; Child; Cisplatin; Clinical; Communities; Cultured Cells; Cyclophosphamide; DNA; DNA Damage; DNA Repair; DNA Sequence Alteration; Disease; Educational process of instructing; Embryo; Endocrine Disruptors; Environmental Risk Factor; Epigenetic Process; Evaluation; Experimental Models; Exposure to; Familial disease; Family; Female; Fertility; Future; Generations; Genetic; Genetic Engineering; Genetic Materials; Genome; Genomic DNA; Genomics; Germ Cells; Health; Hereditary Disease; Heritability; Histones; Hospitals; Human; Human body; Impaired health; Impairment; In Vitro; Industry; Intervention; Knock-out; Knowledge; Laboratories; Laboratory Research; Lesion; Location; Methods; Methylation; Modeling; Mus; Mutagens; Mutate; Mutation; Nonhomologous DNA End Joining; Nucleotides; Organ Culture Techniques; Outcome; Paint; Pattern; Pharmaceutical Preparations; Pluripotent Stem Cells; Poison; Poly-fluoroalkyl substances; Population; Pregnant Women; Prevalence; Production; Proteins; Purines; Pyrimidine; Research; Research Project Grants; Resistance; Risk; Risk Assessment; Running; Seminiferous tubule structure; Serum; Somatic Cell; Specimen; Stains; Stress; Structure; Structure of primordial sex cell; System; Techniques; Testing; Testis; Therapeutic; Toxic Environmental Substances; Toxicology; Variant; base; bisphenol A; bisulfite sequencing; cell type; chemical safety; chemotherapeutic agent; chemotherapy; cigarette smoke; egg; environmental chemical; exposed human population; genome-wide; genotoxicity; high risk population; homologous recombination; human model; in vitro Model; induced pluripotent stem cell; insight; male; nanoparticle; next generation; novel; offspring; oxidation; perfluorooctane sulfonate; perfluorooctanoic acid; phthalates; precursor cell; prevent; programs; reconstitution; screening; senescence; sperm cell; substance use; titanium dioxide; toxicant; trait; transcriptome sequencing; tributyltin; whole genome

Because germ cells are exclusively capable of passing the genetic materials to the progeny, damages to their genome – either involving DNA mutations or only affecting epigenetic machineries – impose a unique risk of initiating heritable disorders. Whereas toxicants that reduce viability of germ cells may diminish fertility, lesions in the genome of survived germ cells may be persistent beyond generations to harm health of the offspring. Because the risk of creating hereditary diseases would increase when the genome of embryonic precursors of germ cells is impaired, and because sensitivities and genotoxic mechanisms of a toxic substance may vary among different cell types, assessments of such a risk would be best performed using human germline precursor cells. However, the lack of cell culture models of human germ cell precursors suitable for in vitro assessments has been a significant obstacle to understanding the realistic risk that chemical exposure initiates heritable diseases as well as to develop strategies for protecting the genome of human germ cells. The large and ever-increasing number of synthetic compounds further raises the hurdle of comprehensive understanding of such exposure-initiated heritable health threats. The Primordial germ cells (PGC) is the most upstream precursor of all germ cells. The human PGC-like cell (hPGCLC) is a pluripotent stem cell-derived cell culture model of human PGCs, but technical difficulties with its in vitro expansion has been preventing its practical use for toxicological assessments. Our laboratory has recently overcome this hurdle by successfully establishing the Long-Term Culture hPGCLC (LTC-hPGCLC), which perpetually expands in a serum/feeder layer-free cell culture condition as a highly homogeneous, senescence-free cell population without losing their PGC-like characteristics. Taking advantage of LTC-hPGCLCs, this project will obtain critical mechanistic insights into how environmental chemicals affect genetic and epigenetic integrity of human PGCs through creating novel nucleotide base mutations (Aim 1) or disrupting the epigenetic integrity (Aim 2). A genetically engineered variant of LTC-hPGCLCs that are resistant to stress-induced cell death will be exposed to toxicants directly or in the context of organ cultures mimicking the micro-environment of human embryonic testis. Aim 1 will examine DNA damaging effects of cigarette smoke mutagens, drugs commonly used for cancer chemotherapy, and nanoparticles whereas Aim 2 will examine epigenetic effects of endocrine disrupting chemicals and the PFAS chemicals, which are used as stain repellants, polishers, paints, and coatings. Attempts will also be made to develop high-throughput compatible assays for evaluation of these genotoxic actions of chemicals in LTC-hPGCLCs. Outcomes of our research will directly inform not only the scientific research community but also the industry and the governmental programs regulating chemical safety about the risk of creating familial diseases via exposure to toxic substances. Our study will also inform the translational toxicology approaches to develop clinical interventions for protection of human germ cell genome.

由于生殖细胞专门能够将遗传物质传递给后代，因此其生殖细胞受到损害 基因组——要么涉及DNA突变，要么只影响表观遗传机制——带来了独特的风险 引起遗传性疾病的毒物会降低生殖细胞的活力，可能会降低生育能力、病变。 幸存生殖细胞的基因组中的基因可能会持续存在几代人之后，从而损害后代的健康。 因为当胚胎前体的基因组发生变化时，产生遗传性疾病的风险就会增加。 生殖细胞受损，并且有毒物质的敏感性和基因毒性机制可能会有所不同 在不同的细胞类型中，最好使用人类种系来评估这种风险 然而，缺乏适合体外的人类生殖细胞前体细胞培养模型。 评估一直是理解化学品暴露引发的现实风险的重大障碍 遗传性疾病以及制定保护人类生殖细胞基因组的策略。 合成化合物数量的不断增加进一步提高了全面理解的障碍 在这种由暴露引起的遗传性健康威胁中，原始生殖细胞（PGC）是最上游的。 人类 PGC 样细胞 (hPGCLC) 是一种多能干细胞衍生的细胞培养物。 人类 PGC 模型，但其体外扩增的技术困难一直阻碍其实际应用 我们的实验室最近成功建立了毒理学评估的方法，克服了这一障碍。 长期培养 hPGCLC (LTC-hPGCLC)，可在无血清/饲养层细胞中永久扩增 培养条件为高度同质、无衰老细胞群，且不会失去其 PGC 样特征 利用 LTC-hPGCLC 的优势，该项目将获得关键的机制见解。 环境化学物质如何通过创造新的影响人类 PGC 的遗传和表观遗传完整性 核苷酸碱基突变（目标 1）或破坏表观遗传完整性（目标 2）。 抵抗应激诱导的细胞死亡的 LTC-hPGCLC 变体将直接暴露于有毒物质或 目标 1 将在模拟人类胚胎睾丸微环境的器官培养中进行。 检查香烟烟雾诱变剂（常用于治疗癌症的药物）对 DNA 的破坏作用 化疗和纳米粒子，而 Aim 2 将检查内分泌干扰的表观遗传效应 化学品和 PFAS 化学品，用作防污剂、抛光剂、油漆和涂料。 还将尝试开发高通量兼容测定法来评估这些基因毒性测定法 我们的研究结果不仅会直接为科学提供信息。 研究界以及行业和政府监管化学品安全的计划 我们的研究还将为转化提供信息。 毒理学方法来开发保护人类生殖细胞基因组的临床干预措施。