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.

由于生殖细胞只能将遗传物质传递给后代， 基因组-无论是涉及DNA突变或只影响表观遗传机制-施加一个独特的风险， 引发遗传性疾病而降低生殖细胞活力的毒物可能会降低生育能力， 在存活的生殖细胞基因组中，可能会持续几代以上，损害后代的健康。 因为当胚胎前体细胞的基因组发生突变时， 生殖细胞受损，并且由于毒性物质的敏感性和遗传毒性机制可能不同， 在不同的细胞类型中，评估这种风险最好使用人类生殖细胞 前体细胞然而，缺乏适合于体外培养的人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一个重大障碍 遗传性疾病以及制定保护人类生殖细胞基因组的战略。大 越来越多的合成化合物进一步增加了全面理解的障碍 这种由基因引发的遗传性健康威胁。原始生殖细胞（PGC）是最上游的 所有生殖细胞的前体。人PGC样细胞（hPGCLC）是一种多能干细胞衍生的细胞培养物 人PGCs模型，但其体外扩增的技术困难一直阻碍其实际应用 进行毒理学评估。我们的实验室最近克服了这一障碍，成功地建立了 长期培养hPGCLC（LTC-hPGCLC），其在无血清/饲养层细胞中永久扩增 培养条件下作为一个高度同质的，无衰老的细胞群体，而不会失去他们的PGC样 特色利用LTC-hPGCLC，该项目将获得关键的机理见解， 环境化学物质如何通过创造新的物质影响人类PGC的遗传和表观遗传完整性 核苷酸碱基突变（Aim 1）或破坏表观遗传完整性（Aim 2）。基因工程 对应激诱导的细胞死亡具有抗性的LTC-hPGCLC的变体将直接暴露于毒物或 在模仿人类胚胎睾丸微环境的器官培养物的背景下。目标1将 研究香烟烟雾诱变剂对DNA的破坏作用， 而Aim 2将研究内分泌干扰素的表观遗传效应， 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相容性试验，用于评价这些遗传毒性。 LTC-hPGCLC中化学物质的作用。我们的研究成果不仅将直接告知科学界， 研究界以及工业界和政府制定的有关化学品安全的计划， 通过接触有毒物质而产生家族性疾病的风险。我们的研究也将告知翻译 毒理学方法，以开发保护人类生殖细胞基因组的临床干预措施。