Long-term expansion, genomic stability and in vivo safety of adult human pancreas organoids
Long-term expansion, genomic stability and in vivo safety of adult human pancreas organoids
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DOI：
10.1186/s12861-020-0209-5
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发表时间：
2020-02-26
影响因子：
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通讯作者：
Huch, Meritxell
中科院分区：
文献类型：
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作者：
Georgakopoulos, Nikitas;Prior, Nicole;Huch, Meritxell
背景胰腺类器官系统最近被描述用于体外培养来自小鼠和人的胰腺导管细胞。小鼠胰腺类器官表现出无限的扩增潜力，而先前报道的人胰腺类器官（hPO）培养物在化学成分确定的无血清培养基中不能有效地长期扩增。我们试图产生一个3D培养系统，用于人胰腺导管细胞作为hPO的长期扩增，以作为研究人胰腺导管上皮、外分泌胰腺疾病和开发用于糖尿病的基因组稳定的替代细胞疗法的基础。结果我们的化学成分确定的、无血清的人胰腺类器官培养基支持从新鲜和冷冻保存的原代组织高效地产生和扩增hPO。hPO可以从单个细胞扩增，使其能够进行遗传操作和产生克隆培养物。在体外扩增数月的hPO保持其导管形态、生物标志物表达和染色体完整性。hPO的异种移植物在移植到免疫缺陷小鼠的胰腺中时在体内长期存活。值得注意的是，小鼠原位移植没有显示出致瘤性的迹象。至关重要的是，我们的培养基还支持在化学定义的、可修饰的和可扩展的仿生水凝胶中建立和扩增hPO。结论在化学成分确定的无血清培养基中，可以从新鲜和冻存的人胰腺组织中长期扩增hPOs，并且没有可检测到的致瘤性。hPO可以克隆扩增，遗传操作，并适合在化学上确定的水凝胶中培养。因此，hPO代表了保留组织来源特征的胰腺导管细胞的丰富来源，这为导管上皮疾病的建模和增加对人类胰腺外分泌生物学的理解以及潜在地产生用于治疗糖尿病的胰岛素分泌细胞开辟了途径。
Background Pancreatic organoid systems have recently been described for the in vitro culture of pancreatic ductal cells from mouse and human. Mouse pancreatic organoids exhibit unlimited expansion potential, while previously reported human pancreas organoid (hPO) cultures do not expand efficiently long-term in a chemically defined, serum-free medium. We sought to generate a 3D culture system for long-term expansion of human pancreas ductal cells as hPOs to serve as the basis for studies of human pancreas ductal epithelium, exocrine pancreatic diseases and the development of a genomically stable replacement cell therapy for diabetes mellitus. Results Our chemically defined, serum-free, human pancreas organoid culture medium supports the generation and expansion of hPOs with high efficiency from both fresh and cryopreserved primary tissue. hPOs can be expanded from a single cell, enabling their genetic manipulation and generation of clonal cultures. hPOs expanded for months in vitro maintain their ductal morphology, biomarker expression and chromosomal integrity. Xenografts of hPOs survive long-term in vivo when transplanted into the pancreas of immunodeficient mice. Notably, mouse orthotopic transplants show no signs of tumorigenicity. Crucially, our medium also supports the establishment and expansion of hPOs in a chemically defined, modifiable and scalable, biomimetic hydrogel. Conclusions hPOs can be expanded long-term, from both fresh and cryopreserved human pancreas tissue in a chemically defined, serum-free medium with no detectable tumorigenicity. hPOs can be clonally expanded, genetically manipulated and are amenable to culture in a chemically defined hydrogel. hPOs therefore represent an abundant source of pancreas ductal cells that retain the characteristics of the tissue-of-origin, which opens up avenues for modelling diseases of the ductal epithelium and increasing understanding of human pancreas exocrine biology as well as for potentially producing insulin-secreting cells for the treatment of diabetes.