Center for Gynepathology Research and Biological Engineering Department, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA, 02138, USA.
Deparment of Pediatrics, Harvard Medical School, 300 Longwood Avenue, Boston, MA, 02115, USA.
Biomaterials. 2020 Sep;254:120125. doi: 10.1016/j.biomaterials.2020.120125. Epub 2020 May 25.
Epithelial organoids derived from human donor tissues are important tools in fields ranging from regenerative medicine to drug discovery. Organoid culture requires expansion of stem/progenitor cells in Matrigel, a tumor-derived extracellular matrix (ECM). An alternative completely synthetic ECM could improve reproducibility, clarify mechanistic phenomena, and enable human implantation of organoids. We designed synthetic ECMs with tunable biomolecular and biophysical properties to identify gel compositions supporting human tissue-derived stem/progenitor epithelial cells as enteroids and organoids starting with single cells rather than tissue fragments. The synthetic ECMs consist of 8-arm PEG-macromers modified with ECM-binding peptides and different combinations of integrin-binding peptides, crosslinked with peptides susceptible to matrix metalloprotease (MMP) degradation, and tuned to exhibit a range of biophysical properties. A gel containing an α2β1 integrin-binding peptide (GFOGER) and matrix binder peptides grafted to a 20 kDa 8-arm PEG macromer showed the most robust support of human duodenal and colon enteroids and endometrial organoids. In this synthetic ECM, human intestinal enteroids and endometrial organoids emerge from single cells and show cell-specific and apicobasal polarity markers upon differentiation. Intestinal enteroids, in addition, retain their proliferative capacity, are functionally responsive to basolateral stimulation, express canonical markers of intestinal crypt cells including Paneth cells, and can be serially passaged. The success of this synthetic ECM in supporting human postnatal organoid culture from multiple different donors and from both the intestine and endometrium suggests it may be broadly useful for other epithelial organoid culture.
从人类供体组织中衍生的上皮类器官是再生医学到药物发现等领域的重要工具。类器官培养需要在 Matrigel（一种肿瘤衍生的细胞外基质）中扩增干细胞/祖细胞。一种完全合成的细胞外基质替代品可以提高重现性、阐明机制现象，并能够实现类器官的人体植入。我们设计了具有可调节生物分子和生物物理特性的合成细胞外基质，以确定凝胶组成，这些凝胶可以支持源自人类组织的干细胞/祖细胞上皮细胞作为肠类器官和类器官，起始材料是单细胞而不是组织碎片。合成细胞外基质由 8 臂 PEG 大分子与 ECM 结合肽修饰而成，并结合了不同组合的整合素结合肽，用易受基质金属蛋白酶（MMP）降解的肽交联，并调整为具有一系列生物物理特性。一种含有 α2β1 整合素结合肽（GFOGER）和基质结合肽的凝胶，连接到 20 kDa 8 臂 PEG 大分子上，对人十二指肠和结肠肠类器官和子宫内膜类器官的支持效果最为显著。在这种合成细胞外基质中，人肠类器官和子宫内膜类器官从单细胞中出现，并在分化时表现出细胞特异性和顶底极性标记。此外，肠类器官保持其增殖能力，对基底外侧刺激具有功能性反应，表达包括潘氏细胞在内的肠隐窝细胞的典型标志物，并且可以连续传代。这种合成细胞外基质在支持来自多个不同供体的人产后类器官培养以及肠和子宫内膜方面的成功表明，它可能对其他上皮类器官培养具有广泛的用途。
