Polymer Supported Directed Differentiation Reveals a Unique Gene Signature Predicting Stable Hepatocyte Performance.
Polymer Supported Directed Differentiation Reveals a Unique Gene Signature Predicting Stable Hepatocyte Performance.
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聚合物支持的定向分化揭示了预测稳定肝细胞性能的独特基因特征。
DOI：
10.1002/adhm.201500391
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发表时间：
2015
影响因子：
10
通讯作者：
Villarin BL
中科院分区：
文献类型：
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作者：
Villarin BL
DOI：10.1002/adhm。 201500391使用，廉价，可扩展，定义，并且能够提供具有可预测性能和长“保质期”的产品。人们已经开发了多种方法，包括使用不同的细胞因子或化学组合、3D细胞聚集和灌注装置来模拟肝脏生态位和结构。[17-20]虽然这些方法取得了重大进展，但其复杂性和/或不确定性限制了该技术的大规模部署。为了解决这个问题，我们特此报告将确定的生物聚合物底物与无血清分化程序结合使用，用于研究级和良好生产规范 (GMP) 级 PSC。重要的是，这些干细胞群体在细胞培养中表现出稳健且可预测的性能，其特点是基质金属蛋白酶 13 (MMP13)、δ 连环蛋白 (CTNND2) 和血小板反应蛋白 2 (THBS2) 的基因表达。H9 人胚胎干细胞 (hESC) 表现出适当的形态并表达干细胞标记物（Oct-4 和 Nanog、SSEA-4、TRA-1-60 和TRA-1–81) 被扩展并用于分化实验。重要的是，这些人群中 SSEA-1 的表达量极小（图 S1A-D，支持信息）。此外，hESC 能够自发分化为所有三个胚层并直接分化为 HLC（图 S1E，支持信息和图 1）。验证后，hESC 群体使用规定的程序直接分化为成肝细胞。 [20]将干细胞衍生的成肝细胞从其基质中取出，并在无血清条件下重新铺板到化学成分确定的聚氨酯表面 (PU) 上。移植后 10、15 和 20 天，通过肝细胞分化标志物 HNF4α 和去分化标志物波形蛋白的免疫染色研究肝细胞分化状态。干细胞衍生的肝细胞在 PU 上表达稳定水平的 HNF4α，培养时间长达 20 天（图 1 A）。 Matrigel (MG) 培养物的情况正好相反，从第 15 天起开始恶化，到第 20 天，表达 HNF4α 的细胞大约减少四倍 (22%±1.8)（图 1 B）。肝细胞去分化标记物的染色也证实了肝细胞表型的丧失。重新接种在 PU 上的干细胞衍生的 HLC 表达低水平的波形蛋白，范围为 23% 至 35%（图 1 A）。相比之下，MG 上的干细胞来源的肝细胞表现出更大的去分化，染色范围为 54% 至 78%（图 1 B）。这些结果得到了免疫染色研究的进一步支持
DOI: 10.1002/adhm. 201500391 use, inexpensive, scalable, defined, and capable of delivering a product with predictable performance and long “shelf life.” There have been a number of approaches developed, including the use of differential cytokine or chemical combinations, 3D cell aggregation, and perfused devices, to mimic the liver niche and architecture.[17–20] While these approaches marked significant progress, their complexity and/or undefined nature has limited large-scale deployment of the technology. In order to tackle this issue, we hereby report on the use of a defined biopolymer substrate in conjunction with a serum-free differentiation procedure with both research and good manufacturing practice (GMP) grade PSCs. Importantly, those stem-derived populations displayed robust and predictable performance in cell culture which was hallmarked by gene expression of matrix metalloproteinase 13 (MMP13), delta catenin (CTNND2), and thrombospondin 2 (THBS2).H9 human embryonic stem cells (hESCs), displaying an appropriate morphology and expressing stem cell markers (Oct-4 and Nanog, SSEA-4, TRA-1–60, and TRA-1–81) were expanded and used for the differentiation experiments. Importantly, SSEA-1 expression was minimal in these populations (Figure S1A–D, Supporting Information). Additionally, hESCs were able to spontaneously differentiate to all the three germ layers and directly differentiate to HLCs (Figure S1E, Supporting Information and Figure 1). Following validation, hESC populations were directly differentiated to hepatoblasts using a defined procedure.[20] Stem cell–derived hepatoblasts were removed from their substrate and replated, under serum-free conditions, onto a chemically defined polyurethane surface (PU). At 10, 15, and 20 d postreplating, hepatocyte differentiation status was studied by immunostaining for a marker of hepatocyte differentiation, HNF4α, and dedifferentiation, vimentin. Stem cell–derived hepatocytes expressed stable levels of HNF4α on the PU for up to 20 d in culture (Figure 1 A). The converse was true for Matrigel (MG) cultures which began to deteriorate from day 15 onward, and by day 20 approximately fourfold fewer cells expressed HNF4α (22%±1.8)(Figure 1 B). The loss of the hepatocyte phenotype was also corroborated by staining for a marker of hepatocellular dedifferentiation. Stem cell–derived HLCs replated on PU expressed low levels of vimentin, ranging from 23% to 35%(Figure 1 A). In contrast, stem cell–derived hepatocytes on MG demonstrated greater dedifferentiation, with staining ranging from 54% to 78%(Figure 1 B). These results were further supported by immunostaining studies