Improvement of Cell Survival During Human Pluripotent Stem Cell Definitive Endoderm Differentiation.
Improvement of Cell Survival During Human Pluripotent Stem Cell Definitive Endoderm Differentiation.
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DOI：
10.1089/scd.2015.0018
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发表时间：
2015-07
影响因子：
4
通讯作者：
Han Wang;Xie Luo;Li Yao;D. Lehman;Pei-Rong Wang
中科院分区：
文献类型：
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作者：
Han Wang;Xie Luo;Li Yao;D. Lehman;Pei-Rong Wang
分泌性内胚层（DE）是肝脏和胰腺等内脏器官的重要前体。将人胚胎干细胞（hESC）或诱导多能干细胞（iPSC）分化为DE的有效方案对于再生医学和疾病建模至关重要;然而，DE分化期间细胞存活率差的问题仍未解决。在这项研究中，我们在改良的分化方案中使用B27补充剂导致了实质性的改善。我们使用SOX 17增强的绿色荧光蛋白（eGFP）报告人胚胎干细胞系比较和修改建立DE分化方案。使用总活细胞数量和eGFP阳性细胞百分比来评估分化效率。在测试的方案中，开发了三种使用无血清B27补充剂的改良方案以产生大量DE细胞。在DE分化过程中避免了大量细胞死亡，DE细胞的百分比仍然很高。当所得DE细胞进一步向胰腺谱系分化时，胰腺特异性标志物的表达显著增加。通过修改的方案，在H1 hESC和iPSC中观察到类似的高DE分化效率。在B27组分中，发现牛血清白蛋白促进DE分化和细胞存活。使用我们修改的DE分化方案，可以产生令人满意的数量的优质DE作为进一步内胚层谱系分化的主要材料。
Definitive endoderm (DE) is a vital precursor for internal organs such as liver and pancreas. Efficient protocol to differentiate human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs) to DE is essential for regenerative medicine and for modeling diseases; yet, poor cell survival during DE differentiation remains unsolved. In this study, our use of B27 supplement in modified differentiation protocols has led to a substantial improvement. We used an SOX17-enhanced green fluorescent protein (eGFP) reporter hESC line to compare and modify established DE differentiation protocols. Both total live cell numbers and the percentages of eGFP-positive cells were used to assess differentiation efficiency. Among tested protocols, three modified protocols with serum-free B27 supplement were developed to generate a high number of DE cells. Massive cell death was avoided during DE differentiation and the percentage of DE cells remained high. When the resulting DE cells were further differentiated toward the pancreatic lineage, the expression of pancreatic-specific markers was significantly increased. Similar high DE differentiation efficiency was observed in H1 hESCs and iPSCs through the modified protocols. In B27 components, bovine serum albumin was found to facilitate DE differentiation and cell survival. Using our modified DE differentiation protocols, satisfactory quantities of quality DE can be produced as primary material for further endoderm lineage differentiation.