Early Differentiation Signatures in Human Induced Pluripotent Stem Cells Determined by Non-Targeted Metabolomics Analysis
Early Differentiation Signatures in Human Induced Pluripotent Stem Cells Determined by Non-Targeted Metabolomics Analysis
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通过非靶向代谢组学分析确定人类诱导多能干细胞的早期分化特征
DOI：
10.1101/2023.03.02.530741
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发表时间：
2023
期刊：
影响因子：
--
通讯作者：
Fujita Takuya
中科院分区：
文献类型：
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作者：
Abdalkader Rodi;Chaleckis Romanas;Fujita Takuya
人类诱导多能干细胞（hiPSC）作为产生多种人类细胞的有价值来源具有巨大潜力，然而监测向特定谱系的早期细胞分化仍然具有挑战性。在这项研究中，我们采用了非靶向代谢组学分析技术来分析小至1微升的样品中存在的细胞外代谢物。hiPSC通过在基础培养基E6下与化学抑制剂（其先前已被报道为单独或与bFGF组合引导向外胚层谱系分化）和糖原激酶3（GSK-3）的抑制组合启动培养来进行分化，所述化学抑制剂例如Wnt/β-连环蛋白和TGF-β激酶/激活素受体，所述糖原激酶3（GSK-3）通常用于使hiPSC转向中胚层谱系。在0 h和48 h，鉴定了117种代谢产物，包括生物学相关代谢产物，如乳酸、乳酸和氨基酸。通过确定多能性标志物OCT 3/4的表达，我们能够将细胞的分化状态与变化的代谢物相关联。经历外胚层分化的细胞组显示出OCT 3/4表达的更大降低。此外，代谢物如马尿酸和犬尿氨酸在外胚层分化条件下显示出显著变化，其中马尿酸消耗增加1-2倍，而犬尿氨酸分泌减少2倍。进一步的代谢物分析揭示了一组与外胚层谱系特异性相关的代谢物，突出了我们的发现在细胞分化期间确定hiPSC特征的潜力，特别是在外胚层谱系条件下。
Human induced pluripotent stem cells (hiPSCs) possess immense potential as a valuable source for the generation of a wide variety of human cells, yet monitoring the early cell differentiation towards a specific lineage remains challenging. In this study, we employed a non-targeted metabolomic analysis technique to analyze the extracellular metabolites present in samples as small as one microliter. The hiPSCs were subjected to differentiation by initiating culture under the basal medium E6 in combination with chemical inhibitors that have been previously reported to direct differentiation towards the ectodermal lineage such as Wnt/β-catenin and TGF-β kinase/activin receptor, alone or in combination with bFGF, and the inhibition of glycogen kinase 3 (GSK-3), which is commonly used for the diversion of hiPSCs towards mesodermal lineage. At 0 h and 48 h, 117 metabolites were identified, including biologically relevant metabolites such as lactic acid, pyruvic acid, and amino acids. By determining the expression of the pluripotency marker OCT3/4, we were able to correlate the differentiation status of cells with the shifted metabolites. The group of cells undergoing ectodermal differentiation showed a greater reduction in OCT3/4 expression. Moreover, metabolites such as pyruvic acid and kynurenine showed dramatic change under ectodermal differentiation conditions where pyruvic acid consumption increased 1–2-fold, while kynurenine secretion decreased 2-fold. Further metabolite analysis uncovered a group of metabolites specifically associated with ectodermal lineage, highlighting the potential of our findings to determine the characteristics of hiPSCs during cell differentiation, particularly under ectodermal lineage conditions.