1 Centre for Genetic Diseases, Hudson Institute of Medical Research , Clayton, Australia .
2 Department of Molecular and Translational Science, Monash University , Clayton, Australia .
Stem Cells Dev. 2017 Oct 15;26(20):1505-1519. doi: 10.1089/scd.2017.0041. Epub 2017 Sep 5.
Mitochondrial deoxyribonucleic acid (mtDNA) copy number is tightly regulated during pluripotency and differentiation. There is increased demand of cellular adenosine triphosphate (ATP) during differentiation for energy-intensive cell types such as hepatocytes and neurons to meet the cell's functional requirements. During hepatocyte differentiation, mtDNA copy number should be synchronously increased to generate sufficient ATP through oxidative phosphorylation. Unlike bone marrow mesenchymal cells, mtDNA copy number failed to increase by 28 days of differentiation of human amniotic epithelial cells (hAEC) into hepatocyte-like cells (HLC) despite their expression of some end-stage hepatic markers. This was due to higher levels of DNA methylation at exon 2 of POLGA, the mtDNA-specific replication factor. Treatment with a DNA demethylation agent, 5-azacytidine, resulted in increased mtDNA copy number, reduced DNA methylation at exon 2 of POLGA, and reduced hepatic gene expression. Depletion of mtDNA followed by subsequent differentiation did not increase mtDNA copy number, but reduced DNA methylation at exon 2 of POLGA and increased expression of hepatic and pluripotency genes. We encapsulated hAEC in barium alginate microcapsules and subsequently differentiated them into HLC. Encapsulation resulted in no net increase of mtDNA copy number but a significant reduction in DNA methylation of POLGA. RNAseq analysis showed that differentiated HLC express hepatocyte-specific genes but also increased expression of inflammatory interferon genes. Differentiation in encapsulated cells showed suppression of inflammatory genes as well as increased expression of genes associated with hepatocyte function pathways and networks. This study demonstrates that an increase in classical hepatic gene expression can be achieved in HLC through encapsulation, although they fail to effectively regulate mtDNA copy number.
线粒体脱氧核糖核酸 (mtDNA) 拷贝数在多能性和分化过程中受到严格调控。在分化过程中，对于需要大量细胞内三磷酸腺苷 (ATP) 的细胞类型（如肝细胞和神经元），ATP 的需求会增加，以满足细胞的功能需求。在肝细胞分化过程中，mtDNA 拷贝数应同步增加，通过氧化磷酸化产生足够的 ATP。与骨髓间充质细胞不同，人羊膜上皮细胞 (hAEC) 分化为肝细胞样细胞 (HLC) 28 天后，mtDNA 拷贝数未能增加，尽管它们表达了一些终末肝标志物。这是由于 mtDNA 特异性复制因子 POLGA 的外显子 2 处的 DNA 甲基化水平较高。用 DNA 去甲基化剂 5-氮杂胞苷处理可导致 mtDNA 拷贝数增加，POLGA 外显子 2 的 DNA 甲基化减少，肝基因表达减少。mtDNA 耗竭后再分化不会增加 mtDNA 拷贝数，但会减少 POLGA 外显子 2 的 DNA 甲基化，并增加肝和多能性基因的表达。我们将 hAEC 包埋在钡藻酸盐微胶囊中，然后将其分化为 HLC。包埋导致 mtDNA 拷贝数没有净增加，但 POLGA 的 DNA 甲基化显著减少。RNAseq 分析显示，分化的 HLC 表达肝细胞特异性基因，但也增加了炎症干扰素基因的表达。在包埋细胞中分化显示出炎症基因的抑制以及与肝细胞功能途径和网络相关的基因表达增加。这项研究表明，通过包埋可以在 HLC 中实现经典肝基因表达的增加，尽管它们不能有效地调节 mtDNA 拷贝数。
