{"title":"人诱导多能干细胞向胰岛素生成细胞分化过程的自噬评价。","authors":"A Sabet, N Azarpira, L Kohan, S Ghavami","doi":"","DOIUrl":null,"url":null,"abstract":"<p><strong>Background: </strong>Autophagy is an intracellular self-degradative homeostasis process which eliminates undesirable and harmful macromolecules and organelles. Autophagy is also involved in self-renewal and differentiation of induced pluripotent stem cell (iPSCs).</p><p><strong>Objective: </strong>In this study, we investigated the expression profile of autophagy marker genes in human iPSCs during their differentiation induction toward insulin producing β-like cells.</p><p><strong>Methods: </strong>Human iPSC line, R1-hiPSC1, was used for differentiation induction toward β-like cells. The mRNA expression of Nanog, OCT4 (pluripotency markers), SOX17, FOXA2 (endodermic markers), PTF1A, NKX6.1 (exocrine/endocrine determinants), and PDX1 were measured during differentiation stages. Autophagy was monitored by genes expression study of four autophagy markers, MAP1LC3B, BECN1, SQSTM1/P62 and ATG5, along with protein expression profile of LC3b-II during differentiation stages.</p><p><strong>Results: </strong>The mRNA expression measurement of pluripotency, endoderm and exocrine/endocrine marker genes confirmed that hiPSCs skipped pluripotency, differentiated into endoderm, passed through the pancreatic lineage commitment stage and successfully generated insulin producing β-like cells. Expression profile of autophagy genes during differentiation stages indicated the decreased expression levels at the early stages (EB and MEI) and then increased at the definitive endoderm stages (DEI 1, DEI 2 and DE) followed by a subtractive pattern toward the end of differentiation. The results of protein expression of LC3b-II were consistent with gene expression data.</p><p><strong>Conclusion: </strong>This study demonstrated the high contribution of key autophagy genes/proteins during the differentiation of hiPSC toward β-like cells. The enhanced autophagy levels were a prominent feature of early stages of differentiation and DE rather than the later stages.</p>","PeriodicalId":14242,"journal":{"name":"International Journal of Organ Transplantation Medicine","volume":null,"pages":null},"PeriodicalIF":0.3000,"publicationDate":"2022-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10460531/pdf/ijotm-13-04.pdf","citationCount":"0","resultStr":"{\"title\":\"Evaluation of Autophagy Process in Differentiation of Human Induced Pluripotent Stem Cells toward Insulin Producing Cells.\",\"authors\":\"A Sabet, N Azarpira, L Kohan, S Ghavami\",\"doi\":\"\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><strong>Background: </strong>Autophagy is an intracellular self-degradative homeostasis process which eliminates undesirable and harmful macromolecules and organelles. Autophagy is also involved in self-renewal and differentiation of induced pluripotent stem cell (iPSCs).</p><p><strong>Objective: </strong>In this study, we investigated the expression profile of autophagy marker genes in human iPSCs during their differentiation induction toward insulin producing β-like cells.</p><p><strong>Methods: </strong>Human iPSC line, R1-hiPSC1, was used for differentiation induction toward β-like cells. The mRNA expression of Nanog, OCT4 (pluripotency markers), SOX17, FOXA2 (endodermic markers), PTF1A, NKX6.1 (exocrine/endocrine determinants), and PDX1 were measured during differentiation stages. Autophagy was monitored by genes expression study of four autophagy markers, MAP1LC3B, BECN1, SQSTM1/P62 and ATG5, along with protein expression profile of LC3b-II during differentiation stages.</p><p><strong>Results: </strong>The mRNA expression measurement of pluripotency, endoderm and exocrine/endocrine marker genes confirmed that hiPSCs skipped pluripotency, differentiated into endoderm, passed through the pancreatic lineage commitment stage and successfully generated insulin producing β-like cells. Expression profile of autophagy genes during differentiation stages indicated the decreased expression levels at the early stages (EB and MEI) and then increased at the definitive endoderm stages (DEI 1, DEI 2 and DE) followed by a subtractive pattern toward the end of differentiation. The results of protein expression of LC3b-II were consistent with gene expression data.</p><p><strong>Conclusion: </strong>This study demonstrated the high contribution of key autophagy genes/proteins during the differentiation of hiPSC toward β-like cells. 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引用次数: 0
摘要
背景:自噬是一种细胞内自我降解的稳态过程,它消除不需要的和有害的大分子和细胞器。自噬也参与诱导多能干细胞(iPSCs)的自我更新和分化。目的:研究自噬标记基因在诱导人iPSCs向胰岛素生成β样细胞分化过程中的表达谱。方法:采用人iPSC细胞系R1-hiPSC1诱导β样细胞分化。在分化阶段检测Nanog、OCT4(多能性标志物)、SOX17、FOXA2(内胚层标志物)、PTF1A、NKX6.1(外分泌/内分泌决定因子)和PDX1的mRNA表达。通过研究MAP1LC3B、BECN1、SQSTM1/P62和ATG5四种自噬标志物的基因表达以及LC3b-II在分化阶段的蛋白表达谱来监测自噬。结果:多能性、内胚层和外分泌/内分泌标记基因mRNA表达量测定证实,hipsc跳过多能性,向内胚层分化,通过胰腺谱系承诺阶段,成功生成产生胰岛素的β样细胞。自噬基因在分化阶段的表达谱表明,自噬基因在早期(EB和MEI)表达水平降低,然后在终末期(DEI 1, DEI 2和DE)表达水平升高,并在分化结束时呈减量模式。LC3b-II蛋白表达结果与基因表达数据一致。结论:本研究表明关键自噬基因/蛋白在hiPSC向β样细胞分化过程中发挥了重要作用。自噬水平的增强是早期分化和DE的显著特征,而不是晚期。
Evaluation of Autophagy Process in Differentiation of Human Induced Pluripotent Stem Cells toward Insulin Producing Cells.
Background: Autophagy is an intracellular self-degradative homeostasis process which eliminates undesirable and harmful macromolecules and organelles. Autophagy is also involved in self-renewal and differentiation of induced pluripotent stem cell (iPSCs).
Objective: In this study, we investigated the expression profile of autophagy marker genes in human iPSCs during their differentiation induction toward insulin producing β-like cells.
Methods: Human iPSC line, R1-hiPSC1, was used for differentiation induction toward β-like cells. The mRNA expression of Nanog, OCT4 (pluripotency markers), SOX17, FOXA2 (endodermic markers), PTF1A, NKX6.1 (exocrine/endocrine determinants), and PDX1 were measured during differentiation stages. Autophagy was monitored by genes expression study of four autophagy markers, MAP1LC3B, BECN1, SQSTM1/P62 and ATG5, along with protein expression profile of LC3b-II during differentiation stages.
Results: The mRNA expression measurement of pluripotency, endoderm and exocrine/endocrine marker genes confirmed that hiPSCs skipped pluripotency, differentiated into endoderm, passed through the pancreatic lineage commitment stage and successfully generated insulin producing β-like cells. Expression profile of autophagy genes during differentiation stages indicated the decreased expression levels at the early stages (EB and MEI) and then increased at the definitive endoderm stages (DEI 1, DEI 2 and DE) followed by a subtractive pattern toward the end of differentiation. The results of protein expression of LC3b-II were consistent with gene expression data.
Conclusion: This study demonstrated the high contribution of key autophagy genes/proteins during the differentiation of hiPSC toward β-like cells. The enhanced autophagy levels were a prominent feature of early stages of differentiation and DE rather than the later stages.
期刊介绍:
The International Journal of Organ Transplantation Medicine (IJOTM) is a quarterly peer-reviewed English-language journal that publishes high-quality basic sciences and clinical research on transplantation. The scope of the journal includes organ and tissue donation, procurement and preservation; surgical techniques, innovations, and novelties in all aspects of transplantation; genomics and immunobiology; immunosuppressive drugs and pharmacology relevant to transplantation; graft survival and prevention of graft dysfunction and failure; clinical trials and population analyses in the field of transplantation; transplant complications; cell and tissue transplantation; infection; post-transplant malignancies; sociological and ethical issues and xenotransplantation.