Evaluation of Autophagy Process in Differentiation of Human Induced Pluripotent Stem Cells toward Insulin Producing Cells.

Abstract

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.