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

Abstract

Introduction: Autophagy is an intracellular self-degradative homeostasis process which eliminates undesirable and harmful cellular components including macromolecules and organelles. Autophagy is also involved in self-renewal and differentiation of induced pluripotent stem cell (iPSCs). In this study, we investigated the expression profile of autophagy marker genes in human iPSCs (hiPSCs) during their differentiation induction toward insulin producing b-like cells.

Method: Human iPSC line, R1-hiPSC1, was used for differentiation induction toward B-like cells. The mRNA expression of Nanog, OCT4, SOX17, FOXA2, PTF1A, NKX6.1, and PDX1 were measured during six stages of differentiation induction process including embryoid body (EB), definitive endoderm (DE), primitive gut (PG), posterior foregut (PF), pancreatic endoderm (PE) and B-like cells. Autophagy was monitored by genes expression of four autophagy markers including, MAP1LC3B, BECN1, SQSTM1/P62 and ATG5 during differentiation stages of EB, mesendoderm induction (MEI), 1^th day of definitive endoderm induction (DEI 1), 2^nd day of definitive endoderm induction (DEI 2), DE, PG, PF, PE and B-like cell.

Result: The mRNA expression measurement of NANOG, OCT4 (pluripotency markers), SOX17, FOXA2 (endodermic markers), PTF1A, NKX6.1 (key determinants of exocrine/endocrine patterning) and PDX1 genes confirmed that hiPSCs skipped pluripotency stage, differentiated into endoderm stage, passed through the pancreatic lineage commitment stage and successfully generated insulin producing B-like cell. Expression level of autophagy genes MAP1LC3B, BECN-1, SQSTM1/P62 and ATG5 during differentiation stages indicated the decreased expression levels at the early stages of differentiation (EB and MEI) and then increased at the DEI 1, DEI 2 and DE stages 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 implication of high autophagy levels and contribution of key regulatory genes/proteins during the differentiation of hiPSC toward b-like cells. The enhanced autophagy levels were a prominent feature of early stages of differentiation and DE rather than the later stages. Further studies are warranted to further elucidate the precise role of autophagy and its interaction with different signaling pathways during the differentiation of hiPSC toward b-like cells.