MicroRNA miRNA Expression Profiling during Hepatic Diffrentiation from Stem Cells

Abstract

MicroRNAs (miRNAs) play an important role in the control of cell fate determination during differentiation. In this study, we used high-throughput sequencing to identify miRNA dynamics during hepatic trans-differentiation of human umbilical cord Wharton s jelly derived mesenchymal stem cells (hUC-MSCs). The protocol employed the use of valproic acid (VPA), a histone deacetylase inhibitor (HDACI) to enhance hepatic trans-differentiation ability of hUC-MSCs. The differentiated hepatocyte like cells (HLCs) from hUC-MSCs shared typical characteristics with mature hepatocytes, including morphology, expression of hepatocyte specific genes at the molecular and cellular level. Moreover, the functionality of HLCs was confirmed through various liver function tests such as periodic acid-Schiff (PAS) stain for glycogen accumulation, enzyme-linked immunosorbent assay (ELISA) for synthesis of albumin and release of urea. newlineLimited regulatory miRNAs have been described to date during hepatic differentiation of stem cells. The present study was designed to identify the signature miRNAs expressed during various days of differentiation. Six small RNA libraries were generated and sequenced simultaneously. The proton torrent sequencing system was used to generate small RNA reads from two biological replicates set (six samples). A total of 75,007,963 raw reads were obtained from six samples and after removal of lower quality reads, 24,317,194 high quality reads were utilized for further analysis. The low abundant miRNA count with less than a five normalized read count was removed across all samples. Further, the number of known miRNAs identified in our study was 880 of 2588 known mature miRNAs from miRBase V21 using miRDeep2 algorithm. The most abundantly expressed miRNAs in hUC-MSCs and HLCs were hsa-miR-21-5p and hsa-miR-122-5p with miRNA count 428,287 and 184,213 respectively.