Abstract
BackgroundDifferentiation of human induced pluripotent stem cells (hiPSCs) into hepatocytes has important clinical significance in providing a new stem cell source for cell therapy of terminal liver disease. The differential gene expression analysis of hiPSCs, induced hepatocyte-like cells (HLCs), and primary human hepatocytes (PHHs) provides valuable information for optimization of an induction scheme and exploration of differentiation mechanisms.MethodsHuman hair follicle-derived iPSCs (hHF-iPSCs) were induced in vitro by mimicking the environment of a developing liver for 19 days. Expression of specific proteins was determined by immunofluorescence staining; the function of HLCs in storage and metabolism was identified by detecting periodic acid–Schiff, indocyanine green, and low-density lipoprotein. Based on the transcriptomics data, the differential gene expression profiles of hHF-iPSCs, HLCs, and PHHs were analyzed by Gene Ontology, Kyoto Encyclopedia of Genes and Genomes pathway, FunRich, and network analysis methods.ResultsHLCs were able to express albumin (ALB), alpha-fetoprotein, CYP3A4, and CYP7A1, and exhibited matured liver cell functions such as glycogen synthesis and storage. Complement and coagulation cascades and metabolic pathways ranked top in the downregulated list of HLCs/PHHs, while the cell cycle ranked top in the upregulated list of HLCs/PHHs. In the protein–protein interaction network, according to the degree rankings, TOP2A, CDK1, etc. were the important upregulated differentially expressed genes (DEGs), while ALB, ACACB, etc. were the major downregulated DEGs in HLCs/PHHs; the module analysis indicated that CDCA8, AURKB, and AURKA were the top upregulated DEGs in HLCs/PHHs.ConclusionsWe presented the differences in gene expression among hHF-iPSCs, HLCs, and PHHs through transcriptome array data and provided new ideas for the optimization of induction.
Highlights
Differentiation of human induced pluripotent stem cells into hepatocytes has important clinical significance in providing a new stem cell source for cell therapy of terminal liver disease
MEFs were cultured in MEF medium (DMEM supplemented with 10% Fetal bovine serum (FBS), 1% nonessential amino acids, 1 mM L -glutamine; all from Gibco, Invitrogen, USA). hHF-iPSCs were acquired by reprogramming human hair follicle-derived MSCs using a cocktail of lentiviruses carrying Yamanaka factors (OCT4, SOX2, C-MYC, and KLF4) [6] and cultured in a T25 culture flask coated with feeder cells in HES medium (80% DMEM/F12 supplemented with 20% KSR, 1% nonessential amino acids, 1 mM L-glutamine, 4 ng/ml human Basic fibroblast growth factor (bFGF), and 0.1 mM β-mercaptoethanol; all from Invitrogen, USA). hHF-iPSCs were separated with 1 mg/ml collagenase type IV (Invitrogen, USA) for 30– 60 min at 37 °C, at a ratio of 1:3 every 6–7 days
Gene Ontology (GO) analysis showed that compared to hHF-iPSCs, upregulated Differentially expressed gene (DEG) of Primary human hepatocyte (PHH) majorly involved the metabolic process of organic acid, oxoacid, carboxylic, etc., and the downregulated DEGs majorly involved in the chromosome organization, mitosis, and cell cycle processes
Summary
Differentiation of human induced pluripotent stem cells (hiPSCs) into hepatocytes has important clinical significance in providing a new stem cell source for cell therapy of terminal liver disease. Human induced pluripotent stem cells (hiPSCs) have similar self-renewal and differentiation potential to embryonic stem (ES) cells, and do not involve ethical issues [3], and they provide a new and suitable seed cell source for liver transplantation. We obtained human hair follicle-derived iPSCs (hHF-iPSCs) possessing the potential for differentiating into HLCs through definitive endoderm (DE) induction via growth factors [5, 6], by means of the identification of protein expression levels, storage function, and the metabolism of glycogen, and determined the function of HLCs and the feasibility of differentiation. We believe that using this method for inducing the differentiation of hESCs into hepatocytes along with the accumulated experience in inducing differentiation of hHF-iPSCs, it would be possible to optimize the technical operation and explore the mechanism of hHF-iPSC differentiation into hepatocytes, which will provide a new way to solve the problem of cell source and lay the foundation for the wide application of iPSCs in the treatment of clinical liver disease
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