(665a) Differential Gene Expression Analysis of Primary Human Hepatocytes and iPSC-Hepatocyte like Cells By RNA Seq

Stem cell research has expanded in the last two decades after the discovery of induced pluripotent stem cells (iPSCs). Advantages to iPSCs include retrieval by non-invasive procedures, maintenance of host genotype, and differentiation into various cell types such as iPSC-hepatocyte-like cells (iHLCs). However, disadvantages to using iPSC-derived cells include teratoma formation, tumorigenic properties, and genetic mutations. Specifically, iHLCs have been reported to perform similar functions as primary human hepatocytes (PHHs) such as glycogen storage, albumin secretion, urea production, hepatocyte morphology, and cytochrome P450 (CYP450) expression. However, these liver functions are lower in iHLCs in comparison to PHHs. Although, PHHs are widely used in in vitro liver models, they are difficult to obtain, require invasive retrieval, and have significant donor variability. For these reasons, iHLCs exhibit significant potential in being used instead of PHHs. Due to the reported differences between iHLCs and PHHs, our goal is to understand the transcriptome-wide gene expression differences between these cell types using an RNA Seq analysis. Our focus was to assess the specific pathways and key genes that are upregulated in PHHs compared to iHLCs, specifically looking at pathways related to hepatic functions and drug metabolism.

Methods

RNA extraction occurred immediately upon thawing iHLCs and PHHs and then 48h after a type 1 collagen sandwich (CS) culture was assembled for both cell types. iHLCs were cultured as monolayers for 7 days to enable maturation. CS cultures were assembled 24h after PHH seeding and 7 days after iHLC seeding. RNA extraction was carried out using the RNeasy Plus Micro Kit (Qiagen). Sequencing was conducted at the Biocomplexity Institute of Virginia Tech. RNA Seq samples from the four groups were compared by using DE-Seq2. The sequenced library was aligned to the Gene Ontology (GO) database and Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Genes with false discovery rate (FDR) adjusted p-values < 0.05 were selected as sets of differentially expressed genes and were selected as over- represented or under-represented in each comparison.

Results

We focus our results primarily on PHHs and iHLCs at 48h after CS assembly to investigate differences in pathways associated with hepatocyte function and drug metabolism aligned with the KEGG database. The analysis of RNASeq data showed an upregulation of 44 out of a total of 80 genes involved in bile secretion. Specific genes in this pathway exhibited up to a 455-fold change increase in PHHs compared to iHLCs. Upon immunostaining, bile canaliculi were shown to be present in PHHs. The TCA cycle in PHHs was upregulated compared to iHLCs with 19 out of a total of 29 genes expressing significantly higher fold-changes. Our experimental analysis shows a 2-2.5-fold increase in normalized urea production in PHHs compared to iHLCs. Additionally, the RNA Seq analysis resulted in three drug metabolism pathways that were upregulated in PHHs compared to iHLCs. Metabolism of xenobiotics by CYP450 enzymes displayed an upregulation of 48 out of a total of 66 genes. Significantly higher fold-changes of 43 out of a total of 61 genes were expressed in the metabolism of drugs by CYP450 enzymes pathway. Lastly, drug metabolism by other enzymes showed upregulation of 46 out of a total of 76 genes. Of note, CYP2E1 in PHHs was significantly higher expressed by 21810-fold. Experimental results show PHHs in CS cultures exhibit a 3.7-fold increase in CYP2E1 activity compared to iHLCs.