Joseph Ignatius Irudayam

Characterization of type I interferon pathway during hepatic differentiation of human pluripotent stem cells and hepatitis C virus infection

Pluripotent stem cells are being actively studied as a cell source for regenerating damaged liver. For long-term survival of engrafting cells in the body, not only do the cells have to execute liver-specific function but also withstand the physical strains and invading pathogens. The cellular innate immune system orchestrated by the interferon (IFN) pathway provides the first line of defense against pathogens. The objective of this study is to assess the innate immune function as well as to systematically profile the IFN-induced genes during hepatic differentiation of pluripotent stem cells. To address this objective, we derived endodermal cells (day 5 post-differentiation), hepatoblast (day 15) and hepatocyte-like cells (day 21) from human embryonic stem cells (hESCs). Day 5, 15 and 21 cells were stimulated with IFN-α and subjected to IFN pathway analysis. Transcriptome analysis was carried out by RNA sequencing. The results showed that the IFN-α treatment activated STAT-JAK pathway in differentiating cells. Transcriptome analysis indicated stage specific expression of classical and non-classical IFN-stimulated genes (ISGs). Subsequent validation confirmed the expression of novel ISGs including RASGRP3, CLMP and TRANK1 by differentiated hepatic cells upon IFN treatment. Hepatitis C virus replication in hESC-derived hepatic cells induced the expression of ISGs--LAMP3, ETV7, RASGRP3, and TRANK1. The hESC-derived hepatic cells contain intact innate system and can recognize invading pathogens. Besides assessing the tissue-specific functions for cell therapy applications, it may also be important to test the innate immune function of engrafting cells to ensure adequate defense against infections and improve graft survival.

Bioartificial Liver Device Based on Induced Pluripotent Stem Cell-Derived Hepatocytes

Decompensated liver disorders require liver transplantation. However, the donor organ shortage is a limiting factor. Harnessing the power of human induced pluripotent stem cell (iPSC) technology in combination with hollow fiber-based bioartificial liver (BAL) device can be beneficial to patients with liver failure. Our goal is to develop a BAL module comprised of iPSC-derived hepatocytes (iHeps) arrayed on the extracapillary space (ECS) of hollow fiber membranous capillaries that allow the flow of blood through the intracapillary space (ICS), thus mimicking the tissue microarchitecture. For the proof-of-concept in vitro study, a cartridge having semipermeable polysulfone membrane fibers was used as an artificial liver device. As a source for human liver cells, we derived metabolically active hepatocytes from iPSCs. The iHeps on microcarrier beads were loaded into the ECS of a hollow fiber bioreactor cartridge and cultured using a closed-circuit continuous flow system. The iHeps secreted human albumin, prothrombin, and apolipoprotein B into the hollow fiber ICS media, and the continuous flow system also improved maturation of iHeps. In conclusion, the iPSC-hepatocytes in the bioartificial liver device maintained the secretory function and exhibited cell maturation. The iPSC-hepatocyte BAL has the potential to be further developed as a liver support device for the treatment of decompensated liver diseases.

Hepatic Tm6sf2 overexpression affects cellular ApoB-trafficking, plasma lipid levels, hepatic steatosis and atherosclerosis

The human transmembrane 6 superfamily member 2 (TM6SF2) gene has been implicated in plasma lipoprotein metabolism, alcoholic and non-alcoholic fatty liver disease and myocardial infarction in multiple genome-wide association studies. To investigate the role of Tm6sf2 in metabolic homeostasis, we generated mice with elevated expression using adeno-associated virus (AAV)-mediated gene delivery. Hepatic overexpression of mouse Tm6sf2 resulted in phenotypes previously observed in Tm6sf2-deficient mice including reduced plasma lipid levels, diminished hepatic triglycerides secretion and increased hepatosteatosis. Furthermore, increased hepatic Tm6sf2 expression protected against the development of atherosclerosis in LDL-receptor/ApoB48-deficient mice. In cultured human hepatocytes, Tm6sf2 overexpression reduced apolipoprotein B secretion and resulted in its accumulation within the endoplasmic reticulum (ER) suggesting impaired ER-to-Golgi trafficking of pre-very low-density lipoprotein (VLDL) particles. Analysis of two metabolic trait-associated coding polymorphisms in the human TM6SF2 gene (rs58542926 and rs187429064) revealed that both variants impact TM6SF2 expression by affecting the rate of protein turnover. These data demonstrate that rs58542926 (E167K) and rs187429064 (L156P) are functional variants and suggest that they influence metabolic traits through altered TM6SF2 protein stability. Taken together, our results indicate that cellular Tm6sf2 level is an important determinant of VLDL metabolism and further implicate TM6SF2 as a causative gene underlying metabolic disease and trait associations at the 19p13.11 locus.

Profile of Inflammation-associated genes during Hepatic Differentiation of Human Pluripotent Stem Cells.

Expression of genes associated with inflammation was analyzed during differentiation of human pluripotent stem cells (PSCs) to hepatic cells. Messenger RNA transcript profiles of differentiated endoderm (day 5), hepatoblast (day 15) and hepatocyte-like cells (day 21) were obtained by RNA sequencing analysis. When compared to endoderm cells an immature cell type, the hepatic cells (days 15 and 21) had significantly higher expression of acute phase protein genes including complement factors, coagulation factors, serum amyloid A and serpins. Furthermore, hepatic phase of cells expressed proinflammatory cytokines IL18 and IL32 as well as cytokine receptors IL18R1, IL1R1, IL1RAP, IL2RG, IL6R, IL6ST and IL10RB. These cells also produced CCL14, CCL15, and CXCL- 1, 2, 3, 16 and 17 chemokines. Endoderm cells had higher levels of chemokine receptors, CXCR4 and CXCR7, than that of hepatic cells. Sirtuin family of genes involved in aging, inflammation and metabolism were differentially regulated in endoderm and hepatic phase cells. Ligands and receptors of the tumor necrosis factor (TNF) family as well as downstream signaling factors TRAF2, TRAF4, FADD, NFKB1 and NFKBIB were differentially expressed during hepatic differentiation.

Modeling Liver Diseases Using Induced Pluripotent Stem Cell (Ipsc)-Derived Hepatocytes

The induced pluripotent stem cells (iPSCs) are reprogrammed somatic cells in a stem state. The iPSCs can give rise to cells of all three germ layers and provide an unlimited supply of tissue-specific differentiated cell types for disease modeling and cell therapy. The generation of patient-specific iPSC lines and studying disease phenotype in a dish using differentiated hepatocytes open up new avenue towards personalized medicine. There has been active investigation on generating homogenous functional human hepatocytes from iPSCs. Liver carries out secretory and metabolic functions. Recent studies showed that iPSC derived-human hepatocytes are useful for in vitro investigation of genetic liver disorders, drug screening and metabolism, hepatitis C viral infection and assessing efficacy of cell therapy. Inherited metabolic disorders, including α1-antitrypsin deficiency (A1AD), familial hypercholesterolemia, glycogen storage disease type 1a and Wilson’s disease have been modeled using disease-specific iPSC lines. The iPSC hepatocytes derived from patients with A1AD were used for drug screening. Advancement made in precise genetic engineering technology using designer nucleases provides a new tool for gene correction, and reverse genetic engineering of disease causing genotype in pluripotent stem cells. Moreover, iPSC-hepatocytes from various genetic backgrounds are valuable resource for evaluating drug interactions and drug metabolism. In this review, we summarize the recent developments on the various applications of iPSC-derived human hepatocytes for disease modeling.

C19ORF66 is an Interferon-Stimulated Gene (ISG) which Inhibits Human Immunodeficiency Virus-1

Innate immunity is the first line of defense against invading microbes1. The type I interferon (IFN) pathway plays a key role in controlling Human Immunodeficiency Virus type 1 (HIV-1) replication2,3. We identified an IFN-α stimulated gene C19ORF66 that we term Suppressor of Viral Activity (SVA). Full length SVA-1 protein inhibits HIV-1 by blocking virion production. SVA splice variants truncated at the C-terminus and/or disrupted at the nuclear export signal (NES) lose antiviral activity and localize to nucleus, while full length SVA-1 co-localizes with HIV-1 p24 protein in the cytoplasmic compartment of infected cells. SVA-1 is structurally and functionally conserved across species, including mouse and chimpanzee. We provide the first description of the effector function of the gene SVA/C190RF66 as an innate immune factor with anti-HIV-1 activity.