CRISPR/Cas9 screen in human iPSC‐derived cortical neurons identifies NEK6 as a novel disease modifier of C9orf72 poly(PR) toxicity

Introduction The most common genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are hexanucleotide repeats in chromosome 9 open reading frame 72 (C9orf72). These repeats produce dipeptide repeat proteins with poly(PR) being the most toxic one. Methods We performed a kinome-wide CRISPR/Cas9 knock-out screen in human induced pluripotent stem cell (iPSC) -derived cortical neurons to identify modifiers of poly(PR) toxicity, and validated the role of candidate modifiers using in vitro, in vivo, and ex-vivo studies. Results Knock-down of NIMA-related kinase 6 (NEK6) prevented neuronal toxicity caused by poly(PR). Knock-down of nek6 also ameliorated the poly(PR)-induced axonopathy in zebrafish and NEK6 was aberrantly expressed in C9orf72 patients. Suppression of NEK6 expression and NEK6 activity inhibition rescued axonal transport defects in cortical neurons from C9orf72 patient iPSCs, at least partially by reversing p53-related DNA damage. Discussion We identified NEK6, which regulates poly(PR)-mediated p53-related DNA damage, as a novel therapeutic target for C9orf72 FTD/ALS

Ontwikkeling van een fysiologisch relevant model voor hepatotrope virussen met behulp van hepatocyten afgeleid van humane pluripotente stamcellen

The pharmaceutical industry is in high need of efficient and physiologically relevant in vitro and in vivo models to study hepatotropic viruses, screen for potential antiviral drugs and test for liver toxicity that might be caused by these and other drugs. Current in vitro models rely on primary human hepatocytes (PHHs) and hepatoma cell lines, which have major drawbacks. As an alternative hepatocyte source, the use of human pluripotent stem cell derived hepatocyte-like cells (hPSC-HLCs) to study hepatotropic viruses has been proposed. Previous studies have shown that these hPSC-HLCs are indeed relevant models to study hepatotropic viruses as they are susceptible for hepatitis C, hepatitis B, hepatitis E and dengue virus. In this thesis, I examined whether hPSC-HLCs could also be a relevant model to study zika virus (ZIKV) infection. ZIKV has recently been linked to microcephaly in newborns. However, ZIKV-infected mouse models and non-human primates do not only have a high viral load in the brain, but also in other organs such as the liver. Additionally, as two reports described the association between ZIKV infection and liver injury and as ZIKV belongs to the same viral family of known hepatotropic viruses, such as hepatitis C and dengue virus, I examined whether human hepatocytes are susceptible to ZIKV infection. I demonstrated that hPSC-HLCs and the hepatoma cell line, Huh7, supports the complete ZIKV life cycle, including the entry, viral replication and the production of novel infectious ZIKV virions. Furthermore, treatment of ZIKV-infected hPSC-HLCs and Huh7 cells with 7-deaza-2'-C-methyladenosine, a known viral RNA-dependent RNA polymerase inhibitor, decreased ZIKV replication significantly in a dose-dependent manner, while 2'-C-methylcytidine and 6-fluoro-3-hydroxypyrazine-2-carboxamide only decreased ZIKV replication in ZIKV-infected Huh7 cells. Finally, ZIKV-infected hPSC-HLCs but not Huh7 cells induced an innate immune and NFκβ response, which may explain the more extensive cytopathic effect observed in ZIKV-infected Huh7 cells. These significant differences between hPSC-HLCs and hepatoma cells in the innate immune response against ZIKV and antiviral drug sensitivity highlight the need to assess ZIKV infection as well as antiviral activity not only in cell line models, but also in more physiologically relevant systems. As hPSC-HLCs used to demonstrate the susceptibility of hepatocytes to ZIKV, resemble fetal hepatocytes rather than adult primary hepatocytes and do not have drug metabolizing activity, I tested if metabolically improved hPSC-HLCs created by the Verfaillie lab at the Stem Cell Institute Leuven, consisting of inducible overexpression of 3 transcription factors (TFs) HNF1α, PROX1 and FOXA3 (termed HC3x-HLCs) and cultured in metabolically optimized differentiation culture medium, would be a suitable model to study hepatitis B virus (HBV) and to test new anti-HBV antivirals that might need to undergo biotransformation to be active. The reason to study HBV infection is that despite an adequate anti-HBV vaccine, 260 million people remain chronically infected with HBV worldwide and available treatments do not cure patients as the covalently closed circular DNA (cccDNA) is retained in the hepatocyte nucleus. Hence, there is an urgent need for novel therapeutics that can eliminate HBV infection in chronically infected patients. I here demonstrated that metabolically improved HC3x-HLCs can efficiently be infected with HBV by immunofluorescence staining of HBV core antigen and surface antigens (HBsAg). Furthermore, I detected the release of HBsAg and HBV e antigen in the supernatant, which increased over time, via ELISA, as well as high titers of infectious virus when back-titrated on HepG2-NTCP cells. This data suggested that functional cccDNA was formed by the HBV-infected HC3x-HLCs. Additionally, I validated the model for use in antiviral drug studies using various known HBV inhibitors. Finally, as the pharmaceutical industry also requires suitable in vivo models to study hepatotropic viruses and test drugs for liver toxicity, I attempted to transplant the metabolically improved HC3x-HLCs and HC6x-HLCs (that overexpress not only HNF1α, PROX1, FOXA3 but also PGC1α, SIRT1 and AMPK) into urokinase plasminogen activator - severe combined immunodeficiency (uPA-SCID) mice to generate a humanized liver mouse model. Six months after transplantation, human cells were detected in the livers of mice transplanted with HC3x-HLCs, but not HC6x-HLCs. However, engraftment efficiencies were low, despite the enhanced maturity of HC3x-HLCs compared to many other HLC populations. Studies will be required to determine if the failure to robustly repopulate murine livers is due to the specific mouse model used or due to failure to home and establish initial engraftment, and/or subsequent expansion of the commonly small numbers of initially engrafted cells.status: publishe

Alternative Cell Sources for Liver Parenchyma Repopulation: Where Do We Stand?

Acute and chronic liver failure is a highly prevalent medical condition with high morbidity and mortality. Currently, the therapy is orthotopic liver transplantation. However, in some instances, chiefly in the setting of metabolic diseases, transplantation of individual cells, specifically functional hepatocytes, can be an acceptable alternative. The gold standard for this therapy is the use of primary human hepatocytes, isolated from livers that are not suitable for whole organ transplantations. Unfortunately, primary human hepatocytes are scarcely available, which has led to the evaluation of alternative sources of functional hepatocytes. In this review, we will compare the ability of most of these candidate alternative cell sources to engraft and repopulate the liver of preclinical animal models with the repopulation ability found with primary human hepatocytes. We will discuss the current shortcomings of the different cell types, and some of the next steps that we believe need to be taken to create alternative hepatocyte progeny capable of regenerating the failing liver.status: publishe

Current Status and Challenges of Human Induced Pluripotent Stem Cell-Derived Liver Models in Drug Discovery

The pharmaceutical industry is in high need of efficient and relevant in vitro liver models, which can be incorporated in their drug discovery pipelines to identify potential drugs and their toxicity profiles. Current liver models often rely on cancer cell lines or primary cells, which both have major limitations. However, the development of human induced pluripotent stem cells (hiPSCs) has created a new opportunity for liver disease modeling, drug discovery and liver toxicity research. hiPSCs can be differentiated to any cell of interest, which makes them good candidates for disease modeling and drug discovery. Moreover, hiPSCs, unlike primary cells, can be easily genome-edited, allowing the creation of reporter lines or isogenic controls for patient-derived hiPSCs. Unfortunately, even though liver progeny from hiPSCs has characteristics similar to their in vivo counterparts, the differentiation of iPSCs to fully mature progeny remains highly challenging and is a major obstacle for the full exploitation of these models by pharmaceutical industries. In this review, we discuss current liver-cell differentiation protocols and in vitro iPSC-based liver models that could be used for disease modeling and drug discovery. Furthermore, we will discuss the challenges that still need to be overcome to allow for the successful implementation of these models into pharmaceutical drug discovery platforms

Alternative Cell Sources for Liver Parenchyma Repopulation: Where Do We Stand?

Acute and chronic liver failure is a highly prevalent medical condition with high morbidity and mortality. Currently, the therapy is orthotopic liver transplantation. However, in some instances, chiefly in the setting of metabolic diseases, transplantation of individual cells, specifically functional hepatocytes, can be an acceptable alternative. The gold standard for this therapy is the use of primary human hepatocytes, isolated from livers that are not suitable for whole organ transplantations. Unfortunately, primary human hepatocytes are scarcely available, which has led to the evaluation of alternative sources of functional hepatocytes. In this review, we will compare the ability of most of these candidate alternative cell sources to engraft and repopulate the liver of preclinical animal models with the repopulation ability found with primary human hepatocytes. We will discuss the current shortcomings of the different cell types, and some of the next steps that we believe need to be taken to create alternative hepatocyte progeny capable of regenerating the failing liver

Recent advances in lineage differentiation from stem cells: hurdles and opportunities? [version 1; referees: 2 approved]

Pluripotent stem cells have the property of long-term self-renewal and the potential to give rise to descendants of the three germ layers and hence all mature cells in the human body. Therefore, they hold the promise of offering insight not only into human development but also for human disease modeling and regenerative medicine. However, the generation of mature differentiated cells that closely resemble their in vivo counterparts remains challenging. Recent advances in single-cell transcriptomics and computational modeling of gene regulatory networks are revealing a better understanding of lineage commitment and are driving modern genome editing approaches. Additional modification of the chemical microenvironment, as well as the use of bioengineering tools to recreate the cellular, extracellular matrix, and physical characteristics of the niche wherein progenitors and mature cells reside, is now being used to further improve the maturation and functionality of stem cell progeny

Recent advances in lineage differentiation from stem cells: hurdles and opportunities?

Pluripotent stem cells have the property of long-term self-renewal and the potential to give rise to descendants of the three germ layers and hence all mature cells in the human body. Therefore, they hold the promise of offering insight not only into human development but also for human disease modeling and regenerative medicine. However, the generation of mature differentiated cells that closely resemble theircounterparts remains challenging. Recent advances in single-cell transcriptomics and computational modeling of gene regulatory networks are revealing a better understanding of lineage commitment and are driving modern genome editing approaches. Additional modification of the chemical microenvironment, as well as the use of bioengineering tools to recreate the cellular, extracellular matrix, and physical characteristics of the niche wherein progenitors and mature cells reside, is now being used to further improve the maturation and functionality of stem cell progeny.status: publishe

Human stem cell-derived hepatocyte-like cells support Zika virus replication and provide a relevant model to assess the efficacy of potential antivirals

Zika virus (ZIKV) infection during pregnancy has been extensively linked to microcephaly in newborns. High levels of ZIKV RNA were, however, also detected in mice and non-human primates in organs other than the brain, such as the liver. As ZIKV is a flavivirus closely related to the dengue and yellow fever virus, which are known to cause hepatitis, we here examined whether human hepatocytes are susceptible to ZIKV infection. We demonstrated that both human pluripotent stem cell (hPSC)-derived hepatocyte-like cells (HLCs) and the Huh7 hepatoma cell line support the complete ZIKV replication cycle. Of three antiviral molecules that inhibit ZIKV infection in Vero cells, only 7-deaza-2'-C-methyladenosine (7DMA) inhibited ZIKV replication in hPSC-HLCs, while all drugs inhibited ZIKV infection in Huh7 cells. ZIKV-infected hPSC-HLCs but not Huh7 cells mounted an innate immune and NFκβ response, which may explain the more extensive cytopathic effect observed in Huh7 cells. In conclusion, ZIKV productively infects human hepatocytes in vitro. However, significant differences in the innate immune response against ZIKV and antiviral drug sensitivity were observed when comparing hPSC-HLCs and hepatoma cells, highlighting the need to assess ZIKV infection as well as antiviral activity not only in hepatoma cells, but also in more physiologically relevant systems.status: publishe

Human stem cell-derived hepatocyte-like cells support Zika virus replication and provide a relevant model to assess the efficacy of potential antivirals.

Zika virus (ZIKV) infection during pregnancy has been extensively linked to microcephaly in newborns. High levels of ZIKV RNA were, however, also detected in mice and non-human primates in organs other than the brain, such as the liver. As ZIKV is a flavivirus closely related to the dengue and yellow fever virus, which are known to cause hepatitis, we here examined whether human hepatocytes are susceptible to ZIKV infection. We demonstrated that both human pluripotent stem cell (hPSC)-derived hepatocyte-like cells (HLCs) and the Huh7 hepatoma cell line support the complete ZIKV replication cycle. Of three antiviral molecules that inhibit ZIKV infection in Vero cells, only 7-deaza-2'-C-methyladenosine (7DMA) inhibited ZIKV replication in hPSC-HLCs, while all drugs inhibited ZIKV infection in Huh7 cells. ZIKV-infected hPSC-HLCs but not Huh7 cells mounted an innate immune and NFκβ response, which may explain the more extensive cytopathic effect observed in Huh7 cells. In conclusion, ZIKV productively infects human hepatocytes in vitro. However, significant differences in the innate immune response against ZIKV and antiviral drug sensitivity were observed when comparing hPSC-HLCs and hepatoma cells, highlighting the need to assess ZIKV infection as well as antiviral activity not only in hepatoma cells, but also in more physiologically relevant systems

Metabolically Improved Stem Cell Derived Hepatocyte-Like Cells Support HBV Life Cycle and Are a Promising Tool for HBV Studies and Antiviral Drug Screenings

More than 300 million people worldwide are diagnosed with a chronic hepatitis B virus (HBV) infection. Nucleos(t)ide viral polymerase inhibitors are available on the market and can efficiently treat patients with chronic HBV. However, life-long treatment is needed as covalently closed circular DNA (cccDNA) persists in the hepatocyte nucleus. Hence, there is a high demand for novel therapeutics that can eliminate cccDNA from the hepatocyte nucleus and cure chronically infected HBV patients. The gold standard for in vitro HBV studies is primary human hepatocytes (PHHs). However, alternatives are needed due to donor organ shortage and high batch-to-batch variability. Therefore, human pluripotent stem cell (hPSC)-derived hepatocyte-like cells (HLCs) are being explored as an in vitro HBV infection model. We recently generated hPSC lines that overexpress three transcription factors (HC3x) and that, upon differentiation in a high amino-acid supplemented maturation medium, generate a more mature hepatocyte progeny (HC3x-AA-HLCs). Here, we demonstrate that HBV can efficiently infect these HC3x-AA-HLCs, as was shown by the presence of HBV core (HBc) and surface antigens. A clear increasing release of HBV surface and e antigens was detected, indicating the formation of functional cccDNA. Moreover, back-titration of culture supernatant of HBV-infected HC3x-AA-HLCs on HepG2-NTCP cells revealed the production of novel infectious HBV particles. Additionally, an increasing number of HBc-positive HC3x-AA-HLCs over time suggests viral spreading is occurring. Finally, the HC3x-AA-HLC model was validated for use in antiviral drug studies using the nucleoside reverse-transcriptase inhibitor, lamivudine, and the HBV entry inhibitor, Myrcludex B