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.

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.

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.

A Novel UPLC-MS Metabolomic Analysis-Based Strategy to Monitor the Course and Extent of iPSC Differentiation to Hepatocytes.

Typical protocols to differentiate induced pluripotent stem cells (iPSCs) from hepatocyte-like cells (HLCs) imply complex strategies that include transfection with key hepatic transcription factors and the addition to culture media of nutrients, growth factors, and cytokines. A main constraint to evaluate the hepatic phenotype achieved arises from the way the grade of differentiation is determined. Currently, it relies on the assessment of the expression of a limited number of hepatic gene transcripts, less frequently by assessing certain hepatic metabolic functions, and rarely by the global metabolic performance of differentiated cells. We envisaged a new strategy to assess the extent of differentiation achieved, based on the analysis of the cellular metabolome along the differentiation process and its quantitative comparison with that of primary human hepatocytes (PHHs). To validate our approach, we examined the changes in the metabolome of three iPSC progenies (transfected with/without key transcription factors), cultured in three differentiation media, and compared them to PHHs. Results revealed consistent metabolome changes along differentiation and evidenced the factors that more strongly promote changes in the metabolome. The integrated dissimilarities between the PHHs and HLCs retrieved metabolomes were used as a numerical reference for quantifying the degree of iPSCs differentiation. This newly developed metabolome-analysis approach evidenced its utility in assisting us to select a cell's source, culture conditions, and differentiation media, to achieve better-differentiated HLCs.

A fully defined matrix to support a pluripotent stem cell derived multi-cell-liver steatohepatitis and fibrosis model.

Chronic liver injury, as observed in non-alcoholic steatohepatitis (NASH), progressive fibrosis, and cirrhosis, remains poorly treatable. Steatohepatitis causes hepatocyte loss in part by a direct lipotoxic insult, which is amplified by derangements in the non-parenchymal cellular (NPC) interactive network wherein hepatocytes reside, including, hepatic stellate cells, liver sinusoidal endothelial cells and liver macrophages. To create an in vitro culture model encompassing all these cells, that allows studying liver steatosis, inflammation and fibrosis caused by NASH, we here developed a fully defined hydrogel microenvironment, termed hepatocyte maturation (HepMat) gel, that supports maturation and maintenance of pluripotent stem cell (PSC) derived hepatocyte- and NPC-like cells for at least one month. The HepMat-based co-culture system modeled key molecular and functional features of TGFß-induced liver fibrosis and fatty-acid induced inflammation and fibrosis better than monocultures of its constituent cell populations. The novel co-culture system should open new avenues for studying mechanisms underlying liver steatosis, inflammation and fibrosis as well as for assessing drugs counteracting these effects.

Patient-Specific Induced Pluripotent Stem Cell-Derived Hepatocyte-Like Cells as a Model to Study Autosomal Recessive Hypercholesterolemia.

Autosomal recessive hypercholesterolemia (ARH) is a rare monogenic disorder caused by pathogenic variants in the low-density lipoprotein receptor (LDLR) adaptor protein 1 (LDLRAP1) gene, encoding for the LDLRAP1 protein, which impairs internalization of hepatic LDLR. There are variable responses of ARH patients to treatment and the pathophysiological mechanism(s) for this variability remains unclear. This is in part caused by absence of reliable cellular models to evaluate the effect of LDLRAP1 mutations on the LDLRAP1 protein function and its role in LDLR internalization. Here, we aimed to validate patient-specific induced pluripotent stem cell (iPSC)-derived hepatocyte-like cells (HLCs) as an appropriate tool to model ARH disease. Fibroblasts from an ARH patient carrying the recently reported nonsense mutation, c.649G>T, were reprogrammed into hiPSCs using Sendai viral vectors. In addition, we used clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) to create an LDLRAP1 gene (also known as ARH) knockout in two different human iPSC lines. ARH patient-derived iPSCs, ARH-knockout iPSC lines, and control iPSCs were efficiently differentiated into HLCs. Western blot analysis demonstrated the absence of LDLRAP1 in HLCs derived from patient and knockout iPSCs, and this was associated with a decreased low-density lipoprotein cholesterol (LDL-C) uptake in ARH-mutant/knockout HLCs compared to control HLCs. In conclusion, we determined that the recently described c.649G>T point mutation in LDLRAP1 induces absence of the LDLRAP1 protein, similar to what is seen following LDLRAP1 knockout. This causes a decreased, although not fully absent, LDL-uptake in ARH-mutant/knockout HLCs. As knockout of LDLRAP1 or presence of the c.649G>T point mutation results in absence of LDLRAP1 protein, residual LDL uptake might be regulated by LDLRAP1-independent internalization mechanisms. Patient-specific iPSC-derived HLCs can therefore be a powerful tool to further decipher LDLRAP1 mutations and function of the protein.

HiPSC-Derived Hepatocyte-like Cells Can Be Used as a Model for Transcriptomics-Based Study of Chemical Toxicity.

Traditional toxicity risk assessment approaches have until recently focussed mainly on histochemical readouts for cell death. Modern toxicology methods attempt to deduce a mechanistic understanding of pathways involved in the development of toxicity, by using transcriptomics and other big data-driven methods such as high-content screening. Here, we used a recently described optimised method to differentiate human induced pluripotent stem cells (hiPSCs) to hepatocyte-like cells (HLCs), to assess their potential to classify hepatotoxic and non-hepatotoxic chemicals and their use in mechanistic toxicity studies. The iPSC-HLCs could accurately classify chemicals causing acute hepatocellular injury, and the transcriptomics data on treated HLCs obtained by TempO-Seq technology linked the cytotoxicity to cellular stress pathways, including oxidative stress and unfolded protein response (UPR). Induction of these stress pathways in response to amiodarone, diclofenac, and ibuprofen, was demonstrated to be concentration and time dependent. The transcriptomics data on diclofenac-treated HLCs were found to be more sensitive in detecting differentially expressed genes in response to treatment, as compared to existing datasets of other diclofenac-treated in vitro hepatocyte models. Hence iPSC-HLCs generated by transcription factor overexpression and in metabolically optimised medium appear suitable for chemical toxicity detection as well as mechanistic toxicity studies.

Comparing in vitro human liver models to in vivo human liver using RNA-Seq.

The liver plays an important role in xenobiotic metabolism and represents a primary target for toxic substances. Many different in vitro cell models have been developed in the past decades. In this study, we used RNA-sequencing (RNA-Seq) to analyze the following human in vitro liver cell models in comparison to human liver tissue: cancer-derived cell lines (HepG2, HepaRG 3D), induced pluripotent stem cell-derived hepatocyte-like cells (iPSC-HLCs), cancerous human liver-derived assays (hPCLiS, human precision cut liver slices), non-cancerous human liver-derived assays (PHH, primary human hepatocytes) and 3D liver microtissues. First, using CellNet, we analyzed whether these liver in vitro cell models were indeed classified as liver, based on their baseline expression profile and gene regulatory networks (GRN). More comprehensive analyses using non-differentially expressed genes (non-DEGs) and differential transcript usage (DTU) were applied to assess the coverage for important liver pathways. Through different analyses, we noticed that 3D liver microtissues exhibited a high similarity with in vivo liver, in terms of CellNet (C/T score: 0.98), non-DEGs (10,363) and pathway coverage (highest for 19 out of 20 liver specific pathways shown) at the beginning of the incubation period (0 h) followed by a decrease during long-term incubation for 168 and 336 h. PHH also showed a high degree of similarity with human liver tissue and allowed stable conditions for a short-term cultivation period of 24 h. Using the same metrics, HepG2 cells illustrated the lowest similarity (C/T: 0.51, non-DEGs: 5623, and pathways coverage: least for 7 out of 20) with human liver tissue. The HepG2 are widely used in hepatotoxicity studies, however, due to their lower similarity, they should be used with caution. HepaRG models, iPSC-HLCs, and hPCLiS ranged clearly behind microtissues and PHH but showed higher similarity to human liver tissue than HepG2 cells. In conclusion, this study offers a resource of RNA-Seq data of several biological replicates of human liver cell models in vitro compared to human liver tissue.

Therapeutic modalities and novel approaches in regenerative medicine for COVID-19.

The recent coronavirus disease 2019 outbreak around the world has had an enormous impact on the global health burden, threatening the lives of many individuals, and has had severe socio-economic consequences. Many pharmaceutical and biotechnology companies have commenced intensive research on different therapeutic strategies, from repurposed antiviral drugs to vaccines and monoclonal antibodies to prevent the spread of the disease and treat infected patients. Among the various strategies, advanced therapeutic approaches including cell- and gene-editing-based therapeutics are also being investigated, and initial results in in-vitro and early phase I studies have been promising. However, further assessments are required. This article reviews the underlying mechanisms for the pathogenesis of severe acute respiratory syndrome coronavirus-2, and discusses available therapeutic candidates and advanced modalities that are being evaluated in in-vitro/in-vivo models and are of note in clinical trials.

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.

Amino acid levels determine metabolism and CYP450 function of hepatocytes and hepatoma cell lines.

Predicting drug-induced liver injury in a preclinical setting remains challenging, as cultured primary human hepatocytes (PHHs), pluripotent stem cell-derived hepatocyte-like cells (HLCs), and hepatoma cells exhibit poor drug biotransformation capacity. We here demonstrate that hepatic functionality depends more on cellular metabolism and extracellular nutrients than on developmental regulators. Specifically, we demonstrate that increasing extracellular amino acids beyond the nutritional need of HLCs and HepG2 cells induces glucose independence, mitochondrial function, and the acquisition of a transcriptional profile that is closer to PHHs. Moreover, we show that these high levels of amino acids are sufficient to drive HLC and HepG2 drug biotransformation and liver-toxin sensitivity to levels similar to those in PHHs. In conclusion, we provide data indicating that extracellular nutrient levels represent a major determinant of cellular maturity and can be utilized to guide stem cell differentiation to the hepatic lineage.

Dystrophin deficiency leads to dysfunctional glutamate clearance in iPSC derived astrocytes.

Duchenne muscular dystrophy (DMD) results, beside muscle degeneration in cognitive defects. As neuronal function is supported by astrocytes, which express dystrophin, we hypothesized that loss of dystrophin from DMD astrocytes might contribute to these cognitive defects. We generated cortical neuronal and astrocytic progeny from induced pluripotent stem cells (PSC) from six DMD subjects carrying different mutations and several unaffected PSC lines. DMD astrocytes displayed cytoskeletal abnormalities, defects in Ca+2 homeostasis and nitric oxide signaling. In addition, defects in glutamate clearance were identified in DMD PSC-derived astrocytes; these deficits were related to a decreased neurite outgrowth and hyperexcitability of neurons derived from healthy PSC. Read-through molecule restored dystrophin expression in DMD PSC-derived astrocytes harboring a premature stop codon mutation, corrected the defective astrocyte glutamate clearance and prevented associated neurotoxicity. We propose a role for dystrophin deficiency in defective astroglial glutamate homeostasis which initiates defects in neuronal development.

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 NFκß 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.

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 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.

HDAC6 inhibition reverses axonal transport defects in motor neurons derived from FUS-ALS patients.

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive neurodegenerative disorder due to selective loss of motor neurons (MNs). Mutations in the fused in sarcoma (FUS) gene can cause both juvenile and late onset ALS. We generated and characterized induced pluripotent stem cells (iPSCs) from ALS patients with different FUS mutations, as well as from healthy controls. Patient-derived MNs show typical cytoplasmic FUS pathology, hypoexcitability, as well as progressive axonal transport defects. Axonal transport defects are rescued by CRISPR/Cas9-mediated genetic correction of the FUS mutation in patient-derived iPSCs. Moreover, these defects are reproduced by expressing mutant FUS in human embryonic stem cells (hESCs), whereas knockdown of endogenous FUS has no effect, confirming that these pathological changes are mutant FUS dependent. Pharmacological inhibition as well as genetic silencing of histone deacetylase 6 (HDAC6) increase α-tubulin acetylation, endoplasmic reticulum (ER)-mitochondrial overlay, and restore the axonal transport defects in patient-derived MNs.Amyotrophic lateral sclerosis (ALS) leads to selective loss of motor neurons. Using motor neurons derived from induced pluripotent stem cells from patients with ALS and FUS mutations, the authors demonstrate that axonal transport deficits that are observed in these cells can be rescued by HDAC6 inhibition.