HDAC6 inhibition restores TDP-43 pathology and axonal transport defects in human motor neurons with TARDBP mutations.

TDP-43 is the major component of pathological inclusions in most ALS patients and in up to 50% of patients with frontotemporal dementia (FTD). Heterozygous missense mutations in TARDBP, the gene encoding TDP-43, are one of the common causes of familial ALS. In this study, we investigate TDP-43 protein behavior in induced pluripotent stem cell (iPSC)-derived motor neurons from three ALS patients with different TARDBP mutations, three healthy controls and an isogenic control. TARDPB mutations induce several TDP-43 changes in spinal motor neurons, including cytoplasmic mislocalization and accumulation of insoluble TDP-43, C-terminal fragments, and phospho-TDP-43. By generating iPSC lines with allele-specific tagging of TDP-43, we find that mutant TDP-43 initiates the observed disease phenotypes and has an altered interactome as indicated by mass spectrometry. Our findings also indicate that TDP-43 proteinopathy results in a defect in mitochondrial transport. Lastly, we show that pharmacological inhibition of histone deacetylase 6 (HDAC6) restores the observed TDP-43 pathologies and the axonal mitochondrial motility, suggesting that HDAC6 inhibition may be an interesting therapeutic target for neurodegenerative disorders linked to TDP-43 pathology.

Breast cancer cells rely on environmental pyruvate to shape the metastatic niche.

The extracellular matrix is a major component of the local environment-that is, the niche-that determines cell behaviour1. During metastatic growth, cancer cells shape the extracellular matrix of the metastatic niche by hydroxylating collagen to promote their own metastatic growth2,3. However, only particular nutrients might support the ability of cancer cells to hydroxylate collagen, because nutrients dictate which enzymatic reactions are active in cancer cells4,5. Here we show that breast cancer cells rely on the nutrient pyruvate to drive collagen-based remodelling of the extracellular matrix in the lung metastatic niche. Specifically, we discovered that pyruvate uptake induces the production of α-ketoglutarate. This metabolite in turn activates collagen hydroxylation by increasing the activity of the enzyme collagen prolyl-4-hydroxylase (P4HA). Inhibition of pyruvate metabolism was sufficient to impair collagen hydroxylation and consequently the growth of breast-cancer-derived lung metastases in different mouse models. In summary, we provide a mechanistic understanding of the link between collagen remodelling and the nutrient environment in the metastatic niche.

Evidence for an alternative fatty acid desaturation pathway increasing cancer plasticity.

Most tumours have an aberrantly activated lipid metabolism1,2 that enables them to synthesize, elongate and desaturate fatty acids to support proliferation. However, only particular subsets of cancer cells are sensitive to approaches that target fatty acid metabolism and, in particular, fatty acid desaturation3. This suggests that many cancer cells contain an unexplored plasticity in their fatty acid metabolism. Here we show that some cancer cells can exploit an alternative fatty acid desaturation pathway. We identify various cancer cell lines, mouse hepatocellular carcinomas, and primary human liver and lung carcinomas that desaturate palmitate to the unusual fatty acid sapienate to support membrane biosynthesis during proliferation. Accordingly, we found that sapienate biosynthesis enables cancer cells to bypass the known fatty acid desaturation pathway that is dependent on stearoyl-CoA desaturase. Thus, only by targeting both desaturation pathways is the in vitro and in vivo proliferation of cancer cells that synthesize sapienate impaired. Our discovery explains metabolic plasticity in fatty acid desaturation and constitutes an unexplored metabolic rewiring in cancers.

Disruption of MAM integrity in mutant FUS oligodendroglial progenitors from hiPSCs.

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive and fatal neurodegenerative disorder, characterized by selective loss of motor neurons (MNs). A number of causative genetic mutations underlie the disease, including mutations in the fused in sarcoma (FUS) gene, which can lead to both juvenile and late-onset ALS. Although ALS results from MN death, there is evidence that dysfunctional glial cells, including oligodendroglia, contribute to neurodegeneration. Here, we used human induced pluripotent stem cells (hiPSCs) with a R521H or a P525L mutation in FUS and their isogenic controls to generate oligodendrocyte progenitor cells (OPCs) by inducing SOX10 expression from a TET-On SOX10 cassette. Mutant and control iPSCs differentiated efficiently into OPCs. RNA sequencing identified a myelin sheath-related phenotype in mutant OPCs. Lipidomic studies demonstrated defects in myelin-related lipids, with a reduction of glycerophospholipids in mutant OPCs. Interestingly, FUSR521H OPCs displayed a decrease in the phosphatidylcholine/phosphatidylethanolamine ratio, known to be associated with maintaining membrane integrity. A proximity ligation assay further indicated that mitochondria-associated endoplasmic reticulum membranes (MAM) were diminished in both mutant FUS OPCs. Moreover, both mutant FUS OPCs displayed increased susceptibility to ER stress when exposed to thapsigargin, and exhibited impaired mitochondrial respiration and reduced Ca2+ signaling from ER Ca2+ stores. Taken together, these results demonstrate a pathological role of mutant FUS in OPCs, causing defects in lipid metabolism associated with MAM disruption manifested by impaired mitochondrial metabolism with increased susceptibility to ER stress and with suppressed physiological Ca2+ signaling. As such, further exploration of the role of oligodendrocyte dysfunction in the demise of MNs is crucial and will provide new insights into the complex cellular mechanisms underlying ALS.

Automated Generation of hiPSC-Derived Hepatic Progeny by Cost-Efficient Compounds.

Human pluripotent stem cell (hPSC)-derived hepatocyte-like cells (HLCs) hold great promise for liver disease modeling, drug discovery, and drug toxicity screens. Yet, several hurdles still need to be overcome, including among others decrease in the cost of goods to generate HLCs and automation of the differentiation process. We here describe that the use of an automated liquid handling system results in highly reproducible HLC differentiation from hPSCs. This enabled us to screen 92 chemicals to replace expensive growth factors at each step of the differentiation protocol to reduce the cost of goods of the differentiation protocol by approximately 79%. In addition, we also evaluated several recombinant extracellular matrices to replace Matrigel. We demonstrated that differentiation of hPSCs on Laminin-521 using an optimized small molecule combination resulted in HLCs that were transcriptionally identical to HLCs generated using the growth factor combinations. In addition, the HLCs created using the optimized small molecule combination secreted similar amounts of albumin and urea, and relatively low concentrations of alfa-fetoprotein, displayed similar CYP3A4 functionality, and a similar drug toxicity susceptibility as HLCs generated with growth factor cocktails. The broad applicability of the new differentiation protocol was demonstrated for 4 different hPSC lines. This allowed the creation of a scalable, xeno-free, and cost-efficient hPSC-derived HLC culture, suitable for high throughput disease modeling and drug screenings, or even for the creation of HLCs for regenerative therapies.

From methylation to myelination: epigenomic and transcriptomic profiling of chronic inactive demyelinated multiple sclerosis lesions.

In the progressive phase of multiple sclerosis (MS), the hampered differentiation capacity of oligodendrocyte precursor cells (OPCs) eventually results in remyelination failure. We have previously shown that DNA methylation of Id2/Id4 is highly involved in OPC differentiation and remyelination. In this study, we took an unbiased approach by determining genome-wide DNA methylation patterns within chronically demyelinated MS lesions and investigated how certain epigenetic signatures relate to OPC differentiation capacity. We compared genome-wide DNA methylation and transcriptional profiles between chronically demyelinated MS lesions and matched normal-appearing white matter (NAWM), making use of post-mortem brain tissue (n = 9/group). DNA methylation differences that inversely correlated with mRNA expression of their corresponding genes were validated for their cell-type specificity in laser-captured OPCs using pyrosequencing. The CRISPR-dCas9-DNMT3a/TET1 system was used to epigenetically edit human-iPSC-derived oligodendrocytes to assess the effect on cellular differentiation. Our data show hypermethylation of CpGs within genes that cluster in gene ontologies related to myelination and axon ensheathment. Cell type-specific validation indicates a region-dependent hypermethylation of MBP, encoding for myelin basic protein, in OPCs obtained from white matter lesions compared to NAWM-derived OPCs. By altering the DNA methylation state of specific CpGs within the promotor region of MBP, using epigenetic editing, we show that cellular differentiation and myelination can be bidirectionally manipulated using the CRISPR-dCas9-DNMT3a/TET1 system in vitro. Our data indicate that OPCs within chronically demyelinated MS lesions acquire an inhibitory phenotype, which translates into hypermethylation of crucial myelination-related genes. Altering the epigenetic status of MBP can restore the differentiation capacity of OPCs and possibly boost (re)myelination.

Selective PDE4 subtype inhibition provides new opportunities to intervene in neuroinflammatory versus myelin damaging hallmarks of multiple sclerosis.

Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system (CNS) characterized by focal inflammatory lesions and prominent demyelination. Even though the currently available therapies are effective in treating the initial stages of disease, they are unable to halt or reverse disease progression into the chronic progressive stage. Thus far, no repair-inducing treatments are available for progressive MS patients. Hence, there is an urgent need for the development of new therapeutic strategies either targeting the destructive immunological demyelination or boosting endogenous repair mechanisms. Using in vitro, ex vivo, and in vivo models, we demonstrate that selective inhibition of phosphodiesterase 4 (PDE4), a family of enzymes that hydrolyzes and inactivates cyclic adenosine monophosphate (cAMP), reduces inflammation and promotes myelin repair. More specifically, we segregated the myelination-promoting and anti-inflammatory effects into a PDE4D- and PDE4B-dependent process respectively. We show that inhibition of PDE4D boosts oligodendrocyte progenitor cells (OPC) differentiation and enhances (re)myelination of both murine OPCs and human iPSC-derived OPCs. In addition, PDE4D inhibition promotes in vivo remyelination in the cuprizone model, which is accompanied by improved spatial memory and reduced visual evoked potential latency times. We further identified that PDE4B-specific inhibition exerts anti-inflammatory effects since it lowers in vitro monocytic nitric oxide (NO) production and improves in vivo neurological scores during the early phase of experimental autoimmune encephalomyelitis (EAE). In contrast to the pan PDE4 inhibitor roflumilast, the therapeutic dose of both the PDE4B-specific inhibitor A33 and the PDE4D-specific inhibitor Gebr32a did not trigger emesis-like side effects in rodents. Finally, we report distinct PDE4D isoform expression patterns in human area postrema neurons and human oligodendroglia lineage cells. Using the CRISPR-Cas9 system, we confirmed that pde4d1/2 and pde4d6 are the key targets to induce OPC differentiation. Collectively, these data demonstrate that gene specific PDE4 inhibitors have potential as novel therapeutic agents for targeting the distinct disease processes of MS.

Transcriptomics analysis of human iPSC-derived dopaminergic neurons reveals a novel model for sporadic Parkinson's disease.

Parkinson's disease (PD) is a progressive, neurodegenerative disease affecting over 1% of the population beyond 65 years of age. Although some PD cases are inheritable, the majority of PD cases occur in a sporadic manner. Risk factors comprise next to heredity, age, and gender also exposure to neurotoxins from for instance pesticides and herbicides. As PD is characterized by a loss of dopaminergic neurons in the substantia nigra, it is nearly impossible to access and extract these cells from patients for investigating disease mechanisms. The emergence of induced pluripotent stem (iPSC) technology allows differentiating and growing human dopaminergic neurons, which can be used for in vitro disease modeling. Here, we differentiated human iPSCs into dopaminergic neurons, and subsequently exposed the cells to increasing concentrations of the neurotoxin MPP+. Temporal transcriptomics analysis revealed a strong time- and dose-dependent response with genes over-represented across pathways involved in PD etiology such as "Parkinson's Disease", "Dopaminergic signaling" and "calcium signaling". Moreover, we validated this disease model by showing robust overlap with a meta-analysis of transcriptomics data from substantia nigra from post-mortem PD patients. The overlap included genes linked to e.g. mitochondrial dysfunction, neuron differentiation, apoptosis and inflammation. Our data shows, that MPP+-induced, human iPSC-derived dopaminergic neurons present molecular perturbations as observed in the etiology of PD. Therefore we propose iPSC-derived dopaminergic neurons as a foundation for a novel sporadic PD model to study the pathomolecular mechanisms of PD, but also to screen for novel anti-PD drugs and to develop and test new treatment strategies.

Niche-Mediated Integrin Signaling Supports Steady-State Hematopoiesis in the Spleen.

Outside-in integrin signaling regulates cell fate decisions in a variety of cell types, including hematopoietic stem cells (HSCs). Our earlier published studies showed that interruption of periostin (POSTN) and integrin-αv (ITGAV) interaction induces faster proliferation in HSCs with developmental stage-dependent functional effects. In this study, we examined the role of POSTN-ITGAV axis in lymphohematopoietic activity in spleen that hosts a rare population of HSCs, the functional regulation of which is not clearly known. Vav-iCre-mediated deletion of Itgav in the hematopoietic system led to higher proliferation rates, resulting in increased frequency of primitive HSCs in the adult spleen. However, in vitro CFU-C assays demonstrated a poorer differentiation potential following Itgav deletion. This also led to a decrease in the white pulp area with a significant decline in the B cell numbers. Systemic deletion of its ligand, POSTN, phenocopied the effects noted in Vav-Itgav-/- mice. Histological examination of Postn-deficient spleen also showed an increase in the spleen trabecular areas. Importantly, these are the myofibroblasts of the trabecular and capsular areas that expressed high levels of POSTN within the spleen tissue. In addition, vascular smooth muscle cells also expressed POSTN. Through CFU-S12 assays, we showed that hematopoietic support potential of stroma in Postn-deficient splenic hematopoietic niche was defective. Overall, we demonstrate that POSTN-ITGAV interaction plays an important role in spleen lymphohematopoiesis.

DJ-1 is an essential downstream mediator in PINK1/parkin-dependent mitophagy.

Loss-of-function mutations in the PRKN, PINK1 and PARK7 genes (encoding parkin, PINK1 and DJ-1, respectively) cause autosomal recessive forms of Parkinson's disease. PINK1 and parkin jointly mediate selective autophagy of damaged mitochondria (mitophagy), but the mechanisms by which loss of DJ-1 induces Parkinson's disease are not well understood. Here, we investigated PINK1/parkin-mediated mitophagy in cultured human fibroblasts and induced pluripotent stem cell-derived neurons with homozygous PARK7 mutations. We found that DJ-1 is essential for PINK1/parkin-mediated mitophagy. Loss of DJ-1 did not interfere with PINK1 or parkin activation after mitochondrial depolarization but blocked mitophagy further downstream by inhibiting recruitment of the selective autophagy receptor optineurin to depolarized mitochondria. By contrast, starvation-induced, non-selective autophagy was not affected by loss of DJ-1. In wild-type fibroblasts and induced pluripotent stem cell-derived dopaminergic neurons, endogenous DJ-1 translocated to depolarized mitochondria in close proximity to optineurin. DJ-1 translocation to depolarized mitochondria was dependent on PINK1 and parkin and did not require oxidation of cysteine residue 106 of DJ-1. Overexpression of DJ-1 did not rescue the mitophagy defect of PINK1- or parkin-deficient cells. These findings position DJ-1 downstream of PINK1 and parkin in the same pathway and suggest that disruption of PINK1/parkin/DJ-1-mediated mitophagy is a common pathogenic mechanism in autosomal recessive Parkinson's disease.

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.

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.

Rectal organoid morphology analysis (ROMA) as a promising diagnostic tool in cystic fibrosis.

Diagnosing cystic fibrosis (CF) when sweat chloride is not in the CF range and less than 2 disease-causing CFTR mutations are found requires physiological CFTR assays, which are not always feasible or available. We developed a new physiological CFTR assay based on the morphological differences between rectal organoids from subjects with and without CF. In organoids from 167 subjects with and 22 without CF, two parameters derived from a semi-automated image analysis protocol (rectal organoid morphology analysis, ROMA) fully discriminated CF subjects with two disease-causing mutations from non-CF subjects (p<0.001). ROMA, feasible at all ages, can be centralised to improve standardisation.

Correction of CFTR function in intestinal organoids to guide treatment of cystic fibrosis.

RATIONALE: Given the vast number of cystic fibrosis transmembrane conductance regulator (CFTR) mutations, biomarkers predicting benefit from CFTR modulator therapies are needed for subjects with cystic fibrosis (CF). OBJECTIVES: To study CFTR function in organoids of subjects with common and rare CFTR mutations and evaluate correlations between CFTR function and clinical data. METHODS: Intestinal organoids were grown from rectal biopsies in a cohort of 97 subjects with CF. Residual CFTR function was measured by quantifying organoid swelling induced by forskolin and response to modulators by quantifying organoid swelling induced by CFTR correctors, potentiator and their combination. Organoid data were correlated with clinical data from the literature. RESULTS: Across 28 genotypes, residual CFTR function correlated (r2=0.87) with sweat chloride values. When studying the same genotypes, CFTR function rescue by CFTR modulators in organoids correlated tightly with mean improvement in lung function (r2=0.90) and sweat chloride (r2=0.95) reported in clinical trials. We identified candidate genotypes for modulator therapy, such as E92K, Q237E, R334W and L159S. Based on organoid results, two subjects started modulator treatment: one homozygous for complex allele Q359K_T360K, and the second with mutation E60K. Both subjects had major clinical benefit. CONCLUSIONS: Measurements of residual CFTR function and rescue of function by CFTR modulators in intestinal organoids correlate closely with clinical data. Our results for reference genotypes concur with previous results. CFTR function measured in organoids can be used to guide precision medicine in patients with CF, positioning organoids as a potential in vitro model to bring treatment to patients carrying rare CFTR mutations.

Gene editing technology for improving life quality: A dream coming true?

The fact that monogenic diseases are related to mutations in one specific gene, make gene correction one of the promising strategies in the future to treat genetic diseases or alleviate their symptoms. From this perspective, and along with recent advances in technology, genome editing tools have gained momentum and developed fast. In fact, clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9), transcription activator-like effector nucleases (TALENs), and zinc-finger nucleases (ZFNs) are regarded as novel technologies which are able to correct a number of genetic aberrations in vitro and in vivo. The number of ongoing clinical trials employing these tools has been increased showing the encouraging outcomes of these tools. However, there are still some major challenges with respect to the safety profile and directed delivery of them. In this paper, we provided updated information regarding the history, nature, methods of delivery, and application of the above-mentioned gene editing tools along with the meganucleases (an older similar tool) based on published in vitro and in vivo studies and introduced clinical trials which employed these technologies.

Integrative and perturbation-based analysis of the transcriptional dynamics of TGFß/BMP system components in transition from embryonic stem cells to neural progenitors.

Cooperative actions of extrinsic signals and cell-intrinsic transcription factors alter gene regulatory networks enabling cells to respond appropriately to environmental cues. Signaling by transforming growth factor type ß (TGFß) family ligands (eg, bone morphogenetic proteins [BMPs] and Activin/Nodal) exerts cell-type specific and context-dependent transcriptional changes, thereby steering cellular transitions throughout embryogenesis. Little is known about coordinated regulation and transcriptional interplay of the TGFß system. To understand intrafamily transcriptional regulation as part of this system's actions during development, we selected 95 of its components and investigated their mRNA-expression dynamics, gene-gene interactions, and single-cell expression heterogeneity in mouse embryonic stem cells transiting to neural progenitors. Interrogation at 24 hour intervals identified four types of temporal gene transcription profiles that capture all stages, that is, pluripotency, epiblast formation, and neural commitment. Then, between each stage we performed esiRNA-based perturbation of each individual component and documented the effect on steady-state mRNA levels of the remaining 94 components. This exposed an intricate system of multilevel regulation whereby the majority of gene-gene interactions display a marked cell-stage specific behavior. Furthermore, single-cell RNA-profiling at individual stages demonstrated the presence of detailed co-expression modules and subpopulations showing stable co-expression modules such as that of the core pluripotency genes at all stages. Our combinatorial experimental approach demonstrates how intrinsically complex transcriptional regulation within a given pathway is during cell fate/state transitions.

Fluorescent tagging of endogenous Heme oxygenase-1 in human induced pluripotent stem cells for high content imaging of oxidative stress in various differentiated lineages.

Tagging of endogenous stress response genes can provide valuable in vitro models for chemical safety assessment. Here, we present the generation and application of a fluorescent human induced pluripotent stem cell (hiPSC) reporter line for Heme oxygenase-1 (HMOX1), which is considered a sensitive and reliable biomarker for the oxidative stress response. CRISPR/Cas9 technology was used to insert an enhanced green fluorescent protein (eGFP) at the C-terminal end of the endogenous HMOX1 gene. Individual clones were selected and extensively characterized to confirm precise editing and retained stem cell properties. Bardoxolone-methyl (CDDO-Me) induced oxidative stress caused similarly increased expression of both the wild-type and eGFP-tagged HMOX1 at the mRNA and protein level. Fluorescently tagged hiPSC-derived proximal tubule-like, hepatocyte-like, cardiomyocyte-like and neuron-like progenies were treated with CDDO-Me (5.62-1000 nM) or diethyl maleate (5.62-1000 µM) for 24 h and 72 h. Multi-lineage oxidative stress responses were assessed through transcriptomics analysis, and HMOX1-eGFP reporter expression was carefully monitored using live-cell confocal imaging. We found that eGFP intensity increased in a dose-dependent manner with dynamics varying amongst lineages and stressors. Point of departure modelling further captured the specific lineage sensitivities towards oxidative stress. We anticipate that the newly developed HMOX1 hiPSC reporter will become a valuable tool in understanding and quantifying critical target organ cell-specific oxidative stress responses induced by (newly developed) chemical entities.

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.

N-acetylcysteine prevents oxidized low-density lipoprotein-induced reduction of MG53 and enhances MG53 protective effect on bone marrow stem cells.

MG53 is an important membrane repair protein and partially protects bone marrow multipotent adult progenitor cells (MAPCs) against oxidized low-density lipoprotein (ox-LDL). The present study was to test the hypothesis that the limited protective effect of MG53 on MAPCs was due to ox-LDL-induced reduction of MG53. MAPCs were cultured with and without ox-LDL (0-20 µg/mL) for up to 48 hours with or without MG53 and antioxidant N-acetylcysteine (NAC). Serum MG53 level was measured in ox-LDL-treated mice with or without NAC treatment. Ox-LDL induced significant membrane damage and substantially impaired MAPC survival with selective inhibition of Akt phosphorylation. NAC treatment effectively prevented ox-LDL-induced reduction of Akt phosphorylation without protecting MAPCs against ox-LDL. While having no effect on Akt phosphorylation, MG53 significantly decreased ox-LDL-induced membrane damage and partially improved the survival, proliferation and apoptosis of MAPCs in vitro. Ox-LDL significantly decreased MG53 level in vitro and serum MG53 level in vivo without changing MG53 clearance. NAC treatment prevented ox-LDL-induced MG53 reduction both in vitro and in vivo. Combined NAC and MG53 treatment significantly improved MAPC survival against ox-LDL. These data suggested that NAC enhanced the protective effect of MG53 on MAPCs against ox-LDL through preventing ox-LDL-induced reduction of MG53.