Effect of hypoxia on aquaporins and hepatobiliary transport systems in human hepatic cells.

OBJECTIVES: Hepatic ischemia and hypoxia are accompanied by reduced bile flow, biliary sludge and cholestasis. Hepatobiliary transport systems, nuclear receptors and aquaporins were studied after hypoxia and reoxygenation in human hepatic cells. METHODS: Expression of Aquaporin 8 (AQP8), Aquaporin 9 (AQP9), Pregnane X receptor (PXR), Farnesoid X receptor (FXR), Organic anion transporting polypeptide 1 (OATP1), and the Multidrug resistance-associated protein 4 (MRP4) were investigated in induced pluripotent stem cells (iPSCs) derived hepatic cells and the immortalized hepatic line HepG2. HepG2 was subjected to combined oxygen and glucose deprivation for 4 h followed by reoxygenation. RESULTS: Expression of AQP8 and AQP9 increased during differentiation in iPSC-derived hepatic cells. Hypoxia did not alter mRNA levels of AQP8, but reoxygenation caused a marked increase in AQP8 mRNA expression. While expression of OATP1 had a transient increase during reoxygenation, MRP4 showed a delayed downregulation. Knock-down of FXR did not alter the expression of AQP8, AQP9, MRP4, or OATP1. Post-hypoxic protein levels of AQP8 were reduced after 68 h of reoxygenation compared to normoxic controls. CONCLUSIONS: Post-transcriptional mechanisms rather than reduced transcription cause reduction in AQP8 protein concentration after hypoxia-reoxygenation in hepatic cells. Expression patterns differed between hepatobiliary transport systems during hypoxia and reoxygenation. IMPACT: Expression of AQP8 and AQP9 increased during differentiation in induced pluripotent stem cells. Expression of hepatobiliary transporters varies during hypoxia and reoxygenation. Post-hypoxic protein levels of AQP8 were reduced after 68 h of reoxygenation. Post-transcriptional mechanisms rather than reduced transcription cause reduction in AQP8 protein concentration after hypoxia-reoxygenation in hepatic cells. Hypoxia and reoxygenation may affect aquaporins in hepatic cells and potentially affect bile composition.

The NAD+ Precursor Nicotinamide Riboside Rescues Mitochondrial Defects and Neuronal Loss in iPSC derived Cortical Organoid of Alpers' Disease.

Alpers' syndrome is an early-onset neurodegenerative disorder usually caused by biallelic pathogenic variants in the gene encoding the catalytic subunit of polymerase-gamma (POLG), which is essential for mitochondrial DNA (mtDNA) replication. The disease is progressive, incurable, and inevitably it leads to death from drug-resistant status epilepticus. The neurological features of Alpers' syndrome are intractable epilepsy and developmental regression, with no effective treatment; the underlying mechanisms are still elusive, partially due to lack of good experimental models. Here, we generated the patient derived induced pluripotent stem cells (iPSCs) from one Alpers' patient carrying the compound heterozygous mutations of A467T (c.1399G>A) and P589L (c.1766C>T), and further differentiated them into cortical organoids and neural stem cells (NSCs) for mechanistic studies of neural dysfunction in Alpers' syndrome. Patient cortical organoids exhibited a phenotype that faithfully replicated the molecular changes found in patient postmortem brain tissue, as evidenced by cortical neuronal loss and depletion of mtDNA and complex I (CI). Patient NSCs showed mitochondrial dysfunction leading to ROS overproduction and downregulation of the NADH pathway. More importantly, the NAD+ precursor nicotinamide riboside (NR) significantly ameliorated mitochondrial defects in patient brain organoids. Our findings demonstrate that the iPSC model and brain organoids are good in vitro models of Alpers' disease; this first-in-its-kind stem cell platform for Alpers' syndrome enables therapeutic exploration and has identified NR as a viable drug candidate for Alpers' disease and, potentially, other mitochondrial diseases with similar causes.

A loss-of-function mutation in human Oxidation Resistance 1 disrupts the spatial-temporal regulation of histone arginine methylation in neurodevelopment.

BACKGROUND: Oxidation Resistance 1 (OXR1) gene is a highly conserved gene of the TLDc domain-containing family. OXR1 is involved in fundamental biological and cellular processes, including DNA damage response, antioxidant pathways, cell cycle, neuronal protection, and arginine methylation. In 2019, five patients from three families carrying four biallelic loss-of-function variants in OXR1 were reported to be associated with cerebellar atrophy. However, the impact of OXR1 on cellular functions and molecular mechanisms in the human brain is largely unknown. Notably, no human disease models are available to explore the pathological impact of OXR1 deficiency. RESULTS: We report a novel loss-of-function mutation in the TLDc domain of the human OXR1 gene, resulting in early-onset epilepsy, developmental delay, cognitive disabilities, and cerebellar atrophy. Patient lymphoblasts show impaired cell survival, proliferation, and hypersensitivity to oxidative stress. These phenotypes are rescued by TLDc domain replacement. We generate patient-derived induced pluripotent stem cells (iPSCs) revealing impaired neural differentiation along with dysregulation of genes essential for neurodevelopment. We identify that OXR1 influences histone arginine methylation by activating protein arginine methyltransferases (PRMTs), suggesting OXR1-dependent mechanisms regulating gene expression during neurodevelopment. We model the function of OXR1 in early human brain development using patient-derived brain organoids revealing that OXR1 contributes to the spatial-temporal regulation of histone arginine methylation in specific brain regions. CONCLUSIONS: This study provides new insights into pathological features and molecular underpinnings associated with OXR1 deficiency in patients.

Scalable production of tissue-like vascularized liver organoids from human PSCs.

The lack of physiological parity between 2D cell culture and in vivo culture has led to the development of more organotypic models, such as organoids. Organoid models have been developed for a number of tissues, including the liver. Current organoid protocols are characterized by a reliance on extracellular matrices (ECMs), patterning in 2D culture, costly growth factors and a lack of cellular diversity, structure, and organization. Current hepatic organoid models are generally simplistic and composed of hepatocytes or cholangiocytes, rendering them less physiologically relevant compared to native tissue. We have developed an approach that does not require 2D patterning, is ECM independent, and employs small molecules to mimic embryonic liver development that produces large quantities of liver-like organoids. Using single-cell RNA sequencing and immunofluorescence, we demonstrate a liver-like cellular repertoire, a higher order cellular complexity, presenting with vascular luminal structures, and a population of resident macrophages: Kupffer cells. The organoids exhibit key liver functions, including drug metabolism, serum protein production, urea synthesis and coagulation factor production, with preserved post-translational modifications such as N-glycosylation and functionality. The organoids can be transplanted and maintained long term in mice producing human albumin. The organoids exhibit a complex cellular repertoire reflective of the organ and have de novo vascularization and liver-like function. These characteristics are a prerequisite for many applications from cellular therapy, tissue engineering, drug toxicity assessment, and disease modeling to basic developmental biology.

Autophagy modulates cell fate decisions during lineage commitment.

Early events during development leading to exit from a pluripotent state and commitment toward a specific germ layer still need in-depth understanding. Autophagy has been shown to play a crucial role in both development and differentiation. This study employs human embryonic and induced pluripotent stem cells to understand the early events of lineage commitment with respect to the role of autophagy in this process. Our data indicate that a dip in autophagy facilitates exit from pluripotency. Upon exit, we demonstrate that the modulation of autophagy affects SOX2 levels and lineage commitment, with induction of autophagy promoting SOX2 degradation and mesendoderm formation, whereas inhibition of autophagy causes SOX2 accumulation and neuroectoderm formation. Thus, our results indicate that autophagy-mediated SOX2 turnover is a determining factor for lineage commitment. These findings will deepen our understanding of development and lead to improved methods to derive different lineages and cell types.Abbreviations: ACTB: Actin, beta; ATG: Autophagy-related; BafA1: Bafilomycin A1; CAS9: CRISPR-associated protein 9; CQ: Chloroquine; DE: Definitive endoderm; hESCs: Human Embryonic Stem Cells; hiPSCs: Human Induced Pluripotent Stem Cells; LAMP1: Lysosomal Associated Membrane Protein 1; MAP1LC3: Microtubule-Associated Protein 1 Light Chain 3; MTOR: Mechanistic Target Of Rapamycin Kinase; NANOG: Nanog Homeobox; PAX6: Paired Box 6; PE: Phosphatidylethanolamine; POU5F1: POU class 5 Homeobox 1; PRKAA2: Protein Kinase AMP-Activated Catalytic Subunit Alpha 2; SOX2: SRY-box Transcription Factor 2; SQSTM1: Sequestosome 1; ULK1: unc-51 like Autophagy Activating Kinase 1; WDFY3: WD Repeat and FYVE Domain Containing 3.

Distinct Mitochondrial Remodeling During Mesoderm Differentiation in a Human-Based Stem Cell Model.

Given the considerable interest in using stem cells for modeling and treating disease, it is essential to understand what regulates self-renewal and differentiation. Remodeling of mitochondria and metabolism, with the shift from glycolysis to oxidative phosphorylation (OXPHOS), plays a fundamental role in maintaining pluripotency and stem cell fate. It has been suggested that the metabolic "switch" from glycolysis to OXPHOS is germ layer-specific as glycolysis remains active during early ectoderm commitment but is downregulated during the transition to mesoderm and endoderm lineages. How mitochondria adapt during these metabolic changes and whether mitochondria remodeling is tissue specific remain unclear. Here, we address the question of mitochondrial adaptation by examining the differentiation of human pluripotent stem cells to cardiac progenitors and further to differentiated mesodermal derivatives, including functional cardiomyocytes. In contrast to recent findings in neuronal differentiation, we found that mitochondrial content decreases continuously during mesoderm differentiation, despite increased mitochondrial activity and higher levels of ATP-linked respiration. Thus, our work highlights similarities in mitochondrial remodeling during the transition from pluripotent to multipotent state in ectodermal and mesodermal lineages, while at the same time demonstrating cell-lineage-specific adaptations upon further differentiation. Our results improve the understanding of how mitochondrial remodeling and the metabolism interact during mesoderm differentiation and show that it is erroneous to assume that increased OXPHOS activity during differentiation requires a simultaneous expansion of mitochondrial content.

Differentiation of human pluripotent stem cells toward pharyngeal endoderm derivatives: Current status and potential.

The pharyngeal apparatus, a transient embryological structure, includes diverse cells from all three germ layers that ultimately contribute to a variety of adult tissues. In particular, pharyngeal endoderm produces cells of the inner ear, palatine tonsils, the thymus, parathyroid and thyroid glands, and ultimobranchial bodies. Each of these structures and organs contribute to vital human physiological processes, including central immune tolerance (thymus) and metabolic homeostasis (parathyroid and thyroid glands, and ultimobranchial bodies). Thus, improper development or damage to pharyngeal endoderm derivatives leads to complicated and severe human maladies, such as autoimmunity, immunodeficiency, hypothyroidism, and/or hypoparathyroidism. To study and treat such diseases, we can utilize human pluripotent stem cells (hPSCs), which differentiate into functionally mature cells in vitro given the proper developmental signals. Here, we discuss current efforts regarding the directed differentiation of hPSCs toward pharyngeal endoderm derivatives. We further discuss model system and therapeutic applications of pharyngeal endoderm cell types produced from hPSCs. Finally, we provide suggestions for improving hPSC differentiation approaches to pharyngeal endoderm derivatives with emphasis on current single cell-omics and 3D culture system technologies.

A method for differentiating human induced pluripotent stem cells toward functional cardiomyocytes in 96-well microplates.

The capacity of pluripotent stem cells both for self-renewal and to differentiate into any cell type have made them a powerful tool for studying human disease. Protocols for efficient differentiation towards cardiomyocytes using defined, serum-free culture medium combined with small molecules have been developed, but thus far, limited to larger formats. We adapted protocols for differentiating human pluripotent stem cells to functional human cardiomyocytes in a 96-well microplate format. The resulting cardiomyocytes expressed cardiac specific markers at the transcriptional and protein levels and had the electrophysiological properties that confirmed the presence of functional cardiomyocytes. We suggest that this protocol provides an incremental improvement and one that reduces the impact of heterogeneity by increasing inter-experimental replicates. We believe that this technique will improve the applicability of these cells for use in developmental biology and mechanistic studies of disease.

Disease-specific phenotypes in iPSC-derived neural stem cells with POLG mutations.

Mutations in POLG disrupt mtDNA replication and cause devastating diseases often with neurological phenotypes. Defining disease mechanisms has been hampered by limited access to human tissues, particularly neurons. Using patient cells carrying POLG mutations, we generated iPSCs and then neural stem cells. These neural precursors manifested a phenotype that faithfully replicated the molecular and biochemical changes found in patient post-mortem brain tissue. We confirmed the same loss of mtDNA and complex I in dopaminergic neurons generated from the same stem cells. POLG-driven mitochondrial dysfunction led to neuronal ROS overproduction and increased cellular senescence. Loss of complex I was associated with disturbed NAD+ metabolism with increased UCP2 expression and reduced phosphorylated SirT1. In cells with compound heterozygous POLG mutations, we also found activated mitophagy via the BNIP3 pathway. Our studies are the first that show it is possible to recapitulate the neuronal molecular and biochemical defects associated with POLG mutation in a human stem cell model. Further, our data provide insight into how mitochondrial dysfunction and mtDNA alterations influence cellular fate determining processes.

Directed reprogramming of comprehensively characterized dental pulp stem cells extracted from natal tooth.

The aim of this study was to extensively characterise natal dental pulp stem cells (nDPSC) and assess their efficiency to generate human induced pluripotent stem cells (hiPSC). A number of distinguishing features prompted us to choose nDPSC over normal adult DPSC, in that they differed in cell surface marker expression and initial doubling time. In addition, nDPSC expressed 17 out of 52 pluripotency genes we analysed, and the level of expression was comparable to human embryonic stem cells (hESC). Ours is the first group to report comprehensive characterization of nDPSC followed by directed reprogramming to a pluripotent stem cell state. nDPSC yielded hiPSC colonies upon transduction with Sendai virus expressing the pluripotency transcription factors POU5F1, SOX2, c-MYC and KLF4. nDPSC had higher reprogramming efficiency compared to human fibroblasts. nDPSC derived hiPSCs closely resembled hESC in terms of their morphology, expression of pluripotency markers and gene expression profiles. Furthermore, nDPSC derived hiPSCs differentiated into the three germ layers when cultured as embryoid bodies (EB) and by directed differentiation. Based on our findings, nDPSC present a unique marker expression profile compared with adult DPSC and possess higher reprogramming efficiency as compared with dermal fibroblasts thus proving to be more amenable for reprogramming.

Rapid Screening of the Endodermal Differentiation Potential of Human Pluripotent Stem Cells.

Human pluripotent stem cells (hPSCs) hold tremendous promise for regenerative medicine, disease modeling, toxicology screening, and developmental biology. These applications are hindered due to inherent differences in differentiation potential observed among different hPSC lines. This is particularly true for the differentiation of hPSCs toward the endodermal lineage. Several groups have developed methods to screen hPSCs for their endodermal differentiation potential (EP). Particularly notable studies include (i) the use of WNT3A expression as a predictive biomarker, (ii) an embryoid body-based screen, and (iii) a transcriptomics-based approach. We recently developed a rapid screen to access the EP of hPSCs solely based on morphological analysis. The screen takes 4 days to perform and yields results that are easy to interpret. As the screen is based on our recently developed small molecule protocol for hepatocyte like cell (HLC) differentiation of hPSCs, this method is extremely cost-effective compared to the aforementioned approaches. © 2017 by John Wiley & Sons, Inc.

Development of an inducible platform for intercellular protein delivery.

A challenge to protein based therapies is the ability to produce biologically active proteins and their ensured delivery. Various approaches have been utilised including fusion of protein transduction domains with a protein or biomolecule of interest. A compounding issue is lack of specificity, efficiency and indeed whether the protein fusions are actually translocated into the cell and not merely an artefact of the fixation process. Here we present a novel platform, allowing the inducible export and uptake of a protein of interest. The system utilises a combination of the Tetracyline repressor system, combined with a fusion protein containing the N-terminal signal peptide from human chorionic gonadotropin beta-subunit, and a C-terminal poly-arginine domain for efficient uptake by target cells. This novel platform was validated using enhanced green fluorescent protein as the gene of interest. Doxycycline efficiently induced expression of the fusion protein. The human chorionic gonadotropin beta-subunit facilitated the export of the fusion protein into the cell culture media. Finally, the fusion protein was able to efficiently enter into neighbouring cells (target cells), mediated by the poly-arginine cell penetrating peptide. Importantly we have addressed the issue of whether the observed uptake is an artefact of the fixation process or indeed genuine translocation. In addition this platform provides a number of potential applications in diverse areas such as stem cell biology, immune therapy and cancer targeting therapies.

Low-dose acetaminophen induces early disruption of cell-cell tight junctions in human hepatic cells and mouse liver.

Dysfunction of cell-cell tight junction (TJ) adhesions is a major feature in the pathogenesis of various diseases. Liver TJs preserve cellular polarity by delimiting functional bile-canalicular structures, forming the blood-biliary barrier. In acetaminophen-hepatotoxicity, the mechanism by which tissue cohesion and polarity are affected remains unclear. Here, we demonstrate that acetaminophen, even at low-dose, disrupts the integrity of TJ and cell-matrix adhesions, with indicators of cellular stress with liver injury in the human hepatic HepaRG cell line, and primary hepatocytes. In mouse liver, at human-equivalence (therapeutic) doses, dose-dependent loss of intercellular hepatic TJ-associated ZO-1 protein expression was evident with progressive clinical signs of liver injury. Temporal, dose-dependent and specific disruption of the TJ-associated ZO-1 and cytoskeletal-F-actin proteins, correlated with modulation of hepatic ultrastructure. Real-time impedance biosensing verified in vitro early, dose-dependent quantitative decreases in TJ and cell-substrate adhesions. Whereas treatment with NAPQI, the reactive metabolite of acetaminophen, or the PKCα-activator and TJ-disruptor phorbol-12-myristate-13-acetate, similarly reduced TJ integrity, which may implicate oxidative stress and the PKC pathway in TJ destabilization. These findings are relevant to the clinical presentation of acetaminophen-hepatotoxicity and may inform future mechanistic studies to identify specific molecular targets and pathways that may be altered in acetaminophen-induced hepatic depolarization.

Small-Molecule-Directed Hepatocyte-Like Cell Differentiation of Human Pluripotent Stem Cells.

Hepatocyte-like cells (HLCs) generated in vitro from human pluripotent stem cells (hPSCs) provide an invaluable resource for basic research, regenerative medicine, drug screening, toxicology, and modeling of liver disease and development. This unit describes a small-molecule-driven protocol for in vitro differentiation of hPSCs into HLCs without the use of growth factors. hPSCs are coaxed through a developmentally relevant route via the primitive streak to definitive endoderm (DE) using the small molecule CHIR99021 (a Wnt agonist), replacing the conventional growth factors Wnt3A and activin A. The small-molecule-derived DE is then differentiated to hepatoblast-like cells in the presence of dimethyl sulfoxide. The resulting hepatoblasts are then differentiated to HLCs with N-hexanoic-Tyr, Ile-6 aminohexanoic amide (Dihexa, a hepatocyte growth factor agonist) and dexamethasone. The protocol provides an efficient and reproducible procedure for differentiation of hPSCs into HLCs utilizing small molecules. © 2016 by John Wiley & Sons, Inc.

Development of a rapid screen for the endodermal differentiation potential of human pluripotent stem cell lines.

A challenge facing the human pluripotent stem cell (hPSC) field is the variability observed in differentiation potential of hPSCs. Variability can lead to time consuming and costly optimisation to yield the cell type of interest. This is especially relevant for the differentiation of hPSCs towards the endodermal lineages. Endodermal cells have the potential to yield promising new knowledge and therapies for diseases affecting multiple organ systems, including lung, thymus, intestine, pancreas and liver, as well as applications in regenerative medicine and toxicology. Providing a means to rapidly, cheaply and efficiently assess the differentiation potential of multiple hPSCs is of great interest. To this end, we have developed a rapid small molecule based screen to assess the endodermal potential (EP) of hPSCs, based solely on definitive endoderm (DE) morphology. This drastically reduces the cost and time to identify lines suitable for use in deriving endodermal lineages. We demonstrate the efficacy of this screen using 10 different hPSCs, including 4 human embryonic stem cell lines (hESCs) and 6 human induced pluripotent stem cell lines (hiPSCs). The screen clearly revealed lines amenable to endodermal differentiation, and only lines that passed our morphological assessment were capable of further differentiation to hepatocyte like cells (HLCs).

Small-molecule-driven hepatocyte differentiation of human pluripotent stem cells.

The differentiation of pluripotent stem cells to hepatocytes is well established, yet current methods suffer from several drawbacks. These include a lack of definition and reproducibility, which in part stems from continued reliance on recombinant growth factors. This has remained a stumbling block for the translation of the technology into industry and the clinic for reasons associated with cost and quality. We have devised a growth-factor-free protocol that relies on small molecules to differentiate human pluripotent stem cells toward a hepatic phenotype. The procedure can efficiently direct both human embryonic stem cells and induced pluripotent stem cells to hepatocyte-like cells. The final population of cells demonstrates marker expression at the transcriptional and protein levels, as well as key hepatic functions such as serum protein production, glycogen storage, and cytochrome P450 activity.

Modelling human disease with pluripotent stem cells.

Recent progress in the field of cellular reprogramming has opened up the doors to a new era of disease modelling, as pluripotent stem cells representing a myriad of genetic diseases can now be produced from patient tissue. These cells can be expanded and differentiated to produce a potentially limitless supply of the affected cell type, which can then be used as a tool to improve understanding of disease mechanisms and test therapeutic interventions. This process requires high levels of scrutiny and validation at every stage, but international standards for the characterisation of pluripotent cells and their progeny have yet to be established. Here we discuss the current state of the art with regard to modelling diseases affecting the ectodermal, mesodermal and endodermal lineages, focussing on studies which have demonstrated a disease phenotype in the tissue of interest. We also discuss the utility of pluripotent cell technology for the modelling of cancer and infectious disease. Finally, we spell out the technical and scientific challenges which must be addressed if the field is to deliver on its potential and produce improved patient outcomes in the clinic.