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• Pisal RV, Suchanek J, Hrebikova H, Bezrou A, Soukup T, Kunke D, Micuda S, Filip S, Siller R, Sullivan GJ and Mokry J. Directed reprogramming of comprehensively characterized dental pulp stem cells extracted from natal tooth.  (2018): Accepted and in Press. Scientific Reports.

• Siller R and Sullivan GJ^*. Simple and rapid screen for human pluripotent stem cell endoderm differentiation potential. Current Protocols in Stem Cell Biology, (2017): 43, 1G.7.1–1G.7.23.doi: 10.1002/cpsc.36. (*corresponding author).

• Rutkovskiy A, Malashicheva A, Sullivan GJ^*, Bogdanova M, Kostareva A, Stensløkken KO, Fianne A, Vaage J. Valve Interstitial Cells: The Key to Understanding the Pathophysiology of Heart Valve Calcification. Journal of the American Heart Association. (2017): 14;6(9) pii: e006339. doi: 10.1161/JAHA.117.006339.(*co-corresponding author).

• Scarfe L, Brillant N, Kumar JD, Ali N, Alrumayh A, Amali M, Barbellion S and et al. Pre-clinical imaging methods for assessing the safety and efficacy of regenerative medicine therapies. Nature Regenerative Medicine. (2017): (2017)2:28; doi:10.1038/s41536-017-0029-9.

• Siller R, Greenhough S, Mathipati S, Si-Tayeb K^* and Sullivan GJ^*. Future challenges in the generation of hepatocyte like cells from human pluripotent stem cells. Current Pathobiology Reports. (2017):5:(3):301-314. (*co-corresponding author).

• Siller R, Dufour E, Wilmut I, Jung Y-W, Park IH and Sullivan GJ^*. Development of an inducible platform for intercellular protein delivery. International Journal of Pharmaceutics. (2017): http://dx.doi.org/10.1016/j.ijpharm.2017.02.067. (*corresponding author).

• Gamal W, Treskes P, Chesne CG, Samuel K, Sullivan GJ, Siller R, Srsen V, Underwood I, Smith S, Hayes PC, Plevris JN, Bagnaninchi PO and Nelson LJ. Low-dose acetaminophen induces early disruption of cell-cell tight junctions in human hepatic cells and mouse liver. Scientific Reports. (2017):DOI: 10.1038/srep37541.

• Siller R, Naumovska E, Mathapati S, Lycke M and Sullivan GJ^*. Development of a rapid screen for the endodermal differentiation potential of human pluripotent stem cell lines. Scientific Reports. (2016):DOI: 10.1038/srep37178. (*corresponding author).   

• Mathapati S, Siller R, Impellizzeri AR, Lycke M, Vegheim K, Almass R and Sullivan GJ^*. Small molecule directed hepatocytes like cells differentiation of human pluripotent stem cells. Curr. Protoc. Stem Cell Biol. (2016): 38:1G.6.1-1G.6.18.doi: 10.1002/cpsc.13. (*corresponding author).   

• Xu M, Stattin E-L, Shaw G, Heinegård D, Sullivan G, Wilmut I, Colman A, Önnerfjord P, Khabut A, Aspberg A, Dockery P, Hardingham T, Murphy M, Barry F. Chondrocytes derived from mesenchymal stromal cells and induced pluripotent cells of patients with familial osteochondritis dissecans exhibit an ER stress response and defective matrix assembly. Stem Cells Transl. Med. (2016): 5:1171-1181.

• Siller R, Greenhough S, Naumovska E and Sullivan GJ^*. Small molecule driven hepatocyte differentiation of human pluripotent stem cells. Stem Cell Reports. (2015): 4:939-952. (*corresponding author).     

• Siller R, Greenhough S, Park IH and Sullivan GJ*. (2013) Modelling human disease with pluripotent stem cells. Current Gene Therapy. 13(2):99-110. (*corresponding author).   
• Dajani R, Koo SE, Sullivan GJ*, Park IH. (2013) Investigation of Rett syndrome using pluripotent stem cells. J Cell Biochem. 114:2446-2453 (*corresponding author).  
• Bilican B, Serio A, Barmada SJ, Nishimura AL, Sullivan GJ, Carrasco M, Phatnani HP, Puddifoot CA, Story D, Fletcher J, Park IH, Friedman BA, Daley GQ, Wyllie DJ, Hardingham GE, Wilmut I, Finkbeiner S, Maniatis T, Shaw CE, Chandran S. (2013) Comment on "Drug screening for ALS using patient-specific induced pluripotent stem cells".  Sci Transl Med. 5(188):188le2.
• Kim KY, Jung YW, Sullivan GJ, Chung L and Park IH. Cellular Reprogramming: a novel tool in investigating ASDs. Trends Mol Med 2012;18:463-71.
• Yamazaki T, Chen S, Yu Y, Yan B, Carrasco MA, Tapia JC, Zhai B, Das R, Lalancette-Hebert M, Sharma A, Chandran S, Sullivan G, Nishimura AL, Shaw CE, Gygi SP, Shneider NA, Maniatis T, and Reed R. FUS-SMN protein interactions link the motor neuron diseases ALS and SMA. Cell Rep. 2012;25;2:799-806. F1000 recommended.
• Bilican B, Serio A, Barmada SJ, Nishimura AL, Sullivan GJ, Carrasco M, Phatnani HP, Puddifoot CA, Story D, Fletcher J, Park IH, Friedman BA, Daley GQ, Wyllie DJ, Hardingham GE, Wilmut I, Finkbeiner S, Maniatis T, Shaw CE, Chandran S. Mutant induced pluripotent stem cell lines recapitulate aspects of TDP-43 proteinopathies and reveal cell-specific vulnerability.   Proc Natl Acad Sci USA 2012;109:5803-8. F1000 recommended.
• Zhou XL, Sullivan GJ, Sun P, and Park IH. Humanized murine model for HBV and HCV using human induced pluripotent stem cells. Archives of Pharmacal Research. 2012;35(2):261-269.
• Hysolli E, Zhou XL, Liu R, Kim JH, Adams B, Sullivan GJ, and Park IH. Role of Pluripotent Stem Cells in Regenerative Medicine. Regenerative Medicine, Stem Cells and the Liver. Book Chapter, March 15, (2012) by Science Publishers.
• Wilmut I, Sullivan G and Chambers I. (2011).  The evolving biology of cell reprogramming. Philosophical Transactions of the Royal Society B. Philosophical Transactions of the Royal Society B 366:2183-2197.
• Wilmut I,  Wongtawan T, Quigley M and Sullivan GJ.  Biomedical and social contributions to sustainability. Philosophical Transactions of the Royal Society A. 2011;369:1730-1747.
• Ruzov A,  Tsenkina Y, Serio A, Dudnakova T, Fletcher J, Bai Y, Chebotareva T, Pells S, Hannoun Z, Sullivan GJ, Chandran S, Hay D, Bradley M, Wilmut I and De Sousa P. (2011) Lineage specific distribution of high levels of genomic 5-hydroxymethylcytosine in mammalian development. Cell Research. 21:1332-42.
• Sullivan GJ, Bai YM, Fletcher J and Wilmut I. (2010) Induced pluripotent stem cells: epigenetic memories and practical implications.  Molecular Human Reproduction 16:880–885.
• Hannoun Z, Filippi C, Sullivan GJ, Hay DC and Iredale JP. (2010) Hepatic Endoderm Differentiation from Human Embryonic Stem Cells. Current Stem Cell Research &Therapy  5:233-244.
• Sullivan GJ*, Hay DC, Park IH, Fletcher J, Hannoun Z, Payne CM, Dalgetty D, Black JR, Ross JA, Samuel K, Wang G, Daley GQ, Lee JH, Church GM, Forbes SJ, Iredale JP and Wilmut I. (2010) Efficient Generation of iPSC-Derived Human Hepatic Endoderm Displaying Liver Specific Metabolism. Hepatology 51:329-335. (*corresponding author). F1000 recommended. Editiorial Hepatology and Press coverage BBC.
• Taylor J, Wilmut I, Sullivan GJ.  What are the limits to cell plasticity? [Research Highlight] Cell Research. 2010;20:502-503.
• Wilmut I, Sullivan GJ and Taylor J. A decade of progress since the birth of Dolly. Reproduction, Fertility and Development. 2009;21, 95–100.
• O’Sullivan A, Sullivan GJ and McStay B.  UBF binding in vivo is not restricted to regulatory sequences within the vertebrate rDNA repeat.  MCB 2002;22(2); 657-668
• Sullivan GJ, Bridger JM, Cuthbert AP, Newbold RF, Bickmore WA, McStay B.  Human acrocentric chromosomes with transcriptionally silent nucleolar organizer regions associate with nucleoli. EMBO J. 2001;20(11):2867-74.
• Sullivan GJ, McStay B.  Dimerization and HMG box domains 1-3 present in Xenopus UBF are sufficient for its role in transcriptional enhancement. Nucleic Acids Res. 1998;26(15):3555-61.
• McStay B, Sullivan GJ, Cairns C.  The Xenopus RNA polymerase I transcription factor, UBF, has a role in transcriptional enhancement distinct from that at the promoter. EMBO J. 1997; 16(2):396-405.
• Woodward MJ, Sullivan GJ.     Nucleotide sequence of a repetitive element isolated from Leptospira interrogans serovar hardjo type hardjo-bovis. J Gen Microbiol. 1991;137:1101-9.
• Woodward MJ, Sullivan GJ, Palmer NM, Woolley JC, Redstone JS.  Development of a PCR test specific for Leptospira hardjo genotype bovis. Vet Rec. 1991;128(12):282-3.
• Book Chapters
• Hysolli E, Zhou XL, Liu R, Kim JH, Adams B, Sullivan GJ, and Park IH. Role of Pluripotent Stem Cells in Regenerative Medicine. Regenerative Medicine, Stem Cells and the Liver. Book Chapter, March 15, (2012) by Science Publishers.

• Chen, Anbin; Yangzom, Tsering; Sullivan, Gareth John & Liang, Kristina Xiao (2026). Impaired NADH-linked mitochondrial respiration disrupts ventral midbrain neuronal programs in POLG disease. Journal of Translational Medicine. 24(1). doi: 10.1186/s12967-026-08706-w. Fulltekst i vitenarkiv Vis sammendrag

  Background POLG (DNA polymerase γ catalytic subunit)-related mitochondrial diseases are among the most severe primary mitochondrial disorders and are characterized by progressive neurodegeneration with prominent dopaminergic involvement. However, the cell type-specific mechanisms linking mitochondrial DNA instability to neuronal vulnerability remain incompletely defined. Methods Using patient-derived midbrain organoids and single-cell RNA sequencing, we investigated how POLG mutations alter mitochondrial and neuronal programs at subtype resolution. We analyzed dopaminergic neuronal populations and ventral midbrain neurons to define disease-associated transcriptional changes. To evaluate therapeutic improvement, POLG organoids were treated chronically with nicotinamide riboside (NR), followed by single-cell transcriptomic profiling and pathway enrichment analysis. Results POLG mutations induced a coordinated downregulation of genes associated with oxidative phosphorylation and synaptic signaling, particularly in terminally differentiated dopaminergic neurons. This transcriptional alteration involved genes encoding respiratory chain complexes I–V, mitochondrial translation machinery, and ATP synthase components, suggesting disruption of mitochondrial bioenergetic programs at the transcriptomic level. Among dopaminergic subtypes, DA2 neurons and ventral midbrain neurons showed the most pronounced transcriptional alterations, indicating maturation-dependent vulnerability. NR treatment was associated with altered expression of genes involved in oxidative phosphorylation, NADH dehydrogenase activity, respiratory chain assembly, and synaptic pathways. Following NR exposure, dopaminergic subpopulations exhibited changes in cell-type proportions and partial normalization of mitochondrial- and synaptic-related transcriptional programs. Conclusions These findings identify transcriptional alterations in pathways related to mitochondrial respiration. The data further suggests that modulation of NAD⁺ metabolism is associated with transcriptional changes in mitochondrial and neuronal pathways in this disease context.

• Roman, Giacomo; Lauritzen, Knut Husø; Harrison, Sean Philip; Bhattacharya, Anindita; Andresen, Marianne Seierstad & Mowinckel, Marie-Christine [Vis alle 16 forfattere av denne artikkelen] (2025). Ex vivo correction of severe coagulation Factor VII deficiency in patient-derived 3D liver organoids. Haematologica. ISSN 0390-6078. doi: 10.3324/haematol.2025.288046. Fulltekst i vitenarkiv Vis sammendrag

  Coagulation factor (F) VII deficiency is the most frequent among the rare, inherited bleeding disorders and is predominantly caused by missense mutations in the F7 gene. The disease phenotype ranges from asymptomatic cases to extremely severe hemorrhagic forms, requiring prophylactic injections with plasma-derived or recombinant FVII concentrates. In response, we have developed an autologous cell-based approach that corrects the disease-causing mutation in patient-derived induced pluripotent stem cells (iPSCs) and generates therapeutic, three-dimensional hepatic organoids (HOs). We report the CRISPRmediated correction of homozygous c.718G>C (p.G240R), a missense mutation associated with a severe, life-threatening bleeding phenotype. The HOs contain all liver cell types and exhibit key liver functions, including coagulation factor production. After correction, our data indicate that the patient-derived HOs secrete consistent amounts of functional FVII protein, resulting in improved thrombin generation times. These results represent a significant milestone toward the establishment of an autologous cell-based therapy for patients with FVII- and other coagulation factor deficiencies.

• Hong, Yu; Zhang, Zhuoyuan; Yangzom, Tsering; Chen, Anbin; Lundberg, Bjørn Christian & Fang, Fei [Vis alle 12 forfattere av denne artikkelen] (2024). The NAD+ Precursor Nicotinamide Riboside Rescues Mitochondrial Defects and Neuronal Loss in iPSC derived Cortical Organoid of Alpers' Disease. International Journal of Biological Sciences. 20(4), s. 1194–1217. doi: 10.7150/ijbs.91624. Fulltekst i vitenarkiv Vis sammendrag

  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.

• Chen, Anbin; Yangzom, Tsering; Hong, Yu; Lundberg, Bjørn Christian; Sullivan, Gareth John & Tzoulis, Charalampos [Vis alle 8 forfattere av denne artikkelen] (2024). Hallmark Molecular and Pathological Features of POLG Disease are Recapitulated in Cerebral Organoids. Advanced Science. 11(18). doi: 10.1002/advs.202307136. Fulltekst i vitenarkiv Vis sammendrag

  In this research, a 3D brain organoid model is developed to study POLG-related encephalopathy, a mitochondrial disease stemming from POLG mutations. Induced pluripotent stem cells (iPSCs) derived from patients with these mutations is utilized to generate cortical organoids, which exhibited typical features of the diseases with POLG mutations, such as altered morphology, neuronal loss, and mitochondiral DNA (mtDNA) depletion. Significant dysregulation is also identified in pathways crucial for neuronal development and function, alongside upregulated NOTCH and JAK-STAT signaling pathways. Metformin treatment ameliorated many of these abnormalities, except for the persistent affliction of inhibitory dopamine-glutamate (DA GLU) neurons. This novel model effectively mirrors both the molecular and pathological attributes of diseases with POLG mutations, providing a valuable tool for mechanistic understanding and therapeutic screening for POLG-related disorders and other conditions characterized by compromised neuronal mtDNA maintenance and complex I deficiency.

• Liang, Xiao; Chen, Anbin; Kianian, Atefeh; Kristiansen, Cecilie Katrin; Yangzom, Tsering Yangzom Tsering & Palmer, Jessica Furriol [Vis alle 13 forfattere av denne artikkelen] (2024). Activation of Neurotoxic Astrocytes Due to Mitochondrial Dysfunction Triggered by POLG Mutation. International Journal of Biological Sciences. 20(8), s. 2860–2880. doi: 10.7150/ijbs.93445. Fulltekst i vitenarkiv Vis sammendrag

  Mitochondrial diseases are associated with neuronal death and mtDNA depletion. Astrocytes respond to injury or stimuli and damage to the central nervous system. Neurodegeneration can cause astrocytes to activate and acquire toxic functions that induce neuronal death. However, astrocyte activation and its impact on neuronal homeostasis in mitochondrial disease remain to be explored. Using patient cells carrying POLG mutations, we generated iPSCs and then differentiated these into astrocytes. POLG astrocytes exhibited mitochondrial dysfunction including loss of mitochondrial membrane potential, energy failure, loss of complex I and IV, disturbed NAD+/NADH metabolism, and mtDNA depletion. Further, POLG derived astrocytes presented an A1-like reactive phenotype with increased proliferation, invasion, upregulation of pathways involved in response to stimulus, immune system process, cell proliferation and cell killing. Under direct and indirect co-culture with neurons, POLG astrocytes manifested a toxic effect leading to the death of neurons. We demonstrate that mitochondrial dysfunction caused by POLG mutations leads not only to intrinsic defects in energy metabolism affecting both neurons and astrocytes, but also to neurotoxic damage driven by astrocytes. These findings reveal a novel role for dysfunctional astrocytes that contribute to the pathogenesis of POLG diseases.

• Hutchinson, Ashlee M.; Appeltant, Ruth; Burdon, Tom; Bao, Qiuye; Bargaje, Rhishikesh & Bodnar, Andrea [Vis alle 41 forfattere av denne artikkelen] (2024). Advancing stem cell technologies for conservation of wildlife biodiversity. Development. ISSN 0950-1991. 151(20), s. 1–9. doi: 10.1242/dev.203116. Fulltekst i vitenarkiv
• Lunova, Mariia; Jirsa, Milan; Dejneka, Alexandr; Sullivan, Gareth John & Lunov, Oleg (2024). Mechanical regulation of mitochondrial morphodynamics in cancer cells by extracellular microenvironment. Biomaterials and Biosystems. 14, s. 1–9. doi: 10.1016/j.bbiosy.2024.100093. Fulltekst i vitenarkiv
• Westerberg, Niklas Starck; Atneosen-Åsegg, Monica; Melheim, Maria; Dugarte, Maria Eugenia Chollet; Harrison, Sean Philip & Siller, Richard [Vis alle 8 forfattere av denne artikkelen] (2024). Effect of hypoxia on aquaporins and hepatobiliary transport systems in human hepatic cells. Pediatric Research. ISSN 0031-3998. doi: 10.1038/s41390-024-03368-0. Fulltekst i vitenarkiv
• Harrison, Sean Philip; Baumgarten, Saphira Felicitas; Dugarte, Maria Eugenia Chollet; Stavik, Benedicte; Bhattacharya, Anindita & Almaas, Runar [Vis alle 7 forfattere av denne artikkelen] (2024). Parenteral nutrition emulsion inhibits CYP3A4 in an iPSC derived liver organoids testing platform. Journal of Pediatric Gastroenterology and Nutrition - JPGN. ISSN 0277-2116. 78, s. 1047–1058. doi: 10.1002/jpn3.12195. Fulltekst i vitenarkiv Vis sammendrag

  Objectives Parenteral nutrition (PN) is used for patients of varying ages with intestinal failure to supplement calories. Premature newborns with low birth weight are at a high risk for developing PN associated liver disease (PNALD) including steatosis, cholestasis, and gallbladder sludge/stones. To optimize nutrition regimens, models are required to predict PNALD. Methods We have exploited induced pluripotent stem cell derived liver organoids to provide a testing platform for PNALD. Liver organoids mimic the developing liver and contain the different hepatic cell types. The organoids have an early postnatal maturity making them a suitable model for premature newborns. To mimic PN treatment we used medium supplemented with either clinoleic (80% olive oil/20% soybean oil) or intralipid (100% soybean oil) for 7 days. Results Homogenous HNF4a staining was found in all organoids and PN treatments caused accumulation of lipids in hepatocytes. Organoids exhibited a dose dependent decrease in CYP3A4 activity and expression of hepatocyte functional genes. The lipid emulsions did not affect overall organoid viability and glucose levels had no contributory effect to the observed results. Conclusions Liver organoids could be utilized as a potential screening platform for the development of new, less hepatotoxic PN solutions. Both lipid treatments caused hepatic lipid accumulation, a significant decrease in CYP3A4 activity and a decrease in the RNA levels of both CYP3A4 and CYP1A2 in a dose dependent manner. The presence of high glucose had no additive effect, while Clinoleic at high dose, caused significant upregulation of interleukin 6 and TLR4 expression.

• Uzhytchak, Mariia; Smolková, Barbora; Frtús, Adam; Stupakov, Alexandr; Lunova, Mariia & Scollo, Federica [Vis alle 11 forfattere av denne artikkelen] (2023). Sensitivity of endogenous autofluorescence in HeLa cells to the application of external magnetic fields. Scientific Reports. 13(1). doi: 10.1038/s41598-023-38015-x. Fulltekst i vitenarkiv Vis sammendrag

  Abstract Dramatically increased levels of electromagnetic radiation in the environment have raised concerns over the potential health hazards of electromagnetic fields. Various biological effects of magnetic fields have been proposed. Despite decades of intensive research, the molecular mechanisms procuring cellular responses remain largely unknown. The current literature is conflicting with regards to evidence that magnetic fields affect functionality directly at the cellular level. Therefore, a search for potential direct cellular effects of magnetic fields represents a cornerstone that may propose an explanation for potential health hazards associated with magnetic fields. It has been proposed that autofluorescence of HeLa cells is magnetic field sensitive, relying on single-cell imaging kinetic measurements. Here, we investigate the magnetic field sensitivity of an endogenous autofluorescence in HeLa cells. Under the experimental conditions used, magnetic field sensitivity of an endogenous autofluorescence was not observed in HeLa cells. We present a number of arguments indicating why this is the case in the analysis of magnetic field effects based on the imaging of cellular autofluorescence decay. Our work indicates that new methods are required to elucidate the effects of magnetic fields at the cellular level.

• Kristiansen, Cecilie Katrin; Furriol, Jessica; Chen, Anbin; Sullivan, Gareth John; Bindoff, Laurence Albert & Liang, Xiao (2023). Deoxyribonucleoside treatment rescues EtBr-induced mtDNA depletion in iPSC-derived neural stem cells with POLG mutations. The FASEB Journal. ISSN 0892-6638. 37(9). doi: 10.1096/fj.202300650RR. Fulltekst i vitenarkiv Vis sammendrag

  Mutations in POLG, the gene encoding the catalytic subunit of the mitochondrial DNA (mtDNA) polymerase gamma (Pol-γ), lead to diseases driven by defective mtDNA maintenance. Despite being the most prevalent cause of mitochondrial disease, treatments for POLG-related disorders remain elusive. In this study, we used POLG patient-induced pluripotent stem cell (iPSC)-derived neural stem cells (iNSCs), one homozygous for the POLG mutation c.2243G>C and one compound heterozygous with c.2243G>C and c.1399G>A, and treated these iNSCs with ethidium bromide (EtBr) to study the rate of depletion and repopulation of mtDNA. In addition, we investigated the effect of deoxyribonucleoside (dNs) supplementation on mtDNA maintenance during EtBr treatment and post-treatment repopulation in the same cells. EtBr-induced mtDNA depletion occurred at a similar rate in both patient and control iNSCs, however, restoration of mtDNA levels was significantly delayed in iNSCs carrying the compound heterozygous POLG mutations. In contrast, iNSC with the homozygous POLG mutation recovered their mtDNA at a rate similar to controls. When we treated cells with dNs, we found that this reduced EtBr-induced mtDNA depletion and significantly increased repopulation rates in both patient iNSCs. These observations are consistent with the hypothesis that mutations in POLG impair mtDNA repopulation also within intact neural lineage cells and suggest that those with compound heterozygous mutation have a more severe defect of mtDNA synthesis. Our findings further highlight the potential for dNs to improve mtDNA replication in the presence of POLG mutations, suggesting that this may offer a new therapeutic modality for mitochondrial diseases caused by disturbed mtDNA homeostasis.

• Wesseler, Milan Finn; Taebnia, Nayere; Harrison, Sean Philip; Youhanna, Sonia; Preiss, Lena C. & Kemas, Aurino M. [Vis alle 11 forfattere av denne artikkelen] (2023). 3D microperfusion of mesoscale human microphysiological liver models improves functionality and recapitulates hepatic zonation. Acta Biomaterialia. ISSN 1742-7061. 171, s. 336–349. doi: 10.1016/j.actbio.2023.09.022. Fulltekst i vitenarkiv
• Almaas, Runar; Atneosen-Åsegg, Monica; Ytre-Arne, Mari Eknes; Melheim, Maria; Sorte, Hanne Sørmo & Cízková, Dana [Vis alle 16 forfattere av denne artikkelen] (2023). Aagenaes syndrome/lymphedema cholestasis syndrome 1 is caused by a founder variant in the 5’-untranslated region of UNC45A. Journal of Hepatology. ISSN 0168-8278. 79(4), s. 945–954. doi: 10.1016/j.jhep.2023.05.037. Fulltekst i vitenarkiv Vis sammendrag

  Background & Aims Lymphedema cholestasis syndrome 1 or Aagenaes syndrome is a condition characterized by neonatal cholestasis, lymphedema, and giant cell hepatitis. The genetic background of this autosomal recessive disease was unknown up to now. Methods A total of 26 patients with Aagenaes syndrome and 17 parents were investigated with whole-genome sequencing and/or Sanger sequencing. PCR and western blot analyses were used to assess levels of mRNA and protein, respectively. CRISPR/Cas9 was used to generate the variant in HEK293T cells. Light microscopy, transmission electron microscopy and immunohistochemistry for biliary transport proteins were performed in liver biopsies. Results One specific variant (c.-98G>T) in the 5’-untranslated region of Unc-45 myosin chaperone A (UNC45A) was identified in all tested patients with Aagenaes syndrome. Nineteen were homozygous for the c.-98G>T variant and seven were compound heterozygous for the variant in the 5’-untranslated region and an exonic loss-of-function variant in UNC45A. Patients with Aagenaes syndrome exhibited lower expression of UNC45A mRNA and protein than controls, and this was reproduced in a CRISPR/Cas9-created cell model. Liver biopsies from the neonatal period demonstrated cholestasis, paucity of bile ducts and pronounced formation of multinucleated giant cells. Immunohistochemistry revealed mislocalization of the hepatobiliary transport proteins BSEP (bile salt export pump) and MRP2 (multidrug resistance-associated protein 2). Conclusions c.-98G>T in the 5’-untranslated region of UNC45A is the causative genetic variant in Aagenaes syndrome. Impact and implications The genetic background of Aagenaes syndrome, a disease presenting with cholestasis and lymphedema in childhood, was unknown until now. A variant in the 5’-untranslated region of the Unc-45 myosin chaperone A (UNC45A) was identified in all tested patients with Aagenaes syndrome, providing evidence of the genetic background of the disease. Identification of the genetic background provides a tool for diagnosis of patients with Aagenaes syndrome before lymphedema is evident.

• Roman, Giacomo; Stavik, Benedicte; Lauritzen, Knut Husø; Sandset, Per Morten; Harrison, Sean & Sullivan, Gareth John [Vis alle 7 forfattere av denne artikkelen] (2023). “iPSC-derived liver organoids and inherited bleeding disorders: Potential and future perspectives”. Frontiers in Physiology. 14. doi: 10.3389/fphys.2023.1094249. Fulltekst i vitenarkiv Vis sammendrag

  The bleeding phenotype of hereditary coagulation disorders is caused by the low or undetectable activity of the proteins involved in hemostasis, due to a broad spectrum of genetic alterations. Most of the affected coagulation factors are produced in the liver. Therefore, two-dimensional (2D) cultures of primary human hepatocytes and recombinant overexpression of the factors in non-human cell lines have been primarily used to mimic disease pathogenesis and as a model for innovative therapeutic strategies. However, neither human nor animal cells fully represent the hepatocellular biology and do not harbor the exact genetic background of the patient. As a result, the inability of the current in vitro models in recapitulating the in vivo situation has limited the studies of these inherited coagulation disorders. Induced Pluripotent Stem Cell (iPSC) technology offers a possible solution to overcome these limitations by reprogramming patient somatic cells into an embryonic-like pluripotent state, thus giving the possibility of generating an unlimited number of liver cells needed for modeling or therapeutic purposes. By combining this potential and the recent advances in the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 technology, it allows for the generation of autologous and gene corrected liver cells in the form of three-dimensional (3D) liver organoids. The organoids recapitulate cellular composition and organization of the liver, providing a more physiological model to study the biology of coagulation proteins and modeling hereditary coagulation disorders. This advanced methodology can pave the way for the development of cell-based therapeutic approaches to treat inherited coagulation disorders. In this review we will explore the use of liver organoids as a state-of-the-art methodology for modeling coagulation factors disorders and the possibilities of using organoid technology to treat the disease.

• Hong, Yu; Kristiansen, Cecilie Katrin; Chen, Anbin; Nido, Gonzalo Sanchez; Høyland, Lena Elise & Ziegler, Mathias [Vis alle 9 forfattere av denne artikkelen] (2023). POLG genotype influences degree of mitochondrial dysfunction in iPSC derived neural progenitors, but not the parent iPSC or derived glia. Experimental Neurology. ISSN 0014-4886. 365. doi: 10.1016/j.expneurol.2023.114429. Fulltekst i vitenarkiv Vis sammendrag

  Diseases caused by POLG mutations are the most common form of mitochondrial diseases and associated with phenotypes of varying severity. Clinical studies have shown that patients with compound heterozygous POLG mutations have a lower survival rate than patients with homozygous mutations, but the molecular mechanisms behind this remain unexplored. Using an induced pluripotent stem cell (iPSC) model, we investigate differences between homozygous and compound heterozygous genotypes in different cell types, including patient-specific fibroblasts, iPSCs, and iPSC-derived neural stem cells (NSCs) and astrocytes. We found that compound heterozygous lines exhibited greater impairment of mitochondrial function in NSCs than homozygous NSCs, but not in fibroblasts, iPSCs, or astrocytes. Compared with homozygous NSCs, compound heterozygous NSCs exhibited more severe functional defects, including reduced ATP production, loss of mitochondrial DNA (mtDNA) copy number and complex I expression, disturbance of NAD+ metabolism, and higher ROS levels, which further led to cellular senescence and activation of mitophagy. RNA sequencing analysis revealed greater downregulation of mitochondrial and metabolic pathways, including the citric acid cycle and oxidative phosphorylation, in compound heterozygous NSCs. Our iPSC-based disease model can be widely used to understand the genotype-phenotype relationship of affected brain cells in mitochondrial diseases, and further drug discovery applications.

• Harrison, Sean Philip; Siller, Richard; Tanaka, Yoshiaki; Dugarte, Maria Eugenia Chollet; Morena-Barrio, María Eugenia de la & Xiang, Yangfei [Vis alle 36 forfattere av denne artikkelen] (2023). Scalable production of tissue-like vascularized liver organoids from human PSCs. Experimental and Molecular Medicine. ISSN 1226-3613. 55, s. 2005–2024. doi: 10.1038/s12276-023-01074-1. Fulltekst i vitenarkiv Vis sammendrag

  Abstract 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.

• Lin, Xiaolin; Wang, Wei; Yang, Mingyi; Damseh, Nadirah; Sousa, Mirta & Jacob, Fadi [Vis alle 29 forfattere av denne artikkelen] (2023). A loss-of-function mutation in human Oxidation Resistance 1 disrupts the spatial–temporal regulation of histone arginine methylation in neurodevelopment. Genome Biology. ISSN 1465-6906. 24(1). doi: 10.1186/s13059-023-03037-1. Fulltekst i vitenarkiv Vis sammendrag

  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.

• Kristiansen, Cecilie Katrin; Chen, Anbin; Høyland, Lena Elise; Ziegler, Mathias; Sullivan, Gareth John & Bindoff, Laurence Albert [Vis alle 7 forfattere av denne artikkelen] (2022). Comparing the mitochondrial signatures in ESCs and iPSCs and their neural derivations. Cell Cycle. ISSN 1538-4101. 21(20), s. 2206–2221. doi: 10.1080/15384101.2022.2092185. Fulltekst i vitenarkiv Vis sammendrag

  Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have distinct origins: ESCs are derived from pre-implanted embryos while iPSCs are reprogrammed somatic cells. Both have their own characteristics and lineage specificity, and both are valuable tools for studying human neurological development and disease. Thus far, few studies have analyzed how differences between stem cell types influence mitochondrial function and mitochondrial DNA (mtDNA) homeostasis during differentiation into neural and glial lineages. In this study, we compared mitochondrial function and mtDNA replication in human ESCs and iPSCs at three different stages − pluripotent, neural progenitor and astrocyte. We found that while ESCs and iPSCs have a similar mitochondrial signature, neural and astrocyte derivations manifested differences. At the neural stem cell (NSC) stage, iPSC-NSCs displayed decreased ATP production and a reduction in mitochondrial respiratory chain (MRC) complex IV expression compared to ESC-NSCs. IPSC-astrocytes showed increased mitochondrial activity including elevated ATP production, MRC complex IV expression, mtDNA copy number and mitochondrial biogenesis relative to those derived from ESCs. These findings show that while ESCs and iPSCs are similar at the pluripotent stage, differences in mitochondrial function may develop during differentiation and must be taken into account when extrapolating results from different cell types.

• Kogler, Stian; Aizenshtadt, Aleksandra; Harrison, Sean; Skottvoll, Frøydis Sved; Berg, Henriette Engen & Abadpour, Shadab [Vis alle 14 forfattere av denne artikkelen] (2022). "Organ-in-a-Column" Coupled On-line with Liquid Chromatography-Mass Spectrometry. Analytical Chemistry. ISSN 0003-2700. 94(50), s. 17677–17684. doi: 10.1021/acs.analchem.2c04530. Fulltekst i vitenarkiv Vis sammendrag

  Organoids, i.e., laboratory-grown organ models developed from stem cells, are emerging tools for studying organ physiology, disease modeling, and drug development. On-line analysis of organoids with mass spectrometry would provide analytical versatility and automation. To achieve these features with robust hardware, we have loaded liquid chromatography column housings with induced pluripotent stem cell (iPSC) derived liver organoids and coupled the “organ-in-a-column” units on-line with liquid chromatography-mass spectrometry (LC-MS). Liver organoids were coloaded with glass beads to achieve an even distribution of organoids throughout the column while preventing clogging. The liver organoids were interrogated “on column” with heroin, followed by on-line monitoring of the drug’s phase 1 metabolism. Enzymatic metabolism of heroin produced in the “organ-in-a-column” units was detected and monitored using a triple quadrupole MS instrument, serving as a proof-of-concept for on-line coupling of liver organoids and mass spectrometry. Taken together, the technology allows direct integration of liver organoids with LC-MS, allowing selective and automated tracking of drug metabolism over time.

• Chen, Anbin; Kristiansen, Cecilie Katrin; Høyland, Lena Elise; Ziegler, Mathias; Wang, Jian & Sullivan, Gareth John [Vis alle 9 forfattere av denne artikkelen] (2022). POLG mutations lead to abnormal mitochondrial remodeling during neural differentiation of human pluripotent stem cells via SIRT3/AMPK pathway inhibition. Cell Cycle. ISSN 1538-4101. 21(11), s. 1178–1193. doi: 10.1080/15384101.2022.2044136. Fulltekst i vitenarkiv Vis sammendrag

  We showed previously that POLG mutations cause major changes in mitochondrial function, including loss of mitochondrial respiratory chain (MRC) complex I, mitochondrial DNA (mtDNA) depletion and an abnormal NAD+/NADH ratio in both neural stem cells (NSCs) and astrocytes differentiated from induced pluripotent stem cells (iPSCs). In the current study, we looked at mitochondrial remodeling as stem cells transit pluripotency and during differentiation from NSCs to both dopaminergic (DA) neurons and astrocytes comparing the process in POLG-mutated and control stem cells. We saw that mitochondrial membrane potential (MMP), mitochondrial volume, ATP production and reactive oxygen species (ROS) changed in similar ways in POLG and control NSCs, but mtDNA replication, MRC complex I and NAD+ metabolism failed to remodel normally. In DA neurons differentiated from NSCs, we saw that POLG mutations caused failure to increase MMP and ATP production and blunted the increase in mtDNA and complex I. Interestingly, mitochondrial remodeling during astrocyte differentiation from NSCs was similar in both POLG-mutated and control NSCs. Further, we showed downregulation of the SIRT3/AMPK pathways in POLG-mutated cells, suggesting that POLG mutations lead to abnormal mitochondrial remodeling in early neural development due to the downregulation of these pathways.

• Bogdanova, Mariia; Zabirnyk, Arsenii; Malashicheva, Anna; Semenova, Daria; Kvitting, John-Peder & Kaljusto, Mari-Liis [Vis alle 12 forfattere av denne artikkelen] (2022). Models and Techniques to Study Aortic Valve Calcification in Vitro, ex Vivo and in Vivo. An Overview. Frontiers in Pharmacology. 13. doi: 10.3389/fphar.2022.835825. Fulltekst i vitenarkiv Vis sammendrag

  Aortic valve stenosis secondary to aortic valve calcification is the most common valve disease in the Western world. Calcification is a result of pathological proliferation and osteogenic differentiation of resident valve interstitial cells. To develop non-surgical treatments, the molecular and cellular mechanisms of pathological calcification must be revealed. In the current overview, we present methods for evaluation of calcification in different ex vivo , in vitro and in vivo situations including imaging in patients. The latter include echocardiography, scanning with computed tomography and magnetic resonance imaging. Particular emphasis is on translational studies of calcific aortic valve stenosis with a special focus on cell culture using human primary cell cultures. Such models are widely used and suitable for screening of drugs against calcification. Animal models are presented, but there is no animal model that faithfully mimics human calcific aortic valve disease. A model of experimentally induced calcification in whole porcine aortic valve leaflets ex vivo is also included. Finally, miscellaneous methods and aspects of aortic valve calcification, such as, for instance, biomarkers are presented.

• Skottvoll, Frøydis Sved; Hansen, Frederik; Harrison, Sean; Boger, Ida Caroline Sneis; Mrsa, Ago & Restan, Magnus Saed [Vis alle 14 forfattere av denne artikkelen] (2021). Electromembrane extraction and mass spectrometry for liver organoid drug metabolism studies. Analytical Chemistry. ISSN 0003-2700. 93, s. 3576–3585. doi: 10.1021/acs.analchem.0c05082. Fulltekst i vitenarkiv Vis sammendrag

  Liver organoids are emerging tools for precision drug development and toxicity screening. We demonstrate that electromembrane extraction (EME) based on electrophoresis across an oil membrane is suited for segregating selected organoid-derived drug metabolites prior to mass spectrometry (MS)-based measurements. EME allowed drugs and drug metabolites to be separated from cell medium components (albumin, etc.) that could interfere with subsequent measurements. Multiwell EME (parallel-EME) holding 100 μL solutions allowed for simple and repeatable monitoring of heroin phase I metabolism kinetics. Organoid parallel-EME extracts were compatible with ultrahigh-performance liquid chromatography (UHPLC) used to separate the analytes prior to detection. Taken together, liver organoids are well-matched with EME followed by MS-based measurements.

• Kim, Jonghun; Sullivan, Gareth & Park, In-Hyun (2021). How well do brain organoids capture your brain? iScience. 24(2), s. 1–14. doi: 10.1016/j.isci.2021.102063. Fulltekst i vitenarkiv Vis sammendrag

  Brain organoids closely recapitulate many features and characteristics of in vivo brain tissue. This technology in turn allows unprecedented possibilities to investigate brain development and function in the dish. Several brain organoid protocols have been established, and the studies have focused on validating the architecture, cellular composition, and function of the organoids. In future, the improved and advanced organoid models will enable us to understand cellular and molecular features of the developing brain. However, several obstacles, such as the quality of the organoids, 3D structural analysis, and measurement of the neural connectivity need to be improved. In this perspective, we will provide an overview of the current state of the art of the brain organoid field, with a focus on protocols and organoid characterization. Additionally, we will address the current limitations of this evolving field and provide an understanding of the current brain organoid landscape and insight toward the next steps.

• Liang, Xiao; Kianian, Atefeh; Chen, Anbin; Kristiansen, Cecilie Katrin; Hong, Yu & Furriol, Jessica [Vis alle 12 forfattere av denne artikkelen] (2021). Stem cell derived astrocytes with POLG mutations and mitochondrial dysfunction including abnormal NAD+ metabolism is toxic for neurons. BioRxiv. ISSN 0362-4331. doi: 10.1101/2020.12.20.423652. Fulltekst i vitenarkiv
• Harrison, Sean; Baumgarten, Saphira Felicitas; Verma, Rajneesh; Lunov, Oleg; Dejneka, Alexandr & Sullivan, Gareth (2021). Liver Organoids: Recent Developments, Limitations and Potential. Frontiers in Medicine. 8. doi: 10.3389/fmed.2021.574047. Fulltekst i vitenarkiv Vis sammendrag

  Liver cell types derived from induced pluripotent stem cells (iPSCs) share the potential to investigate development, toxicity, as well as genetic and infectious disease in ways currently limited by the availability of primary tissue. With the added advantage of patient specificity, which can play a role in all of these areas. Many iPSC differentiation protocols focus on 3 dimensional (3D) or organotypic differentiation, as these offer the advantage of more closely mimicking in vivo systems including; the formation of tissue like architecture and interactions/crosstalk between different cell types. Ultimately such models have the potential to be used clinically and either with or more aptly, in place of animal models. Along with the development of organotypic and micro-tissue models, there will be a need to co-develop imaging technologies to enable their visualization. A variety of liver models termed “organoids” have been reported in the literature ranging from simple spheres or cysts of a single cell type, usually hepatocytes, to those containing multiple cell types combined during the differentiation process such as hepatic stellate cells, endothelial cells, and mesenchymal cells, often leading to an improved hepatic phenotype. These allow specific functions or readouts to be examined such as drug metabolism, protein secretion or an improved phenotype, but because of their relative simplicity they lack the flexibility and general applicability of ex vivo tissue culture. In the liver field these are more often constructed rather than developed together organotypically as seen in other organoid models such as brain, kidney, lung and intestine. Having access to organotypic liver like surrogates containing multiple cell types with in vivo like interactions/architecture, would provide vastly improved models for disease, toxicity and drug development, combining disciplines such as microfluidic chip technology with organoids and ultimately paving the way to new therapies.

• Aprigliano, Rossana; Aksu, Merdane Ezgi; Bradamante, Stefano; Mihaljevic, Boris; Wang, Wei & Rian, Kristin [Vis alle 19 forfattere av denne artikkelen] (2021). Increased p53 signaling impairs neural differentiation in HUWE1-promoted intellectual disabilities. Cell Reports Medicine. 2(4). doi: 10.1016/j.xcrm.2021.100240. Fulltekst i vitenarkiv Vis sammendrag

  Essential E3 ubiquitin ligase HUWE1 (HECT, UBA, and WWE domain containing 1) regulates key factors, such as p53. Although mutations in HUWE1 cause heterogenous neurodevelopmental X-linked intellectual disabilities (XLIDs), the disease mechanisms common to these syndromes remain unknown. In this work, we identify p53 signaling as the central process altered in HUWE1-promoted XLID syndromes. By focusing on Juberg-Marsidi syndrome (JMS), one of the severest XLIDs, we show that increased p53 signaling results from p53 accumulation caused by HUWE1 p.G4310R destabilization. This further alters cell-cycle progression and proliferation in JMS cells. Modeling of JMS neurodevelopment reveals majorly impaired neural differentiation accompanied by increased p53 signaling. The neural differentiation defects can be successfully rescued by reducing p53 levels and restoring the expression of p53 target genes, in particular CDKN1A/p21. In summary, our findings suggest that increased p53 signaling underlies HUWE1-promoted syndromes and impairs XLID JMS neural differentiation.

• Fernandez-Checa, Jose C; Bagnaninchi, Pierre-Olivier; Ye, H; Sancho-Bru, Pau; Falcon-Perez, JM & Royo, Felix [Vis alle 21 forfattere av denne artikkelen] (2021). Advanced preclinical models for evaluation of drug induced liver injury - consensus statement by the european drug-induced liver injury network [PRO-EURO-DILI-NET]. Journal of Hepatology. ISSN 0168-8278. 75(4), s. 935–959. doi: 10.1016/j.jhep.2021.06.021. Fulltekst i vitenarkiv Vis sammendrag

  Drug induced liver injury (DILI) is a major cause of acute liver failure (ALF) and one of the leading indications for liver transplantation in Western societies. Given the wide use of both prescribed and over the counter drugs, DILI has become a major health issue with a pressing need to find novel and effective therapies. Although significant progress has been made in understanding the molecular mechanisms underlying DILI, our incomplete knowledge of its pathogenesis and inability to predict DILI is largely due to both discordance between human and animal DILI in preclinical drug development and a lack of models that faithfully recapitulate complex pathophysiological features of human DILI. This is exemplified by the hepatotoxicity of acetaminophen (APAP) overdose, a major cause ALF due to its extensive worldwide use as an analgesic. Despite intensive efforts utilizing current animal and in vitro models, the mechanisms involved in the hepatotoxicity of APAP are still not fully understood. In this expert Consensus Statement, which is endorsed by the European Drug-Induced Liver Injury Network, we aim to facilitate and outline clinically impactful knowledge discovery by detailing the requirements for more realistic human-based systems to assess hepatotoxicity to guide future drug safety testing. We present novel insights and majorplayers in APAP pathophysiology and describe emerging in vitro and in vivo pre-clinicalmodels, as well as advanced imaging and in silico technologies, which may improveprediction of clinical outcomes of DILI including APAP hepatotoxicity.

• Chen, Anbin; Kristiansen, Cecilie Katrin; Hong, Yu; Kianian, Atefeh; Fang, Fei & Sullivan, Gareth John [Vis alle 10 forfattere av denne artikkelen] (2021). Nicotinamide riboside and metformin ameliorate mitophagy defect in induced pluripotent stem cell-derived astrocytes with POLG mutations. Frontiers in Cell and Developmental Biology. 9, s. 1–20. doi: 10.3389/fcell.2021.737304. Fulltekst i vitenarkiv Vis sammendrag

  Mitophagy specifically recognizes and removes damaged or superfluous mitochondria to maintain mitochondrial homeostasis and proper neuronal function. Defective mitophagy and the resulting accumulation of damaged mitochondria occur in several neurodegenerative diseases. Previously, we showed mitochondrial dysfunction in astrocytes with POLG mutations, and here, we examined how POLG mutations affect mitophagy in astrocytes and how this can be ameliorated pharmacologically. Using induced pluripotent stem cell (iPSC)-derived astrocytes carrying POLG mutations, we found downregulation of mitophagy/autophagy-related genes using RNA sequencing-based KEGG metabolic pathway analysis. We confirmed a deficit in mitochondrial autophagosome formation under exogenous stress conditions and downregulation of the mitophagy receptor p62, reduced lipidation of LC3B-II, and decreased expression of lysosome protein lysosomal-associated membrane protein 2A (LAMP2A). These changes were regulated by the PINK1/Parkin pathway and AKT/mTOR/AMPK/ULK1 signaling pathways. Importantly, we found that double treatment with nicotinamide riboside (NR) and metformin rescued mitophagy defects and mitochondrial dysfunction in POLG-mutant astrocytes. Our findings reveal that impaired mitophagy is involved in the observed mitochondrial dysfunction caused by POLG mutations in astrocytes, potentially contributing to the phenotype in POLG-related diseases. This study also demonstrates the therapeutic potential of NR and metformin in these incurable mitochondrial diseases.

• Convery, Neil; Samardzhieva, Iliyana; Stormonth-Darling, John Moir; Harrison, Sean; Sullivan, Gareth John & Gadegaard, Nikolaj (2021). 3D printed tooling for injection moulded microfluidics. Macromolecular Materials and Engineering. ISSN 1438-7492. 306(11), s. 1–11. doi: 10.1002/mame.202100464. Fulltekst i vitenarkiv Vis sammendrag

  Microfluidics have been used for several decades to conduct a wide range of research in chemistry and the life sciences. The reduced dimensions of these devices give them advantages over classical analysis techniques such as increased sensitivity, shorter analysis times, and lower reagent consumption. However, current manufacturing processes for microfluidic chips either limits them to materials with unwanted properties, or are not cost effective enough for rapid-prototyping approaches. Here we show that inlays for injection moulding can be 3D printed, thus reducing the skills, cost, and time required for tool fabrication. We demonstrate the importance of orientation of the part during 3D printing so that features as small as 100 x 200 μm can be printed. We also demonstrate that the 3D printed inlay is durable enough to fabricate at least 500 parts. Furthermore, devices can be designed, manufactured, and tested within one working day. Finally, as demonstrators we design and mould a microfluidic chip to house a plasmonic biosensor as well as a device to house liver organoids showing how such chips can be used in organ-on-a-chip applications. This new fabrication technique bridges the gap between small production and industrial scale manufacturing, whilst making microfluidics cheaper, and more widely accessible.

• Sharma, Kulbhushan; Asp, Nagham Theres; Harrison, Sean; Siller, Richard; Baumgarten, Saphira Felicitas & Gupta, Swapnil [Vis alle 10 forfattere av denne artikkelen] (2021). Autophagy modulates cell fate decisions during lineage commitment. Autophagy. ISSN 1554-8627. s. 1–17. doi: 10.1080/15548627.2021.2008691. Fulltekst i vitenarkiv Vis sammendrag

  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.

• Mostafavi, Sepideh; Balafkan, Novin; Pettersen, Ina Katrine Nitschke; Nido, Gonzalo Sanchez; Siller, Richard & Tzoulis, Charalampos [Vis alle 8 forfattere av denne artikkelen] (2021). Distinct Mitochondrial Remodeling During Mesoderm Differentiation in a Human-Based Stem Cell Model. Frontiers in Cell and Developmental Biology. 9, s. 1–12. doi: 10.3389/fcell.2021.744777. Fulltekst i vitenarkiv Vis sammendrag

  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.

• Harrison, Sean; Siller, Richard; Tanaka, Yoshiaki; Xiang, Yangfei; Patterson, Benjamin & Kempf, Henning [Vis alle 29 forfattere av denne artikkelen] (2020). Scalable production of tissue-like vascularised liver organoids from human PSCs. BioRxiv. ISSN 0362-4331. doi: 10.1101/2020.12.02.406835. Fulltekst i vitenarkiv
• Balafkan, Novin; Mostafavi, Sepideh; Schubert, Manja; Siller, Richard; Liang, Kristina Xiao & Sullivan, Gareth [Vis alle 7 forfattere av denne artikkelen] (2020). A method for differentiating human induced pluripotent stem cells toward functional cardiomyocytes in 96‐well microplates. Scientific Reports. 10, s. 1–14. doi: 10.1038/s41598-020-73656-2. Fulltekst i vitenarkiv Vis sammendrag

  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.

• Liang, Xiao; Kristiansen, Cecilie Katrin; Mostafavi, Sepideh; Vatne, Guro Helén; Zantingh, Gina Alien & Kianian, Atefeh [Vis alle 17 forfattere av denne artikkelen] (2020). Disease-specific phenotypes in iPSC-derived neural stem cells with POLG mutations. EMBO Molecular Medicine. ISSN 1757-4676. 12(10), s. 1–26. doi: 10.15252/emmm.202012146. Fulltekst i vitenarkiv Vis sammendrag

  Mutations in POLG disrupt mtDNA replication and cause devas- tating 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 dysfunc- tion 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 deter- mining processes.

• Liang, Xiao; Vatne, Guro Helén; Kristiansen, Cecilie Katrin; Ievglevskyi, Oleksandr; Kondratskaya, Elena & Glover, Joel [Vis alle 9 forfattere av denne artikkelen] (2020). N-acetylcysteine amide ameliorates mitochondrial dysfunction and reduces oxidative stress in hiPSC-derived dopaminergic neurons with POLG mutation. Experimental Neurology. ISSN 0014-4886. 337. doi: 10.1016/j.expneurol.2020.113536. Fulltekst i vitenarkiv
• Tanaka, Yoshiaki; Cakir, Bilal; Xiang, Yangfei; Sullivan, Gareth & Park, In-Hyun (2020). Synthetic Analyses of Single-Cell Transcriptomes from Multiple Brain Organoids and Fetal Brain. Cell reports. 30(6), s. 1682–1689. doi: 10.1016/j.celrep.2020.01.038. Fulltekst i vitenarkiv Vis sammendrag

  Human brain organoid systems offer unprecedented opportunities to investigate both neurodevelopmental and neurological disease. Single-cell-based transcriptomics or epigenomics have dissected the cellular and molecular heterogeneity in the brain organoids, revealing a complex organization. Similar but distinct protocols from different labs have been applied to generate brain organoids, providing a large resource to perform a comparative analysis of brain developmental processes. Here, we take a systematic approach to compare the single-cell transcriptomes of various human cortical brain organoids together with fetal brain to define the identity of specific cell types and differentiation routes in each method. Importantly, we identify unique developmental programs in each protocol compared to fetal brain, which will be a critical benchmark for the utility of human brain organoids in the future.

• Xiang, Yangfei; Tanaka, Yoshiaki; Patterson, Benjamin; Hwang, Sun-Min; Hysolli, Eriona & Cakir, Bilal [Vis alle 17 forfattere av denne artikkelen] (2020). Dysregulation of BRD4 Function Underlies the Functional Abnormalities of MeCP2 Mutant Neurons. Molecular Cell. ISSN 1097-2765. 79(1), s. 84–98. doi: 10.1016/j.molcel.2020.05.016. Fulltekst i vitenarkiv Vis sammendrag

  Rett syndrome (RTT), mainly caused by mutations in methyl-CpG binding protein 2 (MeCP2), is one of the most prevalent intellectual disorders without effective therapies. Here, we used 2D and 3D human brain cul- tures to investigate MeCP2 function. We found that MeCP2 mutations cause severe abnormalities in human interneurons (INs). Surprisingly, treatment with a BET inhibitor, JQ1, rescued the molecular and functional phenotypes of MeCP2 mutant INs. We uncovered that abnormal increases in chromatin binding of BRD4 and enhancer-promoter interactions underlie the abnormal transcription in MeCP2 mutant INs, which were recovered to normal levels by JQ1. We revealed cell-type-specific transcriptome impairment in MeCP2 mutant region-specific human brain organoids that were rescued by JQ1. Finally, JQ1 ameliorated RTT- like phenotypes in mice. These data demonstrate that BRD4 dysregulation is a critical driver for RTT etiology and suggest that targeting BRD4 could be a potential therapeutic opportunity for RTT.

• Kogler, Stian; Harrison, Sean; Aizenshtadt, Aleksandra; Skottvoll, Frøydis Sved; Abadpour, Shadab & Krauss, Stefan [Vis alle 10 forfattere av denne artikkelen] (2020). “Organoid-in-a-column” coupled on-line with liquid chromatography-mass spectrometry. BioRxiv. ISSN 0362-4331. doi: 10.1101/2020.09.08.282756. Fulltekst i vitenarkiv
• Lin, Ann; Skottvoll, Frøydis Sved; Rayner, Simon; Pedersen-Bjergaard, Stig; Sullivan, Gareth & Krauss, Stefan [Vis alle 8 forfattere av denne artikkelen] (2019). 3D cell culture models and organ-on-a-chip: Meet separation science and mass spectrometry. Electrophoresis. ISSN 0173-0835. 41, s. 56–64. doi: 10.1002/elps.201900170. Fulltekst i vitenarkiv Vis sammendrag

  In vitro derived simplified 3D representations of human organs or organ functionalities are predicted to play a major role in disease modeling, drug development, and personalized medicine, as they complement traditional cell line approaches and animal models. The cells for 3D organ representations may be derived from primary tissues, embryonic stem cells or induced pluripotent stem cells and come in a variety of formats from aggregates of individual or mixed cell types, self‐organizing in vitro developed “organoids” and tissue mimicking chips. Microfluidic devices that allow long‐term maintenance and combination with other tissues, cells or organoids are commonly referred to as “microphysiological” or “organ‐on‐a‐chip” systems. Organ‐on‐a‐chip technology allows a broad range of “on‐chip” and “off‐chip” analytical techniques, whereby “on‐chip” techniques offer the possibility of real time tracking and analysis. In the rapidly expanding tool kit for real time analytical assays, mass spectrometry, combined with “on‐chip” electrophoresis, and other separation approaches offer attractive emerging tools. In this review, we provide an overview of current 3D cell culture models, a compendium of current analytical strategies, and we make a case for new approaches for integrating separation science and mass spectrometry in this rapidly expanding research field.

• Bogdanova, Mariia; Zabirnyk, Arsenii; Malashicheva, Anna; Zihlavnikova, Katarina; Karlsen, Tommy Aleksander & Kaljusto, Mari-Liis [Vis alle 13 forfattere av denne artikkelen] (2019). Interstitial cells in calcified aortic valves have reduced differentiation potential and stem cell-like properties. Scientific Reports. 9:12934, s. 1–13. doi: 10.1038/s41598-019-49016-0. Fulltekst i vitenarkiv Vis sammendrag

  Valve interstitial cells (VICs) are crucial in the development of calcific aortic valve disease. The purpose of the present investigation was to compare the phenotype, differentiation potential and stem cell-like properties of cells from calcified and healthy aortic valves. VICs were isolated from human healthy and calcified aortic valves. Calcification was induced with osteogenic medium. Unlike VICs from healthy valves, VICs from calcified valves cultured without osteogenic medium stained positively for calcium deposits with Alizarin Red confirming their calcific phenotype. Stimulation of VICs from calcified valves with osteogenic medium increased calcification (p = 0.02), but not significantly different from healthy VICs. When stimulated with myofibroblastic medium, VICs from calcified valves had lower expression of myofibroblastic markers, measured by flow cytometry and RT-qPCR, compared to healthy VICs. Contraction of collagen gel (a measure of myofibroblastic activity) was attenuated in cells from calcified valves (p = 0.04). Moreover, VICs from calcified valves, unlike cells from healthy valves had lower potential to differentiate into adipogenic pathway and lower expression of stem cell-associated markers CD106 (p = 0.04) and aldehyde dehydrogenase (p = 0.04). In conclusion, VICs from calcified aortic have reduced multipotency compared to cells from healthy valves, which should be considered when investigating possible medical treatments of aortic valve calcification.

• Pisal, Rishikaysh V.; Suchánek, Jakub; Siller, Richard; Soukup, Tomáš; Hrebíková, Hana & Bezrouk, Ales [Vis alle 11 forfattere av denne artikkelen] (2018). Directed reprogramming of comprehensively characterized dental pulp stem cells extracted from natal tooth. Scientific Reports. 8(1). doi: 10.1038/s41598-018-24421-z. Fulltekst i vitenarkiv Vis sammendrag

  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.

• Bogdanova, Mariia; Kostina, Aleksandra; Zihlavnikova, Katarina; Zabirnyk, Arsenii; Malashicheva, Anna & Stensløkken, Kåre-Olav [Vis alle 12 forfattere av denne artikkelen] (2018). Inflammation and Mechanical Stress Stimulate Osteogenic Differentiation of Human Aortic Valve Interstitial Cells. Frontiers in Physiology. 9. doi: 10.3389/fphys.2018.01635. Fulltekst i vitenarkiv Vis sammendrag

  Background: Aortic valve calcification is an active proliferative process, where interstitial cells of the valve transform into either myofibroblasts or osteoblast-like cells causing valve deformation, thickening of cusps and finally stenosis. This process may be triggered by several factors including inflammation, mechanical stress or interaction of cells with certain components of extracellular matrix. The matrix is different on the two sides of the valve leaflets. We hypothesize that inflammation and mechanical stress stimulate osteogenic differentiation of human aortic valve interstitial cells (VICs) and this may depend on the side of the leaflet. Methods: Interstitial cells isolated from healthy and calcified human aortic valves were cultured on collagen or elastin coated plates with flexible bottoms, simulating the matrix on the aortic and ventricular side of the valve leaflets, respectively. The cells were subjected to 10% stretch at 1 Hz (FlexCell bioreactor) or treated with 0.1 μg/ml lipopolysaccharide, or both during 24 h. Gene expression of myofibroblast- and osteoblast-specific genes was analyzed by qPCR. VICs cultured in presence of osteogenic medium together with lipopolysaccharide, 10% stretch or both for 14 days were stained for calcification using Alizarin Red. Results: Treatment with lipopolysaccharide increased expression of osteogenic gene bone morphogenetic protein 2 (BMP2) (5-fold increase from control; p = 0.02) and decreased expression of mRNA of myofibroblastic markers: α-smooth muscle actin (ACTA2) (50% reduction from control; p = 0.0006) and calponin (CNN1) (80% reduction from control; p = 0.0001) when cells from calcified valves were cultured on collagen, but not on elastin. Mechanical stretch of VICs cultured on collagen augmented the effect of lipopolysaccharide. Expression of periostin (POSTN) was inhibited in cells from calcified donors after treatment with lipopolysaccharide on collagen (70% reduction from control, p = 0.001), but not on elastin. Lipopolysaccharide and stretch both enhanced the pro-calcific effect of osteogenic medium, further increasing the effect when combined for cells cultured on collagen, but not on elastin. Conclusion: Inflammation and mechanical stress trigger expression of osteogenic genes in VICs in a side-specific manner, while inhibiting the myofibroblastic pathway. Stretch and lipopolysaccharide synergistically increase calcification.

• Kim, Kun-Yong; Tanaka, Yoshiaki; Su, Juan; Cakir, Bilal; Xiang, Yangfei & Patterson, Benjamin [Vis alle 20 forfattere av denne artikkelen] (2018). Uhrf1 regulates active transcriptional marks at bivalent domains in pluripotent stem cells through Setd1a. Nature Communications. 9:2583, s. 1–13. doi: 10.1038/s41467-018-04818-0. Fulltekst i vitenarkiv Vis sammendrag

  Embryonic stem cells (ESCs) maintain pluripotency through unique epigenetic states. When ESCs commit to a specific lineage, epigenetic changes in histones and DNA accompany the transition to specialized cell types. Investigating how epigenetic regulation controls lineage specification is critical in order to generate the required cell types for clinical applications. Uhrf1 is a widely known hemi-methylated DNA-binding protein, playing a role in DNA methylation through the recruitment of Dnmt1 and in heterochromatin formation alongside G9a, Trim28, and HDACs. Although Uhrf1 is not essential in ESC self-renewal, it remains elusive how Uhrf1 regulates cell specification. Here we report that Uhrf1 forms a complex With the active trithorax group, the Setd1a/COMPASS complex, to maintain bivalent histone marks, particularly those associated with neuroectoderm and mesoderm specification. Overall, our data demonstrate that Uhrf1 safeguards proper differentiation via bivalent histone modifications.

• Siller, Richard; Greenhough, Sebastian; Mathapati, Santosh Sadashiv; Si-Tayeb, K & Sullivan, Gareth (2017). Future challenges in the generation of hepatocyte like cells from human pluripotent stem cells. Current Pathobiology Reports. 5(3), s. 301–314. doi: 10.1007/s40139-017-0150-x. Fulltekst i vitenarkiv
• Rutkovskiy, Arkady; Malashicheva, Anna; Sullivan, Gareth; Bogdanova, Mariia; Kostareva, Anna & Stensløkken, Kåre-Olav [Vis alle 8 forfattere av denne artikkelen] (2017). Valve interstitial cells: The key to understanding the pathophysiology of heart valve calcification. Journal of the American Heart Association (JAHA). 6(9), s. 1–24. doi: 10.1161/JAHA.117.006339. Fulltekst i vitenarkiv Vis sammendrag

  Aortic valve stenosis due to calcification of the valve leaflets is the most common valve disease in the developed world. It is the third leading cause of cardiovascular disease.1 Risk factors include male gender, smoking, diabetes mellitus, hypertension, high levels of circulating lipids, and metabolic syndrome.2 Calcification was earlier believed to be a passive degenerative process, but it is now recognized as an active disease process driven by the cells native to the aortic valve.3, 4 The only option for treatment is heart surgery with implantation of a valve prosthesis. The heart valve prostheses are either mechanical, requiring life‐long anticoagulation treatment, or based on biological material, which will degenerate and calcify after 10 to 15 years. Implantation of heart valve prostheses has been characterized as “replacing one disease with another.” Understanding the cellular and molecular processes behind valve calcification may possibly lead to nonsurgical treatment.

• Siller, Richard; Dufour, Eric; Lycke, Eric Maximilian; Wilmut, Ian; Jung, Yong Wook & Park, In-Hyun [Vis alle 7 forfattere av denne artikkelen] (2017). Development of an inducible platform for intercellular protein delivery. International Journal of Pharmaceutics. ISSN 0378-5173. 522(1-2), s. 1–10. doi: 10.1016/j.ijpharm.2017.02.067. Fulltekst i vitenarkiv
• Gamal, Wesam; Treskes, Philipp; Samuel, Kay; Sullivan, Gareth; Siller, Richard & Srsen, Vlastimil [Vis alle 20 forfattere av denne artikkelen] (2017). Low-dose acetaminophen induces early disruption of cell-cell tight junctions in human hepatic cells and mouse liver. Scientific Reports. 7:37541, s. 1–16. doi: 10.1038/srep37541. Fulltekst i vitenarkiv
• Sullivan, Gareth & Siller, Richard (2017). Simple and rapid screen for human pluripotent stem cell endoderm differentiation potential. Current protocols in stem cell biology. ISSN 1941-7322. 43, s. 1G.7.1–1G.7.23. doi: 10.1002/cpsc.36. Fulltekst i vitenarkiv
• Scarfe, Lauren; Brillant, Nathalie; Kumar, J. Dinesh; Ali, Noura; Alrumayh, Ahmed & Amali, Mohammed [Vis alle 39 forfattere av denne artikkelen] (2017). Pre-clinical imaging methods for assessing the safety and efficacy of regenerative medicine therapies. npj Regenerative Medicine. 2(28). doi: 10.1038/s41536-017-0029-9. Fulltekst i vitenarkiv Vis sammendrag

  Regenerative medicine therapies hold enormous potential for a variety of currently incurable conditions with high unmet clinical need. Most progress in this field to date has been achieved with cell-based regenerative medicine therapies, with over a thousand clinical trials performed up to 2015. However, lack of adequate safety and efficacy data is currently limiting wider uptake of these therapies. To facilitate clinical translation, non-invasive in vivo imaging technologies that enable careful evaluation and characterisation of the administered cells and their effects on host tissues are critically required to evaluate their safety and efficacy in relevant preclinical models. This article reviews the most common imaging technologies available and how they can be applied to regenerative medicine research. We cover details of how each technology works, which cell labels are most appropriate for different applications, and the value of multi-modal imaging approaches to gain a comprehensive understanding of the responses to cell therapy in vivo.

• Siller, Richard; Naumovska, Elena; Mathapati, Santosh Sadashiv; Lycke, Max; Greenhough, Sebastian & Sullivan, Gareth (2016). Development of a rapid screen for the endodermal differentiation potential of human pluripotent stem cell lines. Scientific Reports. 6. doi: 10.1038/srep37178. Fulltekst i vitenarkiv Vis sammendrag

  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).

• XU, m; Stattin, EL; Shaw, G; Heinegård, Dick; Sullivan, Gareth & Wilmut, Ian [Vis alle 14 forfattere av denne artikkelen] (2016). Chondrocytes derived from mesenchymal stromal cells and induced pluripotent cells of patients with familial osteochondritis dissecans exhibit an ER stress response and defective matrix assembly. Stem Cells Translational Medicine. ISSN 2157-6564. 5(9), s. 1171–1181. doi: 10.5966/sctm.2015-0384. Fulltekst i vitenarkiv
• Mathapati, Santosh Sadashiv; Siller, Richard; Impellizzeri, Agata Antonina Rita; Lycke, Eric Maximilian; Vegheim, Karianne & Almaas, Runar [Vis alle 7 forfattere av denne artikkelen] (2016). Small-molecule-directed hepatocyte-like cell differentiation of human pluripotent stem cells. Current protocols in stem cell biology. ISSN 1941-7322. 38, s. 1G.6.1–1G.6.18. doi: 10.1002/cpsc.13. Fulltekst i vitenarkiv
• Siller, Richard; Greenhough, Sebastian; Naumovska, Elena & Sullivan, Gareth (2015). Small-molecule-driven hepatocyte differentiation of human pluripotent stem cells. Stem Cell Reports. ISSN 2213-6711. 4(5), s. 939–952. doi: 10.1016/j.stemcr.2015.04.001. Fulltekst i vitenarkiv Vis sammendrag

  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.

• Siller, Richard; Greenhough, Sebastian; Park, IH & Sullivan, Gareth (2013). Modelling Human Disease with Pluripotent Stem Cells. Current Gene Therapy. ISSN 1566-5232. 13(2), s. 99–110. doi: 10.2174/1566523211313020004. Fulltekst i vitenarkiv
• Dajani, Rana; Koo, Sung-Eun; Sullivan, Gareth & Park, In-Hyun (2013). Investigation of Rett Syndrome Using Pluripotent Stem Cells. Journal of Cellular Biochemistry. ISSN 0730-2312. 114(11), s. 2446–2453. doi: 10.1002/jcb.24597. Fulltekst i vitenarkiv
• Kim, Kun-Yong; Jung, Yong Wook; Sullivan, Gareth; Chung, Leeyup & Park, In-Hyun (2012). Cellular reprogramming: a novel tool for investigating autism spectrum disorders. Trends in Molecular Medicine. ISSN 1471-4914. 18(8), s. 463–471. doi: 10.1016/j.molmed.2012.06.002. Fulltekst i vitenarkiv
• Zhou, Xiao-Ling; Sullivan, Gareth; Sun, Pingnan & Park, In-Hyung (2012). Humanized Murine Model for HBV and HCV Using Human Induced Pluripotent Stem Cells. Archives of pharmacal research. ISSN 0253-6269. 35(2), s. 261–269. doi: 10.1007/s12272-012-0206-8. Fulltekst i vitenarkiv

Se alle arbeider i NVA

• Dugarte, Maria Eugenia Chollet; Bhattacharya, Anindita; Roman, Giacomo; Harrison, Sean Philip; Baumgarten, Saphira Felicitas & Andersen, Elisabeth [Vis alle 12 forfattere av denne artikkelen] (2025). Autologous gene-corrected hepatic organoids for the treatment of FVII deficiency. Research and Practice in Thrombosis and Haemostasis (RPTH). 9(S2), s. 265–266. doi: 10.1016/j.rpth.2025.102925. Fulltekst i vitenarkiv
• Stavik, Benedicte; Bhattacharya, Anindita; Lauritzen, Knut Husø; Baumgarten, Saphira Felicitas; barrio, maria eugenia & corral, javier [Vis alle 10 forfattere av denne artikkelen] (2025). Human serum enhances the expression and production of antithrombin by hepatic organoids. Research and Practice in Thrombosis and Haemostasis (RPTH). 9(S2), s. 405–406. doi: 10.1016/j.rpth.2025.102923. Fulltekst i vitenarkiv
• Roman, Giacomo; Lauritzen, Knut Husø; Harrison, Sean Philip; Bhattacharya, Anindita; Mowinckel, Marie-Christine & Andresen, Marianne Seierstad [Vis alle 14 forfattere av denne artikkelen] (2025). Gene editing in patient-derived iPSCs restores coagulation FVII deficiency in 3D liver organoids. Research and Practice in Thrombosis and Haemostasis (RPTH). 9(S2), s. 950–951. doi: 10.1016/j.rpth.2025.102923. Fulltekst i vitenarkiv
• Bhattacharya, Anindita; Roman, Giacomo; Harrison, Sean Philip; Sullivan, Gareth John; Mowinckel, Marie-Christine & Sandset, Per Morten [Vis alle 10 forfattere av denne artikkelen] (2024). Developing cell-based genome editing approach for antithrombin deficiency: a therapeutic prospect. Research and Practice in Thrombosis and Haemostasis (RPTH). doi: 10.1016/j.rpth.2024.102504. Fulltekst i vitenarkiv
• Dugarte, Maria Eugenia Chollet; Bhattacharya, Anindita; Roman, Giacomo; Harrison, Sean; Baumgarten, Saphira Felicitas & Andersen, Elisabeth [Vis alle 12 forfattere av denne artikkelen] (2024). Autologous gene-corrected stem cell-derived hepatic organoids for the treatment of FVII deficiency. HemaSphere. doi: 10.1002/hem3.104. Fulltekst i vitenarkiv
• Dugarte, Maria Eugenia Chollet; Andersen, Elisabeth; Harrison, Sean Philip; Morena-Barrio, Maria Eugenia de la; Baumgarten, Saphira Felicitas & Bhattacharya, Anindita [Vis alle 14 forfattere av denne artikkelen] (2023). OC 07.5 Modeling and Correcting FVII Deficiency using Human Stem Cell-Derived Hepatic Organoids. Research and Practice in Thrombosis and Haemostasis (RPTH). 7(Oral communications ISTH 2023). doi: 10.1016/j.rpth.2023.100470. Fulltekst i vitenarkiv
• Roman, Giacomo; Lauritzen, Knut Husø; Harrison, Sean Philip; Sullivan, Gareth John; Iversen, Nina & Henriksson, Carola Elisabeth [Vis alle 10 forfattere av denne artikkelen] (2023). OC 07.1 Autologous CRISPR/Cas9 Correction of a Missense Variant from a Patient with Severe, Inherited FVII Deficiency. Research and Practice in Thrombosis and Haemostasis (RPTH). 7(Oral communications ISTH 2023). doi: 10.1016/j.rpth.2023.100475. Fulltekst i vitenarkiv
• Kogler, Stian; Aizenshtadt, Aleksandra; Harrison, Sean Philip; Sullivan, Gareth John; Lundanes, Elsa & Bogen, Inger Lise [Vis alle 9 forfattere av denne artikkelen] (2023). Organ-in-a-column – automated on-line incubation, sampling, and LC-MS analysis for measurement of drug metabolites. Fulltekst i vitenarkiv
• Roman, Giacomo; Lauritzen, Knut Husø; Harrison, Sean; Sullivan, Gareth John; Iversen, Nina & Henriksson, Carola Elisabeth [Vis alle 10 forfattere av denne artikkelen] (2023). Autologous CRISPR/Cas9 correction of a missense variant from a patient with severe, inherited FVII deficiency. Fulltekst i vitenarkiv
• Roman, Giacomo; Lauritzen, Knut Husø; Harrison, Sean Philip; Sullivan, Gareth John; Iversen, Nina & Henriksson, Carola Elisabeth [Vis alle 10 forfattere av denne artikkelen] (2023). OC 07.1 Autologous CRISPR/Cas9 Correction of a Missense Variant from a Patient with Severe, Inherited FVII Deficiency. Research and Practice in Thrombosis and Haemostasis (RPTH). doi: 10.1016/j.rpth.2023.100475. Fulltekst i vitenarkiv
• Dugarte, Maria Eugenia Chollet; Harrison, Sean; Baumgarten, Saphira Felicitas; Morena-Barrio, Maria Eugenia de la; Bravo-Pérez, Carlos & Mowinckel, Marie-Christine [Vis alle 12 forfattere av denne artikkelen] (2022). Human iPSC-derived hepatic organoids – a model for blood coagulation research and therapy. Human Gene Therapy. ISSN 1043-0342. doi: 10.1089/hum.2022.29225.abstracts. Fulltekst i vitenarkiv
• Dugarte, Maria Eugenia Chollet; andersen, elisabeth; Morena-Barrio, Maria Eugenia de la; Bravo-Perez, Carlos; Mowinckel, Marie-Christine & Myklebust, Christiane Fillion [Vis alle 12 forfattere av denne artikkelen] (2020). Characterizing Coagulation FVII from iPSC-Hepatocytes-like Cells: Setting the Basis for Cell Therapy Development. Blood. ISSN 0006-4971. 136. doi: 10.1182/blood-2020-139211. Fulltekst i vitenarkiv
• Skottvoll, Frøydis Sved; Harrison, Sean; Hansen, Frederik; Pedersen-Bjergaard, Stig; Sullivan, Gareth & Bogen, Inger Lise [Vis alle 8 forfattere av denne artikkelen] (2020). Liver mini-organ analyses using liquid chromatography mass spectrometry. Fulltekst i vitenarkiv Vis sammendrag

  Organoids (self-organized multi-cellular tissue derived from e.g. patient-derived stem cells) are emerging in vitro 3D representations of human organ functionality, predicted to have a game-changing impact on drug discovery, disease modeling and personalized medicine [1]. Integrating the organoids in a microfluidic chip (i.e. organoids-on-a-chip) would allow for controllable perfusion of nutrients and manipulation of the organoids by the addition of e.g. drugs [2]. Coupling the organoid-on-a-chip online to liquid chromatography mass spectrometry (LC-MS) would enable real-time measurements of the biochemical processes that occur on-chip. However, limited sample sizes, complex matrices and the presence of low abundant analytes also create analytical challenges [3]. Moreover, the organoids are still at the developmental stage, thus fundamental questions on the functionality of the developed organoids remain to be answered. Liver organoid functionality was assessed by protein characterization of adult liver tissue and liver organoids using nanoLC-MS/MS. The proteins identified from the liver organoids covered 70-84% (1003 proteins) of the liver protein expression from adult liver tissue derived from five patients. The proteins that were shared between the adult liver tissue and liver organoids could also relate to central liver pathways (e.g. amino acid biosynthesis and glycolysis). The sample preparation technique electromembrane extraction (i.e. electrophoretic separation across an oil membrane, EME) has shown to have on-chip analytical potential [4] and was thus used in the study of heroin drug metabolism in liver organoids. Multi-well EME (100 µL solutions) allowed for simple and repeatable monitoring of heroin metabolism kinetics without interference from cell medium components (e.g. albumin). The EME extracts were also compatible with different analytical approaches (LC-MS, nanoLC-MS, capillary electrophoresis-UV). Hence, the hyphenation of EME with nanoLC-MS would be a natural next step. References 1. Method of the Year 2017: Organoids. Nature Methods. Vol. 15 (2018) 1-1. 2. Park, S.E., A. Georgescu, and D. Huh, Organoids-on-a-chip. Science. Vol. 364 (2019) 960. 3. Lin, A., F. Sved Skottvoll, S. Rayner, S. Pedersen-Bjergaard, G. Sullivan, S. Krauss, S. Ray Wilson, and S. Harrison, 3D cell culture models and organ-on-a-chip: Meet separation science and mass spectrometry. ELECTROPHORESIS. Vol. 0 (2019). 4. Hansen, F.A., D. Sticker, J.P. Kutter, N.J. Petersen, and S. Pedersen-Bjergaard, Nanoliter-Scale Electromembrane Extraction and Enrichment in a Microfluidic Chip. Analytical Chemistry. Vol. 90 (2018) 9322-9329.

• Mrša, Ago; Skottvoll, Frøydis Sved; Harrison, Sean; Lundanes, Elsa; Sullivan, Gareth & Wilson, Steven Ray Haakon (2020). Miniaturized liquid chromatography systems for small molecule analysis of organoids. Fulltekst i vitenarkiv Vis sammendrag

  Liver organoids are three-dimensional tissue models (typically in the millimetre scale or below) derived from human-induced pluripotent stem cells [1]. They are intended to e.g. represent the physiological functions of a patient’s liver. The liver is the main metabolizing organ in the human body [2]; thus, an important application of liver organoids is to map drug metabolism in vitro, as liver organoids can be more patient-specific compared to traditional biomaterials. The aim of the study is to develop miniaturized liquid chromatography (LC) systems capable of analyzing very small samples, e.g secretion of liver organoids. Emphasis is placed on the determination of small molecules, i.e metabolites. Due to the small size of organoids, there are significant challenges in performing sensitive analysis of the organoid itself and it's minute secretion. Hence a nanoLC system has been explored, capable of sensitive analysis of model substance heroin and its well-known metabolites morphine and 6-Monoacetylmorphine (6-MAM). Using a 50 µm x 5 cm C18 pre-column and a 50 µm x 12 cm C18 analytical column, coupled up with a triple quadrupole mass spectrometer (TQMS) with electrospray ionization, a limit of detection of 0.7 fg was achieved for heroin. Further method development was done using commercially packed C18 columns typically used in proteomics. Separation and detection of heroin, morphine and 6-MaM were achieved in less than 8 minutes and with detection limits in the sub fg area. The method was successfully transferred to liver organoid samples that had been incubated with 1.5 µM heroin for 1 hour, cleaned using electromembrane extraction and further diluted 1:1000. Deuterated internal standards were implemented into the method for future work with organoid samples and the next step is to map the metabolism of liver organoids incubated within various time frames. Taken together, nanoLC-MS proves to be a viable tool for analysis of small molecules and the achievd results are promising for further work with organoid samples.

• Boger, Ida Caroline Sneis; Nerem, Elisabeth; Skottvoll, Frøydis Sved; Harrison, Sean; Sullivan, Gareth & Lundanes, Elsa [Vis alle 8 forfattere av denne artikkelen] (2020). Determination of liver organoid-induced drug metabolites using liquid chromatography-mass spectrometry (LC-MS)”. Fulltekst i vitenarkiv Vis sammendrag

  Liver organoids are three-dimensional tissue models typically derived from adult and human induced pluripotent stem cells. They are intended to e.g. represent the physiological functions of a patient´s liver [1]. The liver is the main metabolizing organ in the human body [2]; thus, an important application of liver organoids is to map drug metabolism in vitro as liver organoids can be more patient-specific compared to traditional biomaterials, e.g. human liver microsomes (HLM). The aim of this study is to explore liver organoid drug metabolism in vitro using liquid chromatography-mass spectrometry (LC-MS). To establish a standardized conventional approach for metabolism studies for later comparison with organoids, heroin metabolism studies in HLMs and S9 fraction were carried out. Quantification of model substance heroin and its well-known metabolites, 6-Monoacetylmorphine (6-MAM) and morphine, was done using UHPLC-MS/MS. The heroin metabolism method was miniaturized to make it transferable to the small organoid samples. This was done by decreasing the microsome amount as much as possible while still observing heroin metabolism (HLM amount decreased from 0.2 mg to 0.01 mg; Heroin concentration was decreased from 10 µM to 0.1 µM). This miniaturized experiment was also transferred to S9 fraction Preliminary experiments with organoids showed that the organoids metabolize heroin to 6-MAM and morphine, as well as phase II biotransformation metabolites morphine-3-glucuronide and morphine-6-glucuronide. These results show that the studied organoids have metabolizing properties. Taken together, LC-MS can be a valuable tool for studying metabolism properties of organoids.

• Skottvoll, Frøydis Sved; Boger, Ida Caroline Sneis; Harrison, Sean; Nerem, Elisabeth; Bogen, Inger Lise & Sullivan, Gareth [Vis alle 7 forfattere av denne artikkelen] (2019). LC-MS approaches to liver organoid analyses. Fulltekst i vitenarkiv Vis sammendrag

  Liquid chromatography coupled to mass spectrometry (LC-MS) is an analytical tool used to separate and measure the mass (m/z) and concentration of compounds in matrices like water, blood, and tumors. Drugs, metabolites and proteins are all measurable with LC-MS. Unfortunately, the sample is diluted during chromatography, hurting the sensitivity. Thus, the limited sample volumes and low abundant compounds of organ-on-a-chip and organoids demands miniaturization of LC-MS to enable measurements of high sensitivity. By narrowing the inner diameter (ID) of the separation column, the sample dilution is vastly reduced, adding to sensitivity, e.g. enabling analysis of limited sample volumes and low abundant compounds of the organ-on-a-chip and organoids. The bioanalytical group at the Department of Chemistry (UiO) is currently developing miniaturized systems for proteins and small molecules for assessing the properties of liver organoids. These efforts will be expanded to testing islet and adipose organoids, and will also be incorporated in to on-line systems, e.g. organ on a chip systems. We believe that LC-MS and organoid analysis will be an increasingly natural partnering.

• Mrša, Ago; Boger, Ida Caroline Sneis; Nerem, Elisabeth; Skottvoll, Frøydis Sved; Harrison, Sean & Lundanes, Elsa [Vis alle 9 forfattere av denne artikkelen] (2019). Studying organoid drug metabolism using UHPLC/nanoLC-MS. Fulltekst i vitenarkiv Vis sammendrag

  Liver organoids are three-dimensional tissue models typically derived from adult and human induced pluripotent stem cells. They are intended to e.g. represent the physiological functions of a patient´s liver [1]. The liver is the main metabolizing organ in the human body [2]; thus, an important application of liver organoids is to map drug metabolism in vitro as liver organoids can be more patient-specific compared to traditional biomaterials, e.g. human liver microsomes (HLM). The aim of this study is to explore liver organoid drug metabolism in vitro using liquid chromatography-mass spectrometry (LC-MS). To establish a standardized conventional approach for metabolism studies for later comparison with organoids, heroin metabolism studies in HLMs were carried out. Quantification of model substance heroin and its well-known metabolites, 6-Monoacetylmorphine (6-MAM) and morphine, was done using UHPLC-MS/MS. The heroin metabolism method was miniaturized to make it transferable to the small organoid samples. This was done by decreasing the microsome amount as much as possible while still observing heroin metabolism (HLM amount decreased from 0.2 mg to 0.01 mg; Heroin concentration was decreased from 10 µM to 0.1 µM). Preliminary experiments with organoids showed that the organoids metabolize heroin to 6-MAM and morphine, as well as phase II biotransformation metabolites morphine-3-glucuronide and morphine-6-glucuronide. These results show that the studied organoids have metabolizing properties. We are also exploring sensitive nano-LC-MS (traditionally used for proteomics) for studying organoid drug metabolism. Using a 50 µm (I.D) x 5 cm C18 pre-column and a 50 µm (I.D) x 12 cm C18 analytical column, coupled up with a triple quadrupole MS (QMS) using electrospray ionization (ESI), a LoD of 0.7 fg heroin was achieved. The LoD of the nano-LC-MS method gives a good foundation for future work with detection of trace liver-organoid induced drug metabolites. Taken together, LC-MS can be a valuable tool for studying metabolism properties of organoids. [1] Eisenstein, M., Organoids: the body builders, Nature Methods, (2018) 19. [2] Brandon, E. F., Raap, C. D., Meijerman, I., Beijnen, J. H., Schellens, J. H., An update on in vitro test methods in human hepatic drug biotransformation research: pros and cons, Toxicology and applied pharmacology, (2003) 233-246

• Dugarte, Maria Eugenia Chollet; Andersen, Elisabeth; Skarpen, Ellen; Mowinckel, Marie-Christine; Stavik, Benedicte & Sullivan, Gareth [Vis alle 7 forfattere av denne artikkelen] (2019). FVII: A human pluripotent stem cell derived hepatocyte model with potential therapeutic applications. Research and Practice in Thrombosis and Haemostasis (RPTH). 3. Fulltekst i vitenarkiv
• Skottvoll, Frøydis Sved; Nilsen, Ola; Krauss, Stefan; Sullivan, Gareth & Wilson, Steven Ray Haakon (2018). Integrating highly miniaturized LC-MS systems with “Organ on a Chip”; the future of preclinical modelling. Fulltekst i vitenarkiv
• Impellizzeri, Agata Antonina Rita; Szabo, Attila; Fjerdingstad, Hege Brincker; Sullivan, Gareth; Glover, Joel & Andreassen, Ole Andreas [Vis alle 7 forfattere av denne artikkelen] (2017). Using Stem Cells to Understand the Underlying Mechanisms of Polygenic Risk of Severe Mental Disorders. Fulltekst i vitenarkiv

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