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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.

• Liang, Xiao; Chen, Anbin; Kianian, Atefeh; Kristiansen, Cecilie Katrin; Tsering Yangzom, Tsering Yangzom & Furriol Palmer, Jessica [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. ISSN 1449-2288. 20(8), s. 2860–2880. doi: 10.7150/ijbs.93445.
• Harrison, Sean Philip; Baumgarten, Saphira Felicitas; Chollet Dugarte, Maria Eugenia; 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. doi: 10.1002/jpn3.12195.
• 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. ISSN 2666-5344. 14. doi: 10.1016/j.bbiosy.2024.100093.
• 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. ISSN 2198-3844. doi: 10.1002/advs.202307136.
• 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. ISSN 1449-2288. 20(4), s. 1194–1217. doi: 10.7150/ijbs.91624.
• 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.
• 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
• Harrison, Sean Philip; Siller, Richard; Tanaka, Yoshiaki; Chollet Dugarte, Maria Eugenia; de la Morena-Barrio, María Eugenia & 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
• 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
• 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
• 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. ISSN 2045-2322. 13(1). doi: 10.1038/s41598-023-38015-x. Fulltekst i vitenarkiv
• Hong, Yu; Kristiansen, Cecilie Katrin; Chen, Anbin; Sanchez Nido, Gonzalo; 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
• 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. ISSN 1664-042X. 14. doi: 10.3389/fphys.2023.1094249. Fulltekst i vitenarkiv
• 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
• 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. ISSN 1663-9812. 13. doi: 10.3389/fphar.2022.835825. Fulltekst i vitenarkiv
• 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
• 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.

• 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
• 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. ISSN 2296-634X. 9, s. 1–12. doi: 10.3389/fcell.2021.744777. Fulltekst i vitenarkiv
• 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. ISSN 2296-634X. 9, s. 1–20. doi: 10.3389/fcell.2021.737304. Fulltekst i vitenarkiv
• 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.

• 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.

• Harrison, Sean; Baumgarten, Saphira Felicitas; Verma, Rajneesh; Lunov, Oleg; Dejneka, Alexandr & Sullivan, Gareth (2021). Liver Organoids: Recent Developments, Limitations and Potential. Frontiers in medicine. ISSN 2296-858X. 8. doi: 10.3389/fmed.2021.574047. Fulltekst i vitenarkiv
• 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. ISSN 2666-3791. 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.

• 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 2692-8205. doi: 10.1101/2020.12.20.423652.
• 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
• Kim, Jonghun; Sullivan, Gareth & Park, In-Hyun (2021). How well do brain organoids capture your brain? iScience. ISSN 2589-0042. 24(2), s. 1–14. doi: 10.1016/j.isci.2021.102063. Fulltekst i vitenarkiv
• 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 2692-8205. doi: 10.1101/2020.12.02.406835.
• 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.
• 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 2692-8205. doi: 10.1101/2020.09.08.282756.
• 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. ISSN 2045-2322. 10, s. 1–14. doi: 10.1038/s41598-020-73656-2. Fulltekst i vitenarkiv
• 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.

• 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.

• 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. ISSN 2211-1247. 30(6), s. 1682–1689. doi: 10.1016/j.celrep.2020.01.038. 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
• 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. ISSN 2045-2322. 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.

• 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. ISSN 1664-042X. 9. doi: 10.3389/fphys.2018.01635. Fulltekst i vitenarkiv
• 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. ISSN 2041-1723. 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.

• 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. ISSN 2045-2322. 8(1). doi: 10.1038/s41598-018-24421-z. Fulltekst i vitenarkiv
• 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. ISSN 2167-485X. 5(3), s. 301–314. doi: 10.1007/s40139-017-0150-x.
• 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. Nature partner journals regenerative medicine. ISSN 2057-3995. 2(28). doi: 10.1038/s41536-017-0029-9. 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.

• 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.
• 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). ISSN 2047-9980. 6(9), s. 1–24. doi: 10.1161/JAHA.117.006339.
• 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.
• 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. ISSN 2045-2322. 7:37541, s. 1–16. doi: 10.1038/srep37541.
• 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.
• 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.
• 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. ISSN 2045-2322. 6. doi: 10.1038/srep37178. 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
• 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.
• 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.
• 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.
• 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.

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• Chollet Dugarte, Maria Eugenia; Andersen, Elisabeth; Harrison, Sean Philip; de la Morena-Barrio, Maria Eugenia; 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). ISSN 2475-0379. 7(Oral communications ISTH 2023). doi: 10.1016/j.rpth.2023.100470.
• 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). ISSN 2475-0379. 7(Oral communications ISTH 2023). doi: 10.1016/j.rpth.2023.100475.
• Lauritzen, Knut Husø; Sullivan, Gareth John; Harrison, Sean Philip; Iversen, Nina; Henriksson, Carola Elisabeth & Glosli, Heidi [Vis alle 9 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). ISSN 2475-0379. doi: 10.1016/j.rpth.2023.100475.
• 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.
• Dugarte, Maria Eugenia Chollet; Harrison, Sean; Baumgarten, Saphira Felicitas; de la Morena-Barrio, Maria Eugenia; 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.
• Dugarte, Maria Eugenia Chollet; andersen, elisabeth; de la Morena-Barrio, Maria Eugenia; 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.
• 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. 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.

• 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)”. 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.

• 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. 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.

• 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). ISSN 2475-0379. 3.
• 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. 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

• 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. 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.

• 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 .
• 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.

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