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4. Locational memory of macrovessel vascular cells is transcriptionally imprinted

Author

Spanjersberg, Talitha C F, Oosterhoff, Loes A, Kruitwagen, Hedwig S, van den Dungen, Noortje A M, Vernooij, Johannes C M, Asselbergs, Folkert W, Mokry, Michal, Spee, Bart, Harakalova, Magdalena, van Steenbeek, Frank G, Spanjersberg, Talitha C F, Oosterhoff, Loes A, Kruitwagen, Hedwig S, van den Dungen, Noortje A M, Vernooij, Johannes C M, Asselbergs, Folkert W, Mokry, Michal, Spee, Bart, Harakalova, Magdalena, and van Steenbeek, Frank G

Abstract

Vascular pathologies show locational predisposition throughout the body; further insights into the transcriptomics basis of this vascular heterogeneity are needed. We analyzed transcriptomes from cultured endothelial cells and vascular smooth muscle cells from nine adult canine macrovessels: the aorta, coronary artery, vena cava, portal vein, femoral artery, femoral vein, saphenous vein, pulmonary vein, and pulmonary artery. We observed that organ-specific expression patterns persist in vitro, indicating that these genes are not regulated by blood flow or surrounding cell types but are likely fixed in the epigenetic memory. We further demonstrated the preserved location-specific expression of GATA4 protein in cultured cells and in the primary adult vessel. On a functional level, arterial and venous endothelial cells differed in vascular network morphology as the arterial networks maintained a higher complexity. Our findings prompt the rethinking of the extrapolation of results from single-origin endothelial cell systems.

Published
2023

5. Locational memory of macrovessel vascular cells is transcriptionally imprinted

Author

Internal Medicine of Companion Animals, Interne geneeskunde GD, FAH Evidence based Veterinary Medicine, CS_STEAM, CS_Genetics, Genetics, STEAM, Welfare & Emerging Diseases, Spanjersberg, Talitha C F, Oosterhoff, Loes A, Kruitwagen, Hedwig S, van den Dungen, Noortje A M, Vernooij, Johannes C M, Asselbergs, Folkert W, Mokry, Michal, Spee, Bart, Harakalova, Magdalena, van Steenbeek, Frank G, Internal Medicine of Companion Animals, Interne geneeskunde GD, FAH Evidence based Veterinary Medicine, CS_STEAM, CS_Genetics, Genetics, STEAM, Welfare & Emerging Diseases, Spanjersberg, Talitha C F, Oosterhoff, Loes A, Kruitwagen, Hedwig S, van den Dungen, Noortje A M, Vernooij, Johannes C M, Asselbergs, Folkert W, Mokry, Michal, Spee, Bart, Harakalova, Magdalena, and van Steenbeek, Frank G

Published
2023

6. Exploring intrinsic variability between cultured nasal and bronchial epithelia in cystic fibrosis

Author

Longziekten onderzoek 2, Regenerative Medicine and Stem Cells, CTI, Longziekten patientenzorg, Child Health, Speerpunt Child Health, Infection & Immunity, Onderzoek, Rodenburg, Lisa W, Metzemaekers, Mieke, van der Windt, Isabelle S, Smits, Shannon M A, den Hertog-Oosterhoff, Loes A, Kruisselbrink, Evelien, Brunsveld, Jesse E, Michel, Sabine, de Winter-de Groot, Karin M, van der Ent, Cornelis K, Stadhouders, Ralph, Beekman, Jeffrey M, Amatngalim, Gimano D, Longziekten onderzoek 2, Regenerative Medicine and Stem Cells, CTI, Longziekten patientenzorg, Child Health, Speerpunt Child Health, Infection & Immunity, Onderzoek, Rodenburg, Lisa W, Metzemaekers, Mieke, van der Windt, Isabelle S, Smits, Shannon M A, den Hertog-Oosterhoff, Loes A, Kruisselbrink, Evelien, Brunsveld, Jesse E, Michel, Sabine, de Winter-de Groot, Karin M, van der Ent, Cornelis K, Stadhouders, Ralph, Beekman, Jeffrey M, and Amatngalim, Gimano D

Published
2023

7. Locational memory of macrovessel vascular cells is transcriptionally imprinted

Author

Onderzoek Precision medicine, CDL Research analisten, Cancer, CDL Onderzoek Pasterkamp, Experimentele Afd. Cardiologie 1, Child Health, Circulatory Health, Spanjersberg, Talitha C F, Oosterhoff, Loes A, Kruitwagen, Hedwig S, van den Dungen, Noortje A M, Vernooij, Johannes C M, Asselbergs, Folkert W, Mokry, Michal, Spee, Bart, Harakalova, Magdalena, van Steenbeek, Frank G, Onderzoek Precision medicine, CDL Research analisten, Cancer, CDL Onderzoek Pasterkamp, Experimentele Afd. Cardiologie 1, Child Health, Circulatory Health, Spanjersberg, Talitha C F, Oosterhoff, Loes A, Kruitwagen, Hedwig S, van den Dungen, Noortje A M, Vernooij, Johannes C M, Asselbergs, Folkert W, Mokry, Michal, Spee, Bart, Harakalova, Magdalena, and van Steenbeek, Frank G

Published
2023

8. Protocol for generating airway organoids from 2D air liquid interface-differentiated nasal epithelia for use in a functional CFTR assay

Author

Longziekten onderzoek 2, Cancer, Onderzoek, Child Health, Regenerative Medicine and Stem Cells, Rodenburg, Lisa W, van der Windt, Isabelle S, Dreyer, Henriette H M, Smits, Shannon M A, den Hertog-Oosterhoff, Loes A, Aarts, Ellen M, Beekman, Jeffrey M, Amatngalim, Gimano D, Longziekten onderzoek 2, Cancer, Onderzoek, Child Health, Regenerative Medicine and Stem Cells, Rodenburg, Lisa W, van der Windt, Isabelle S, Dreyer, Henriette H M, Smits, Shannon M A, den Hertog-Oosterhoff, Loes A, Aarts, Ellen M, Beekman, Jeffrey M, and Amatngalim, Gimano D

Published
2023

9. High level of polarized engraftment of porcine intrahepatic cholangiocyte organoids in decellularized liver scaffolds

Author

Krüger, Melanie, primary, Samsom, Roos‐Anne, additional, Oosterhoff, Loes A., additional, van Wolferen, Monique E., additional, Kooistra, Hans S., additional, Geijsen, Niels, additional, Penning, Louis C., additional, Kock, Linda M., additional, Sainz‐Arnal, Pilar, additional, Baptista, Pedro M., additional, and Spee, Bart, additional

Published
2022
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10. High level of polarized engraftment of porcine intrahepatic cholangiocyte organoids in decellularized liver scaffolds

Author

Krüger, Melanie, Samsom, Roos-Anne, Oosterhoff, Loes, van Wolferen, Monique, Kooistra, Hans, Geijsen, Niels, Penning, Louis, Kock, Linda M, Sainz-Arnal, Pilar, Baptista, Pedro Miguel, Spee, Bart, Krüger, Melanie, Samsom, Roos-Anne, Oosterhoff, Loes, van Wolferen, Monique, Kooistra, Hans, Geijsen, Niels, Penning, Louis, Kock, Linda M, Sainz-Arnal, Pilar, Baptista, Pedro Miguel, and Spee, Bart

Abstract

In Europe alone, each year 5500 people require a life-saving liver transplantation, but 18% die before receiving one due to the shortage of donor organs. Whole organ engineering, utilizing decellularized liver scaffolds repopulated with autologous cells, is an attractive alternative to increase the pool of available organs for transplantation. The development of this technology is hampered by a lack of a suitable large-animal model representative of the human physiology and a reliable and continuous cell source. We have generated porcine intrahepatic cholangiocyte organoids from adult stem cells and demonstrate that these cultures remained stable over multiple passages whilst retaining the ability to differentiate into hepatocyte- and cholangiocyte-like cells. Recellularization onto porcine scaffolds was efficient and the organoids homogeneously differentiated, even showing polarization. Our porcine intrahepatic cholangiocyte system, combined with porcine liver scaffold paves the way for developing whole liver engineering in a relevant large-animal model.

Published
2022

11. High level of polarized engraftment of porcine intrahepatic cholangiocyte organoids in decellularized liver scaffolds

Author

Interne geneeskunde GD, CS_Welfare & emerging diseases, CS_STEM, Krüger, Melanie, Samsom, Roos-Anne, Oosterhoff, Loes, van Wolferen, Monique, Kooistra, Hans, Geijsen, Niels, Penning, Louis, Kock, Linda M, Sainz-Arnal, Pilar, Baptista, Pedro Miguel, Spee, Bart, Interne geneeskunde GD, CS_Welfare & emerging diseases, CS_STEM, Krüger, Melanie, Samsom, Roos-Anne, Oosterhoff, Loes, van Wolferen, Monique, Kooistra, Hans, Geijsen, Niels, Penning, Louis, Kock, Linda M, Sainz-Arnal, Pilar, Baptista, Pedro Miguel, and Spee, Bart

Published
2022

12. Bioprinting of Human Liver-derived Epithelial Organoids for Toxicity Studies

Author

Bouwmeester, Manon C, Bernal, Paulina Nunez, Oosterhoff, Loes A, van Wolferen, Monique, Lehmann, Vivian, Vermaas, Monique, Buchholz, Maj-Britt, Peiffer, Quentin, Malda, Jos, van der Laan, Luc J W, Kramer, Nynke I, Schneeberger, Kerstin, Levato, Riccardo, Spee, Bart, Interne geneeskunde GD, dCSCA RMSC-1, Equine Musculoskeletal Biology, dES RMSC, dIRAS RA-1, CS_STEM, Interne geneeskunde GD, dCSCA RMSC-1, Equine Musculoskeletal Biology, dES RMSC, dIRAS RA-1, CS_STEM, and Surgery

Subjects
Polymers and Plastics, Cell, Cell Culture Techniques, in vitro modeling, Bioengineering, 02 engineering and technology, Toxicology, drug induced liver injury, Biomaterials, 03 medical and health sciences, SDG 3 - Good Health and Well-being, medicine, Organoid, Materials Chemistry, Humans, Viability assay, Cells, Cultured, Toxicologie, organoids, 030304 developmental biology, Liver injury, 0303 health sciences, Human liver, Tissue Engineering, Chemistry, Bioprinting, 021001 nanoscience & nanotechnology, medicine.disease, In vitro, Cell biology, medicine.anatomical_structure, Liver, Toxicity, Printing, Three-Dimensional, extrusion-based bioprinting, 0210 nano-technology, Biofabrication, Biotechnology
Abstract

There is a need for long-lived hepatic in vitro models to better predict drug induced liver injury (DILI). Human liver-derived epithelial organoids are a promising cell source for advanced in vitro models. Here, organoid technology is combined with biofabrication techniques, which holds great potential for the design of in vitro models with complex and customizable architectures. Here, porous constructs with human hepatocyte-like cells derived from organoids are generated using extrusion-based printing technology. Cell viability of bioprinted organoids remains stable for up to ten days (88–107% cell viability compared to the day of printing). The expression of hepatic markers, transporters, and phase I enzymes increased compared to undifferentiated controls, and is comparable to non-printed controls. Exposure to acetaminophen, a well-known hepatotoxic compound, decreases cell viability of bioprinted liver organoids to 21–51% (p < 0.05) compared to the start of exposure, and elevated levels of damage marker miR-122 are observed in the culture medium, indicating the potential use of the bioprinted constructs for toxicity testing. In conclusion, human liver-derived epithelial organoids can be combined with a biofabrication approach, thereby paving the way to create perfusable, complex constructs which can be used as toxicology- and disease-models.

Published
2021

13. Bioprinting of Human Liver-derived Epithelial Organoids for Toxicity Studies

Author

Interne geneeskunde GD, dCSCA RMSC-1, Equine Musculoskeletal Biology, dES RMSC, dIRAS RA-1, CS_STEM, Bouwmeester, Manon C, Bernal, Paulina Nunez, Oosterhoff, Loes A, van Wolferen, Monique, Lehmann, Vivian, Vermaas, Monique, Buchholz, Maj-Britt, Peiffer, Quentin, Malda, Jos, van der Laan, Luc J W, Kramer, Nynke I, Schneeberger, Kerstin, Levato, Riccardo, Spee, Bart, Interne geneeskunde GD, dCSCA RMSC-1, Equine Musculoskeletal Biology, dES RMSC, dIRAS RA-1, CS_STEM, Bouwmeester, Manon C, Bernal, Paulina Nunez, Oosterhoff, Loes A, van Wolferen, Monique, Lehmann, Vivian, Vermaas, Monique, Buchholz, Maj-Britt, Peiffer, Quentin, Malda, Jos, van der Laan, Luc J W, Kramer, Nynke I, Schneeberger, Kerstin, Levato, Riccardo, and Spee, Bart

Published
2021

14. Bioprinting of Human Liver-Derived Epithelial Organoids for Toxicity Studies

Author

Bouwmeester, Manon C., Bernal, Paulina N., Oosterhoff, Loes A., van Wolferen, Monique E., Lehmann, Vivian, Vermaas, Monique, Buchholz, Maj Britt, Peiffer, Quentin C., Malda, Jos, van der Laan, Luc J.W., Kramer, Nynke I., Schneeberger, Kerstin, Levato, Riccardo, Spee, Bart, Bouwmeester, Manon C., Bernal, Paulina N., Oosterhoff, Loes A., van Wolferen, Monique E., Lehmann, Vivian, Vermaas, Monique, Buchholz, Maj Britt, Peiffer, Quentin C., Malda, Jos, van der Laan, Luc J.W., Kramer, Nynke I., Schneeberger, Kerstin, Levato, Riccardo, and Spee, Bart

Abstract

There is a need for long-lived hepatic in vitro models to better predict drug induced liver injury (DILI). Human liver-derived epithelial organoids are a promising cell source for advanced in vitro models. Here, organoid technology is combined with biofabrication techniques, which holds great potential for the design of in vitro models with complex and customizable architectures. Here, porous constructs with human hepatocyte-like cells derived from organoids are generated using extrusion-based printing technology. Cell viability of bioprinted organoids remains stable for up to ten days (88–107% cell viability compared to the day of printing). The expression of hepatic markers, transporters, and phase I enzymes increased compared to undifferentiated controls, and is comparable to non-printed controls. Exposure to acetaminophen, a well-known hepatotoxic compound, decreases cell viability of bioprinted liver organoids to 21–51% (p < 0.05) compared to the start of exposure, and elevated levels of damage marker miR-122 are observed in the culture medium, indicating the potential use of the bioprinted constructs for toxicity testing. In conclusion, human liver-derived epithelial organoids can be combined with a biofabrication approach, thereby paving the way to create perfusable, complex constructs which can be used as toxicology- and disease-models.

Published
2021

15. Bioprinting of Human Liver-Derived Epithelial Organoids for Toxicity Studies

Author

Orthopaedie Onderzoek, Metabole ziekten onderzoek 2, Regenerative Medicine and Stem Cells, MDL onderzoek 2, Other research (not in main researchprogram), Bouwmeester, Manon C., Bernal, Paulina N., Oosterhoff, Loes A., van Wolferen, Monique E., Lehmann, Vivian, Vermaas, Monique, Buchholz, Maj Britt, Peiffer, Quentin C., Malda, Jos, van der Laan, Luc J.W., Kramer, Nynke I., Schneeberger, Kerstin, Levato, Riccardo, Spee, Bart, Orthopaedie Onderzoek, Metabole ziekten onderzoek 2, Regenerative Medicine and Stem Cells, MDL onderzoek 2, Other research (not in main researchprogram), Bouwmeester, Manon C., Bernal, Paulina N., Oosterhoff, Loes A., van Wolferen, Monique E., Lehmann, Vivian, Vermaas, Monique, Buchholz, Maj Britt, Peiffer, Quentin C., Malda, Jos, van der Laan, Luc J.W., Kramer, Nynke I., Schneeberger, Kerstin, Levato, Riccardo, and Spee, Bart

Published
2021

16. Cellulose Nanofibril Hydrogel Promotes Hepatic Differentiation of Human Liver Organoids

Author

Krüger, Melanie, Oosterhoff, Loes A, van Wolferen, Monique E, Schiele, Simon A, Walther, Andreas, Geijsen, Niels, De Laporte, Laura, van der Laan, Luc J W, Kock, Linda M, Spee, Bart, Interne geneeskunde GD, dCSCA RMSC-1, CS_STEM, Orthopaedic Biomechanics, Interne geneeskunde GD, dCSCA RMSC-1, CS_STEM, and Surgery

Subjects
Adult, Scaffold, Hydrogels/pharmacology, Cellular differentiation, Cell, Biomedical Engineering, Pharmaceutical Science, 02 engineering and technology, 010402 general chemistry, cellulose nanofibril hydrogels, 01 natural sciences, Biomaterials, Mice, Matrigel, Tissue engineering, medicine, Organoid, Humans, Animals, clinical‐grade scaffolds, Cellulose, liver organoid scaffolds, Basement membrane, clinical-grade scaffolds, liver organoids, engineered tissues, Chemistry, Hydrogels, Cell Differentiation, 021001 nanoscience & nanotechnology, 0104 chemical sciences, Cell biology, Transplantation, Organoids, medicine.anatomical_structure, Liver, 0210 nano-technology
Abstract

To replicate functional liver tissue in vitro for drug testing or transplantation, 3D tissue engineering requires representative cell models as well as scaffolds that not only promote tissue production but also are applicable in a clinical setting. Recently, adult liver-derived liver organoids are found to be of much interest due to their genetic stability, expansion potential, and ability to differentiate toward a hepatocyte-like fate. The current standard for culturing these organoids is a basement membrane hydrogel like Matrigel (MG), which is derived from murine tumor material and apart from its variability and high costs, possesses an undefined composition and is therefore not clinically applicable. Here, a cellulose nanofibril (CNF) hydrogel is investigated with regard to its potential to serve as an alternative clinical grade scaffold to differentiate liver organoids. The results show that its mechanical properties are suitable for differentiation with overall, either equal or improved, functionality of the hepatocyte-like cells compared to MG. Therefore, and because of its defined and tunable chemical definition, the CNF hydrogel presents a viable alternative to MG for liver tissue engineering with the option for clinical use.

Published
2020

17. A Chemically Defined Hydrogel for Human Liver Organoid Culture

Author

Ye, Shicheng, Boeter, Jochem W.B., Mihajlovic, Marko, van Steenbeek, Frank G., van Wolferen, Monique E., Oosterhoff, Loes A., Marsee, Ary, Caiazzo, Massimiliano, van der Laan, Luc J.W., Penning, Louis C., Vermonden, Tina, Spee, Bart, Schneeberger, Kerstin, Ye, Shicheng, Boeter, Jochem W.B., Mihajlovic, Marko, van Steenbeek, Frank G., van Wolferen, Monique E., Oosterhoff, Loes A., Marsee, Ary, Caiazzo, Massimiliano, van der Laan, Luc J.W., Penning, Louis C., Vermonden, Tina, Spee, Bart, and Schneeberger, Kerstin

Abstract

End-stage liver diseases are an increasing health burden, and liver transplantations are currently the only curative treatment option. Due to a lack of donor livers, alternative treatments are urgently needed. Human liver organoids are very promising for regenerative medicine; however, organoids are currently cultured in Matrigel, which is extracted from the extracellular matrix of the Engelbreth-Holm-Swarm mouse sarcoma. Matrigel is poorly defined, suffers from high batch-to-batch variability and is of xenogeneic origin, which limits the clinical application of organoids. Here, a novel hydrogel based on polyisocyanopeptides (PIC) and laminin-111 is described for human liver organoid cultures. PIC is a synthetic polymer that can form a hydrogel with thermosensitive properties, making it easy to handle and very attractive for clinical applications. Organoids in an optimized PIC hydrogel proliferate at rates comparable to those observed with Matrigel; proliferation rates are stiffness-dependent, with lower stiffnesses being optimal for organoid proliferation. Moreover, organoids can be efficiently differentiated toward a hepatocyte-like phenotype with key liver functions. This proliferation and differentiation potential maintain over at least 14 passages. The results indicate that PIC is very promising for human liver organoid culture and has the potential to be used in a variety of clinical applications including cell therapy and tissue engineering.

Published
2020

18. Long-Term Survival of Transplanted Autologous Canine Liver Organoids in a COMMD1-Deficient Dog Model of Metabolic Liver Disease

Author

Kruitwagen, Hedwig S, Oosterhoff, Loes A, van Wolferen, Monique E, Chen, Chen, Nantasanti Assawarachan, Sathidpak, Schneeberger, Kerstin, Kummeling, Anne, van Straten, Giora, Akkerdaas, Ies C, Vinke, Christel R, van Steenbeek, Frank G, van Bruggen, Leonie W L, Wolfswinkel, Jeannette, Grinwis, Guy C M, Fuchs, Sabine A, Gehart, Helmuth, Geijsen, Niels, Vries, Robert G, Clevers, Hans, Rothuizen, Jan, Schotanus, Baukje A, Penning, Louis C, Spee, Bart, Kruitwagen, Hedwig S, Oosterhoff, Loes A, van Wolferen, Monique E, Chen, Chen, Nantasanti Assawarachan, Sathidpak, Schneeberger, Kerstin, Kummeling, Anne, van Straten, Giora, Akkerdaas, Ies C, Vinke, Christel R, van Steenbeek, Frank G, van Bruggen, Leonie W L, Wolfswinkel, Jeannette, Grinwis, Guy C M, Fuchs, Sabine A, Gehart, Helmuth, Geijsen, Niels, Vries, Robert G, Clevers, Hans, Rothuizen, Jan, Schotanus, Baukje A, Penning, Louis C, and Spee, Bart

Abstract

The shortage of liver organ donors is increasing and the need for viable alternatives is urgent. Liver cell (hepatocyte) transplantation may be a less invasive treatment compared with liver transplantation. Unfortunately, hepatocytes cannot be expanded in vitro, and allogenic cell transplantation requires long-term immunosuppression. Organoid-derived adult liver stem cells can be cultured indefinitely to create sufficient cell numbers for transplantation, and they are amenable to gene correction. This study provides preclinical proof of concept of the potential of cell transplantation in a large animal model of inherited copper toxicosis, such as Wilson's disease, a Mendelian disorder that causes toxic copper accumulation in the liver. Hepatic progenitors from five COMMD1-deficient dogs were isolated and cultured using the 3D organoid culture system. After genetic restoration of COMMD1 expression, the organoid-derived hepatocyte-like cells were safely delivered as repeated autologous transplantations via the portal vein. Although engraftment and repopulation percentages were low, the cells survived in the liver for up to two years post-transplantation. The low engraftment was in line with a lack of functional recovery regarding copper excretion. This preclinical study confirms the survival of genetically corrected autologous organoid-derived hepatocyte-like cells in vivo and warrants further optimization of organoid engraftment and functional recovery in a large animal model of human liver disease.

Published
2020

19. Large-scale Production of LGR5-positive Bipotential Human Liver Stem Cells.

Author

Schneeberger, Kerstin, Sánchez-Romero, Natalia, Ye, Shicheng, van Steenbeek, Frank G, Oosterhoff, Loes A, Pla Palacin, Iris, Chen, Chen, van Wolferen, Monique E, van Tienderen, Gilles, Lieshout, Ruby, Colemonts-Vroninks, Haaike, Schene, Imre, Hoekstra, Ruurdtje, Verstegen, Monique M A, van der Laan, Luc J W, Penning, Louis C, Fuchs, Sabine A, Clevers, Hans, De Kock, Joery, Baptista, Pedro M, Spee, Bart, Schneeberger, Kerstin, Sánchez-Romero, Natalia, Ye, Shicheng, van Steenbeek, Frank G, Oosterhoff, Loes A, Pla Palacin, Iris, Chen, Chen, van Wolferen, Monique E, van Tienderen, Gilles, Lieshout, Ruby, Colemonts-Vroninks, Haaike, Schene, Imre, Hoekstra, Ruurdtje, Verstegen, Monique M A, van der Laan, Luc J W, Penning, Louis C, Fuchs, Sabine A, Clevers, Hans, De Kock, Joery, Baptista, Pedro M, and Spee, Bart

Abstract

The gap between patients on transplant waiting lists and available donor organs is steadily increasing. Human organoids derived from Leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5)-positive adult stem cells represent an exciting new cell source for liver regeneration; however, culturing large numbers of organoids with current protocols is tedious and the level of hepatic differentiation is limited. Here, we established a new method for the expansion of large quantities of human liver organoids in spinner flasks. Due to improved oxygenation in the spinner flasks, organoids rapidly proliferated and reached an average 40-fold cell expansion after two weeks, compared to 6-fold expansion in static cultures. The organoids repopulated decellularized liver discs and formed liver-like tissue. After differentiation in spinner flasks, mature hepatocyte markers were highly upregulated compared to static organoid cultures, and cytochrome p450 activity reached levels equivalent to hepatocytes. CONCLUSION: We established a highly efficient method for culturing large numbers of LGR5-positive stem cells in the form of organoids, which paves the way for the application of organoids for tissue engineering and liver transplantation.

Published
2020

20. Identification of potential drugs for treatment of hepatic lipidosis in cats using an in vitro feline liver organoid system

Author

Haaker, Maya W, Kruitwagen, Hedwig S, Vaandrager, Arie B, Houweling, Martin, Penning, Louis C, Molenaar, Martijn R, van Wolferen, Monique E, Oosterhoff, Loes A, Spee, Bart, Helms, J Bernd, Haaker, Maya W, Kruitwagen, Hedwig S, Vaandrager, Arie B, Houweling, Martin, Penning, Louis C, Molenaar, Martijn R, van Wolferen, Monique E, Oosterhoff, Loes A, Spee, Bart, and Helms, J Bernd

Published
2020

21. Cellulose Nanofibril Hydrogel Promotes Hepatic Differentiation of Human Liver Organoids

Author

Interne geneeskunde GD, dCSCA RMSC-1, Krüger, Melanie, Oosterhoff, Loes A, van Wolferen, Monique E, Schiele, Simon A, Walther, Andreas, Geijsen, Niels, De Laporte, Laura, van der Laan, Luc J W, Kock, Linda M, Spee, Bart, Interne geneeskunde GD, dCSCA RMSC-1, Krüger, Melanie, Oosterhoff, Loes A, van Wolferen, Monique E, Schiele, Simon A, Walther, Andreas, Geijsen, Niels, De Laporte, Laura, van der Laan, Luc J W, Kock, Linda M, and Spee, Bart

Published
2020

22. Long-Term Survival of Transplanted Autologous Canine Liver Organoids in a COMMD1-Deficient Dog Model of Metabolic Liver Disease

Author

Interne geneeskunde GD, dCSCA RMSC-1, Onderzoek, Chirurgie, dCSCA AVR, LS Interne geneeskunde, Operatiekamer en sterilisatie, Diagnostische beeldvorming, VPDC pathologie, dPB CR, Faculteit Diergeneeskunde, CS_Genetics, Kruitwagen, Hedwig S, Oosterhoff, Loes A, van Wolferen, Monique E, Chen, Chen, Nantasanti Assawarachan, Sathidpak, Schneeberger, Kerstin, Kummeling, Anne, van Straten, Giora, Akkerdaas, Ies C, Vinke, Christel R, van Steenbeek, Frank G, van Bruggen, Leonie W L, Wolfswinkel, Jeannette, Grinwis, Guy C M, Fuchs, Sabine A, Gehart, Helmuth, Geijsen, Niels, Vries, Robert G, Clevers, Hans, Rothuizen, Jan, Schotanus, Baukje A, Penning, Louis C, Spee, Bart, Interne geneeskunde GD, dCSCA RMSC-1, Onderzoek, Chirurgie, dCSCA AVR, LS Interne geneeskunde, Operatiekamer en sterilisatie, Diagnostische beeldvorming, VPDC pathologie, dPB CR, Faculteit Diergeneeskunde, CS_Genetics, Kruitwagen, Hedwig S, Oosterhoff, Loes A, van Wolferen, Monique E, Chen, Chen, Nantasanti Assawarachan, Sathidpak, Schneeberger, Kerstin, Kummeling, Anne, van Straten, Giora, Akkerdaas, Ies C, Vinke, Christel R, van Steenbeek, Frank G, van Bruggen, Leonie W L, Wolfswinkel, Jeannette, Grinwis, Guy C M, Fuchs, Sabine A, Gehart, Helmuth, Geijsen, Niels, Vries, Robert G, Clevers, Hans, Rothuizen, Jan, Schotanus, Baukje A, Penning, Louis C, and Spee, Bart

Published
2020

23. Identification of potential drugs for treatment of hepatic lipidosis in cats using an in vitro feline liver organoid system

Author

LS Veterinaire biochemie, dB&C FR-RMSC RMSC, LS Interne geneeskunde, dCSCA RMSC-1, Onderzoek, dCSCA AVR, Sub Biochemie Algemeen, Biochemisch laboratorium, Dep Biochemie en Celbiologie, dB&C FR-RMSC FR, Haaker, Maya W, Kruitwagen, Hedwig S, Vaandrager, Arie B, Houweling, Martin, Penning, Louis C, Molenaar, Martijn R, van Wolferen, Monique E, Oosterhoff, Loes A, Spee, Bart, Helms, J Bernd, LS Veterinaire biochemie, dB&C FR-RMSC RMSC, LS Interne geneeskunde, dCSCA RMSC-1, Onderzoek, dCSCA AVR, Sub Biochemie Algemeen, Biochemisch laboratorium, Dep Biochemie en Celbiologie, dB&C FR-RMSC FR, Haaker, Maya W, Kruitwagen, Hedwig S, Vaandrager, Arie B, Houweling, Martin, Penning, Louis C, Molenaar, Martijn R, van Wolferen, Monique E, Oosterhoff, Loes A, Spee, Bart, and Helms, J Bernd

Published
2020

24. A Chemically Defined Hydrogel for Human Liver Organoid Culture

Author

Afd Pharmaceutics, Interne geneeskunde GD, dCSCA AVR, dCSCA RMSC-1, Pharmaceutics, CS_STEM, CS_Genetics, Ye, Shicheng, Boeter, Jochem W.B., Mihajlovic, Marko, van Steenbeek, Frank G., van Wolferen, Monique E., Oosterhoff, Loes A., Marsee, Ary, Caiazzo, Massimiliano, van der Laan, Luc J.W., Penning, Louis C., Vermonden, Tina, Spee, Bart, Schneeberger, Kerstin, Afd Pharmaceutics, Interne geneeskunde GD, dCSCA AVR, dCSCA RMSC-1, Pharmaceutics, CS_STEM, CS_Genetics, Ye, Shicheng, Boeter, Jochem W.B., Mihajlovic, Marko, van Steenbeek, Frank G., van Wolferen, Monique E., Oosterhoff, Loes A., Marsee, Ary, Caiazzo, Massimiliano, van der Laan, Luc J.W., Penning, Louis C., Vermonden, Tina, Spee, Bart, and Schneeberger, Kerstin

Published
2020

25. Large-scale Production of LGR5-positive Bipotential Human Liver Stem Cells.

Author

Onderzoek, dCSCA RMSC-1, Sub General Pharmacology, dCSCA AVR, Biochemisch laboratorium, Faculteit Diergeneeskunde, Schneeberger, Kerstin, Sánchez-Romero, Natalia, Ye, Shicheng, van Steenbeek, Frank G, Oosterhoff, Loes A, Pla Palacin, Iris, Chen, Chen, van Wolferen, Monique E, van Tienderen, Gilles, Lieshout, Ruby, Colemonts-Vroninks, Haaike, Schene, Imre, Hoekstra, Ruurdtje, Verstegen, Monique M A, van der Laan, Luc J W, Penning, Louis C, Fuchs, Sabine A, Clevers, Hans, De Kock, Joery, Baptista, Pedro M, Spee, Bart, Onderzoek, dCSCA RMSC-1, Sub General Pharmacology, dCSCA AVR, Biochemisch laboratorium, Faculteit Diergeneeskunde, Schneeberger, Kerstin, Sánchez-Romero, Natalia, Ye, Shicheng, van Steenbeek, Frank G, Oosterhoff, Loes A, Pla Palacin, Iris, Chen, Chen, van Wolferen, Monique E, van Tienderen, Gilles, Lieshout, Ruby, Colemonts-Vroninks, Haaike, Schene, Imre, Hoekstra, Ruurdtje, Verstegen, Monique M A, van der Laan, Luc J W, Penning, Louis C, Fuchs, Sabine A, Clevers, Hans, De Kock, Joery, Baptista, Pedro M, and Spee, Bart

Published
2020

26. Cellulose Nanofibril Hydrogel Promotes Hepatic Differentiation of Human Liver Organoids

Author

Hubrecht Institute with UMC, Krüger, Melanie, Oosterhoff, Loes A, van Wolferen, Monique E, Schiele, Simon A, Walther, Andreas, Geijsen, Niels, De Laporte, Laura, van der Laan, Luc J W, Kock, Linda M, Spee, Bart, Hubrecht Institute with UMC, Krüger, Melanie, Oosterhoff, Loes A, van Wolferen, Monique E, Schiele, Simon A, Walther, Andreas, Geijsen, Niels, De Laporte, Laura, van der Laan, Luc J W, Kock, Linda M, and Spee, Bart

Published
2020

27. Large-scale Production of LGR5-positive Bipotential Human Liver Stem Cells

Author

MDL onderzoek 2, Onderzoek Precision medicine, Metabole ziekten onderzoek 1, Metabole ziekten patientenzorg, Regenerative Medicine and Stem Cells, Child Health, CMM Sectie Molecular Cancer Research, Cancer, Hubrecht Institute with UMC, Schneeberger, Kerstin, Sánchez-Romero, Natalia, Ye, Shicheng, van Steenbeek, Frank G, Oosterhoff, Loes A, Pla Palacin, Iris, Chen, Chen, van Wolferen, Monique E, van Tienderen, Gilles, Lieshout, Ruby, Colemonts-Vroninks, Haaike, Schene, Imre, Hoekstra, Ruurdtje, Verstegen, Monique M A, van der Laan, Luc J W, Penning, Louis C, Fuchs, Sabine A, Clevers, Hans, De Kock, Joery, Baptista, Pedro M, Spee, Bart, MDL onderzoek 2, Onderzoek Precision medicine, Metabole ziekten onderzoek 1, Metabole ziekten patientenzorg, Regenerative Medicine and Stem Cells, Child Health, CMM Sectie Molecular Cancer Research, Cancer, Hubrecht Institute with UMC, Schneeberger, Kerstin, Sánchez-Romero, Natalia, Ye, Shicheng, van Steenbeek, Frank G, Oosterhoff, Loes A, Pla Palacin, Iris, Chen, Chen, van Wolferen, Monique E, van Tienderen, Gilles, Lieshout, Ruby, Colemonts-Vroninks, Haaike, Schene, Imre, Hoekstra, Ruurdtje, Verstegen, Monique M A, van der Laan, Luc J W, Penning, Louis C, Fuchs, Sabine A, Clevers, Hans, De Kock, Joery, Baptista, Pedro M, and Spee, Bart

Published
2020

28. Long-Term Survival of Transplanted Autologous Canine Liver Organoids in a COMMD1-Deficient Dog Model of Metabolic Liver Disease

Author

Kruitwagen, Hedwig S., primary, Oosterhoff, Loes A., additional, van Wolferen, Monique E., additional, Chen, Chen, additional, Nantasanti Assawarachan, Sathidpak, additional, Schneeberger, Kerstin, additional, Kummeling, Anne, additional, van Straten, Giora, additional, Akkerdaas, Ies C., additional, Vinke, Christel R., additional, van Steenbeek, Frank G., additional, van Bruggen, Leonie W.L., additional, Wolfswinkel, Jeannette, additional, Grinwis, Guy C.M., additional, Fuchs, Sabine A., additional, Gehart, Helmuth, additional, Geijsen, Niels, additional, Vries, Robert G., additional, Clevers, Hans, additional, Rothuizen, Jan, additional, Schotanus, Baukje A., additional, Penning, Louis C., additional, and Spee, Bart, additional

Published
2020
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30. Long-Term Adult Feline Liver Organoid Cultures for Disease Modeling of Hepatic Steatosis

Author

Kruitwagen, Hedwig S., Oosterhoff, Loes A., Vernooij, Ingrid G W H, Schrall, Ingrid M., van Wolferen, Monique E., Bannink, Farah, Roesch, Camille, van Uden, Lisa, Molenaar, Martijn R., Helms, J. Bernd, Grinwis, Guy C.M., Verstegen, Monique M A, van der Laan, Luc J W, Huch, Meritxell, Geijsen, Niels, Vries, Robert R G, Clevers, Hans, Rothuizen, Jan, Schotanus, Baukje A., Penning, Louis C., Spee, Bart, dB&C FR-RMSC FR, dCSCA RMSC-1, dPB CR, dCSCA AVR, Onderzoek, Biochemisch laboratorium, LS Pathologie, Sub Biochemie Algemeen, Dep Biochemie en Celbiologie, LS Veterinaire biochemie, Veterinair Pathologisch Diagnostisch Cnt, PB AVM, Applied Veterinary Research, Dep Pathobiologie, Faculteit Diergeneeskunde, LS Interne geneeskunde, Surgery, Hubrecht Institute for Developmental Biology and Stem Cell Research, dB&C FR-RMSC FR, dCSCA RMSC-1, dPB CR, dCSCA AVR, Onderzoek, Biochemisch laboratorium, LS Pathologie, Sub Biochemie Algemeen, Dep Biochemie en Celbiologie, LS Veterinaire biochemie, Veterinair Pathologisch Diagnostisch Cnt, PB AVM, Applied Veterinary Research, Dep Pathobiologie, Faculteit Diergeneeskunde, LS Interne geneeskunde, Huch Ortega, Meritxell [0000-0002-1545-5265], and Apollo - University of Cambridge Repository

Subjects
0301 basic medicine, Male, Pathology, species differences, Biochemistry, Liver disease, 0302 clinical medicine, Lipid droplet, disease modeling, Non-U.S. Gov't, lcsh:QH301-705.5, lcsh:R5-920, Research Support, Non-U.S. Gov't, hepatic steatosis, Fatty liver, Cell Differentiation, 3. Good health, Organoids, Adult Stem Cells, Liver, 030220 oncology & carcinogenesis, Female, Stem cell, lcsh:Medicine (General), Adult stem cell, Feline hepatic lipidosis, medicine.medical_specialty, feline liver organoids, Biology, Research Support, 03 medical and health sciences, Organ Culture Techniques, Report, Internal medicine, Genetics, medicine, Organoid, Journal Article, Animals, Animal, Cell Biology, medicine.disease, Fatty Liver, Disease Models, Animal, adult liver stem cells, 030104 developmental biology, Endocrinology, lcsh:Biology (General), Disease Models, Cats, Hepatocytes, feline hepatic lipidosis, Steatosis, Developmental Biology
Abstract

Summary Hepatic steatosis is a highly prevalent liver disease, yet research is hampered by the lack of tractable cellular and animal models. Steatosis also occurs in cats, where it can cause severe hepatic failure. Previous studies demonstrate the potential of liver organoids for modeling genetic diseases. To examine the possibility of using organoids to model steatosis, we established a long-term feline liver organoid culture with adult liver stem cell characteristics and differentiation potential toward hepatocyte-like cells. Next, organoids from mouse, human, dog, and cat liver were provided with fatty acids. Lipid accumulation was observed in all organoids and interestingly, feline liver organoids accumulated more lipid droplets than human organoids. Finally, we demonstrate effects of interference with β-oxidation on lipid accumulation in feline liver organoids. In conclusion, feline liver organoids can be successfully cultured and display a predisposition for lipid accumulation, making them an interesting model in hepatic steatosis research., Highlights • Feline organoids can be cultured from fresh and frozen liver and from needle biopsy • A feline-specific liver culture medium allows organoid expansion up to 32 passages • When fed fatty acids, feline liver organoids store more lipids than human organoids • Feline liver organoids can be used to model hepatic steatosis, In this study Kruitwagen and colleagues establish and characterize a feline liver organoid culture, which has adult stem cell properties and can be differentiated toward hepatocyte-like cells. They propose liver organoids as a tool to model hepatic steatosis and show that feline liver organoids accumulate more lipids than human organoids when provided with excess fatty acids.

Published
2017

31. A Versatile Biosynthetic Hydrogel Platform for Engineering of Tissue Analogues

Author

Klotz, Barbara J, Oosterhoff, Loes A, Utomo, Lizette, Lim, Khoon S, Vallmajo-Martin, Queralt, Clevers, Hans, Woodfield, Tim B F, Rosenberg, Antoine J W P, Malda, Jos, Ehrbar, Martin, Spee, Bart, Gawlitta, Debby, Afd Pharmaceutics, Biochemisch laboratorium, dCSCA RMSC-1, Chirurgie, Faculteit Diergeneeskunde, LS Equine Muscoskeletal Biology, dES RMSC, Onderzoek, University of Zurich, Gawlitta, Debby, Afd Pharmaceutics, Biochemisch laboratorium, dCSCA RMSC-1, Chirurgie, Faculteit Diergeneeskunde, LS Equine Muscoskeletal Biology, dES RMSC, Onderzoek, and Hubrecht Institute for Developmental Biology and Stem Cell Research

Subjects
Swine, Cellular differentiation, 3003 Pharmaceutical Science, Pharmaceutical Science, Biocompatible Materials, 02 engineering and technology, 01 natural sciences, Gelatin, Polyethylene Glycols, chemistry.chemical_compound, Tissue engineering, matrigel, FXIII, Tissue Scaffolds, Chemistry, Cell Differentiation, Hydrogels, 021001 nanoscience & nanotechnology, Organoids, Drug Combinations, Liver, polyethylene glycol, Proteoglycans, Collagen, Stem cell, 0210 nano-technology, food.ingredient, Cell Survival, Biomedical Engineering, 2204 Biomedical Engineering, 610 Medicine & health, Polyethylene glycol, macromolecular substances, 010402 general chemistry, Bone and Bones, Catalysis, Article, osteogenesis, vasculogenesis, gelatin, Biomaterials, food, Matrigel, PEG ratio, Organoid, Journal Article, Animals, Humans, 10026 Clinic for Obstetrics, liver organoids, Tissue Engineering, 2502 Biomaterials, technology, industry, and agriculture, Mesenchymal Stem Cells, Culture Media, 0104 chemical sciences, Biophysics, Laminin, Peptides
Abstract

For creating functional tissue analogues in tissue engineering, stem cells require very specific 3D microenvironments to thrive and mature. Demanding (stem) cell types that are used nowadays can find such an environment in a heterogeneous protein mixture with the trade name Matrigel. Several variations of synthetic hydrogel platforms composed of poly(ethylene glycol) (PEG), which are spiked with peptides, have been recently developed and shown equivalence to Matrigel for stem cell differentiation. Here a clinically relevant hydrogel platform, based on PEG and gelatin, which even outperforms Matrigel when targeting 3D prevascularized bone and liver organoid tissue engineering models is presented. The hybrid hydrogel with natural and synthetic components stimulates efficient cell differentiation, superior to Matrigel models. Furthermore, the strength of this hydrogel lies in the option to covalently incorporate unmodified proteins. These results demonstrate how a hybrid hydrogel platform with intermediate biological complexity, when compared to existing biological materials and synthetic PEG-peptide approaches, can efficiently support tissue development from human primary cells.

Published
2019

32. Characterization of Endothelial and Smooth Muscle Cells From Different Canine Vessels

Author

Oosterhoff, Loes A, Kruitwagen, Hedwig S, van Wolferen, Monique E, van Balkom, Bas W M, Mokry, Michal, Lansu, Nico, van den Dungen, Noortje A M, Penning, Louis C, Spanjersberg, Talitha C F, de Graaf, Johannes W, Veenendaal, Tomas, Zomerdijk, Flin, Fledderus, Joost O, Spee, Bart, van Steenbeek, Frank G, Biochemisch laboratorium, dCSCA RMSC-1, LS Interne geneeskunde, Sub Biomol.Mass Spect. and Proteomics, dCSCA AVR, Onderzoek, Undergraduate School, Biochemisch laboratorium, dCSCA RMSC-1, LS Interne geneeskunde, Sub Biomol.Mass Spect. and Proteomics, dCSCA AVR, Onderzoek, and Undergraduate School

Subjects
0301 basic medicine, Cell type, Vascular smooth muscle, Endothelium, Physiology, Angiogenesis, Biology, lcsh:Physiology, Transcriptome, 03 medical and health sciences, angiogenesis, 0302 clinical medicine, Tissue engineering, cell model system, vascular cell interaction, Physiology (medical), medicine, vascular smooth muscle cells, vascular smooth, Tissue homeostasis, Original Research, lcsh:QP1-981, Vascular disease, medicine.disease, endothelial cells, Cell biology, 030104 developmental biology, medicine.anatomical_structure, 030220 oncology & carcinogenesis, cardiovascular system
Abstract

Vasculature performs a critical function in tissue homeostasis, supply of oxygen and nutrients, and the removal of metabolic waste products. Vascular problems are implicated in a large variety of pathologies and accurate in vitro models resembling native vasculature are of great importance. Unfortunately, existing in vitro models do not sufficiently reflect their in vivo counterpart. The complexity of vasculature requires the examination of multiple cell types including endothelial cells (ECs) and vascular smooth muscle cells (VSMCs), as well as vessel location in the body from which they originate. The use of canine blood vessels provides a way to study vasculature with similar vessel size and physiology compared to human vasculature. We report an isolation procedure that provides the possibility to isolate both the endothelial and smooth muscle cells from the same vessels simultaneously, enabling new opportunities in investigating vasculature behavior. Canine primary ECs and VSMCs were isolated from the vena cava, vena porta and aorta. All tissue sources were derived from three donors for accurate comparison and to reduce inter-animal variation. The isolation and purification of the two distinct cell types was confirmed by morphology, gene- and protein-expression and function. As both cell types can be derived from the same vessel, this approach allows accurate modeling of vascular diseases and can also be used more widely, for example, in vascular bioreactors and tissue engineering designs. Additionally, we identified several new genes that were highly expressed in canine ECs, which may become candidate genes for novel EC markers. In addition, we observed transcriptional and functional differences between arterial- and venous-derived endothelium. Further exploration of the transcriptome and physiology of arteriovenous differentiation of primary cells may have important implications for a better understanding of the fundamental behavior of the vasculature and pathogenesis of vascular disease.

Published
2019

33. A Versatile Biosynthetic Hydrogel Platform for Engineering of Tissue Analogues

Author

Afd Pharmaceutics, Biochemisch laboratorium, dCSCA RMSC-1, Chirurgie, Faculteit Diergeneeskunde, LS Equine Muscoskeletal Biology, dES RMSC, Onderzoek, Klotz, Barbara J, Oosterhoff, Loes A, Utomo, Lizette, Lim, Khoon S, Vallmajo-Martin, Queralt, Clevers, Hans, Woodfield, Tim B F, Rosenberg, Antoine J W P, Malda, Jos, Ehrbar, Martin, Spee, Bart, Gawlitta, Debby, Afd Pharmaceutics, Biochemisch laboratorium, dCSCA RMSC-1, Chirurgie, Faculteit Diergeneeskunde, LS Equine Muscoskeletal Biology, dES RMSC, Onderzoek, Klotz, Barbara J, Oosterhoff, Loes A, Utomo, Lizette, Lim, Khoon S, Vallmajo-Martin, Queralt, Clevers, Hans, Woodfield, Tim B F, Rosenberg, Antoine J W P, Malda, Jos, Ehrbar, Martin, Spee, Bart, and Gawlitta, Debby

Published
2019

34. Large-scale Production of LGR5-positive Bipotential Human Liver Stem Cells

Author

Schneeberger, Kerstin, Sánchez-Romero, Natalia, Ye, Shicheng, van Steenbeek, Frank G, Oosterhoff, Loes A, Pla Palacin, Iris, Chen, Chen, van Wolferen, Monique E, van Tienderen, Gilles, Lieshout, Ruby, Colemonts-Vroninks, Haaike, Schene, Imre, Hoekstra, Ruurdtje, Verstegen, Monique M A, van der Laan, Luc J W, Penning, Louis C, Fuchs, Sabine A, Clevers, Hans, De Kock, Joery, Baptista, Pedro M, Spee, Bart, Schneeberger, Kerstin, Sánchez-Romero, Natalia, Ye, Shicheng, van Steenbeek, Frank G, Oosterhoff, Loes A, Pla Palacin, Iris, Chen, Chen, van Wolferen, Monique E, van Tienderen, Gilles, Lieshout, Ruby, Colemonts-Vroninks, Haaike, Schene, Imre, Hoekstra, Ruurdtje, Verstegen, Monique M A, van der Laan, Luc J W, Penning, Louis C, Fuchs, Sabine A, Clevers, Hans, De Kock, Joery, Baptista, Pedro M, and Spee, Bart

Abstract

The gap between patients on transplant waiting lists and available donor organs is steadily increasing. Human organoids derived from Leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5)-positive adult stem cells represent an exciting new cell source for liver regeneration; however, culturing large numbers of organoids with current protocols is tedious and the level of hepatic differentiation is limited. Here, we established a new method for the expansion of large quantities of human liver organoids in spinner flasks. Due to improved oxygenation in the spinner flasks, organoids rapidly proliferated and reached an average 40-fold cell expansion after two weeks, compared to 6-fold expansion in static cultures. The organoids repopulated decellularized liver discs and formed liver-like tissue. After differentiation in spinner flasks, mature hepatocyte markers were highly upregulated compared to static organoid cultures, and cytochrome p450 activity reached levels equivalent to hepatocytes. CONCLUSION: We established a highly efficient method for culturing large numbers of LGR5-positive stem cells in the form of organoids, which paves the way for the application of organoids for tissue engineering and liver transplantation.

Published
2019

35. Characterization of Endothelial and Smooth Muscle Cells From Different Canine Vessels

Author

Biochemisch laboratorium, dCSCA RMSC-1, LS Interne geneeskunde, Sub Biomol.Mass Spect. and Proteomics, dCSCA AVR, Onderzoek, Undergraduate School, Oosterhoff, Loes A, Kruitwagen, Hedwig S, van Wolferen, Monique E, van Balkom, Bas W M, Mokry, Michal, Lansu, Nico, van den Dungen, Noortje A M, Penning, Louis C, Spanjersberg, Talitha C F, de Graaf, Johannes W, Veenendaal, Tomas, Zomerdijk, Flin, Fledderus, Joost O, Spee, Bart, van Steenbeek, Frank G, Biochemisch laboratorium, dCSCA RMSC-1, LS Interne geneeskunde, Sub Biomol.Mass Spect. and Proteomics, dCSCA AVR, Onderzoek, Undergraduate School, Oosterhoff, Loes A, Kruitwagen, Hedwig S, van Wolferen, Monique E, van Balkom, Bas W M, Mokry, Michal, Lansu, Nico, van den Dungen, Noortje A M, Penning, Louis C, Spanjersberg, Talitha C F, de Graaf, Johannes W, Veenendaal, Tomas, Zomerdijk, Flin, Fledderus, Joost O, Spee, Bart, and van Steenbeek, Frank G

Published
2019

36. A Versatile Biosynthetic Hydrogel Platform for Engineering of Tissue Analogues

Author

Regenerative Medicine and Stem Cells, MKA/BT Onderzoek, CMM Sectie Molecular Cancer Research, Child Health, Cancer, Hubrecht Institute with UMC, Orthopaedie Onderzoek, Klotz, Barbara J, Oosterhoff, Loes A, Utomo, Lizette, Lim, Khoon S, Vallmajo-Martin, Queralt, Clevers, Hans, Woodfield, Tim B F, Rosenberg, Antoine J W P, Malda, Jos, Ehrbar, Martin, Spee, Bart, Gawlitta, Debby, Regenerative Medicine and Stem Cells, MKA/BT Onderzoek, CMM Sectie Molecular Cancer Research, Child Health, Cancer, Hubrecht Institute with UMC, Orthopaedie Onderzoek, Klotz, Barbara J, Oosterhoff, Loes A, Utomo, Lizette, Lim, Khoon S, Vallmajo-Martin, Queralt, Clevers, Hans, Woodfield, Tim B F, Rosenberg, Antoine J W P, Malda, Jos, Ehrbar, Martin, Spee, Bart, and Gawlitta, Debby

Published
2019

37. Characterization of endothelial and smooth muscle cells from different canine vessels

Author

UMC Utrecht, MS Nefrologie, Circulatory Health, Dagcentrum, Child Health, MDL onderzoek 1, Regenerative Medicine and Stem Cells, Oosterhoff, Loes A., Kruitwagen, Hedwig S., Van Wolferen, Monique E., Van Balkom, Bas W.M., Mokry, Michal, Lansu, Nico, Lansu, NR, Van Den Dungen, Noortje A.M., Penning, Louis C., Spanjersberg, Talitha C.F., De Graaf, Johannes W., Veenendaal, Tomas, Zomerdijk, Flin, Fledderus, Joost O., Spee, Bart, Van Steenbeek, Frank G., UMC Utrecht, MS Nefrologie, Circulatory Health, Dagcentrum, Child Health, MDL onderzoek 1, Regenerative Medicine and Stem Cells, Oosterhoff, Loes A., Kruitwagen, Hedwig S., Van Wolferen, Monique E., Van Balkom, Bas W.M., Mokry, Michal, Lansu, Nico, Lansu, NR, Van Den Dungen, Noortje A.M., Penning, Louis C., Spanjersberg, Talitha C.F., De Graaf, Johannes W., Veenendaal, Tomas, Zomerdijk, Flin, Fledderus, Joost O., Spee, Bart, and Van Steenbeek, Frank G.

Published
2019

39. DYRK1A Is a Regulator of S-Phase Entry in Hepatic Progenitor Cells

Author

Kruitwagen, Hedwig S., Westendorp, Bart, Viebahn, Cornelia S., Post, Krista, van Wolferen, Monique E., Oosterhoff, Loes A., Egan, David A., Delabar, Jean-Maurice, Toussaint, Mathilda J., Schotanus, Baukje A., de Bruin, Alain, Rothuizen, Jan, Penning, Louis C., Spee, Bart, Kruitwagen, Hedwig S., Westendorp, Bart, Viebahn, Cornelia S., Post, Krista, van Wolferen, Monique E., Oosterhoff, Loes A., Egan, David A., Delabar, Jean-Maurice, Toussaint, Mathilda J., Schotanus, Baukje A., de Bruin, Alain, Rothuizen, Jan, Penning, Louis C., and Spee, Bart

Abstract

Plant cells contain two major pools of K+, one in the vacuole and one in the cytosol. The behavior of K+ concentrations in these pools is fundamental to understand-ing the way this nutrient affects plantigrowth. Triple-barreled microelectrodes have been used to obtain the first fully quantitative measurements of the changes in K+ activity (ay) in the vacuole and cytosol of barley (Hordeum vulgare L.) root cells grown in different K+ concentrations. The electrodes incorporate a pH-selective barrel allowing each measurement to be assigned to either the cytosol or vacuole. The measure-ments revealed that vacuolar aK declined linearly with de-creases in tissue K+ concentration, whereas cytosolic aK initially remained constant in both epidermal and cortical cells but then declined at different rates in each cell type. An unexpected finding was that cytoplasmic pH declined in parallel with cytosolic aK, but acidification of the cytosol with butyrate did not reveal any short-term link between these two parameters. These measurements show the very different responses of the vacuolar and cytosolic K+ pools to changes in K+ availability and also show that cytosolic K+ homeostasis differs quantitatively in different cell types. The data have

Published
2018

40. Hepatocyte-like cells generated by direct reprogramming from murine somatic cells can repopulate decellularized livers

Author

Chen, Chen, Pla-Palacín, Iris, Baptista, Pedro M, Shang, Peng, Oosterhoff, Loes A, van Wolferen, Monique E, Penning, Louis C, Geijsen, Niels, Spee, Bart, Chen, Chen, Pla-Palacín, Iris, Baptista, Pedro M, Shang, Peng, Oosterhoff, Loes A, van Wolferen, Monique E, Penning, Louis C, Geijsen, Niels, and Spee, Bart

Abstract

Direct reprogramming represents an easy technique to generate hepatocyte-like cells (iHeps) from somatic cells. However, current protocols are accompanied by several drawbacks as iHeps are heterogenous and lack fully mature phenotypes of primary hepatocytes. Here, we established a polycistronic expression system to induce the direct reprogramming of mouse embryonic fibroblasts towards hepatocytes. The resulting iHeps are homogenous and display key properties of primary hepatocytes, such as expression of hepatocyte markers, albumin secretion, and presence of liver transaminases. iHeps also possess the capacity to repopulate decellularized liver tissue and exhibit enhanced hepatic maturation. As such, we present a novel strategy to generate homogenous and functional hepatocyte-like cells for applications in tissue engineering and cell therapy. This article is protected by copyright. All rights reserved.

Published
2018

41. DYRK1A Is a Regulator of S-Phase Entry in Hepatic Progenitor Cells

Author

dCSCA RMSC-1, Onderzoek, LS Pathobiologie, dPB RMSC, LS Interne geneeskunde, LS Algemene chirurgie, Biochemisch laboratorium, dCSCA AVR, Kruitwagen, Hedwig S., Westendorp, Bart, Viebahn, Cornelia S., Post, Krista, van Wolferen, Monique E., Oosterhoff, Loes A., Egan, David A., Delabar, Jean-Maurice, Toussaint, Mathilda J., Schotanus, Baukje A., de Bruin, Alain, Rothuizen, Jan, Penning, Louis C., Spee, Bart, dCSCA RMSC-1, Onderzoek, LS Pathobiologie, dPB RMSC, LS Interne geneeskunde, LS Algemene chirurgie, Biochemisch laboratorium, dCSCA AVR, Kruitwagen, Hedwig S., Westendorp, Bart, Viebahn, Cornelia S., Post, Krista, van Wolferen, Monique E., Oosterhoff, Loes A., Egan, David A., Delabar, Jean-Maurice, Toussaint, Mathilda J., Schotanus, Baukje A., de Bruin, Alain, Rothuizen, Jan, Penning, Louis C., and Spee, Bart

Published
2018

42. Hepatocyte-like cells generated by direct reprogramming from murine somatic cells can repopulate decellularized livers

Author

Hubrecht Institute with UMC, Chen, Chen, Pla-Palacín, Iris, Baptista, Pedro M., Shang, Peng, Oosterhoff, Loes A., van Wolferen, Monique E., Penning, Louis C., Geijsen, Niels, Spee, Bart, Hubrecht Institute with UMC, Chen, Chen, Pla-Palacín, Iris, Baptista, Pedro M., Shang, Peng, Oosterhoff, Loes A., van Wolferen, Monique E., Penning, Louis C., Geijsen, Niels, and Spee, Bart

Published
2018

43. DYRK1A Is a Regulator of S-Phase Entry in Hepatic Progenitor Cells

Author

Kruitwagen, Hedwig S., Westendorp, Bart, Viebahn, Cornelia S., Post, Krista, van Wolferen, Monique E., Oosterhoff, Loes A., Egan, David A., Delabar, Jean-Maurice, Toussaint, Mathilda J., Schotanus, Baukje A., de Bruin, Alain, Rothuizen, Jan, Penning, Louis C., Spee, Bart, dCSCA RMSC-1, Onderzoek, LS Pathobiologie, dPB RMSC, LS Interne geneeskunde, LS Algemene chirurgie, Biochemisch laboratorium, and dCSCA AVR

Subjects
0301 basic medicine, Small interfering RNA, Transcription, Genetic, proliferation, Biology, Protein Serine-Threonine Kinases, Cell Line, S Phase, 03 medical and health sciences, RNA interference screen, Organoid, Humans, Progenitor cell, E2F, hepatic progenitor cells, Cell Biology, Hematology, DYRK1A, Cell cycle, Protein-Tyrosine Kinases, Liver regeneration, Cell biology, Adult Stem Cells, 030104 developmental biology, Liver, Cell culture, cell cycle, Stem cell, Developmental Biology
Abstract

Hepatic progenitor cells (HPCs) are adult liver stem cells that act as second line of defense in liver regeneration. They are normally quiescent, but in case of severe liver damage, HPC proliferation is triggered by external activation mechanisms from their niche. Although several important proproliferative mechanisms have been described, it is not known which key intracellular regulators govern the switch between HPC quiescence and active cell cycle. We performed a high-throughput kinome small interfering RNA (siRNA) screen in HepaRG cells, a HPC-like cell line, and evaluated the effect on proliferation with a 5-ethynyl-2'-deoxyuridine (EdU) incorporation assay. One hit increased the percentage of EdU-positive cells after knockdown: dual specificity tyrosine phosphorylation regulated kinase 1A (DYRK1A). Although upon DYRK1A silencing, the percentage of EdU- and phosphorylated histone H3 (pH3)-positive cells was increased, and total cell numbers were not increased, possibly through a subsequent delay in cell cycle progression. This phenotype was confirmed with chemical inhibition of DYRK1A using harmine and with primary HPCs cultured as liver organoids. DYRK1A inhibition impaired Dimerization Partner, RB-like, E2F, and multivulva class B (DREAM) complex formation in HPCs and abolished its transcriptional repression on cell cycle progression. To further analyze DYRK1A function in HPC proliferation, liver organoid cultures were established from mBACtgDyrk1A mice, which harbor one extra copy of the murine Dyrk1a gene (Dyrk+++). Dyrk+++ organoids had both a reduced percentage of EdU-positive cells and reduced proliferation compared with wild-type organoids. This study provides evidence for an essential role of DYRK1A as balanced regulator of S-phase entry in HPCs. An exact gene dosage is crucial, as both DYRK1A deficiency and overexpression affect HPC cell cycle progression.

Published
2017

44. Identification of potential drugs for treatment of hepatic lipidosis in cats using an in vitro feline liver organoid system.

Author

Haaker, Maya W., Kruitwagen, Hedwig S., Vaandrager, Arie B., Houweling, Martin, Penning, Louis C., Molenaar, Martijn R., Wolferen, Monique E., Oosterhoff, Loes A., Spee, Bart, and Helms, J. Bernd

Subjects
ACYLTRANSFERASES, LIPIDOSES, FREE fatty acids, ADENOSINE monophosphate, CATS, LIVER
Abstract

Background: Hepatic lipidosis is increasing in incidence in the Western world, with cats being particularly sensitive. When cats stop eating and start utilizing their fat reserves, free fatty acids (FFAs) increase in blood, causing an accumulation of triacylglycerol (TAG) in the liver. Objective: Identifying potential new drugs that can be used to treat hepatic lipidosis in cats using a feline hepatic organoid system. Animals: Liver organoids obtained from 6 cats. Methods: Eight different drugs were tested, and the 2 most promising were further studied using a quantitative TAG assay, lipid droplet staining, and qPCR. Results: Both T863 (a diacylglycerol O‐acyltransferase 1 [DGAT1] inhibitor) and 5‐aminoimidazole‐4‐carboxamide 1‐β‐D‐ribofuranoside (AICAR; an adenosine monophosphate kinase activator) decreased TAG accumulation by 55% (P <.0001) and 46% (P =.0003), respectively. Gene expression of perilipin 2 (PLIN2) increased upon the addition of FFAs to the medium and decreased upon treatment with AICAR but not significantly after treatment with T863. Conclusions and Clinical Importance: Two potential drugs useful in the treatment of hepatic lipidosis in cats were identified. The drug T863 inhibits DGAT1, indicating that DGAT1 is the primary enzyme responsible for TAG synthesis from external fatty acids in cat organoids. The drug AICAR may act as a lipid‐lowering compound via decreasing PLIN2 mRNA. Liver organoids can be used as an in vitro tool for drug testing in a species‐specific system and provide the basis for further clinical testing of drugs to treat steatosis. [ABSTRACT FROM AUTHOR]

Published
2020
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45. Reduced FXR Target Gene Expression in Copper-Laden Livers of COMMD1-Deficient Dogs.

Author

Xiaoyan Wu, Chien, Hsiaotzu, van Wolferen, Monique E., Kruitwagen, Hedwig S., Oosterhoff, Loes A., and Penning, Louis C.

Subjects
HEPATOLENTICULAR degeneration, DOG diseases, GENE expression, NUCLEAR receptors (Biochemistry), APOLIPOPROTEIN E
Abstract

Wilson's disease (WD), an autosomal recessive disorder, results in copper accumulation in the liver as a consequence of mutations in the gene ATPase copper transporting beta (ATP7B). The disease is characterized by chronic hepatitis, eventually resulting in liver cirrhosis. Recent studies have shown that dysregulation of nuclear receptors (NR) by high hepatic copper levels is an important event in the pathogenesis of liver disease in WD. Intracellular trafficking of ATP7B is mediated by COMMD1 and, in Bedlington terriers, a mutation in the COMMD1 gene results in high hepatic copper levels. Here, we demonstrate a reduced Farnesoid X nuclear receptor (FXR)-activity in liver biopsies of COMMD1-deficient dogs with copper toxicosis, a unique large animal model of WD. FXR-induced target genes, small heterodimer partner (SHP), and apolipoprotein E (ApoE) were down-regulated in liver samples from COMMD1-deficient dogs with hepatic copper accumulation. In contrast, the relative mRNA levels of the two CYP-enzymes (reduced by FXR activity) was similar in both groups. These data are in line with the previously observed reduced FXR activity in livers of ATP7B/mice and WD patients. Therefore, these data further corroborate on the importance of the COMMD1-deficient dogs as a large animal model for WD. [ABSTRACT FROM AUTHOR]

Published
2019
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46. Long-Term Adult Feline Liver Organoid Cultures for Disease Modeling of Hepatic Steatosis

Author

dB&C FR-RMSC FR, dCSCA RMSC-1, dPB CR, dCSCA AVR, Onderzoek, Biochemisch laboratorium, LS Pathologie, Sub Biochemie Algemeen, Dep Biochemie en Celbiologie, LS Veterinaire biochemie, Veterinair Pathologisch Diagnostisch Cnt, PB AVM, Applied Veterinary Research, Dep Pathobiologie, Faculteit Diergeneeskunde, LS Interne geneeskunde, Kruitwagen, Hedwig S., Oosterhoff, Loes A., Vernooij, Ingrid G W H, Schrall, Ingrid M., van Wolferen, Monique E., Bannink, Farah, Roesch, Camille, van Uden, Lisa, Molenaar, Martijn R., Helms, J. Bernd, Grinwis, Guy C.M., Verstegen, Monique M A, van der Laan, Luc J W, Huch, Meritxell, Geijsen, Niels, Vries, Robert R G, Clevers, Hans, Rothuizen, Jan, Schotanus, Baukje A., Penning, Louis C., Spee, Bart, dB&C FR-RMSC FR, dCSCA RMSC-1, dPB CR, dCSCA AVR, Onderzoek, Biochemisch laboratorium, LS Pathologie, Sub Biochemie Algemeen, Dep Biochemie en Celbiologie, LS Veterinaire biochemie, Veterinair Pathologisch Diagnostisch Cnt, PB AVM, Applied Veterinary Research, Dep Pathobiologie, Faculteit Diergeneeskunde, LS Interne geneeskunde, Kruitwagen, Hedwig S., Oosterhoff, Loes A., Vernooij, Ingrid G W H, Schrall, Ingrid M., van Wolferen, Monique E., Bannink, Farah, Roesch, Camille, van Uden, Lisa, Molenaar, Martijn R., Helms, J. Bernd, Grinwis, Guy C.M., Verstegen, Monique M A, van der Laan, Luc J W, Huch, Meritxell, Geijsen, Niels, Vries, Robert R G, Clevers, Hans, Rothuizen, Jan, Schotanus, Baukje A., Penning, Louis C., and Spee, Bart

Published
2017

47. Long-Term Adult Feline Liver Organoid Cultures for Disease Modeling of Hepatic Steatosis

Author

Hubrecht Institute with UMC, CMM Sectie Molecular Cancer Research, Cancer, Regenerative Medicine and Stem Cells, Child Health, Kruitwagen, Hedwig S., Oosterhoff, Loes A., Vernooij, Ingrid G.W.H., Schrall, Ingrid M., van Wolferen, Monique E., Bannink, Farah, Roesch, Camille, van Uden, Lisa, Molenaar, Martijn R., Helms, J. Bernd, Grinwis, Guy C.M., Verstegen, Monique M.A., van der Laan, Luc J.W., Huch, Meritxell, Geijsen, Niels, Vries, Robert G., Clevers, Hans, Rothuizen, Jan, Schotanus, Baukje A., Penning, Louis C., Spee, Bart, Hubrecht Institute with UMC, CMM Sectie Molecular Cancer Research, Cancer, Regenerative Medicine and Stem Cells, Child Health, Kruitwagen, Hedwig S., Oosterhoff, Loes A., Vernooij, Ingrid G.W.H., Schrall, Ingrid M., van Wolferen, Monique E., Bannink, Farah, Roesch, Camille, van Uden, Lisa, Molenaar, Martijn R., Helms, J. Bernd, Grinwis, Guy C.M., Verstegen, Monique M.A., van der Laan, Luc J.W., Huch, Meritxell, Geijsen, Niels, Vries, Robert G., Clevers, Hans, Rothuizen, Jan, Schotanus, Baukje A., Penning, Louis C., and Spee, Bart

Published
2017

48. Isolation and Culture of Primary Endothelial Cells from Canine Arteries and Veins

Author

Oosterhoff, Loes A, Kruitwagen, Hedwig S, Spee, Bart, van Steenbeek, Frank G, dCSCA RMSC-1, dCSCA AVR, dCSCA RMSC-1, and dCSCA AVR

Subjects
0301 basic medicine, CD31, Umbilical Veins, Pathology, medicine.medical_specialty, vasculature, Endothelium, Angiogenesis, Endothelial cells, General Chemical Engineering, Myocytes, Smooth Muscle, Primary Cell Culture, Cell Separation, Umbilical Arteries, Umbilical vein, General Biochemistry, Genetics and Molecular Biology, angiogenesis, 03 medical and health sciences, Dogs, Vasculogenesis, medicine.artery, Issue 117, medicine, Animals, Myocyte, Cells, Cultured, General Immunology and Microbiology, business.industry, General Neuroscience, Umbilical artery, 030104 developmental biology, medicine.anatomical_structure, Cell culture, dog, cardiovascular system, Medicine, Endothelium, Vascular, Medicine, Issue 117, Endothelial cells, vasculature, isolation, angiogenesis, dog, CD31, business, isolation
Abstract

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study vasculogenesis, (tumor) angiogenesis, and atherosclerosis. The current standard for in vitro research on human endothelial cells (ECs) is the use of Human Umbilical Vein Endothelial Cells (HUVECs) and Human Umbilical Artery Endothelial Cells (HUAECs). For canine endothelial research, only one cell line (CnAOEC) is available, which is derived from canine aortic endothelium. Although currently not completely understood, there is a difference between ECs originating from either arteries or veins. For a more direct approach to in vitro functionality studies on ECs, we describe a new method for isolating Canine Primary Endothelial Cells (CaPECs) from a variety of vessels. This technique reduces the chance of contamination with fast-growing cells such as fibroblasts and smooth muscle cells, a problem that is common in standard isolation methods such as flushing the vessel with enzymatic solutions or mincing the vessel prior to digestion of the tissue containing all cells. The technique we describe was optimized for the canine model, but can easily be utilized in other species such as human.

Published
2016

50. Disease Modeling and Gene Therapy of Copper Storage Disease in Canine Hepatic Organoids

Author

Nantasanti, Sathidpak, Spee, Bart, Kruitwagen, Hedwig S, Chen, Chen, Geijsen, Niels, Oosterhoff, Loes A, van Wolferen, Monique E, Pelaez, Nicolas, Fieten, Hille, Wubbolts, Richard W, Grinwis, Guy C, Chan, Jefferson, Huch, Meritxell, Vries, Robert R G, Clevers, Hans, de Bruin, Alain, Rothuizen, Jan, Penning, Louis C, Schotanus, Baukje A, Nantasanti, Sathidpak, Spee, Bart, Kruitwagen, Hedwig S, Chen, Chen, Geijsen, Niels, Oosterhoff, Loes A, van Wolferen, Monique E, Pelaez, Nicolas, Fieten, Hille, Wubbolts, Richard W, Grinwis, Guy C, Chan, Jefferson, Huch, Meritxell, Vries, Robert R G, Clevers, Hans, de Bruin, Alain, Rothuizen, Jan, Penning, Louis C, and Schotanus, Baukje A

Abstract

The recent development of 3D-liver stem cell cultures (hepatic organoids) opens up new avenues for gene and/or stem cell therapy to treat liver disease. To test safety and efficacy, a relevant large animal model is essential but not yet established. Because of its shared pathologies and disease pathways, the dog is considered the best model for human liver disease. Here we report the establishment of a long-term canine hepatic organoid culture allowing undifferentiated expansion of progenitor cells that can be differentiated toward functional hepatocytes. We show that cultures can be initiated from fresh and frozen liver tissues using Tru-Cut or fine-needle biopsies. The use of Wnt agonists proved important for canine organoid proliferation and inhibition of differentiation. Finally, we demonstrate that successful gene supplementation in hepatic organoids of COMMD1-deficient dogs restores function and can be an effective means to cure copper storage disease.

Published
2015

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