Martin Hoogduijn is a medical biologist and did his PhD on skin pigmentation at the University of Bradford, UK, before working as a postdoc on mesenchymal stem cells and bone regeneration at the University of York, UK. Since 2006, he is employed at the Erasmus Medical Center Transplant Institute, Department of Internal Medicine, in Rotterdam, The Netherlands. His research focuses on kidney regeneration. Research projects include studies to the biology of mesenchymal stem cells and use of these cells for immune regulatory and regenerative therapy for kidney (transplant) patients. An increasing part of his team is studying the differentiation of pluripotent stem cells into kidney organoids. These kidney organoids are used as a kidney disease model, for drug testing and for replacement of specific kidney functions, in particular the hormonal function of the kidney. More translational work involves studies to the physiology of transplant kidneys on machine perfusion. All these studies are carried out in collaboration with researchers from various departments of the Erasmus Medical Center and with international collaborators.
Generation of EPO-overexpressing kidney organoids by DNA vectors
Zhaoyu Du1, Amanda Bas-Cristóbal Menéndez2, Manuela Urban3, Thierry PP van den Bosch4, Marian Clahsen-van Groningen4, Joost Gribnau5, Marlies EJ Reinders1, Carla C Baan1, Richard P Harbottle3, Martin Hoogduijn1.
1Erasmus MC Transplant Institute, Department of Internal Medicine, Erasmus University Medical Center, Rotterdam, Netherlands; 2Department of Pediatrics, Sophia Children’s Hospital, Erasmus University Medical Center, Rotterdam, Netherlands; 3DNA Vectors Lab, German Cancer Research Center (DKFZ), Heidelberg, Germany; 4Department of Pathology, Erasmus University Medical Center, Rotterdam, Netherlands; 5Department of Developmental Biology and iPS Core Facility, Erasmus University Medical Center, Rotterdam, Netherlands
Background: Human induced pluripotent stem cell (iPSC) derived kidney organoids are a tool for studying kidney development, physiology and disease. The kidney plays an important endocrine role, including the secretion of erythropoietin (EPO) under hypoxia. We aimed to generate stable EPO-overexpressing kidney organoids by using DNA vectors to study the effects of EPO on kidney development and to examine the physiological effects of EPO-producing organoids.
Methods: A scaffold matrix attachment region (S/MAR) DNA vector was used to genetically modify iPSC, which induced robust and persistent expression of EPO at all stages of differentiation. The EPO-overexpressing (EPO-oe) organoids were characterized using markers including PODXL (podocyte), Villin-1 (proximal tubule) and E-cadherin (distal tubule). To study whether EPO-oe organoids had physiological effects, EPO-oe kidney organoids were subcutaneously implanted in immunodeficient mice and mouse hematocrit levels were measured after 1 month.
Results: In control organoids, EPO was undetectable at the transcript and protein levels. After 24 hours in hypoxia (1% O2), EPO was detected at 0.43 (interquartile range [IQR] 0.34-0.59) mIU/ml in the supernatant. This low level of EPO was inadequate for functional studies. EPO-oe kidney organoids produced constitutive high levels of EPO mRNA expression for over 25 days. EPO levels in the supernatant were 933 mIU/ day per EPO-oe kidney organoid. Immunohistochemistry staining showed that EPO overexpressing kidney organoids formed similar kidney structures compared to control organoids and the expression of the markers PODXL, Villin-1 and E-cadherin were not changed. One month after implantation of organoids in mice, hematocrit levels were significantly increased in mice that received EPO-oe organoids (0.48, IQR 0.44-0.55) compared to mice that received control organoids (0.40, IQR 0.40-0.41) (p=0.0092, n=9). Meanwhile, implanted EPO-oe organoids showed 6.5-fold higher CD31 expression than control organoids and displayed endothelial structures with an elongated morphology.
Conclusions: S/MAR DNA vectors can be used to generate robust and stable transgene expression in iPSC throughout their differentiation into kidney organoids. EPO-oe human kidney organoids showed high levels of sustained and functional EPO release. The implantation of EPO-oe organoids illustrates the potential of using human gene-edited iPSC-derived kidney organoids for cell therapy.
