Erythropoietin delivery through kidney organoids engineered with an episomal DNA vector

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Du, A. Bas-Cristóbal Menéndez, M. Urban, A. Hartley, D. Ratsma, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5534834/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 12 Apr, 2025 Read the published version in Stem Cell Research & Therapy → Version 1 posted 5 You are reading this latest preprint version Abstract Background The kidney's endocrine function is essential for maintaining body homeostasis. Erythropoietin (EPO) is one of the key endocrine factors produced by the kidney, and kidney disease patients frequently experience anemia due to impaired EPO production. In the present study we explored the potential of human induced pluripotent stem cell (iPSC)-derived kidney organoids to restore EPO production. Methods EPO secretion by kidney organoids was examined under 1% and 20% oxygen levels. To increase the EPO secreting capacity of kidney organoids, iPSC were genetically engineered with a non-integrating scaffold/matrix attachment region (S/MAR) DNA vector containing the EPO gene and generated EPO-overexpressing (EPO+) kidney organoids. To assess the physiological effects of EPO + organoids, 2–8 organoids were implanted subcutaneously in immunodeficient mice. Results Kidney organoids produced low amounts of EPO under 1% oxygen. EPO S/MAR DNA vectors persisted and continued to robustly express EPO during iPSC expansion and kidney organoid differentiation without interfering with cellular proliferation. EPO + iPSC demonstrated efficient differentiation into kidney organoids. One-month post-implantation, EPO + organoids displayed continuously elevated EPO mRNA levels and significantly increased endothelial cell numbers compared to control organoids. Hematocrit levels were notably elevated in mice implanted with EPO + organoids in an organoid number-dependent manner. EPO + organoids furthermore influenced bone homeostasis in their hosts, evidenced by a change in trabecular bone composition. Conclusion Kidney organoids modified by EPO S/MAR DNA vector allow stable long-term delivery of EPO. The observed physiological effects following the implantation of EPO + organoids underscore the potential of gene-edited kidney organoids for endocrine restoration therapy. DNA vector Erythropoietin Kidney Organoids Pluripotent stem cells Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Chronic kidney disease (CKD) is characterized by a decreased glomerular filtration rate (GFR) and, in addition, by a decline of endocrine function of the kidney [ 1 , 2 ]. The kidney is the main source of EPO, and CKD-induced fibrosis leads to irreversible loss of renal EPO-producing cells. This results in insufficient EPO production and to complications such as anemia and disturbed bone homeostasis [ 3 – 6 ]. Repeated injection of recombinant human EPO and its derivatives has been shown to be effective in preventing anaemia and has been used as a treatment for decades [ 7 , 8 ]. However, fixed-dose EPO supplementation causes gastroenterological problems in up to 30% of patients, is associated with increased risk for hypertension and thromboembolism [ 9 ], and negatively impacts bone mineral density [ 10 ]. Prolyl-hydroxylase inhibitors are a novel alternative to elevate EPO levels [ 11 ], but their actions are not restricted to the kidney, which may cause adverse effects. Regenerative therapies based on the differentiation of induced pluripotent stem cells (iPSC) may represent a curative approach for restoring EPO-producing capacity. Human iPSC-derived kidney organoids contain self-organized 3D nephron-like structures surrounded by renal stromal cells [ 12 ]. It has recently been demonstrated that iPSC can be differentiated in renal interstitial progenitor cells that develop in EPO-producing cells [ 13 ], and we showed previously that kidney organoids have endocrine capacity and contain pericyte-like cells that produce renin in response to cAMP stimulation [ 14 ]. Furthermore, kidney organoids have the ability to convert inactive 25-hydroxyvitamin D3 to its active form [ 15 ]. Upon implantation under the kidney capsule, subcutaneously, or intracelomic in chicken embryos, kidney organoids become vascularized and maintain their kidney-like phenotype and endocrine function, highlighting their potential for restoring the endocrine function of the kidney [ 14 , 16 , 17 ]. However, due to their small size kidney organoids are unlikely to be able to produce effective doses of hormones. The use of gene editing can potentially overcome this limitation. For such therapy to make an impact, it would have to induce efficient and long-term gene overexpression in a safe manner. In the present proof-of-concept study, we used an episomal DNA-vector platform comprising a scaffold/matrix attachment region (S/MAR) [ 18 , 19 ], to generate EPO-overexpressing (EPO+) iPSC and differentiate them into kidney organoids. S/MAR vectors do not integrate in the genome and are therefore an attractive tool for potential therapeutic applications where the risk of insertional mutagenesis is a concern. It is however not known whether S/MAR vectors can provide long-term stable gene expression. In the present study we investigated the stability of EPO S/MAR DNA vector expression and its effect on kidney organoid morphology and examined the physiological effects of EPO + kidney organoids on hematocrit and bone composition after subcutaneous implantation in mice. Methods Human iPSC culture Human iPSC were generated from human primary skin fibroblasts isolated from human skin that became available as rest material, as approved by the Medical Ethics Committee of the Erasmus University Medical Center at the time of iPSC generation (MEC-2017-248) [ 20 ]. iPSC were cultured on Geltrex LDEV-Free hESC-qualified basement membrane matrix (Gibco, USA) using complete Essential 8 medium (Gibco, USA). Cells were refreshed daily and dissociated as cell aggregates using 0.5mM UltraPure EDTA (Invitrogen, USA) for routine passaging every three days. The split ratios were between 1:5 and 1:10 to reach 20 ~ 30% seeding density. DPBS without calcium and magnesium (Gibco, NL) was used for cell rinsing before dissociation. DNA vector transfection A CAG-GFP/hEPO-S/MAR DNA vector was designed, and iPSC were transfected using a protocol that was adapted from earlier studies [ 18 ]. In brief, 24h after single-cell plating of iPSC, medium was refreshed and a transfection mix consisting of 5µL lipofectamine stem (Invitrogen, USA) and 2000ng DNA vector was added. Cells were refreshed daily and routinely passaged for 6 ~ 8 days, after which GFP + cells were enriched by FACS using a FACSAria (BD Biosciences, USA). A second cell sorting for GFP + cells was performed three weeks later. Cells were then expanded and cryopreserved at -150°C. Kidney organoid differentiation Human iPSC-derived kidney organoids were generated based on existing protocols [ 21 , 22 ]. iPSC were dissociated into single cells by TrypLE Select Enzyme (Gibco, DK), and 300k cells were seeded per well of a 6-well plate and cultured overnight in Essential 8 medium supplemented with 1x Revitacell (Gibco, USA). The next day when the cells were stably attached, Essential 8 media was replaced by advanced RPMI 1640 media (Gibco, NL), supplemented with GlutaMax (Gibco, UK) and 100U/ml Penicillin-Streptomycin. 8µM of the GSK-3 inhibitor CHIR-99021 (Tocris, UK) was added for 3 days with daily refreshment. Subsequently, cells were cultured in advanced RPMI 1640 media supplemented with 200ng/ml recombinant human Fibroblast growth factor 9 (FGF9) (Peprotech, USA), 10ng/ml Activin A (R&D system, USA) and 1 µg/ml heparin sodium salt (Sigma Aldrich, USA) for 1 day. The next day, the cells were dissociated into single cells by TryplE Select (Gibco, DK), and resuspended in advanced RPMI 1640 supplemented with 3µM CHIR-99021, 200ng/ml FGF9, and 1µg/ml heparin. The cell suspension was adjusted to 500,000 cells per 150µl, and 150µl suspension was pipetted in each well of a 96-well conical (V) bottom plate (Nunc, DK). The plates were centrifuged at 300×g for 3 min and kept in the incubator for 48h without refreshing. After this period of self-aggregation, the cell spheroids were carefully transferred on to 0.4µm PET membrane inserts (cellQART, DE), and advanced RPMI 1640 with 3µM CHIR, 200ng/ml FGF9, and 1µg/ml heparin was added to the base of the inserts, so that the organoids were at an air–liquid interface. The next day, media was replaced with advanced RPMI 1640 with 200ng/ml FGF9 and 1 µg/ml heparin and refreshed every other day for 4 days. Finally, medium was replaced by advanced RPMI 1640 without further additions and refreshed every other day for 8 days. Real time RT-PCR Total RNA from iPSC and kidney organoids was isolated with RNeasy Kits (Qiagen, Germany). Complementary DNA (cDNA) was synthesized using Moloney Murine Leukemia Virus Reverse Transcriptase Kit (Invitrogen, USA), random primers (Promega, USA) and RNasin® Ribonuclease Inhibitor (Promega, USA). TaqMan Gene Expression Master Mix (Applied Biosystems, USA) and TaqMan Gene Expression Assays (Life technologies, USA) (Table 1) were used for the RT-PCR reaction. CT values were measured by a StepOnePlus Real-Time PCR System (Applied Biosystems, Singapore). EPO measurement EPO was measured by Access® Erythropoietin chemiluminescence immunoassay (Beckman Coulter, CA, USA) on the Access 2 Immunoassay System (Beckman Coulter, CA, USA). Once a month, three external controls from UK NEQAS were measured for inter-laboratory standardization. The detection range of the immunoassay is 0.5–750 mIU/mL. Western blot analysis Organoid pellets were lysed in ice-cold radioimmunoprecipitation assay buffer (RIPA buffer) under constant agitation for 30 min. A small volume of protein lysate was used to perform a BCA protein-assay (Thermo Scientific, USA) to determine protein concentration. The rest of the samples were mixed with 4 × loading buffer (Bio-rad, USA) and denatured. 25ug of each sample was loaded into 4–15% Mini-PROTEAN® TGX™ Precast Protein Gels (Bio-rad, USA) and proteins separated by molecular weight. Using Trans-blot Turbo System (Bio-rad, USA) at 1.3A up to 25V for 7min, proteins were transferred to Immun-blot PVDF membrane (Bio-rad, USA). After blocking in Tris-buffered 5% non-fat milk, membranes were incubated with anti-HIF2α monoclonal antibody (1:1000 dilution, NB100-132, Novus, USA) or anti-β-actin monoclonal antibody (1:5000 dilution, Cell signaling technology, USA). Horseradish peroxidase (HRP) conjugated anti-mouse IgG secondary antibody and chemiluminescence detection kits (Bio-rad, USA) were used for signal detection. The quantification was performed in Image J2 version: 2.9.0/1.53t (NIH, USA). Immunohistochemical (IHC) and multiplex immunofluorescence staining Immunohistochemistry was performed on a BenchMark ULTRA IHC/ISH System using ultraView (UV) Universal DAB Detection Kit (#760 − 600) or optiView (OV) Universal DAB detection Kit (#760 − 700, all Ventana Medical System, USA). After deparaffinization, rehydration and antigen retrieval with CC1 (#950 − 500, Ventana), the tissue were incubated with antibody of interest (Table 2). Incubation was followed by optiview detection and hematoxylin II counter stain for 8 minutes followed by a blue coloring reagent for 8 minutes according to the manufactures instructions (Ventana). For triple immunofluorescence staining of Podocalyxin-like protein 1 (PODXL), VILLIN1 and E-cadherin (ECAD) the Discovery Ultra (Ventana) was used. In brief, following deparaffinization and heat-induced antigen retrieval with CC1 (#950 − 224, Ventana) for 32 min, anti-PODXL was incubated for 32 min at 37°C followed by omnimap anti-rabbit HRP (#760–4311, Ventana) and detection with Cy5 (#760 − 238, Ventana) for 8 min. An antibody denaturation step was performed with CC2 (#950 − 123, Ventana) at 100°C for 20 min. Secondly, incubation with anti-ECAD was performed for 20 min at 37°C, followed by omnimap anti-mouse HRP (#760–4310, Ventana) and detection with with Red610 (#760 − 245, Ventana). An antibody denaturation step followed with CC2 at 100°C for 20 min. Thirdly, anti-Villin1 was incubated for 32 min at 37°C, followed by omnimap anti-rabbit HRP (#760–4311, Ventana) and detection with detection with FAM (#760 − 243, Ventana). Finally, slides were washed in phosphate-buffered saline and mounted with Vectashield containing 4’,6-diamidino-2-phenylindole (Vector laboratories, Peterborough, UK). Slides were imaged with Axioscan Zeiss. Images were analyzed using QuPath software [ 23 ]. A first threshold was set to determine organoid area using the average channels. Thereafter, color deconvolution was used to determine positive staining, followed by thresholding. Results were obtained as positive percentage of total organoid area. Subcutaneous implantation of kidney organoids Immuno-deficient mice Rag2 −/− /IL2rγ −/− double knock out (BALB/c) of 8–15 weeks old were acclimatized for at least 1 week before start of the experiments. For organoids implantation, inhaled anesthesia was induced by 4–5% isoflurane and maintained by 1–2% isoflurane with 500-600ml/min oxygen. Two kidney organoids were encapsuled in 40µl semisolid Geltrex and placed subcutaneously through 0.5cm skin incisions into the flanks. To study whether EPO + organoids had a dose-dependent effect, animals were implanted with 2, 4, 6 or 8 EPO + organoids. Control animals received either no organoids or control organoids. Animals were randomly allocated to the groups in a no blinded fashion, but male and female mice were equally distributed over the groups to control for potential sex differences. For the group receiving 8 organoids, 10 animals received EPO + organoids and 9 animals received control organoids. In total 33 animal were used. Wounds were closed with 2 or 3 sutures. Mice were checked daily and weighed weekly. Four weeks after organoid implantation day, blood was collected via cardiac puncture under isofluran anesthesia followed by euthanasia through cervical dislocation. Organoids were retrieved and snap frozen in liquid nitrogen for RT-PCR analysis or fixed in 4% paraformaldehyde for histological analysis. Long bones were collected for microcomputed tomography (µCT) analysis or bone marrow was collected and snap frozen in liquid nitrogen for RT-PCR analysis. The work has been reported in line with the ARRIVE guidelines 2.0 [ 24 ]. Whole blood analysis The whole blood of the mice was transferred and mixed in MiniCollect® K3EDTA tubes (Greiner, Austria). Within 4 hours after collection, two 50µl aliquots of blood from each mouse were measured by automated hematology analyzer XP-300 (Sysmex, Japan) using the whole blood mode to determine hematocrit levels. Microcomputed tomography (µCT) analysis Femurs were scanned at a resolution of 9 µm, using a SkyScan 1076 system (Bruker, Kontich, Belgium) in a blinded fashion. According to the published guidelines [ 25 ], the following settings were used: X-Ray power and tube current were 40 kV and 250 µA, respectively. Beam hardening was reduced using a 1 mm aluminium filter, exposure time was 2.3 seconds, and an average of three pictures was taken at each angle with steps of 0.8° to generate final images. Segmentation of the reconstructed images was done on basis of global thresholding. Using software packages from Bruker (NRecon, CtAn, and Dataviewer), bone microarchitecture parameters were assessed in trabecular and cortical bones of all mice. The trabecular bone parameters trabecular bone volume fraction (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), trabecular separation (Tb.Sp), trabecular patterning factor (Tb.Pf) and structure model index (SMI; 0 = plate-like, 3 = rod-like) were determined in the distal metaphysis of the femur (region of interest (ROI) of 0.9 mm from distal growth plate towards diaphysis). Statistical analysis The unpaired t-test with Welch’s correction was used to test the RT-PCR results for significance. The Mann-Whitney test was used to test the quantifications of the immunohistochemical staining for significance, as this data was not normally distributed. Statistical significance was considered for p values less than 0.05 (*), 0.01 (**) and 0.001 (***). Data analysis was performed using GraphPad Prism version 8.0.0 (GraphPad Software, La Jolla, USA). Results Kidney organoids are capable of producing EPO under hypoxia. iPSC-derived kidney organoids generated at an air-liquid interface contained PODXL expressing glomerular structures, VILLIN expressing proximal tubular structures, and ECAD expressing distal tubular structures (Fig. 1A-C). In addition, kidney organoids contained PDGFRβ + interstitial cells situated adjacent to the nephron structures (Fig. 1D), which marks the EPO-producing cells in the kidney [ 26 ]. EPO expression is driven by oxygen-sensitive hypoxia-inducible transcription factor 2 alpha (HIF2α) and we observed that culture of kidney organoids under hypoxia (1% oxygen) for four hours triggered the stabilization of HIF2α (Fig. 1E and Supplementary Fig. 1). This was accompanied by an increased release of EPO from levels under the detection limit at 20% oxygen to 0.20 mIU/organoid/day at 1% oxygen (Fig. 1F). These results demonstrate that kidney organoids have the capacity to secrete EPO. The amounts of EPO secreted per kidney organoid are however very low considering normal EPO concentrations range between 4–26 mIU/ml blood. EPO productin by organoids would therefore be insufficient to compensate for the loss of EPO-production capacity in kidney disease. We thus proceeded to increase the EPO production capacity of organoids through the use of DNA vectors. EPO + kidney organoids show stably elevated EPO expression. To increase the EPO-producing capacity of kidney organoids, iPSC were genetically engineered with S/MAR DNA vectors carrying the human EPO and GFP genes (EPO + iPSC) (Fig. 2A). GFP expression was maintained throughout the iPSC expansion period of 67 days (Fig. 2B). 0.5 million EPO + iPSC released 1398 mIU/day of EPO, which is more than 4500 times higher than the EPO release rate of control iPSC (Fig. 2C). GFP-reported expression of the DNA vector was preserved after differentiation of iPSC into kidney organoids (Fig. 2D), and EPO + kidney organoids produced strongly elevated levels of EPO mRNA compared with control organoids (Fig. 2E). This resulted in highly elevated EPO release rates in EPO + organoids (1204 mIU/day/organoid) compared to control organoids (0.006 mIU/day/organoid) (Fig. 2F). EPO + kidney organoids develop nephron structures. To determine whether EPO overexpression affected kidney organoid development, mRNA expression analysis of key nephron markers was performed. mRNA expression levels of the podocyte marker PODXL, the proximal tubular marker VILLIN-1 and the endothelial cell marker PECAM1 were not altered in EPO + organoids. However, the distal tubular marker ECAD was downregulated in EPO + organoids compared to control organoids (Fig. 3A). Immunohistochemistry confirmed the development of nephron structures in EPO + organoids (Fig. 3B). Quantification of immunohistochemical staining demonstrated increased PODXL + structures in EPO + organoids, whereas there was a significant decrease in ECAD + structures in EPO + kidney organoids (Fig. 3C). Interestingly, the area of CD31+ (PECAM1 gene) endothelial cells was increased from 1.7–4.9% in EPO + kidney organoids. EPO + kidney organoids show enhanced vascularization following implantation. To investigate the physiological effects of EPO + kidney organoids, 2, 4, 6, or 8 organoids were implanted subcutaneously in the flanks of immunodeficient mice and retrieved after 4 weeks (Fig. 4A). Kidney organoids were connected to the vascular system of the host mice, as evidenced by the macroscopically visible growth of blood vessels of the skin into the organoids (Fig. 4B). EPO + organoids continued to express high levels of EPO mRNA 4 weeks after implantation (Fig. 4C). Immunohistochemical staining showed that nephron structures were maintained in the implanted organoids, although the interstitial space had expanded (Fig. 4D). We observed no morphological differences in nephron structures between control and EPO + organoids, but there was an increase in CD31 positive structures in the EPO + organoids (Fig. 4D-E). Human CD31 showed an increased area staining from 0.10% in control organoids to 0.48% in EPO + organoids, indicating an increase in endothelial structures in EPO + organoids. This was supported by increased mRNA expression of PECAM-1 in EPO + organoids (Fig. 4F). In addition, PODXL mRNA expression was increased in EPO + organoids. EPO + organoids increase hematocrit levels. Four weeks after kidney organoid implantation, hyperemic ears were observed in mice that received EPO + organoids (Fig. 5A). In addition, the spleen size of mice that received EPO + organoids were increased in comparison to that of mice implanted with control organoids (Fig. 5B). There was no effect of EPO + organoids on body weight (Supplementary Fig. 2A). Whole blood analysis demonstrated that hematocrit levels in mice implanted with EPO + organoids were increased. Increases in hematocrit were positively correlated with the number of implanted EPO + organoids (Supplementary Fig. 2B). Mice implanted with eight EPO + organoids showed a significant increase in hematocrit levels from 0.40 L/L to 0.59 L/L compared to mice implanted with control organoids (Fig. 5C). Hematocrit levels in mice implanted with control organoids did not differ from mice that had no organoids implanted. These results demonstrate that EPO production by human kidney organoids has a physiological effect on hosts. EPO + organoids affect trabecular bone structure. As EPO supplementation is known to affect bone homeostasis, we investigated whether organoid-derived EPO affected bone composition in host mice. We observed no difference in bone volume fraction (BV/TV%), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp) or trabecular number (Tb.N) between mice implanted with 8 control organoids or EPO + organoids (Fig. 6A). However, the trabecular bone pattern factor (Tb.Pf) and the structure model index (SMI) were decreased in the EPO + organoid group, indicating a more dense trabecular spatial structure, which suggests an increased bone strength. Cortical bone parameters were not different between the groups. Interestingly, Fgf23 mRNA expression was increased in bone marrow of the EPO + organoid group (Fig. 6B). FGF23 acts on the kidney to increase phosphate excretion and suppress 1,25(OH) 2 vitamin D synthesis. The increased FGF23 expression thus further demonstrates that EPO + organoids affect endocrine systems in the host. Representative images of a femoral metaphysis of a control mouse and an EPO + mouse are shown in Fig. 6C. Discussion We demonstrated that S/MAR DNA vector technology applied to human iPSC-derived kidney organoids enables the generation of EPO-overexpressing organoids that demonstrate long-term EPO secretion capacity, evidenced by physiological effects on host mice after implantation. These results show for the first time the potential of such a platform to restore the renal EPO endocrine system in kidney disease. By using episomal DNA vectors, we aimed to cause minimal impact on cellular processes by reducing innate immunity and avoiding random genomic integration [ 27 ], and furthermore to provide stable expression of EPO in human iPSC. Throughout iPSC expansion, differentiation of kidney organoids and subsequent implantation in mice, EPO was stably expressed. EPO overexpression enhanced the development of podocyte-like cells and endothelial cells during in vitro kidney organoid differentiation, while it reduced the formation of distal tubular cells. The enhanced presence of podocyte-like and endothelial cells was still evident in organoids retrieved from host mice, but a difference in distal tubular cells was no longer observed. This data indicates that high levels of EPO in developing kidney organoids impact cellular differentiation. This is interesting as the EPO receptor is widely expressed in fetal tissue [ 28 ]. Kidney organoids thus represent a relevant model to study potential therapeutic or adverse effects of EPO in the developing kidney. The increased numbers of endothelial cells in EPO + organoids is of interest for vascularization purposes. We and others have demonstrated that organoid-derived endothelial cells contribute, together with host-derived endothelial cell, to the formation of endothelial structures in kidney organoids after implantation in animal models [ 14 , 16 ]. Increased presence of endothelial cells may enhance the speed of vascularization and improved branching of vascular structures within the organoids. This may lead to a decreased period of anoxia in organoids after implantation, and a more functional endocrine interaction of organoids with the host. The physiological effects of EPO + kidney organoids were obvious in our model. Hematocrit levels were strongly elevated in an organoid number-dependent manner and spleens were enlarged. EPO + kidney organoids furthermore had a beneficial effect on trabecular bone structure and predicted bone strength. In our model, human EPO efficiently interacted with the mouse EPO receptor. EPO and EPO receptors are evolutionarily conserved, however their interaction might be less efficient in a cross-species setting [ 29 ]. It is therefore not unlikely that in a fully human setting, the effects of EPO + organoids may be even more efficient than in our model. This becomes relevant when determining how many organoids are needed to support the renal EPO production. In our model, EPO production in kidney organoids was constitutive and not cell type restricted, which is a limitation of the study. Uncontrolled increase in hematocrit levels would not be suitable in a clinical setting as it can lead to coagulation. Therefore future studies shall focus on generation of regulatable EPO-producing kidney organoids. EPO-producing cells in the kidney can be identified through their expression of platelet-derived-growth-factor-receptor-β (PDGFRβ) and reside around the peritubular capillaries, which is one of the most oxygen-deprived sites of the body [ 30 ]. When oxygen levels at this site drop below a certain threshold, the transcription factor hypoxia-inducible-factor-2α is activated in these cells and stimulates EPO production. To truly mimic EPO regulation of the kidney it is instrumental to restrict EPO secretion to PDGFRβ + cells, and to place these cells at a site with a similar morphology as the kidney. Kidney organoids provide such a morphology. Furthermore, DNA vectors could be designed to be active exclusively in PDGFRβ + cells. Future studies should in addition focus on the long-term effects of EPO + organoids. The present study was limited to four weeks, but is is essential to evaluate whether S/MAR vector-mediated EPO production remains functional after months or years. Furthermore, it is essential to examine whether changes in organoid size or cellular composition over time occur and affect EPO release. Finally, future research should study the effects of EPO + organoids in an anemic model to examine whether organoid-derived EPO is sufficient to restore failing EPO production by the host. Conclusion In conclusion, the generation of safe, specific, and effective EPO-producing kidney organoids is a promising tool for the restoration of EPO production capacity in CKD. The present manuscript demonstrates the potential of such therapy and indicates the issues that require further research. Abbreviations BV/TV Bone volume fraction CKD Chronic kidney disease is characterized by a decreased ECAD E-cadherin EPO Erythropoietin EPO+ Erythropoietin overexpression FGF9 Fibroblast growth factor 9 FGF23 Fibroblast growth factor 23 GFR glomerular filtration rate HIF2α hypoxia-inducible transcription factor 2 alpha iPSC induced pluripotent stem cell PDGFRβ platelet-derived-growth-factor-receptor-β Podocalyxin-like protein 1 PODXL S/MAR scaffold/matrix attachment region SMI structure model index Tb.N trabecular number Tb.Pf trabecular bone pattern factor Tb.Sp trabecular separation Tb.Th trabecular thickness Declarations Ethics approval and consent to participate iPSC were generated from human donors as part of the project ‘Creation of disease model systems to understand and correct genetic diseases through gene or other therapy using iPS cell derived from somatic cells: IPSC protocol Rotterdam’ approved by the Erasmus MC Medical Ethical Committee under approval number MEC-2017-248, which was published 29 th March 2018 and last updated 15 th April 2024. Ethics statement on animal experiments The animal studies were performed after receiving approval of the Dutch central committee animal experiments under licence AVD101002016635. Consent for publication Not applicable Availability of data and materials The raw data files belonging to this manuscript are assessable on DataverseNL via https://doi.org/10.34894/GZPSB6. Competing interests The authors have no financial or personal relationships with other people or organisations that could inappropriately influence (bias) their work. Funding Z. Du is funded by a grant of the Chinese Scholarschip Council. Use of Artificial Intelligence (AI) The authors declare that they have not use AI-generated work in this manuscript. Author contributions Z. Du: Conception and design, collection and/or assembly of data, data analysis and interpretation, manuscript writing A. Bas-Cristóbal Menéndez: Collection and/or assembly of data, data analysis and interpretation M. Urban : Collection and/or assembly of data, data analysis and interpretation A. Hartley: Collection and/or assembly of data, data analysis and interpretation D. Ratsma: Collection and/or assembly of data, data analysis and interpretation M. Koedam: Collection and/or assembly of data T.P.P. van den Bosch: Collection and/or assembly of data, data analysis and interpretation M. Clahsen-van Groningen: Final approval of manuscript J. Gribnau: Provision of study material, final approval of manuscript J. Mulder: Final approval of manuscript M.E.J. Reinders: Final approval of manuscript C.C. Baan: Final approval of manuscript B. van der Eerden: Data analysis and interpretation, final approval of manuscript R.P. Harbottle: Conception and design, provision of study material, data analysis and interpretation, final approval of manuscript M.J. Hoogduijn: Conception and design, data analysis and interpretation, manuscript writing References Babitt JL, Lin HY. Mechanisms of anemia in CKD [in eng]. 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Nature. 2015;526(7574):564–8. Tsujimoto H, Hoshina A, Mae SI, et al. Selective induction of human renal interstitial progenitor-like cell lineages from iPSCs reveals development of mesangial and EPO-producing cells [in eng]. Cell Rep. 2024;43(2):113602. Shankar AS, Du Z, Mora HT, et al. Human kidney organoids produce functional renin [in eng]. Kidney Int. 2021;99(1):134–47. Shankar AS, van den Berg SAA, Tejeda Mora H, et al. Vitamin D metabolism in human kidney organoids [in eng]. Nephrol Dial Transpl. 2021;37(1):190–3. van den Berg CW, Ritsma L, Avramut MC, et al. Renal Subcapsular Transplantation of PSC-Derived Kidney Organoids Induces Neo-vasculogenesis and Significant Glomerular and Tubular Maturation In Vivo [in eng]. Stem Cell Rep. 2018;10(3):751–65. Koning M, Lievers E, Jaffredo T et al. Efficient Vascularization of Kidney Organoids through Intracelomic Transplantation in Chicken Embryos [in eng]. J Vis Exp 2023(192). Roig-Merino A, Urban M, Bozza M, et al. An episomal DNA vector platform for the persistent genetic modification of pluripotent stem cells and their differentiated progeny [in eng]. Stem Cell Rep. 2022;17(1):143–58. Bozza M, De Roia A, Correia MP et al. A nonviral, nonintegrating DNA nanovector platform for the safe, rapid, and persistent manufacture of recombinant T cells [in eng]. Sci Adv 2021;7(16). de Esch CE, Ghazvini M, Loos F, et al. Epigenetic characterization of the FMR1 promoter in induced pluripotent stem cells from human fibroblasts carrying an unmethylated full mutation [in eng]. Stem Cell Rep. 2014;3(4):548–55. Garreta E, Prado P, Tarantino C, et al. Fine tuning the extracellular environment accelerates the derivation of kidney organoids from human pluripotent stem cells [in eng]. Nat Mater. 2019;18(4):397–405. Selfa IL, Gallo M, Montserrat N, Garreta E. Directed Differentiation of Human Pluripotent Stem Cells for the Generation of High-Order Kidney Organoids [in eng]. Methods Mol Biol. 2021;2258:171–92. Bankhead P, Loughrey MB, Fernandez JA, et al. QuPath: Open source software for digital pathology image analysis [in eng]. Sci Rep. 2017;7(1):16878. Percie du Sert N, Hurst V, Ahluwalia A, et al. The ARRIVE guidelines 2.0: updated guidelines for reporting animal research [in eng]. BMJ Open Sci. 2020;4(1):e100115. Bouxsein ML, Boyd SK, Christiansen BA, et al. Guidelines for assessment of bone microstructure in rodents using micro-computed tomography [in eng]. J Bone Min Res. 2010;25(7):1468–86. Gerl K, Nolan KA, Karger C, et al. Erythropoietin production by PDGFR-beta(+) cells [in eng]. Pflugers Arch. 2016;468(8):1479–87. Bozza M, Green EW, Espinet E, et al. Novel Non-integrating DNA Nano-S/MAR Vectors Restore Gene Function in Isogenic Patient-Derived Pancreatic Tumor Models [in eng]. Mol Ther Methods Clin Dev. 2020;17:957–68. Juul SE, Yachnis AT, Christensen RD. Tissue distribution of erythropoietin and erythropoietin receptor in the developing human fetus [in eng]. Early Hum Dev. 1998;52(3):235–49. Divoky V, Prchal JT. Mouse surviving solely on human erythropoietin receptor (EpoR): model of human EpoR-linked disease [in eng]. Blood. 2002;99(10):3873–4. author reply 3874–3875. Bauer C, Kurtz A. Oxygen sensing in the kidney and its relation to erythropoietin production [in eng]. Annu Rev Physiol. 1989;51:845–56. Tables Table 1 and 2 are not available with this version. Supplementary Files supplementaryfigure1.jpg Supplementary figure 1. Western blot of HIF2α and Actin in kidney organoids cultured for 4 hours under 20% O2 and 1% O2. Data are quantified in Figure 1E. supplementaryfigure2.jpg Supplementary figure 2. A. Weights of mice implanted with 8 control or 8 EPO+ organoids. B. Hematocrit (HCT) in mice implanted with 4, 6 or 8 EPO+ organoids at 4 weeks after implantation. ARRIVEchecklistEPOorganoidimplantations.docx Cite Share Download PDF Status: Published Journal Publication published 12 Apr, 2025 Read the published version in Stem Cell Research & Therapy → Version 1 posted Reviewers agreed at journal 17 Jan, 2025 Reviewers invited by journal 17 Jan, 2025 Editor assigned by journal 06 Jan, 2025 First submitted to journal 03 Jan, 2025 Editorial decision: Major Revision 09 Dec, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5534834","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":403612210,"identity":"1c7c6695-fc12-4427-90c1-55e758a74479","order_by":0,"name":"Z. Du","email":"","orcid":"","institution":"Erasmus Medical Centre: Erasmus MC","correspondingAuthor":false,"prefix":"","firstName":"Z.","middleName":"","lastName":"Du","suffix":""},{"id":403612211,"identity":"5783fb9b-d3b5-44f3-a135-f6d2a8d7dd31","order_by":1,"name":"A. Bas-Cristóbal Menéndez","email":"","orcid":"","institution":"Erasmus Medical Centre: Erasmus MC","correspondingAuthor":false,"prefix":"","firstName":"A.","middleName":"Bas-Cristóbal","lastName":"Menéndez","suffix":""},{"id":403612212,"identity":"304d7da3-83cf-4cdb-9874-d16a71a6b85f","order_by":2,"name":"M. Urban","email":"","orcid":"","institution":"German Cancer Research Centre: Deutsches Krebsforschungszentrum","correspondingAuthor":false,"prefix":"","firstName":"M.","middleName":"","lastName":"Urban","suffix":""},{"id":403612213,"identity":"d8f1d3a9-6496-469a-bb39-049a896950ee","order_by":3,"name":"A. Hartley","email":"","orcid":"","institution":"German Cancer Research Centre: Deutsches Krebsforschungszentrum","correspondingAuthor":false,"prefix":"","firstName":"A.","middleName":"","lastName":"Hartley","suffix":""},{"id":403612214,"identity":"3a240801-5509-492e-b046-4fc87b642955","order_by":4,"name":"D. Ratsma","email":"","orcid":"","institution":"Erasmus Medical Centre: Erasmus MC","correspondingAuthor":false,"prefix":"","firstName":"D.","middleName":"","lastName":"Ratsma","suffix":""},{"id":403612215,"identity":"ea822ace-c8f3-4a38-bcd1-9a7b98969538","order_by":5,"name":"M. Koedam","email":"","orcid":"","institution":"Erasmus Medical Centre: Erasmus MC","correspondingAuthor":false,"prefix":"","firstName":"M.","middleName":"","lastName":"Koedam","suffix":""},{"id":403612216,"identity":"f9b09c9f-a8b0-4c70-b4a7-278ca609b909","order_by":6,"name":"T.P.P. van den Bosch","email":"","orcid":"","institution":"Erasmus Medical Centre: Erasmus MC","correspondingAuthor":false,"prefix":"","firstName":"T.P.P.","middleName":"van den","lastName":"Bosch","suffix":""},{"id":403612217,"identity":"5529cfa3-0ce2-4486-89d4-930c860d01dc","order_by":7,"name":"M. Clahsen-van Groningen","email":"","orcid":"","institution":"Erasmus Medical Centre: Erasmus MC","correspondingAuthor":false,"prefix":"","firstName":"M.","middleName":"Clahsen-van","lastName":"Groningen","suffix":""},{"id":403612218,"identity":"5d60fee7-a8bd-4ed4-b1e9-bfd5b1b4f12e","order_by":8,"name":"J. Gribnau","email":"","orcid":"","institution":"Erasmus Medical Centre: Erasmus MC","correspondingAuthor":false,"prefix":"","firstName":"J.","middleName":"","lastName":"Gribnau","suffix":""},{"id":403612219,"identity":"04b3f8a5-adf1-494e-bd91-a4ebc5cd920f","order_by":9,"name":"J. Mulder","email":"","orcid":"","institution":"Erasmus Medical Centre: Erasmus MC","correspondingAuthor":false,"prefix":"","firstName":"J.","middleName":"","lastName":"Mulder","suffix":""},{"id":403612220,"identity":"a7ee1e11-b130-479b-8cac-b539b4ccf3d2","order_by":10,"name":"M.E.J. Reinders","email":"","orcid":"","institution":"Erasmus Medical Centre: Erasmus MC","correspondingAuthor":false,"prefix":"","firstName":"M.E.J.","middleName":"","lastName":"Reinders","suffix":""},{"id":403612221,"identity":"820ba0c2-0dc0-4efb-9442-0c04cb1d6ffc","order_by":11,"name":"C.C. Baan","email":"","orcid":"","institution":"Erasmus Medical Centre: Erasmus MC","correspondingAuthor":false,"prefix":"","firstName":"C.C.","middleName":"","lastName":"Baan","suffix":""},{"id":403612222,"identity":"1cb76cf1-a75f-47c6-9d62-850d9b6bafc1","order_by":12,"name":"B. van der Eerden","email":"","orcid":"","institution":"Erasmus Medical Centre: Erasmus MC","correspondingAuthor":false,"prefix":"","firstName":"B.","middleName":"van der","lastName":"Eerden","suffix":""},{"id":403612223,"identity":"58c7e090-fc4a-4cd5-b933-d77865934ef9","order_by":13,"name":"R.P. Harbottle","email":"","orcid":"","institution":"German Cancer Research Centre: Deutsches Krebsforschungszentrum","correspondingAuthor":false,"prefix":"","firstName":"R.P.","middleName":"","lastName":"Harbottle","suffix":""},{"id":403612224,"identity":"82b42707-8698-44eb-84cb-efc42198a55b","order_by":14,"name":"Martin Hoogduijn","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8klEQVRIiWNgGAWjYFACHhDBzMDGcIDxAbK4DDFamA0wxPFqAQI2CaK06DbwHnzw4Zd1Yh/j2WMVH/fY2evOSH+64QfDHZxazA7wJRvO7EtPbGM4l3ZzxrPkxG03csxu9jA8w6OFx0yat+cwUMsZs9s8B5gTzG7ksN1mYDhMnJbiPwfq7c1upD8jrIXnB0QLM8OBw4zbbiSY4ddymMfYcGZDujFQi7Fkz4HjidvOvAH6xQCPluM9hg8+/LGWnT/jjOGHHweq7c2Opz+78aPisBwuLeAYYWwDEhIHkIUNsCpGAn+AmL+BkKpRMApGwSgYqQAA9LtdMVBbnJwAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-0217-8254","institution":"Erasmus Medical Centre: Erasmus MC","correspondingAuthor":true,"prefix":"","firstName":"Martin","middleName":"","lastName":"Hoogduijn","suffix":""}],"badges":[],"createdAt":"2024-11-27 11:19:22","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5534834/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5534834/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13287-025-04282-w","type":"published","date":"2025-04-12T16:04:52+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":74427580,"identity":"964fb432-8c00-48b6-bf88-b441bb78b5b2","added_by":"auto","created_at":"2025-01-22 08:10:40","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1062595,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHuman kidney organoids produce EPO under hypoxia.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Schematic overview of the kidney organoid differentiation procedure. B. Bright-field image of a kidney organoid. C. Immunofluorescence image of a kidney organoid section stained for the glomerular marker PODXL (white), the proximal tubular marker Villin-1 (yellow) and the distal tubular marker E-cadherin (red). D. Immunohistochemical staining of kidney organoid section for PDGFRβ. E. HIF2α protein levels corrected for actin in kidney organoids cultured for 1 hour or 4 hours under 20% O\u003csub\u003e2\u003c/sub\u003e and 1% O\u003csub\u003e2\u003c/sub\u003e. F. EPO release by kidney organoids cultured under 20% O\u003csub\u003e2\u003c/sub\u003e and 1% O\u003csub\u003e2\u003c/sub\u003e. Scale bars represents 100µm.\u003c/p\u003e","description":"","filename":"figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5534834/v1/f4b9c0c63396ab31ca60ec04.jpg"},{"id":74427984,"identity":"40243a1e-3b8f-4a00-8419-7fe5adf87cc5","added_by":"auto","created_at":"2025-01-22 08:18:41","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":667772,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eS/MAR DNA vector induced EPO overexpression.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Schematic overview of the generation of EPO and GFP overexpressing iPSC using S/MAR DNA vectors. B. GFP expression in iPSC over a period of 67 days of expansion. C. EPO release by control and EPO+ iPSC. D. GFP expression in kidney organoid at day 24 of differentiation. E. Relative EPO mRNA expression in control and EPO+ kidney organoids. F. EPO release in culture medium by control and EPO+ kidney organoids.\u003c/p\u003e","description":"","filename":"figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5534834/v1/4a4bc60308b215b97941cb0a.jpg"},{"id":74429125,"identity":"3f8fb261-6000-4af0-bc75-5678e997e255","added_by":"auto","created_at":"2025-01-22 08:26:41","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1491316,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEPO affects kidney organoid differentiation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. mRNA expression of PODXL, Villin-1, E-cadherin and PECAM1 in control organoids and EPO+ organoids. B. Immunohistochemical staining of PODXL, Villin-1, E-cadherin and CD31 (PECAM1) in control organoids and EPO+ organoids. C. Quantification of PODXL, Villin-1, E-cadherin and CD31 immunohistochemistry in control organoids and EPO+ organoids.\u003c/p\u003e","description":"","filename":"figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5534834/v1/efc295ae0468470e47b2ed08.jpg"},{"id":74427982,"identity":"846c1a01-5742-4474-861e-fc0c7f5558eb","added_by":"auto","created_at":"2025-01-22 08:18:41","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1079959,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImplantation of EPO+ organoids\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Cartoon depicting the implantation of control and EPO+ organoids. B. Subcutaneous localization of a kidney organoid 4 weeks after implantation, showing the connection with the dermal vascular system of the host. C. EPO mRNA expression in control and EPO+ kidney organoids 4 weeks after implantation. D. Immunohistochemical staining of PODXL, Villin-1, E-cadherin and CD31 (PECAM1) in control organoids and EPO+ organoids 4 weeks after implantation. E. Quantification of PODXL, Villin-1, E-cadherin and CD31 immunohistochemistry in control organoids and EPO+ organoids. F. mRNA expression of PODXL, Villin-1, E-cadherin and PECAM1 in control organoids and EPO+ organoids 4 weeks after implantation.\u003c/p\u003e","description":"","filename":"figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5534834/v1/98ebdce28df8b46834c18ee9.jpg"},{"id":74427583,"identity":"0b391729-b00a-4321-af8f-a7b910cb30fb","added_by":"auto","created_at":"2025-01-22 08:10:40","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1326850,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhysiological effects of EPO+ kidney organoids\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Ears of mice implanted with 8 control or EPO+ organoids 4 weeks after implantation. B. Spleen size of mice implanted with 8 control or EPO+ organoids 4 weeks after implantation. C. Hematocrit (HCT) of control mice (no organoids) and mice implanted with control or EPO+ organoids 4 weeks after implantation.\u003c/p\u003e","description":"","filename":"figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5534834/v1/a0306b198f76cddebb3d29ba.jpg"},{"id":74427591,"identity":"33fb61a0-04ab-4e8f-ad87-8e7cf53ca74c","added_by":"auto","created_at":"2025-01-22 08:10:41","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2293656,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of EPO+ kidney organoids on bone\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Bone volume fraction (BV/TV), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), trabecular number (Tb.N), trabecular bone pattern factor (Tb.Pf) and structure model index (SMI) in mice implanted with 8 control organoids or EPO+ organoids 4 weeks after implantation. B. FGF23 mRNA expression in bone marrow of mice implanted with 8 control or EPO+ organoids. C. Representative images of a µCT scan of a femoral metaphysis of a control mouse and a mouse implanted with EPO+ organoids.\u003c/p\u003e","description":"","filename":"figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5534834/v1/c4da7859175d53a251d0d2bc.jpg"},{"id":80558607,"identity":"cdfffcae-5450-4316-af81-0a2535278c1f","added_by":"auto","created_at":"2025-04-14 16:14:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7694321,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5534834/v1/33286c3a-cb6e-4135-8645-0be906d0eea5.pdf"},{"id":74427581,"identity":"932c7725-d5bb-4781-a9b8-33c5ebe6bb1e","added_by":"auto","created_at":"2025-01-22 08:10:40","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":637504,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary figure 1.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWestern blot of HIF2α and Actin in kidney organoids cultured for 4 hours under 20% O2 and 1% O2. Data are quantified in Figure 1E.\u003c/p\u003e","description":"","filename":"supplementaryfigure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5534834/v1/d63e98110d31547646f43de4.jpg"},{"id":74427586,"identity":"61415794-a598-4ea0-bd8c-25827e7b5d3c","added_by":"auto","created_at":"2025-01-22 08:10:41","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1473521,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary figure 2.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Weights of mice implanted with 8 control or 8 EPO+ organoids. B. Hematocrit (HCT) in mice implanted with 4, 6 or 8 EPO+ organoids at 4 weeks after implantation.\u003c/p\u003e","description":"","filename":"supplementaryfigure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5534834/v1/bd36dd9a665c037712db38c6.jpg"},{"id":74427582,"identity":"2423e28e-2ad5-4fb4-94b6-c0586be00f27","added_by":"auto","created_at":"2025-01-22 08:10:40","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":24680,"visible":true,"origin":"","legend":"","description":"","filename":"ARRIVEchecklistEPOorganoidimplantations.docx","url":"https://assets-eu.researchsquare.com/files/rs-5534834/v1/7c054ae4a90c257da27f6335.docx"}],"financialInterests":"","formattedTitle":"Erythropoietin delivery through kidney organoids engineered with an episomal DNA vector","fulltext":[{"header":"Introduction","content":"\u003cp\u003eChronic kidney disease (CKD) is characterized by a decreased glomerular filtration rate (GFR) and, in addition, by a decline of endocrine function of the kidney [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The kidney is the main source of EPO, and CKD-induced fibrosis leads to irreversible loss of renal EPO-producing cells. This results in insufficient EPO production and to complications such as anemia and disturbed bone homeostasis [\u003cspan additionalcitationids=\"CR4 CR5\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Repeated injection of recombinant human EPO and its derivatives has been shown to be effective in preventing anaemia and has been used as a treatment for decades [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. However, fixed-dose EPO supplementation causes gastroenterological problems in up to 30% of patients, is associated with increased risk for hypertension and thromboembolism [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], and negatively impacts bone mineral density [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Prolyl-hydroxylase inhibitors are a novel alternative to elevate EPO levels [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], but their actions are not restricted to the kidney, which may cause adverse effects. Regenerative therapies based on the differentiation of induced pluripotent stem cells (iPSC) may represent a curative approach for restoring EPO-producing capacity.\u003c/p\u003e \u003cp\u003eHuman iPSC-derived kidney organoids contain self-organized 3D nephron-like structures surrounded by renal stromal cells [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. It has recently been demonstrated that iPSC can be differentiated in renal interstitial progenitor cells that develop in EPO-producing cells [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], and we showed previously that kidney organoids have endocrine capacity and contain pericyte-like cells that produce renin in response to cAMP stimulation [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Furthermore, kidney organoids have the ability to convert inactive 25-hydroxyvitamin D3 to its active form [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Upon implantation under the kidney capsule, subcutaneously, or intracelomic in chicken embryos, kidney organoids become vascularized and maintain their kidney-like phenotype and endocrine function, highlighting their potential for restoring the endocrine function of the kidney [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. However, due to their small size kidney organoids are unlikely to be able to produce effective doses of hormones. The use of gene editing can potentially overcome this limitation. For such therapy to make an impact, it would have to induce efficient and long-term gene overexpression in a safe manner.\u003c/p\u003e \u003cp\u003eIn the present proof-of-concept study, we used an episomal DNA-vector platform comprising a scaffold/matrix attachment region (S/MAR) [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], to generate EPO-overexpressing (EPO+) iPSC and differentiate them into kidney organoids. S/MAR vectors do not integrate in the genome and are therefore an attractive tool for potential therapeutic applications where the risk of insertional mutagenesis is a concern. It is however not known whether S/MAR vectors can provide long-term stable gene expression. In the present study we investigated the stability of EPO S/MAR DNA vector expression and its effect on kidney organoid morphology and examined the physiological effects of EPO\u0026thinsp;+\u0026thinsp;kidney organoids on hematocrit and bone composition after subcutaneous implantation in mice.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eHuman iPSC culture\u003c/h2\u003e \u003cp\u003eHuman iPSC were generated from human primary skin fibroblasts isolated from human skin that became available as rest material, as approved by the Medical Ethics Committee of the Erasmus University Medical Center at the time of iPSC generation (MEC-2017-248) [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. iPSC were cultured on Geltrex LDEV-Free hESC-qualified basement membrane matrix (Gibco, USA) using complete Essential 8 medium (Gibco, USA). Cells were refreshed daily and dissociated as cell aggregates using 0.5mM UltraPure EDTA (Invitrogen, USA) for routine passaging every three days. The split ratios were between 1:5 and 1:10 to reach 20\u0026thinsp;~\u0026thinsp;30% seeding density. DPBS without calcium and magnesium (Gibco, NL) was used for cell rinsing before dissociation.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eDNA vector transfection\u003c/h3\u003e\n\u003cp\u003eA CAG-GFP/hEPO-S/MAR DNA vector was designed, and iPSC were transfected using a protocol that was adapted from earlier studies [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In brief, 24h after single-cell plating of iPSC, medium was refreshed and a transfection mix consisting of 5\u0026micro;L lipofectamine stem (Invitrogen, USA) and 2000ng DNA vector was added. Cells were refreshed daily and routinely passaged for 6\u0026thinsp;~\u0026thinsp;8 days, after which GFP\u0026thinsp;+\u0026thinsp;cells were enriched by FACS using a FACSAria (BD Biosciences, USA). A second cell sorting for GFP\u0026thinsp;+\u0026thinsp;cells was performed three weeks later. Cells were then expanded and cryopreserved at -150\u0026deg;C.\u003c/p\u003e\n\u003ch3\u003eKidney organoid differentiation\u003c/h3\u003e\n\u003cp\u003eHuman iPSC-derived kidney organoids were generated based on existing protocols [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. iPSC were dissociated into single cells by TrypLE Select Enzyme (Gibco, DK), and 300k cells were seeded per well of a 6-well plate and cultured overnight in Essential 8 medium supplemented with 1x Revitacell (Gibco, USA). The next day when the cells were stably attached, Essential 8 media was replaced by advanced RPMI 1640 media (Gibco, NL), supplemented with GlutaMax (Gibco, UK) and 100U/ml Penicillin-Streptomycin. 8\u0026micro;M of the GSK-3 inhibitor CHIR-99021 (Tocris, UK) was added for 3 days with daily refreshment. Subsequently, cells were cultured in advanced RPMI 1640 media supplemented with 200ng/ml recombinant human Fibroblast growth factor 9 (FGF9) (Peprotech, USA), 10ng/ml Activin A (R\u0026amp;D system, USA) and 1 \u0026micro;g/ml heparin sodium salt (Sigma Aldrich, USA) for 1 day. The next day, the cells were dissociated into single cells by TryplE Select (Gibco, DK), and resuspended in advanced RPMI 1640 supplemented with 3\u0026micro;M CHIR-99021, 200ng/ml FGF9, and 1\u0026micro;g/ml heparin. The cell suspension was adjusted to 500,000 cells per 150\u0026micro;l, and 150\u0026micro;l suspension was pipetted in each well of a 96-well conical (V) bottom plate (Nunc, DK). The plates were centrifuged at 300\u0026times;g for 3 min and kept in the incubator for 48h without refreshing. After this period of self-aggregation, the cell spheroids were carefully transferred on to 0.4\u0026micro;m PET membrane inserts (cellQART, DE), and advanced RPMI 1640 with 3\u0026micro;M CHIR, 200ng/ml FGF9, and 1\u0026micro;g/ml heparin was added to the base of the inserts, so that the organoids were at an air\u0026ndash;liquid interface. The next day, media was replaced with advanced RPMI 1640 with 200ng/ml FGF9 and 1 \u0026micro;g/ml heparin and refreshed every other day for 4 days. Finally, medium was replaced by advanced RPMI 1640 without further additions and refreshed every other day for 8 days.\u003c/p\u003e\n\u003ch3\u003eReal time RT-PCR\u003c/h3\u003e\u003cp\u003eTotal RNA from iPSC and kidney organoids was isolated with RNeasy Kits (Qiagen, Germany). Complementary DNA (cDNA) was synthesized using Moloney Murine Leukemia Virus Reverse Transcriptase Kit (Invitrogen, USA), random primers (Promega, USA) and RNasin\u0026reg; Ribonuclease Inhibitor (Promega, USA). TaqMan Gene Expression Master Mix (Applied Biosystems, USA) and TaqMan Gene Expression Assays (Life technologies, USA) (Table\u0026nbsp;1) were used for the RT-PCR reaction. CT values were measured by a StepOnePlus Real-Time PCR System (Applied Biosystems, Singapore).\u003c/p\u003e\n\u003ch3\u003eEPO measurement\u003c/h3\u003e\n\u003cp\u003eEPO was measured by Access\u0026reg; Erythropoietin chemiluminescence immunoassay (Beckman Coulter, CA, USA) on the Access 2 Immunoassay System (Beckman Coulter, CA, USA). Once a month, three external controls from UK NEQAS were measured for inter-laboratory standardization. The detection range of the immunoassay is 0.5\u0026ndash;750 mIU/mL.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot analysis\u003c/h2\u003e \u003cp\u003eOrganoid pellets were lysed in ice-cold radioimmunoprecipitation assay buffer (RIPA buffer) under constant agitation for 30 min. A small volume of protein lysate was used to perform a BCA protein-assay (Thermo Scientific, USA) to determine protein concentration. The rest of the samples were mixed with 4 \u0026times; loading buffer (Bio-rad, USA) and denatured. 25ug of each sample was loaded into 4\u0026ndash;15% Mini-PROTEAN\u0026reg; TGX\u0026trade; Precast Protein Gels (Bio-rad, USA) and proteins separated by molecular weight. Using Trans-blot Turbo System (Bio-rad, USA) at 1.3A up to 25V for 7min, proteins were transferred to Immun-blot PVDF membrane (Bio-rad, USA). After blocking in Tris-buffered 5% non-fat milk, membranes were incubated with anti-HIF2α monoclonal antibody (1:1000 dilution, NB100-132, Novus, USA) or anti-β-actin monoclonal antibody (1:5000 dilution, Cell signaling technology, USA). Horseradish peroxidase (HRP) conjugated anti-mouse IgG secondary antibody and chemiluminescence detection kits (Bio-rad, USA) were used for signal detection. The quantification was performed in Image J2 version: 2.9.0/1.53t (NIH, USA).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eImmunohistochemical (IHC) and multiplex immunofluorescence staining\u003c/h3\u003e\n\u003cp\u003eImmunohistochemistry was performed on a BenchMark ULTRA IHC/ISH System using ultraView (UV) Universal DAB Detection Kit (#760\u0026thinsp;\u0026minus;\u0026thinsp;600) or optiView (OV) Universal DAB detection Kit (#760\u0026thinsp;\u0026minus;\u0026thinsp;700, all Ventana Medical System, USA). After deparaffinization, rehydration and antigen retrieval with CC1 (#950\u0026thinsp;\u0026minus;\u0026thinsp;500, Ventana), the tissue were incubated with antibody of interest (Table\u0026nbsp;2). Incubation was followed by optiview detection and hematoxylin II counter stain for 8 minutes followed by a blue coloring reagent for 8 minutes according to the manufactures instructions (Ventana).\u003c/p\u003e \u003cp\u003eFor triple immunofluorescence staining of Podocalyxin-like protein 1 (PODXL), VILLIN1 and E-cadherin (ECAD) the Discovery Ultra (Ventana) was used. In brief, following deparaffinization and heat-induced antigen retrieval with CC1 (#950\u0026thinsp;\u0026minus;\u0026thinsp;224, Ventana) for 32 min, anti-PODXL was incubated for 32 min at 37\u0026deg;C followed by omnimap anti-rabbit HRP (#760\u0026ndash;4311, Ventana) and detection with Cy5 (#760\u0026thinsp;\u0026minus;\u0026thinsp;238, Ventana) for 8 min. An antibody denaturation step was performed with CC2 (#950\u0026thinsp;\u0026minus;\u0026thinsp;123, Ventana) at 100\u0026deg;C for 20 min. Secondly, incubation with anti-ECAD was performed for 20 min at 37\u0026deg;C, followed by omnimap anti-mouse HRP (#760\u0026ndash;4310, Ventana) and detection with with Red610 (#760\u0026thinsp;\u0026minus;\u0026thinsp;245, Ventana). An antibody denaturation step followed with CC2 at 100\u0026deg;C for 20 min. Thirdly, anti-Villin1 was incubated for 32 min at 37\u0026deg;C, followed by omnimap anti-rabbit HRP (#760\u0026ndash;4311, Ventana) and detection with detection with FAM (#760\u0026thinsp;\u0026minus;\u0026thinsp;243, Ventana). Finally, slides were washed in phosphate-buffered saline and mounted with Vectashield containing 4\u0026rsquo;,6-diamidino-2-phenylindole (Vector laboratories, Peterborough, UK). Slides were imaged with Axioscan Zeiss.\u003c/p\u003e \u003cp\u003eImages were analyzed using QuPath software [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. A first threshold was set to determine organoid area using the average channels. Thereafter, color deconvolution was used to determine positive staining, followed by thresholding. Results were obtained as positive percentage of total organoid area.\u003c/p\u003e\n\u003ch3\u003eSubcutaneous implantation of kidney organoids\u003c/h3\u003e\n\u003cp\u003eImmuno-deficient mice Rag2\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e/IL2rγ\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e double knock out (BALB/c) of 8\u0026ndash;15 weeks old were acclimatized for at least 1 week before start of the experiments. For organoids implantation, inhaled anesthesia was induced by 4\u0026ndash;5% isoflurane and maintained by 1\u0026ndash;2% isoflurane with 500-600ml/min oxygen. Two kidney organoids were encapsuled in 40\u0026micro;l semisolid Geltrex and placed subcutaneously through 0.5cm skin incisions into the flanks. To study whether EPO\u0026thinsp;+\u0026thinsp;organoids had a dose-dependent effect, animals were implanted with 2, 4, 6 or 8 EPO\u0026thinsp;+\u0026thinsp;organoids. Control animals received either no organoids or control organoids. Animals were randomly allocated to the groups in a no blinded fashion, but male and female mice were equally distributed over the groups to control for potential sex differences. For the group receiving 8 organoids, 10 animals received EPO\u0026thinsp;+\u0026thinsp;organoids and 9 animals received control organoids. In total 33 animal were used. Wounds were closed with 2 or 3 sutures. Mice were checked daily and weighed weekly. Four weeks after organoid implantation day, blood was collected via cardiac puncture under isofluran anesthesia followed by euthanasia through cervical dislocation. Organoids were retrieved and snap frozen in liquid nitrogen for RT-PCR analysis or fixed in 4% paraformaldehyde for histological analysis. Long bones were collected for microcomputed tomography (\u0026micro;CT) analysis or bone marrow was collected and snap frozen in liquid nitrogen for RT-PCR analysis. The work has been reported in line with the ARRIVE guidelines 2.0 [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eWhole blood analysis\u003c/h2\u003e \u003cp\u003eThe whole blood of the mice was transferred and mixed in MiniCollect\u0026reg; K3EDTA tubes (Greiner, Austria). Within 4 hours after collection, two 50\u0026micro;l aliquots of blood from each mouse were measured by automated hematology analyzer XP-300 (Sysmex, Japan) using the whole blood mode to determine hematocrit levels.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eMicrocomputed tomography (\u0026micro;CT) analysis\u003c/h2\u003e \u003cp\u003eFemurs were scanned at a resolution of 9 \u0026micro;m, using a SkyScan 1076 system (Bruker, Kontich, Belgium) in a blinded fashion. According to the published guidelines [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], the following settings were used: X-Ray power and tube current were 40 kV and 250 \u0026micro;A, respectively. Beam hardening was reduced using a 1 mm aluminium filter, exposure time was 2.3 seconds, and an average of three pictures was taken at each angle with steps of 0.8\u0026deg; to generate final images. Segmentation of the reconstructed images was done on basis of global thresholding. Using software packages from Bruker (NRecon, CtAn, and Dataviewer), bone microarchitecture parameters were assessed in trabecular and cortical bones of all mice. The trabecular bone parameters trabecular bone volume fraction (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), trabecular separation (Tb.Sp), trabecular patterning factor (Tb.Pf) and structure model index (SMI; 0\u0026thinsp;=\u0026thinsp;plate-like, 3\u0026thinsp;=\u0026thinsp;rod-like) were determined in the distal metaphysis of the femur (region of interest (ROI) of 0.9 mm from distal growth plate towards diaphysis).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe unpaired t-test with Welch\u0026rsquo;s correction was used to test the RT-PCR results for significance. The Mann-Whitney test was used to test the quantifications of the immunohistochemical staining for significance, as this data was not normally distributed. Statistical significance was considered for p values less than 0.05 (*), 0.01 (**) and 0.001 (***). Data analysis was performed using GraphPad Prism version 8.0.0 (GraphPad Software, La Jolla, USA).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eKidney organoids are capable of producing EPO under hypoxia.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eiPSC-derived kidney organoids generated at an air-liquid interface contained PODXL expressing glomerular structures, VILLIN expressing proximal tubular structures, and ECAD expressing distal tubular structures (Fig.\u0026nbsp;1A-C). In addition, kidney organoids contained PDGFRβ\u0026thinsp;+\u0026thinsp;interstitial cells situated adjacent to the nephron structures (Fig.\u0026nbsp;1D), which marks the EPO-producing cells in the kidney [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. EPO expression is driven by oxygen-sensitive hypoxia-inducible transcription factor 2 alpha (HIF2α) and we observed that culture of kidney organoids under hypoxia (1% oxygen) for four hours triggered the stabilization of HIF2α (Fig.\u0026nbsp;1E and Supplementary Fig.\u0026nbsp;1). This was accompanied by an increased release of EPO from levels under the detection limit at 20% oxygen to 0.20 mIU/organoid/day at 1% oxygen (Fig.\u0026nbsp;1F). These results demonstrate that kidney organoids have the capacity to secrete EPO. The amounts of EPO secreted per kidney organoid are however very low considering normal EPO concentrations range between 4\u0026ndash;26 mIU/ml blood. EPO productin by organoids would therefore be insufficient to compensate for the loss of EPO-production capacity in kidney disease. We thus proceeded to increase the EPO production capacity of organoids through the use of DNA vectors.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEPO\u0026thinsp;+\u0026thinsp;kidney organoids show stably elevated EPO expression.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo increase the EPO-producing capacity of kidney organoids, iPSC were genetically engineered with S/MAR DNA vectors carrying the human EPO and GFP genes (EPO\u0026thinsp;+\u0026thinsp;iPSC) (Fig.\u0026nbsp;2A). GFP expression was maintained throughout the iPSC expansion period of 67 days (Fig.\u0026nbsp;2B). 0.5\u0026nbsp;million EPO\u0026thinsp;+\u0026thinsp;iPSC released 1398 mIU/day of EPO, which is more than 4500 times higher than the EPO release rate of control iPSC (Fig.\u0026nbsp;2C). GFP-reported expression of the DNA vector was preserved after differentiation of iPSC into kidney organoids (Fig.\u0026nbsp;2D), and EPO\u0026thinsp;+\u0026thinsp;kidney organoids produced strongly elevated levels of EPO mRNA compared with control organoids (Fig.\u0026nbsp;2E). This resulted in highly elevated EPO release rates in EPO\u0026thinsp;+\u0026thinsp;organoids (1204 mIU/day/organoid) compared to control organoids (0.006 mIU/day/organoid) (Fig.\u0026nbsp;2F).\u003c/p\u003e \u003cp\u003e \u003cb\u003eEPO\u0026thinsp;+\u0026thinsp;kidney organoids develop nephron structures.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo determine whether EPO overexpression affected kidney organoid development, mRNA expression analysis of key nephron markers was performed. mRNA expression levels of the podocyte marker PODXL, the proximal tubular marker VILLIN-1 and the endothelial cell marker PECAM1 were not altered in EPO\u0026thinsp;+\u0026thinsp;organoids. However, the distal tubular marker ECAD was downregulated in EPO\u0026thinsp;+\u0026thinsp;organoids compared to control organoids (Fig.\u0026nbsp;3A). Immunohistochemistry confirmed the development of nephron structures in EPO\u0026thinsp;+\u0026thinsp;organoids (Fig.\u0026nbsp;3B). Quantification of immunohistochemical staining demonstrated increased PODXL\u0026thinsp;+\u0026thinsp;structures in EPO\u0026thinsp;+\u0026thinsp;organoids, whereas there was a significant decrease in ECAD\u0026thinsp;+\u0026thinsp;structures in EPO\u0026thinsp;+\u0026thinsp;kidney organoids (Fig.\u0026nbsp;3C). Interestingly, the area of CD31+ (PECAM1 gene) endothelial cells was increased from 1.7\u0026ndash;4.9% in EPO\u0026thinsp;+\u0026thinsp;kidney organoids.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEPO\u0026thinsp;+\u0026thinsp;kidney organoids show enhanced vascularization following implantation.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo investigate the physiological effects of EPO\u0026thinsp;+\u0026thinsp;kidney organoids, 2, 4, 6, or 8 organoids were implanted subcutaneously in the flanks of immunodeficient mice and retrieved after 4 weeks (Fig.\u0026nbsp;4A). Kidney organoids were connected to the vascular system of the host mice, as evidenced by the macroscopically visible growth of blood vessels of the skin into the organoids (Fig.\u0026nbsp;4B). EPO\u0026thinsp;+\u0026thinsp;organoids continued to express high levels of EPO mRNA 4 weeks after implantation (Fig.\u0026nbsp;4C). Immunohistochemical staining showed that nephron structures were maintained in the implanted organoids, although the interstitial space had expanded (Fig.\u0026nbsp;4D). We observed no morphological differences in nephron structures between control and EPO\u0026thinsp;+\u0026thinsp;organoids, but there was an increase in CD31 positive structures in the EPO\u0026thinsp;+\u0026thinsp;organoids (Fig.\u0026nbsp;4D-E). Human CD31 showed an increased area staining from 0.10% in control organoids to 0.48% in EPO\u0026thinsp;+\u0026thinsp;organoids, indicating an increase in endothelial structures in EPO\u0026thinsp;+\u0026thinsp;organoids. This was supported by increased mRNA expression of PECAM-1 in EPO\u0026thinsp;+\u0026thinsp;organoids (Fig.\u0026nbsp;4F). In addition, PODXL mRNA expression was increased in EPO\u0026thinsp;+\u0026thinsp;organoids.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEPO\u0026thinsp;+\u0026thinsp;organoids increase hematocrit levels.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eFour weeks after kidney organoid implantation, hyperemic ears were observed in mice that received EPO\u0026thinsp;+\u0026thinsp;organoids (Fig.\u0026nbsp;5A). In addition, the spleen size of mice that received EPO\u0026thinsp;+\u0026thinsp;organoids were increased in comparison to that of mice implanted with control organoids (Fig.\u0026nbsp;5B). There was no effect of EPO\u0026thinsp;+\u0026thinsp;organoids on body weight (Supplementary Fig.\u0026nbsp;2A). Whole blood analysis demonstrated that hematocrit levels in mice implanted with EPO\u0026thinsp;+\u0026thinsp;organoids were increased. Increases in hematocrit were positively correlated with the number of implanted EPO\u0026thinsp;+\u0026thinsp;organoids (Supplementary Fig.\u0026nbsp;2B). Mice implanted with eight EPO\u0026thinsp;+\u0026thinsp;organoids showed a significant increase in hematocrit levels from 0.40 L/L to 0.59 L/L compared to mice implanted with control organoids (Fig.\u0026nbsp;5C). Hematocrit levels in mice implanted with control organoids did not differ from mice that had no organoids implanted. These results demonstrate that EPO production by human kidney organoids has a physiological effect on hosts.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEPO\u0026thinsp;+\u0026thinsp;organoids affect trabecular bone structure.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAs EPO supplementation is known to affect bone homeostasis, we investigated whether organoid-derived EPO affected bone composition in host mice. We observed no difference in bone volume fraction (BV/TV%), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp) or trabecular number (Tb.N) between mice implanted with 8 control organoids or EPO\u0026thinsp;+\u0026thinsp;organoids (Fig.\u0026nbsp;6A). However, the trabecular bone pattern factor (Tb.Pf) and the structure model index (SMI) were decreased in the EPO\u0026thinsp;+\u0026thinsp;organoid group, indicating a more dense trabecular spatial structure, which suggests an increased bone strength. Cortical bone parameters were not different between the groups. Interestingly, \u003cem\u003eFgf23\u003c/em\u003e mRNA expression was increased in bone marrow of the EPO\u0026thinsp;+\u0026thinsp;organoid group (Fig.\u0026nbsp;6B). FGF23 acts on the kidney to increase phosphate excretion and suppress 1,25(OH)\u003csub\u003e2\u003c/sub\u003e vitamin D synthesis. The increased FGF23 expression thus further demonstrates that EPO\u0026thinsp;+\u0026thinsp;organoids affect endocrine systems in the host. Representative images of a femoral metaphysis of a control mouse and an EPO\u0026thinsp;+\u0026thinsp;mouse are shown in Fig.\u0026nbsp;6C.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eWe demonstrated that S/MAR DNA vector technology applied to human iPSC-derived kidney organoids enables the generation of EPO-overexpressing organoids that demonstrate long-term EPO secretion capacity, evidenced by physiological effects on host mice after implantation. These results show for the first time the potential of such a platform to restore the renal EPO endocrine system in kidney disease.\u003c/p\u003e \u003cp\u003eBy using episomal DNA vectors, we aimed to cause minimal impact on cellular processes by reducing innate immunity and avoiding random genomic integration [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], and furthermore to provide stable expression of EPO in human iPSC. Throughout iPSC expansion, differentiation of kidney organoids and subsequent implantation in mice, EPO was stably expressed. EPO overexpression enhanced the development of podocyte-like cells and endothelial cells during \u003cem\u003ein vitro\u003c/em\u003e kidney organoid differentiation, while it reduced the formation of distal tubular cells. The enhanced presence of podocyte-like and endothelial cells was still evident in organoids retrieved from host mice, but a difference in distal tubular cells was no longer observed. This data indicates that high levels of EPO in developing kidney organoids impact cellular differentiation. This is interesting as the EPO receptor is widely expressed in fetal tissue [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Kidney organoids thus represent a relevant model to study potential therapeutic or adverse effects of EPO in the developing kidney.\u003c/p\u003e \u003cp\u003eThe increased numbers of endothelial cells in EPO\u0026thinsp;+\u0026thinsp;organoids is of interest for vascularization purposes. We and others have demonstrated that organoid-derived endothelial cells contribute, together with host-derived endothelial cell, to the formation of endothelial structures in kidney organoids after implantation in animal models [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Increased presence of endothelial cells may enhance the speed of vascularization and improved branching of vascular structures within the organoids. This may lead to a decreased period of anoxia in organoids after implantation, and a more functional endocrine interaction of organoids with the host.\u003c/p\u003e \u003cp\u003eThe physiological effects of EPO\u0026thinsp;+\u0026thinsp;kidney organoids were obvious in our model. Hematocrit levels were strongly elevated in an organoid number-dependent manner and spleens were enlarged. EPO\u0026thinsp;+\u0026thinsp;kidney organoids furthermore had a beneficial effect on trabecular bone structure and predicted bone strength. In our model, human EPO efficiently interacted with the mouse EPO receptor. EPO and EPO receptors are evolutionarily conserved, however their interaction might be less efficient in a cross-species setting [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. It is therefore not unlikely that in a fully human setting, the effects of EPO\u0026thinsp;+\u0026thinsp;organoids may be even more efficient than in our model. This becomes relevant when determining how many organoids are needed to support the renal EPO production.\u003c/p\u003e \u003cp\u003eIn our model, EPO production in kidney organoids was constitutive and not cell type restricted, which is a limitation of the study. Uncontrolled increase in hematocrit levels would not be suitable in a clinical setting as it can lead to coagulation. Therefore future studies shall focus on generation of regulatable EPO-producing kidney organoids. EPO-producing cells in the kidney can be identified through their expression of platelet-derived-growth-factor-receptor-β (PDGFRβ) and reside around the peritubular capillaries, which is one of the most oxygen-deprived sites of the body [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. When oxygen levels at this site drop below a certain threshold, the transcription factor hypoxia-inducible-factor-2α is activated in these cells and stimulates EPO production. To truly mimic EPO regulation of the kidney it is instrumental to restrict EPO secretion to PDGFRβ\u003csup\u003e+\u003c/sup\u003e cells, and to place these cells at a site with a similar morphology as the kidney. Kidney organoids provide such a morphology. Furthermore, DNA vectors could be designed to be active exclusively in PDGFRβ\u0026thinsp;+\u0026thinsp;cells.\u003c/p\u003e \u003cp\u003eFuture studies should in addition focus on the long-term effects of EPO\u0026thinsp;+\u0026thinsp;organoids. The present study was limited to four weeks, but is is essential to evaluate whether S/MAR vector-mediated EPO production remains functional after months or years. Furthermore, it is essential to examine whether changes in organoid size or cellular composition over time occur and affect EPO release. Finally, future research should study the effects of EPO\u0026thinsp;+\u0026thinsp;organoids in an anemic model to examine whether organoid-derived EPO is sufficient to restore failing EPO production by the host.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, the generation of safe, specific, and effective EPO-producing kidney organoids is a promising tool for the restoration of EPO production capacity in CKD. The present manuscript demonstrates the potential of such therapy and indicates the issues that require further research.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBV/TV\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eBone volume fraction\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCKD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eChronic kidney disease is characterized by a decreased\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eECAD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eE-cadherin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eEPO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eErythropoietin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eEPO+\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eErythropoietin overexpression\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFGF9\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFibroblast growth factor 9\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFGF23\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFibroblast growth factor 23\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGFR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eglomerular filtration rate\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHIF2α\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehypoxia-inducible transcription factor 2 alpha\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eiPSC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003einduced pluripotent stem cell\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePDGFRβ\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eplatelet-derived-growth-factor-receptor-β\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePodocalyxin-like protein 1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePODXL\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eS/MAR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003escaffold/matrix attachment region\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSMI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003estructure model index\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTb.N\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003etrabecular number\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTb.Pf\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003etrabecular bone pattern factor\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTb.Sp\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003etrabecular separation\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTb.Th\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003etrabecular thickness\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eiPSC were generated from human donors as part of the project \u0026lsquo;Creation of disease model systems to understand and correct genetic diseases through gene or other therapy using iPS cell derived from somatic cells: IPSC protocol Rotterdam\u0026rsquo; approved by the Erasmus MC Medical Ethical Committee under approval number MEC-2017-248, which was published 29\u003csup\u003eth\u003c/sup\u003e March 2018 and last updated 15\u003csup\u003eth\u003c/sup\u003e April 2024.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics statement on animal experiments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe animal studies were performed after receiving approval of the Dutch central committee animal experiments under licence AVD101002016635.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe raw data files belonging to this manuscript are assessable on DataverseNL via https://doi.org/10.34894/GZPSB6.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no financial or personal relationships with other people or organisations that could inappropriately influence (bias) their work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eZ. Du is funded by a grant of the Chinese Scholarschip Council.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eUse of Artificial Intelligence (AI)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have not use AI-generated work in this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eZ. Du: Conception and design,\u0026nbsp;collection and/or assembly of data,\u0026nbsp;data analysis and interpretation,\u0026nbsp;manuscript\u0026nbsp;writing\u003c/p\u003e\n\u003cp\u003eA. Bas-Crist\u0026oacute;bal Men\u0026eacute;ndez:\u0026nbsp;Collection and/or assembly of data,\u0026nbsp;data analysis and interpretation\u003c/p\u003e\n\u003cp\u003eM. Urban :\u0026nbsp;Collection and/or assembly of data,\u0026nbsp;data analysis and interpretation\u003c/p\u003e\n\u003cp\u003eA. Hartley:\u0026nbsp;Collection and/or assembly of data,\u0026nbsp;data analysis and interpretation\u003c/p\u003e\n\u003cp\u003eD. Ratsma:\u0026nbsp;Collection and/or assembly of data,\u0026nbsp;data analysis and interpretation\u003c/p\u003e\n\u003cp\u003eM. Koedam:\u0026nbsp;Collection and/or assembly of data\u003c/p\u003e\n\u003cp\u003eT.P.P. van den Bosch:\u0026nbsp;Collection and/or assembly of data,\u0026nbsp;data analysis and interpretation\u003c/p\u003e\n\u003cp\u003eM. Clahsen-van Groningen:\u0026nbsp;Final approval of manuscript\u003c/p\u003e\n\u003cp\u003eJ. Gribnau:\u0026nbsp;Provision of study material,\u0026nbsp;final approval of manuscript\u003c/p\u003e\n\u003cp\u003eJ. Mulder:\u0026nbsp;Final approval of manuscript\u003c/p\u003e\n\u003cp\u003eM.E.J. Reinders:\u0026nbsp;Final approval of manuscript\u003c/p\u003e\n\u003cp\u003eC.C. Baan:\u0026nbsp;Final approval of manuscript\u003c/p\u003e\n\u003cp\u003eB. van der Eerden:\u0026nbsp;Data analysis and interpretation, final approval of manuscript\u003c/p\u003e\n\u003cp\u003eR.P. Harbottle: Conception and design, provision of study material, data analysis and interpretation, final approval of manuscript\u003c/p\u003e\n\u003cp\u003eM.J. Hoogduijn: Conception and design, data analysis and interpretation, manuscript writing\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBabitt JL, Lin HY. Mechanisms of anemia in CKD [in eng]. J Am Soc Nephrol. 2012;23(10):1631\u0026ndash;4.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePortoles J, Martin L, Broseta JJ, Cases A. Anemia in Chronic Kidney Disease: From Pathophysiology and Current Treatments, to Future Agents [in eng]. Front Med (Lausanne). 2021;8:642296.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWebster AC, Nagler EV, Morton RL, Masson P. Chronic Kidney Disease [in eng]. Lancet. 2017;389(10075):1238\u0026ndash;52.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLappin KM, Mills KI, Lappin TR. Erythropoietin in bone homeostasis-Implications for efficacious anemia therapy [in eng]. Stem Cells Transl Med. 2021;10(6):836\u0026ndash;43.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSouma T, Suzuki N, Yamamoto M. Renal erythropoietin-producing cells in health and disease [in eng]. Front Physiol. 2015;6:167.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSuresh S, Lee J, Noguchi CT. Erythropoietin signaling in osteoblasts is required for normal bone formation and for bone loss during erythropoietin-stimulated erythropoiesis [in eng]. FASEB J. 2020;34(9):11685\u0026ndash;97.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEschbach JW, Egrie JC, Downing MR, et al. Correction of the anemia of end-stage renal disease with recombinant human erythropoietin. Results of a combined phase I and II clinical trial [in eng]. N Engl J Med. 1987;316(2):73\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWinearls CG, Oliver DO, Pippard MJ, et al. Effect of human erythropoietin derived from recombinant DNA on the anaemia of patients maintained by chronic haemodialysis [in eng]. Lancet. 1986;2(8517):1175\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCollister D, Rigatto C, Tangri N. Anemia management in chronic kidney disease and dialysis: a narrative review [in eng]. Curr Opin Nephrol Hypertens. 2017;26(3):214\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCheng CY, Kuo YJ. Single-centre cross-sectional study on the impact of cumulative erythropoietin on bone mineral density in maintenance dialysis patients [in eng]. BMJ Open. 2022;12(4):e056390.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLocatelli F, Del Vecchio L. Are prolyl-hydroxylase inhibitors potential alternative treatments for anaemia in patients with chronic kidney disease? [in eng]. Nephrol Dial Transpl. 2020;35(6):926\u0026ndash;32.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTakasato M, Er PX, Chiu HS, et al. Kidney organoids from human iPS cells contain multiple lineages and model human nephrogenesis [in eng]. Nature. 2015;526(7574):564\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTsujimoto H, Hoshina A, Mae SI, et al. Selective induction of human renal interstitial progenitor-like cell lineages from iPSCs reveals development of mesangial and EPO-producing cells [in eng]. Cell Rep. 2024;43(2):113602.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShankar AS, Du Z, Mora HT, et al. Human kidney organoids produce functional renin [in eng]. Kidney Int. 2021;99(1):134\u0026ndash;47.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShankar AS, van den Berg SAA, Tejeda Mora H, et al. Vitamin D metabolism in human kidney organoids [in eng]. Nephrol Dial Transpl. 2021;37(1):190\u0026ndash;3.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003evan den Berg CW, Ritsma L, Avramut MC, et al. Renal Subcapsular Transplantation of PSC-Derived Kidney Organoids Induces Neo-vasculogenesis and Significant Glomerular and Tubular Maturation In Vivo [in eng]. Stem Cell Rep. 2018;10(3):751\u0026ndash;65.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKoning M, Lievers E, Jaffredo T et al. Efficient Vascularization of Kidney Organoids through Intracelomic Transplantation in Chicken Embryos [in eng]. J Vis Exp 2023(192).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRoig-Merino A, Urban M, Bozza M, et al. An episomal DNA vector platform for the persistent genetic modification of pluripotent stem cells and their differentiated progeny [in eng]. Stem Cell Rep. 2022;17(1):143\u0026ndash;58.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBozza M, De Roia A, Correia MP et al. A nonviral, nonintegrating DNA nanovector platform for the safe, rapid, and persistent manufacture of recombinant T cells [in eng]. Sci Adv 2021;7(16).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ede Esch CE, Ghazvini M, Loos F, et al. Epigenetic characterization of the FMR1 promoter in induced pluripotent stem cells from human fibroblasts carrying an unmethylated full mutation [in eng]. Stem Cell Rep. 2014;3(4):548\u0026ndash;55.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGarreta E, Prado P, Tarantino C, et al. Fine tuning the extracellular environment accelerates the derivation of kidney organoids from human pluripotent stem cells [in eng]. Nat Mater. 2019;18(4):397\u0026ndash;405.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSelfa IL, Gallo M, Montserrat N, Garreta E. Directed Differentiation of Human Pluripotent Stem Cells for the Generation of High-Order Kidney Organoids [in eng]. Methods Mol Biol. 2021;2258:171\u0026ndash;92.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBankhead P, Loughrey MB, Fernandez JA, et al. QuPath: Open source software for digital pathology image analysis [in eng]. Sci Rep. 2017;7(1):16878.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePercie du Sert N, Hurst V, Ahluwalia A, et al. The ARRIVE guidelines 2.0: updated guidelines for reporting animal research [in eng]. BMJ Open Sci. 2020;4(1):e100115.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBouxsein ML, Boyd SK, Christiansen BA, et al. Guidelines for assessment of bone microstructure in rodents using micro-computed tomography [in eng]. J Bone Min Res. 2010;25(7):1468\u0026ndash;86.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGerl K, Nolan KA, Karger C, et al. Erythropoietin production by PDGFR-beta(+) cells [in eng]. Pflugers Arch. 2016;468(8):1479\u0026ndash;87.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBozza M, Green EW, Espinet E, et al. Novel Non-integrating DNA Nano-S/MAR Vectors Restore Gene Function in Isogenic Patient-Derived Pancreatic Tumor Models [in eng]. Mol Ther Methods Clin Dev. 2020;17:957\u0026ndash;68.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJuul SE, Yachnis AT, Christensen RD. Tissue distribution of erythropoietin and erythropoietin receptor in the developing human fetus [in eng]. Early Hum Dev. 1998;52(3):235\u0026ndash;49.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDivoky V, Prchal JT. Mouse surviving solely on human erythropoietin receptor (EpoR): model of human EpoR-linked disease [in eng]. Blood. 2002;99(10):3873\u0026ndash;4. author reply 3874\u0026ndash;3875.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBauer C, Kurtz A. Oxygen sensing in the kidney and its relation to erythropoietin production [in eng]. Annu Rev Physiol. 1989;51:845\u0026ndash;56.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 and 2 are not available with this version.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"stem-cell-research-and-therapy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scrt","sideBox":"Learn more about [Stem Cell Research \u0026 Therapy](http://stemcellres.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/scrt/default.aspx","title":"Stem Cell Research \u0026 Therapy","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"DNA vector, Erythropoietin, Kidney, Organoids, Pluripotent stem cells","lastPublishedDoi":"10.21203/rs.3.rs-5534834/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5534834/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eThe kidney's endocrine function is essential for maintaining body homeostasis. Erythropoietin (EPO) is one of the key endocrine factors produced by the kidney, and kidney disease patients frequently experience anemia due to impaired EPO production. In the present study we explored the potential of human induced pluripotent stem cell (iPSC)-derived kidney organoids to restore EPO production.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eEPO secretion by kidney organoids was examined under 1% and 20% oxygen levels. To increase the EPO secreting capacity of kidney organoids, iPSC were genetically engineered with a non-integrating scaffold/matrix attachment region (S/MAR) DNA vector containing the EPO gene and generated EPO-overexpressing (EPO+) kidney organoids. To assess the physiological effects of EPO\u0026thinsp;+\u0026thinsp;organoids, 2\u0026ndash;8 organoids were implanted subcutaneously in immunodeficient mice.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eKidney organoids produced low amounts of EPO under 1% oxygen. EPO S/MAR DNA vectors persisted and continued to robustly express EPO during iPSC expansion and kidney organoid differentiation without interfering with cellular proliferation. EPO\u0026thinsp;+\u0026thinsp;iPSC demonstrated efficient differentiation into kidney organoids. One-month post-implantation, EPO\u0026thinsp;+\u0026thinsp;organoids displayed continuously elevated EPO mRNA levels and significantly increased endothelial cell numbers compared to control organoids. Hematocrit levels were notably elevated in mice implanted with EPO\u0026thinsp;+\u0026thinsp;organoids in an organoid number-dependent manner. EPO\u0026thinsp;+\u0026thinsp;organoids furthermore influenced bone homeostasis in their hosts, evidenced by a change in trabecular bone composition.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eKidney organoids modified by EPO S/MAR DNA vector allow stable long-term delivery of EPO. The observed physiological effects following the implantation of EPO\u0026thinsp;+\u0026thinsp;organoids underscore the potential of gene-edited kidney organoids for endocrine restoration therapy.\u003c/p\u003e","manuscriptTitle":"Erythropoietin delivery through kidney organoids engineered with an episomal DNA vector","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-22 08:10:36","doi":"10.21203/rs.3.rs-5534834/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-01-17T15:45:08+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-01-17T15:41:18+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-01-06T08:36:59+00:00","index":"","fulltext":""},{"type":"submitted","content":"Stem Cell Research \u0026 Therapy","date":"2025-01-03T07:10:18+00:00","index":"","fulltext":""},{"type":"decision","content":"Major Revision","date":"2024-12-10T01:24:13+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"stem-cell-research-and-therapy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scrt","sideBox":"Learn more about [Stem Cell Research \u0026 Therapy](http://stemcellres.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/scrt/default.aspx","title":"Stem Cell Research \u0026 Therapy","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1fa784f9-88e5-43ed-9571-fa22bcd044ec","owner":[],"postedDate":"January 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-04-14T16:09:40+00:00","versionOfRecord":{"articleIdentity":"rs-5534834","link":"https://doi.org/10.1186/s13287-025-04282-w","journal":{"identity":"stem-cell-research-and-therapy","isVorOnly":false,"title":"Stem Cell Research \u0026 Therapy"},"publishedOn":"2025-04-12 16:04:52","publishedOnDateReadable":"April 12th, 2025"},"versionCreatedAt":"2025-01-22 08:10:36","video":"","vorDoi":"10.1186/s13287-025-04282-w","vorDoiUrl":"https://doi.org/10.1186/s13287-025-04282-w","workflowStages":[]},"version":"v1","identity":"rs-5534834","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5534834","identity":"rs-5534834","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}