Erythropoietin delivery through kidney organoids engineered with an episomal DNA vector

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.

1. 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 erythropoietin (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 anemia 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.

It has been demonstrated that modification of protocols for differentiation of iPSC into the hepatic lineage leads to the generation of EPO-producing cells [12]. These cells show erythropoietic activity in vitro and in vivo upon implantation under the kidney capsule through EPO secretion. The finding that CD140b and CD73 mark iPSC-derived erythropoietin-producing cells facilitates the identification of these cells for purification purposes [13]. The responsiveness of the cells to retinoic acid through upregulation of EPO production cooperatively offers the ability to regulate EPO release [14]. Other work showed that iPSC can be differentiated into neural crest cells capable of producing functional EPO [15]. In their natural niche in the kidney, EPO-producing cells reside at a low oxygen location, which allows them to efficiently monitor oxygen levels. Tsujimoto et al. explored recreation of the natural niche by combining EPO-producing cells with hiPSC-derived nephron progenitor cells and nephric duct cells to form kidney organoids [16].