Abstract

Regenerative medicine has received increasing attention as a new therapeutic approach due to the scarcity of treatments for kidney diseases. Recent advances in renal regeneration using human induced pluripotent stem cells (hiPSCs) are noteworthy. Based on the knowledge of kidney development, hiPSCs directionally differentiate into two types of embryonic kidney progenitor cells, nephron metabolic progenitor cells (NPCs) and ureteral buds (UB), capable of generating nephrons and collecting duct-like organs. In addition, human kidney tissue can be generated from these hiPSC-derived progenitor cells in which NPC-derived glomeruli and tubules and UB-derived collecting ducts are interconnected. The induced kidney tissue is further vascularized after transplantation into immunodeficient mice. In addition to renal reconstruction for transplantation, cell therapy with hiPSC-derived NPC has been shown to ameliorate acute kidney injury (AKI) in mice. Disease modeling and drug discovery studies using disease-specific hiPSCs have also been strongly used in refractory kidney diseases such as autosomal dominant polycystic kidney disease (ADPKD). To address the complications associated with renal diseases, hiPSC-derived erythropoietin (EPO)-producing cells have been successfully generated for drug discovery and development of cellular therapies for renal anemia. This article reviews the current and future perspectives of renal developmental biology and regenerative medicine for renal diseases based on iPSC technology.

Keywords

iPSC ;Kidney regeneration ;Nephron progenitor cell;Ureteric bud;Cell therapy ; Disease modeling;Cistanche tubulosa

Introduction

Kidney disease poses a huge medical problem and economic burden worldwide, but due to the severe shortage of donor organs, there are few treatment options other than kidney transplantation. One solution is to develop regenerative medicine strategies using human pluripotent stem cells (hPSCs), such as embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs). Since hPSCs have the potential to proliferate indefinitely and differentiate into any cell type, including renal cells, they are expected to serve as a source of cells for regenerative medicine, such as renal reconstruction and cell therapy. In addition, disease-specific hPSCs with genetic susceptibility to cause specific diseases can be used to develop pathological analyses and drug discovery models in which damaged cell types differentiated from hPSCs replicate disease phenotypes in vitro. In this article, I summarize recent advances in developmental biology-based kidney regeneration research and describe future prospects for regenerative medicine and disease modeling in kidney disease.

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Kidney development

The kidney is derived from the early embryonic blastoderm, the intermediate mesoderm (IM) (Figure 1a). In vertebrates, the IM gives rise to three kidneys in sequence, the primary, intermediate, and posterior kidneys (Figure 1b). The middle kidney is the adult kidney of fish and amphibians, and the posterior kidney is the adult kidney of reptiles, birds, and mammals. Although these three kidneys are similar because they consist of nephrons, which are the functional units of the kidney, they have different numbers of nephrons. The adult mammalian kidney post-nephron is composed of two embryonic tissues, the post-nephric mesenchyme (MM) and the ureteral bud (UB; Figure 1 c). the MM forms the nephron and mesenchyme of the adult kidney, and the UB differentiates to form the lower urinary tract from the collecting duct to part of the bladder.

Figure 1: Directed differentiation of kidney lineage cells. a-c Schematic drawings showing mesoderm specification (a), the formation of three kidneys (b) metanephros development (c). IM: intermediate mesoderm; MM: metanephric mesenchyme; UB: ureteric bud.

By generating a new clonogenic assay, we demonstrated for the first time that MM contains multipotent progenitor cells that can differentiate into the multiple epithelial cell types that make up the nephron, such as glomerular foot cells and tubular epithelial cells. Later, genealogical tracing experiments revealed that these progenitor cells are labeled by the transcription factor Six2. These progenitor cells are now referred to as renal metabolic progenitor cells (NPC). Taguchi et al. demonstrated that IM is divided into anterior and posterior domains, giving rise to UB and MM, respectively. based on these findings in kidney development, efforts have been initiated to regenerate renal genealogy cells from hPSCs.

Directed differentiation of hPSCs into kidney lineages

As a preliminary measure to differentiate hiPSCs directly into kidney lineages by mimicking kidney development, our group focused on the generation of IM cells and generated reporter hiPSC lines with the OSR1 gene, a specific marker for IM, by gene editing. Using a quantitative assessment system and reporter hiPSC lines, we developed an efficient differentiation scheme to induce hiPSCs into IM cells expressing osr1. These induced IM cells showed developmental potential for further differentiation into adult kidney cell types, such as glomerular podocytes and tubular cells, and formed three-dimensional (3D) tubular structures by co-culture with mouse posterior kidney cells.

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Taguchi et al. developed the first method for selective differentiation of mouse ESCs (mESCs) and hiPSCs via posterior IM to induce NPCs and generated renal unit-like organs containing glomeruli and tubules from induced NPCs in vitro. Takasato et al. reported the generation of renal-like organs containing multiple renal cell types, such as glomeruli, tubules, collecting ducts, interstitial cells, and vascular cells. By developing a 2D culture system, Morizane et al. efficiently generated NPC from hiPSCs and then generated renal metabolic organoids from NPC. Recently, our group developed a stepwise differentiation method that efficiently induces hiPSCs into NPC having differentiation potential to form renal unit-like organs (Figure 2 d, e). Our differentiation method consists of six steps and better summarizes the natural developmental process of NPC than the methods reported by Takasato et al. and Morizane et al. and generates NPC in 2D differentiation format more efficiently than the method using 3D culture by Taguchi et al.

Concerning the directed differentiation of UB lineage, Taguchi and Nishinakamura differentiated mESCs and hiPSCs into UB-like structures through the anterior IM and renal tubules (ND). However, ND epithelial cells were induced with low efficiency, and subsequent analysis required the purification of ND cells by flow cytometry. We developed a more efficient 2D differentiation method that generates ND epithelial cells from hiPSCs by pre-IM and includes a subsequent differentiation step without purification (Figure 2 f, g). In 3D culture, induced ND cells formed UB-like structures with RET(+) tips and CK8(+) stem structural domains. However, the generated UB-like structures from both groups showed limited branching potential.

Figure 2: d: Immunostaining of hiPSC-derived nephron progenitor cells (NPCs) for OSR1, SIX2, and HOXD11. e: Immunostaining of a nephron organoid formed from hiPSC-derived NPCs after 10 days of air–liquid interface culture. PODXL: Podocalyxin (podocyte marker; white); LTL: Lotus tetragonolobus lectin (proximal tubule marker; red); CDH1: CADHERIN 1 (distal tubule marker; green). f, g: Immunostaining of anterior IM cells for OSR1 (green) and GATA3 (red; f) and a nephric duct cell aggregate for E-CADHERIN (green), GATA3 (red) and nuclei (blue; g).h: Morphological change during the reconstruction of branching iUB organoids for 7 days.

Recently, by modifying our UB differentiation method, we successfully generated induced UB (iUB)-like organs with epithelial polarity, tubular lumen, and repetitive branching morphogenesis (Figure2,3 h-j). Furthermore, we successfully induced the differentiation of these iUB-like organs into their in vivo counterparts by collecting duct-like organs in human embryos at the 7th week of gestation (Figure 3k).

Figure 3:i: Immunostaining of an iUB organoid for RET (green), CK8 (red), and PAX2 (blue). j Toluidine blue staining of an iUB organoid showing tubular lumens. k: Bright-field (left) and immunostaining images (middle and right) of collecting duct-like tubular structures derived from an iUB organoid for FOXA1 (white), AQP2 (red), and GATA3 (green). Scale bars, 100 μm. (d) and (e) are adapted from Tsujimoto et al. [12], (f) and (g)–(k) are adapted from Mae et al. respectively

Expansion of kidney progenitors

In order to provide a large number of kidney cells for basic and clinical research, methods for in vitro expansion of embryonic kidney progenitor cells in culture were investigated. Brown et al. and Tanigawa et al. reported in vitro expansion of NPC in mouse embryos. NPC from mouse and human embryos were expanded and maintained in vitro for 17 and 7 months, respectively, using 3D cell aggregation culture by Li et al. The group also used the same method to expand NPC differentiated from hiPSCs in vitro for 2 months. Although all three methods use bone morphogenetic protein (BMP), the role of BMP7 in the expansion of NPCs remains unknown. Through compound screening, we identified a JAK3 inhibitor, TCS21311, as an alternative to BMP7 in the bb0 expansion culture developed by Li et al. and revealed the inhibitory effect of BMP7 on nasopharyngeal carcinoma expansion in the JAK3- stat3 signaling pathway. In addition, the addition of TCS21311 to the expansion culture improved the proliferation rate of mouse embryos and hiPSC-derived NPC.

Recently, we developed an expansion culture of hiPSC-derived UB cells in which single cells isolated from iUB-like organs proliferated to form colonies expressing UB tip markers. These tip colonies can reconstitute iUB-like organs in a repetitive branching potential, and this reconstitution process can be repeated at least three times.

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Kidney reconstruction

Early work on reconstructing renal structures used the putative amphibian fertilized egg ectodermal region, called the animal cap, which is a pluripotent cell mass (Figure 4 a). Moriya et al. reported that a combination of activin A and retinoic acid (RA) treatment induced the differentiation of the animal cap into prorenal tubules in vitro. Brennan et al. also induced prorenal angiosperms in exosomes. We demonstrated that the induced exosomes contain prerenal tubules and can regenerate prerenal tissue from amphibian pluripotent cells in vitro (Figure 4 b-d). Although the in vitro regeneration system of the pre-renal cannot be directly translated to clinical studies, it can serve as a simple and useful system for studying kidney development.

Figure 4: Reconstruction of kidney structures. a: A schematic showing the generation of pronephros structures from the animal cap of Xenopus embryos. b–d: Whole mount (b)and section double immunostaining images (c, d): of stage 42 equivalent Xenopus explant (b, c)and a stage 40 larvae (d)using a pronephric tubule-specific antibody (3G8, red) and a pronephric duct-specific antibody (4A6, blue).

In contrast to generating kidney unit-like organs from mESC- and hPSC-derived NPC and collecting duct-like organs from hPSC-derived UB cells, Taguchi et al. generated mouse kidney-like organs by combining mESC-derived NPC and UBs and mesenchymal progenitor cells removed from mouse embryos, which contain glomeruli, tubules, and collecting ducts, as described above. We generated human kidney-like organs in vitro by co-culture of NPC and UB cells induced by hiPSCs, respectively, in which NPC-derived glomeruli and tubules and UB-derived collecting ducts were interconnected (Figure 5 e, f). When transplanted into the subepithelial space of immunodeficient mice, these hiPSCs-derived kidney-like organs integrated with the vasculature of the host mice (Figure 5 g-i).

Figure 5:e: A schematic showing the in vitro reconstruction of kidney structures from hiPSCs. f: Triple immunostaining of day 20 kidney organoids for markers of podocytes (PODXL), proximal tubules (LTL) and distal tubules and collecting ducts (CDH1; left), and for PODXL and markers of distal tubules and collecting ducts (AVPR2) and collecting ducts only (CALB1; right). Note that weak CDH1 signals were also found in parts of the LTL+ proximal tubules in the left panel. g: A schematic showing the in vivo reconstruction of kidney structures from hiPSCs. h: A lower magnification image showing the whole host kidney and hiPSC-derived kidney graft (green) after rhodamine B-conjugated dextran administration through the tail vein of the host mouse. i: An intravital multiphoton microscopic image after tail vein injection of rhodamine B-conjugated dextran showing the vessel lumens of host mice penetrate into the hiPSC-derived glomerulus-like structure (green). Scale bars, 100 μm in (b)–(d) and (f), 500 μm in (h), and 40 μm in (i). (b)–(d) and (e)–(i) are adapted from Osafune et al. and Tsujimoto et al., respectively

For the transplantation of renal organs with urinary tracts, methods using experimental animal bodies, such as interspecies blastocyst complementation and organ ecotone methods, were investigated. Goto et al. injected wild-type mESCs into blastocysts of anemic Sall1(-/-) rats and successfully generated mouse kidneys in the host rats. Fujimoto et al. developed a cell transplantation method in which hiPSC-derived NPCs were transplanted into the developing kidney region (i.e., organ ecotone) of mouse uterine embryos, and the transplanted NPCs co-formed chimeric cap mesenchyme with host NPCs attached to the host mouse UB. However, although mm-derived glomeruli and tubules were derived from injected PSCs or NPCs, the remaining renal constituent cell types, such as UB-derived collecting ducts and inferior urethral and vascular cells, were derived from the host animal in both strategies.

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