Various types of cells are considered to be able to either contribute directly to renal repair after damage or substantially improve renal injury without direct contributing to the renal epithelium. The proper regulation of these cells and successful delivery are important for the development of kidney regeneration. There are four possible main origins for cells contributing to kidney repair: (1) interstitial cell transdifferentiation to epithelium [12], (2) recruitment of stem cells from the bone marrow [13–17], (3) tubular cell dedifferentiation and proliferation in response to injury [12, 18, 19], and (4) repopulation of the renal tubules by a resident kidney stem/progenitor cells [20–22]. The first two options involve nonepithelial cell transdifferentiation into renal epithelium, whereas the other two options involve epithelial cells within the renal epithelium itself. The theory that renal repair involves cells within the renal epithelium of the nephron was supported by lineage studies showing no evidence of dilution of the cap mesenchyme-derived (Six2+) tubular epithelium with a nonepithelial cell source [23]. However, this study did not answer whether any cell within the epithelium can contribute to repair or whether repair relied on a resident stem cell population within the tubules.

Soluble factors involved in kidney development have been identified by gene targeting techniques, in vitro tubulogenesis models, and organ culture systems. The regeneration process after renal injury resembles the kidney organogenesis. Most of these factors have been proved to regulate renal recovery as potential renotropic factors by using animal kidney injury models. These renotropic factors are as follows: hepatocyte growth factor (HGF) [61], epidermal growth factor (EGF) [62], heparin-binding EGF-like growth factor (HB-EGF) [63, 64], bone morphogenetic protein-7 (BMP-7) [65, 66], insulin-like growth factor-I (IGF-I) [67, 68], platelet-derived growth factor (PDGF) [69], and uterine sensitization-associated gene-1 (USAG-1) [70]. In addition, several key regulatory molecules required for renal organogenesis, such as Pax-2, leukemia inhibitory factor, and Wnt4, are reactivated in regenerating tubular cells after ischemic renal injury [71–73].

Recently, the role of these renotropic factors in kidney injury has been demonstrated. Chen et al. showed the importance of EGF receptor activation in the recovery phase after acute kidney injury through mice with a specific EGF receptor deletion in renal proximal tubules [74]. Deletion of the BMP receptor activin-like kinase 3 in the tubular epithelium enhances epithelial damage and fibrosis [75]. Conditional knockout mice lacking the HGF receptor, c-met, specifically in renal tubules demonstrated the antiapoptotic of anti-inflammatory effect of c-met signaling in renal protection after acute kidney injury [76].

A negative regulatory factor of kidney repair has also been identified. Several studies using transgenic mice expressing truncated activin type II receptor, an in vitro tubulogenesis model, the Wolffian duct culture, and isolated rat embryonic kidney culture showed that activin A is an endogenous inhibitor of renal organogenesis [77–83]. Activin A is also a potent inhibitor of renal regeneration after kidney injury [84].

Most of these renotropic factors can induce tubular cell proliferation after kidney injury when exogenously administered. However, it is still not well known whether these factors are involved in cell maturation, modulation of renal blood flow, neutrophil infiltration, and restoration of polarity. It will be interesting to examine if these renotropic factors promote renal regeneration via the activation of intrinsic renal stem cells.

Generally, macrophages have a function to clear foreign pathogens in the body that can disrupt tissue homeostasis [85] and also play an important role to mediate kidney repair. Although macrophages are well known to promote the fibrotic and inflammation reaction during renal disease, they have been also demonstrated to have a protective role in acute and chronic kidney diseases [86]. Moreover, Wyburn et al. reported that macrophages can ameliorate allograft rejection [87].

Macrophages represent a heterogeneous population with distinct functions depending on resident tissue and phase of tissue injury or disease. In some cases, macrophages display classical activation through the release of nitric oxide, reactive oxygen species, and proinflammatory cytokines, termed an M1 response or phenotype [88, 89]. In another stage of disease, macrophages can play antifibrotic and anti-inflammatory roles, termed M2 phenotype [90, 91]. These M2 macrophages have a reparative function in ischemia-reperfusion injury and obstructive nephropathy, induced by unilateral ureteric obstruction [92–95]. Many studies reported that changes in the local microenvironment are important to trigger an M1 phenotype macrophages, such as macrophage colony-stimulating factor, which can be released by tubular epithelial cells to stimulate this process [96, 97]. Based on these findings, macrophages were directed toward an M2 phenotype via genetic manipulation or cytokine alteration, such as overexpression of interleukin 4 (IL-4), IL-10, inducible nitric oxide synthase, or heme oxygenase, resulting in improved renal function [98–106]. In terms of regenerative medicine, macrophages are considered to have therapeutic potential and be one of the options for cellular or cytokine/growth factor-based therapies in kidney diseases.

Many cells, such as renal progenitor cells, endogenous renal tubular epithelial cells, bone marrow-derived stem cells, and macrophages, are involved in kidney regeneration. However, the exact mechanism of cell to cell interaction and influencing factors for cell reaction involving regenerative process after kidney injury is still not exactly known. Although several studies suggested possible pathways involved in kidney regeneration, further research to identify the factors in specific signaling pathways will be needed.

Early studies using stem cells for kidney regeneration usually have shown that the predominant recovery mechanism of the acute kidney injury was mediated through the transdifferentiation of the infused mesenchymal stem cells into specific renal cell types, especially podocytes, renal tubular cells, glomerular cells, and mesangial cells [13, 125–127]. More recent studies support the concept that cytokines and/or growth factors secreted from the injected mesenchymal stem cells stimulate endogenous cells to proliferate and regenerate the infused renal tissues [128–131]. Several studies suggested that the effect of mesenchymal stem cells in acute kidney injury models is associated with paracrine mechanisms, such as immune-related response and mitogenic, antiapoptotic, and vasoprotective effect [132, 133]. It was evidenced by the results of several studies that bone marrow-derived mesenchymal stem cell-cultured medium contains microvesicles and growth factors that reduce inflammation and enhance renal repair through interactions with renal progenitors [15, 125, 134, 135].

Bone marrow-derived mesenchymal stem cells are multipotent, migrate across tissues, are easily harvested, are produced throughout life, and contribute to the repair of organs [136]. The administration of bone marrow stem cells has been shown to promote neovascularization, reduce inflammation, inhibit apoptosis, and stimulate differentiation and proliferation in multiple systems [137]. Poulsom et al. first reported the effect of engrafted bone marrow stem cells into the damaged kidney, improving repair of renal tissues after acute kidney injury [17]. However, despite these benefits of bone marrow stem cells, clinical application is still in question because negative results were also reported. Bone marrow stem cells have been shown to promote interstitial fibrosis in mouse models, and direct renal injection of bone marrow stem cells induced the development of angiomyeloproliferative lesions of unknown neoplastic potential [138, 139]. Therefore, further safety studies are necessary before clinical applications to ensure safe and successful outcomes for kidney regeneration using bone marrow stem cells.

Another promising cell source of mesenchymal stem cells is adipose-derived stem cells. Several studies reported the effectiveness of adipose-derived stem cells to improve renal functions in animal models, including mouse, rat, and pig [140–143]. Administration of adipose-derived stem cells into an acute kidney injury model reduced renal fibrosis at 6 weeks [144], and intrarenal injection of adipose-derived stem cells showed improved angiogenesis and preserved the renal structure integrity, which restored renal function at 14 days [145].

Amniotic fluid-derived stem cells are easily harvested and cultured with lower risk of tumorigenicity and similar multipotency to bone marrow stem cells. Rota et al. demonstrated the therapeutic potential of human amniotic fluid stem cells [146]. In their study, engrafted amniotic fluid stem cells localized primarily to the peritubular region, limiting tubular damage, improving renal function, and prolonging survival of the animals. They also reported that amniotic fluid stem cells that were preconditioned with glial cell line-derived neurotrophic factor were observed to promote better functional recovery by contributing to renal regeneration in acute kidney injury models when compared to amniotic fluid stem cells without treatment. The authors suggested that a combination of transdifferentiation of amniotic fluid stem cells and paracrine signaling involved the regenerative effect through this study. Hauser et al. reported that intravenous infusion of amniotic fluid stem cells induced more rapid recovery of renal function in an animal model of acute kidney injury, when compared to that of bone marrow stem cells. In this study, bone marrow stem cells showed higher potential for proliferation, whereas amniotic fluid stem cells showed better capacity for antiapoptosis and persistence within the peritubular capillaries. In addition, they isolated different cytokines and growth factors from bone marrow and amniotic fluid stem cells, suggesting different modes of action between both cell sources.

Induced pluripotent stem cells (iPSCs) have gained increasing interest in the field of regenerative medicine, as they possess pluripotent capability and potentially provide an inexhaustible source of patient- and tissue-specific stem cells. Recently, iPSCs have been successfully generated from human renal sources, such as kidney mesangial cells, proximal tubular cells, podocytes, and epithelial cells derived from urine [147–149]. IPSCs have several advantages compared with embryonic stem cell, such as absence of ethical issues related to cell sourcing, fewer immune rejection, and less potential for abnormal tissue formation. Moreover, they also have more capabilities to facilitate targeted, organ-specific differentiation due to retaining the epigenetic pattern of the parent cell [150]. Recent studies reported the improvement of renal functions in an acute kidney injury rodent model after administration of iPSCs [151, 152]. However, iPSCs have been shown to express abnormal genes and induce T-cell-dependent immune reaction in syngeneic mice [153]. This unexpected immune response is a big hurdle of iPSCs for clinical application.

Aforementioned renal progenitor cells are also promising cell sources for the treatment of acute kidney injury. It has been demonstrated that CD24+/CD133+/CD106- renal progenitor cells are resistant to apoptosis and promote recovery of tubular injury in the rhabdomyolysis-induced acute kidney injury rat model [22]. Neural cell adhesion molecule (NCAM)-positive cells that were identified from human kidney tissue showed clonogenic and renal progenitor properties [154]. After engraftment into the chick embryo, NCAM+ human nephron progenitor cells formed a renal-like structure. Administration of human nephron progenitor cells into the kidneys of acute kidney injury rat model induced inhibition of disease progression and improvement of renal function. Taken together, these results support the concept that renal progenitor cells have the capacity to migrate and proliferate to improve structural and functional damage after acute kidney injury.

Although advancement of stem cell technology has showed promising results in the field of kidney regeneration, stem cells themselves have limitations to maintain the structure of the kidney. Thus, many researchers tried to develop the cellular approach by incorporating principles of developmental biology. This investigative field sought to create, implant, and maintain a renal structure that mimics the physical and physiological characteristics of the native kidney.

The concept of early investigations in this field endeavored to infuse new nephrons to developing kidneys by implantation of embryonic metanephric tissue into the renal cortex of neonatal mice [155]. Metanephros is the embryological precursor of the adult kidney, characterized by mesenchyme and ureteric buds (Fig. 7.2) [156]. Transplantation of primitive metanephric tissue over fully developed kidney may have some advantages, such as reduced immunogenicity due to the absence of native vasculature and antigen-presenting cells [156, 157]. Thus, metanephros can be maintained in vivo without host immunosuppression. Although transplanted metanephric tissue was successfully differentiated and developed into functional nephrons in the host kidney of neonate, it was not initially reproduced in host kidneys of adult due to lack of differentiation and acute graft rejection [158]. However, another study reported successful implantation and differentiation of metanephroi into adult hosts, with demonstrated glomerular filtration and plasma clearance after modifying the protocol [156]. Encouraged by these results, further studies to investigate the possibility of xenotransplantation have been performed. More information related to xenotransplantation will be discussed in another section.

Extracellular matrix is the naturally occurring scaffold material secreted and manufactured by the resident cells of each organ, and it plays a critical role in development and repair of organ [161, 162]. The extracellular matrix is a complex three-dimensional framework of structural and functional proteins in a state of dynamic equilibrium within the surrounding tissues and provides the means by which cells communicate with each other and the external environment [163]. The extracellular matrix contains growth factors and bioinductive cytokines, which facilitate the reparative process, cell attachment, and tissue integration [164, 165]. Consequently, the roles of extracellular matrix for organogenesis and regeneration can be categorized by providing a three-dimensional scaffold for the spatial organization of cells, secreting and storing growth factors and cytokines, and regulating signal transduction [166].

The kidney has approximately 20 different specific cell types. For efficient kidney regeneration, all cells must be characterized for repopulation. Although many attempts have been performed to establish a reliable cell source and optimal cell growth conditions to provide adequate enrichment for achieving stable renal cell expansion systems, it is still challenging. It is essential to isolate feasible stem or progenitor cells for cell expansion and differentiation in vitro, and renal stem/progenitor cells are one of the good candidates. There are five different methods which have been used to isolate renal stem/progenitor cells from the adult kidney.

Recent advances in stem cell biology and cell culture techniques have facilitated the development of cell therapy for clinical translation [206, 207]. Compared with other approaches for kidney regeneration, this method using cellular approach can be more practically applied to kidney tissue regeneration due to the relatively simple cell manipulation process, easy access to the target site in a less invasive manner, and effective integration of administered cells with the host tissues. Consequently, many studies have been performed using this cellular approach to treat renal failure [3, 41, 206]. In this section, cellular approach for kidney tissue regeneration, including various cell sources used as well recent advances made in preclinical and clinical studies, will be discussed.

There have been many efforts to generate mature cells using pluripotent stem cells in vitro. Several studies reported successful generation of mature cells, which have characteristics of liver, pancreas, and intestine, from embryonic stem cells or iPSCs using stepwise protocols mimicking the mechanism of embryonic development [263, 265, 267, 270, 271]. Furthermore, autonomous formation of three-dimensional adenohypophysis, polarized cortical tissues, optic cap, and retina structures using three-dimensional culture systems of embryonic stem cells suggested the possibility of organogenesis for therapeutic regeneration using this approach [301–304].

It was demonstrated that a single multipotent progenitor cell from embryonic mouse kidney can express Sall-1 and differentiate into several types of renal cells, such as podocytes and tubular epithelial cells, and eventually reconstruct a three-dimensional kidney structure [305]. Another study also reported that single-cell suspensions from embryonic kidney reaggregated and formed organotypic renal structures [306]. Because intermediate mesoderm cells differentiated into renal progenitor cells and then into maturing renal cells, if pluripotent stem cells can be differentiated into intermediate mesoderm, all kinds of renal cells may be generated from embryonic stem cells or iPSCs. The protocols to differentiate human iPSCs into intermediate mesoderm cells were established using bacterial artificial chromosome-based vectors and single-nucleotide polymorphism array-based detection [274]. These cells expressed intermediated mesoderm marker genes and could mature into various types of cells including those found in organs, such as the kidney, adrenal gland, and gonad. These results suggested that three-dimensional kidney structure could be constructed if pluripotent stem cells can be made to generate renal progenitor cells.

Blastocyst is an early developmental stage of the embryo that occurs 5 days after fertilization. Injection of pluripotent stem cells into blastocysts synchronizes the development of two line cells, and the combined blastocyst can generate a chimeric body. The chimera can also be produced by injecting pluripotent stem cells into xenoblastocyst, which lacks potential to form any particular cell lineage. The chimera is then transplanted into a foster uterus, and the deficient cells are exclusively derived from injected pluripotent stem cells. It was first reported that injection of normal embryonic stem cells into the blastocysts of recombination-activating gene 2-deficient mice, which have no mature B or T lymphocytes, generated somatic chimeras with embryonic stem cell-derived mature B and T cells [307]. Recent several studies demonstrated that several types of tissues and organs, such as the thymic epithelium, yolk sac, germ cells, hepatocytes, heart, pancreas, and kidney, could be reconstructed using this blastocyst complementation system [308–315]. Kobayashi et al. successfully generated a functional pancreas using this approach [310]. In this study, iPSCs of rat were injected into Pdx-/- (pancreatogenesis-disabled) blastocysts of mouse that produced newborn rat/mouse chimeras with a pancreas which originated almost entirely from the injected iPSCs of rat. The mouse and rat iPSC-derived pancreas produced insulin, and the transplantation of this iPSC-derived pancreas islets improved hyperglycemia in a diabetic rodent model [217]. This study suggested that iPSC-derived cellular progeny could aggregate and differentiate into the deficient organ in a vacant developmental niche. Moreover, these results also demonstrated that interspecific blastocyst complementation could be used to generate organs derived from donor pluripotent stem cells in vivo using a xenogeneic environment. This study group generated a Pdx-/- pig and succeeded in generating bigger pancreas using this technique, suggesting that human-scale organs could theoretically be generated [311]. Likewise, chimeric mice carrying a liver derived from injected iPSCs of mouse were also generated from blastocysts with fumarylacetoacetate hydrolase deficiency [308]. Hepatocytes derived from the injected iPSCs also had a proliferative characteristic of normal hepatocytes.

Kidney reconstruction using this blastocyst complementation system has been attempted. Usui et al. injected normal mouse iPSCs into blastocysts from kidney-deficient mice lacking the SAL-like 1 protein, which is essential for kidney development; eventually, the neonatal mice had kidneys derived from injected iPSCs [315]. However, the vascular and nervous systems were not constructed from iPSC-originated cells; thus, the kidney was not completely complemented. Immunohistochemical analysis of the regenerated kidney showed that the vascular system, including renal segmental, lobar, interlobar, arcuate, and interlobular arterioles, was a chimeric structure originating from both donor iPSCs and host cells. Whether urine was produced by this regenerated kidney was not certain due to the lack of precise urine analysis. In addition, generation of rat kidneys in mice failed after injection of rat iPSCs into kidney-deficient mouse blastocysts. This result indicates that the key molecules in mice involved in interactions between the metanephric mesenchyme and ureteric bud do not cross-react with those in rats. Therefore, the generation of xenogeneic organs using interspecific blastocysts requires a host animal strain lacking all renal lineages.

Although this strategy using blastocyst complementation has showed promising results for kidney regeneration, there are several limitations for clinical applications. It is difficult to generate interspecific chimeras in livestock. In addition, tissue rejection also should be overcome. Moreover, ethical issue is one of the most serious problems. There has been always a concern about the possibility of generating interspecific chimeras containing brain derived from injected pluripotent stem cells.

The embryonic metanephros (Fig. 7.3), which constitutes the primordial mammalian kidney, has been used for the investigation of kidney regeneration and showed promising results as a potential source for the generation of a functional whole kidney [156, 316–324]. If a metanephros is transplanted into the renal cortex of a host mouse, it can continue to grow [155]. The transplanted metanephros contains vascularized glomeruli and mature proximal tubules endowed with a capacity for glomerular filtration. Collecting duct-like structures also appeared and extended from the transplant toward the papilla of the host. Although there is no direct evidence that these collecting duct-like structures connect with the collecting system of the host or that the transplanted metanephros functions in a manner similar to that of native kidney, these results offer a rationale for the existence of renal stem cells in the metanephros from embryos, which can be a potential source of transplantable regenerated kidney. However, there are still several unsolved problems related to this technique, including questions as to the suitability of the renal capsule of patients with dialysis as a transplant site, given the significant disruptions to this area, such as the vasculature, and the fact that space limitations beneath the renal capsule may inhibit the growth of transplanted metanephros. To overcome these concerns, the metanephroi were transplanted into a host omentum in another study [156]. The transplanted metanephroi into the omentum were not confined by a tight organ capsule or disturbed by dialysis. The metanephroi transplanted into a host omentum also could differentiate into mature vascularized glomeruli and proximal tubules and produce urine. Moreover, after an intact ureteroureterostomy with the ureter of the native kidney that was removed, anephric host animals started to void and showed prolonged survival. These metanephroi developed in host animals exhibit metabolic renal function and produce erythropoietin and renin leading to detectable levels of these proteins in serum [319, 323, 324]. Furthermore, metanephroi from porcine embryos were transplanted into either the omentum of mice in which costimulation was blocked or area under the kidney capsules of immunodeficient mice and developed to generate fully functional nephrons [316, 320]. The average levels of creatinine and urea nitrogen were higher in the cystic fluid that arose from the transplanted tissue than in the sera of the transplanted host mice. These successful xenotransplantation of metanephroi between different species was based on previous studies showing minimal immunogenicity in tissues, including the metanephros harvested at earlier gestational ages [325]. In cases of allotransplantation, such as rat metanephros to rat omentum, transplants assumed a kidney-like shape in situ that was approximately one-third the diameter of the native kidney, and the transplants contained well-differentiated kidney structure in histological analysis. In addition, the transplantation can be performed without immunosuppression. However, in xenotransplantation, although transplanted metanephros grew and differentiated into renal tissue in the host omentum, immunosuppressants were required. Without these agents, the transplants disappeared soon after transplantation.

Transplantation of metanephros was also shown to maintain blood pressure in anephric rats with induced acute hypotension and reduce vascular calcification in rats with chronic renal failure, indicating that transplanted metanephroi have multiple renal functions as well as urine production [323, 324]. Although the omentum has been preferred because a tight renal capsule does not confine it and the host vessels can be preferentially integrated into the transplanted metanephros, the low hydrostatic pressure in the omentum may influence the acquisition of some renal function in the developed metanephros. Indeed, it was reported that the para-aortic area is better for the establishment of renin-producing tissue [319].

Recently, it was reported that xenotransplanted metanephros could supply endogenous mesenchymal stem cells with a niche for differentiation into erythropoietin-producing renal tissues [318]. Polymerase chain reaction (PCR) using species-specific primers and sequence analysis showed that xenotransplanted metanephroi expressed host animal-originated erythropoietin. This result implied that the erythropoietin-producing cells were originated from the host animals and developed to produce erythropoietin in the transplanted metanephros. It was also shown that the erythropoietin-producing cells were not originated from integrating vessels but from circulating host mesenchymal stem cells mobilized from the bone marrow.

Conventional transplantation of metanephros requires strong and persistent immunosuppression to avoid humoral rejection associated with the xenogeneic barrier, which can cause several adverse effects, such as carcinogenicity and severe rejection. Thus, for safety, when the xenotransplant is not needed anymore, it should be discarded by introducing a cell fate-regulating system in which a suicide gene is expressed on demand. To avoid the xenogeneic barrier, metanephroi isolated from transgenic ER-E2F1 mice that are expressing E2F1 under the control of tamoxifen can be used. E2F1 is a transcription factor which regulates cell proliferation and ectopic expression of which induces apoptosis. The administration of tamoxifen in host rats wherein metanephroi of ER-E2Fa mice were transplanted activated the suicide gene, and the xenocompartments of transplanted metanephroi were ablated by apoptosis, leaving the autologous erythropoietin-producing tissues. In addition, it was also confirmed that the metanephros established by this method no longer needed immunosuppressants while maintaining erythropoietin production [318]. These results indicate that xenometanephroi per se could acquire some renal functions in the host omentum as well as supply a niche for host stem cells to regenerate renal tissues and then be rebuilt to constitute only host cell components. This technique can be helpful to reduce the adverse effects of long-term administration of immunosuppressants and to moderate the ethical issues surrounding xenotransplantation.

One of the most promising methods for kidney engineering is the generation of fetal or mature renal tissues using intrinsic proficiency of cells to organize themselves into fully self-derived sophisticated structures [301, 303]. Several early studies using this strategy demonstrated that renal epithelial cell lines cultured in extracellular matrix gels could be induced to create branching tubules either by the whole embryonic kidney or by the metanephric mesenchyme [326]. Advancement of this methodology showed that cells isolated from ureteric bud (Fig. 7.4) of mice at embryonic day 11.5 (E11.5) and propagated in vitro could undergo branching tubulogenesis when stimulated by a conditioned medium from a metanephric mesenchyme-derived cell line [327]. Similarly, a tubular epithelial cell line or mesenchymal stem cells generated glomerular and tubular structures in subcutaneous spaces when stimulated by a media from vascular endothelial and tubular cells [328]. These results suggested that simple cultures of cells derived from undifferentiated metanephric mesenchyme and unbranched ureteric bud could be used to create branching tubular structures similar to those that form in normal metanephros.

Generation of three-dimensional renal epithelia which contain glomerular-like structures in vitro was attempted using suspensions of murine metanephric mesenchymal E11.5 progenitor cells selected for a high expression of Sall-1, which is essential during kidney development [305]. This cell preparation required coculture with an exogenous spinal cord cell layer, as has been performed in traditional developmental investigations [329]. Although the functional capacity of these structures has not been investigated in vivo, this approach suggested that dispersed cell suspensions can be used as starting material to obtain rudimental three-dimensional kidney tissues. Further development of this method enabled the generation of fetal kidney tissue from suspensions of E11.5 kidney cells without using any exogenous tissue [306]. While this method successfully reproduced the structures and differentiation states of nephrons and stroma, collecting ducts developed as a multitude of very small collecting duct trees rather than a continuous one as would happen in a normal kidney. Combination of reaggregated single-cell suspensions of ureteric bud and metanephric mesenchyme allowed the formation of nephrons arranged around a single collecting duct tree [330]. However, this endeavor did not achieve development of glomeruli to any meaningful extent due to avascular environment in vitro. This limitation inhibits further progress of research related to maturation potential of tissue obtained from a simple culture or suspension of precursor cells. Therefore, the capacity of these cells to generate nephros with functional properties still remained unclear.

Using simple suspensions of singe kidney cells, “organoids” that could carry out renal functions when implanted into a living animal, such as glomerular filtration, tubular reabsorption of macromolecules, and production of erythropoietin, were finally constructed [322]. These organoids were generated from single-cell suspensions derived from E11.5 mouse kidneys and then implanted under the renal capsule of an athymic rat. The main procedure to obtain complete glomerulogenesis consisted of soaking the organoids in a solution containing VEGF, and then injection of VEGF into the recipient animals after the organoids had been implanted in the kidney. This method restituted the efficacy of the podocyte VEGF axis that is necessary for glomerular capillary endothelial induction and resulted in the formation of vascularized glomeruli with fully differentiated glomerular capillary walls, including endothelial fenestrae, slit diaphragms, and foot processes. These organoids could develop glomeruli with normal-appearing slits in vivo, wherein the proportion of diaphragms were comparable to that found in the adult glomerulus. When the ultrafiltering function of the intragraft nephrons was tested by injecting fluorescent dextrans of increasingly high molecular weight into the host blood system, the proximal tubular cells concentrated dextrans only of lower molecular weights from the lumen, indicating efficient ultrafiltration.

Approaches starting from the metanephros and dissociating down to the single-cell level offer a valuable source for performing pharmacologic or genetic modifications in individual cells prior to the in vitro aggregation step [322, 331]. The single-cell stage allows the use of genetic engineering approaches to humanize cells, by introducing immunomodulatory genes or by switching off others, to reduce the possibility of rejection and facilitate the xenotransplantation of animal renal tissues [331]. These self-forming organoids can be exploited to generate human renal tissue by constructing chimeric kidneys that combine animal progenitor cells and human stem cells, followed by the selective elimination of animal cells after transplantation. Previous studies have shown that the organoids could incorporate cells of another source, such as human amniotic fluid stem cells or iPSC-derived OSR1-positive cells, to build chimeric tissue in vitro [253, 274]. Although several technical problems remain to be overcome, these systems are useful to examine the mechanisms of renal progenitor differentiation and suggest the possibility of developing a whole kidney from a single stem cell.

The idea of generating human organs in animals was previously suggested by several studies [332, 333]. Regeneration of a whole functional kidney that produced urine and renal hormones using a developing heterozoic embryo as an organ factory has been endeavored [334–336]. This strategy is based on the concept of “borrowing” the developing program of a growing xenoembryo by applying stem cells at the niche of organogenesis. During development of the metanephros, the metanephric mesenchyme initially forms from the caudal portion of the nephrogenic cord and secretes GDNF, which induces the nearby Wolffian duct to produce a ureteric bud [337]. Then, the metanephric mesenchyme consequently forms the glomerulus, proximal and distal tubule, loop of Henle, and the interstitium, as a result of reciprocal epithelial-mesenchymal induction between the ureteric bud and metanephric mesenchyme [338]. For this epithelial-mesenchymal induction to occur, GDNF must interact with its receptor, c-ret, which is expressed in the Wolffian duct. Thus, it can be hypothesized that GDNF-expressing stem cells can differentiate into kidney structures if they are placed at the budding site and stimulated by several factors spatially and temporally identical to those found in the developmental milieu.