Kidney cells with mesenchymal stem characteristics have been derived and propagated in vitro from the postnatal kidney. Subsequently, resident MSCs have been isolated and characterized from human glomeruli. ^, It was found that human glomeruli deprived of the Bowman capsule contain a population of CD133 ^– CD146 ⁺ cells expressing MSC markers, as well as the renal stem cell markers CD24 and Pax-2. These renal stem cell markers are not found in bone marrow stem cells (BMSCs). These cells exhibited a capacity for self-renewal, clonogenicity, and multipotency. These cells also acquired mesangial cell markers such as α-smooth muscle actin (α-SMA) and angiotensin II (AT-II) receptor I when cultured in appropriate conditions. It was concluded that a multipotent mesenchymal cell population is resident in human glomeruli and may have a role in physiologic cell turnover and/or in response to glomerular injury. ^, A further report demonstrated that CD133 ⁺ CD24 ⁺ cells in the adult human kidney consist of a hierarchical population of progenitors that are arranged in a precise sequence within the Bowman capsule and exhibit heterogeneous potential for differentiation and regeneration. These cells improved chronic glomerular damage in a model of doxorubicin (Adriamycin)-induced nephropathy in mice. CD133 ⁺ CD24 ⁺ CD106 ⁺ cells exhibited a high proliferative rate and could differentiate toward the podocyte, as well as the tubular lineage. CD133 ⁺ CD24 ⁺ CD106 ^– cells showed a lower proliferative capacity and displayed a committed phenotype toward the tubular lineage. Both these populations displayed the capacity to engraft within the kidney, generate new tubular cells, and improve renal function after injection into SCID mice with acute tubular injury. CD133 ⁺ CD24 ⁺ CD106 ^– cells proliferate upon tubular injury, becoming the predominant component of the regenerating epithelium in patients with acute or chronic tubular damage. Thus CD133 ⁺ CD24 ⁺ CD106 ^– cells represent tubular-committed progenitors that display resistance to apoptotic stimuli and exert regenerative potential for injured tubular tissue. Further studies have confirmed that CD24 ⁺ CD133 ⁺ cells in the Bowman capsule contribute to tubular regeneration.

The presence of stem cells in kidney tubules is well reported. In 2003, Maeshima and colleagues identified renal progenitor-like tubular cells that participate in regeneration processes after kidney injury. These renal progenitor-like tubular cells actively proliferate and eventually differentiate into epithelial cells during tubular regeneration. Gupta and colleagues reported a population of cells with characteristics of renal stem cells isolated from rat kidneys. The features of these cells include spindle-shaped morphology; self-renewal for >200 population doublings without evidence for senescence; normal karyotype and DNA analysis; and expression of vimentin, CD90 (thy1.1), Pax-2, and Oct4 but not cytokeratin, MHC class I or II, or other markers of more differentiated cells. These cells can differentiate into renal tubules when injected under the capsule of an uninjured kidney or intraarterially after renal ischemia-reperfusion injury (IPI). Challen and colleagues reported that adult kidney side population (SP) cells demonstrated multilineage differentiation in vitro, whereas microinjection into mouse metanephroi showed that SP cells had a 3.5- to 13-fold greater potential to contribute to developing kidneys than the non-SP main population cells. Many studies have shown that CD24 ⁺ CD133 ⁺ stem/progenitor cells in the tubules can regenerate tubular cells and improve renal function after kidney injury. ^{, , ,} Lazzeri and colleagues identified and characterized renal multipotent progenitors in human embryonic kidneys that share CD24 and CD133 surface expression with adult renal progenitors. These cells have the capacity for self-renewal and multilineage differentiation, regenerate different portions of the nephron, reduce tissue necrosis and fibrosis, and significantly improve renal function after injection into SCID mice with AKI.

Kidney resident MSCs in adult kidneys have been isolated, and their characteristics are like those of ESCs. These cells can differentiate into various lineages including mesodermal lineages, endothelial cells (ECs), and erythropoietin-producing fibroblasts. These cells promote improvement of renal function following kidney injury. As reviewed by Bruno and colleagues, an MSC-like population has been demonstrated when kidney injury occurs. These cells are considered to provide support for kidney regeneration by recapitulating the role they have in embryonic life.

It is well accepted that bone marrow–derived stem/progenitor cells (BMSCs) can be released into the peripheral blood and then distributed in all organs and tissues of the body. Patschan and colleagues confirmed endothelial progenitor cell (EPC) mobilization and homing during acute renal ischemia. Hematopoietic stem cells contribute to the regeneration of renal tubules after renal IRI in mice. It is reported that EPCs are mobilized into the damaged glomeruli and are likely to be involved in glomerular capillary repair in damaged glomeruli in experimental glomerulonephritis in mice. Human CD34 ⁽⁺⁾ hematopoietic stem/progenitor cells (HSPCs) have been demonstrated to promote kidney repair and regeneration by paracrine mechanisms, rather than replacement of vasculature in an IRI model in mice. By studying the time course of EPC and HSC stimulation and homing following induction of selective EC injury in the mouse kidney, along with various angiogenic factors potentially involved in EC repair and progenitor cell stimulation, it was found that selective EC injury leads to a marked stimulation of EPCs, HSCs, and various angiogenic factors that orchestrate microvascular repair.

BMSCs have been identified in both histologically normal mouse kidneys and human transplanted kidneys with damage from a variety of causes, which suggested that bone marrow cells contribute to both normal turnover of renal epithelia and regeneration after damage. It is reported that bone marrow–derived cells may have the potential to differentiate into glomerular mesangial cells. A report showed that recruitment of bone marrow–derived cells into glomeruli was dramatically facilitated in response to mesangiolysis evoked in anti-Thy1 antibody-mediated glomerulonephritis. In the regenerated glomeruli, 11% to 12% of glomerular cells were derived from the transplanted bone marrow and bone marrow–derived Thy1(+) cells kept increasing in number until the remodeling ceased, finally making up 7% to 8% of glomerular cells. These results indicate the contribution of bone marrow–derived cells to repopulating mesangial cells during glomerular remodeling in vivo. BMSCs can be induced to differentiate along podocytic lines in vitro. Thus BMSCs may be useful in the development of stem cell–based reconstitution of glomeruli that are damaged by disease. As well reviewed by Imai and Ito, bone marrow–derived cells have the capacity to differentiate into renal cells including mesangial cells, ECs podocytes, and tubular cells in the kidney, although controversy exists.

Stem cell–based therapy in kidney disease was described briefly earlier. Here the tissue-repairing function of stem cells in acute and chronic kidney injury is reviewed.

An efficient system of homologous recombination of human pluripotent stem cells has been established to generate human intermediate mesoderm (IM) (an embryonic germ layer that gives rise to kidneys), which has the ability to differentiate into multiple cell types that constitute adult kidneys, and to form renal tubule–like structures. An alternative system for differentiating hESCs and hiPSCs into cells expressing markers of the IM has been established through fibroblast growth factor signaling to induce IM differentiation in hPSCs, with features characteristic of kidney lineage cells. Three-dimensional (3D) structures of the kidney including glomeruli with podocytes, as well as renal tubules with proximal and distal regions and clear lumina, have been successfully developed in vitro from PSCs by reevaluating the developmental origins of metanephric progenitors. It is reported that the renal progenitors generated from hiPSCs are capable of reconstituting 3D proximal renal tubule–like structures in vitro and in vivo and have the potential to ameliorate AKI (AKI). Transplantation of iPSCs without c-Myc reduced the expression of oxidative substances, proinflammatory cytokines, and apoptotic factors in kidney tissues subjected to IRI and eventually improved survival in rats subjected to ischemic AKI. iPSC-derived conditioned medium has also been reported to attenuate AKI by downregulating the oxidative stress–related pathway following ischemia-reperfusion injury in rats.

EPC-based therapies have been studied in different models of AKI and CKD. Injection of renal artery–derived vascular progenitor cells (RAPCs) is associated with decreased serum creatinine after ischemia/reperfusion, reduced albuminuria, and improved blood flow in both animal models of acute ischemic and hyperperfusion injuries, with a small population of RAPC being shown to integrate with the renal microvasculature in the kidney. In vitro studies have also shown that RAPCs can promote local endothelial migration in co-culture. Systemically injected early endothelial progenitor cells (eEPCs) reduced serum creatinine levels in C57/Bl6N mice subjected to unilateral renal ischemia post uninephrectomy, significantly diminished interstitial fibrosis and prevented the loss of peritubular capillaries. The effect of EPC treatment on the progression of CKD was determined in C57BL/6 mice with 5/6 nephrectomy-induced CKD. Renal function deteriorated steadily over time in control mice, whereas the EPC-treated CKD group showed less deterioration of renal function and reduced proteinuria, along with relatively preserved kidney structure. The expression of proinflammatory cytokines and adhesion molecules was decreased in the kidneys of the EPC-CKD group. These results indicate that EPCs trafficked into the injured kidney protected the kidney from inflammation and consequently resulted in functional and structural renal preservation. Additional studies have shown that peripheral blood-derived endothelial progenitor cell therapy effectively inhibits the propagation of CKD and deterioration of renal function through enhancement of angiogenesis, blood flow, and antioxidative capacity, as well as suppression of inflammation, oxidative stress, apoptosis, and fibrosis in kidneys of rats subjected to 5/6 nephrectomy.

The renoprotective potential of pluripotent and adult stem cell therapy in experimental models of acute and chronic kidney injury has been intensively studied. MSCs are clearly shown to be the most efficient cells to initiate regeneration in damaged kidneys. AKI-CKD transition is one of the major contributors to CKD. Yet there is still no reliable therapeutic approach for treatment of the AKI-CKD transition. MSCs have the potential to at least partially block AKI-CKD transition. Treatment with MSC resulted in reduced inflammatory cytokines synthesis and a significant increase in the synthesis of adenosine triphosphate and number of renal cells undergoing mitosis (26%) in ex vivo kidneys (human kidney allografts from donation after cardiac death). MSCs can differentiate into multiple kidney cells including epithelial, mesangial, and ECs. For example, MSC GFP-positive cells, injected intravenously, homed to the kidney of mice with glycerol-induced AKI but not the kidney of normal mice. These MSCs localized to the tubular epithelial lining and expressed cytokeratin, differentiated into tubular epithelial cells, and promoted morphologic and functional recovery. Moreover, these MSCs enhanced tubular proliferation. hMSCs transdifferentiate to renal tubule epithelium after injection into the kidneys of newborn mice. Gene transcripts during the differentiation of MSC into mesangial cells have been identified through systematic transcriptomic profiling. MSCs have also been successfully induced to differentiate into endothelial lineage cells with capacity to develop capillary networks and progressively form vessel-like structures. Hence MSCs can differentiate into multiple renal cells to effect renal repair and regeneration.

In vivo infusion of MSCs protected mice from AKI and subsequent renal function impairment and severe tubular injury in a model generated by glycerol-induced rhabdomyolysis, which was accompanied by a time-dependent increase in CD206-positive M2 macrophage infiltration. A further study demonstrated that treatment with synthetic erythropoietin–pretreated BM-MSCs resulted in significantly reduced levels of serum IL-1β and TNF-α and significantly increased levels of IL-10 in rats following ischemia/reperfusion-induced AKI, compared with control rats infused with untreated BM-MSCs. Synthetic erythropoietin–BM-MSCs improved renal function as indicated by reduced serum creatinine, blood urea nitrogen, and improved pathologic scores. It was also reported that infusion of MSCs enhanced recovery of renal function in I/R-induced AKI in rats. MSCs were located in the kidney cortex after injection, and histologically, MSCs were predominantly located in glomerular capillaries. MSC therapy has also been shown to promote renal repair by limiting glomerular podocyte and progenitor cell dysfunction in experimental Adriamycin-induced nephropathy. It has been shown that BM-MSCs can differentiate into mesangial cells in vivo in athymic mice with glomerular injury and accelerate glomerular healing in experimental glomerulonephritis. ^, In addition to supporting glomerular repair, human BM-MSCs have been shown to decrease proximal tubular epithelial cell injury and ameliorate renal dysfunction, resulting in reduced mortality in a mouse model of cisplatin-induced AKI. BM-MSCs were predominantly localized in peritubular areas and shown to reduce renal cell apoptosis and increase proliferation. BM-MSCs protect against AKI-related peritubular capillary pathology, including EC abnormalities, such as cytoplasmic swelling, marked reduction in volume density of ECs and capillary lumen, leukocyte infiltration, and low EC and lumen volume density. The effect of MSC treatment for CKD has been reviewed elsewhere. ^,

Infusion of AD-MSCs has been reported to ameliorate renal dysfunction and kidney injury in a rat model of AKI induced by cisplatin, with a parallel increased survival in treated rats. AD-MSCs reduced apoptotic cell death, activation of p53, c-Jun N-terminal Kinase (JNK), extracellular signal–regulated kinase, and the expression of inflammation-related molecules in the injured kidney. These results show that human AD-MSCs exert a paracrine-protective effect on cisplatin nephrotoxicity at multiple target sites. It was also reported that human adipose tissue–derived stromal cells (hASCs) cultured with low (2%) serum showed a greater potential for tissue regeneration in folic acid–induced AKI in rats through paracrine effects, with hepatocyte growth factor (HGF) being one of the key mediators. hAD-MSCs have been shown to reduce oxidative stress markers and have a renoprotective effect against cisplatin-induced AKI in Sprague-Dawley rats. These studies raise the possibility that easily obtained autologous AD-MSCs could be used when AKI is expected to occur, such as in patients undergoing chemotherapy with platinum-based therapies.

An isolation protocol has been developed to obtain MSCs from the umbilical cord matrix. The isolated cells expressing all core markers for MSCs are clonogenic, retain long telomeres, can undergo several population doublings in vitro, and can be differentiated into mature mesenchyme, such as bone and adipose tissue. UC-MSCs are reported to be more potent immunosuppressors and less immunogenic than BM-MSCs, which may be due to increased levels of immunomodulatory surface proteins, such as CD200, CD273, CD274, and cytokines like IL1β, IL-8, LIF, and TGFβ2. UC-MSCs have a greater proliferative capacity compared with AD-MSCs and BM-MSCs. ^, UC-MSCs have been shown to traffic into the postischemic kidney and attenuate renal functional decline and tubular injury in mouse models of IRI. UC-MSCs reduced expression of interferon-γ but enhanced expression of vascular endothelial growth factor (VEGF) in the injured kidney. UC-MSCs after IRI in male rats improved glomerular filtration (decreased plasma urea and creatinine and increased creatinine clearance) and histologically had higher expression of aquaporin 2 and less macrophage infiltration in the kidneys. The IRI-induced pro-oxidative and senescence-related proteins and microRNAs and reduction in Klotho expression were reversed in the kidney after IRI by UC-MSC treatment. Further studies have confirmed the renoprotective role of UC-MSCs in animal models of CKD. ^, The number of UC-MSCs recruited to the injured kidneys was increased significantly in a rat model of streptozotocin-induced diabetic kidney disease (DKD), and UC-MSC treatment ameliorated the increase in 24-hour urinary protein, serum creatinine, urea nitrogen, and renal hypertrophy index and significantly reduced renal vacuole degeneration, inflammatory cell infiltration, and renal interstitial fibrosis. In in vitro experiments, UC-MSC conditioned medium and UC-MSC–derived exosomes decreased the production of proinflammatory cytokines (IL-6, IL-1β, and TNF-α) and profibrotic factor (TGF-β) in renal tubular epithelial cells and glomerular ECs exposed to high glucose. Moreover, UC-MSCs secrete large amounts of growth factors considered to be renoprotective including EGF, FGF, HGF, and VEGF. These results suggested UC-MSCs can effectively improve the renal function and inhibit inflammation and fibrosis in an animal model of diabetes-induced CKD. The low immunogenicity and immunoregulatory activity of UC-MSCs make them particularly attractive for therapeutic exploitation. However, the current method to isolate UC-MSCs results in no specific phenotype and the isolation and culture should be improved and standardized. The malignant tendency and malignant transformation after long-term cultivation of UC-MSCs also need further investigation.

BM-MSCs promote renal regeneration through ECVs such as BM-MSC–derived exosomes. It has been shown that MSC-derived exosomes significantly reduce their intrinsic regenerative potential in a mouse model of glycerol-induced AKI. ^, BM-MSCs exert a renoprotective role through their paracrine antiinflammatory and antifibrotic properties. BM-MSCs recruited to the site of injury may reprogram the damaged cells, stimulating their proliferation, and favoring tissue regeneration through the contribution of growth factors and transfer of genetic material by exosomes locally released by BM-MSCs. The effect of these exosomes on the recovery of AKI was similar to that of human MSCs. MSC-derived exosomes were reported to protect kidneys from IRI-induced AKI in rats by inhibiting apoptosis and stimulating tubular epithelial cell proliferation. Consequently, exosomes significantly improved the impairment of renal function and reduced transition of AKI to CKD.

Recent studies have demonstrated crucial roles of exosomes and tunneling nanotubes (TNTs) in long-range intercellular transfer of cellular materials. TNTs have been accepted as an emerging route of long-range intercellular communication, and cells can exchange cargo and organelles through TNTs. It has been reported that enhanced mitochondrial transfer between human corneal epithelial cells through TNTs promotes the regeneration of corneal epithelial cells in dry eye disease. Microglia exposed to α-synuclein established a cellular network through TNTs, and lowering the α-synuclein burden attenuated the inflammatory profile of microglia and improved their survival. As reviewed and summarized, TNTs mediate intercellular trafficking of cellular cargo and organelles. ^, The synergistic interplay between exosomes and TNTs in long-range intercellular transfer could be a critical repair mechanism in kidney injury.

The role of exosomes in renal inflammation has been examined in a porcine model of metabolic syndrome and renal artery stenosis, which was established in swine with unilateral renovascular disease complicated by metabolic syndrome (MetS). A single intrarenal delivery of exosomes harvested from autologous adipose tissue–derived MSCs was performed after 16 weeks of diet-induced MetS and renovascular disease, 4 weeks after exosome delivery. Stenotic-kidney renal blood flow and glomerular filtration rate (GFR) were measured in vivo (fast computed tomography), while characteristics of exosomes, renal microvascular architecture (micro-computed tomography), and injury pathways were analyzed ex vivo. The exosome fragments colocalized with stenotic-kidney tubular cells and macrophages and exosomes delivery attenuated renal inflammation and improved medullary oxygenation and fibrosis. The fall of renal blood flow and GFR in pigs with metabolic syndrome and renal artery stenosis was restored by exosome injection. This research group has further demonstrated that adipose tissue MSC-derived exosomes elicit better preservation of the stenotic-kidney microvasculature and greater attenuation of renal injury and fibrosis compared to percutaneous transluminal renal angioplasty (PTRA), possibly partly attributed to their cargo of vasculoprotective genes. It has been reported that adipose tissue MSC-derived exosomes protected renal function in mice with AKI induced by cecal ligation and puncture (CLP) through the sirtuin 1 (SIRT1) pathway. Adipose MSC-derived exosomes protect against LPS-induced AKI more significantly compared to BM-MSC–derived exosomes and improved kidney function and structure by inhibiting oxidative stress and inflammation.

Endothelial progenitor cell–derived exosomes have been reported to prevent AKI in a rat model of IRI by enhancing tubular cell proliferation, reducing apoptosis, and leukocyte infiltration. These exosomes also protected against progression of chronic kidney damage by inhibiting capillary rarefaction, glomerulosclerosis, and tubulointerstitial fibrosis. After injection in rats with Thy1.1 glomerulonephritis, exosomes derived from EPCs localized within injured glomeruli and inhibited mesangial cell activation, leucocyte infiltration, and apoptosis; increased serum complement hemolytic activity (CH50); decreased proteinuria; and ameliorated renal dysfunction, which suggest these exosomes exerted a protective effect by inhibition of antibody- and complement-mediated injury of mesangial cells.

iPSC-derived exosomes reduce cell death and inflammatory responses, protect functional mitochondria, and regulate several genes associated with oxidative stress in renal tubule cells with hypoxia-reoxygenation injury and in a rat model of AKI induced by IRI. It was reported recently that iPSC-derived exosomes enhance the survival of HK-2 cells undergoing cisplatin-induced cell death in cell culture. In mice with cisplatin-induced AKI, iPSC-derived exosomes reduce renal functional impairment as shown by reduced blood urea nitrogen and creatinine, in association with decreased infiltration of immune cells, reduced expression of inflammatory genes in M1-induced THP-1 cells, and enhanced capillary density in the kidney of AKI mice. These exosomes suppressed inflammatory responses, inhibited tubular cell apoptosis, and reversed the activation of extracellular signal–regulated kinase 1/2 signaling in the kidney of mice with AKI. Another study demonstrated that hiPSC exosomes protect against renal injury in AKI induced by IRI via delivering SP1 into target renal cells and intracellularly, activating the expression of sphingosine kinase 1 and generation of sphingosine-1-phosphate (S1P).

The renoprotective role of MSC-derived exosomes in DKD has been widely studied. ^, It was reported that MSC exosomes attenuate diabetic nephropathy in a rat model of streptozotocin-induced diabetes mellitus through enhancement of autophagy activity. It was also reported that MSC-derived exosomes could attenuate DKD by decreasing cell apoptosis and inhibiting epithelial-to-mesenchymal transition (EMT) in diabetic kidneys in db/db mice. In vitro studies showed MSC-derived exosomes could reverse high glucose-induced apoptosis and EMT in HK2 cells, and miR-424-5p derived from MSC-derived exosomes inhibited YAP1 activation in HK2 cells, resulting in alleviation of high glucose-induced cell apoptosis and EMT. Recent reports have demonstrated that MSC-derived exosomes attenuate inflammation including the expression of IL-6, IL-1β, IL-18, and TNF-α; depress the activation of the NLRP3 signaling pathway in podocytes under high glucose conditions and in diabetic mice; and ameliorate kidney injury. Knocking down miR-22-3p from MSC exosomes abolished their antiinflammation activity and beneficial function both in vitro and in vivo. These results indicate that MSC exosomes transferring miR-22-3p protected the podocytes and diabetic mice from inflammation, including by targeting the NLRP3 inflammasome. ^, Adipose-derived stem cell exosomes regulate Nrf2/Keap1 in diabetic nephropathy by targeting FAM129B. ADSC-derived exosomes reversed oxidative stress and inflammation in podocytes and kidney tissues of diabetic mice induced by high glucose levels in vitro and in vivo, and FAM129B siRNA blocked the inhibitory effect of ADSC-derived exosomes on intracellular reactive oxygen species and malondialdehyde upregulation induced by high glucose in podocytes. The renoprotective role of MSC-derived exosomes has been well reviewed elsewhere. ^, As summarized in these review articles, MSC-derived exosomes reduce ECM accumulation, thus improving fibrosis while protecting podocytes in models of DKD. However, the exosomes produced by various kidney resident cells are both crucial signaling mediators and potential biomarkers of DKD. These kidney cell exosomes facilitate cell-cell crosstalk as a contributing factor in DKD. Moreover, exosomes can be detected in urine and blood and have emerged as promising noninvasive biomarkers for the progress of DKD.

After kidney transplantation, rats treated with MSC-derived exosomes showed improved renal function and survival. MSC-derived exosomes mitigated renal cell apoptosis, enhanced proliferation, alleviated inflammation at the early stage, and reduced renal fibrosis at the late stage of transplantation. MSC-derived exosomes also decreased the number of CD68 ⁺ macrophages and expression levels of α-SMA and TGF-β1 proteins while increasing the protein expression level of HGF in the kidney. MSC-derived exosomes also prevented ischemic injury in isolated rat kidneys, as MSC-derived exosomes upregulate the expression of three genes encoding enzymes known to improve cell energy metabolism and three genes encoding proteins involved in ion membrane transport. It is suggested that MSCs-derived exosomes protect the kidney from ischemic injury by preserving the enzymatic machinery essential for cell viability and protecting the kidney from reperfusion injury.

In 2015, an efficient, chemically defined protocol for differentiating hPSCs into multipotent nephron progenitor cells (NPCs) that can form nephron-like structures was reported. In 3D culture, NPCs form kidney organoids containing epithelial nephron-like structures expressing markers of podocytes, proximal tubules, loops of Henle, and distal tubules in an organized, continuous arrangement that resembles the nephron in vivo. In the same year, kidney organoids that contained nephrons associated with a collecting duct network surrounded by renal interstitium and ECs were generated. The distal and proximal tubules, early loops of Henle, and glomeruli containing podocytes elaborating foot processes and undergoing vascularization were present in the nephrons of these organoids. The transcription profiles of these kidney organoids showed highest congruence with the first trimester of human fetal kidney. It is well accepted that dysfunction of podocytes and proximal tubular cells mainly contribute to glomerulosclerosis and tubular interstitial fibrosis, respectively, in CKD. Thus iPSC-derived kidney organoids have emerged as advanced in vitro models of kidney development, physiology, and disease. Current conventional research models, such as static cell cultures and animal models, are insufficient to reflect the complex human in vivo situation or lack translational value. Kidney organoids resemble the human kidney in a cell culture dish. Thus they can be applied in regenerative medicine, human kidney disease models, and drug development (“clinical trials in a dish”) and greatly reduce the usage of animals.

In comparison with immortalized 2-dimensional human podocyte cell lines, 3D human glomeruli sieved from iPSC-derived kidney organoids show improved podocyte-specific gene expression and an improved glomerular basement membrane matrisome. Hence human iPSC-derived organoid glomeruli represent an accessible approach to the in vitro modeling of human podocytopathies and screening for podocyte toxicity. The applications of kidney organoids are currently limited due to organoid variability, nephron immaturity, low throughput, and limited scale. Extrusion-based 3D cellular bioprinting to deliver rapid and high-throughput generation of kidney organoids with highly reproducible cell number and viability has been tested. The automated extrusion-based bioprinting for kidney organoid production delivers improvements in throughput, quality control, scale, and structure, facilitating in vitro and in vivo applications of stem cell–derived human kidney tissue. Kidney organoids that transcriptomically match second-trimester human fetal kidneys have been generated in an in vivo microenvironment that promotes the growth and differentiation of implanted kidney organoids, as well as providing a vascular component, which demonstrates that soft hydrogel accelerates the differentiation of hPSC-derived kidney organoids.

Primary kidney tubular epithelial organoids have been established from human and mouse kidney tissue and can be expanded for at least 20 passages while retaining a normal number of chromosomes. These organoids can also be established from human urine–derived stem cells (USCs). These human organoids represent proximal and distal nephron segments and have been used to model infectious, malignant, and hereditary kidney diseases in a personalized fashion. A 3D in vitro human renal organoid model has been developed from adult differentiated cells without gene modification. It is composed of multiple cell types and has been evaluated for morphology, viability, and erythropoietin production. This renal organoid is proposed as a model for drug screening and nephrotoxicity assays. The generation of an expandable, 3D branching ureteric bud (UB) organoid culture model that can be derived from primary UB progenitors from mouse and human fetal kidneys or generated de novo from human pluripotent stem cells has also been reported. Aggregating 3D-cultured NPCs with UB organoids in vitro results in a reiterative process of branching morphogenesis and nephron induction, similar to kidney development. Thus autologous ASC-derived organoids from patients may prove a useful tool for precision medicine.

The reduced survival time and small size of kidney organoids have led to challenges that have limited its capacity to study renal regeneration. It has been reported that kidney progenitors expressing nephrons develop from human pluripotent stem cells (hPSCs) subcutaneously implanted into immunodeficient mice. After 12 weeks, implants formed organ-like masses, which included perfused glomeruli containing human capillaries, podocytes with regions of mature basement membrane, and mesangial cells. After intravenous injection of fluorescent dextran, signal was detected in tubules, demonstrating uptake from glomerular filtrate. The functioning nephrons formed from hPSC-derived kidney precursors in vivo have been developed to model and treat kidney diseases. Similarly, vascularized 3D kidney organoids have been generated using dynamic modulation of WNT signaling to control the relative proportion of proximal versus distal nephron segments, producing a correlative level of vascular endothelial growth factor A (VEGFA) to define a resident vascular network. These kidney organoids undergo further structural and functional maturation upon implantation.

Despite great efforts from international researchers and rapid development and advancements in kidney organoid generation, there are challenges and limitations in clinical applications. For example, kidney organoids lack some critical cell types (e.g., infiltrating immune cells) and structures (e.g., blood vessels), lack of maturity, lack of ongoing nephron formation, and lack of systemic hemodynamic-related influences, as well as exposure to complex metabolic milieus in diabetic and other metabolic diseases. Another challenge lies in reproducibility due to technical variations in the study of kidney disease using kidney organoids. However, the robustness of the protocols and simplicity of organoid generation provide great benefits by using human/patient-based approaches to study kidney disease compared with 2-dimensional immortalized or primary cell line approaches. ASC-derived organoids to date have been more useful to define therapies for kidney-specific problems, such as autosomal dominant polycystic kidney disease (ADPKD). Hence modeling for personalized therapies to date has largely included ADPKD compared with glomerular and metabolic disease, for which systemic immune/inflammatory/metabolic mechanisms are more at play.

Regenerative medicine aims to replace or regenerate damaged cells, tissues, and ultimately the kidney to completely restore normal renal structure and function. As described earlier, kidney organoids that contain nephrons associated with a collecting duct network surrounded by renal interstitium and ECs have been generated, but significant limitations exist. Despite various strategies to establish functional kidney organoids, the therapeutic benefit of organoids before translation to clinical use warrants many further studies. As well summarized by Vives and Batlle-Morera: “The efficacy and safety of organoid-based therapies will require appropriate cellular composition, proper engraftment and vascularization into the host, and the demonstration of functional activity. Translation of organoid technologies into the clinic may benefit from the emergence of human leukocyte antigen (HLA)-homozygous iPSC initiatives, which hold the potential to foster the development of patient-compatible regenerative therapeutic approaches, and the blending of organoid technology with 3D bioprinting and vascularization approaches may lead to macrostructures with the desired cellular composition for successful transplantation.”

3D bioprinting has the potential to be used to “rebuild” a kidney. Bioprinting is a technology to allow the precise deposition of biomaterial inks layer by layer to fabricate complex tissues or organ models. Cellular extrusion–based 3D bioprinting has been used to deliver rapid and high-throughput generation of kidney organoids with highly reproducible cell number and viability. 3D bioprinting enables precise manipulation of biophysical properties including organoid size, cell number, and conformation, with modification of organoid conformation substantially increasing nephron yield per starting cell number, which facilitates the manufacture of uniformly patterned kidney tissue sheets with functional proximal tubular segments. Using Organovo’s proprietary 3D bioprinting platform, King and colleagues developed a fully cellular human in vitro model of the proximal tubule interstitial interface comprising renal fibroblasts, ECs and primary human renal proximal tubule epithelial cells to enable a more accurate prediction of tissue-level clinical outcomes. Histologically, the extensive microvascular networks are supported by endogenous extracellular matrix deposition, the epithelial cells of the 3D proximal tubule tissues demonstrated tight junction formation and expression of renal uptake and efflux transporters, and the polarized localization and function of P-glycoprotein (P-gp) and sodium-glucose transport protein 2 (SGLT2) were confirmed. The tissues also demonstrated nephrotoxicity of cisplatin and a fibrotic response to TGFβ. This research has successfully opened a door to kidney regeneration and organ transplantation. However, the challenge for 3D bioprinting of kidneys is to develop “renal” printing ink that will resemble complex renal structures, ECM composition, and both glomerular and tubular functionality. Despite advances in 3D bioprinting technology, there are still limitations in clinical applications, such as the ability of cells to survive in the printing process, the print speed, temperature, and pressure, all of which will need to be optimized for different cell types. Additionally, once developed, these complex 3D tissues can only be maintained for a relatively short period. ^,

Kidney decellularization is an alternative strategy to develop a kidney scaffold, which can then theoretically be used to “rebuild” a kidney. Briefly, all cells are removed throughout the whole kidney from the host (autologous kidney) with detergent, and then various sources of cells such as stem cells or adult differentiated cells from the host are injected to differentiate into mature cells in the decellularized scaffold with the extracellular matrix (ECM) to form a recellularized kidney, with functionality and no immune response after in vivo transplantation on the host. Ross and colleagues decellularized intact rat kidneys in a manner that preserved the intricate architecture and seeded them with pluripotent murine embryonic stem cells (mESCs). Similar studies reported that the decellularized intact rat kidney scaffolds preserve the 3D architecture of blood vessels, glomeruli, and tubuli, and histologically, collagen IV, laminin, and fibronectin staining of decellularized scaffolds were similar to those of native kidney tissues. Urine production from the recellularized kidneys was reported. Additionally, kidneys were recellularized by ex vivo infusion of hiPSC-ECs through the renal artery and vein of acellular kidneys, which resulted in the uniform distribution of the cells in all the vasculature compartments, from glomerular capillaries to peritubular capillaries and small vessels, and the presence of continuously distributed cells along the vessel wall was evidenced. The biomimetic scaffold for kidney regeneration was assessed in a rat kidney cortical defect model. Despite early promise, this strategy has not been translated into viable options for organ regeneration due to the complexity of kidney structure and influences of systemic factors.