Kidney tissue consists of more than 20 specialized cell types that are structurally organized into anatomically and functionally distinct compartments [21]. Primary kidney cells can be harvested from normal and diseased kidney tissue and expanded in culture while maintaining the phenotype and function from which they are derived. Among different types of renal cells in the native kidney, proximal tubular cells (PTCs) play important roles in kidney functions [22,23]. Such roles of PTC include reabsorption of proteins and electrolytes, hydrolase activity, and EPO production. In normal kidney, the PTCs occupy the highest percentage of cell population (≈60% of total cell population) among the other types, which include distal tubular cells, descending Loop of Henle cells, collecting duct cells and podocytes [24]. Therefore, isolation and expansion of functional primary PTCs from kidney tissues can be considered as an attractive renal cell source for cell-based therapy. Primary PTC cultures have many advantages and are more representative of normal PTC physiology than immortalized cell lines; however, primary renal cells, including PTCs, lose expression of specific genes during culture and are limited to only 2-5 passages [25]. The optimal combination of high purity, consistently well-preserved proliferation, and differentiation is observed at passage 2-3 [26].

Our group established a cell culture method that enabled expansion of primary kidney cells from human kidney tissues [12]. Histological analyses show that the majority of the cultured cells retained a proximal tubular phenotype while distal tubular cells and podocytes were present in a lower percentage of the entire cell population. Additionally, when the expanded cells were cultured under a 3D environment, the cultured cells form tubule-like structures with functional properties. These results demonstrate that the established cell harvesting and culture method may potentially be developed as an effective cell-based therapy for patients with renal failure.

While numerous studies have been conducted on PTC culture and physiology, several protocols have been established on other kidney cell types. Presnell et al. [27] established a culture method for primary cell cultures isolated from all major compartments of the kidney. In particular, two subpopulations, the tubular cell-enriched subpopulation and the EPO-producing subpopulation, were reproducibly developed from both normal and diseased kidneys. Owing to recent advances in immunomagnetic cell isolation, Baer and Geiger [28] isolated additional subtypes of kidney-derived epithelial cells. Human renal epithelial cells were separated from the ascending limb and the distal tubule using glycoproteincoated magnetic beads. The results demonstrate a successful in vitro system to study the thick ascending limb of Henle's loop and early distal tubule as well as a promising cell source for treatment of renal failure.

Existence and identification of renal stem or progenitor cells in the adult kidney tissues have been somewhat controversial [19]. Numerous peer-reviewed articles have identified progenitor cells in the Bowman's capsule [29], glomeruli [30], proximal tubules [31,32,33,34], and renal papilla [35]. Human renal cells were isolated from unused donor kidneys, expanded in culture, and maintained their renal phenotypes through numerous passages [12]. This result may support that differentiated renal epithelial cells isolated from the kidney tubule structures have proliferation capacity through dedifferentiation of the surviving epithelia during cell culture. Recent evidence demonstrates that kidney nephrons contain a rare cell population with proliferative capability [31,34,36,37,38]. Those stem or progenitor cells express several stem cell markers such as CD24, CD133, CD106, vimentin and Pax-2, and they possess high clonogenic potential, self-renewal ability, and differentiate into specific renal cell lineages, which suggest a potential therapeutic cell source for renal regeneration.

Embryonic stem (ES) cells have been studied as a cell source for various cell-based therapies [39]. ES cells exhibit two remarkable properties: the ability to grow in an undifferentiated state (self-renewal) and the capacity to differentiate to several cell types of the mesodermal, endodermal, and ectodermal lineages (multidifferentiation). This makes ES cells an attractive cell source for TE and RM. Renal differentiation of ES cells has been reported by several investigators [40,41,42,43]. Interestingly, using a versatile in vitro culture system, Steenhard et al. [42] demonstrated that integration of ES cells into embryonic metanephric kidney produced kidney-like organs in vitro. This efficient integration and creation of new kidney tissue strongly supports the therapeutic feasibility of the use of ES cells; however, several limitations decreased the enthusiasm for active development. These include uncontrolled growth and developing into teratoma formation in vivo as well as legal and ethical problems associated with the use of embryonic tissue.

Another cell source that possesses pluripotent capability is the induced pluripotent stem cell (iPS), which was first developed by Takahashi and Yamanaka [44] through reprogramming human fibroblasts by introducing four genes (Oct3/4, Sox2, c-Myc, and Klf4). Recent advances in the production of iPS cells from both mesangial and epithelial cells derived from urine [45] has encouraged the application of iPS cells to the treatment of kidney diseases. In addition, iPS cells have been generated from other renal sources such as PTCs and podocytes [46], which demonstrate the high universality of iPS cell technology. When compared with ES cells, the use of iPS cells has advantages for RM that includes the absence of ethical issues related to tissue sourcing and fewer immune rejection complications. Therefore, iPS cells may be a promising cell source for treatment of kidney diseases in a clinical setting once safety and other issues, including control of outgrowth and teratoma formation of undifferentiated iPS cells, are resolved. Several studies have demonstrated that iPS cells express abnormal genes and induce T-cell dependent immune response in syngeneic recipients [47]. This unexpected immune response by iPS cell infusion should be clarified before clinical application. Therefore, sophisticated renal differentiation protocols should be established by defining optimal cell culture conditions and additional factors such as a choice of a target cell type and reprogramming options [20].

The idea of cell-based therapy to treat renal failure originated from the discovery of the natural regenerative ability of the damaged kidney [53]. After their early study, Cuppage and Tate [54] developed an acute renal injury model by treating rats with mercury chloride and examined the reparative phase of the injury to demonstrate the correlation of structural and functional parameters of recovery. Their studies showed that squamous cells attached to the basement membrane were able to regenerate the tubular epithelium in AKI and suggested that the basement membrane could be a site of origin of the regenerating cells and a key regulator of kidney regeneration. Importantly, these researchers discuss the requirement of an upregulation of protein synthesis within the activated microenvironment for the efficacy of renal regeneration. Therefore, it can be considered that the success of cellbased therapy in the treatment of renal disease depends on the efficient integration of the transplanted cells to the activated renal microenvironment and subsequent growth to induce renal regeneration after injury.

Most preclinical studies use rodents to create AKI. As a cell source, BM-MSCs have been utilized extensively in a wide range of studies [55,56,57,58,59,60,61,62]. Early studies have shown that the predominant recovery mechanism of the AKI was mediated through the trans-differentiation of the infused MSCs into renal specific cell types, particularly renal tubular cells [63], podocytes [64], mesanglial cells [65], and glomerular cells [66]. More recent studies support the concept that trophic factors secreted from the injected MSCs stimulate endogenous cells to proliferate and regenerate the infused renal tissues [61,67,68,69]. In terms of autocrine and paracrine effects of the secreted factors on the renal regeneration, immune-related response [70], antiapoptotic, mitogenic, and vaso-protective effect [62] were suggested. Several studies suggest that the effect of BM-MSCs in acute renal injury models is mediated by paracrine mechanisms. As evidenced by reports that BM-MSC-conditioned medium contains microvesicles and growth factors that reduce inflammation and accelerate renal repair through interactions with renal progenitors [66,71,72,73]. While numerous positive effects have been observed using BM-MSCs for renal regeneration, negative results were reported following infusion of BMMSCs into the renal disease model that promoted interstitial fibrosis [74] and development of angiomyelproliferative effect [75]. Therefore, safety studies to evaluate complications are needed to ensure that successful clinical outcomes are obtained with the use of BM-MSCs.

Another source of MSCs, adipose-derived MSCs (ADMSCs), has been tested in the renal failure model. De Almeida et al. [76] demonstrated that infusion of ADMSCs into an acute renal injury model reduced renal fibrosis at six weeks. Chen et al. [77] also showed that an intrarenal injection of ADMSCs resulted in improved angiogenesis and preserved the renal structure integrity, which restored renal function at 14 days. Several other studies also demonstrated the effectiveness of ADSC on the improved renal functions using mouse [78], rat [79], and pig [80,81] models.

As described earlier, primary kidney progenitor cells are promising cell sources for the treatment of renal disease. Recent developments in stem cell biology have provided researchers with better isolation and culture techniques that have enabled identification of stem or progenitor cells from kidney tissues. Bussolati et al. [36] reported that CD133⁺-cells could be isolated from the normal adult kidney tissue and cultured in vitro. When the CD133⁺-cells were intravenously injected in severe combined immunodeficiency mice with glycerol-induced tubulonecrosis, the expanded kidney-derived CD133⁺-cells were grafted into the injured kidney and integrated into kidney tubules. These researchers proposed that CD133⁺-cells isolated from normal kidney represent a multipotent adult resident stem cell population that may contribute to the repair of tissue damaged by renal injury. Using a more specific set of surface markers, Angelotti et al. [31] also reported that CD24⁺-CD133⁺-CD106-renal progenitor cells are resistant to apoptosis and provide regenerative potential for tubular tissue damage in the rhabdomyolysis-induced AKI model of rats. Harari-Steinberg et al. [82] identified nephron progenitor cells (hNPCs) in human kidney tissue. Neural cell adhesion molecule (NCAM1)-positive cells showed clonogenic and renal progenitor properties. After transplantation into the chick embryo, NCAM-positive hNPCs formed a renal-like structure. After hNPCs were infused into the kidneys of rats with renal failure, it was observed that disease progression was inhibited and renal function was increased. Together, these results support the idea that kidney-derived stem or progenitor cells have the capacity to migrate and proliferate to restore structural and functional kidney tissue.

Recent studies by our group have demonstrated that primary renal cells isolated from human kidneys facilitate beneficial effects toward the recovery of renal functions. As previously described, we developed a cell isolation and culture system to obtain sufficient numbers of human primary renal cells for cell therapy [12]. We have established two rodent kidney injury models by varying the length of the renal ischemic time [83]. In particular, we demonstrated that the use of a longer renal ischemic time (75 and 90 minutes) could be used to evaluate novel therapies for acute renal disease, whereas a shorter renal ischemic time (60 minutes) could be appropriate to study therapies for chronic renal failure [83]. To investigate the therapeutic effects of primary kidney cells on the improvement of renal functions, cultured renal cells were harvested and infused into a CKD rat model [83], and renal functions were evaluated. In particular, several reports focused on a cell population of EPO-positive renal cells. EPO, a cytokine produced by fibroblast-like cells in the kidney [84,85], has recently been shown to act as a renoprotective factor with anti-inflammatory and antioxidant activity. Our group established a cell purification protocol to enrich the EPOpositive renal cell population [85]. The results showed that intra-renal delivery of an EPO-positive-enriched cell population facilitated beneficial effects in the treatment of renal injury, with respect to inflammation and oxidative stress, compared to unpurified cell cultures in a CKD model. The results demonstrated that EPO-positive cells from primary kidney cells can be considered as a potential cell population for treating degenerative kidney diseases. Another CKD model using two-step 5/6 nephrectomy in rats was used to demonstrate the beneficial effects of primary renal cells on the recovery of renal functions [86]. In this study, the authors demonstrated that the tubular cells-enriched population provided better therapeutic effects compared to unfractionated heterogeneous renal cells by attenuating canonical pathways of profibrotic extracellular matrix production.

The use of hAFSCs to treat renal disease shows promise toward restoration of normal kidney functions. Hauser et al. [87] have shown that intravenous infusion of hAFSCs resulted in more rapid recovery of renal function in an AKI model compared to infusion of BM-MSCs. The authors of the study suggest that a range of cytokine and growth factors produced by hAFSCs gives them the advantage in renal regeneration. In addition, the preconditioning effects of hAFSCs were evaluated for renal function recovery. Rota et al. [88] demonstrated that hAFSCs 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 models when compared to hAFSCs alone. Also, other kidney failure model such as Alport syndrome was used to examine the effect of AFSC on the improved renal function [89]. Moreover, a large porcine animal model was evaluated for efficiency of renal regeneration. Baulier et al. [90] reported that hAFSCs were capable of preventing fibrosis and preserved renal function in a porcine model of renal transplantation. As a pluripotent cell source, iPS cells have shown improvement of renal functions using an AKI rodent model [91,92].

To date, few clinical trials have been performed to evaluate the safety and efficacy of cell injection for renal disease [17], particularly targeting amelioration of renal transplantation-related complications. In most cases, MSCs have been used due to their low immunomodulatory effects and high regenerative capability. Reinders et al. [93] performed a safety and feasibility study on kidney allograft patients. Autologous BM-MSCs were administrated into the patients, and clinical and immune reaction was monitored up to 24 weeks after MSC injections. Results of this trial showed that the autologous injection improved resolution of interstitial fibrosis/tubular atrophy (IF/TA), and the authors concluded that autologous treatment with BM-MSCs in kidney transplant recipients with subclinical rejection and IF/TA is clinically feasible and safe; furthermore, the findings are suggestive of systemic immunosuppression. Similarly, autologous MSCs were infused into a group of kidney recipients to evaluate acute rejection in the event of graft-versus-host-disease as compared to the non-cell-injected groups. Tan et al. [94] demonstrated that the use of autologous MSCs compared to no treatment facilitated lower incidence of acute rejection, decreased risk of infection, and better estimated renal function at 1 year. In another clinical trial composed of two kidney recipients, autologous MSCs were evaluated for feasibility of cell injection and therapeutic effects. Following kidney transplant, Perico et al. [95] demonstrated positive effects of infusion of autologous MSCs through the reduction of immune rejection through the enlargement of regulatory T cells (Treg) in the peripheral blood and control of CD8⁺ T cell function.

In addition to the clinical outcomes in the treatment of acute rejection from kidney transplant patients through infusion of MSCs, as described above, a few clinical trials have begun to examine the effects of cell-based therapy on restoration of renal functions in the AKI and CKD. According to a recent clinical report [96], several companies will initiate Phase I/II clinical trials to treat acute kidney failure using autologous MSCs or other cell types.