A potentially exciting possibility for deriving

A potentially exciting possibility for deriving podocytes has been created by studies by Romagnani and co-workers, who identified and isolated renal progenitor cells (RPCs) from the parietal epithelium of Bowman\'s capsule of the adult kidney (Ronconi et al., 2009). This CD133+CD24+ cell population, which represents 1 to 4% of all renal cells, exhibits heterogeneous potential for differentiation into different renal cells. In this cell population, the subset of CD133+CD24+Podocalyxin− cells displayed the potential to differentiate into podocytes and tubular cells in vitro and to functionally improve glomerular and tubulointerstitial injury in a model of adriamycin-induced renal injury. Despite promising results, difficulties with accessing human RPCs from kidney biopsies has pushed research towards searching for a new source of RPCs. Taking into account that renal cells are naturally lost in urine, urine itself may represent a possible source of renal progenitor cells. To this end, the same group (Lazzeri et al., 2015) did establish RPC cultures from the urine of children with glomerular genetic disorders with the aim of obtaining podocytes and tubular cells. However, the major limitation of this technique is that the success rate for achieving a culture ranges from 8% to 51%, according to the phase of the disease, and drops to 0% with healthy subjects (Lazzeri et al., 2015). The breakthrough discovery of induced pluripotent stem cells (iPSCs) makes it possible to generate cells in vitro with an overall genetic and epigenetic background identical to donor cells, making iPSCs the ideal tool for in vitro disease modelling (Ye et al., 2013). The derivation of podocytes from pluripotent stem cells is an attractive alternative and an inexhaustible source of podocytes. Recently, different protocols for iPSC commitment towards renal progenitor cells through the activation of Wnt, bone morphogenic protein (BMP), fibroblast growth factor (FGF) and retinoic KPT-185 (RA) pathways involved in the induction of the intermediate mesoderm (IM) and subsequently in the metanephric mesenchyme and ureteric bud cells have been reported (Batchelder et al., 2009; Imberti et al., 2015; Kim and Dressler, 2005; Mae et al., 2010; Mae et al., 2013; Oeda et al., 2013; Taguchi et al., 2014; Takasato et al., 2014; Xia et al., 2013). The feasibility of deriving more mature kidney cells from pluripotent stem cells has also been demonstrated (Kang and Han, 2014; Kobayashi et al., 2005; Lam et al., 2014; Song et al., 2012). Here, we propose a simple and robust three-stage protocol based on single cell differentiation in chemically defined and feeder-free conditions, allowing for the highly efficient generation of human iPSC-derived podocytes. The podocytes generated are mature cells expressing the main podocyte markers and are able to respond to Angiotensin II, one of the major players in podocyte injury, and to internalize BSA and to integrate into WT1 positive structures in an ex-vivo model of developing kidney.
Materials and methods
Results A highly efficient and chemically defined protocol for differentiating human iPS cells into fully mature podocytes was attempted by trying to recapitulate kidney developmental stages in vitro. The differentiation protocol is based on three different stages, which include the induction phase into intermediate mesoderm, commitment towards nephron precursors, and specification into podocytes (Fig. 1A).
Discussion From the developmental perspective, the kidney is a mesodermal organ derived from the embryonic metanephros, which is composed of the ureteric bud, whose maturation produces the collecting ducts, the renal pelvis and ureters, and the intermediate mesoderm-derived metanephric mesenchyme, from which renal tubules and glomeruli originate. Over the last decade, a number of groups worldwide have tested the feasibility of deriving renal cells from embryonic stem cells or iPSCs. However, most of them generated mixed populations of progenitor cells committed to the ureteric bud or metanephric mesenchyme fate. So far, few studies have demonstrated the ability of progenitor cells to differentiate into more mature renal cells. Kobayashi et al. (2005), and more recently Lam et al. (2014), promoted stem cell differentiation into renal tubular cells. Two groups have attempted to derive podocytes from hiPSCs, but both studies have some limitations. Kang and Han described the generation of podocytes as just the ability of nephron progenitors to differentiate into more mature cells without giving any evidence of podocyte functionality (Kang and Han, 2014). Song and colleagues generated podocytes from iPSCs which, in contrast with their in vivo counterparts, still maintained proliferative capacity, a feature typical of immature cells (Song et al., 2012). Moreover, in this latter study the efficiency and robustness of the protocol were not discussed. Here, we developed a three-stage protocol that allowed us to obtain mature podocytes from iPSCs, passing through a nephron progenitor cell generation step. Our protocol induces the generation of intermediate mesoderm cells, which then acquire metanephric mesenchyme cell markers to finally mature into a podocyte-lineage cell population.