Human induced pluripotent stem cells (iPSCs) have been generated with varied efficiencies from multiple tissues. Yet, acquiring donor cells is, in most instances, an invasive procedure that requires laborious isolation. Here we present a detailed protocol for generating human iPSCs from exfoliated renal epithelial cells present in urine. This method is advantageous in many circumstances, as the isolation of urinary cells is simple (30 ml of urine are sufficient), cost-effective and universal (can be applied to any age, gender and race). Moreover, the entire procedure is reasonably quick--around 2 weeks for the urinary cell culture and 3-4 weeks for the reprogramming--and the yield of iPSC colonies is generally high--up to 4% using retroviral delivery of exogenous factors. Urinary iPSCs (UiPSCs) also show excellent differentiation potential, and thus represent a good choice for producing pluripotent cells from normal individuals or patients with genetic diseases, including those affecting the kidney.

MicroRNAs (miRNAs) are emerging critical regulators of cell function that frequently reside in clusters throughout the genome. They influence a myriad of cell functions, including the generation of induced pluripotent stem cells, also termed reprogramming. Here, we have successfully delivered entire miRNA clusters into reprogramming fibroblasts using retroviral vectors. This strategy avoids caveats associated with transient transfection of chemically synthesized miRNA mimics. Overexpression of 2 miRNA clusters, 106a-363 and in particular 302-367, allowed potent increases in induced pluripotent stem cell generation efficiency in mouse fibroblasts using 3 exogenous factors (Sox2, Klf4, and Oct4). Pathway analysis highlighted potential relevant effectors, including mesenchymal-to-epithelial transition, cell cycle, and epigenetic regulators. Further study showed that miRNA cluster 302-367 targeted TGF␤ receptor 2, promoted increased E-cadherin expression, and accelerated mesenchymal-to-epithelial changes necessary for colony formation. Our work thus provides an interesting alternative for improving reprogramming using miRNAs and adds new evidence for the emerging relationship between pluripotency and the epithelial phenotype.Pluripotent and differentiated cell fates are determined at least in part by tissue-specific transcription factors that impose a concrete genetic program (1). In addition to coding RNAs, noncoding RNAs (2) are an integral part of the genetic programs that specify cell fate, regulating, for example, the expression of key cell-specific transcription factors (3) and chromatin stability (4) and therefore cell-specific properties. miRNAs 4 are 21-23-nucleotide-long noncoding RNAs that, by inducing degradation and/or preventing translation of target mRNAs (2), modulate a plethora of cell functions, including those related to ESC self-renewal/differentiation (5) and cell cycle progression (6). In this context, it is not only expected that miRNAs can enhance reprogramming but also tempting to speculate that, in the right combination, they might be able to reset somatic cells into iPSCs without added factors. Blelloch and co-workers (7) observed that, in the original mixture devised by Takahashi and Yamanaka (1), c-Myc can be substituted by components of the miR-290 cluster or by miR-302d. Elimination of c-Myc is desirable because it reduces tumor formation but has a negative effect on reprogramming. However, use of chemically synthesized oligonucleotides involves repeated transfection, and this implies a transient effect, toxicity, and an inability to pool large numbers of miRNAs without reducing their concentration beyond an optimal threshold. The latter is a concern because many ESC-specific miRNAs reside clustered within the same genome locus, and one could argue that delivering some or all these miRNA clusters together should be optimal for producing iPSCs. In this regard, we demonstrate herein that stable overexpression of entire endogenous miRNA clusters can potently improve reprogramming and be a...