MicroRNA Cluster 302–367 Enhances Somatic Cell Reprogramming by Accelerating a Mesenchymal-to-Epithelial Transition

Abstract

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...

Embryonic stem cell and induced pluripotent stem cell: an epigenetic perspective

“…Upregulation of epithelial genes, such as Cdh1 and Epcam, and downregulation of mesenchymal genes, such as Snai1/2 and Zeb1/2, take place early in reprogramming [7,115,116]. Consistent with this, factors promoting the epithelial state, such as TGF-β inhibitors, BMPs, microRNA miR200s and miR302/367, and Cdh1, enhance iPSC generation, and in some cases, are able to substitute for reprogramming factors [116,[157][158][159][160][161][162]. In contrast, factors that suppress the epithelial state (e.g., TGF-β) or depletion of key epithelial adhesion molecules (e.g., Figure 1 The path from a somatic cell to a refined iPSC and the putative epigenetic barriers during the process.…”

MicroRNA-mediated iPS cells

“…[4][5][6][7] The UPenn researchers went a step further and showed that lentiviral-mediated expression of the miRNA-302/367 cluster of miRNAs reprogrammed mouse embryonic fibroblasts into iPS cells with higher efficiency than the standard four transcription factor reprogramming protocol. At 8 and 10 days following viral transduction, cell counting showed that the miRNA reprogramming protocol generated about two orders of magnitude more iPS cells than the standard protocol.…”

Urine-derived induced pluripotent stem cells as a modeling tool to study rare human diseases

“…HA-iPSCs are generated from patients' urine cells using an integrationfree transfection technique (46,70). Differentiated hepatocyte-like cells derived from HA-iPSCs (HAiPSC-Hep) display the phenotype of the defective FVIII found in selected patients.…”