MicroRNAs Are Indispensable for Reprogramming Mouse Embryonic Fibroblasts into Induced Stem Cell-Like Cells

MicroRNAs play a pivotal role in cellular maintenance, proliferation, and differentiation. They have also been implicated to play a key role in disease pathogenesis, and more recently, cellular reprogramming. Certain microRNA clusters can enhance or even directly induce reprogramming, while repressing key proteins involved in microRNA processing decreases reprogramming efficiency. Although microRNAs clearly play important roles in cellular reprogramming, it remains unknown whether microRNAs are absolutely necessary. We endeavored to answer this fundamental question by attempting to reprogram Dicer-null mouse embryonic fibroblasts (MEFs) that lack almost all functional microRNAs using a defined set of transcription factors. Transduction of reprogramming factors using either lentiviral or piggyBac transposon vector into two, independently derived lines of Dicer-null MEFs failed to produce cells resembling embryonic stem cells (ESCs). However, expression of human Dicer in the Dicer-null MEFs restored their reprogramming potential. Our study demonstrates for the first time that microRNAs are indispensable for dedifferentiation reprogramming

Nanog induces suppression of senescence through downregulation of p27KIP1^{KIP1} expression

A comprehensive analysis of the molecular network of cellular factors establishing and maintaining pluripotency as well as self renewal of pluripotent stem cells is key for further progress in understanding basic stem cell biology. Nanog is necessary for the natural induction of pluripotency in early mammalian development but dispensable for both its maintenance and its artificial induction. To gain further insight into the molecular activity of Nanog, we analyzed the outcomes of Nanog gain-of-function in various cell models employing a recently developed biologically active recombinant cell-permeant protein, Nanog-TAT. We found that Nanog enhances the proliferation of both NIH 3T3 and primary fibroblast cells. Nanog transduction into primary fibroblasts results in suppression of senescence-associated beta-galactosidase activity. Investigation of cell cycle factors revealed that transient activation of Nanog correlates with consistent downregulation of the cell cycle inhibitor p27$^{KIP1}$ (also known as CDKN1B). By performing chromatin immunoprecipitation analysis, we confirmed bona fide Nanog-binding sites upstream of the p27$^{KIP1}$ gene, establishing a direct link between physical occupancy and functional regulation. Our data demonstrates that Nanog enhances proliferation of fibroblasts through transcriptional regulation of cell cycle inhibitor p27 gene

Human <i>Dicer</i> expression in <i>Dicer</i>-null MEFs allows generation of iPSCs.

<p>(<b>A</b>) Timeline of reprogramming <i>Dicer</i>-null MEFs rescued with human <i>Dicer</i>. Once human <i>Dicer</i> cDNA integrated into the <i>Dicer</i>^Δ/Δ MEF genome, reprogramming became possible even when factors were transduced 6 days after Cre induction. (<b>B</b>) Rescued iPSCs (ResDcr iPSC) lacked mouse <i>Dicer</i> (<i>mDcr</i>), but instead expressed human <i>Dicer</i> (<i>hDcr</i>) gene, verified by RT-PCR. (<b>C, D, E</b>) <i>Dicer</i>^Δ/Δ MEFs expressing human <i>Dicer</i> can reprogram to become iPSCs. Rescued iPSCs expressing human <i>Dicer</i> showed ESC morphology and stained for alkaline phosphatase (boxed areas represent magnified view) (<b>C</b>), and expressed stem cell markers tested by RT-PCR (<b>D</b>) and immunofluorescence (<b>E</b>). (<b>F</b>) Rescued iPSC promoters for stem cell genes <i>Oct4</i> and <i>Nanog</i> became demethylated, resembling wild-type ESCs. (<b>G</b>) Upon subcutaneous injection into SCID mice, rescued iPSCs formed teratomas that showed differentiation into all three germ layers. (<b>H</b>) Human Dicer can cleave mouse pre-miRNAs into mature miRNAs. qPCR for a panel of mature miRNAs in rescued iPSCs (ResDcr) lacking mouse <i>Dicer</i> demonstrated comparable expression levels to that of wild-type ESCs (W4). In contrast, mature miRNAs were completely depleted in <i>Dicer</i>-null ESCs (Dicer^Δ/Δ). Each value is represented relative to an assigned W4 value of 1.0 for that miRNA. Data are presented as mean +/− SD.</p

<i>Dicer</i>-null MEFs lacking miRNAs fail to reprogram.

<p>(<b>A, B</b>) Timelines of attempt at reprogramming <i>Dicer</i>-null MEFs. The main difference between the two strategies is that (<b>A</b>) transduces reprogramming transcription factors (TFs) 6 days post induction with Cre, while (<b>B</b>) transduces TFs 1 day post induction with Cre. <i>Dicer</i>-null MEFs could not be reprogrammed when reprogramming factors were transduced 6 days after induction. However, reprogramming <i>Dicer</i>-null MEFs was possible when reprogramming factors were transduced 1 day post induction with Cre. (<b>C</b>) <i>Dicer</i>^+/+, <i>Dicer</i>^Δ/+, and <i>Dicer</i>^f/f MEFs consistently reprogrammed into iPSCs with reprogramming factors. These iPSCs stained for alkaline phosphatase. <i>Dicer</i>^Δ/Δ MEFs reprogrammed to form induced stem cell-like cell colonies that stained for alkaline phosphatase when reprogramming factors were transduced 1 day post induction with Cre (Δ/Δ-1dpi). Transducing reprogramming factors 6 dpi (Δ/Δ-6dpi) or not transducing any factors (No TF) never reprogrammed <i>Dicer</i>-null MEFs. Boxed areas represent magnified view. (<b>D</b>) Genomic PCR confirmed induced stem cell-like cell colonies formed by transducing reprogramming factors 1 day post induction with Cre (Δ/Δ-1dpi) having <i>Dicer</i>^Δ/Δ genotype (floxed DNA band). Control iPSC colonies (f/f iPSC) formed without Cre induction had <i>Dicer</i>^f/f genotype (flox DNA band). (<b>E</b>) Residual Dicer protein is still present 1 day after deletion of <i>Dicer</i> gene. By 6 days post induction (dpi) with Cre, residual Dicer protein is completely degraded, inhibiting cellular reprogramming. (<b>F, G</b>) Wild-type ESCs, <i>Dicer</i>^f/f iPSCs (f/f iPSC), and <i>Dicer</i>^Δ/Δ induced stem cell-like cells generated by transducing reprogramming factors 1 day post induction with Cre (Δ/Δ-1dpi) expressed all stem cell markers tested by RT-PCR (<b>F</b>), and immunofluorescence (<b>G</b>). (<b>H</b>) <i>Dicer</i>^f/f iPSCs and <i>Dicer</i>^Δ/Δ induced stem cell-like cells acquired ESC methylation patterns in <i>Oct4</i> and <i>Nanog</i> promoters.</p