Mir-302 Cluster Governs Complete Nuclear Reprogramming Through Suppression of MBD2 in iPSCs Derived From CD34+ Cord Blood Cells Stored Frozen for More Than 20 Years

Abstract 1265

Precise control of gene expression is of paramount importance in the process of proper cell reprogramming. Although a number of microRNAs (miRNAs) have been implicated in fine-tuning mRNA translation during early mammalian development, the regulatory role of miRNAs, particularly in connection with functional targets involved during reversion of a differentiated cell to a pluripotent state, is not completely understood. To gain further insight into the role of miRNAs in nuclear reprogramming we performed miRNA PCR-array analysis on partially reprogrammed cells and completely reprogrammed human induced pluripotent stem cells (iPSC) that we obtained during our recent reprogramming experiments with CD34+ cells isolated from umbilical cord blood that had been stored frozen for 21 years. We found significant downregulation of human embryonic stem cells (hESCs)-specific miRNAs, miR-302 and 371 clusters, in the incompletely reprogrammed iPSCs, which was comparable to that of differentiated cells; i.e. opposite to pluripotent hESCs. Therefore, we hypothesized that miR-302 and 371 clusters may promote a transition from partially to completely reprogrammed iPSC through regulation of methylation status. To test this hypothesis, we over-expressed miRNA clusters of miR-302 and 371 in partially reprogrammed cells, and determined whether these miRNAs converted partially reprogrammed to completely reprogrammed iPSCs. Our results indicated that over expression of miR-302 clusters resulted in conversion of more than 15%±2 of partially reprogrammed to fully reprogrammed iPSCs. These new fully reprogrammed iPSCs possessed the same characteristics as cells that were initially completely reprogrammed without enforced expression of miR-302 cluster including acquisition of typical hESCs-specific pluripotent markers and the ability to differentiate into three germ layers. We then demonstrated that this more complete reprogramming mechanism functioned through miR-302 cluster-targeted suppression of an epigenetic regulator methyl-DNA binding domain protein 2 (MBD2), which suppresses gene transcription through binding to methylated CpG islands of target genes. While MBD2 expression was unobservable, or found at very low levels, in hESCs, hiPSCs, and human embryocarcinoma cells (hECCs), MBD2 was highly expressed in incompletely reprogrammed cells as well as in differentiated cells derived from human pluripotent stem cells. We showed that MBD2 was directly bound to the DNA methylation region of the NANOG promoter, as revealed by ChIP analysis, and miR-302 cluster directly reduced expression of MBD2 as shown by luciferase activity in reporter assays containing wild-type 3' UTR MBD2 by 50% in hECCs. Thus, enhanced expression of the exogenous miR-302 cluster in the incompletely reprogrammed cells may play an active role in expression of endogenous pluripotent genes by inhibiting MBD2 expression, thereby promoting more complete nuclear reprogramming. Taken together, our results provide strong support for a regulatory role of miR-302 in epigenetic regulation of core pluripotent specific genes involved in self-renewal and reprogramming of somatic cells.