MicroRNAs (miRNAs) are posttranscriptional modulators of gene expression and play an important role in many developmental processes. We report here that expression of microRNA-145 (miR-145) is low in self-renewing human embryonic stem cells (hESCs) but highly upregulated during differentiation. We identify the pluripotency factors OCT4, SOX2, and KLF4 as direct targets of miR-145 and show that endogenous miR-145 represses the 3' untranslated regions of OCT4, SOX2, and KLF4. Increased miR-145 expression inhibits hESC self-renewal, represses expression of pluripotency genes, and induces lineage-restricted differentiation. Loss of miR-145 impairs differentiation and elevates OCT4, SOX2, and KLF4. Furthermore, we find that the miR-145 promoter is bound and repressed by OCT4 in hESCs. This work reveals a direct link between the core reprogramming factors and miR-145 and uncovers a double-negative feedback loop involving OCT4, SOX2, KLF4, and miR-145.

SUMMARY Self-renewal of human embryonic stem (ES) cells is promoted by FGF and TGFβ/Activin signaling, and differentiation is promoted by BMP signaling, but how these signals regulate genes critical to the maintenance of pluripotency has been unclear. Using a defined medium, we show here that both TGFβ and FGF signals synergize to inhibit BMP signaling, sustain expression of pluripotency-associated genes such as NANOG, OCT4, and SOX2, and promote long-term undifferentiated proliferation of human ES cells. We also show that both TGFβ- and BMP-responsive SMADs can bind with the NANOG proximal promoter. NANOG promoter activity is enhanced by TGFβ/Activin and FGF signaling, and is decreased by BMP signaling. Mutation of putative SMAD binding elements reduces NANOG promoter activity to basal levels, and makes NANOG unresponsive to BMP and TGFβ signaling. These results suggest that direct binding of TGFβ/Activin-responsive SMADs to the NANOG promoter plays an essential role in sustaining human ES cell self-renewal.

Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) resets the epigenome to an embryonic-like state. Vitamin C enhances the reprogramming process, but the underlying mechanisms are unclear. Here we show that the histone demethylases Jhdm1a/1b are key effectors of somatic cell reprogramming downstream of vitamin C. We first observed that vitamin C induces H3K36me2/3 demethylation in mouse embryonic fibroblasts in culture and during reprogramming. We then identified Jhdm1a/1b, two known vitamin-C-dependent H3K36 demethylases, as potent regulators of reprogramming through gain- and loss-of-function approaches. Furthermore, we found that Jhdm1b accelerates cell cycle progression and suppresses cell senescence during reprogramming by repressing the Ink4/Arf locus. Jhdm1b also cooperates with Oct4 to activate the microRNA cluster 302/367, an integral component of the pluripotency machinery. Our results therefore reveal a role for H3K36me2/3 in cell fate determination and establish a link between histone demethylases and vitamin-C-induced reprogramming.

SUMMARY Here we show that as human embryonic stem (ES) cells exit the pluripotent state, NANOG can play a key role in determining lineage outcome. It has previously been reported that BMPs induce differentiation of human ES cells into extraembryonic lineages. Here we find that FGF2, acting through the MEK-ERK pathway, switches BMP4 induced human ES cell differentiation outcome to mesendoderm, characterized by the uniform expression of T (brachyury) and other primitive streak markers. We also find that MEK-ERK signaling prolongs NANOG expression during BMP-induced differentiation; that forced NANOG expression results in FGF independent BMP4 induction of mesendoderm; and that knockdown of NANOG greatly reduces T induction. Together, our results demonstrate that FGF2 signaling switches the outcome of BMP4 induced differentiation of human ES cells by maintaining NANOG levels through the MEKERK pathway.

Vitamin C, a micronutrient known for its anti-scurvy activity in humans, promotes the generation of induced pluripotent stem cells (iPSCs) through the activity of histone demethylating dioxygenases. TET hydroxylases are also dioxygenases implicated in active DNA demethylation. Here we report that TET1 either positively or negatively regulates somatic cell reprogramming depending on the absence or presence of vitamin C. TET1 deficiency enhances reprogramming, and its overexpression impairs reprogramming in the context of vitamin C by modulating the obligatory mesenchymal-to-epithelial transition (MET). In the absence of vitamin C, TET1 promotes somatic cell reprogramming independent of MET. Consistently, TET1 regulates 5-hydroxymethylcytosine (5hmC) formation at loci critical for MET in a vitamin C-dependent fashion. Our findings suggest that vitamin C has a vital role in determining the biological outcome of TET1 function at the cellular level. Given its benefit to human health, vitamin C should be investigated further for its role in epigenetic regulation.

Present practices for reprogramming somatic cells to induced pluripotent stem cells involve simultaneous introduction of reprogramming factors. Here we report that a sequential introduction protocol (Oct4-Klf4 first, then c-Myc and finally Sox2) outperforms the simultaneous one. Surprisingly, the sequential protocol activates an early epithelial-to-mesenchymal transition (EMT) as indicated by the upregulation of Slug and N-cadherin followed by a delayed mesenchymal-to-epithelial transition (MET). An early EMT induced by 1.5-day TGF-β treatment enhances reprogramming with the simultaneous protocol, whereas 12-day treatment blocks reprogramming. Consistent results were obtained when the TGF-β antagonist Repsox was applied in the sequential protocol. These results reveal a time-sensitive role of individual factors for optimal reprogramming and a sequential EMT-MET mechanism at the start of reprogramming. Our studies provide a rationale for further optimizing reprogramming, and introduce the concept of a sequential EMT-MET mechanism for cell fate decision that should be investigated further in other systems, both in vitro and in vivo.

Human neural stem cells hold great promise for research and therapy in neural disease. We describe the generation of integration-free and expandable human neural progenitor cells (NPCs). We combined an episomal system to deliver reprogramming factors with a chemically defined culture medium to reprogram epithelial-like cells from human urine into NPCs (hUiNPCs). These transgene-free hUiNPCs can self-renew and can differentiate into multiple functional neuronal subtypes and glial cells in vitro. Although functional in vivo analysis is still needed, we report that the cells survive and differentiate upon transplant into newborn rat brain.

Mitogen-activated protein kinase (MAPK) and PUF (for Pumilio and FBF [fem-3 binding factor]) RNA-binding proteins control many cellular processes critical for animal development and tissue homeostasis. In the present work, we report that PUF proteins act directly on MAPK/ERK-encoding mRNAs to downregulate their expression in both the Caenorhabditis elegans germline and human embryonic stem cells. In C. elegans, FBF/PUF binds regulatory elements in the mpk-1 3′ untranslated region (3′ UTR) and coprecipitates with mpk-1 mRNA; moreover, mpk-1 expression increases dramatically in FBF mutants. In human embryonic stem cells, PUM2/PUF binds 3′UTR elements in both Erk2 and p38α mRNAs, and PUM2 represses reporter constructs carrying either Erk2 or p38α 3′ UTRs. Therefore, the PUF control of MAPK expression is conserved. Its biological function was explored in nematodes, where FBF promotes the self-renewal of germline stem cells, and MPK-1 promotes oocyte maturation and germ cell apoptosis. We found that FBF acts redundantly with LIP-1, the C. elegans homolog of MAPK phosphatase (MKP), to restrict MAPK activity and prevent apoptosis. In mammals, activated MAPK can promote apoptosis of cancer cells and restrict stem cell self-renewal, and MKP is upregulated in cancer cells. We propose that the dual negative regulation of MAPK by both PUF repression and MKP inhibition may be a conserved mechanism that influences both stem cell maintenance and tumor progression.