SUMMARY N6-methyl-adenosine (m6A) is the most abundant modification on messenger RNAs and is linked to human diseases, but its functions in mammalian development are poorly understood. Here we reveal the evolutionary conservation and function of m6A by mapping the m6A methylome in mouse and human embryonic stem cells. Thousands of messenger and long noncoding RNAs show conserved m6A modification, including transcripts encoding core pluripotency transcription factors. m6A is enriched over 3′ untranslated regions at defined sequence motifs, and marks unstable transcripts, including transcripts turned over upon differentiation. Genetic inactivation or depletion of mouse and human Mettl3, one of the m6A methylases, led to m6A erasure on select target genes, prolonged Nanog expression upon differentiation, and impaired ESC’s exit from self-renewal towards differentiation into several lineages in vitro and in vivo. Thus, m6A is a mark of transcriptome flexibility required for stem cells to differentiate to specific lineages.

We recently showed that defined sets of transcription factors are sufficient to convert mouse and human fibroblasts directly into cells resembling functional neurons, referred to as "induced neuronal" (iN) cells. For some applications however, it would be desirable to convert fibroblasts into proliferative neural precursor cells (NPCs) instead of neurons. We hypothesized that NPC-like cells may be induced using the same principal approach used for generating iN cells. Toward this goal, we infected mouse embryonic fibroblasts derived from Sox2-EGFP mice with a set of 11 transcription factors highly expressed in NPCs. Twenty-four days after transgene induction, Sox2-EGFP + colonies emerged that expressed NPC-specific genes and differentiated into neuronal and astrocytic cells. Using stepwise elimination, we found that Sox2 and FoxG1 are capable of generating clonal self-renewing, bipotent induced NPCs that gave rise to astrocytes and functional neurons. When we added the Pou and Homeobox domain-containing transcription factor Brn2 to Sox2 and FoxG1, we were able to induce tripotent NPCs that could be differentiated not only into neurons and astrocytes but also into oligodendrocytes. The transcription factors FoxG1 and Brn2 alone also were capable of inducing NPC-like cells; however, these cells generated less mature neurons, although they did produce astrocytes and even oligodendrocytes capable of integration into dysmyelinated Shiverer brain. Our data demonstrate that direct lineage reprogramming using target cell-type-specific transcription factors can be used to induce NPC-like cells that potentially could be used for autologous cell transplantation-based therapies in the brain or spinal cord.induced neural precursor cells D uring development, the creation of distinct cell types depends upon tightly regulated spatiotemporal expression of lineage-specific transcription factors. A key question is whether cells retain their competence to respond to such transcription factors even after differentiation and after their cell-type-specific phenotype has been stabilized by epigenetic mechanisms (1). A number of classic and recent studies have provided powerful evidence that the differentiated state of at least some somatic cells is more flexible than assumed. For instance, transfer of somatic nuclei into oocytes has been shown to impose an early embryonic program on somatic cells (2, 3). Similarly, aberrant cell-type-specific genes could be induced following cell fusion (4), and misexpression of defined transcription factors has been shown to induce conversion of cells in closely related cell types (5). For instance, the basic helix-loop-helix (bHLH) transcription factor MyoD has been shown to induce muscle-specific properties in fibroblasts but not in hepatocytes (6, 7); expression of Cebpα in B cells induces features of macrophages (8); loss of Pax5 in B cells induces dedifferentiation to a common lymphoid progenitor (9); and the bHLH transcription factor Ngn3 or NeuroD1, in combination with Pdx1 and MafA, efficientl...

SUMMARY To analyze cellular reprogramming at the single-cell level, mass cytometry was used to simultaneously measure markers of pluripotency, differentiation, cell-cycle status, and cellular signaling throughout the reprogramming process. Time-resolved progression analysis of the resulting data sets was used to construct a continuous molecular roadmap for three independent reprogramming systems. Although these systems varied substantially in Oct4, Sox2, Klf4, and c-Myc stoichiometry, they presented a common set of reprogramming landmarks. Early in the reprogramming process, Oct4highKlf4high cells transitioned to a CD73highCD104highCD54low partially reprogrammed state. Ki67low cells from this intermediate population reverted to a MEF-like phenotype, but Ki67high cells advanced through the M-E-T and then bifurcated into two distinct populations: an ESC-like NanoghighSox2highCD54high population and a mesendoderm-like NanoglowSox2lowLin28high CD24highPDGFR-αhigh population. The methods developed here for time-resolved, single-cell progression analysis may be used for the study of additional complex and dynamic systems, such as cancer progression and embryonic development.

Stem cells self-renew and generate specialized progeny through differentiation, but vary in the range of cells and tissues they generate, a property called developmental potency. Pluripotent stem cells produce all cells of an organism, while multipotent or unipotent stem cells regenerate only specific lineages or tissues. Defining stem-cell potency relies upon functional assays and diagnostic transcriptional, epigenetic and metabolic states. Here we describe functional and molecular hallmarks of pluripotent stem cells, propose a checklist for their evaluation, and illustrate how forensic genomics can validate their provenance.