Human induced pluripotent stem (iPS) cells are remarkably similar to embryonic stem (ES) cells, but recent reports suggest that there may be important differences between them. We performed a systematic comparison of human iPS cells generated from hepatocytes (representative of endoderm), skin fibroblasts (mesoderm) and melanocytes (ectoderm). All low passage iPS cells analyzed retain a transcriptional memory of the original cells. The persistent expression of somatic genes can be partially explained by incomplete promoter DNA methylation. This epigenetic mechanism underlies a robust form of memory that can be found in iPS cells generated by multiple laboratories using different methods, including RNA transfection. Incompletely silenced genes tend to be isolated from other genes that are repressed during reprogramming, indicating that recruitment of the silencing machinery may be inefficient at isolated genes. Knockdown of the incompletely reprogrammed gene C9orf64 reduces the efficiency of human iPS cell generation, suggesting that somatic memory genes may be functionally relevant during reprogramming.

It is largely recognized that microRNAs (miRNAs) function to silence gene expression by targeting 3′UTR regions. However, miRNAs have also been implicated to positively-regulate gene expression by targeting promoter elements, a phenomenon known as RNA activation (RNAa). In the present study, we show that expression of mouse Cyclin B1 (Ccnb1) is dependent on key factors involved in miRNA biogenesis and function (i.e. Dicer, Drosha, Ago1 and Ago2). In silico analysis identifies highly-complementary sites for 21 miRNAs in the Ccnb1 promoter. Experimental validation identified three miRNAs (miR-744, miR-1186 and miR-466d-3p) that induce Ccnb1 expression in mouse cell lines. Conversely, knockdown of endogenous miR-744 led to decreased Ccnb1 levels. Chromatin immunoprecipitation (ChIP) analysis revealed that Ago1 was selectively associated with the Ccnb1 promoter and miR-744 increased enrichment of RNA polymerase II (RNAP II) and trimethylation of histone 3 at lysine 4 (H3K4me3) at the Ccnb1 transcription start site. Functionally, short-term overexpression of miR-744 and miR-1186 resulted in enhanced cell proliferation, while prolonged expression caused chromosomal instability and in vivo tumor suppression. Such phenotypes were recapitulated by overexpression of Ccnb1. Our findings reveal an endogenous system by which miRNA functions to activate Ccnb1 expression in mouse cells and manipulate in vivo tumor development/growth.

Significance Patients homozygous for the C-C chemokine receptor type 5 (CCR5) gene with 32-bp deletions (Δ32) are resistant to HIV infection. Using the piggyBac technology plus transcription activator-like effector nucleases or clustered regularly interspaced short palindromic repeats-Cas9, the authors report, to our knowledge, for the first time in induced pluripotent stem cells (iPSCs) the efficient and seamless derivation of a homozygous CCR5Δ32 mutation, exactly mimicking the natural mutation. Monocytes and macrophages differentiated from these mutated iPSCs in vitro are resistant to HIV infection. This approach can be applied in the future toward the functional cure of HIV infection. The findings are also of great interest to researchers in many fields who wish to correct or introduce mutations in specific genes.

SUMMARY Studying 830 pre-B ALL cases from four clinical trials, we found that human ALL can be divided into two fundamentally distinct subtypes based on pre-BCR function. While absent in the majority of ALL cases, tonic pre-BCR signaling was found in 112 cases (13.5%). In these cases, tonic pre-BCR signaling induced activation of BCL6, which in turn increased pre-BCR signaling output at the transcriptional level. Interestingly, inhibition of pre-BCR-related tyrosine kinases reduced constitutive BCL6 expression and selectively killed patient-derived pre-BCR+ ALL cells. These findings identify a genetically and phenotypically distinct subset of human ALL that critically depends on tonic pre-BCR signaling. In vivo treatment studies suggested that pre-BCR tyrosine kinase inhibitors are useful for the treatment of patients with pre-BCR+ ALL.

SUMMARY The human naive pluripotent stem cell (PSC) state, corresponding to a pre-implantation stage of development, has been difficult to capture and sustain in vitro. We report that the Hippo pathway effector YAP is nuclearly localized in the inner cell mass of human blastocysts. Overexpression of YAP in human embryonic stem cells (ESCs) and induced PSCs (iPSCs) promotes the generation of naive PSCs. Lysophosphatidic acid (LPA) can partially substitute for YAP to generate transgene-free human naive PSCs. YAP- or LPA-induced naive PSCs have a rapid clonal growth rate, a normal karyotype, the ability to form teratomas, transcriptional similarities to human pre-implantation embryos, reduced heterochromatin levels, and other hallmarks of the naive state. YAP/LPA act in part by suppressing differentiation-inducing effects of GSK3 inhibition. CRISPR/Cas9-generated YAP−/− cells have an impaired ability to form colonies in naive but not primed conditions. These results uncover an unexpected role for YAP in the human naive state, with implications for early human embryology.

Argonaute proteins are often credited for their cytoplasmic activities in which they function as central mediators of the RNAi platform and microRNA (miRNA)-mediated processes. They also facilitate heterochromatin formation and establishment of repressive epigenetic marks in the nucleus of fission yeast and plants. However, the nuclear functions of Ago proteins in mammalian cells remain elusive. In the present study, we combine ChIP-seq (chromatin immunoprecipitation coupled with massively parallel sequencing) with biochemical assays to show that nuclear Ago1 directly interacts with RNA Polymerase II and is widely associated with chromosomal loci throughout the genome with preferential enrichment in promoters of transcriptionally active genes. Additional analyses show that nuclear Ago1 regulates the expression of Ago1-bound genes that are implicated in oncogenic pathways including cell cycle progression, growth, and survival. Our findings reveal the first landscape of human Ago1-chromosomal interactions, which may play a role in the oncogenic transcriptional program of cancer cells.

Pluripotent stem cells are derived from culture of early embryos or the germline and can be induced by reprogramming of somatic cells. Barriers to reprogramming that stabilize the differentiated state and have tumor suppression functions are expected to exist. However, we have a limited understanding of what such barriers might be. To find novel barriers to reprogramming to pluripotency, we compared the transcriptional profiles of the mouse germline with pluripotent and somatic cells, in vivo and in vitro. There is a remarkable global expression of the transcriptional program for pluripotency in primordial germ cells (PGCs). We identify parallels between PGC reprogramming to pluripotency and human germ cell tumorigenesis, including the loss of LATS2, a tumor suppressor kinase of the Hippo pathway. We show that knockdown of LATS2 increases the efficiency of induction of pluripotency in human cells. LATS2 RNAi, unlike p53 RNAi, specifically enhances the generation of fully reprogrammed iPS cells without accelerating cell proliferation. We further show that LATS2 represses reprogramming in human cells by post-transcriptionally antagonizing TAZ but not YAP, two downstream effectors of the Hippo pathway. These results reveal transcriptional parallels between germ cell transformation and the generation of iPS cells and indicate that the Hippo pathway constitutes a barrier to cellular reprogramming.

Cystic fibrosis (CF) is a recessive inherited disease associated with multiorgan damage that compromises epithelial and inflammatory cell function. Induced pluripotent stem cells (iPSCs) have significantly advanced the potential of developing a personalized cell-based therapy for diseases like CF by generating patient-specific stem cells that can be differentiated into cells that repair tissues damaged by disease pathology. The F508del mutation in airway epithelial cell-derived CF-iPSCs was corrected with small/short DNA fragments (SDFs) and sequence-specific TALENs. An allele-specific PCR, cyclic enrichment strategy gave ~100-fold enrichment of the corrected CF-iPSCs after six enrichment cycles that facilitated isolation of corrected clones. The seamless SDF-based gene modification strategy used to correct the CF-iPSCs resulted in pluripotent cells that, when differentiated into endoderm/airway-like epithelial cells showed wild-type (wt) airway epithelial cell cAMP-dependent Cl ion transport or showed the appropriate cell-type characteristics when differentiated along mesoderm/hematopoietic inflammatory cell lineage pathways.