Identification of novel genes and networks governing hematopoietic stem cell development.

Hematopoietic stem cells (HSCs) are capable of giving rise to all blood cell lineages throughout adulthood, and the generation of engraftable HSCs from human pluripotent stem cells is a major goal for regenerative medicine. Here, we describe a functional genome-wide RNAi screen to identify genes required for the differentiation of embryonic stem cell (ESC) into hematopoietic stem/progenitor cells (HSPCs) in vitro We report the discovery of novel genes important for the endothelial-to-hematopoietic transition and subsequently for HSPC specification. High-throughput sequencing and bioinformatic analyses identified twelve groups of genes, including a set of 351 novel genes required for HSPC specification. As in vivo proof of concept, four of these genes, Ap2a1, Mettl22, Lrsam1, and Hal, are selected for validation, confirmed to be essential for HSPC development in zebrafish and for maintenance of human HSCs. Taken together, our results not only identify a number of novel regulatory genes and pathways essential for HSPC development but also serve as valuable resource for directed differentiation of therapy grade HSPCs using human pluripotent stem cells.

Polycomb Group Protein Pcgf6 Acts as a Master Regulator to Maintain Embryonic Stem Cell Identity.

The polycomb repressive complex 1 (PRC1) is a multi-subunit complex that plays critical roles in the epigenetic modulation of gene expression. Here, we show that the PRC1 component polycomb group ring finger 6 (Pcgf6) is required to maintain embryonic stem cell (ESC) identity. In contrast to canonical PRC1, Pcgf6 acts as a positive regulator of transcription and binds predominantly to promoters bearing active chromatin marks. Pcgf6 is expressed at high levels in ESCs, and knockdown reduces the expression of the core ESC regulators Oct4, Sox2, and Nanog. Conversely, Pcgf6 overexpression prevents downregulation of these factors and impairs differentiation. In addition, Pcgf6 enhanced reprogramming in both mouse and human somatic cells. The genomic binding profile of Pcgf6 is highly similar to that of trithorax group proteins, but not of PRC1 or PRC2 complexes, suggesting that Pcgf6 functions atypically in ESCs. Our data reveal novel roles for Pcgf6 in directly regulating Oct4, Nanog, Sox2, and Lin28 expression to maintain ESC identity.

Genome-wide functional analysis reveals factors needed at the transition steps of induced reprogramming.

Although transcriptome analysis can uncover the molecular changes that occur during induced reprogramming, the functional requirements for a given factor during stepwise cell-fate transitions are left unclear. Here, we used a genome-wide RNAi screen and performed integrated transcriptome analysis to identify key genes and cellular events required at the transition steps in reprogramming. Genes associated with cell signaling pathways (e.g., Itpr1, Itpr2, and Pdia3) constitute the major regulatory networks before cells acquire pluripotency. Activation of a specific gene set (e.g., Utf1 or Tdgf1) is important for mature induced pluripotent stem cell formation. Strikingly, a major proportion of RNAi targets (∼ 53% to 70%) includes genes whose expression levels are unchanged during reprogramming. Among these non-differentially expressed genes, Dmbx1, Hnf4g, Nobox, and Asb4 are important, whereas Nfe2, Cdkn2aip, Msx3, Dbx1, Lzts1, Gtf2i, and Ankrd22 are roadblocks to reprogramming. Together, our results provide a wealth of information about gene functions required at transition steps during reprogramming.

Kinome-wide functional analysis highlights the role of cytoskeletal remodeling in somatic cell reprogramming.

The creation of induced pluripotent stem cells (iPSCs) from somatic cells by ectopic expression of transcription factors has galvanized the fields of regenerative medicine and developmental biology. Here, we report a kinome-wide RNAi-based analysis to identify kinases that regulate somatic cell reprogramming to iPSCs. We prepared 3,686 small hairpin RNA (shRNA) lentiviruses targeting 734 kinase genes covering the entire mouse kinome and individually examined their effects on iPSC generation. We identified 59 kinases as barriers to iPSC generation and characterized seven of them further. We found that shRNA-mediated knockdown of the serine/threonine kinases TESK1 or LIMK2 promoted mesenchymal-to-epithelial transition, decreased COFILIN phosphorylation, and disrupted Actin filament structures during reprogramming of mouse embryonic fibroblasts. Similarly, knockdown of TESK1 in human fibroblasts also promoted reprogramming to iPSCs. Our study reveals the breadth of kinase networks regulating pluripotency and identifies a role for cytoskeletal remodeling in modulating the somatic cell reprogramming process.

An evolutionarily conserved long noncoding RNA TUNA controls pluripotency and neural lineage commitment.

Here, we generated a genome-scale shRNA library targeting long intergenic noncoding RNAs (lincRNAs) in the mouse. We performed an unbiased loss-of-function study in mouse embryonic stem cells (mESCs) and identified 20 lincRNAs involved in the maintenance of pluripotency. Among these, TUNA (Tcl1 Upstream Neuron-Associated lincRNA, or megamind) was required for pluripotency and formed a complex with three RNA-binding proteins (RBPs). The TUNA-RBP complex was detected at the promoters of Nanog, Sox2, and Fgf4, and knockdown of TUNA or the individual RBPs inhibited neural differentiation of mESCs. TUNA showed striking evolutionary conservation of both sequence- and CNS-restricted expression in vertebrates. Accordingly, knockdown of tuna in zebrafish caused impaired locomotor function, and TUNA expression in the brains of Huntington's disease patients was significantly associated with disease grade. Our results suggest that the lincRNA TUNA plays a vital role in pluripotency and neural differentiation of ESCs and is associated with neurological function of adult vertebrates.

Discovery of nonsteroidal anti-inflammatory drug and anticancer drug enhancing reprogramming and induced pluripotent stem cell generation.

Recent breakthroughs in creating induced pluripotent stem cells (iPSCs) provide alternative means to obtain embryonic stem-like cells without destroying embryos by introducing four reprogramming factors (Oct3/4, Sox2, and Klf4/c-Myc or Nanog/Lin28) into somatic cells. iPSCs are versatile tools for investigating early developmental processes and could become sources of tissues or cells for regenerative therapies. Here, for the first time, we describe a strategy to analyze genomics datasets of mouse embryonic fibroblasts (MEFs) and embryonic stem cells to identify genes constituting barriers to iPSC reprogramming. We further show that computational chemical biology combined with genomics analysis can be used to identify small molecules regulating reprogramming. Specific downregulation by small interfering RNAs (siRNAs) of several key MEF-specific genes encoding proteins with catalytic or regulatory functions, including WISP1, PRRX1, HMGA2, NFIX, PRKG2, COX2, and TGFß3, greatly increased reprogramming efficiency. Based on this rationale, we screened only 17 small molecules in reprogramming assays and discovered that the nonsteroidal anti-inflammatory drug Nabumetone and the anticancer drug 4-hydroxytamoxifen can generate iPSCs without Sox2. Nabumetone could also produce iPSCs in the absence of c-Myc or Sox2 without compromising self-renewal and pluripotency of derived iPSCs. In summary, we report a new concept of combining genomics and computational chemical biology to identify new drugs useful for iPSC generation. This hypothesis-driven approach provides an alternative to shot-gun screening and accelerates understanding of molecular mechanisms underlying iPSC induction.

microRNAs modulate iPS cell generation.

Although induced pluripotent stem cells (iPSCs) hold great promise for customized regenerative medicine, the molecular basis of reprogramming is largely unknown. Overcoming barriers that maintain cell identities is a critical step in the reprogramming of differentiated cells. Since microRNAs (miRNAs) modulate target genes tissue-specifically, we reasoned that distinct mouse embryonic fibroblast (MEF)-enriched miRNAs post-transcriptionally modulate proteins that function as reprogramming barriers. Inhibiting these miRNAs should influence cell signaling to lower those barriers. Here we show that depleting miR-21 and miR-29a enhances reprogramming efficiency in MEFs. We also show that the p53 and ERK1/2 pathways are regulated by miR-21 and miR-29a and function in reprogramming. In addition, we provide the first evidence that c-Myc enhances reprogramming partly by repressing MEF-enriched miRNAs, such as miR-21 and miR-29a. Our results demonstrate the significance of miRNA function in regulating multiple signaling networks involved in iPSC generation. These studies should facilitate development of clinically applicable reprogramming strategies.

Small RNA-mediated regulation of iPS cell generation.

Somatic cells can be reprogrammed to an ES-like state to create induced pluripotent stem cells (iPSCs) by ectopic expression of four transcription factors, Oct4, Sox2, Klf4 and cMyc. Here, we show that cellular microRNAs (miRNAs) regulate iPSC generation. Knock-down of key microRNA pathway proteins resulted in significant decreases in reprogramming efficiency. Three miRNA clusters, miR-17∼92, miR-106b∼25 and miR-106a∼363, were shown to be highly induced during early reprogramming stages. Several miRNAs, including miR-93 and miR-106b, which have very similar seed regions, greatly enhanced iPSC induction and modulated mesenchymal-to-epithelial transition step in the initiation stage of reprogramming, and inhibiting these miRNAs significantly decreased reprogramming efficiency. Moreover, miR-iPSC clones reached the fully reprogrammed state. Further analysis revealed that Tgfbr2 and p21 are directly targeted by these miRNAs and that siRNA knock-down of both genes indeed enhanced iPSC induction. Here, for the first time, we demonstrate that miR-93 and its family members directly target TGF-ß receptor II to enhance iPSC generation. Overall, we demonstrate that miRNAs function in the reprogramming process and that iPSC induction efficiency can be greatly enhanced by modulating miRNA levels in cells.

Gefitinib reverses chemotherapy resistance in gefitinib-insensitive multidrug resistant cancer cells expressing ATP-binding cassette family protein.

Gefitinib inhibits the ATP-binding site of the tyrosine kinase associated with the epidermal growth factor receptor. It is conceivable that gefitinib may inhibit functions of ATP-binding cassette (ABC) transporters by binding at their ATP-binding sites. The aim of this study is to systematically explore the combined effect of gefitinib and chemotherapeutic agents in gefitinib-insensitive multidrug resistant (MDR) cells that overexpress ABC transporters. MCF7 breast carcinoma cells and CL1 lung adenocarcinoma cells were both insensitive to gefitinib. MDR cancer cells were developed by stepwise escalating concentrations of each chemotherapeutic agent in culture media. Cells that overexpress P-glycoprotein (MCF7/Adr and CL1/Pac), breast cancer-resistant protein (MCF7/TPT and CL1/Tpt), and MDR-associated protein 1 (MCF7/Vp) were used in this study. All resistant mutants were insensitive to gefitinib. Gefitinib (0.3-3 micromol/L) added to culture media had no effect on IC50 values of paclitaxel, topotecan, doxorubicin, or etoposide in wild-type MCF7 or CL1 cells. In contrast, these concentrations of gefitinib caused a dose-dependent reversal of resistance to paclitaxel in CL1/Pac cells, to doxorubicin in MCF7/ADR cells, and to topotecan in CL1/Tpt and MCF7/TPT cells. Gefitinib had no influence on sensitivity to etoposide in MDR-associated protein1 overexpressing MCF7/VP cells. Topotecan efflux was inhibited and accumulation was partially restored in CL1/Tpt and MCF7/TPT cells when cells were incubated simultaneously with gefitinib. Our results suggest that the interaction of gefitinib and chemotherapeutic agents does occur in cells expressing one of these two proteins.