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.

Learning the molecular mechanisms of the reprogramming factors: let's start from microRNAs.

Induced reprogramming of somatic cells has had a great impact on stem cell research, and the reprogramming technologies have evolved from four transgenic factors (Oct4, Sox2, Klf4, and c-Myc; OSKM) to just a few microRNAs (mainly miR-290/302 seed family). Despite these advances, the molecular events occurring during various stages of reprogramming remain largely unknown. Here, we concisely review current knowledge of miRNA regulation from the initiation phase of OSKM-induced reprogramming, through the transitional stage, to final maturation. At the start of reprogramming, the microRNAs miR-21, miR-29a, let-7a, and miR-34 act as guards to secure the somatic identity and genomic integrity of the cell of origin. As reprogramming proceeds, miR-155, miR-10b, miR-205, and miR-429 modulate the epithelial-mesenchymal/mesenchymal-epithelial transition (EMT/MET), which is a critical step towards transformed pluripotent status. Finally, the pluripotency regulatory network is secured in the iPSCs and fine-tuned by a group of miRNAs belonging to the miR-290/302 seed family. Among the four reprogramming factors, c-Myc plays the dominant role in regulating the miRNAs under reprogramming-specific conditions. Accumulating evidence suggests that the reprogramming efficiency can be improved by either blocking barrier miRNAs or introducing helper miRNAs. Intriguingly, induced pluripotency can be obtained by introducing a single miR-302 cluster, although the supportive molecular mechanism is still lacking. In the near future, we may be able to realize the broad potential of miRNAs in the stem cell field, such as altering cell identities with high efficiency through the transient introduction of tissue-specific miRNAs.

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.

Nanotubes functionalized with lipids and natural amino acid dendrimers: a new strategy to create nanomaterials for delivering systemic RNAi.

Single-walled carbon nanotubes (SWNT) have unique electronic, mechanical, and structural properties as well as chemical stability that make them ideal nanomaterials for applications in materials science and medicine. Here, we report the design and creation of a novel strategy for functionalizing SWNT to systemically silence a target gene in mice by delivering siRNA at doses of <1 mg/kg. SWNT were functionalized with lipids and natural amino acid-based dendrimers (TOT) and complexed to siRNA. Our model study of the silencing efficiency of the TOT-siRNA complex showed that, in mice injected at 0.96 mg/kg, an endogenous gene for apoliproprotein B (ApoB) was silenced in liver, plasma levels of ApoB decreased, and total plasma cholesterol decreased. TOT-siRNA treatment was nontoxic and did not induce an immune response. Most (80%) of the RNA trigger molecules assembled with TOT were cleared from the body 48 h after injection, suggesting that the nanotubes did not cause siRNA aggregation or inhibit biodegradation and drug clearance in vivo. These results provide the first evidence that nanotubes can be functionalized with lipids and amino acids to systemically deliver siRNA. This new technology not only can be used for systemic RNAi, but may also be used to deliver other drugs in vivo.

Vortex array laser beam generation from a Dove prism-embedded unbalanced Mach-Zehnder interferometer.

This paper proposes a new scheme for generating vortex laser beams from a laser. The proposed system consists of a Dove prism embedded in an unbalanced Mach-Zehnder interferometer configuration. This configuration allows controlled construction of p x p vortex array beams from Ince-Gaussian modes, IG(e) (p,p) modes. An incident IG(e)(p,p) laser beam of variety order p can easily be generated from an end-pumped solid-state laser system with an off-axis pumping mechanism. This study simulates this type of vortex array laser beam generation, analytically derives the vortex positions of the resulting vortex array laser beams, and discusses beam propagation effects. The resulting vortex array laser beam can be applied to optical tweezers and atom traps in the form of two-dimensional arrays, or used to study the transfer of angular momentum to micro particles or atoms (Bose-Einstein condensate).

Evolutionary emergence of microRNAs in human embryonic stem cells.

Human embryonic stem (hES) cells have unique abilities to divide indefinitely without differentiating and potential to differentiate into more than 200 cell types. These properties make hES cells an ideal model system for understanding early human development and for regenerative medicine. Molecular mechanisms including cellular signaling and transcriptional regulation play important roles in hES cell differentiation. However, very little information is available on posttranscriptional regulation of hES cell pluripotency, self-renewal, and early decisions about cell fate. microRNAs (miRNAs), 22-nt long non-coding small RNAs found in plants and animals, regulate gene expression by targeting mRNAs for cleavage or translation repression. In hES cells we found that 276 miRNAs were expressed; of these, a set of 30 miRNAs had significantly changed expression during differentiation. Using a representative example, miR-302b, we show that miRNAs in human ES cells assemble into a bona fide RISC that contains Ago2 and can specifically cleave perfectly matched target RNA. Our results demonstrate that human ES cell differentiation is accompanied by changes in the expression of a unique set of miRNAs, providing a glimpse of a new molecular circuitry that may regulate early development in humans. Chromosomes 19 and X contained 98 and 40 miRNA genes, respectively, indicating that majority of miRNA genes in hES cells were expressed from these two chromosomes. Strikingly, distribution analysis of miRNA gene loci across six species including dog, rat, mouse, rhesus, chimpanzee, and human showed that miRNA genes encoded in chromosome 19 were drastically increased in chimpanzees and humans while miRNA gene loci on other chromosomes were decreased as compared with dog, rat, and mouse. Comparative genomic studies showed 99% conservation of chromosome 19 miRNA genes between chimpanzees and humans. Together, these findings reveal the evolutionary emergence, approximately 5 million years ago, of miRNAs involved in regulating early human development. One could imagine that this burst of miRNA gene clusters at specific chromosomes was part of an evolutionary event during species divergence.

Low temperature synthesis of single-walled carbon nanotubes in an inductively coupled plasma chemical vapor deposition system.

In this work, we present a parametric study on the low temperature synthesis of single-walled carbon nanotubes (SWNTs) in an inductively coupled plasma (ICP) CVD system using dry bi-layered catalytic thin-films (Fe/Al and Ni/Al, deposited by electron-beam evaporation method) as the catalysts. With a low substrate temperature of 550 degrees C and above, SWNTs were successfully synthesized on both catalysts, as revealed from the characteristic peaks of SWNTs in the micro-Raman spectra. By the reduction of plasma power and the shortening of the process times, the lowest synthesis temperature of SWNTs achieved in our system was approached to 500 degrees C on Ni/Al catalysts; on the other hands, the lowest temperature for Fe/Al catalysts was 550 degrees C. Our results suggest that as compared with Fe/Al, Ni/Al is more favorable for plasma-enhanced CVD (PECVD) synthesis of SWNTs at low temperatures. This work can be used for further improvements and better understanding on the production processes of SWNTs by PECVD methods.

Therapeutic gene silencing delivered by a chemically modified small interfering RNA against mutant SOD1 slows amyotrophic lateral sclerosis progression.

Inherited neurodegenerative diseases, such as Huntington disease and subset of Alzheimer disease, Parkinson disease, and amyotrophic lateral sclerosis, are caused by the mutant genes that have gained undefined properties that harm cells in the nervous system, causing neurodegeneration and clinical phenotypes. Lowering the mutant gene expression is predicted to slow the disease progression and produce clinical benefit. Administration of small interfering RNA (siRNA) can silence specific genes. However, long term delivery of siRNA to silence the mutant genes, a requirement for treatment of these chronic central nervous system (CNS) diseases, remains a critical unsolved issue. Here we designed and tested a chemically stabilized siRNA against human Cu,Zn-superoxide dismutase (SOD1) in a mouse model for amyotrophic lateral sclerosis. We show that the modified siRNA has enhanced stability and retains siRNA activity. Administration of this siRNA at the disease onset by long term infusion into the CNS resulted in widespread distribution of this siRNA, knocked down the mutant SOD1 expression, slowed the disease progression, and extended the survival. These results bring RNA interference therapy one step closer to its clinical application for treatment of chronic, devastating, and fatal CNS disorders.

Design and creation of new nanomaterials for therapeutic RNAi.

RNA interference is an evolutionarily conserved gene-silencing phenomenon that shows great promise for developing new therapies. However, the development of small interfering RNA (siRNA)-based therapies needs to overcome two barriers and be able to (i) identify chemically stable and effective siRNA sequences and (ii) efficiently silence target genes with siRNA doses that will be clinically feasible in humans. Here, we report the design and creation of interfering nanoparticles (iNOPs) as new systemic gene-silencing agents. iNOPs have two subunits: (i) a well-defined functionalized lipid nanoparticle as a delivery agent and (ii) a chemically modified siRNA for sustained silencing in vivo. When we injected iNOPs containing only 1-5 mg kg(-1) siRNA into mice, an endogenous gene for apolipoprotein B (apoB) was silenced in liver, plasma levels of apoB decreased, and total plasma cholesterol was lowered. iNOP treatment was nontoxic and did not induce an immune response. Our results show that these iNOPs can silence disease-related endogenous genes in clinically acceptable and therapeutically affordable doses.

Effects of bulk dissipation on the critical exponents of a sandpile.

Bulk dissipation of a sandpile on a square lattice with the periodic boundary condition is investigated through a dissipating probability f during each toppling process. We find that the power-law behavior is broken for f>10(-1) and not evident for 10(-1)}>f>10(-2). In the range 10(-2)>or=f>or=10(-5), numerical simulations for the toppling size exponents of all, dissipative, and last waves have been studied. Two kinds of definitions for exponents are considered: the exponents obtained from the direct fitting of data and the exponents defined by the simple scaling. Our result shows that the exponents from these two definitions may be different. Furthermore, we propose analytic expressions of the exponents for the direct fitting, and it is consistent with the numerical result. Finally, we point out that small dissipation drives the behavior of this model toward the simple scaling.

Direct synthesis of single-walled carbon nanotubes selectively suspended on tips of vertically aligned silicon nanostructures fabricated by hydrogen plasma etching.

Here we present a method to synthesize single-walled carbon nanotubes (SWNTs) selectively suspended on tips of silicon-based nanostructure (Si-ns) templates. The Si-ns templates vertically aligned to the substrates are fabricated via an anisotropic etch process using reactive hydrogen plasmas, in which the etch-resistive nanomasks are the nanosized particles formed by thermal annealing of multi-layered catalytic thin films. After plasma etching, the nanosized self-masks remaining at the tips of the Si-ns directly serve as the catalysts for SWNT growth by thermal chemical vapour deposition. Consequently, the synthesized SWNTs are selectively suspended on the tips of the Si-ns, as revealed by characterizations using scanning electron microscopy and resonance Raman spectroscopy. This methodology provides a simple and straightforward approach to assemble two different nanomaterials, i.e., Si-ns and suspended SWNTs, together as a building block for constructing nanodevices for possible applications.

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.