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Designer Stem Cells Despite their promise for use as disease models and in regenerative medicine, the generation of human-induced pluripotent stem (iPS) cells has been hindered by the integration of vector and transgenes in the host cell genome. Recent studies using the Cre/LoxP recombination strategy and the piggyBac transposon approach have approached this objective. However, Yu et al. (p. 797 , published online 26 March) now show the derivation of human iPS cells from postnatal foreskin fibroblasts using the nonintegrating oriP/EBNA1-based episomal vectors. The resultant iPS cells show characteristics of human embryonic stem cells and are free of vector and transgenes.

Self-renewal of human embryonic stem cells (ESCs) is promoted by FGF and TGFbeta/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 TGFbeta 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 ESCs. We also show that both TGFbeta- and BMP-responsive SMADs can bind with the NANOG proximal promoter. NANOG promoter activity is enhanced by TGFbeta/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 TGFbeta signaling. These results suggest that direct binding of TGFbeta/Activin-responsive SMADs to the NANOG promoter plays an essential role in sustaining human ESC self-renewal.

Potassium (K) channels regulate cell membrane po- tential and modulate a number of important cellular functions. KCNA10 is a cyclic nucleotide-gated, voltage-activated K channel that is detected in kidney, heart, and aorta by Northern blot and postulated to participate in renal K metabolism and to regulate vascular tone. The aim of this study was to establish the cellular and subcellular localization of KCNA10 in kidney and vascular tissues. An anti-KCNA10 polyclonal antibody was generated, and immunocytochemical studies were per- formed on rat kidney. KCNA10 protein was easily detectable at the apical membrane of rat proximal tubular cells, and a weaker signal was also evident in the glomerulus. In situ hybridization experiments confirmed the immunocytochemical studies and revealed KCNA10 expression in human proximal tubular cells, glomerular and vascular endothelial cells, and also in vascular smooth muscle cells. The data suggest that KCNA10 may facilitate proximal tubular sodium absorption by stabilizing cell membrane voltage. Furthermore, its presence in endothelial and vascular smooth muscle cells supports the notion that it also regulates vascular tone. Potassium (K) channels are membrane proteins that participate in many cellular processes primarily by regulating membrane potential. KCNA10 is a voltage-gated K (Kv) channel gene related to the Shaker superfamily (1), and its most distinguish- ing feature relates to the presence of a putative cyclic nucle- otide-binding (CNB) domain at the carboxy terminus. A few other K channels also contain CNB domains and may belong to new subclass of K channels with structural features common to

Voltage-gated K+ (Kv) channels are heteromultimeric complexes consisting of pore-forming α-subunits and accessory β-subunits. Several β-subunits have been identified and shown to interact with specific α-subunits to modify their levels of expression or some of their kinetic properties. The aim of the present study was to isolate accessory proteins for KCNA10, a novel Kv channel α-subunit functionally related to Kv and cyclic nucleotide-gated cation channels. Because one distinguishing feature of KCNA10 is a putative cyclic nucleotide-binding domain located at the COOH terminus, the entire COOH-terminal region was used to probe a human cardiac cDNA library using the yeast two-hybrid system. Interacting clones were then rescreened in a functional assay by coinjection with KCNA10 in Xenopus oocytes. One of these clones (KCNA4B), when injected alone in oocytes, produced no detectable current. However, when coinjected with KCNA10, it increased KCNA10 current expression by nearly threefold. In addition, the current became more sensitive to activation by cAMP. KCNA4B can be coimmunoprecipitated with the COOH terminus of KCNA10 and full-length KCNA10. It encodes a soluble protein (141 aa) with no amino acid homology to known β-subunits but with limited structural similarity to the NAD(P)H-dependent oxidoreductase superfamily. KCNA4Bis located on chromosome 13 and spans ∼16 kb, and its coding region is made up of five exons. In conclusion, KCNA4B represents the first member of a new class of accessory proteins that modify the properties of Kv channels.

Our laboratory previously cloned a novel rabbit gene ( Kcn1), expressed in kidney, heart, and aorta, and predicted to encode a protein with 58% amino acid identity with the K channel Shaker Kv1.3 (Yao X et al. Proc Natl Acad Sci USA 92: 11711–11715, 1995). Because Kcn1 did not express well (peak current in Xenopus laevis oocytes of 0.3 μA at +60 mV), the human homolog (KCNA10) was isolated, and its expression was optimized in oocytes. KCNA10 mediates voltage-gated K+currents that exhibit minimal steady-state inactivation. Ensemble currents of 5–10 μA at +40 mV were consistently recorded from injected oocytes. Channels are closed at the holding potential of −80 mV but are progressively activated by depolarizations more positive than −30 mV, with half-activation at +3.5 ± 2.5 mV. The channel displays an unusual inhibitor profile because, in addition to being blocked by classical K channel blockers (barium tetraethylammonium and 4-aminopyridine), it is also sensitive to inhibitors of cyclic nucleotide-gated (CNG) cation channels (verapamil and pimozide). Tail-current analysis shows a reversal potential shift of 47 mV/decade change in K concentration, indicating a K-to-Na selectivity ratio of at least 15:1. The phorbol ester phorbol 12-myristate 13-acetate, an activator of protein kinase C, inhibited whole cell current by 42%. Analysis of single-channel currents reveals a conductance of ∼11 pS. We conclude KCNA10 is a novel human voltage-gated K channel with features common to both K-selective and CNG cation channels. Given its distribution in renal blood vessels and heart, we speculate that KCNA10 may be involved in regulating the tone of renal vascular smooth muscle and may also participate in the cardiac action potential.

Somatic cell nuclear transfer allows trans-acting factors present in the mammalian oocyte to reprogram somatic cell nuclei to an undifferentiated state. We show that four factors ( OCT4, SOX2, NANOG , and LIN28 ) are sufficient to reprogram human somatic cells to pluripotent stem cells that exhibit the essential characteristics of embryonic stem (ES) cells. These induced pluripotent human stem cells have normal karyotypes, express telomerase activity, express cell surface markers and genes that characterize human ES cells, and maintain the developmental potential to differentiate into advanced derivatives of all three primary germ layers. Such induced pluripotent human cell lines should be useful in the production of new disease models and in drug development, as well as for applications in transplantation medicine, once technical limitations (for example, mutation through viral integration) are eliminated.

The salamander has the remarkable ability to regenerate its limb after amputation. Cells at the site of amputation form a blastema and then proliferate and differentiate to regrow the limb. To better understand this process, we performed deep RNA sequencing of the blastema over a time course in the axolotl, a species whose genome has not been sequenced. Using a novel comparative approach to analyzing RNA-seq data, we characterized the transcriptional dynamics of the regenerating axolotl limb with respect to the human gene set. This approach involved de novo assembly of axolotl transcripts, RNA-seq transcript quantification without a reference genome, and transformation of abundances from axolotl contigs to human genes. We found a prominent burst in oncogene expression during the first day and blastemal/limb bud genes peaking at 7 to 14 days. In addition, we found that limb patterning genes, SALL genes, and genes involved in angiogenesis, wound healing, defense/immunity, and bone development are enriched during blastema formation and development. Finally, we identified a category of genes with no prior literature support for limb regeneration that are candidates for further evaluation based on their expression pattern during the regenerative process., Author Summary Salamanders such as the axolotl can fully regenerate a limb upon amputation, making them the vertebrate champions of regeneration. On the other hand, humans and other mammals possess a very limited ability to regenerate limb structures. Learning about the genes, gene networks, and pathways activated in the salamander during limb regeneration will provide cues to improving the regenerative response in mammals. Elucidating these genes, networks, and pathways is difficult, however, because the axolotl does not yet have its genome sequenced and because it has diverged evolutionarily from species with a sequenced genome. Here, we produce a set of gene transcripts via RNA sequencing (RNA-seq) for the axolotl and provide information on the nature of the genes activated during regeneration. To determine the identity of these axolotl genes, we use comparative transcriptomics techniques to match the axolotl transcript data to that of the well-annotated human gene set. Supporting previous studies, we find upregulation of many genes previously found to be involved in limb development and regeneration. In addition, we find a burst of cancer-related genes during the first phase of regeneration and identify a set of genes previously not associated with the regeneration process.

Abstract 1277 Animal studies demonstrated that the first HSCs are generated in the aorta-gonad-mesonephros from a unique population of endothelial cells, hemogenic endothelium (HE), through endothelial-hematopoietic transition. However, the identity of HE remains obscure and the specific features that distinguish HE from non-HE and the discrete stages of endothelial transition into hematopoietic cells are not characterized. Here we employed hESC differentiation system in coculture with OP9 to define the distinct population of HE, and to develop an experimental system for analysis of endothelial-hematopoietic transition in vitro. Previously, we demonstrated that endothelial cells could be distinguished from hematopoietic progenitors based on the lack of CD43 expression (Vodyanik et al., Blood 2006;108:2095). Here, we analyzed the earliest stages of endothelial development from hESCs before the typical hematopoietic CFCs could be detected. We found that the first CD144+ endothelial cells appear on day 4 of differentiation within a population of APLNR+ cells expressing a high level of KDR. Based on expression of CD235a/CD43, CD73 and CD41a, we identified three major subsets within the emerging CD144+ cells: 1)CD235a/CD43−CD73+, 2)CD235a/CD43−CD73−, and 3)CD235a/CD43+CD41a−. Although all three subsets formed monolayer endothelial cells when grown on fibronectin in endothelial media, only CD235a/CD43+CD41a− cells displayed hematopoietic CFC potential, which required serum-free medium and were dependent on hematopoietic cytokines and FGF2. After culture with OP9, both CD144+CD235a/CD43−CD73− and CD144+CD235a/CD43+CD41a− cells generated CD31+CD43/45− endothelial cells and a significant amount of CD43+ blood cells. In contrast CD144+CD235a/CD43−CD73+ cells formed almost exclusively endothelial cells. Single cell deposition experiments demonstrated that CD144+CD235a/CD43+CD41a− cells formed predominantly hematopoietic cells, while most CD144+CD235a/CD43−CD73− cells gave rise to either hematopoietic or endothelial clusters with approximately 2.5% of cells forming hematoendothelial clusters, indicating the presence of bipotential progenitors within this population. Though all three CD144+ subsets had a very similar molecular signature and expressed typical endothelial genes and genes associated with angiohematopoietic and HSC development, CD235a/CD43+CD41a− showed the higher expression of hematopoietic genes and lower expression of CDH5, CDH2, CAV1, and APOLD1 typical endothelial genes. Based on the functional, phenotypic and molecular properties we designated the identity of subsets as follows: 1) CD144+CD235a/CD43+CD41a− angiogenic blood cells, i.e. cells that possessed primary hematopoietic characteristics but were also capable of generating endothelial cells; 2) CD144+CD235a/CD43−CD73+ endothelial progenitors, i.e. cells that had all functional and molecular features of endothelial cells and endothelial colony-forming potential on OP9; 3) CD144+CD235a/CD43−CD73− HE cells, i.e. cells with primary endothelial characteristics lacking hematopoietic CFC potential and surface markers, but capable of generating blood and endothelial cells after coculture with stromal cells. To find out the direct precursors of HE and blood cells, we analyzed the differentiation potential of major subsets of mesodermal cells isolated on day 4 of hESC coculture with OP9. We found that only APLNR+KDRbrightCD140alow/− mesodermal cells lacking expression of typical hematopoietic (CD43, CD45), endothelial (CD31, CD144), and mesenchymal (CD73, CD105) markers had the potential to generate both blood and endothelium. Moreover, single cell deposition experiments demonstrated that APLNR+KDRbrightCD140alow/− cells formed HE clusters with a high frequency (about 1/10 cells), strongly indicating that these cells represent the direct precursors of HE. Because APLNR+KDRbrightCD140alow/− cells upregulated expression LMO2, TAL1, CBFB, GATA2, and FLI1 transcriptional regulators of hematopoietic and endothelial development, but were lacking expression of primitive streak genes, we designated these cells as angioblasts. Together these studies provide a hESC-based platform for identification of factors required for acquisition of HSC potential by blood cells following transition from endothelium. Disclosures: Thomson: CDI: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Slukvin:CDI: Consultancy, Equity Ownership.

SummaryAmong the three embryonic germ layers, the mesoderm is a major source of the mesenchymal precursors giving rise to skeletal and connective tissues, but these precursors have not previously been identified and characterized. Using human embryonic stem cells directed toward mesendodermal differentiation, we show that mesenchymal stem/stromal cells (MSCs) originate from a population of mesodermal cells identified by expression of apelin receptor. In semisolid medium, these precursors form FGF2-dependent compact spheroid colonies containing mesenchymal cells with a transcriptional profile representative of mesoderm-derived embryonic mesenchyme. When transferred to adherent cultures, individual colonies give rise to MSC lines with chondro-, osteo-, and adipogenic differentiation potentials. Although the MSC lines lacked endothelial potential, endothelial cells could be derived from the mesenchymal colonies, suggesting that, similar to hematopoietic cells, MSCs arise from precursors with angiogenic potential. Together, these studies identified a common precursor of mesenchymal and endothelial cells, mesenchymoangioblast, as the source of mesoderm-derived MSCs.

Five mappers and five variant callers used in the study. Table S2. SNP and INDEL calling precision rate in simulated data. Table S3. Percent of SNP calling sensitivity for three callers in simulated data. Table S4. INDEL calling sensitivity for two callers in simulated data. Table S5. Known INDELs in the HLA region of NA12878. Table S6. Novel INDELs in the HLA region of NA12878. Table S7. Number of novel SNPs in 22 CLL samples. Table S8. Number of INDELs in 22 CLL samples. (PDF 170 kb)

Five mappers and five variant callers used in the study. Table S2. SNP and INDEL calling precision rate in simulated data. Table S3. Percent of SNP calling sensitivity for three callers in simulated data. Table S4. INDEL calling sensitivity for two callers in simulated data. Table S5. Known INDELs in the HLA region of NA12878. Table S6. Novel INDELs in the HLA region of NA12878. Table S7. Number of novel SNPs in 22 CLL samples. Table S8. Number of INDELs in 22 CLL samples. (PDF 170 kb)

Background GATK Best Practices workflows are widely used in large-scale sequencing projects and recommend post-alignment processing before variant calling. Two key post-processing steps include the computationally intensive local realignment around known INDELs and base quality score recalibration (BQSR). Both have been shown to reduce erroneous calls; however, the findings are mainly supported by the analytical pipeline that incorporates BWA and GATK UnifiedGenotyper. It is not known whether there is any benefit of post-processing and to what extent the benefit might be for pipelines implementing other methods, especially given that both mappers and callers are typically updated. Moreover, because sequencing platforms are upgraded regularly and the new platforms provide better estimations of read quality scores, the need for post-processing is also unknown. Finally, some regions in the human genome show high sequence divergence from the reference genome; it is unclear whether there is benefit from post-processing in these regions. Results We used both simulated and NA12878 exome data to comprehensively assess the impact of post-processing for five or six popular mappers together with five callers. Focusing on chromosome 6p21.3, which is a region of high sequence divergence harboring the human leukocyte antigen (HLA) system, we found that local realignment had little or no impact on SNP calling, but increased sensitivity was observed in INDEL calling for the Stampy + GATK UnifiedGenotyper pipeline. No or only a modest effect of local realignment was detected on the three haplotype-based callers and no evidence of effect on Novoalign. BQSR had virtually negligible effect on INDEL calling and generally reduced sensitivity for SNP calling that depended on caller, coverage and level of divergence. Specifically, for SAMtools and FreeBayes calling in the regions with low divergence, BQSR reduced the SNP calling sensitivity but improved the precision when the coverage is insufficient. However, in regions of high divergence (e.g., the HLA region), BQSR reduced the sensitivity of both callers with little gain in precision rate. For the other three callers, BQSR reduced the sensitivity without increasing the precision rate regardless of coverage and divergence level. Conclusions We demonstrated that the gain from post-processing is not universal; rather, it depends on mapper and caller combination, and the benefit is influenced further by sequencing depth and divergence level. Our analysis highlights the importance of considering these key factors in deciding to apply the computationally intensive post-processing to Illumina exome data. Electronic supplementary material The online version of this article (doi:10.1186/s12859-016-1279-z) contains supplementary material, which is available to authorized users.