Question: Cell autonomous sex determination in somatic cells does not occur in Galliformes.

Title: Somatic sex identity is cell-autonomous in the chicken Content: In the mammalian model of sex determination, embryos are considered to be sexually indifferent until the transient action of a sex-determining gene initiates gonadal differentiation. Although this model is thought to apply to all vertebrates, this has yet to be established. Here we have examined three lateral gynandromorph chickens (a rare, naturally occurring phenomenon in which one side of the animal appears male and the other female) to investigate the sex-determining mechanism in birds. These studies demonstrated that gynandromorph birds are genuine male:female chimaeras, and indicated that male and female avian somatic cells may have an inherent sex identity. To test this hypothesis, we transplanted presumptive mesoderm between embryos of reciprocal sexes to generate embryos containing male:female chimaeric gonads. In contrast to the outcome for mammalian mixed-sex chimaeras, in chicken mixed-sex chimaeras the donor cells were excluded from the functional structures of the host gonad. In an example where female tissue was transplanted into a male host, donor cells contributing to the developing testis retained a female identity and expressed a marker of female function. Our study demonstrates that avian somatic cells possess an inherent sex identity and that, in birds, sexual differentiation is substantively cell autonomous.

Title: The embryonic cell lineage of the nematode Caenorhabditis elegans. Content: The embryonic cell lineage of Caenorhabditis elegans has been traced from zygote to newly hatched larva, with the result that the entire cell lineage of this organism is now known. During embryogenesis 671 cells are generated; in the hermaphrodite 113 of these (in the male 111) undergo programmed death and the remainder either differentiate terminally or become postembryonic blast cells. The embryonic lineage is highly invariant, as are the fates of the cells to which it gives rise. In spite of the fixed relationship between cell ancestry and cell fate, the correlation between them lacks much obvious pattern. Thus, although most neurons arise from the embryonic ectoderm, some are produced by the mesoderm and a few are sisters to muscles; again, lineal boundaries do not necessarily coincide with functional boundaries. Nevertheless, cell ablation experiments (as well as previous cell isolation experiments) demonstrate substantial cell autonomy in at least some sections of embryogenesis. We conclude that the cell lineage itself, complex as it is, plays an important role in determining cell fate. We discuss the origin of the repeat units (partial segments) in the body wall, the generation of the various orders of symmetry, the analysis of the lineage in terms of sublineages, and evolutionary implications.

Title: Nanog safeguards pluripotency and mediates germline development Content: Nanog is a divergent homeodomain protein found in mammalian pluripotent cells and developing germ cells. Deletion of Nanog causes early embryonic lethality, whereas constitutive expression enables autonomous self-renewal of embryonic stem cells. Nanog is accordingly considered a core element of the pluripotent transcriptional network. However, here we report that Nanog fluctuates in mouse embryonic stem cells. Transient downregulation of Nanog appears to predispose cells towards differentiation but does not mark commitment. By genetic deletion we show that, although they are prone to differentiate, embryonic stem cells can self-renew indefinitely in the permanent absence of Nanog. Expanded Nanog null cells colonize embryonic germ layers and exhibit multilineage differentiation both in fetal and adult chimaeras. Although they are also recruited to the germ line, primordial germ cells lacking Nanog fail to mature on reaching the genital ridge. This defect is rescued by repair of the mutant allele. Thus Nanog is dispensible for expression of somatic pluripotency but is specifically required for formation of germ cells. Nanog therefore acts primarily in construction of inner cell mass and germ cell states rather than in the housekeeping machinery of pluripotency. We surmise that Nanog stabilizes embryonic stem cells in culture by resisting or reversing alternative gene expression states.

Title: Memory and Modularity in Cell-Fate Decision Making Content: Genetically identical cells sharing an environment can display markedly different phenotypes. It is often unclear how much of this variation derives from chance, external signals, or attempts by individual cells to exert autonomous phenotypic programs. By observing thousands of cells for hundreds of consecutive generations under constant conditions, we dissect the stochastic decision between a solitary, motile state and a chained, sessile state in Bacillus subtilis. We show that the motile state is 'memoryless', exhibiting no autonomous control over the time spent in the state. In contrast, the time spent as connected chains of cells is tightly controlled, enforcing coordination among related cells in the multicellular state. We show that the three-protein regulatory circuit governing the decision is modular, as initiation and maintenance of chaining are genetically separable functions. As stimulation of the same initiating pathway triggers biofilm formation, we argue that autonomous timing allows a trial commitment to multicellularity that external signals could extend.

Title: Adult Neurogenesis Is Sustained by Symmetric Self-Renewal and Differentiation. Content: Somatic stem cells have been identified in multiple adult tissues. Whether self-renewal occurs symmetrically or asymmetrically is key to understanding long-term stem cell maintenance and generation of progeny for cell replacement. In the adult mouse brain, neural stem cells (NSCs) (B1 cells) are retained in the walls of the lateral ventricles (ventricular-subventricular zone [V-SVZ]). The mechanism of B1 cell retention into adulthood for lifelong neurogenesis is unknown. Using multiple clonal labeling techniques, we show that the vast majority of B1 cells divide symmetrically. Whereas 20%-30% symmetrically self-renew and can remain in the niche for several months before generating neurons, 70%-80% undergo consuming divisions generating progeny, resulting in the depletion of B1 cells over time. This cellular mechanism decouples self-renewal from the generation of progeny. Limited rounds of symmetric self-renewal and consuming symmetric differentiation divisions can explain the levels of neurogenesis observed throughout life.

Title: All dosage compensation is local: Gene-by-gene regulation of sex-biased expression on the chicken Z chromosome Content: Recent reports have suggested that birds lack a mechanism of wholesale dosage compensation for the Z sex chromosome. This discovery was rather unexpected, as all other animals investigated with chromosomal mechanisms of sex determination have some method to counteract the effects of gene dosage of the dominant sex chromosome in males and females. Despite the lack of a global mechanism of avian dosage compensation, the pattern of gene expression difference between males and females varies a great deal for individual Z-linked genes. This suggests that some genes may be individually dosage compensated, and that some less-than-global pattern of dosage compensation, such as local or temporal, exists on the avian Z chromosome. We used global gene expression profiling in males and females for both somatic and gonadal tissue at several time points in the life cycle of the chicken to assess the pattern of sex-biased gene expression on the Z chromosome. Average fold-change between males and females varied somewhat among tissue time-point combinations, with embryonic brain samples having the smallest gene dosage effects, and adult gonadal tissue having the largest degree of male bias. Overall, there were no neighborhoods of overall dosage compensation along the Z. Taken together, this suggests that dosage compensation is regulated on the Z chromosome entirely on a gene-by-gene level, and can vary during the life cycle and by tissue type. This regulation may be an indication of how critical a given gene's functionality is, as the expression level for essential genes will be tightly regulated in order to avoid perturbing important pathways and networks with differential expression levels in males and females.

Title: Direct conversion of patient fibroblasts demonstrates non-cell autonomous toxicity of astrocytes to motor neurons in familial and sporadic ALS. Content: Amyotrophic lateral sclerosis (ALS) causes motor neuron degeneration, paralysis, and death. Accurate disease modeling, identifying disease mechanisms, and developing therapeutics is urgently needed. We previously reported motor neuron toxicity through postmortem ALS spinal cord-derived astrocytes. However, these cells can only be harvested after death, and their expansion is limited. We now report a rapid, highly reproducible method to convert adult human fibroblasts from living ALS patients to induced neuronal progenitor cells and subsequent differentiation into astrocytes (i-astrocytes). Non-cell autonomous toxicity to motor neurons is found following coculture of i-astrocytes from familial ALS patients with mutation in superoxide dismutase or hexanucleotide expansion in C9orf72 (ORF 72 on chromosome 9) the two most frequent causes of ALS. Remarkably, i-astrocytes from sporadic ALS patients are as toxic as those with causative mutations, suggesting a common mechanism. Easy production and expansion of i-astrocytes now enables rapid disease modeling and high-throughput drug screening to alleviate astrocyte-derived toxicity.

Title: Male Japanese quails with female brains do not show male sexual behaviors. Content: During embryonic development, gonadal steroid hormones (androgens and estrogens) are thought to organize the sexual differentiation of the brain in the heterogametic sexes of higher vertebrates (males in mammals, females in birds). Brain differentiation of the homogametic sexes is thought to proceed by default, not requiring sex hormones for sex-specific organization. In gallinaceous birds such as the Japanese quail, female brain organization is thought to develop via estrogen-dependent demasculinization of a default male brain phenotype. We performed male donor-to-female host (MF), female-to-male (FM), male-to-male (MM), and female-to-female (FF) isotopic, isochronic transplantation of the forebrain primordium in Japanese quail embryos before gonadal differentiation had occurred; brain chimeras had a forebrain (including the hypothalamus) originating exclusively from donor cells. MM, FF, and MF chimeras all showed sexual behavior governed by the genetic sex of the host. In contrast, FM chimeras (genetically female forebrain, all other tissues genetically male) showed no mounting and only rudimentary crowing behavior. Although MM, FF, MF, and FM chimeras all showed host-typical production of steroid hormones during embryonic life, only FM chimeras were hypogonadal, had atypical low levels of circulating testosterone in adulthood, and showed reduction (crowing) or absence (mounting) of reproductive behaviors. Morphological features of the medial preoptic nucleus (a sexually dimorphic brain area) also were not male-like in FM males. These data demonstrate a brain-intrinsic, genetically determined component that organizes the sex-typical production of gonadal hormones in adulthood and call for a reevaluation of the mechanisms underlying brain sexual differentiation in other higher-vertebrate species.

Title: Interface Contractility between Differently Fated Cells Drives Cell Elimination and Cyst Formation Content: Although cellular tumor-suppression mechanisms are widely studied, little is known about mechanisms that act at the level of tissues to suppress the occurrence of aberrant cells in epithelia. We find that ectopic expression of transcription factors that specify cell fates causes abnormal epithelial cysts in Drosophila imaginal discs. Cysts do not form cell autonomously but result from the juxtaposition of two cell populations with divergent fates. Juxtaposition of wild-type and aberrantly specified cells induces enrichment of actomyosin at their entire shared interface, both at adherens junctions as well as along basolateral interfaces. Experimental validation of 3D vertex model simulations demonstrates that enhanced interface contractility is sufficient to explain many morphogenetic behaviors, which depend on cell cluster size. These range from cyst formation by intermediate-sized clusters to segregation of large cell populations by formation of smooth boundaries or apical constriction in small groups of cells. In addition, we find that single cells experiencing lateral interface contractility are eliminated from tissues by apoptosis. Cysts, which disrupt epithelial continuity, form when elimination of single, aberrantly specified cells fails and cells proliferate to intermediate cell cluster sizes. Thus, increased interface contractility functions as error correction mechanism eliminating single aberrant cells from tissues, but failure leads to the formation of large, potentially disease-promoting cysts. Our results provide a novel perspective on morphogenetic mechanisms, which arise from cell-fate heterogeneities within tissues and maintain or disrupt epithelial homeostasis.

Title: Astrocytes from Familial and Sporadic ALS Patients are Toxic to Motor Neurons Content: Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disease, with astrocytes implicated as contributing substantially to motor neuron death in familial (F)ALS. However, the proposed role of astrocytes in the pathology of ALS derives in part from rodent models of FALS based upon dominant mutations within the superoxide dismutase 1 (SOD1) gene, which account for <2% of all ALS cases. Their role in sporadic (S)ALS, which affects >90% of ALS patients, remains to be established. Using astrocytes generated from postmortem tissue from both FALS and SALS patients, we show that astrocytes derived from both patient groups are similarly toxic to motor neurons. We also demonstrate that SOD1 is a viable target for SALS, as its knockdown significantly attenuates astrocyte-mediated toxicity toward motor neurons. Our data highlight astrocytes as a non-cell autonomous component in SALS and provide an in vitro model system to investigate common disease mechanisms and evaluate potential therapies for SALS and FALS.

Title: Molecular features of cellular reprogramming and development Content: Differentiating somatic cells are progressively restricted to specialized functions during ontogeny, but they can be experimentally directed to form other cell types, including those with complete embryonic potential. Early nuclear reprogramming methods, such as somatic cell nuclear transfer (SCNT) and cell fusion, posed significant technical hurdles to precise dissection of the regulatory programmes governing cell identity. However, the discovery of reprogramming by ectopic expression of a defined set of transcription factors, known as direct reprogramming, provided a tractable platform to uncover molecular characteristics of cellular specification and differentiation, cell type stability and pluripotency. We discuss the control and maintenance of cellular identity during developmental transitions as they have been studied using direct reprogramming, with an emphasis on transcriptional and epigenetic regulation.

Title: Reprogramming somatic cells towards pluripotency by cellular fusion. Content: Pluripotent cells arise within the inner cell mass (ICM) of mammals and have the potential to generate all cell types of the adult organism through a process of commitment and ordered differentiation. Despite many decades of investigation, the mechanisms that guide and stabilise cell fate choice as well as those that can be engineered to promote its reversal, remain only partially resolved. Reprogramming of somatic cells towards a pluripotent-like state can be achieved by several different experimental routes including nuclear transfer, the supply of a defined cocktail of transcription factors, or by fusing somatic cells with a pluripotent stem cell partner. These approaches have been used to demonstrate the remarkable intrinsic epigenetic plasticity of many terminally differentiated cell types, as well as to define the factors that are required for pluripotent conversion. In this review we summarise some recent advances using cell fusion-based studies to better understand the basis of pluripotency and the epigenetic mechanisms that promote cell type inter-conversion.

Title: Integrated maps in quail (Coturnix japonica) confirm the high degree of synteny conservation with chicken (Gallus gallus) despite 35 million years of divergence Content: BackgroundBy comparing the quail genome with that of chicken, chromosome rearrangements that have occurred in these two galliform species over 35 million years of evolution can be detected. From a more practical point of view, the definition of conserved syntenies helps to predict the position of genes in quail, based on information taken from the chicken sequence, thus enhancing the utility of this species in biological studies through a better knowledge of its genome structure. A microsatellite and an Amplified Fragment Length Polymorphism (AFLP) genetic map were previously published for quail, as well as comparative cytogenetic data with chicken for macrochromosomes. Quail genomics will benefit from the extension and the integration of these maps. ResultsThe integrated linkage map presented here is based on segregation analysis of both anonymous markers and functional gene loci in 1,050 quail from three independent F2 populations. Ninety-two loci are resolved into 14 autosomal linkage groups and a Z chromosome-specific linkage group, aligned with the quail AFLP map. The size of linkage groups ranges from 7.8 cM to 274.8 cM. The total map distance covers 904.3 cM with an average spacing of 9.7 cM between loci. The coverage is not complete, as macrochromosome CJA08, the gonosome CJAW and 23 microchromosomes have no marker assigned yet. Significant sequence identities of quail markers with chicken enabled the alignment of the quail linkage groups on the chicken genome sequence assembly. This, together with interspecific Fluorescence In Situ Hybridization (FISH), revealed very high similarities in marker order between the two species for the eight macrochromosomes and the 14 microchromosomes studied. ConclusionIntegrating the two microsatellite and the AFLP quail genetic maps greatly enhances the quality of the resulting information and will thus facilitate the identification of Quantitative Trait Loci (QTL). The alignment with the chicken chromosomes confirms the high conservation of gene order that was expected between the two species for macrochromosomes. By extending the comparative study to the microchromosomes, we suggest that a wealth of information can be mined in chicken, to be used for genome analyses in quail.

Title: Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro Content: We describe the derivation of pluripotent embryonic stem (ES) cells from human blastocysts. Two diploid ES cell lines have been cultivated in vitro for extended periods while maintaining expression of markers characteristic of pluripotent primate cells. Human ES cells express the transcription factor Oct-4, essential for development of pluripotential cells in the mouse. When grafted into SCID mice, both lines give rise to teratomas containing derivatives of all three embryonic germ layers. Both cell lines differentiate in vitro into extraembryonic and somatic cell lineages. Neural progenitor cells may be isolated from differentiating ES cell cultures and induced to form mature neurons. Embryonic stem cells provide a model to study early human embryology, an investigational tool for discovery of novel growth factors and medicines, and a potential source of cells for use in transplantation therapy.

Title: Autocrine VEGF Signaling Synergizes with EGFR in Tumor Cells to Promote Epithelial Cancer Development Content: It is established that tumor cell-derived VEGF acts on endothelial cells to promote angiogenesis and tumor growth. Here, we demonstrate that in K5-SOS-dependent mouse skin tumors, autocrine VEGF is required for tumor cell proliferation in a cell-autonomous and angiogenesis-independent manner. VEGF is upregulated in SOS-expressing tumors, and its deletion in epidermal cells delays tumorigenesis by suppressing angiogenesis and tumor cell proliferation. Epidermis-specific Flt1 deletion also impairs tumorigenesis and proliferation. Surprisingly, complete tumor inhibition occurs in the absence of VEGF in EGFR mutant mice, demonstrating that VEGFR and EGFR synergize in neoplastic cells to promote tumor growth. Mechanistically, K5-SOS upregulates VEGF, Flt1, and Neuropilin-1 in an Erk-dependent manner, thereby activating an autocrine proliferation loop, whereas EGFR prevents tumor cells from apoptosis. Moreover, Flt1 is upregulated in human SCC, and its inhibition in SCC cells impairs proliferation. Thus, in addition to regulating angiogenesis, VEGF has to be considered as a potent growth factor for epidermal tumors.

Title: Induced pluripotent stem cell lines derived from human somatic cells. Content: 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.

Title: MicroRNA-Containing T-Regulatory-Cell-Derived Exosomes Suppress Pathogenic T Helper 1 Cells Content: Foxp3(+) T regulatory (Treg) cells prevent inflammatory disease but the mechanistic basis of suppression is not understood completely. Gene silencing by RNA interference can act in a cell-autonomous and non-cell-autonomous manner, providing mechanisms of intercellular regulation. Here, we demonstrate that non-cell-autonomous gene silencing, mediated by miRNA-containing exosomes, is a mechanism employed by Treg cells to suppress T-cell-mediated disease. Treg cells transferred microRNAs (miRNA) to various immune cells, including T helper 1 (Th1) cells, suppressing Th1 cell proliferation and cytokine secretion. Use of Dicer-deficient or Rab27a and Rab27b double-deficient Treg cells to disrupt miRNA biogenesis or the exosomal pathway, respectively, established a requirement for miRNAs and exosomes for Treg-cell-mediated suppression. Transcriptional analysis and miRNA inhibitor studies showed that exosome-mediated transfer of Let-7d from Treg cell to Th1 cells contributed to suppression and prevention of systemic disease. These studies reveal a mechanism of Treg-cell-mediated suppression mediated by miRNA-containing exosomes.

Title: Evidence of a pluripotent human embryonic stem cell line derived from a cloned blastocyst. Content: Somatic cell nuclear transfer (SCNT) technology has recently been used to generate animals with a common genetic composition. In this study, we report the derivation of a pluripotent embryonic stem (ES) cell line (SCNT-hES-1) from a cloned human blastocyst. The SCNT-hES-1 cells displayed typical ES cell morphology and cell surface markers and were capable of differentiating into embryoid bodies in vitro and of forming teratomas in vivo containing cell derivatives from all three embryonic germ layers in severe combined immunodeficient mice. After continuous proliferation for more than 70 passages, SCNT-hES-1 cells maintained normal karyotypes and were genetically identical to the somatic nuclear donor cells. Although we cannot completely exclude the possibility that the cells had a parthenogenetic origin, imprinting analyses support a SCNT origin of the derived human ES cells.

Title: The cancer stem cell hypothesis: a guide to potential molecular targets. Content: Common cancer theories hold that tumor is an uncontrolled somatic cell proliferation caused by the progressive addition of random mutations in critical genes that control cell growth. Nevertheless, various contradictions related to the mutation theory have been reported previously. These events may be elucidated by the persistence of residual tumor cells, called Cancer Stem Cells (CSCs) responsible for tumorigenesis, tumor maintenance, tumor spread, and tumor relapse. Herein, we summarize the current understanding of CSCs, with a focus on the possibility to identify specific markers of CSCs, and discuss the clinical application of targeting CSCs for cancer treatment.

Title: Reprogramming factor expression initiates widespread targeted chromatin remodeling. Content: Despite rapid progress in characterizing transcription factor-driven reprogramming of somatic cells to an induced pluripotent stem cell (iPSC) state, many mechanistic questions still remain. To gain insight into the earliest events in the reprogramming process, we systematically analyzed the transcriptional and epigenetic changes that occur during early factor induction after discrete numbers of divisions. We observed rapid, genome-wide changes in the euchromatic histone modification, H3K4me2, at more than a thousand loci including large subsets of pluripotency-related or developmentally regulated gene promoters and enhancers. In contrast, patterns of the repressive H3K27me3 modification remained largely unchanged except for focused depletion specifically at positions where H3K4 methylation is gained. These chromatin regulatory events precede transcriptional changes within the corresponding loci. Our data provide evidence for an early, organized, and population-wide epigenetic response to ectopic reprogramming factors that clarify the temporal order through which somatic identity is reset during reprogramming.

Title: Autophagy and mTORC1 regulate the stochastic phase of somatic cell reprogramming Content: We describe robust induction of autophagy during the reprogramming of mouse fibroblasts to induced pluripotent stem cells by four reprogramming factors (Sox2, Oct4, Klf4 and c-Myc), henceforth 4F. This process occurs independently of p53 activation, and is mediated by the synergistic downregulation of mechanistic target of rapamycin complex 1 (mTORC1) and the induction of autophagy-related genes. The 4F coordinately repress mTORC1, but bifurcate in their regulation of autophagy-related genes, with Klf4 and c-Myc inducing them but Sox2 and Oct4 inhibiting them. On one hand, inhibition of mTORC1 facilitates reprogramming by promoting cell reshaping (mitochondrial remodelling and cell size reduction). On the other hand, mTORC1 paradoxically impairs reprogramming by triggering autophagy. Autophagy does not participate in cell reshaping in reprogramming but instead degrades p62, whose accumulation in autophagy-deficient cells facilitates reprogramming. Our results thus reveal a complex signalling network involving mTORC1 inhibition and autophagy induction in the early phase of reprogramming, whose delicate balance ultimately determines reprogramming efficiency.

Title: TGF-β and Insulin Signaling Regulate Reproductive Aging via Oocyte and Germline Quality Maintenance Content: Reproductive cessation is perhaps the earliest aging phenotype that humans experience. Similarly, reproduction of Caenorhabditis elegans ceases in mid-adulthood. Although somatic aging has been studied in both worms and humans, mechanisms regulating reproductive aging are not yet understood. Here, we show that TGF-β Sma/Mab and Insulin/IGF-1 signaling regulate C. elegans reproductive aging by modulating multiple aspects of the reproductive process, including embryo integrity, oocyte fertilizability, chromosome segregation fidelity, DNA damage resistance, and oocyte and germline morphology. TGF-β activity regulates reproductive span and germline/oocyte quality noncell-autonomously and is temporally and transcriptionally separable from its regulation of growth. Chromosome segregation, cell cycle, and DNA damage response genes are upregulated in TGF-β mutant oocytes, decline in aged mammalian oocytes, and are critical for oocyte quality maintenance. Our data suggest that C. elegans and humans share many aspects of reproductive aging, including the correlation between reproductive aging and declining oocyte quality and mechanisms determining oocyte quality.

Title: Human oocytes reprogram adult somatic nuclei of a type 1 diabetic to diploid pluripotent stem cells Content: The transfer of somatic cell nuclei into oocytes can give rise to pluripotent stem cells that are consistently equivalent to embryonic stem cells, holding promise for autologous cell replacement therapy. Although methods to induce pluripotent stem cells from somatic cells by transcription factors are widely used in basic research, numerous differences between induced pluripotent stem cells and embryonic stem cells have been reported, potentially affecting their clinical use. Because of the therapeutic potential of diploid embryonic stem-cell lines derived from adult cells of diseased human subjects, we have systematically investigated the parameters affecting efficiency of blastocyst development and stem-cell derivation. Here we show that improvements to the oocyte activation protocol, including the use of both kinase and translation inhibitors, and cell culture in the presence of histone deacetylase inhibitors, promote development to the blastocyst stage. Developmental efficiency varied between oocyte donors, and was inversely related to the number of days of hormonal stimulation required for oocyte maturation, whereas the daily dose of gonadotropin or the total number of metaphase II oocytes retrieved did not affect developmental outcome. Because the use of concentrated Sendai virus for cell fusion induced an increase in intracellular calcium concentration, causing premature oocyte activation, we used diluted Sendai virus in calcium-free medium. Using this modified nuclear transfer protocol, we derived diploid pluripotent stem-cell lines from somatic cells of a newborn and, for the first time, an adult, a female with type 1 diabetes.

Title: Induction of pluripotent stem cells from mouse embryonic fibroblasts by Oct4 and Klf4 with small-molecule compounds. Content: Somatic cells can be induced into pluripotent stem cells (iPSCs) with a combination of four transcription factors, Oct4/Sox2/Klf4/c-Myc or Oct4/Sox2/Nanog/LIN28. This provides an enabling platform to obtain patient-specific cells for various therapeutic and research applications. However, several problems remain for this approach to be therapeutically relevant due to drawbacks associated with efficiency and viral genome integration. Recently, it was shown that neural progenitor cells (NPCs) transduced with Oct4/Klf4 can be reprogrammed into iPSCs. However, NPCs express Sox2 endogenously, possibly facilitating reprogramming in the absence of exogenous Sox2. In this study, we identified a small-molecule combination, BIX-01294 and BayK8644, that enables reprogramming of Oct4/Klf4-transduced mouse embryonic fibroblasts, which do not endogenously express the factors essential for reprogramming. This study demonstrates that small molecules identified through a phenotypic screen can compensate for viral transduction of critical factors, such as Sox2, and improve reprogramming efficiency.

Title: Promiscuous gene expression in medullary thymic epithelial cells mirrors the peripheral self Content: Expression of peripheral antigens in the thymus has been implicated in T cell tolerance and autoimmunity. Here we identified medullary thymic epithelial cells as being a unique cell type that expresses a diverse range of tissue-specific antigens. We found that this promiscuous gene expression was a cell-autonomous property of medullary epithelial cells and was maintained during the entire period of thymic T cell output. It may facilitate tolerance induction to self-antigens that would otherwise be temporally or spatially secluded from the immune system. However, the array of promiscuously expressed self-antigens appeared random rather than selected and was not confined to secluded self-antigens.

Title: Clonal hematopoiesis of indeterminate potential and its distinction from myelodysplastic syndromes. Content: Recent genetic analyses of large populations have revealed that somatic mutations in hematopoietic cells leading to clonal expansion are commonly acquired during human aging. Clonally restricted hematopoiesis is associated with an increased risk of subsequent diagnosis of myeloid or lymphoid neoplasia and increased all-cause mortality. Although myelodysplastic syndromes (MDS) are defined by cytopenias, dysplastic morphology of blood and marrow cells, and clonal hematopoiesis, most individuals who acquire clonal hematopoiesis during aging will never develop MDS. Therefore, acquisition of somatic mutations that drive clonal expansion in the absence of cytopenias and dysplastic hematopoiesis can be considered clonal hematopoiesis of indeterminate potential (CHIP), analogous to monoclonal gammopathy of undetermined significance and monoclonal B-cell lymphocytosis, which are precursor states for hematologic neoplasms but are usually benign and do not progress. Because mutations are frequently observed in healthy older persons, detection of an MDS-associated somatic mutation in a cytopenic patient without other evidence of MDS may cause diagnostic uncertainty. Here we discuss the nature and prevalence of CHIP, distinction of this state from MDS, and current areas of uncertainty regarding diagnostic criteria for myeloid malignancies.

Title: Histone Demethylase Expression Enhances Human Somatic Cell Nuclear Transfer Efficiency and Promotes Derivation of Pluripotent Stem Cells. Content: The extremely low efficiency of human embryonic stem cell (hESC) derivation using somatic cell nuclear transfer (SCNT) limits its potential application. Blastocyst formation from human SCNT embryos occurs at a low rate and with only some oocyte donors. We previously showed in mice that reduction of histone H3 lysine 9 trimethylation (H3K9me3) through ectopic expression of the H3K9me3 demethylase Kdm4d greatly improves SCNT embryo development. Here we show that overexpression of a related H3K9me3 demethylase KDM4A improves human SCNT, and that, as in mice, H3K9me3 in the human somatic cell genome is an SCNT reprogramming barrier. Overexpression of KDM4A significantly improves the blastocyst formation rate in human SCNT embryos by facilitating transcriptional reprogramming, allowing efficient derivation of SCNT-derived ESCs using adult Age-related Macular Degeneration (AMD) patient somatic nuclei donors. This conserved mechanistic insight has potential applications for improving SCNT in a variety of contexts, including regenerative medicine.

Title: Human somatic cell nuclear transfer using adult cells. Content: Derivation of patient-specific human pluripotent stem cells via somatic cell nuclear transfer (SCNT) has the potential for applications in a range of therapeutic contexts. However, successful SCNT with human cells has proved challenging to achieve, and thus far has only been reported with fetal or infant somatic cells. In this study, we describe the application of a recently developed methodology for the generation of human ESCs via SCNT using dermal fibroblasts from 35- and 75-year-old males. Our study therefore demonstrates the applicability of SCNT for adult human cells and supports further investigation of SCNT as a strategy for regenerative medicine.

Title: Somatic coding mutations in human induced pluripotent stem cells Content: Defined transcription factors can induce epigenetic reprogramming of adult mammalian cells into induced pluripotent stem cells. Although DNA factors are integrated during some reprogramming methods, it is unknown whether the genome remains unchanged at the single nucleotide level. Here we show that 22 human induced pluripotent stem (hiPS) cell lines reprogrammed using five different methods each contained an average of five protein-coding point mutations in the regions sampled (an estimated six protein-coding point mutations per exome). The majority of these mutations were non-synonymous, nonsense or splice variants, and were enriched in genes mutated or having causative effects in cancers. At least half of these reprogramming-associated mutations pre-existed in fibroblast progenitors at low frequencies, whereas the rest occurred during or after reprogramming. Thus, hiPS cells acquire genetic modifications in addition to epigenetic modifications. Extensive genetic screening should become a standard procedure to ensure hiPS cell safety before clinical use.

Title: Ablation of NF1 function in neurons induces abnormal development of cerebral cortex and reactive gliosis in the brain. Content: Neurofibromatosis type 1 (NF1) is a prevalent genetic disorder that affects growth properties of neural-crest-derived cell populations. In addition, approximately one-half of NF1 patients exhibit learning disabilities. To characterize NF1 function both in vitro and in vivo, we circumvent the embryonic lethality of NF1 null mouse embryos by generating a conditional mutation in the NF1 gene using Cre/loxP technology. Introduction of a Synapsin I promoter driven Cre transgenic mouse strain into the conditional NF1 background has ablated NF1 function in most differentiated neuronal populations. These mice have abnormal development of the cerebral cortex, which suggests that NF1 has an indispensable role in this aspect of CNS development. Furthermore, although they are tumor free, these mice display extensive astrogliosis in the absence of conspicuous neurodegeneration or microgliosis. These results indicate that NF1-deficient neurons are capable of inducing reactive astrogliosis via a non-cell autonomous mechanism.

Title: Reprogramming of human somatic cells to pluripotency with defined factors Content: Pluripotency pertains to the cells of early embryos that can generate all of the tissues in the organism. Embryonic stem cells are embryo-derived cell lines that retain pluripotency and represent invaluable tools for research into the mechanisms of tissue formation. Recently, murine fibroblasts have been reprogrammed directly to pluripotency by ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and Myc) to yield induced pluripotent stem (iPS) cells. Using these same factors, we have derived iPS cells from fetal, neonatal and adult human primary cells, including dermal fibroblasts isolated from a skin biopsy of a healthy research subject. Human iPS cells resemble embryonic stem cells in morphology and gene expression and in the capacity to form teratomas in immune-deficient mice. These data demonstrate that defined factors can reprogramme human cells to pluripotency, and establish a method whereby patient-specific cells might be established in culture.

Title: The emergence of order in the Drosophila pupal retina. Content: During pupation, long-range order is imposed on the autonomously developing ommatidia which compose the Drosophila eye. To accomplish this, eight additional cell types arise: the primary, secondary, and tertiary pigment cells, and the four cells that form the bristle. These cells form an interweaving lattice between ommatidia. The lattice is refined when excess cells are removed to bring neighboring ommatidia into register. Recent evidence suggests that in larval development, local contacts direct cell fate. The same appears to be true during pupal development: the contacts a cell makes predict the cell type it will become. Cells which contact the anterior or posterior cone cells in an ommatidium invariably become primary pigment cells. Cells which contact primary pigment cells from different ommatidia become secondary and tertiary pigment cells. Bristle development is in several ways distinct from ommatidial development. The four cells of each bristle group appear to be immediate descendents of a single founder cell. During their early differentiation, they do not make stereotyped contacts with surrounding ommatidial cells, but do make particular contacts within the bristle group. And unlike the surrounding ommatidia, differentiation of the bristles radiates from the center of the eye to the edges. As cells are removed during two stages of programmed cell death, the bristles are brought into their final position. When all cells in the lattice have achieved their final position, a second stage of retinal development begins as structures specific to each cell type are produced. This paper follows these various stages of pupal development, and suggests how local cell-cell contacts may produce the cells needed for a functional retina.

Title: Hotspots of aberrant epigenomic reprogramming in human induced pluripotent stem cells Content: Induced pluripotent stem cells (iPSCs) offer immense potential for regenerative medicine and studies of disease and development. Somatic cell reprogramming involves epigenomic reconfiguration, conferring iPSCs with characteristics similar to embryonic stem (ES) cells. However, it remains unknown how complete the reestablishment of ES-cell-like DNA methylation patterns is throughout the genome. Here we report the first whole-genome profiles of DNA methylation at single-base resolution in five human iPSC lines, along with methylomes of ES cells, somatic cells, and differentiated iPSCs and ES cells. iPSCs show significant reprogramming variability, including somatic memory and aberrant reprogramming of DNA methylation. iPSCs share megabase-scale differentially methylated regions proximal to centromeres and telomeres that display incomplete reprogramming of non-CG methylation, and differences in CG methylation and histone modifications. Lastly, differentiation of iPSCs into trophoblast cells revealed that errors in reprogramming CG methylation are transmitted at a high frequency, providing an iPSC reprogramming signature that is maintained after differentiation.

Title: Gene expression during gonadogenesis in the chicken embryo. Content: Genes implicated in vertebrate sex determination and differentiation were studied in embryonic chicken gonads using reverse transcription and the polymerase chain reaction (RT-PCR). Expression profiles were obtained during gonadal sex differentiation for AMH, SOX9, SOX3, the Wilm's Tumour gene, WT1, and the orphan nuclear receptor genes, SF1 and DAX1. Some of these genes showed sexually dimorphic expression profiles during gonadal development, whereas others were expressed at similar levels in both sexes. The gene encoding Anti-Müllerian hormone (AMH) was expressed in both sexes prior to and during sexual differentiation of the gonads, with levels of expression consistently higher in males than in females. SOX9 expression was male-specific, and was up-regulated after the detection of AMH transcripts. SOX3 expression was observed prior to clear SOX9 expression and was up-regulated in both sexes at the onset of gonadal sex differentiation (but declined later in development). The WT1 gene was highly expressed in both sexes, whereas SF1 expression was clearly higher in developing ovaries compared to testes. DAX1 transcripts were observed in both sexes at all stages examined, but expression appeared somewhat higher in developing ovaries. These expression profiles are analysed in terms of current theories of vertebrate sex determination.

Title: Concentration Sensing by the Moving Nucleus in Cell Fate Determination: A Computational Analysis Content: During development of the vertebrate neuroepithelium, the nucleus in neural progenitor cells (NPCs) moves from the apex toward the base and returns to the apex (called interkinetic nuclear migration) at which point the cell divides. The fate of the resulting daughter cells is thought to depend on the sampling by the moving nucleus of a spatial concentration profile of the cytoplasmic Notch intracellular domain (NICD). However, the nucleus executes complex stochastic motions including random waiting and back and forth motions, which can expose the nucleus to randomly varying levels of cytoplasmic NICD. How nuclear position can determine daughter cell fate despite the stochastic nature of nuclear migration is not clear. Here we derived a mathematical model for reaction, diffusion, and nuclear accumulation of NICD in NPCs during interkinetic nuclear migration (INM). Using experimentally measured trajectory-dependent probabilities of nuclear turning, nuclear waiting times and average nuclear speeds in NPCs in the developing zebrafish retina, we performed stochastic simulations to compute the nuclear trajectory-dependent probabilities of NPC differentiation. Comparison with experimentally measured nuclear NICD concentrations and trajectory-dependent probabilities of differentiation allowed estimation of the NICD cytoplasmic gradient. Spatially polarized production of NICD, rapid NICD cytoplasmic consumption and the time-averaging effect of nuclear import/export kinetics are sufficient to explain the experimentally observed differentiation probabilities. Our computational studies lend quantitative support to the feasibility of the nuclear concentration-sensing mechanism for NPC fate determination in zebrafish retina.

Title: Conservation of methylation reprogramming in mammalian development: aberrant reprogramming in cloned embryos. Content: Mouse embryos undergo genome-wide methylation reprogramming by demethylation in early preimplantation development, followed by remethylation thereafter. Here we show that genome-wide reprogramming is conserved in several mammalian species and ask whether it also occurs in embryos cloned with the use of highly methylated somatic donor nuclei. Normal bovine, rat, and pig zygotes showed a demethylated paternal genome, suggesting active demethylation. In bovine embryos methylation was further reduced during cleavage up to the eight-cell stage, and this reduction in methylation was followed by de novo methylation by the 16-cell stage. In cloned one-cell embryos there was a reduction in methylation consistent with active demethylation, but no further demethylation occurred subsequently. Instead, de novo methylation and nuclear reorganization of methylation patterns resembling those of differentiated cells occurred precociously in many cloned embryos. Cloned, but not normal, morulae had highly methylated nuclei in all blastomeres that resembled those of the fibroblast donor cells. Our study shows that epigenetic reprogramming occurs aberrantly in most cloned embryos; incomplete reprogramming may contribute to the low efficiency of cloning.

Title: Targeted inhibition of tumor-specific glutaminase diminishes cell-autonomous tumorigenesis. Content: Glutaminase (GLS), which converts glutamine to glutamate, plays a key role in cancer cell metabolism, growth, and proliferation. GLS is being explored as a cancer therapeutic target, but whether GLS inhibitors affect cancer cell-autonomous growth or the host microenvironment or have off-target effects is unknown. Here, we report that loss of one copy of Gls blunted tumor progression in an immune-competent MYC-mediated mouse model of hepatocellular carcinoma. Compared with results in untreated animals with MYC-induced hepatocellular carcinoma, administration of the GLS-specific inhibitor bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (BPTES) prolonged survival without any apparent toxicities. BPTES also inhibited growth of a MYC-dependent human B cell lymphoma cell line (P493) by blocking DNA replication, leading to cell death and fragmentation. In mice harboring P493 tumor xenografts, BPTES treatment inhibited tumor cell growth; however, P493 xenografts expressing a BPTES-resistant GLS mutant (GLS-K325A) or overexpressing GLS were not affected by BPTES treatment. Moreover, a customized Vivo-Morpholino that targets human GLS mRNA markedly inhibited P493 xenograft growth without affecting mouse Gls expression. Conversely, a Vivo-Morpholino directed at mouse Gls had no antitumor activity in vivo. Collectively, our studies demonstrate that GLS is required for tumorigenesis and support small molecule and genetic inhibition of GLS as potential approaches for targeting the tumor cell-autonomous dependence on GLS for cancer therapy.

Title: The homeobox gene goosecoid controls cell migration in Xenopus embryos. Content: Goosecoid (gsc), a homeobox gene expressed specifically in the dorsal blastopore lip of the Xenopus gastrula, is considered to play an important role in Spemann's organizer phenomenon. Lineage tracing and time-lapse microscopy were used to follow the fate of embryonic cells microinjected with gsc mRNA. Microinjected gsc has non-cell autonomous effects, recruiting neighboring uninjected cells into a twinned dorsal axis. Ectopic expression of gsc mRNA in ventral blastomeres as well as overexpression of gsc in dorsal blastomeres leads to cell movement toward the anterior of the embryo. The results suggest a function for gsc in the control of gastrulation movements in groups of cells, but not in dissociated cells, and demonstrate that a vertebrate homeobox gene can regulate region-specific cell migration.

Title: Identification of secreted proteins that mediate cell-cell interactions in an in vitro model of the lung cancer microenvironment. Content: Non-small cell lung cancer (NSCLC) cells with somatic mutations in K-ras recruit to the tumor a variety of cell types (hereafter collectively termed "stromal cells") that can promote or inhibit tumorigenesis by mechanisms that have not been fully elucidated. Here, we postulated that stromal cells in the tumor microenvironment alter the tumor cell secretome, including those proteins required for tumor growth and dissemination, and we developed an in vitro model to test this hypothesis. Coculturing a murine K-ras mutant lung adenocarcinoma cell line (LKR-13) with a murine lung stromal cell (macrophage, endothelial cell, or fibroblast) enhanced stromal cell migration, induced endothelial tube formation, increased LKR-13 cell proliferation, and regulated the secretion of proteins involved in angiogenesis, inflammation, cell proliferation, and epithelial-to-mesenchymal transition. Among these proteins, CXCL1 has been reported to promote NSCLC development, whereas interleukin-18 (IL-18) has an undefined role. Genetic and pharmacologic strategies to inhibit CXCL1 and IL-18 revealed that stromal cell migration, LKR-13 cell proliferation, and LKR-13 cell tumorigenicity required one or both of these proteins. We conclude that stromal cells enhanced LKR-13 cell tumorigenicity partly through their effects on the secretome of LKR-13 cells. Strategies to inhibit tumor/stromal cell interactions may be useful as therapeutic approaches in NSCLC patients.

Title: Complex Tissue and Disease Modeling using hiPSCs. Content: Defined genetic models based on human pluripotent stem cells have opened new avenues for understanding disease mechanisms and drug screening. Many of these models assume cell-autonomous mechanisms of disease but it is possible that disease phenotypes or drug responses will only be evident if all cellular and extracellular components of a tissue are present and functionally mature. To derive optimal benefit from such models, complex multicellular structures with vascular components that mimic tissue niches will thus likely be necessary. Here we consider emerging research creating human tissue mimics and provide some recommendations for moving the field forward.

Title: Cell type of origin influences the molecular and functional properties of mouse induced pluripotent stem cells Content: Induced pluripotent stem cells (iPSCs) have been derived from various somatic cell populations through ectopic expression of defined factors. It remains unclear whether iPSCs generated from different cell types are molecularly and functionally similar. Here we show that iPSCs obtained from mouse fibroblasts, hematopoietic and myogenic cells exhibit distinct transcriptional and epigenetic patterns. Moreover, we demonstrate that cellular origin influences the in vitro differentiation potentials of iPSCs into embryoid bodies and different hematopoietic cell types. Notably, continuous passaging of iPSCs largely attenuates these differences. Our results suggest that early-passage iPSCs retain a transient epigenetic memory of their somatic cells of origin, which manifests as differential gene expression and altered differentiation capacity. These observations may influence ongoing attempts to use iPSCs for disease modeling and could also be exploited in potential therapeutic applications to enhance differentiation into desired cell lineages.

Title: A review of the methods for human iPSC derivation. Content: The ability to reprogram somatic cells to induced pluripotent stem cells (iPSCs) offers an opportunity to generate pluripotent patient-specific cell lines that can help model human diseases. These iPSC lines could also be powerful tools for drug discovery and the development of cellular transplantation therapies. Many methods exist for generating iPSC lines but those best suited for use in studying human diseases and developing therapies must be of adequate efficiency to produce iPSCs from samples that may be of limited abundance, capable of reprogramming cells from both skin fibroblasts and blood, and footprint-free. Several reprogramming techniques meet these criteria and can be utilized to derive iPSCs in projects with both basic scientific and therapeutic goals. Combining these reprogramming methods with small molecule modulators of signaling pathways can lead to successful generation of iPSCs from even the most recalcitrant patient-derived somatic cells.

Title: Adult Mammalian Neural Stem Cells and Neurogenesis: Five Decades Later. Content: Adult somatic stem cells in various organs maintain homeostatic tissue regeneration and enhance plasticity. Since its initial discovery five decades ago, investigations of adult neurogenesis and neural stem cells have led to an established and expanding field that has significantly influenced many facets of neuroscience, developmental biology, and regenerative medicine. Here we review recent progress and focus on questions related to adult mammalian neural stem cells that also apply to other somatic stem cells. We further discuss emerging topics that are guiding the field toward better understanding adult neural stem cells and ultimately applying these principles to improve human health.

Title: Specific killing of multiple myeloma cells by (-)-epigallocatechin-3-gallate extracted from green tea: biologic activity and therapeutic implications. Content: Epigallocatechin-3-gallate (EGCG), a polyphenol extracted from green tea, is an antioxidant with chemopreventive and chemotherapeutic actions. Based on its ability to modulate growth factor-mediated cell proliferation, we evaluated its efficacy in multiple myeloma (MM). EGCG induced both dose- and time-dependent growth arrest and subsequent apoptotic cell death in MM cell lines including IL-6-dependent cells and primary patient cells, without significant effect on the growth of peripheral blood mononuclear cells (PBMCs) and normal fibroblasts. Treatment with EGCG also led to significant apoptosis in human myeloma cells grown as tumors in SCID mice. EGCG interacts with the 67-kDa laminin receptor 1 (LR1), which is significantly elevated in myeloma cell lines and patient samples relative to normal PBMCs. RNAi-mediated inhibition of LR1 resulted in abrogation of EGCG-induced apoptosis in myeloma cells, indicating that LR1 plays an important role in mediating EGCG activity in MM while sparing PBMCs. Evaluation of changes in gene expression profile indicates that EGCG treatment activates distinct pathways of growth arrest and apoptosis in MM cells by inducing the expression of death-associated protein kinase 2, the initiators and mediators of death receptor-dependent apoptosis (Fas ligand, Fas, and caspase 4), p53-like proteins (p73, p63), positive regulators of apoptosis and NF-kappaB activation (CARD10, CARD14), and cyclin-dependent kinase inhibitors (p16 and p18). Expression of related genes at the protein level were also confirmed by Western blot analysis. These data demonstrate potent and specific antimyeloma activity of EGCG and provide the rationale for its clinical evaluation.

Title: Divergent reprogramming routes lead to alternative stem-cell states Content: Pluripotency is defined by the ability of a cell to differentiate to the derivatives of all the three embryonic germ layers: ectoderm, mesoderm and endoderm. Pluripotent cells can be captured via the archetypal derivation of embryonic stem cells or via somatic cell reprogramming. Somatic cells are induced to acquire a pluripotent stem cell (iPSC) state through the forced expression of key transcription factors, and in the mouse these cells can fulfil the strictest of all developmental assays for pluripotent cells by generating completely iPSC-derived embryos and mice. However, it is not known whether there are additional classes of pluripotent cells, or what the spectrum of reprogrammed phenotypes encompasses. Here we explore alternative outcomes of somatic reprogramming by fully characterizing reprogrammed cells independent of preconceived definitions of iPSC states. We demonstrate that by maintaining elevated reprogramming factor expression levels, mouse embryonic fibroblasts go through unique epigenetic modifications to arrive at a stable, Nanog-positive, alternative pluripotent state. In doing so, we prove that the pluripotent spectrum can encompass multiple, unique cell states.

Title: Reprogramming of human fibroblasts to pluripotency with lineage specifiers. Content: Since the initial discovery that OCT4, SOX2, KLF4, and c-MYC overexpression sufficed for the induction of pluripotency in somatic cells, methodologies replacing the original factors have enhanced our understanding of the reprogramming process. However, unlike in mouse, OCT4 has not been replaced successfully during reprogramming of human cells. Here we report on a strategy to accomplish this replacement. Through a combination of transcriptome and bioinformatic analysis we have identified factors previously characterized as being lineage specifiers that are able to replace OCT4 and SOX2 in the reprogramming of human fibroblasts. Our results show that it is possible to replace OCT4 and SOX2 simultaneously with alternative lineage specifiers in the reprogramming of human cells. At a broader level, they also support a model in which counteracting lineage specification networks underlies the induction of pluripotency.

Title: Direct reprogramming of fibroblasts into neural stem cells by defined factors. Content: Recent studies have shown that defined sets of transcription factors can directly reprogram differentiated somatic cells to a different differentiated cell type without passing through a pluripotent state, but the restricted proliferative and lineage potential of the resulting cells limits the scope of their potential applications. Here we show that a combination of transcription factors (Brn4/Pou3f4, Sox2, Klf4, c-Myc, plus E47/Tcf3) induces mouse fibroblasts to directly acquire a neural stem cell identity-which we term as induced neural stem cells (iNSCs). Direct reprogramming of fibroblasts into iNSCs is a gradual process in which the donor transcriptional program is silenced over time. iNSCs exhibit cell morphology, gene expression, epigenetic features, differentiation potential, and self-renewing capacity, as well as in vitro and in vivo functionality similar to those of wild-type NSCs. We conclude that differentiated cells can be reprogrammed directly into specific somatic stem cell types by defined sets of specific transcription factors.

Title: Interactions between SRY and SOX genes in mammalian sex determination. Content: The SRY gene on the mammalian Y chromosome undoubtedly acts to determine testis, but it is still quite unclear how. It was originally supposed that SRY acts directly to activate other genes in the testis-determining pathway. This paper presents an alternative hypothesis that SRY functions indirectly, by interacting with related genes SOX3 (from which SRY evolved) and SOX9 (which appears to be intimately involved in vertebrate gonad differentiation). Specifically, I propose that in females SOX3 inhibits SOX9 function, but in males, SRY inhibits SOX3 and permits SOX9 to enact its testis-determining role. This hypothesis makes testable predictions of the phenotypes of XX and XY individuals with deficiencies or overproduction of any of the three genes, and is able to account for the difficult cases of XX(SRY-) males and transdifferentiation in the absence of SRY. The hypothesis also suggests a way that the dominant SRY sex-determining system of present-day mammals may have evolved from an ancient system relying on SOX3 dosage.

Title: Reprogramming fibroblasts into bipotential hepatic stem cells by defined factors. Content: Recent studies have demonstrated direct reprogramming of fibroblasts into a range of somatic cell types, but to date stem or progenitor cells have only been reprogrammed for the blood and neuronal lineages. We previously reported generation of induced hepatocyte-like (iHep) cells by transduction of Gata4, Hnf1α, and Foxa3 in p19 Arf null mouse embryonic fibroblasts (MEFs). Here, we show that Hnf1β and Foxa3, liver organogenesis transcription factors, are sufficient to reprogram MEFs into induced hepatic stem cells (iHepSCs). iHepSCs can be stably expanded in vitro and possess the potential of bidirectional differentiation into both hepatocytic and cholangiocytic lineages. In the injured liver of fumarylacetoacetate hydrolase (Fah)-deficient mice, repopulating iHepSCs become hepatocyte-like cells. They also engraft as cholangiocytes into bile ducts of mice with DDC-induced bile ductular injury. Lineage conversion into bipotential expandable iHepSCs provides a strategy to enable efficient derivation of both hepatocytes and cholangiocytes for use in disease modeling and tissue engineering.

Title: Induction of human neuronal cells by defined transcription factors Content: Somatic cell nuclear transfer, cell fusion, or expression of lineage-specific factors have been shown to induce cell-fate changes in diverse somatic cell types. We recently observed that forced expression of a combination of three transcription factors, Brn2 (also known as Pou3f2), Ascl1 and Myt1l, can efficiently convert mouse fibroblasts into functional induced neuronal (iN) cells. Here we show that the same three factors can generate functional neurons from human pluripotent stem cells as early as 6 days after transgene activation. When combined with the basic helix-loop-helix transcription factor NeuroD1, these factors could also convert fetal and postnatal human fibroblasts into iN cells showing typical neuronal morphologies and expressing multiple neuronal markers, even after downregulation of the exogenous transcription factors. Importantly, the vast majority of human iN cells were able to generate action potentials and many matured to receive synaptic contacts when co-cultured with primary mouse cortical neurons. Our data demonstrate that non-neural human somatic cells, as well as pluripotent stem cells, can be converted directly into neurons by lineage-determining transcription factors. These methods may facilitate robust generation of patient-specific human neurons for in vitro disease modelling or future applications in regenerative medicine.

Title: Immunogenicity of induced pluripotent stem cells Content: Induced pluripotent stem cells (iPSCs), reprogrammed from somatic cells with defined factors, hold great promise for regenerative medicine as the renewable source of autologous cells. Whereas it has been generally assumed that these autologous cells should be immune-tolerated by the recipient from whom the iPSCs are derived, their immunogenicity has not been vigorously examined. We show here that, whereas embryonic stem cells (ESCs) derived from inbred C57BL/6 (B6) mice can efficiently form teratomas in B6 mice without any evident immune rejection, the allogeneic ESCs from 129/SvJ mice fail to form teratomas in B6 mice due to rapid rejection by recipients. B6 mouse embryonic fibroblasts (MEFs) were reprogrammed into iPSCs by either retroviral approach (ViPSCs) or a novel episomal approach (EiPSCs) that causes no genomic integration. In contrast to B6 ESCs, teratomas formed by B6 ViPSCs were mostly immune-rejected by B6 recipients. In addition, the majority of teratomas formed by B6 EiPSCs were immunogenic in B6 mice with T cell infiltration, and apparent tissue damage and regression were observed in a small fraction of teratomas. Global gene expression analysis of teratomas formed by B6 ESCs and EiPSCs revealed a number of genes frequently overexpressed in teratomas derived from EiPSCs, and several such gene products were shown to contribute directly to the immunogenicity of the B6 EiPSC-derived cells in B6 mice. These findings indicate that, in contrast to derivatives of ESCs, abnormal gene expression in some cells differentiated from iPSCs can induce T-cell-dependent immune response in syngeneic recipients. Therefore, the immunogenicity of therapeutically valuable cells derived from patient-specific iPSCs should be evaluated before any clinic application of these autologous cells into the patients.

Title: A novel immortalized human endometrial stromal cell line with normal progestational response. Content: Obtaining primary human endometrial stromal cells (HESCs) for in vitro studies is limited by the scarcity of adequate human material and the inability to passage these cells in culture for long periods. Immortalization of these cells would greatly facilitate studies; however, the process of immortalization often results in abnormal karyotypes and aberrant functional characteristics. To meet this need, we have introduced telomerase into cultured HESCs to prevent the normal shortening of telomeres observed in adult somatic cells during mitosis. We have now developed and analyzed a newly immortalized HESC line that contains no clonal chromosomal structural or numerical abnormalities. In addition, when compared with the primary unpassaged parent cells, the new cell line displayed similar biochemical endpoints after treatment with ovarian steroids. Classical decidualization response to estradiol plus medroxyprogesterone acetate were seen in both morphologically, and progestin was seen to induce or regulate the expression of IGF binding protein-1, fibronectin, prolactin, tissue factor, plasminogen activator inhibitor-1, and Fas/Fas ligand. In summary, an immortalized HESC line has been developed that is karyotypically, morphologically, and phenotypically similar to the primary parent cells, and it is a powerful and consistent resource for in vitro work.

Title: Direct reprogramming of mouse fibroblasts to neural progenitors. Content: The simple yet powerful technique of induced pluripotency may eventually supply a wide range of differentiated cells for cell therapy and drug development. However, making the appropriate cells via induced pluripotent stem cells (iPSCs) requires reprogramming of somatic cells and subsequent redifferentiation. Given how arduous and lengthy this process can be, we sought to determine whether it might be possible to convert somatic cells into lineage-specific stem/progenitor cells of another germ layer in one step, bypassing the intermediate pluripotent stage. Here we show that transient induction of the four reprogramming factors (Oct4, Sox2, Klf4, and c-Myc) can efficiently transdifferentiate fibroblasts into functional neural stem/progenitor cells (NPCs) with appropriate signaling inputs. Compared with induced neurons (or iN cells, which are directly converted from fibroblasts), transdifferentiated NPCs have the distinct advantage of being expandable in vitro and retaining the ability to give rise to multiple neuronal subtypes and glial cells. Our results provide a unique paradigm for iPSC-factor-based reprogramming by demonstrating that it can be readily modified to serve as a general platform for transdifferentiation.

Title: An efficient nonviral method to generate integration-free human-induced pluripotent stem cells from cord blood and peripheral blood cells. Content: The generation of induced pluripotent stem cells (iPSCs) provides the opportunity to use patient-specific somatic cells, which are a valuable source for disease modeling and drug discovery. To promote research involving these cells, it is important to make iPSCs from easily accessible and less invasive tissues, like blood. We have recently reported the efficient generation of human iPSCs from adult fibroblasts using a combination of plasmids encoding OCT3/4, SOX2, KLF4, L-MYC, LIN28, and shRNA for TP53. We herein report a modified protocol enabling efficient iPSC induction from CD34+ cord blood cells and from peripheral blood isolated from healthy donors using these plasmid vectors. The original plasmid mixture could induce iPSCs; however, the efficiency was low. The addition of EBNA1, an essential factor for episomal amplification of the vectors, by an extra plasmid greatly increased the efficiency of iPSC induction, especially when the induction was performed from αβT cells. This improvement enabled the establishment of blood-derived iPSCs from seven healthy donors ranging in age from their 20s to their 60s. This induction method will be useful for the derivation of patient-specific integration-free iPSCs and would also be applicable to the generation of clinical-grade iPSCs in the future.

Title: Directly reprogrammed fibroblasts show global epigenetic remodeling and widespread tissue contribution. Content: Ectopic expression of the four transcription factors Oct4, Sox2, c-Myc, and Klf4 is sufficient to confer a pluripotent state upon the fibroblast genome, generating induced pluripotent stem (iPS) cells. It remains unknown if nuclear reprogramming induced by these four factors globally resets epigenetic differences between differentiated and pluripotent cells. Here, using novel selection approaches, we have generated iPS cells from fibroblasts to characterize their epigenetic state. Female iPS cells showed reactivation of a somatically silenced X chromosome and underwent random X inactivation upon differentiation. Genome-wide analysis of two key histone modifications indicated that iPS cells are highly similar to ES cells. Consistent with these observations, iPS cells gave rise to viable high-degree chimeras with contribution to the germline. These data show that transcription factor-induced reprogramming leads to the global reversion of the somatic epigenome into an ES-like state. Our results provide a paradigm for studying the epigenetic modifications that accompany nuclear reprogramming and suggest that abnormal epigenetic reprogramming does not pose a problem for the potential therapeutic applications of iPS cells.

Title: BMP receptor IA is required in mammalian neural crest cells for development of the cardiac outflow tract and ventricular myocardium. Content: The neural crest is a multipotent, migratory cell population arising from the border of the neural and surface ectoderm. In mouse, the initial migratory neural crest cells occur at the five-somite stage. Bone morphogenetic proteins (BMPs), particularly BMP2 and BMP4, have been implicated as regulators of neural crest cell induction, maintenance, migration, differentiation and survival. Mouse has three known BMP2/4 type I receptors, of which Bmpr1a is expressed in the neural tube sufficiently early to be involved in neural crest development from the outset; however, earlier roles in other domains obscure its requirement in the neural crest. We have ablated Bmpr1a specifically in the neural crest, beginning at the five-somite stage. We find that most aspects of neural crest development occur normally; suggesting that BMPRIA is unnecessary for many aspects of early neural crest biology. However, mutant embryos display a shortened cardiac outflow tract with defective septation, a process known to require neural crest cells and to be essential for perinatal viability. Surprisingly, these embryos die in mid-gestation from acute heart failure, with reduced proliferation of ventricular myocardium. The myocardial defect may involve reduced BMP signaling in a novel, minor population of neural crest derivatives in the epicardium, a known source of ventricular myocardial proliferation signals. These results demonstrate that BMP2/4 signaling in mammalian neural crest derivatives is essential for outflow tract development and may regulate a crucial proliferation signal for the ventricular myocardium.

Title: Harnessing transposons for cancer gene discovery Content: Recently, it has become possible to mobilize the Tc1/mariner transposon, Sleeping Beauty (SB), in mouse somatic cells at frequencies high enough to induce cancer. Tumours result from SB insertional mutagenesis of cancer genes, thus facilitating the identification of the genes and signalling pathways that drive tumour formation. A conditional SB transposition system has also been developed that makes it possible to limit where SB mutagenesis occurs, providing a means to selectively model many types of human cancer. SB mutagenesis has already identified a large collection of known cancer genes in addition to a plethora of new candidate cancer genes and potential drug targets.

Title: Establishment of human iPSC-based models for the study and targeting of glioma initiating cells Content: Glioma tumour-initiating cells (GTICs) can originate upon the transformation of neural progenitor cells (NPCs). Studies on GTICs have focused on primary tumours from which GTICs could be isolated and the use of human embryonic material. Recently, the somatic genomic landscape of human gliomas has been reported. RTK (receptor tyrosine kinase) and p53 signalling were found dysregulated in ∼90% and 86% of all primary tumours analysed, respectively. Here we report on the use of human-induced pluripotent stem cells (hiPSCs) for modelling gliomagenesis. Dysregulation of RTK and p53 signalling in hiPSC-derived NPCs (iNPCs) recapitulates GTIC properties in vitro. In vivo transplantation of transformed iNPCs leads to highly aggressive tumours containing undifferentiated stem cells and their differentiated derivatives. Metabolic modulation compromises GTIC viability. Last, screening of 101 anti-cancer compounds identifies three molecules specifically targeting transformed iNPCs and primary GTICs. Together, our results highlight the potential of hiPSCs for studying human tumourigenesis.

Title: Stem Cells and Early Lineage Development Content: The recent derivation of pluripotent stem cell lines from a number of different sources, including reprogrammed adult somatic cells, raises the issue of the developmental equivalence of these different pluripotent states. At least two different states representing the epiblast progenitors in the blastocyst and the pluripotent progenitors of the later gastrulating embryo have been recognized. Understanding the initial developmental status of the different pluripotent lines is critical for defining starting conditions for differentiation toward therapeutically relevant cell types.

Title: Production of cloned mice and ES cells from adult somatic cells by nuclear transfer: how to improve cloning efficiency? Content: Although it has now been 10 years since the first cloned mammals were generated from somatic cells using nuclear transfer (NT), most cloned embryos usually undergo developmental arrest prior to or soon after implantation, and the success rate for producing live offspring by cloning remains below 5%. The low success rate is believed to be associated with epigenetic errors, including abnormal DNA hypermethylation, but the mechanism of "reprogramming" is unclear. We have been able to develop a stable NT method in the mouse in which donor nuclei are directly injected into the oocyte using a piezo-actuated micromanipulator. Especially in the mouse, only a few laboratories can make clones from adult somatic cells, and cloned mice are never successfully produced from most mouse strains. However, this technique promises to be an important tool for future research in basic biology. For example, NT can be used to generate embryonic stem (NT-ES) cell lines from a patient's own somatic cells. We have shown that NT-ES cells are equivalent to ES cells derived from fertilized embryos and that they can be generated relatively easily from a variety of mouse genotypes and cell types of both sexes, even though it may be more difficult to generate clones directly. In general, NT-ES cell techniques are expected to be applied to regenerative medicine; however, this technique can also be applied to the preservation of genetic resources of mouse strain instead of embryos, oocytes and spermatozoa. This review describes how to improve cloning efficiency and NT-ES cell establishment and further applications.

Title: IAP antagonists induce anti-tumor immunity in multiple myeloma Content: The cellular inhibitors of apoptosis (cIAP) 1 and 2 are amplified in about 3% of cancers and have been identified in multiple malignancies as being potential therapeutic targets as a result of their role in the evasion of apoptosis. Consequently, small-molecule IAP antagonists, such as LCL161, have entered clinical trials for their ability to induce tumor necrosis factor (TNF)-mediated apoptosis of cancer cells. However, cIAP1 and cIAP2 are recurrently homozygously deleted in multiple myeloma (MM), resulting in constitutive activation of the noncanonical nuclear factor (NF)-κB pathway. To our surprise, we observed robust in vivo anti-myeloma activity of LCL161 in a transgenic myeloma mouse model and in patients with relapsed-refractory MM, where the addition of cyclophosphamide resulted in a median progression-free-survival of 10 months. This effect was not a result of direct induction of tumor cell death, but rather of upregulation of tumor-cell-autonomous type I interferon (IFN) signaling and a strong inflammatory response that resulted in the activation of macrophages and dendritic cells, leading to phagocytosis of tumor cells. Treatment of a MM mouse model with LCL161 established long-term anti-tumor protection and induced regression in a fraction of the mice. Notably, combination of LCL161 with the immune-checkpoint inhibitor anti-PD1 was curative in all of the treated mice.

Title: A role for interleukin-1β in determining the lineage fate of embryonic rat hippocampal neural precursor cells. Content: Neurogenesis occurs in the hippocampus of the developing and adult brain due to the presence of multipotent stem cells and restricted precursor cells at different stages of differentiation. It has been proposed that they may be of potential benefit for use in cell transplantation approaches for neurodegenerative disorders and trauma. Prolonged release of interleukin-1β (IL-1β) from activated microglia has a deleterious effect on hippocampal neurons and is implicated in the impaired neurogenesis and cognitive dysfunction associated with aging, Alzheimer's disease and depression. This study assessed the effect of IL-1β on the proliferation and differentiation of embryonic rat hippocampal NPCs in vitro. We show that IL-1R1 is expressed on proliferating NPCs and that IL-1β treatment decreases cell proliferation and neurosphere growth. When NPCs were differentiated in the presence of IL-1β, a significant reduction in the percentages of newly-born neurons and post-mitotic neurons and a significant increase in the percentage of astrocytes was observed in these cultures. These effects were attenuated by IL-1 receptor antagonist. These data reveal that IL-1β exerts an anti-proliferative, anti-neurogenic and pro-gliogenic effect on embryonic hippocampal NPCs, which is mediated by IL-1R1. The present results emphasise the consequences of an inflammatory environment during NPC development, and indicate that strategies to inhibit IL-1β signalling may be necessary to facilitate effective cell transplantation approaches or in conditions where endogenous hippocampal neurogenesis is impaired.

Title: Human artificial chromosomes containing chromosome 17 alphoid DNA maintain an active centromere in murine cells but are not stable. Content: Human artificial chromosomes (HACs) are autonomous molecules that can function and segregate as normal chromosomes in human cells. De novo HACs have successfully been used as gene expression vectors to complement genetic deficiencies in human cultured cells. HACs now offer the possibility of studying the regulation and expression of large genes in a variety of cell types from different tissues and correcting gene deficiencies caused by human inherited diseases. Complementary gene expression studies in mice, especially in mouse models of human genetic diseases, are also important in determining if large human transgenes can be expressed appropriately from artificial chromosomes. Toward this aim we are establishing artificial chromosomes in murine cells as novel gene expression vectors. Initially we transferred HAC vectors into murine cells, but were unable to generate de novo HACs at a reasonable frequency. We then transferred HACs previously established in human HT1080 cells to three different murine cell types by microcell fusion, followed by positive selection. We observed that the HACs in murine cells bound centromere protein C (CENP-C), a marker of active centromeres, and were detected under selection but rapidly lost when selection was removed. These results suggest that the HACs maintain at least a partially functional centromere complex in murine cells, but other factors are required for stability and segregation. Artificial chromosomes containing mouse centromeric sequences may be required for better stability and maintenance in murine cells.

Title: Genotoxic Stress Abrogates Renewal of Melanocyte Stem Cells by Triggering Their Differentiation Content: Somatic stem cell depletion due to the accumulation of DNA damage has been implicated in the appearance of aging-related phenotypes. Hair graying, a typical sign of aging in mammals, is caused by the incomplete maintenance of melanocyte stem cells (MSCs) with age. Here, we report that irreparable DNA damage, as caused by ionizing radiation, abrogates renewal of MSCs in mice. Surprisingly, the DNA-damage response triggers MSC differentiation into mature melanocytes in the niche, rather than inducing their apoptosis or senescence. The resulting MSC depletion leads to irreversible hair graying. Furthermore, deficiency of Ataxia-telangiectasia mutated (ATM), a central transducer kinase of the DNA-damage response, sensitizes MSCs to ectopic differentiation, demonstrating that the kinase protects MSCs from their premature differentiation by functioning as a "stemness checkpoint" to maintain the stem cell quality and quantity.

Title: Mutant SOD1 inhibits ER-Golgi transport in amyotrophic lateral sclerosis. Content: Cu/Zn-superoxide dismutase is misfolded in familial and sporadic amyotrophic lateral sclerosis, but it is not clear how this triggers endoplasmic reticulum (ER) stress or other pathogenic processes. Here, we demonstrate that mutant SOD1 (mSOD1) is predominantly found in the cytoplasm in neuronal cells. Furthermore, we show that mSOD1 inhibits secretory protein transport from the ER to Golgi apparatus. ER-Golgi transport is linked to ER stress, Golgi fragmentation and axonal transport and we also show that inhibition of ER-Golgi trafficking preceded ER stress, Golgi fragmentation, protein aggregation and apoptosis in cells expressing mSOD1. Restoration of ER-Golgi transport by over-expression of coatomer coat protein II subunit Sar1 protected against inclusion formation and apoptosis, thus linking dysfunction in ER-Golgi transport to cellular pathology. These findings thus link several cellular events in amyotrophic lateral sclerosis into a single mechanism occurring early in mSOD1 expressing cells.

Title: Sex differences in sex chromosome gene expression in mouse brain. Content: A major question is whether genes encoded on the sex chromosomes act directly in non-gonadal tissues to cause sex differences in development or function, or whether all sex differences in somatic tissues are induced by gonadal secretions. As part of this question we asked whether mouse X-Y homologous gene pairs are expressed in brain in a sex-specific fashion. Using RT-PCR and northern blot analysis, we assessed mRNA expression in brain of eight Y-linked genes as well as their X-linked homologues, at three ages: 13.5 days post coitum, the day of birth (P1) and adult. Transcripts of six Y genes were expressed at one or more ages: Usp9y, Ube1y, Smcy, Eif2s3y, Uty and Dby. Their expression also occurred in XY female brain, and therefore does not require testicular secretions. Six X-linked homologues (Usp9x, Ube1x, Smcx, Eif2s3x, Utx and Dbx) were also expressed in brain, and in adulthood all of these transcripts were expressed at significantly higher levels in brains of females than in brains of males, irrespective of their X-inactivation status. For five of these gene pairs, the expression of the Y-linked homologue in males was not sufficient to compensate for the female bias in X gene expression. Three X-Y gene pairs, Usp9x/y, Ube1x/y and Eif2s3x/y, appeared to be differentially regulated (expressed in brain in a different age- or tissue-dependent pattern), and hence may not be functionally equivalent. These sex differences in X-Y gene expression suggest several mechanisms by which these genes may participate in sex differences in brain development and function.

Title: Cancer cell–autonomous contribution of type I interferon signaling to the efficacy of chemotherapy Content: Some of the anti-neoplastic effects of anthracyclines in mice originate from the induction of innate and T cell–mediated anticancer immune responses. Here we demonstrate that anthracyclines stimulate the rapid production of type I interferons (IFNs) by malignant cells after activation of the endosomal pattern recognition receptor Toll-like receptor 3 (TLR3). By binding to IFN-α and IFN-β receptors (IFNARs) on neoplastic cells, type I IFNs trigger autocrine and paracrine circuitries that result in the release of chemokine (C-X-C motif) ligand 10 (CXCL10). Tumors lacking Tlr3 or Ifnar failed to respond to chemotherapy unless type I IFN or Cxcl10, respectively, was artificially supplied. Moreover, a type I IFN–related signature predicted clinical responses to anthracycline-based chemotherapy in several independent cohorts of patients with breast carcinoma characterized by poor prognosis. Our data suggest that anthracycline-mediated immune responses mimic those induced by viral pathogens. We surmise that such 'viral mimicry' constitutes a hallmark of successful chemotherapy.

Title: 2D and 3D Stem Cell Models of Primate Cortical Development Identify Species-Specific Differences in Progenitor Behavior Contributing to Brain Size. Content: Variation in cerebral cortex size and complexity is thought to contribute to differences in cognitive ability between humans and other animals. Here we compare cortical progenitor cell output in humans and three nonhuman primates using directed differentiation of pluripotent stem cells (PSCs) in adherent two-dimensional (2D) and organoid three-dimensional (3D) culture systems. Clonal lineage analysis showed that primate cortical progenitors proliferate for a protracted period of time, during which they generate early-born neurons, in contrast to rodents, where this expansion phase largely ceases before neurogenesis begins. The extent of this additional cortical progenitor expansion differs among primates, leading to differences in the number of neurons generated by each progenitor cell. We found that this mechanism for controlling cortical size is regulated cell autonomously in culture, suggesting that primate cerebral cortex size is regulated at least in part at the level of individual cortical progenitor cell clonal output.

Title: Human iPSC-based modeling of late-onset disease via progerin-induced aging. Content: Reprogramming somatic cells to induced pluripotent stem cells (iPSCs) resets their identity back to an embryonic age and, thus, presents a significant hurdle for modeling late-onset disorders. In this study, we describe a strategy for inducing aging-related features in human iPSC-derived lineages and apply it to the modeling of Parkinson's disease (PD). Our approach involves expression of progerin, a truncated form of lamin A associated with premature aging. We found that expression of progerin in iPSC-derived fibroblasts and neurons induces multiple aging-related markers and characteristics, including dopamine-specific phenotypes such as neuromelanin accumulation. Induced aging in PD iPSC-derived dopamine neurons revealed disease phenotypes that require both aging and genetic susceptibility, such as pronounced dendrite degeneration, progressive loss of tyrosine hydroxylase (TH) expression, and enlarged mitochondria or Lewy-body-precursor inclusions. Thus, our study suggests that progerin-induced aging can be used to reveal late-onset age-related disease features in hiPSC-based disease models.

Title: Efficient Generation of Fully Reprogrammed Human iPS Cells via Polycistronic Retroviral Vector and a New Cocktail of Chemical Compounds Content: Direct reprogramming of human somatic cells into induced pluripotent stem (iPS) cells by defined transcription factors (TFs) provides great potential for regenerative medicine and biomedical research. This procedure has many challenges, including low reprogramming efficiency, many partially reprogrammed colonies, somatic coding mutations in the genome, etc. Here, we describe a simple approach for generating fully reprogrammed human iPS cells by using a single polycistronic retroviral vector expressing four human TFs in a single open reading frame (ORF), combined with a cocktail containing three small molecules (Sodium butyrate, SB431542, and PD0325901). Our results demonstrate that human iPS cells generated by this approach express human ES cells markers and exhibit pluripotency demonstrated by their abilities to differentiate into the three germ layers in vitro and in vivo. Notably, this approach not only provides a much faster reprogramming process but also significantly diminishes partially reprogrammed iPS cell colonies, thus facilitating efficient isolation of desired fully reprogrammed iPS cell colonies.

Title: The histone demethylases Jhdm1a/1b enhance somatic cell reprogramming in a vitamin-C-dependent manner. Content: Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) resets the epigenome to an embryonic-like state. Vitamin C enhances the reprogramming process, but the underlying mechanisms are unclear. Here we show that the histone demethylases Jhdm1a/1b are key effectors of somatic cell reprogramming downstream of vitamin C. We first observed that vitamin C induces H3K36me2/3 demethylation in mouse embryonic fibroblasts in culture and during reprogramming. We then identified Jhdm1a/1b, two known vitamin-C-dependent H3K36 demethylases, as potent regulators of reprogramming through gain- and loss-of-function approaches. Furthermore, we found that Jhdm1b accelerates cell cycle progression and suppresses cell senescence during reprogramming by repressing the Ink4/Arf locus. Jhdm1b also cooperates with Oct4 to activate the microRNA cluster 302/367, an integral component of the pluripotency machinery. Our results therefore reveal a role for H3K36me2/3 in cell fate determination and establish a link between histone demethylases and vitamin-C-induced reprogramming.

Title: Transcriptional Signature and Memory Retention of Human-Induced Pluripotent Stem Cells Content: Genetic reprogramming of somatic cells to a pluripotent state (induced pluripotent stem cells or iPSCs) by over-expression of specific genes has been accomplished using mouse and human cells. However, it is still unclear how similar human iPSCs are to human Embryonic Stem Cells (hESCs). Here, we describe the transcriptional profile of human iPSCs generated without viral vectors or genomic insertions, revealing that these cells are in general similar to hESCs but with significant differences. For the generation of human iPSCs without viral vectors or genomic insertions, pluripotent factors Oct4 and Nanog were cloned in episomal vectors and transfected into human fetal neural progenitor cells. The transient expression of these two factors, or from Oct4 alone, resulted in efficient generation of human iPSCs. The reprogramming strategy described here revealed a potential transcriptional signature for human iPSCs yet retaining the gene expression of donor cells in human reprogrammed cells free of viral and transgene interference. Moreover, the episomal reprogramming strategy represents a safe way to generate human iPSCs for clinical purposes and basic research.

Title: Sequential histone-modifying activities determine the robustness of transdifferentiation Content: Natural interconversions between distinct somatic cell types have been reported in species as diverse as jellyfish and mice. The efficiency and reproducibility of some reprogramming events represent unexploited avenues in which to probe mechanisms that ensure robust cell conversion. We report that a conserved H3K27me3/me2 demethylase, JMJD-3.1, and the H3K4 methyltransferase Set1 complex cooperate to ensure invariant transdifferentiation (Td) of postmitotic Caenorhabditis elegans hindgut cells into motor neurons. At single-cell resolution, robust conversion requires stepwise histone-modifying activities, functionally partitioned into discrete phases of Td through nuclear degradation of JMJD-3.1 and phase-specific interactions with transcription factors that have conserved roles in cell plasticity and terminal fate selection. Our results draw parallels between epigenetic mechanisms underlying robust Td in nature and efficient cell reprogramming in vitro.

Title: PRC2 complexes with JARID2, MTF2, and esPRC2p48 in ES cells to modulate ES cell pluripotency and somatic cell reprogramming. Content: Polycomb repressive complex two (PRC2) has been implicated in embryonic stem (ES) cell pluripotency; however, the mechanistic roles of this complex are unclear. It was assumed that ES cells contain PRC2 with the same subunit composition as that identified in HeLa cells and Drosophila embryos. Here, we report that PRC2 in mouse ES cells contains at least three additional subunits: JARID2, MTF2, and a novel protein denoted esPRC2p48. JARID2, MTF2, and esPRC2p48 are highly expressed in mouse ES cells compared to differentiated cells. Importantly, knockdowns of JARID2, MTF2, or esPRC2p48 alter the level of PRC2-mediated H3K27 methylation and result in the expression of differentiation-associated genes in ES cells. Interestingly, expression of JARID2, MTF2, and esPRC2p48 together, but not individually, enhances Oct4/Sox2/Klf4-mediated reprogramming of mouse embryonic fibroblasts (MEFs) into induced pluripotent stem cells, whereas knockdown or knockout of JARID2, MTF2, or esPRC2p48 significantly inhibits reprogramming. JARID2, MTF2, and esPRC2p48 modulate H3K27 methylation and facilitate repression of lineage-associated gene expression when transduced into MEFs, and synergistically stimulate the histone methyltransferase activity of PRC2 in vitro. Therefore, these studies identify JARID2, MTF2, and esPRC2p48 as important regulatory subunits of PRC2 in ES cells and reveal critical functions of these subunits in modulating PRC2's activity and gene expression both in ES cells and during somatic cell reprogramming.

Title: Colorectal Cancers from Distinct Ancestral Populations Show Variations in BRAF Mutation Frequency Content: It has been demonstrated for some cancers that the frequency of somatic oncogenic mutations may vary in ancestral populations. To determine whether key driver alterations might occur at different frequencies in colorectal cancer, we applied a high-throughput genotyping platform (OncoMap) to query 385 mutations across 33 known cancer genes in colorectal cancer DNA from 83 Asian, 149 Black and 195 White patients. We found that Asian patients had fewer canonical oncogenic mutations in the genes tested (60% vs Black 79% (P = 0.011) and White 77% (P = 0.015)), and that BRAF mutations occurred at a higher frequency in White patients (17% vs Asian 4% (P = 0.004) and Black 7% (P = 0.014)). These results suggest that the use of genomic approaches to elucidate the different ancestral determinants harbored by patient populations may help to more precisely and effectively treat colorectal cancer.

Title: Invariant natural killer T cells: bridging innate and adaptive immunity Content: Cells of the innate immune system interact with pathogens via conserved pattern-recognition receptors, whereas cells of the adaptive immune system recognize pathogens through diverse, antigen-specific receptors that are generated by somatic DNA rearrangement. Invariant natural killer T (iNKT) cells are a subset of lymphocytes that bridge the innate and adaptive immune systems. Although iNKT cells express T cell receptors that are generated by somatic DNA rearrangement, these receptors are semi-invariant and interact with a limited set of lipid and glycolipid antigens, thus resembling the pattern-recognition receptors of the innate immune system. Functionally, iNKT cells most closely resemble cells of the innate immune system, as they rapidly elicit their effector functions following activation, and fail to develop immunological memory. iNKT cells can become activated in response to a variety of stimuli and participate in the regulation of various immune responses. Activated iNKT cells produce several cytokines with the capacity to jump-start and modulate an adaptive immune response. A variety of glycolipid antigens that can differentially elicit distinct effector functions in iNKT cells have been identified. These reagents have been employed to test the hypothesis that iNKT cells can be harnessed for therapeutic purposes in human diseases. Here, we review the innate-like properties and functions of iNKT cells and discuss their interactions with other cell types of the immune system.

Title: Optogenetics reveal delayed afferent synaptogenesis on grafted human-induced pluripotent stem cell-derived neural progenitors. Content: Reprogramming of somatic cells into pluripotency stem cell state has opened new opportunities in cell replacement therapy and disease modeling in a number of neurological disorders. It still remains unknown, however, to what degree the grafted human-induced pluripotent stem cells (hiPSCs) differentiate into a functional neuronal phenotype and if they integrate into the host circuitry. Here, we present a detailed characterization of the functional properties and synaptic integration of hiPSC-derived neurons grafted in an in vitro model of hyperexcitable epileptic tissue, namely organotypic hippocampal slice cultures (OHSCs), and in adult rats in vivo. The hiPSCs were first differentiated into long-term self-renewing neuroepithelial stem (lt-NES) cells, which are known to form primarily GABAergic neurons. When differentiated in OHSCs for 6 weeks, lt-NES cell-derived neurons displayed neuronal properties such as tetrodotoxin-sensitive sodium currents and action potentials (APs), as well as both spontaneous and evoked postsynaptic currents, indicating functional afferent synaptic inputs. The grafted cells had a distinct electrophysiological profile compared to host cells in the OHSCs with higher input resistance, lower resting membrane potential, and APs with lower amplitude and longer duration. To investigate the origin of synaptic afferents to the grafted lt-NES cell-derived neurons, the host neurons were transduced with Channelrhodopsin-2 (ChR2) and optogenetically activated by blue light. Simultaneous recordings of synaptic currents in grafted lt-NES cell-derived neurons using whole-cell patch-clamp technique at 6 weeks after grafting revealed limited synaptic connections from host neurons. Longer differentiation times, up to 24 weeks after grafting in vivo, revealed more mature intrinsic properties and extensive synaptic afferents from host neurons to the lt-NES cell-derived neurons, suggesting that these cells require extended time for differentiation/maturation and synaptogenesis. However, even at this later time point, the grafted cells maintained a higher input resistance. These data indicate that grafted lt-NES cell-derived neurons receive ample afferent input from the host brain. Since the lt-NES cells used in this study show a strong propensity for GABAergic differentiation, the host-to-graft synaptic afferents may facilitate inhibitory neurotransmitter release, and normalize hyperexcitable neuronal networks in brain diseases, for example, such as epilepsy.

Title: Forebrain engraftment by human glial progenitor cells enhances synaptic plasticity and learning in adult mice. Content: Human astrocytes are larger and more complex than those of infraprimate mammals, suggesting that their role in neural processing has expanded with evolution. To assess the cell-autonomous and species-selective properties of human glia, we engrafted human glial progenitor cells (GPCs) into neonatal immunodeficient mice. Upon maturation, the recipient brains exhibited large numbers and high proportions of both human glial progenitors and astrocytes. The engrafted human glia were gap-junction-coupled to host astroglia, yet retained the size and pleomorphism of hominid astroglia, and propagated Ca2+ signals 3-fold faster than their hosts. Long-term potentiation (LTP) was sharply enhanced in the human glial chimeric mice, as was their learning, as assessed by Barnes maze navigation, object-location memory, and both contextual and tone fear conditioning. Mice allografted with murine GPCs showed no enhancement of either LTP or learning. These findings indicate that human glia differentially enhance both activity-dependent plasticity and learning in mice.

Title: Epigenetic reprogramming and induced pluripotency. Content: The cloning of animals from adult cells has demonstrated that the developmental state of adult cells can be reprogrammed into that of embryonic cells by uncharacterized factors within the oocyte. More recently, transcription factors have been identified that can induce pluripotency in somatic cells without the use of oocytes, generating induced pluripotent stem (iPS) cells. iPS cells provide a unique platform to dissect the molecular mechanisms that underlie epigenetic reprogramming. Moreover, iPS cells can teach us about principles of normal development and disease, and might ultimately facilitate the treatment of patients by custom-tailored cell therapy.

Title: Reprogramming factor stoichiometry influences the epigenetic state and biological properties of induced pluripotent stem cells. Content: We compared two genetically highly defined transgenic systems to identify parameters affecting reprogramming of somatic cells to a pluripotent state. Our results demonstrate that the level and stoichiometry of reprogramming factors during the reprogramming process strongly influence the resulting pluripotency of iPS cells. High expression of Oct4 and Klf4 combined with lower expression of c-Myc and Sox2 produced iPS cells that efficiently generated "all-iPSC mice" by tetraploid (4n) complementation, maintained normal imprinting at the Dlk1-Dio3 locus, and did not create mice with tumors. Loss of imprinting (LOI) at the Dlk1-Dio3 locus did not strictly correlate with reduced pluripotency though the efficiency of generating "all-iPSC mice" was diminished. Our data indicate that stoichiometry of reprogramming factors can influence epigenetic and biological properties of iPS cells. This concept complicates efforts to define a "generic" epigenetic state of iPSCs and ESCs and should be considered when comparing different iPS and ES cell lines.

Title: Proteomic and genomic approaches reveal critical functions of H3K9 methylation and Heterochromatin Protein-1γ in reprogramming to pluripotency Content: Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) involves a marked reorganization of chromatin. To identify post-translational histone modifications that change in global abundance during this process, we have applied a quantitative mass-spectrometry-based approach. We found that iPSCs, compared with both the starting fibroblasts and a late reprogramming intermediate (pre-iPSCs), are enriched for histone modifications associated with active chromatin, and depleted for marks of transcriptional elongation and a subset of repressive modifications including H3K9me2/me3. Dissecting the contribution of H3K9 methylation to reprogramming, we show that the H3K9 methyltransferases Ehmt1, Ehmt2 and Setdb1 regulate global H3K9me2/me3 levels and that their depletion increases iPSC formation from both fibroblasts and pre-iPSCs. Similarly, we find that inhibition of heterochromatin protein-1γ (Cbx3), a protein known to recognize H3K9 methylation, enhances reprogramming. Genome-wide location analysis revealed that Cbx3 predominantly binds active genes in both pre-iPSCs and pluripotent cells but with a strikingly different distribution: in pre-iPSCs, but not in embryonic stem cells, Cbx3 associates with active transcriptional start sites, suggesting a developmentally regulated role for Cbx3 in transcriptional activation. Despite largely non-overlapping functions and the predominant association of Cbx3 with active transcription, the H3K9 methyltransferases and Cbx3 both inhibit reprogramming by repressing the pluripotency factor Nanog. Together, our findings demonstrate that Cbx3 and H3K9 methylation restrict late reprogramming events, and suggest that a marked change in global chromatin character constitutes an epigenetic roadblock for reprogramming.

Title: Human IRGM Regulates Autophagy and Its Cell-Autonomous Immunity Functions Through Mitochondria Content: IRGM, a human immunity-related GTPase, confers autophagic defence against intracellular pathogens by an unknown mechanism. Here, we report an unexpected mode of IRGM action. IRGM demonstrated differential affinity for the mitochondrial lipid cardiolipin, translocated to mitochondria, affected mitochondrial fission and induced autophagy. Mitochondrial fission was necessary for autophagic control of intracellular mycobacteria by IRGM. IRGM influenced mitochondrial membrane polarization and cell death. Overexpression of IRGMd, but not IRGMb splice isoforms, caused mitochondrial depolarization and autophagy-independent, but Bax/Bak-dependent, cell death. By acting on mitochondria, IRGM confers autophagic protection or cell death, explaining IRGM action both in defence against tuberculosis and in the damaging inflammation caused by Crohn's disease.

Title: Chronic pancreatitis is essential for induction of pancreatic ductal adenocarcinoma by K-Ras oncogenes in adult mice. Content: Pancreatic ductal adenocarcinoma (PDA), one of the deadliest human cancers, often involves somatic activation of K-Ras oncogenes. We report that selective expression of an endogenous K-Ras(G12V) oncogene in embryonic cells of acinar/centroacinar lineage results in pancreatic intraepithelial neoplasias (PanINs) and invasive PDA, suggesting that PDA originates by differentiation of acinar/centroacinar cells or their precursors into ductal-like cells. Surprisingly, adult mice become refractory to K-Ras(G12V)-induced PanINs and PDA. However, if these mice are challenged with a mild form of chronic pancreatitis, they develop the full spectrum of PanINs and invasive PDA. These observations suggest that, during adulthood, PDA stems from a combination of genetic (e.g., somatic K-Ras mutations) and nongenetic (e.g., tissue damage) events.

Title: Tet and TDG mediate DNA demethylation essential for mesenchymal-to-epithelial transition in somatic cell reprogramming. Content: Tet-mediated DNA oxidation is a recently identified mammalian epigenetic modification, and its functional role in cell-fate transitions remains poorly understood. Here, we derive mouse embryonic fibroblasts (MEFs) deleted in all three Tet genes and examine their capacity for reprogramming into induced pluripotent stem cells (iPSCs). We show that Tet-deficient MEFs cannot be reprogrammed because of a block in the mesenchymal-to-epithelial transition (MET) step. Reprogramming of MEFs deficient in TDG is similarly impaired. The block in reprogramming is caused at least in part by defective activation of key miRNAs, which depends on oxidative demethylation promoted by Tet and TDG. Reintroduction of either the affected miRNAs or catalytically active Tet and TDG restores reprogramming in the knockout MEFs. Thus, oxidative demethylation to promote gene activation appears to be functionally required for reprogramming of fibroblasts to pluripotency. These findings provide mechanistic insight into the role of epigenetic barriers in cell-lineage conversion.

Title: Direct reprogramming of mouse fibroblasts to cardiomyocyte-like cells using Yamanaka factors on engineered poly(ethylene glycol) (PEG) hydrogels. Content: Direct reprogramming strategies enable rapid conversion of somatic cells to cardiomyocytes or cardiomyocyte-like cells without going through the pluripotent state. A recently described protocol couples Yamanaka factor induction with pluripotency inhibition followed by BMP4 treatment to achieve rapid reprogramming of mouse fibroblasts to beating cardiomyocyte-like cells. The original study was performed using Matrigel-coated tissue culture polystyrene (TCPS), a stiff material that also non-specifically adsorbs serum proteins. Protein adsorption-resistant poly(ethylene glycol) (PEG) materials can be covalently modified to present precise concentrations of adhesion proteins or peptides without the unintended effects of non-specifically adsorbed proteins. Here, we describe an improved protocol that incorporates custom-engineered materials. We first reproduced the Efe et al. protocol on Matrigel-coated TCPS (the original material), reprogramming adult mouse tail-tip mouse fibroblasts (TTF) and mouse embryonic fibroblasts (MEF) to cardiomyocyte-like cells that demonstrated striated sarcomeric α-actinin staining, spontaneous calcium transients, and visible beating. We then designed poly(ethylene glycol) culture substrates to promote MEF adhesion via laminin and RGD-binding integrins. PEG hydrogels improved proliferation and reprogramming efficiency (evidenced by beating patch number and area, gene expression, and flow cytometry), yielding almost twice the number of sarcomeric α-actinin positive cardiomyocyte-like cells as the originally described substrate. These results illustrate that cellular reprogramming may be enhanced using custom-engineered materials.

Title: Opposing Effects of PKCθ and WASp on Symmetry Breaking and Relocation of the Immunological Synapse Content: The immunological synapse (IS) is a junction between the T cell and antigen-presenting cell and is composed of supramolecular activation clusters (SMACs). No studies have been published on naive T cell IS dynamics. Here, we find that IS formation during antigen recognition comprises cycles of stable IS formation and autonomous naive T cell migration. The migration phase is driven by PKCtheta, which is localized to the F-actin-dependent peripheral (p)SMAC. PKCtheta(-/-) T cells formed hyperstable IS in vitro and in vivo and, like WT cells, displayed fast oscillations in the distal SMAC, but they showed reduced slow oscillations in pSMAC integrity. IS reformation is driven by the Wiscott Aldrich Syndrome protein (WASp). WASp(-/-) T cells displayed normal IS formation but were unable to reform IS after migration unless PKCtheta was inhibited. Thus, opposing effects of PKCtheta and WASp control IS stability through pSMAC symmetry breaking and reformation.

Title: Generation of induced pluripotent stem cells from Asian patients with chronic neurodegenerative diseases. Content: Induced pluripotent stem (iPS) cells derived from disease patients are an invaluable resource for biomedical research and may provide a source for replacement therapies. In this study, we have generated iPS cells from Asian patients with chronic degenerative diseases of the nervous system, including spinal muscular atrophy (SMA), Parkinson disease (PD) and amyotrophic lateral sclerosis (ALS) by transduction with four factors (KLF4, SOX2, OCT4 and c-MYC). All of the iPS cells showed pluripotency similar to that of human embryonic stem cells (hESCs) and were able to differentiate into various somatic cell types in vitro and in vivo. Furthermore, the iPS cells also can be committed to differentiate into neural cells, the cell type that is affected in chronic degenerative diseases. Therefore, the patient-specific iPS cells we generated offer a cellular model in which to investigate disease mechanisms, discover and test novel drugs and develop new therapies for chronic neurodegenerative diseases.

Title: Prox1 Is Required for Granule Cell Maturation and Intermediate Progenitor Maintenance During Brain Neurogenesis Content: The dentate gyrus has an important role in learning and memory, and adult neurogenesis in the subgranular zone of the dentate gyrus may play a role in the acquisition of new memories. The homeobox gene Prox1 is expressed in the dentate gyrus during embryonic development and adult neurogenesis. Here we show that Prox1 is necessary for the maturation of granule cells in the dentate gyrus during development and for the maintenance of intermediate progenitors during adult neurogenesis. We also demonstrate that Prox1-expressing intermediate progenitors are required for adult neural stem cell self-maintenance in the subgranular zone; thus, we have identified a previously unknown non-cell autonomous regulatory feedback mechanism that controls adult neurogenesis in this region of the mammalian brain. Finally, we show that the ectopic expression of Prox1 induces premature differentiation of neural stem cells.

Title: A core Klf circuitry regulates self-renewal of embryonic stem cells Content: Embryonic stem (ES) cells are unique in their ability to self-renew indefinitely and maintain pluripotency. These properties require transcription factors that specify the gene expression programme of ES cells. It has been possible to reverse the highly differentiated state of somatic cells back to a pluripotent state with a combination of four transcription factors: Klf4 is one of the reprogramming factors required, in conjunction with Oct4, Sox2 and c-Myc. Maintenance of self-renewal and pluripotency of ES cells requires Oct4, Sox2 and c-Myc, but Klf4 is dispensable. Here, we show that Krüppel-like factors are required for the self-renewal of ES cells. Simultaneous depletion of Klf2, Klf4 and Klf5 lead to ES cell differentiation. Chromatin immunoprecipitation coupled to microarray assay reveals that these Klf proteins share many common targets of Nanog, suggesting a close functional relationship between these factors. Expression analysis after triple RNA interference (RNAi) of the Klfs shows that they regulate key pluripotency genes, such as Nanog. Taken together, our study provides new insight into how the core Klf circuitry integrates into the Nanog transcriptional network to specify gene expression that is unique to ES cells.

Title: Tissue-specific expression and regulation of sexually dimorphic genes in mice. Content: We report a comprehensive analysis of gene expression differences between sexes in multiple somatic tissues of 334 mice derived from an intercross between inbred mouse strains C57BL/6J and C3H/HeJ. The analysis of a large number of individuals provided the power to detect relatively small differences in expression between sexes, and the use of an intercross allowed analysis of the genetic control of sexually dimorphic gene expression. Microarray analysis of 23,574 transcripts revealed that the extent of sexual dimorphism in gene expression was much greater than previously recognized. Thus, thousands of genes showed sexual dimorphism in liver, adipose, and muscle, and hundreds of genes were sexually dimorphic in brain. These genes exhibited highly tissue-specific patterns of expression and were enriched for distinct pathways represented in the Gene Ontology database. They also showed evidence of chromosomal enrichment, not only on the sex chromosomes, but also on several autosomes. Genetic analyses provided evidence of the global regulation of subsets of the sexually dimorphic genes, as the transcript levels of a large number of these genes were controlled by several expression quantitative trait loci (eQTL) hotspots that exhibited tissue-specific control. Moreover, many tissue-specific transcription factor binding sites were found to be enriched in the sexually dimorphic genes.

Title: Direct conversion of fibroblasts to functional neurons by defined factors Content: Cellular differentiation and lineage commitment are considered to be robust and irreversible processes during development. Recent work has shown that mouse and human fibroblasts can be reprogrammed to a pluripotent state with a combination of four transcription factors. This raised the question of whether transcription factors could directly induce other defined somatic cell fates, and not only an undifferentiated state. We hypothesized that combinatorial expression of neural-lineage-specific transcription factors could directly convert fibroblasts into neurons. Starting from a pool of nineteen candidate genes, we identified a combination of only three factors, Ascl1, Brn2 (also called Pou3f2) and Myt1l, that suffice to rapidly and efficiently convert mouse embryonic and postnatal fibroblasts into functional neurons in vitro. These induced neuronal (iN) cells express multiple neuron-specific proteins, generate action potentials and form functional synapses. Generation of iN cells from non-neural lineages could have important implications for studies of neural development, neurological disease modelling and regenerative medicine.

Title: Highly efficient miRNA-mediated reprogramming of mouse and human somatic cells to pluripotency. Content: Transcription factor-based cellular reprogramming has opened the way to converting somatic cells to a pluripotent state, but has faced limitations resulting from the requirement for transcription factors and the relative inefficiency of the process. We show here that expression of the miR302/367 cluster rapidly and efficiently reprograms mouse and human somatic cells to an iPSC state without a requirement for exogenous transcription factors. This miRNA-based reprogramming approach is two orders of magnitude more efficient than standard Oct4/Sox2/Klf4/Myc-mediated methods. Mouse and human miR302/367 iPSCs display similar characteristics to Oct4/Sox2/Klf4/Myc-iPSCs, including pluripotency marker expression, teratoma formation, and, for mouse cells, chimera contribution and germline contribution. We found that miR367 expression is required for miR302/367-mediated reprogramming and activates Oct4 gene expression, and that suppression of Hdac2 is also required. Thus, our data show that miRNA and Hdac-mediated pathways can cooperate in a powerful way to reprogram somatic cells to pluripotency.

Title: Lentiviral vector design and imaging approaches to visualize the early stages of cellular reprogramming. Content: Induced pluripotent stem cells (iPSCs) can be derived from somatic cells by gene transfer of reprogramming transcription factors. Expression levels of these factors strongly influence the overall efficacy to form iPSC colonies, but additional contribution of stochastic cell-intrinsic factors has been proposed. Here, we present engineered color-coded lentiviral vectors in which codon-optimized reprogramming factors are co-expressed by a strong retroviral promoter that is rapidly silenced in iPSC, and imaged the conversion of fibroblasts to iPSC. We combined fluorescence microscopy with long-term single cell tracking, and used live-cell imaging to analyze the emergence and composition of early iPSC clusters. Applying our engineered lentiviral vectors, we demonstrate that vector silencing typically occurs prior to or simultaneously with the induction of an Oct4-EGFP pluripotency marker. Around 7 days post-transduction (pt), a subfraction of cells in clonal colonies expressed Oct4-EGFP and rapidly expanded. Cell tracking of single cell-derived iPSC colonies supported the concept that stochastic epigenetic changes are necessary for reprogramming. We also found that iPSC colonies may emerge as a genetic mosaic originating from different clusters. Improved vector design with continuous cell tracking thus creates a powerful system to explore the subtle dynamics of biological processes such as early reprogramming events.

Title: B-cell self-tolerance in humans. Content: Two mechanisms account for generation of the human antibody repertoire; V(D)J recombination during the early stages of B-cell development in the bone marrow and somatic mutation of immunoglobulin genes in mature B cells responding to antigen in the periphery. V(D)J recombination produces diversity by random joining of gene segments and somatic mutation by introducing random point mutations. Both are required to attain the degree of antigen receptor diversification that is necessary for immune protection: defects in either mechanism are associated with increased susceptibility to infection. However, the downside of producing enormous random diversity in the antibody repertoire is the generation of autoantibodies. To prevent autoimmunity B cells expressing autoantibodies are regulated by strict mechanisms that either modify the specificity of autoantibodies or the fate of cells expressing such antibodies. Abnormalities in B-cell self-tolerance are associated with a large number of autoimmune diseases, but the precise nature of the defects is less well defined. Here we summarize recent data on the self-reactive B-cell repertoire in healthy humans and in patients with autoimmunity.

Title: Normal and leukemic stem cell niches: insights and therapeutic opportunities. Content: Hematopoietic stem cells (HSCs) rely on instructive cues from the bone marrow (BM) niche to maintain their quiescence and adapt blood production to the organism's needs. Alterations in the BM niche are commonly observed in blood malignancies and directly contribute to the aberrant function of disease-initiating leukemic stem cells (LSCs). Here, we review recent insights into the cellular and molecular determinants of the normal HSC niche and describe how genetic changes in stromal cells and leukemia-induced BM niche remodeling contribute to blood malignancies. Moreover, we discuss how these findings can be applied to non-cell-autonomous therapies targeting the LSC niche.

Title: Metabolic rescue in pluripotent cells from patients with mtDNA disease Content: Mitochondria have a major role in energy production via oxidative phosphorylation, which is dependent on the expression of critical genes encoded by mitochondrial (mt)DNA. Mutations in mtDNA can cause fatal or severely debilitating disorders with limited treatment options. Clinical manifestations vary based on mutation type and heteroplasmy (that is, the relative levels of mutant and wild-type mtDNA within each cell). Here we generated genetically corrected pluripotent stem cells (PSCs) from patients with mtDNA disease. Multiple induced pluripotent stem (iPS) cell lines were derived from patients with common heteroplasmic mutations including 3243A>G, causing mitochondrial encephalomyopathy and stroke-like episodes (MELAS), and 8993T>G and 13513G>A, implicated in Leigh syndrome. Isogenic MELAS and Leigh syndrome iPS cell lines were generated containing exclusively wild-type or mutant mtDNA through spontaneous segregation of heteroplasmic mtDNA in proliferating fibroblasts. Furthermore, somatic cell nuclear transfer (SCNT) enabled replacement of mutant mtDNA from homoplasmic 8993T>G fibroblasts to generate corrected Leigh-NT1 PSCs. Although Leigh-NT1 PSCs contained donor oocyte wild-type mtDNA (human haplotype D4a) that differed from Leigh syndrome patient haplotype (F1a) at a total of 47 nucleotide sites, Leigh-NT1 cells displayed transcriptomic profiles similar to those in embryo-derived PSCs carrying wild-type mtDNA, indicative of normal nuclear-to-mitochondrial interactions. Moreover, genetically rescued patient PSCs displayed normal metabolic function compared to impaired oxygen consumption and ATP production observed in mutant cells. We conclude that both reprogramming approaches offer complementary strategies for derivation of PSCs containing exclusively wild-type mtDNA, through spontaneous segregation of heteroplasmic mtDNA in individual iPS cell lines or mitochondrial replacement by SCNT in homoplasmic mtDNA-based disease.

Title: Chromatin modifying enzymes as modulators of reprogramming Content: Generation of induced pluripotent stem cells (iPSCs) by somatic cell reprogramming involves global epigenetic remodelling. Whereas several proteins are known to regulate chromatin marks associated with the distinct epigenetic states of cells before and after reprogramming, the role of specific chromatin-modifying enzymes in reprogramming remains to be determined. To address how chromatin-modifying proteins influence reprogramming, we used short hairpin RNAs (shRNAs) to target genes in DNA and histone methylation pathways, and identified positive and negative modulators of iPSC generation. Whereas inhibition of the core components of the polycomb repressive complex 1 and 2, including the histone 3 lysine 27 methyltransferase EZH2, reduced reprogramming efficiency, suppression of SUV39H1, YY1 and DOT1L enhanced reprogramming. Specifically, inhibition of the H3K79 histone methyltransferase DOT1L by shRNA or a small molecule accelerated reprogramming, significantly increased the yield of iPSC colonies, and substituted for KLF4 and c-Myc (also known as MYC). Inhibition of DOT1L early in the reprogramming process is associated with a marked increase in two alternative factors, NANOG and LIN28, which play essential functional roles in the enhancement of reprogramming. Genome-wide analysis of H3K79me2 distribution revealed that fibroblast-specific genes associated with the epithelial to mesenchymal transition lose H3K79me2 in the initial phases of reprogramming. DOT1L inhibition facilitates the loss of this mark from genes that are fated to be repressed in the pluripotent state. These findings implicate specific chromatin-modifying enzymes as barriers to or facilitators of reprogramming, and demonstrate how modulation of chromatin-modifying enzymes can be exploited to more efficiently generate iPSCs with fewer exogenous transcription factors.

Title: G1 arrest and differentiation can occur independently of Rb family function Content: The ability of progenitor cells to exit the cell cycle is essential for proper embryonic development and homeostasis, but the mechanisms governing cell cycle exit are still not fully understood. Here, we tested the requirement for the retinoblastoma (Rb) protein and its family members p107 and p130 in G0/G1 arrest and differentiation in mammalian cells. We found that Rb family triple knockout (TKO) mouse embryos survive until days 9-11 of gestation. Strikingly, some TKO cells, including in epithelial and neural lineages, are able to exit the cell cycle in G0/G1 and differentiate in teratomas and in culture. This ability of TKO cells to arrest in G0/G1 is associated with the repression of key E2F target genes. Thus, G1 arrest is not always dependent on Rb family members, which illustrates the robustness of cell cycle regulatory networks during differentiation and allows for the identification of candidate pathways to inhibit the expansion of cancer cells with mutations in the Rb pathway.

Title: Mechanosensitivity in the enteric nervous system Content: The enteric nervous system (ENS) autonomously controls gut muscle activity. Mechanosensitive enteric neurons (MEN) initiate reflex activity by responding to mechanical deformation of the gastrointestinal wall. MEN throughout the gut primarily respond to compression or stretch rather than to shear force. Some MEN are multimodal as they respond to compression and stretch. Depending on the region up to 60% of the entire ENS population responds to mechanical stress. MEN fire action potentials after mechanical stimulation of processes or soma although they are more sensitive to process deformation. There are at least two populations of MEN based on their sensitivity to different modalities of mechanical stress and on their firing pattern. (1) Rapidly, slowly and ultra-slowly adapting neurons which encode compressive forces. (2) Ultra-slowly adapting stretch-sensitive neurons encoding tensile forces. Rapid adaptation of firing is typically observed after compressive force while slow adaptation or ongoing spike discharge occurs often during tensile stress (stretch). All MEN have some common properties: they receive synaptic input, are low fidelity mechanoreceptors and are multifunctional in that some serve interneuronal others even motor functions. Consequently, MEN possess processes with mechanosensitive as well as efferent functions. This raises the intriguing hypothesis that MEN sense and control muscle activity at the same time as servo-feedback loop. The mechanosensitive channel(s) or receptor(s) expressed by the different MEN populations are unknown. Future concepts have to incorporate compressive and tensile-sensitive MEN into neural circuits that controls muscle activity. They may interact to control various forms of a particular motor pattern or regulate different motor patterns independently from each other.

Title: Stages of the pathologic process in Alzheimer disease: age categories from 1 to 100 years. Content: Two thousand three hundred and thirty two nonselected brains from 1- to 100-year-old individuals were examined using immunocytochemistry (AT8) and Gallyas silver staining for abnormal tau; immunocytochemistry (4G8) and Campbell-Switzer staining were used for the detection ofβ-amyloid. A total of 342 cases was negative in the Gallyas stain but when restaged for AT8 only 10 were immunonegative. Fifty-eight cases had subcortical tau predominantly in the locus coeruleus, but there was no abnormal cortical tau (subcortical Stages a-c). Cortical involvement (abnormal tau in neurites) was identified first in the transentorhinal region (Stage 1a, 38 cases). Transentorhinal pyramidal cells displayed pretangle material (Stage 1b, 236 cases). Pretangles gradually became argyrophilic neurofibrillary tangles (NFTs) that progressed in parallel with NFT Stages I to VI. Pretangles restricted to subcortical sites were seen chiefly at younger ages. Of the total cases, 1,031 (44.2%) had β-amyloid plaques. The first plaques occurred in the neocortex after the onset of tauopathy in the brainstem. Plaques generally developed in the 40s in 4% of all cases, culminating in their tenth decade (75%). β-amyloid plaques and NFTs were significantly correlated (p < 0.0001). These data suggest that tauopathy associated with sporadic Alzheimer disease may begin earlier than previously thought and possibly in the lower brainstem rather than in the transentorhinal region.