query: Autologous transplantation of mesenchymal stem cells causes fewer opportunistic infections than induction therapy with anti-interleukin-2 receptor antibodies.

positive: Induction therapy with autologous mesenchymal stem cells in living-related kidney transplants: a randomized controlled trial. CONTEXT Antibody-based induction therapy plus calcineurin inhibitors (CNIs) reduce acute rejection rates in kidney recipients; however, opportunistic infections and toxic CNI effects remain challenging. Reportedly, mesenchymal stem cells (MSCs) have successfully treated graft-vs-host disease. OBJECTIVE To assess autologous MSCs as replacement of antibody induction for patients with end-stage renal disease who undergo ABO-compatible, cross-match-negative kidney transplants from a living-related donor. DESIGN, SETTING, AND PATIENTS One hundred fifty-nine patients were enrolled in this single-site, prospective, open-label, randomized study from February 2008-May 2009, when recruitment was completed. INTERVENTION Patients were inoculated with marrow-derived autologous MSC (1-2 x 10(6)/kg) at kidney reperfusion and two weeks later. Fifty-three patients received standard-dose and 52 patients received low-dose CNIs (80% of standard); 51 patients in the control group received anti-IL-2 receptor antibody plus standard-dose CNIs. MAIN OUTCOME MEASURES The primary measure was 1-year incidence of acute rejection and renal function (estimated glomerular filtration rate [eGFR]); the secondary measure was patient and graft survival and incidence of adverse events. RESULTS Patient and graft survival at 13 to 30 months was similar in all groups. After 6 months, 4 of 53 patients (7.5%) in the autologous MSC plus standard-dose CNI group (95% CI, 0.4%-14.7%; P = .04) and 4 of 52 patients (7.7%) in the low-dose group (95% CI, 0.5%-14.9%; P = .046) compared with 11 of 51 controls (21.6%; 95% CI, 10.5%-32.6%) had biopsy-confirmed acute rejection. None of the patients in either autologous MSC group had glucorticoid-resistant rejection, whereas 4 patients (7.8%) in the control group did (95% CI, 0.6%-15.1%; overall P = .02). Renal function recovered faster among both MSC groups showing increased eGFR levels during the first month after surgery than the control group. Patients receiving standard-dose CNI had a mean difference of 6.2 mL/min per 1.73 m(2) (95% CI, 0.4-11.9; P=.04) and those in the low-dose CNI of 10.0 mL/min per 1.73 m(2) (95% CI, 3.8-16.2; P=.002). Also, during the 1-year follow-up, combined analysis of MSC-treated groups revealed significantly decreased risk of opportunistic infections than the control group (hazard ratio, 0.42; 95% CI, 0.20-0.85, P=.02)   CONCLUSION Among patients undergoing renal transplant, the use of autologous MSCs compared with anti-IL-2 receptor antibody induction therapy resulted in lower incidence of acute rejection, decreased risk of opportunistic infection, and better estimated renal function at 1 year. TRIAL REGISTRATION clinicaltrials.gov Identifier: NCT00658073.

negative: Diversification in the HIV-1 Envelope Hyper-variable Domains V2, V4, and V5 and Higher Probability of Transmitted/Founder Envelope Glycosylation Favor the Development of Heterologous Neutralization Breadth A recent study of plasma neutralization breadth in HIV-1 infected individuals at nine International AIDS Vaccine Initiative (IAVI) sites reported that viral load, HLA-A*03 genotype, and subtype C infection were strongly associated with the development of neutralization breadth. Here, we refine the findings of that study by analyzing the impact of the transmitted/founder (T/F) envelope (Env), early Env diversification, and autologous neutralization on the development of plasma neutralization breadth in 21 participants identified during recent infection at two of those sites: Kigali, Rwanda (n = 9) and Lusaka, Zambia (n = 12). Single-genome analysis of full-length T/F Env sequences revealed that all 21 individuals were infected with a highly homogeneous population of viral variants, which were categorized as subtype C (n = 12), A1 (n = 7), or recombinant AC (n = 2). An extensive amino acid sequence-based analysis of variable loop lengths and glycosylation patterns in the T/F Envs revealed that a lower ratio of NXS to NXT-encoded glycan motifs correlated with neutralization breadth. Further analysis comparing amino acid sequence changes, insertions/deletions, and glycan motif alterations between the T/F Env and autologous early Env variants revealed that extensive diversification focused in the V2, V4, and V5 regions of gp120, accompanied by contemporaneous viral escape, significantly favored the development of breadth. These results suggest that more efficient glycosylation of subtype A and C T/F Envs through fewer NXS-encoded glycan sites is more likely to elicit antibodies that can transition from autologous to heterologous neutralizing activity following exposure to gp120 diversification. This initiates an Env-antibody co-evolution cycle that increases neutralization breadth, and is further augmented over time by additional viral and host factors. These findings suggest that understanding how variation in the efficiency of site-specific glycosylation influences neutralizing antibody elicitation and targeting could advance the design of immunogens aimed at inducing antibodies that can transition from autologous to heterologous neutralizing activity.

negative: Macrophages induce COX-2 expression in breast cancer cells: role of IL-1β autoamplification. Tumor-associated macrophages and high levels of cyclooxygenase-2 (COX-2) are associated with poor prognosis in breast cancer patients, but their potential interdependence has not been evaluated. The objective of this study was to determine whether macrophages regulate COX-2 expression in breast cancer cells. For this purpose, THP-1 cells were cocultured with HCC1954 breast cancer cells. Coculture led to increased COX-2 expression in the HCC1954 cells and elevated prostaglandin E(2) levels in conditioned media. Similar results were observed when THP-1 cells were incubated with HCC1937 breast cancer cells or when human monocyte-derived macrophages were cocultured with HCC1954 cells. Coculture triggered production of reactive oxygen species (ROS) in HCC1954 cells. COX-2 induction was blocked in cells preincubated with an reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor or by silencing p67PHOX, a subunit of NADPH oxidase. ROS production triggered activation of Src and mitogen-activated protein kinases (MAPKs). Blocking Src or MAPK activities or antagonizing the activator protein-1 (AP-1) transcription factor attenuated COX-2 induction in HCC1954 cells. Coculture caused rapid induction of interleukin-1β (IL-1β) in both breast cancer cells and macrophages. Increased IL-1β expression was blocked by an interleukin-1 receptor antagonist (IL-1Ra), suggesting autocrine and paracrine effects. Importantly, macrophage-induced COX-2 expression was blocked in HCC1954 cells preincubated with IL-1Ra or anti-IL-1β IgG. Together, these results indicate that macrophage-mediated induction of COX-2 in breast cancer cells is a consequence of IL-1β-mediated stimulation of ROS→Src→MAPK→AP-1 signaling. IL-1β-dependent induction of COX-2 in breast cancer cells provides a mechanism whereby macrophages contribute to tumor progression and potential therapeutic targets in breast cancer.

negative: Synergistic Activation of HIV-1 Expression by Deacetylase Inhibitors and Prostratin: Implications for Treatment of Latent Infection The persistence of transcriptionally silent but replication-competent HIV-1 reservoirs in Highly Active Anti-Retroviral Therapy (HAART)-treated infected individuals, represents a major hurdle to virus eradication. Activation of HIV-1 gene expression in these cells together with an efficient HAART has been proposed as an adjuvant therapy aimed at decreasing the pool of latent viral reservoirs. Using the latently-infected U1 monocytic cell line and latently-infected J-Lat T-cell clones, we here demonstrated a strong synergistic activation of HIV-1 production by clinically used histone deacetylase inhibitors (HDACIs) combined with prostratin, a non-tumor-promoting nuclear factor (NF)- kappaB inducer. In J-Lat cells, we showed that this synergism was due, at least partially, to the synergistic recruitment of unresponsive cells into the expressing cell population. A combination of prostratin+HDACI synergistically activated the 5' Long Terminal Repeat (5'LTR) from HIV-1 Major group subtypes representing the most prevalent viral genetic forms, as shown by transient transfection reporter assays. Mechanistically, HDACIs increased prostratin-induced DNA-binding activity of nuclear NF-kappaB and degradation of cytoplasmic NF-kappaB inhibitor, IkappaBalpha . Moreover, the combined treatment prostratin+HDACI caused a more pronounced nucleosomal remodeling in the U1 viral promoter region than the treatments with the compounds alone. This more pronounced remodeling correlated with a synergistic reactivation of HIV-1 transcription following the combined treatment prostratin+HDACI, as demonstrated by measuring recruitment of RNA polymerase II to the 5'LTR and both initiated and elongated transcripts. The physiological relevance of the prostratin+HDACI synergism was shown in CD8(+)-depleted peripheral blood mononuclear cells from HAART-treated patients with undetectable viral load. Moreover, this combined treatment reactivated viral replication in resting CD4(+) T cells isolated from similar patients. Our results suggest that combinations of different kinds of proviral activators may have important implications for reducing the size of latent HIV-1 reservoirs in HAART-treated patients.

negative: High levels of viral replication contrast with only transient changes in CD4(+) and CD8(+) cell numbers during the early phase of experimental infection with simian immunodeficiency virus SIVmnd-1 in Mandrillus sphinx. Early events during human immunodeficiency virus infections are considered to reflect the capacity of the host to control infection. We have studied early virus and host parameters during the early phase of simian immunodeficiency virus SIVmnd-1 nonpathogenic infection in its natural host, Mandrillus sphinx. Four mandrills were experimentally infected with a primary SIVmnd-1 strain derived from a naturally infected mandrill. Two noninfected control animals were monitored in parallel. Blood and lymph nodes were collected at three time points before infection, twice a week during the first month, and at days 60, 180, and 360 postinfection (p.i.). Anti-SIVmnd-1 antibodies were detected starting from days 28 to 32 p.i. Neither elevated temperature nor increased lymph node size were observed. The viral load in plasma peaked between days 7 to 10 p.i. (2 x 10(6) to 2 x 10(8) RNA equivalents/ml). Viremia then decreased 10- to 1,000-fold, reaching the viral set point between days 30 to 60 p.i. The levels during the chronic phase of infection were similar to that in the naturally infected donor mandrill (2 x 10(5) RNA equivalents/ml). The CD4(+) cell numbers and percentages in blood and lymph nodes decreased slightly (<10%) during primary infection, and CD8(+) cell numbers increased transiently. All values returned to preinfection infection levels by day 30 p.i. CD8(+) cell numbers or percentages, in peripheral blood and lymph nodes, did not increase during the 1 year of follow-up. In conclusion, SIVmnd-1 has the capacity for rapid and extensive replication in mandrills. Despite high levels of viremia, CD4(+) and CD8(+) cell numbers remained stable in the post-acute phase of infection, raising questions regarding the susceptibility of mandrill T cells to activation and/or cell death in response to SIVmnd-1 infection in vivo.

negative: Dual role of interleukin-10 in the regulation of respiratory syncitial virus (RSV)-induced lung inflammation. RSV lower respiratory tract infections (LRTI) are among the most common diseases necessitating hospital admission in children. In addition to causing acute respiratory failure, RSV infections are associated with sequelae such as secondary bacterial infections and reactive airway disease. One characteristic host response observed in severe RSV-induced LRTI and/or subsequent development of asthma is increased expression of interleukin (IL)-10. However, contradictory results have been reported regarding whether IL-10 inhibits asthmatic responses or intensifies the disease. We aimed to reconcile these discordant observations by elucidating the role of IL-10 in regulating the host response to RSV LRTI. In this study, we used a lung-specific, inducible IL-10 over-expression (OE) transgenic mouse model to address this question. Our results showed that the presence of IL-10 at the time of RSV infection not only attenuated acute inflammatory process (i.e. 24 h post-infection), but also late inflammatory changes [characterized by T helper type 2 (Th2) cytokine and chemokine expression]. While this result appears contradictory to some clinical observations where elevated IL-10 levels are observed in asthmatic patients, we also found that delaying IL-10 OE until the late immune response to RSV infection, additive effects rather than inhibitory effects were observed. Importantly, in non-infected, IL-10 OE mice, IL-10 OE alone induced up-regulation of Th2 cytokine (IL-13 and IL-5) and Th2-related chemokine [monocyte chemoattractant protein 1 (MCP-1), chemokine (C-C motif) ligand 3 (CCL3) and regulated upon activation normal T cell expressed and secreted (RANTES)] expression. We identified a subset of CD11b(+)CD11c(+)CD49b(+)F4/80(-)Gr-1(-) myeloid cells as a prinicipal source of IL-10-induced IL-13 production. Therefore, the augmented pathological responses observed in our 'delayed' IL-10 over-expression model could be attributed to IL-10 OE alone. Taken together, our study indicated dual roles of IL-10 on RSV-induced lung inflammation which appear to depend upon the timing of when elevated IL-10 is expressed in the lung.


query: The combination of H3K4me3 and H3K79me2 is found in quiescent hair follicle stem cells.

positive: Genome-wide maps of histone modifications unwind in vivo chromatin states of the hair follicle lineage. Using mouse skin, where bountiful reservoirs of synchronized hair follicle stem cells (HF-SCs) fuel cycles of regeneration, we explore how adult SCs remodel chromatin in response to activating cues. By profiling global mRNA and chromatin changes in quiescent and activated HF-SCs and their committed, transit-amplifying (TA) progeny, we show that polycomb-group (PcG)-mediated H3K27-trimethylation features prominently in HF-lineage progression by mechanisms distinct from embryonic-SCs. In HF-SCs, PcG represses nonskin lineages and HF differentiation. In TA progeny, nonskin regulators remain PcG-repressed, HF-SC regulators acquire H3K27me3-marks, and HF-lineage regulators lose them. Interestingly, genes poised in embryonic stem cells, active in HF-SCs, and PcG-repressed in TA progeny encode not only key transcription factors, but also signaling regulators. We document their importance in balancing HF-SC quiescence, underscoring the power of chromatin mapping in dissecting SC behavior. Our findings explain how HF-SCs cycle through quiescent and activated states without losing stemness and define roles for PcG-mediated repression in governing a fate switch irreversibly.

negative: Assaying proliferation and differentiation capacity of stem cells using disaggregated adult mouse epidermis In this protocol, we describe how to isolate keratinocytes from adult mouse epidermis, fractionate them into different sub-populations on the basis of cell surface markers and examine their function in an in vivo skin reconstitution assay with disaggregated neonatal dermal cells. We also describe how the isolated keratinocytes can be subjected to clonal analysis in vitro and in vivo and how to enrich for hair follicle-inducing dermal papilla cells in the dermal preparation. Using these approaches, it is possible to compare the capacity of different populations of adult epidermal stem cells to proliferate and to generate progeny that differentiate along the different epidermal lineages. Isolating, fractionating and grafting cells for the skin reconstitution assay is normally spread over 2 d. Clonal growth in culture is assessed after 14 d, while evaluation of the grafts is carried out after 4–5 weeks.

negative: Thrombopoietin/MPL signaling regulates hematopoietic stem cell quiescence and interaction with the osteoblastic niche. Maintenance of hematopoietic stem cells (HSCs) depends on interaction with their niche. Here we show that the long-term (LT)-HSCs expressing the thrombopoietin (THPO) receptor, MPL, are a quiescent population in adult bone marrow (BM) and are closely associated with THPO-producing osteoblastic cells. THPO/MPL signaling upregulated beta1-integrin and cyclin-dependent kinase inhibitors in HSCs. Furthermore, inhibition and stimulation of THPO/MPL pathway by treatments with anti-MPL neutralizing antibody, AMM2, and with THPO showed reciprocal regulation of quiescence of LT-HSC. AMM2 treatment reduced the number of quiescent LT-HSCs and allowed exogenous HSC engraftment without irradiation. By contrast, exogenous THPO transiently increased quiescent HSC population and subsequently induced HSC proliferation in vivo. Altogether, these observations suggest that THPO/MPL signaling plays a critical role of LT-HSC regulation in the osteoblastic niche.

negative: RBP2 Belongs to a Family of Demethylases, Specific for Tri-and Dimethylated Lysine 4 on Histone 3 Methylation of histones has been regarded as a stable modification defining the epigenetic program of the cell, which regulates chromatin structure and transcription. However, the recent discovery of histone demethylases has challenged the stable nature of histone methylation. Here we demonstrate that the JARID1 proteins RBP2, PLU1, and SMCX are histone demethylases specific for di- and trimethylated histone 3 lysine 4 (H3K4). Consistent with a role for the JARID1 Drosophila homolog Lid in regulating expression of homeotic genes during development, we show that RBP2 is displaced from Hox genes during embryonic stem (ES) cell differentiation correlating with an increase of their H3K4me3 levels and expression. Furthermore, we show that mutation or RNAi depletion of the C. elegans JARID1 homolog rbr-2 leads to increased levels of H3K4me3 during larval development and defects in vulva formation. Taken together, these results suggest that H3K4me3/me2 demethylation regulated by the JARID1 family plays an important role during development.

negative: Quiescent and active hippocampal neural stem cells with distinct morphologies respond selectively to physiological and pathological stimuli and aging. New neurons are generated in the adult hippocampus throughout life by neural stem/progenitor cells (NSCs), and neurogenesis is a plastic process responsive to external stimuli. We show that canonical Notch signaling through RBP-J is required for hippocampal neurogenesis. Notch signaling distinguishes morphologically distinct Sox2(+) NSCs, and within these pools subpopulations can shuttle between mitotically active or quiescent. Radial and horizontal NSCs respond selectively to neurogenic stimuli. Physical exercise activates the quiescent radial population whereas epileptic seizures induce expansion of the horizontal NSC pool. Surprisingly, reduced neurogenesis correlates with a loss of active horizontal NSCs in aged mice rather than a total loss of stem cells, and the transition to a quiescent state is reversible to rejuvenate neurogenesis in the brain. The discovery of multiple NSC populations with Notch dependence but selective responses to stimuli and reversible quiescence has important implications for the mechanisms of adaptive learning and also for regenerative therapy.

negative: GATA-3 regulates hematopoietic stem cell maintenance and cell-cycle entry. Maintaining hematopoietic stem cell (HSC) quiescence is a critical property for the life-long generation of blood cells. Approximately 75% of cells in a highly enriched long-term repopulating HSC (LT-HSC) pool (Lin(-)Sca1(+)c-Kit(hi)CD150(+)CD48(-)) are quiescent, with only a small percentage of the LT-HSCs in cycle. Transcription factor GATA-3 is known to be vital for the development of T cells at multiple stages in the thymus and for Th2 differentiation in the peripheral organs. Although it is well documented that GATA-3 is expressed in HSCs, a role for GATA-3 in any prethymic progenitor cell has not been established. In the present study, we show that Gata3-null mutant mice generate fewer LT-HSCs and that fewer Gata3-null LT-HSCs are in cycle. Furthermore, Gata3 mutant hematopoietic progenitor cells fail to be recruited into an increased cycling state after 5-fluorouracil-induced myelosuppression. Therefore, GATA-3 is required for the maintenance of a normal number of LT-HSCs and for their entry into the cell cycle.

negative: The developmental potential of iPSCs is greatly influenced by reprogramming factor selection. Induced pluripotent stem cells (iPSCs) are commonly generated by transduction of Oct4, Sox2, Klf4, and Myc (OSKM) into cells. Although iPSCs are pluripotent, they frequently exhibit high variation in terms of quality, as measured in mice by chimera contribution and tetraploid complementation. Reliably high-quality iPSCs will be needed for future therapeutic applications. Here, we show that one major determinant of iPSC quality is the combination of reprogramming factors used. Based on tetraploid complementation, we found that ectopic expression of Sall4, Nanog, Esrrb, and Lin28 (SNEL) in mouse embryonic fibroblasts (MEFs) generated high-quality iPSCs more efficiently than other combinations of factors including OSKM. Although differentially methylated regions, transcript number of master regulators, establishment of specific superenhancers, and global aneuploidy were comparable between high- and low-quality lines, aberrant gene expression, trisomy of chromosome 8, and abnormal H2A.X deposition were distinguishing features that could potentially also be applicable to human.

negative: Deletion of the developmentally essential gene ATR in adult mice leads to age-related phenotypes and stem cell loss. Developmental abnormalities, cancer, and premature aging each have been linked to defects in the DNA damage response (DDR). Mutations in the ATR checkpoint regulator cause developmental defects in mice (pregastrulation lethality) and humans (Seckel syndrome). Here we show that eliminating ATR in adult mice leads to defects in tissue homeostasis and the rapid appearance of age-related phenotypes, such as hair graying, alopecia, kyphosis, osteoporosis, thymic involution, fibrosis, and other abnormalities. Histological and genetic analyses indicate that ATR deletion causes acute cellular loss in tissues in which continuous cell proliferation is required for maintenance. Importantly, thymic involution, alopecia, and hair graying in ATR knockout mice were associated with dramatic reductions in tissue-specific stem and progenitor cells and exhaustion of tissue renewal and homeostatic capacity. In aggregate, these studies suggest that reduced regenerative capacity in adults via deletion of a developmentally essential DDR gene is sufficient to cause the premature appearance of age-related phenotypes.

negative: Sprouty1 Regulates Reversible Quiescence of a Self-Renewing Adult Muscle Stem Cell Pool during Regeneration Satellite cells are skeletal muscle stem cells capable of self-renewal and differentiation after transplantation, but whether they contribute to endogenous muscle fiber repair has been unclear. The transcription factor Pax7 marks satellite cells and is critical for establishing the adult satellite cell pool. By using a lineage tracing approach, we show that after injury, quiescent adult Pax7(+) cells enter the cell cycle; a subpopulation returns to quiescence to replenish the satellite cell compartment, while others contribute to muscle fiber formation. We demonstrate that Sprouty1 (Spry1), a receptor tyrosine kinase signaling inhibitor, is expressed in quiescent Pax7(+) satellite cells in uninjured muscle, downregulated in proliferating myogenic cells after injury, and reinduced as Pax7(+) cells re-enter quiescence. We show that Spry1 is required for the return to quiescence and homeostasis of the satellite cell pool during repair. Our results therefore define a role for Spry1 in adult muscle stem cell biology and tissue repair.

negative: Combined Single-Cell Functional and Gene Expression Analysis Resolves Heterogeneity within Stem Cell Populations Heterogeneity within the self-renewal durability of adult hematopoietic stem cells (HSCs) challenges our understanding of the molecular framework underlying HSC function. Gene expression studies have been hampered by the presence of multiple HSC subtypes and contaminating non-HSCs in bulk HSC populations. To gain deeper insight into the gene expression program of murine HSCs, we combined single-cell functional assays with flow cytometric index sorting and single-cell gene expression assays. Through bioinformatic integration of these datasets, we designed an unbiased sorting strategy that separates non-HSCs away from HSCs, and single-cell transplantation experiments using the enriched population were combined with RNA-seq data to identify key molecules that associate with long-term durable self-renewal, producing a single-cell molecular dataset that is linked to functional stem cell activity. Finally, we demonstrated the broader applicability of this approach for linking key molecules with defined cellular functions in another stem cell system.

negative: Chromatin signatures of pluripotent cell lines. Epigenetic genome modifications are thought to be important for specifying the lineage and developmental stage of cells within a multicellular organism. Here, we show that the epigenetic profile of pluripotent embryonic stem cells (ES) is distinct from that of embryonic carcinoma cells, haematopoietic stem cells (HSC) and their differentiated progeny. Silent, lineage-specific genes replicated earlier in pluripotent cells than in tissue-specific stem cells or differentiated cells and had unexpectedly high levels of acetylated H3K9 and methylated H3K4. Unusually, in ES cells these markers of open chromatin were also combined with H3K27 trimethylation at some non-expressed genes. Thus, pluripotency of ES cells is characterized by a specific epigenetic profile where lineage-specific genes may be accessible but, if so, carry repressive H3K27 trimethylation modifications. H3K27 methylation is functionally important for preventing expression of these genes in ES cells as premature expression occurs in embryonic ectoderm development (Eed)-deficient ES cells. Our data suggest that lineage-specific genes are primed for expression in ES cells but are held in check by opposing chromatin modifications.

negative: Oct4 links multiple epigenetic pathways to the pluripotency network Oct4 is a well-known transcription factor that plays fundamental roles in stem cell self-renewal, pluripotency, and somatic cell reprogramming. However, limited information is available on Oct4-associated protein complexes and their intrinsic protein-protein interactions that dictate Oct4's critical regulatory activities. Here we employed an improved affinity purification approach combined with mass spectrometry to purify Oct4 protein complexes in mouse embryonic stem cells (mESCs), and discovered many novel Oct4 partners important for self-renewal and pluripotency of mESCs. Notably, we found that Oct4 is associated with multiple chromatin-modifying complexes with documented as well as newly proved functional significance in stem cell maintenance and somatic cell reprogramming. Our study establishes a solid biochemical basis for genetic and epigenetic regulation of stem cell pluripotency and provides a framework for exploring alternative factor-based reprogramming strategies.

negative: The histone demethylases Jhdm1a/1b enhance somatic cell reprogramming in a vitamin-C-dependent manner. 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.

negative: Induction of cell cycle entry eliminates human leukemia stem cells in a mouse model of AML Cancer stem cells have been proposed to be important for initiation, maintenance and recurrence of various malignancies, including acute myeloid leukemia (AML). We have previously reported that CD34+CD38− human primary AML stem cells residing in the endosteal region of the bone marrow are relatively chemotherapy resistant. Using a NOD/SCID/IL2rγnull mouse model of human AML, we now show that the AML stem cells in the endosteal region are cell cycle quiescent and that these stem cells can be induced to enter the cell cycle by treatment with granulocyte colony-stimulating factor (G-CSF). In combination with cell cycle-dependent chemotherapy, G-CSF treatment significantly enhances induction of apoptosis and elimination of human primary AML stem cells in vivo. The combination therapy leads to significantly increased survival of secondary recipients after transplantation of leukemia cells compared with chemotherapy alone.

negative: Chromatin modifying enzymes as modulators of reprogramming 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.

negative: The neural crest is a source of mesenchymal stem cells with specialized hematopoietic stem cell niche function Mesenchymal stem cells (MSCs) and osteolineage cells contribute to the hematopoietic stem cell (HSC) niche in the bone marrow of long bones. However, their developmental relationships remain unclear. In this study, we demonstrate that different MSC populations in the developing marrow of long bones have distinct functions. Proliferative mesoderm-derived nestin(-) MSCs participate in fetal skeletogenesis and lose MSC activity soon after birth. In contrast, quiescent neural crest-derived nestin(+) cells preserve MSC activity, but do not generate fetal chondrocytes. Instead, they differentiate into HSC niche-forming MSCs, helping to establish the HSC niche by secreting Cxcl12. Perineural migration of these cells to the bone marrow requires the ErbB3 receptor. The neonatal Nestin-GFP(+) Pdgfrα(-) cell population also contains Schwann cell precursors, but does not comprise mature Schwann cells. Thus, in the developing bone marrow HSC niche-forming MSCs share a common origin with sympathetic peripheral neurons and glial cells, and ontogenically distinct MSCs have non-overlapping functions in endochondrogenesis and HSC niche formation.

negative: Ovarian Surface Epithelium in Patients with Severe Ovarian Infertility: A Potential Source of Cells Expressing Markers of Pluripotent/Multipotent Stem Cells The aim of this study was to confirm the presence of stem cells in the ovarian surface epithelium of patients with premature ovarian failure and no mature follicles and oocytes. In these patients, small round cells of unknown origin expressing SOX-2 marker of pluripotency were observed among the epithelial cells just after the ovarian surface epithelium scraping. These cells were an integral part of the ovarian surface epithelium. When the scraped cells were cultured in a medium with added follicular fluid to provide some ovarian niche, primitive oocyte-like cells and typical round-shaped cell clusters positively stained on alkaline phosphatase, and markers of pluripotency, such as SOX-2 and SSEA-4, were developed. These markers were expressed early and also later in the culture. Single oocyte-like cells expressed genes OCT4A, SOX-2, NANOG, NANOS, STELLA, CD9, LIN28, KLF4, GDF3, and MYC, characteristic for pluripotent stem cells. The results of this study confirmed the presence of putative stem cells in the ovarian surface epithelium of these patients and provided some basis to create a stem cell line in the future.

negative: Proteomic and genomic approaches reveal critical functions of H3K9 methylation and Heterochromatin Protein-1γ in reprogramming to pluripotency 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.

negative: Neuronal circuitry mechanism regulating adult quiescent neural stem cell fate decision Adult neurogenesis arises from neural stem cells within specialized niches. Neuronal activity and experience, presumably acting on this local niche, regulate multiple stages of adult neurogenesis, from neural progenitor proliferation to new neuron maturation, synaptic integration and survival. It is unknown whether local neuronal circuitry has a direct impact on adult neural stem cells. Here we show that, in the adult mouse hippocampus, nestin-expressing radial glia-like quiescent neural stem cells (RGLs) respond tonically to the neurotransmitter γ-aminobutyric acid (GABA) by means of γ2-subunit-containing GABAA receptors. Clonal analysis of individual RGLs revealed a rapid exit from quiescence and enhanced symmetrical self-renewal after conditional deletion of γ2. RGLs are in close proximity to terminals expressing 67-kDa glutamic acid decarboxylase (GAD67) of parvalbumin-expressing (PV+) interneurons and respond tonically to GABA released from these neurons. Functionally, optogenetic control of the activity of dentate PV+ interneurons, but not that of somatostatin-expressing or vasoactive intestinal polypeptide (VIP)-expressing interneurons, can dictate the RGL choice between quiescence and activation. Furthermore, PV+ interneuron activation restores RGL quiescence after social isolation, an experience that induces RGL activation and symmetrical division. Our study identifies a niche cell–signal–receptor trio and a local circuitry mechanism that control the activation and self-renewal mode of quiescent adult neural stem cells in response to neuronal activity and experience.

negative: Targeted therapy for cancer stem cells: the patched pathway and ABC transporters Data from certain leukemias as well as brain and breast cancer indicate that there is a small population of tumor cells with ‘stem cell’ characteristics and the capacity for self-renewal. The self-renewing cells have many of the properties of normal stem cells and have been termed ‘cancer stem cells’. These cancer stem cells make up as few as 1% of the cells in a tumor, making them difficult to detect and study. Like normal stem cells, cancer stem cells have a number of properties permitting them to survive traditional cancer chemotherapy and radiation therapy. These cells express high levels of ATP-binding cassette (ABC) drug transporters, providing for a level of resistance; are relatively quiescent; have higher levels of DNA repair and a lowered ability to enter apoptosis. Combined cancer therapy approaches targeting the cancer stem cells and the non-stem cells may be developed with increased efficacy. Efforts to target the Hedgehog/Patched pathway, critical to embryonic growth and differentiation, and the ABCG2 drug efflux transporter will be presented.

negative: Histone Demethylase Expression Enhances Human Somatic Cell Nuclear Transfer Efficiency and Promotes Derivation of Pluripotent Stem Cells. 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.

negative: Induction of pluripotent stem cells from mouse embryonic fibroblasts by Oct4 and Klf4 with small-molecule compounds. 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.

negative: Sequential histone-modifying activities determine the robustness of transdifferentiation 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.

negative: The transcriptome profile of human embryonic stem cells as defined by SAGE. Human embryonic stem (ES) cell lines that have the ability to self-renew and differentiate into specific cell types have been established. The molecular mechanisms for self-renewal and differentiation, however, are poorly understood. We determined the transcriptome profiles for two proprietary human ES cell lines (HES3 and HES4, ES Cell International), and compared them with murine ES cells and other human tissues. Human and mouse ES cells appear to share a number of expressed gene products although there are numerous notable differences, including an inactive leukemia inhibitory factor pathway and the high preponderance of several important genes like POU5F1 and SOX2 in human ES cells. We have established a list of genes comprised of known ES-specific genes and new candidates that can serve as markers for human ES cells and may also contribute to the "stemness" phenotype. Of particular interest was the downregulation of DNMT3B and LIN28 mRNAs during ES cell differentiation. The overlapping similarities and differences in gene expression profiles of human and mouse ES cells provide a foundation for a detailed and concerted dissection of the molecular and cellular mechanisms governing their pluripotency, directed differentiation into specific cell types, and extended ability for self-renewal.

negative: Human epidermal stem cell function is regulated by circadian oscillations. Human skin copes with harmful environmental factors that are circadian in nature, yet how circadian rhythms modulate the function of human epidermal stem cells is mostly unknown. Here we show that in human epidermal stem cells and their differentiated counterparts, core clock genes peak in a successive and phased manner, establishing distinct temporal intervals during the 24 hr day period. Each of these successive clock waves is associated with a peak in the expression of subsets of transcripts that temporally segregate the predisposition of epidermal stem cells to respond to cues that regulate their proliferation or differentiation, such as TGFβ and calcium. Accordingly, circadian arrhythmia profoundly affects stem cell function in culture and in vivo. We hypothesize that this intricate mechanism ensures homeostasis by providing epidermal stem cells with environmentally relevant temporal functional cues during the course of the day and that its perturbation may contribute to aging and carcinogenesis.

negative: Drosophila oocyte localization is mediated by differential cadherin-based adhesion. In a Drosophila follicle the oocyte always occupies a posterior position among a group of sixteen germline cells. Although the importance of this cell arrangement for the subsequent formation of the anterior-posterior axis of the embryo is well documented, the molecular mechanism responsible for the posterior localization of the oocyte was unknown. Here we show that the homophilic adhesion molecule DE-cadherin mediates oocyte positioning. During follicle biogenesis, DE-cadherin is expressed in germline (including oocyte) and surrounding follicle cells, with the highest concentration of DE-cadherin being found at the interface between oocyte and posterior follicle cells. Mosaic analysis shows that DE-cadherin is required in both germline and follicle cells for correct oocyte localization, indicating that germline-soma interactions may be involved in this process. By analysing the behaviour of the oocyte in follicles with a chimaeric follicular epithelium, we find that the position of the oocyte is determined by the position of DE-cadherin-expressing follicle cells, to which the oocyte attaches itself selectively. Among the DE-cadherin positive follicle cells, the oocyte preferentially contacts those cells that express higher levels of DE-cadherin. On the basis of these data, we propose that in wild-type follicles the oocyte competes successfully with its sister germline cells for contact to the posterior follicle cells, a sorting process driven by different concentrations of DE-cadherin. This is, to our knowledge, the first in vivo example of a cell-sorting process that depends on differential adhesion mediated by a cadherin.

negative: Chromatin structure and gene expression programs of human embryonic and induced pluripotent stem cells. Knowledge of both the global chromatin structure and the gene expression programs of human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) should provide a robust means to assess whether the genomes of these cells have similar pluripotent states. Recent studies have suggested that ESCs and iPSCs represent different pluripotent states with substantially different gene expression profiles. We describe here a comparison of global chromatin structure and gene expression data for a panel of human ESCs and iPSCs. Genome-wide maps of nucleosomes with histone H3K4me3 and H3K27me3 modifications indicate that there is little difference between ESCs and iPSCs with respect to these marks. Gene expression profiles confirm that the transcriptional programs of ESCs and iPSCs show very few consistent differences. Although some variation in chromatin structure and gene expression was observed in these cell lines, these variations did not serve to distinguish ESCs from iPSCs.

negative: Dnmt3a and Dnmt3b Associate with Enhancers to Regulate Human Epidermal Stem Cell Homeostasis. The genome-wide localization and function of endogenous Dnmt3a and Dnmt3b in adult stem cells are unknown. Here, we show that in human epidermal stem cells, the two proteins bind in a histone H3K36me3-dependent manner to the most active enhancers and are required to produce their associated enhancer RNAs. Both proteins prefer super-enhancers associated to genes that either define the ectodermal lineage or establish the stem cell and differentiated states. However, Dnmt3a and Dnmt3b differ in their mechanisms of enhancer regulation: Dnmt3a associates with p63 to maintain high levels of DNA hydroxymethylation at the center of enhancers in a Tet2-dependent manner, whereas Dnmt3b promotes DNA methylation along the body of the enhancer. Depletion of either protein inactivates their target enhancers and profoundly affects epidermal stem cell function. Altogether, we reveal novel functions for Dnmt3a and Dnmt3b at enhancers that could contribute to their roles in disease and tumorigenesis.

negative: Embryonic Origin of Postnatal Neural Stem Cells Adult neural stem/progenitor (B1) cells within the walls of the lateral ventricles generate different types of neurons for the olfactory bulb (OB). The location of B1 cells determines the types of OB neurons they generate. Here we show that the majority of mouse B1 cell precursors are produced between embryonic days (E) 13.5 and 15.5 and remain largely quiescent until they become reactivated postnatally. Using a retroviral library carrying over 100,000 genetic tags, we found that B1 cells share a common progenitor with embryonic cells of the cortex, striatum, and septum, but this lineage relationship is lost before E15.5. The regional specification of B1 cells is evident as early as E11.5 and is spatially linked to the production of neurons that populate different areas of the forebrain. This study reveals an early embryonic regional specification of postnatal neural stem cells and the lineage relationship between them and embryonic progenitor cells.

negative: The Drosophila hindgut lacks constitutively active adult stem cells but proliferates in response to tissue damage. The adult Drosophila hindgut was recently reported to contain active, tissue-replenishing stem cells, like those of the midgut, but located within an anterior ring so as to comprise a single giant crypt. In contrast to this view, we observed no active stem cells and little cell turnover in adult hindgut tissue based on clonal marking and BrdU incorporation studies. Again contradicting the previous proposal, we showed that the adult hindgut is not generated by anterior stem cells during larval/pupal development. However, severe tissue damage within the hindgut elicits cell proliferation within a ring of putative quiescent stem cells at the anterior of the pylorus. Thus, the hindgut does not provide a model of tissue maintenance by constitutively active stem cells, but has great potential to illuminate mechanisms of stress-induced tissue repair.

negative: Division-coupled astrocytic differentiation and age-related depletion of neural stem cells in the adult hippocampus. Production of new neurons in the adult hippocampus decreases with age; this decline may underlie age-related cognitive impairment. Here we show that continuous depletion of the neural stem cell pool, as a consequence of their division, may contribute to the age-related decrease in hippocampal neurogenesis. Our results indicate that adult hippocampal stem cells, upon exiting their quiescent state, rapidly undergo a series of asymmetric divisions to produce dividing progeny destined to become neurons and subsequently convert into mature astrocytes. Thus, the decrease in the number of neural stem cells is a division-coupled process and is directly related to their production of new neurons. We present a scheme of the neurogenesis cascade in the adult hippocampus that includes a proposed "disposable stem cell" model and accounts for the disappearance of hippocampal neural stem cells, the appearance of new astrocytes, and the age-related decline in the production of new neurons.

negative: Identification of vitronectin as an extrinsic inducer of cancer stem cell differentiation and tumor formation. There is mounting evidence that tumors are initiated by a rare subset of cells called cancer stem cells (CSCs). CSCs are generally quiescent, self-renew, form tumors at low numbers, and give rise to the heterogeneous cell types found within a tumor. CSCs isolated from multiple tumor types differentiate both in vivo and in vitro when cultured in serum, yet the factors responsible for their differentiation have not yet been identified. Here we show that vitronectin is the component of human serum driving stem cell differentiation through an integrin alpha V beta 3-dependent mechanism. CSCs cultured on vitronectin result in downregulation of stem cell genes, modulation of differentiation markers, and loss of beta-catenin nuclear localization. Blocking integrin alpha V beta 3 inhibits differentiation and subsequently tumor formation. Thus, CSCs must be engaged by one or more extracellular signals to differentiate and initiate tumor formation, defining a new axis for future novel therapies aimed at both the extrinsic and intracellular pathways.

negative: Clonally derived human embryonic stem cell lines maintain pluripotency and proliferative potential for prolonged periods of culture. Embryonic stem (ES) cell lines derived from human blastocysts have the developmental potential to form derivatives of all three embryonic germ layers even after prolonged culture. Here we describe the clonal derivation of two human ES cell lines, H9.1 and H9.2. At the time of the clonal derivation of the H9.1 and H9.2 ES cell lines, the parental ES cell line, H9, had already been continuously cultured for 6 months. After an additional 8 months of culture, H9.1 and H9.2 ES cell lines continued to: (1) actively proliferate, (2) express high levels of telomerase, and (3) retain normal karyotypes. Telomere lengths, while somewhat variable, were maintained between 8 and 12 kb in high-passage H9.1 and H9.2 cells. High-passage H9.1 and H9.2 cells both formed teratomas in SCID-beige mice that included differentiated derivatives of all three embryonic germ layers. These results demonstrate the pluripotency of single human ES cells, the maintenance of pluripotency during an extended period of culture, and the long-term self-renewing properties of cultured human ES cells. The remarkable developmental potential, proliferative capacity, and karyotypic stability of human ES cells distinguish them from adult cells.