Suwannee Thet

Meis1 regulates postnatal cardiomyocyte cell cycle arrest

The neonatal mammalian heart is capable of substantial regeneration following injury through cardiomyocyte proliferation. However, this regenerative capacity is lost by postnatal day 7 and the mechanisms of cardiomyocyte cell cycle arrest remain unclear. The homeodomain transcription factor Meis1 is required for normal cardiac development but its role in cardiomyocytes is unknown. Here we identify Meis1 as a critical regulator of the cardiomyocyte cell cycle. Meis1 deletion in mouse cardiomyocytes was sufficient for extension of the postnatal proliferative window of cardiomyocytes, and for re-activation of cardiomyocyte mitosis in the adult heart with no deleterious effect on cardiac function. In contrast, overexpression of Meis1 in cardiomyocytes decreased neonatal myocyte proliferation and inhibited neonatal heart regeneration. Finally, we show that Meis1 is required for transcriptional activation of the synergistic CDK inhibitors p15, p16 and p21. These results identify Meis1 as a critical transcriptional regulator of cardiomyocyte proliferation and a potential therapeutic target for heart regeneration.

The Oxygen-Rich Postnatal Environment Induces Cardiomyocyte Cell-Cycle Arrest through DNA Damage Response

The mammalian heart has a remarkable regenerative capacity for a short period of time after birth, after which the majority of cardiomyocytes permanently exit cell cycle. We sought to determine the primary postnatal event that results in cardiomyocyte cell-cycle arrest. We hypothesized that transition to the oxygen-rich postnatal environment is the upstream signal that results in cell-cycle arrest of cardiomyocytes. Here, we show that reactive oxygen species (ROS), oxidative DNA damage, and DNA damage response (DDR) markers significantly increase in the heart during the first postnatal week. Intriguingly, postnatal hypoxemia, ROS scavenging, or inhibition of DDR all prolong the postnatal proliferative window of cardiomyocytes, whereas hyperoxemia and ROS generators shorten it. These findings uncover a protective mechanism that mediates cardiomyocyte cell-cycle arrest in exchange for utilization of oxygen-dependent aerobic metabolism. Reduction of mitochondrial-dependent oxidative stress should be an important component of cardiomyocyte proliferation-based therapeutic approaches.

Prospective isolation of skeletal muscle stem cells with a Pax7 reporter.

Muscle regeneration occurs through activation of quiescent satellite cells whose progeny proliferate, differentiate, and fuse to make new myofibers. We used a transgenic Pax7‐ZsGreen reporter mouse to prospectively isolate stem cells of skeletal muscle by flow cytometry. We show that Pax7‐expressing cells (satellite cells) in the limb, head, and diaphragm muscles are homogeneous in size and granularity and uniformly labeled by certain cell surface markers, including CD34 and CD29. The frequency of the satellite cells varies between muscle types and with age. Clonal analysis demonstrated that all colonies arising from single cells within the Pax7‐sorted fraction have myogenic potential. In response to injury, Pax7+ cells reduce CD34, CD29, and CXCR4 expression, increase in size, and acquire Sca‐1. When directly isolated and cultured in vitro, Pax7+ cells display the hallmarks of activation and proliferate, initially as suspension aggregates and later distributed between suspension and adherence. During in vitro expansion, Pax7 (ZsGreen) and CD34 expression decline, whereas expression of PSA‐NCAM is acquired. The nonmyogenic, Pax7neg cells expand as Sca1+ PDGRα+ PSA‐NCAMneg cells. Satellite cells expanded exclusively in suspension can engraft and produce dystrophin+ fibers in mdx−/− mice. These results establish a novel animal model for the study of muscle stem cell physiology and a culture system for expansion of engraftable muscle progenitors.

Hypoxia fate mapping identifies cycling cardiomyocytes in the adult heart

Although the adult mammalian heart is incapable of meaningful functional recovery following substantial cardiomyocyte loss, it is now clear that modest cardiomyocyte turnover occurs in adult mouse and human hearts, mediated primarily by proliferation of pre-existing cardiomyocytes. However, fate mapping of these cycling cardiomyocytes has not been possible thus far owing to the lack of identifiable genetic markers. In several organs, stem or progenitor cells reside in relatively hypoxic microenvironments where the stabilization of the hypoxia-inducible factor 1 alpha (Hif-1α) subunit is critical for their maintenance and function. Here we report fate mapping of hypoxic cells and their progenies by generating a transgenic mouse expressing a chimaeric protein in which the oxygen-dependent degradation (ODD) domain of Hif-1α is fused to the tamoxifen-inducible CreERT2 recombinase. In mice bearing the creERT2-ODD transgene driven by either the ubiquitous CAG promoter or the cardiomyocyte-specific α myosin heavy chain promoter, we identify a rare population of hypoxic cardiomyocytes that display characteristics of proliferative neonatal cardiomyocytes, such as smaller size, mononucleation and lower oxidative DNA damage. Notably, these hypoxic cardiomyocytes contributed widely to new cardiomyocyte formation in the adult heart. These results indicate that hypoxia signalling is an important hallmark of cycling cardiomyocytes, and suggest that hypoxia fate mapping can be a powerful tool for identifying cycling cells in adult mammals.

Meis1 regulates the metabolic phenotype and oxidant defense of hematopoietic stem cells

The role of Meis1 in leukemia is well established, but its role in hematopoietic stem cells (HSCs) remains poorly understood. Previously, we showed that HSCs use glycolytic metabolism to meet their energy demands. However, the mechanism of regulation of HSC metabolism, and the importance of maintaining this distinct metabolic phenotype on HSC function has not been determined. More importantly, the primary function of Meis1 in HSCs remains unknown. Here, we examined the effect of loss of Meis1 on HSC function and metabolism. Inducible Meis1 deletion in adult mouse HSCs resulted in loss of HSC quiescence, and failure of bone marrow repopulation after transplantation. While we previously showed that Meis1 regulates Hif-1α transcription in vitro, we demonstrate here that loss of Meis1 results in down-regulation of both Hif-1α and Hif-2α in HSCs. This resulted in a shift to mitochondrial metabolism, increased reactive oxygen species production, and apoptosis of HSCs. Finally, we demonstrate that the effect of Meis1 knockout on HSCs is entirely mediated through reactive oxygen species where treatment of the Meis1 knockout mice with the scavenger N-acetylcystein restored HSC quiescence and rescued HSC function. These results uncover an important transcriptional network that regulates metabolism, oxidant defense, and maintenance of HSCs.

Hypoxic metabolism in human hematopoietic stem cells

BackgroundAdult hematopoietic stem cells (HSCs) are maintained in a microenvironment, known as niche in the endosteal regions of the bone marrow. This stem cell niche with low oxygen tension requires HSCs to adopt a unique metabolic profile. We have recently demonstrated that mouse long-term hematopoietic stem cells (LT-HSCs) utilize glycolysis instead of mitochondrial oxidative phosphorylation as their main energy source. However, the metabolic phenotype of human hematopoietic progenitor and stem cells (HPSCs) remains unknown.ResultsWe show that HPSCs have a similar metabolic phenotype, as shown by high rates of glycolysis, and low rates of oxygen consumption. Fractionation of human mobilized peripheral blood cells based on their metabolic footprint shows that cells with a low mitochondrial potential are highly enriched for HPSCs. Remarkably, low MP cells had much better repopulation ability as compared to high MP cells. Moreover, similar to their murine counterparts, we show that Hif-1α is upregulated in human HPSCs, where it is transcriptionally regulated by Meis1. Finally, we show that Meis1 and its cofactors Pbx1 and HoxA9 play an important role in transcriptional activation of Hif-1α in a cooperative manner.ConclusionsThese findings highlight the unique metabolic properties of human HPSCs and the transcriptional network that regulates their metabolic phenotype.

IFN type I and type II independent enhancement of B cell TLR7 expression by natural killer cells

The PRR TLR7 plays a key role in the activation of autoantigen‐reactive B cells. This response is increased markedly by IFN‐α, produced by accessory cells, as a result of the up‐regulation of TLR7. We report herein an alternative pathway by which TLR7 expression can be augmented. This finding was derived from continuation of ongoing studies to uncover interactions between NK and B cells. Here, we have compared gene expression profiles by microarray analysis of B cells before and after their interaction with purified NK cells. The most outstanding alteration of genes transcribed in B cells is a significant increase in the expression of many members of the ISG family, among which is TLR7. Further analysis revealed that the enhancement of TLR7 on B cells is not mediated via type I or type II IFN but by another cytokine, IL‐28, a type III IFN, which acts in concert with contact‐mediated interactions with NK cells. This increased expression allows B cells to respond more readily upon stimulation by its ligand and may increase in vivo responses to other TLR7 ligands, such as autoantigens, prior to or jointly with stimulation by other cytokines.

The role of NK cells in the development of autoantibodies

The systemic lupus erythematosus (Sle1) interval from the NZM2410 mouse strain has been shown to be responsible for high levels of autoantibody production against antinuclear antibodies (ANA) when transferred into C57BL/6 mice. B cells derived from the B6.Sle1 strain are required for the production but help from both T-dependent and independent sources have been documented. Using radiation chimeras constructed in a strain of mice that is chronically depleted of Natural killer (NK) cells, but not NKT cells, we have examined the role of NK cells in the development of ANA in this context. Our results show that in the presence of intact T cell help depletion of NK cells does not affect ANA production. However, when T cell help is compromised, the prevalence of animals producing ANA is significantly decreased suggesting that NK cells can provide help for the T-independent production of ANA. Further experiments provide a possible mechanism for the NK-cell dependence.

Enhancement of Antigen-Specific Immunoglobulin G Responses by Anti-CD48

CD48 is a glycosylphosphatidylinositol-anchored protein expressed ubiquitously on many cell types. Despite the poor ability to signal on its own, CD48 can activate cells via interaction with its counter receptors CD2 and CD244 as well as influence the function of other cell surface molecules by costimulatory activities. We show, herein, that injection of anti-CD48 antibodies into mice can augment the antibody response to a T-independent antigen, NP-Ficoll, that is representative of antigenic determinants expressed on the surface of various pathogens, such as Streptococcus pneumoniae. In C57BL/6 mice, enhancement of the response is dependent on natural killer (NK) cells as well as on the presence of CD2 and CD244, ligands for CD48, suggesting a requirement for direct interaction between NK and B cells. Interestingly, in this case, despite a similar augmentation by anti-CD48 in BALB/C mice, the response is independent of NK or T cells, suggesting that help for this response can be derived from other innate cell types. These results provide a pathway by which CD48, when appropriately activated, can influence the course of an antigen-specific antibody response via the innate system.

Minor Contribution of Cardiac Progenitor Cells in Neonatal Heart Regeneration

The adult mammalian heart is incapable of regeneration after injury, as shown by the limited amount of cardiomyocyte proliferation and poor neovascularization. We recently showed that neonatal mice have a remarkable ability to regenerate damaged heart after apical resection or myocardial infarction (MI), which includes complete reconstruction of myocardial wall with vascular network [2, 3]. Although lineage tracing showed that the main source of newly formed cardiomyocyte is preexisting cardiomyocytes, it is still possible that there is a minor contribution of other types of cells to the cardiomyocyte. In addition, lineage origin of the newly formed vasculature during postnatal cardiac maturation and neonatal heart regeneration remains unclear (Fig. 50.1).