Summary Stem cell regulation and hierarchical organization of human skeletal progenitors remain largely unexplored. Here, we report the isolation of a self-renewing and multipotent human skeletal stem cell (hSSC) that generates progenitors of bone, cartilage, and stroma, but not fat. Self-renewing and multipotent hSSCs are present in fetal and adult bones and can also be derived from BMP2-treated human adipose stroma (B-HAS) and induced pluripotent stem cells (iPSCs). Gene expression analysis of individual hSSCs reveals overall similarity between hSSCs obtained from different sources and partially explains skewed differentiation towards cartilage in fetal and iPSC-derived hSSCs. hSSCs undergo local expansion in response to acute skeletal injury. In addition, hSSC-derived stroma can maintain human hematopoietic stem cells (hHSCs) in serum-free culture conditions. Finally, we combine gene expression and epigenetic data of mouse skeletal stem cells (mSSCs) and hSSCs to identify evolutionarily conserved and divergent pathways driving SSC-mediated skeletogenesis.

Dravet Syndrome is an intractable form of childhood epilepsy associated with deleterious mutations in SCN1A, the gene encoding neuronal sodium channel Nav1.1. Earlier studies using human induced pluripotent stem cells (iPSCs) have produced mixed results regarding the importance of Nav1.1 in human inhibitory versus excitatory neurons. We studied a Nav1.1 mutation (p.S1328P) identified in a pair of twins with Dravet Syndrome and generated iPSC-derived neurons from these patients. Characterization of the mutant channel revealed a decrease in current amplitude and hypersensitivity to steady-state inactivation. We then differentiated Dravet-Syndrome and control iPSCs into telencephalic excitatory neurons or medial ganglionic eminence (MGE)-like inhibitory neurons. Dravet inhibitory neurons showed deficits in sodium currents and action potential firing, which were rescued by a Nav1.1 transgene, whereas Dravet excitatory neurons were normal. Our study identifies biophysical impairments underlying a deleterious Nav1.1 mutation and supports the hypothesis that Dravet Syndrome arises from defective inhibitory neurons.DOI: http://dx.doi.org/10.7554/eLife.13073.001

Summary Brown adipose tissue (BAT) possesses the inherent ability to dissipate metabolic energy as heat through uncoupled mitochondrial respiration. An essential component of the mitochondrial electron transport chain is coenzyme Q (CoQ). While cells mostly synthesize CoQ endogenously, exogenous supplementation with CoQ has been successful as a therapy for patients with CoQ deficiency. However, which tissues depend on exogenous CoQ uptake as well as the mechanism by which CoQ is taken up by cells and the role of this process in BAT function is not well understood. Here we report that the scavenger receptor CD36 drives the uptake of CoQ by BAT and is required for normal BAT function. BAT from mice lacking CD36 displays CoQ deficiency, impaired CoQ uptake, hypertrophy, altered lipid metabolism, mitochondrial dysfunction, and defective non-shivering thermogenesis. Together, these data reveal an important new role for the systemic transport of CoQ to BAT and its function in thermogenesis.

The mitogen-activated protein kinase (MAPK) Erk1/2 has been implicated to modulate the activity of nuclear receptors, including peroxisome proliferator activator receptors (PPARs) and liver X receptor, to alter the ability of cells to export cholesterol. Here, we investigated if the Ras-Raf-Mek-Erk1/2 signaling cascade could affect reverse cholesterol transport via modulation of scavenger receptor class BI (SR-BI) levels. We demonstrate that in Chinese hamster ovary (CHO) and human embryonic kidney (HEK293) cells, Mek1/2 inhibition reduces PPAR␣-inducible SR-BI protein expression and activity, as judged by reduced efflux onto high density lipoprotein (HDL). Ectopic expression of constitutively active H-Ras and Mek1 increases SR-BI protein levels, which correlates with elevated PPAR␣ Ser-21 phosphorylation and increased cholesterol efflux. In contrast, SR-BI levels are insensitive to Mek1/2 inhibitors in PPAR␣-depleted cells. Most strikingly, Mek1/2 inhibition promotes SR-BI degradation in SR-BI-overexpressing CHO cells and human HuH7 hepatocytes, which is associated with reduced uptake of radiolabeled and 1,1 -dioctadecyl-3,3,3 ,3 -tetramethylindocarbocyane-labeled HDL. Loss of Mek1/2 kinase activity reduces SR-BI expression in the presence of bafilomycin, an inhibitor of lysosomal degradation, indicating down-regulation of SR-BI via proteasomal pathways. In conclusion, Mek1/2 inhibition enhances the PPAR␣-dependent degradation of SR-BI in hepatocytes.Anti-atherosclerotic properties of HDL and the major HDL apolipoprotein, apoA-I, are believed to include their ability to induce signaling events that promote cholesterol export from peripheral cells to the liver for disposal. However, the signal transduction pathways that contribute to stimulate reverse cholesterol transport in macrophages and hepatocytes are not fully understood (1-3). HDL binding to receptors such as SR-BI 6 activates various cellular processes, including endothelial nitric-oxide synthase activation in endothelial cells (1-3) and proliferation in smooth muscle cells (4) but also cell surface localization of SR-BI in hepatocytes (5). Downstream targets of HDL include Src family kinases, phospholipase C and D, Ras, phosphatidylinositol 3-kinase (PI3K), Akt, the mitogen-activated protein kinase (MAPK) pathway (Mek1/2 and Erk1/2), and Rac/Rho GTPases (1-8). Other kinases implicated in HDL-or apoAI-inducible cholesterol transport include protein kinase C (PKC), protein kinase A (PKA), c-Jun N-terminal kinase (JNK), and p38 MAPK (9 -14).Alternatively, signaling pathways can modulate the activity of nuclear receptors, including PPAR and LXR, to alter the ability of cells to transport cholesterol (15-18). Indeed, post-translational phosphorylation via various kinases, including Erk1/2, can alter PPAR␣ and PPAR␣ co-activator activity in a liganddependent and -independent manner (17-25). Recent findings link Erk1/2 kinases with nuclear receptors and lipid export via ABCA1. First, enhanced Erk1/2 signaling increases ABCA1 expression and ABCA1-mediated phos...

A pool of murine cytomegalovirus (MCMV) mutants was previously generated by using a Tn3-based transposon mutagenesis approach (X. Zhan, M. Lee, J. Xiao, and F. Liu, J. Virol. 74:7411-7421, 2000). In this study, one of the MCMV mutants, Rvm155, which contained the transposon insertion in open reading frame m155, was characterized in vitro for its replication in tissue culture and in vivo for its growth and virulence in immunodeficient SCID mice. Compared to the wild-type strain and a rescued virus that restored the m155 region, the mutant is significantly deficient in growth in many organs of the infected animals. At 21 days postinfection the titers of Rvm155 in the salivary glands, lungs, spleens, livers, and kidneys of the intraperitoneally infected SCID mice were lower than the titers of the wild-type virus and the rescued virus by 50-, 1,000-, 500-, 100-, and 500-fold, respectively. Moreover, the viral mutant was attenuated in killing the SCID mice, as none of the SCID mice that were intraperitoneally infected with Rvm155 died until 38 days postinfection while all the animals infected with the wild-type and rescued viruses died at 27 days postinfection. Our results provide the first direct evidence that a disruption of m155 expression leads to attenuation of viral virulence and growth in animals. Moreover, these results suggest that m155 is a viral determinant for optimal MCMV growth and virulence in vivo.Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus that causes mild or subclinical diseases in immunocompetent adults but may lead to severe complications in neonates and immunocompromised individuals (18, 23). Disseminated HCMV infection, common in AIDS patients, is usually associated with gastroenteritis, pneumonia, and retinitis (8,22). HCMV infection continues to be a major cause of morbidity and mortality in bone marrow and solid-organ transplant recipients (18, 23). Studies on the functions of viral genes in HCMV replication in vivo are essential for understanding viral pathogenesis and developing new strategies to combat the viral infection. However, there are presently no suitable animal models for HCMV infection. HCMV only propagates in human cells and grows slowly due to a long lytic replication cycle (18,23). These properties of HCMV have hampered the studies of HCMV pathogenesis and gene function.Infection of the mouse with murine cytomegalovirus (MCMV) provides a valuable in vivo model for studying the biology of CMV infection. This is because infection of mice by MCMV resembles in many ways its human counterpart with respect to pathogenesis (11,12,18,23). For example, tropism for the spleen and liver is believed to be important in the infection of both HCMV and MCMV, as the spleen and liver are the sites for primary acute infections as well as persistent and latent infections (18,23). The MCMV genome is 230 kb long and is predicted to encode more than 170 open reading frames, 78 of which have extensive homology to those of HCMV (4,25). A clear understanding of the biology of MCMV and the ...