Background and Purpose Muse cells are endogenous non-tumorigenic stem cells with pluripotency harvestable as pluripotent marker SSEA-3+ cells from the bone marrow (BM) from cultured BM-mesenchymal stem cells (MSCs). After transplantation into neurological disease models, Muse cells exert repair effects, but the exact mechanism remains inconclusive. Methods We conducted mechanism-based experiments by transplanting serum/xeno-free cultured-human BM-Muse cells into the peri-lesion brain at two weeks after lacunar infarction in immunodeficient mice. Results Approximately 28% of initially transplanted Muse cells remained in the host brain at 8 weeks, spontaneously differentiated into cells expressing NeuN (~62%), MAP2 (~30%), and GST-pi (~12%). Dextran tracing revealed connections between host neurons and Muse cells at the lesioned motor cortex and the anterior horn. Muse cells extended neurites through the ipsilateral pyramidal tract, crossed to contralateral side and reached to the pyramidal tract in the dorsal funiculus of spinal cord. Muse-transplanted stroke mice displayed significant recovery in cylinder tests, which was reverted by the human-selective diphtheria toxin. At 10 months post-transplantation, human specific Alu sequence was detected only in the brain but not in other organs, with no evidence of tumor formation. Conclusions Transplantation at the delayed subacute phase showed Muse cells differentiated into neural cells, facilitated neural reconstruction, improved functions, and displayed solid safety outcomes over prolonged graft maturation period, indicating their therapeutic potential for lacunar stroke.

Rationale: Cerebrovascular function is critical for brain health, and endogenous vascular-protective pathways may provide therapeutic targets for neurological disorders. Sphingosine 1-phosphate (S1P) signaling coordinates vascular functions in other organs, and S1P receptor-1 (S1P 1 ) modulators including fingolimod show promise for the treatment of ischemic and hemorrhagic stroke. However, S1P 1 also coordinates lymphocyte trafficking, and lymphocytes are currently viewed as the principal therapeutic target for S1P 1 modulation in stroke. Objective: To address roles and mechanisms of engagement of endothelial cell (EC) S1P 1 in the naïve and ischemic brain and its potential as a target for cerebrovascular therapy. Methods and Results: Using spatial modulation of S1P provision and signaling, we demonstrate a critical vascular protective role for endothelial S1P 1 in the mouse brain. With an S1P 1 signaling reporter, we reveal that abluminal polarization shields S1P 1 from circulating endogenous and synthetic ligands after maturation of the blood-neural barrier, restricting homeostatic signaling to a subset of arteriolar ECs. S1P 1 signaling sustains hallmark endothelial functions in the naïve brain, and expands during ischemia by engagement of cell-autonomous S1P provision. Disrupting this pathway by EC-selective deficiency in S1P production, export, or the S1P 1 receptor substantially exacerbates brain injury in permanent and transient models of ischemic stroke. By contrast, profound lymphopenia induced by loss of lymphocyte S1P 1 provides modest protection only in the context of reperfusion. In the ischemic brain, EC S1P 1 supports blood-brain barrier (BBB) function, microvascular patency, and the rerouting of blood to hypo-perfused brain tissue through collateral anastomoses. Selective S1P 1 agonism counteracts cortical infarct expansion after middle cerebral artery occlusion by engaging the endothelial receptor pool after BBB penetration. Conclusions: This study provides genetic evidence to support a pivotal role for the endothelium in maintaining perfusion and microvascular patency in the ischemic penumbra that is coordinated by S1P signaling and can be harnessed for neuroprotection with BBB-penetrating S1P 1 agonists.

Objective: Muse cells reside as pre-existing pluripotent-like stem cells within the fibroblasts, are nontumorigenic, exhibit differentiation capacity into triploblastic-lineage cells, and replenish lost cells when transplanted in injury models. Cell fate and function of human skin fibroblast-derived Muse cells were evaluated in a rat stroke model. Methods: Muse cells (30,000), collected by pluripotent surface marker stage-specific embryonic antigen-3, were injected stereotaxically into three deposits within the rat ischemic cortex at 2 days after transient middle cerebral artery occlusion, and the cells' biological effects were examined for more than 84 days. Results: Muse cells spontaneously and promptly committed to neural/ neuronal-lineage cells when cocultured with stroke brain slices. Muse-transplanted stroke rats exhibited significant improvements in neurological and motor functions compared to control groups at chronic days 70 and 84, without a reduction in the infarct size. Muse cells survived in the host brain for up to 84 days and differentiated into NeuN (~65%), MAP-2 (~32%), calbindin (~28%), and GST-p (~25%)-positive cells in the cortex, but glial fibrillary acidic proteinpositive cells were rare. Tumor formation was not observed. Muse cells integrated into the sensory-motor cortex, extended their neurites into cervical spinal cord, and displayed normalized hind limb somatosensory evoked potentials. Interpretation: Muse cells are unique from other stem cells in that they differentiate with high ratio into neuronal cells after integration with host brain microenvironment, possibly reconstructing the neuronal circuit to mitigate stroke symptoms. Human fibroblast-derived Muse cells pose as a novel source of transplantable stem cells, circumventing the need for gene manipulations, especially when contemplating autologous cell therapy for stroke. STEM CELLS 2016;34:160-173 SIGNIFICANCE STATEMENTHuman dermal fibroblast-derived Muse cells have practical advantages for regenerative medicine; they are non-tumorigenic, easily accessed both commercially and from skin biopsies of patients, and can be easily collected as SSEA-3(1) cells either by FACS or magnetic-activated cell sorting. Most importantly, Muse cells do not need to be induced to attain stemness and to commit into specific lineages prior to transplantation, because they already display inherent stem cell properties upon isolation, and after transplantation spontaneously home into the damaged site and differentiate into cells compatible with the target tissue, rendering the need for gene manipulation and intricate cell induction protocols unnecessary. As we envision an autologous stem cell product in the clinic, Muse cells will simply involve a two-step process, namely routine collection from fibroblasts and immediate transplantation to the patient. Finally, with the present functional outcomes achieved with only 30,000 Muse cells, such low effective cell dose indicates excellent therapeutic potency of Muse cells compared to general MSC ...

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A novel type of non-tumorigenic pluripotent stem cell, the Muse cell (multi-lineage, differentiating stress enduring cell), resides in the connective tissue and in cultured mesenchymal stem cells (MSCs) and is reported to differentiate into multiple cell types according to the microenvironment to repair tissue damage. We examined the efficiency of Muse cells in a mouse intracerebral hemorrhage (ICH) model. Seventy μl of cardiac blood was stereotactically injected into the left putamen of immunodeficient mice. Five days later, 2 × 10 of human bone marrow MSC-derived Muse cells (n = 6) or cells other than Muse cells in MSCs (non-Muse, n = 6) or the same volume of PBS (n = 11) was injected into the ICH cavity. Water maze and motor function tests were implemented for 68 days, and immunohistochemistry for NeuN, MAP2 and GFAP was done. The Muse group showed impressive recovery: Recovery was seen in the water maze after day 19, and motor functions after 5 days was compared with the other two groups, with a significant statistical difference (p < 0.05). The survival rate of the engrafted cells in the Muse group was significantly higher than in the non-Muse group (p < 0.05) at day 69, and those cells showed positivity for NeuN (~57%) and MAP-2 (~41.6%). Muse cells could remain in the ICH brain, differentiate into neural-lineage cells and restore functions without inducing them into neuronal cells by gene introduction and cytokine treatment prior to transplantation. A simple collection of Muse cells and their supply to the brain in naïve state facilitates regenerative therapy in ICH.

Neural stem/progenitor cells (NSPCs) persist over the lifespan while encountering constant challenges from age or injury related brain environmental changes like elevated oxidative stress. But how oxidative stress regulates NSPC and its neurogenic differentiation is less clear. Here we report that acutely elevated cellular oxidative stress in NSPCs modulates neurogenic differentiation through induction of Forkhead box protein O3 (FOXO3)-mediated cGAS/STING and type I interferon (IFN-I) responses. We show that oxidative stress activates FOXO3 and its transcriptional target glycine-N-methyltransferase (GNMT) whose upregulation triggers depletion of s-adenosylmethionine (SAM), a key co-substrate involved in methyl group transfer reactions. Mechanistically, we demonstrate that reduced intracellular SAM availability disrupts carboxymethylation and maturation of nuclear lamin, which induce cytosolic release of chromatin fragments and subsequent activation of the cGAS/STING-IFN-I cascade to suppress neurogenic differentiation. Together, our findings suggest the FOXO3-GNMT/SAM-lamin-cGAS/STING-IFN-I signaling cascade as a critical stress response program that regulates long-term regenerative potential.

A B S T R A C TObjectives: The aim of this study was to describe the emergence in Nepal of clinical isolates of Klebsiella pneumoniae harboring both bla NDM-5 and bla OXA-181/-232 . Methods: Six clinical isolates of K. pneumoniae highly resistant to carbapenems and aminoglycosides were obtained from inpatients in Nepal. Their whole genomes were sequenced by a next generation sequencer.Results: The minimum inhibitory concentrations of meropenem, amikacin and ciprofloxacin were 128 m g/ml, >1024 mg/ml and 256 mg/ml, respectively. All six isolates co-harbored bla NDM-5 , bla OXA-181 or -232 and rmtB. Of them, 1 also harbored rmtF. The bla NDM-5 , bla OXA-232 and rmtB in all six isolates were located on plasmids. Of the six isolates tested, one isolate harbored two copies of bla OXA-181 and rmtF on the chromosome. Conclusions: This is the first report of clinical isolates of K. pneumoniae co-harboring bla NDM-5 , bla OXA-181 or -232 and rmtB in Nepal. These strains were highly carbapenem-and aminoglycoside-resistant, and belonged to ST147 or ST395. Of them, ST147 isolate harbored two copies of bla OXA-181 on the chromosome.

The emergence of multidrug-resistant (MDR) Pseudomonas aeruginosa has become a serious worldwide medical problem. This study was designed to clarify the genetic and epidemiological properties of MDR P. aeruginosa strains isolated from hospitals in Myanmar. Forty-five MDR P. aeruginosa isolates obtained from different patients in seven hospitals in Myanmar were screened using the broth microdilution method. The whole genomes of the MDR isolates were sequenced using a MiSeq platform (Illumina). Phylogenetic trees were constructed from single nucleotide polymorphism concatemers. Multilocus sequence types were deduced, and drug resistance genes were identified. Of the 45 isolates, 38 harbored genes encoding carbapenemases, including DIM-1, IMP-1, NDM-1, VIM-2, and VIM-5, and 9 isolates had genes encoding 16S rRNA methylases, including RmtB, RmtD3, RmtE, and RmtF2. Most MDR P. aeruginosa strains isolated in Myanmar belonged to sequence type 1047 (ST1047). This is the first molecular epidemiological analysis of MDR P. aeruginosa clinical isolates in Myanmar. These findings strongly suggest that P. aeruginosa ST1047 strains harboring carbapenemases, including DIM-, IMP-, NDM-, and VIM-type metallo-␤lactamases, have been spreading throughout medical settings in Myanmar.