Yoshinori Okada

A gene encoding a putative GTPase regulator is mutated in familial amyotrophic lateral sclerosis 2

Amyotrophic lateral sclerosis 2 (ALS2) is an autosomal recessive form of juvenile ALS and has been mapped to human chromosome 2q33. Here we report the identification of two independent deletion mutations linked to ALS2 in the coding exons of the new gene ALS2. These deletion mutations result in frameshifts that generate premature stop codons. ALS2 is expressed in various tissues and cells, including neurons throughout the brain and spinal cord, and encodes a protein containing multiple domains that have homology to RanGEF as well as RhoGEF. Deletion mutations are predicted to cause a loss of protein function, providing strong evidence that ALS2 is the causative gene underlying this form of ALS.

Growth and differentiation potential of main- and side-population cells derived from murine skeletal muscle

Skeletal muscle-derived CD34+/45- (Sk-34) cells were identified as a new candidate for stem cells. However, the relationship between Sk-34 cells and side-population (SP) cells is unknown. Here, we demonstrate that Sk-34 cells prepared from murine skeletal muscles consist wholly of main-population (MP) cells. The Sk-34 cells included only a few SP cells (1:1000, SP:MP). Colony-forming units of Sk-34 cells of both SP and MP possessed the same potential to differentiate into adipocytes, endothelial, and myogenic cells and showed the same colony-forming activity (1.6%). In addition, the colony-forming units of the CD34-/45- (double negative: DN) population were found to begin CD34 expression and to possess the potential to differentiate into myogenic and endothelial cells. We also found that expression of CD34 antigen precedes MyoD expression during the myogenic process of DN cells. Furthermore, both Sk-34 and DN cell populations were mostly negative for CD73 (93-95%), whereas the CD45+ cell population was >25% positive for CD73, and this trend was also seen in bone marrow-derived CD45+ cells. These results indicate that the MP cell population is about 99.9% responsible for the reported in vitro myogenic-endothelial responses of skeletal muscle-derived cells.

Reduction of Immunocompetent T Cells Followed by Prolonged Lymphopenia in Severe Sepsis in the Elderly.

Objective:To investigate the immunological changes caused by severe sepsis in elderly patients. Design:One-year, prospective observational study. Setting:Emergency department and intensive care unit of a single university hospital. Patients:Seventy-three patients with severe sepsis and 72 healthy donors. Measurements and Main Results:In elderly septic patients (aged 65 yr and over), 3-month survival was significantly reduced compared with that for adult patients (18–64 yr) (60% vs. 89%, p < 0.01). We found that lymphopenia was prolonged for at least 21 days in elderly nonsurvivors of sepsis, while the number of lymphocytes recovered in both adult and elderly survivors of sepsis. In order to examine the immunological status of septic patients, blood samples were collected within 48 hrs of diagnosis of severe sepsis, and peripheral blood mononuclear cells were purified for flow cytometric analysis. T cell levels were significantly reduced in both adult and elderly septic patients, compared with those in healthy donors (56% and 57% reduction, respectively). Interestingly, the immunocompetent CD28+ subset of CD4+ T cells decreased, whereas the immunosuppressive PD-1+ T cells and the percentage of regulatory T cells (CD4+ T cells that are both Foxp3+ and CD25+) increased in elderly patients, especially nonsurvivors, presumably reflecting the initial signs of immunosuppression. Conclusion:Reduction of immunocompetent T cells followed by prolonged lymphopenia may be associated with poor prognosis in elderly septic patients.

Clonal multipotency of skeletal muscle-derived stem cells between mesodermal and ectodermal lineage.

The differentiation potential of skeletal muscle‐derived stem cells (MDSCs) after in vitro culture and in vivo transplantation has been extensively studied. However, the clonal multipotency of MDSCs has yet to be fully determined. Here, we show that single skeletal muscle‐derived CD34−/CD45− (skeletal muscle‐derived double negative [Sk‐DN]) cells exhibit clonal multipotency that can give rise to myogenic, vasculogenic, and neural cell lineages after in vivo single cell‐derived single sphere implantation and in vitro clonal single cell culture. Muscles from green fluorescent protein (GFP) transgenic mice were enzymatically dissociated and sorted based on CD34 and CD45. Sk‐DN cells were clone‐sorted into a 96‐well plate and were cultured in collagen‐based medium with basic fibroblast growth factor and epidermal growth factor for 14 days. Individual colony‐forming units (CFUs) were then transplanted directly into severely damaged muscle together with 1 × 105 competitive carrier Sk‐DN cells obtained from wild‐type mice muscle expanded for 5 days under the same culture conditions using 35‐mm culture dishes. Four weeks after transplantation, implanted GFP+ cells demonstrated differentiation into endothelial, vascular smooth muscle, skeletal muscle, and neural cell (Schwann cell) lineages. This multipotency was also confirmed by expression of mRNA markers for myogenic (MyoD, myf5), neural (Musashi‐1, Nestin, neural cell adhesion molecule‐1, peripheral myelin protein‐22, Nucleostemin), and vascular (α‐smooth muscle actin, smoothelin, vascular endothelial‐cadherin, tyrosine kinase‐endothelial) stem cells by clonal (single‐cell derived) single‐sphere reverse transcription‐polymerase chain reaction. Approximately 70% of clonal CFUs exhibited expression of all three cell lineages. These findings support the notion that Sk‐DN cells are a useful tool for damaged muscle‐related tissue reconstitution by synchronized vasculogenesis, myogenesis, and neurogenesis.

Cardiomyocyte Formation by Skeletal Muscle-Derived Multi-Myogenic Stem Cells after Transplantation into Infarcted Myocardium

BACKGROUNDnCellular cardiomyoplasty for myocardial infarction has been developed using various cell types. However, complete differentiation and/or trans-differentiation into cardiomyocytes have never occurred in these transplant studies, whereas functional contributions were reported.nnnMETHODS AND RESULTSnSkeletal muscle interstitium-derived CD34(+)/CD45(-) (Sk-34) cells were purified from green fluorescent protein transgenic mice by flowcytometory. Cardiac differentiation of Sk-34 cells was examined by in vitro clonal culture and co-culture with embryonic cardiomyocytes, and in vivo transplantation into a nude rat myocardial infarction (MI) model (left ventricle). Lower relative expression of cardiomyogenic transcription factors, such as GATA-4, Nkx2-5, Isl-1, Mef2 and Hand2, was seen in clonal cell culture. However, vigorous expression of these factors was seen on co-culture with embryonic cardiomyocytes, together with formation of gap-junctions and synchronous contraction following sphere-like colony formation. At 4 weeks after transplantation of freshly isolated Sk-34 cells, donor cells exhibited typical cardiomyocyte structure with formation of gap-junctions, as well as intercalated discs and desmosomes, between donor and recipient and/or donor and donor cells. Fluorescence in situ hybridization (FISH) analysis detecting the rat and mouse genomic DNA and immunoelectron microscopy using anti-GFP revealed donor-derived cells. Transplanted Sk-34 cells were incorporated into infarcted portions of recipient muscles and contributed to cardiac reconstitution. Significant improvement in left ventricular function, as evaluated by transthoracic echocardiography and micro-tip conductance catheter, was also observed.nnnCONCLUSIONS AND SIGNIFICANCEnSkeletal muscle-derived multipotent Sk-34 cells that can give rise to skeletal and smooth muscle cells as reported previously, also give rise to cardiac muscle cells as multi-myogenic stem cells, and thus are a potential source for practical cellular cardiomyoplasty.

Synchronized reconstitution of muscle fibers, peripheral nerves and blood vessels by murine skeletal muscle-derived CD34 − /45 − cells

In order to establish the practical isolation and usage of skeletal muscle-derived stem cells (MDSCs), we determined reconstitution capacity of CD34−/CD45− (Sk-DN) cells as a candidate somatic stem cell source for transplantation. Sk-DN cells were enzymatically isolated from GFP transgenic mice (C57/BL6N) skeletal muscle and sorted using fluorescence activated cell sorting (FACS), and expanded by collagen gel-based cell culture with bFGF and EGF. The number of Sk-DN cells was small after sorting (2–8xa0×xa0104); however, the number increased 10–20 fold (2–16xa0×xa0105) after 6xa0days of expansion culture, and the cells maintained immature state and multipotency, expressing mRNAs for mesodermal and ectodermal cell lineages. Transplantation of expanded Sk-DN cells into the severe muscle damage model (C57/BL6N wild-type) resulted in the synchronized reconstitution of blood vessels, peripheral nerves and muscle fibers following significant recovery of total muscle mass (57%) and contractile function (55%), whereas the non-cell-transplanted control group showed around 20% recovery in both factors. These reconstitution capacities were supported by the intrinsic plasticity of Sk-DN cells that can differentiate into muscular (skeletal muscle), vascular (pericyte, endothelial cell and smooth muscle) and peripheral nerve (Schwann cells and perineurium) cell lineages that was revealed by transplantation to non-muscle tissue (beneath renal capsule) and fluorescence in situ hybridization (FISH) analysis.

Overexpression of GRP78 protects glial cells from endoplasmic reticulum stress

Endoplasmic reticulum (ER) stress induces apoptotic cell death by causing the accumulation of structurally abnormal proteins. The 78-kDa glucose-regulated protein (GRP78) is an ER chaperone that regulates protein folding in the ER and has been suggested to contribute to cell survival. Using the rat C6 glioma cell line and flow cytometry, we assessed GRP78 expression following tunicamycin- and glutamate-induced ER stress. The results showed that GRP78 expression is upregulated following ER stress and has protective effects on injured glial cells. Annexin V and propidium iodide labeling revealed cells transiently expressing GRP78 prior to injury were protected against high-concentrations of tunicamycin and glutamate within 72 h. Our findings support the hypothesis that GRP78 inhibits cell death associated with ER stress.

A dopamine receptor antagonist L-745,870 suppresses microglia activation in spinal cord and mitigates the progression in ALS model mice.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by a selective loss of motor neurons in the motor cortex, brainstem, and spinal cord. It has been shown that oxidative stress plays a pivotal role in the progression of this motor neuron loss. We have previously reported that L-745,870, a dopamine D4 receptor antagonist, selectively inhibits oxidative stress-induced cell death in vitro and exerts a potent neuroprotective effect against ischemia-induced neural cell damage in gerbil. To investigate the efficacy of L-745,870 in the treatment of ALS, we here conducted a chronic administration of L-745,870 to transgenic mice expressing a mutated form of human superoxide dismutase gene (SOD1(H46R)); a mouse model of familial ALS, and assessed whether the mice benefit from this treatment. The pre-onset administration of L-745,870 significantly delayed the onset of motor deficits, slowed the disease progression, and extended a life span in transgenic mice. These animals showed a delayed loss of anterior horn cells in the spinal cord concomitant with a reduced level of microglial activation at a late symptomatic stage. Further, the post-onset administration of L-745,870 to the SOD1(H46R) transgenic mice remarkably slowed the disease progression and extended their life spans. Taken together, our findings in a rodent model of ALS may have implication that L-745,870 is a possible novel therapeutic means to the treatment of ALS.

A dopamine D4 receptor antagonist attenuates ischemia-induced neuronal cell damage via upregulation of neuronal apoptosis inhibitory protein

Neuronal apoptosis inhibitory protein (NAIP/BIRC1), the inhibitor of apoptosis protein (IAP) family member, suppresses neuronal cell death induced by a variety of insults, including cell death from ischemia and stroke. The goal of the present study was to develop an efficient method for identification of compounds with the ability to upregulate endogenous NAIP and to determine the effects on these compounds on the cellular response to ischemia. A novel NAIP-enzyme-linked immunosorbent assay (ELISA)-based in vitro drug-screening system is established. Use of this system identified an antagonist of dopamine D4 receptor, termed L-745,870, with a potent NAIP upregulatory effect. L-745,870-mediated NAIP upregulation in neuronal and nonneuronal cultured cells resulted in decreased vulnerability to oxidative stress-induced apoptosis. Reducing NAIP expression via RNA interference techniques resulted in prevention of L-745,870-mediated protection from oxidative stress. Further, systemic administration of L-745,870 attenuated ischemia-induced damage of the hippocampal CA1 neurons and upregulated NAIP expression in the rescued hippocampal CA1 neurons in a gerbil model. These data suggest that the NAIP upregulating compound, L-745,870, has therapeutic potential in acute ischemic disorders and that our NAIP-ELISA-based drug screening may facilitate the discovery of novel neuroprotective compounds.

Vasculogenic Conditioning of Peripheral Blood Mononuclear Cells Promotes Endothelial Progenitor Cell Expansion and Phenotype Transition of Anti-Inflammatory Macrophage and T Lymphocyte to Cells With Regenerative Potential

Background Cell‐based therapies involving mononuclear cells (MNCs) have been developed for vascular regeneration to treat ischemic diseases; however, quality control of therapeutic MNCs has not been evaluated. We investigated the therapeutic potential of peripheral blood (PB) MNCs, operated by recently developed quality and quantity (QQ) culture of endothelial progenitor cells (EPCs). Methods and Results PBs were collected from healthy volunteers; peripheral blood mononuclear cells (PBMNCs) isolated from these PBs were subjected to QQ culture for 7 days with medium containing stem cell factor, thrombopoietin, Flt‐3 ligand, vascular endothelial growth factor, and interleukin‐6. The resulting cells (QQMNCs) in EPC colony‐forming assay generated significantly more definitive EPC colonies than PBMNCs. In flow cytometry, macrophages and helper T lymphocytes of QQMNCs became phenotypically polarized into angiogenic, anti‐inflammatory, and regenerative subsets: classical M1 to alternative M2; T helper (Th)1 to Th2; angiogenic or regulatory T‐cell expansion. Quantitative real‐time polymerase chain reaction (qRT‐PCR) assay revealed the predominant proangiogenic gene expressions in QQMNCs versus PBMNCs. Using murine ischemic hindlimb models, the efficacy of QQMNC intramuscular transplantation (Tx) was compared to that of PBMNCTx, cultured “early EPC” Tx (eEPCTx), and granulocyte colony‐stimulating factor mobilized CD34+ cell Tx (GmCD34Tx). Laser Doppler imaging revealed the blood perfusion recovery in ischemic hindlimbs after QQMNCTx superior to after PBMNCTx and eEPCTx, but also earlier than after GmCD34Tx. Histological evaluations and qRT‐PCR assays in ischemic hindlimbs demonstrated that QQMNCTx, similarly to GmCD34Tx, enhanced angiovasculogenesis and myogenesis, whereas it preponderantly inhibited inflammation and fibrosis versus PBMNCTx and eEPCTx. Conclusions QQ culture potentiates the ability of PBMNCs to promote regeneration of injured tissue; considering the feasible cell preparation, QQ culture‐treated PBMNCs may provide a promising therapeutic option for ischemic diseases. Clinical Trial Registration URL: irb.med.u-tokai.ac.jp/d/2/monthly/2010.html; IRB No.: 10R‐020. URL: irb.med.u-tokai.ac.jp/d/2/monthly/201312.html; IRB No.: 13R228.