Although not tested here, it is assumed that exogenous cells also proceed to functional integration into the host pan\glial network during repair. It is known that Cx43/Cx47 co\expression enables astrocytes to fuel oligodendrocytes the necessary energy supply through GJ\mediated communication (Giaume, Leybaert, Naus, & Saez, 2013; Hirrlinger & Nave, 2014). Hepes (5?mM), FGF2 (20?ng/ml), and EGF (20?ng/ml). NPCs were dissociated once a week and reseeded at the density of 106 cells/T75 flask in 10 ml medium. Immunocytochemistry showed that miPS\NPCs similar to their embryonic counterparts expressed essentially Olig2 (97%), nestin (98%), and Ki67 (71%) while they were negative for OPC markers PDGFR and O4 or neuronal marker MAP2 in vitro, before engraftment (Mozafari et Ginsenoside F3 al., 2015). For in vivo cell tracking, NPCs were transduced with a third\generation lentiviral vector encoding the green fluorescent protein GFP. More than 80% of the cells Ginsenoside F3 were labeled with this method (Laterza et al., 2013). 2.1.3. Human iPSC\OPCs The methods to generate and maintain the human OPCs from iPSCs were previously described (Ehrlich et al., 2017). Briefly, iPSCs were differentiated into NPCs by treatment with small molecules as described (Ehrlich et al., 2017; Reinhardt et al., 2013). Differentiation of NPCs into O4+ oligodendroglial cells was achieved with a poly\cistronic lentiviral vector containing the coding regions of the human transcription factors SOX10, OLIG2, and NKX6.2 (SON) followed by an IRES\pac cassette allowing puromycin selection for 16?hr (Ehrlich et al., 2017). Human NPCs were seeded at 1.5 ?105 cells/well in 12\well plates, allowed to attach overnight and transduced with SON lentiviral particles and 5 g/ml protamine sulfate in fresh NPC medium. After extensive washing, viral medium was replaced with glial induction medium (GIM). GIM was replaced after 4?days with differentiation medium. After 12?days of differentiation, cells were dissociated by accutase treatment for 10 min at 37C, washed with PBS, resuspended in PBS/0.5% BSA buffer, and singularized cells filtered through a 70?m cell strainer (BD Falcon). Cells were incubated with mouse IgM anti\O4\APC antibody (Miltenyi Biotech) following the manufacturer’s protocol, washed, resuspended in PBS/0.5% BSA buffer (5 ?106 cells/ml), and purified by magnetic cell sorting using anti\O4 MicroBeads (Miltenyi Biotec) following the manufacturer’s protocol. Media details were provided in Ehrlich et al. (2017). In vitro characterization by immunocytochemistry revealed that the human iPSC\OPCs were NG2+ and highly expressed GALC and O4 (70%) after 28?days in Ginsenoside F3 vitro (Ehrlich et al., 2017). MACS\purified O4+ cells cultured for 14?days in vitro were used for in vivo studies. 2.2. Animals To study the dynamic expression of oligodendrocyte Cx47 following demyelination and remyelination after engraftment, we used two different immunodeficient mouse strains: nude mice with normal myelination and with dysmyelination (MBP deficient mice) backgrounds as previously published (Mozafari et al., 2015). were adult immunodeficient mice (=?20, 8C9?weeks of age, Janvier). Shiverer mice were crossed to Rag2 null immunodeficient mice (Shinkai et al., 1992) to generate a Ginsenoside F3 line of dysmyelinating immunodeficient Ginsenoside F3 mice (=?21, 8C9?weeks of age). Mice were housed under standard conditions of 12\hr light/12?hr dark with ad libitum access to dry food and water cycle at ICM institute’s animal facility. Experiments were performed according to European Community regulations and were approved by the National Ethic’s Committee (authorization 75\348; April 20, 2005) and local Darwin Ethic’s Committee. 2.3. Demyelination and cell transplantation To induce demyelination, mice were anaesthetized by intraperitoneal injection of a mixture of 100?mg/kg ketamine (Alcyon) and 10 mg/kg xylazine (Alcyon). Focal demyelination was performed as previously described (Blanchard et al., 2013; Buchet, Garcia, Deboux, Nait\Oumesmar, & Baron\Van Evercooren, 2011; Mozafari et al., 2015) by stereotaxic injection of 1 l of 1% LPC (Sigma\Aldrich) in 0.9% NaCl into the dorsal funiculus of the spinal cord at the level of the 13th thoracic vertebrate. Forty\eight hours after demyelination, mice received a single injection (1 l, 105/l) of GFP+ miPSC\NPCs (=?6/genotype) or mouse embryonic NPCs (mE\NPCs; =?6/genotype) or the same amount of medium (=?6/genotype) at the site of demyelination as previously described (Mozafari et al., 2015). All injections (LPC, medium or cells) were performed at low speed (1 l/2 min) using a stereotaxic frame equipped with a micromanipulator and a Hamilton syringe. For sacrifice, mice were perfused transcardially with a Rabbit Polyclonal to HTR7 solution of 1X PBS and 4% paraformaldehyde. Nude mice (=?18).
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- Each adjustable was stratified the following: 0: absent, or zero alterations; +: mild; ++: moderate; +++: intense
- Finish mounting quickly within 30 s?1 min
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