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Induction of Neuronal and Glial Phenotypes in Human Neural Stem Cells Michael L. Moeller, MS, PhD Field Application Scientist III Bioscience Division EMD Millipore A Division of Merck KGaA Darmstadt Germany The Subventricular Zone (SVZ) Is The Source of New Neurons For The Olfactory Bulb 2 Presentation title in footer | 00 Month 0000 www.crulrg.ulaval.ca “Neural Stem Cells” (NSCs) Are Multipotent Progenitor Cells Present in CNS Germinal Zones Such As The SVZ NSCs Neurons www.nih.gov Astrocytes www.sciencematter.files.worldpress.com Information exchange; processing Metabolic support; of information wound healing; volume regulation 3 Presentation title in footer | 00 Month 0000 Oligodendrocytes www.udel.edu Insulation of neurons The Cells of the SVZ Are Organized As “Cell Nests”, Which Have Distinctive Architectures Glioblasts- Type B Neuroblasts- Type A Uncommitted Progenitors- Type C Transit-Amplifying Cells Ependymal Surface of Ventricles Wood, H. 2004. Nature Reviews Neuroscience 5. 4 Presentation title in footer | 00 Month 0000 Early NSCs Are FGF2-Responsive and Neuronal; Later NSCs Are EGFResponsive and Glial 1. 2. 5 Presentation title in footer | 00 Month 0000 1. Temple, S. 2001. Nature 414(6859). 2. Bertrand, N. et al. 2002. Nature Reviews Neuroscience 3(7). Neural Progenitor Isolation Differentiate with RA CX VM Expand using bFGF & EGF Multipotent Neural Progenitors/Nestin 6 Presentation title in footer | 00 Month 0000 Post-mitotic Neurons/ MAP2A/B Astrocytes/GFAP Oligodendrocytes/ bGalactocerebroside 18 Gene and Protein Characterization of Neural Progenitor Cells V or C Myc Transgene Pos Cobblestone Morphology 30 hour doubling time Nestin Positive Neural Progenitors 7 ReNcell human neural stem cell lines • Cultured in defined, serum-free medium • CX and VM cells are grown as monolayers • Display “cobblestone” morphology • Rapid doubling time: 24-48 hrs • Express NSC markers • Retain normal diploid karyotype > 45 passages Normal Male Karyotype Is Observed in ReNcell VM Source: Dr. Carol Tang, National Neuroscience Institute, Singapore 8 19 ReNCell VM Default Differentiation A. C. B. Transgelin (TAGL2)- member of calponin family of cytoskeletal proteins; associated with actin polymerization; 3fold upregulation after 4 days of differentiation. 9 Multipotential Phenotypes from ReNcell VM Scale = 100um Neuron βIII-tubulin (green) Astrocytes βIII-tubulin (green) GFAP-(red) Oligodendrocyte O1 (green) Oligodendrocyte GalC( green) 10 Donato et. al BMC Neuroscience 2007 ReNcell VM can differentiate into TH+ neurons TH (Red) bIII Tubulin (Green) Differentiation using Pre-Aggregation Differentiation protocol Induce differentiation with 1 mM dibutyrl-cAMP & 2ng/mL GDNF High numbers of TH+ neurons derived from ReNcell VM but not CX. Appears that the brain region from where ReNcells were derived affects differentiation capacities 11 22 ReNcell VM can differentiate into TH+ neurons-2 βIII Tubulin Tyrosine Hydroxylase 12 Two Different Ways to Culture Neural Stem Cells Neurospheres in suspension culture Monolayers in adherent culture 13 Presentation title in footer | 00 Month 0000 ReNCell CX, p6 ReNCell VM, p6 Isolation of ES Cells Isolation/ Generation Culture/ Characterization Differentiation Isolation of Embryonic Stem Cells (ES) Fertilized Oocyte – donated from In Vitro Fertilization (IVF) Inner cell mass from blastocyst –isolated by immunosurgery Plate isolated cells on Mouse Embryonic Fibroblasts (MEFs) NIH Approved ES Cell Lines: – H1, H13, and H14 – normal XY Karyotype – H7 and H9 – normal XX Karyotype • Thomson et al. 1998 (Wisconsin – Madison) 14 Purification Delivery Traditional Human ES Culture Well of Tissue Culture Plate Knock Out Serum replacement (KOSR) Basic Fibroblast Growth Factor (bFGF) ES Cells Media Media Mouse or Human Feeder Cells 15 Embryonic Stem Cell Feeder Free Culture Well of Tissue Culture Plate Conditioned Media – from feeder cells ES Cells Media Media Matrigel or Other ECM Coating 16 Membrane Based Co-Culture Well of Tissue Culture Plate Cell Culture Insert Cells Microporous Membrane Media Media Feeder Cells / Co-culture 17 Differentiation of ES/iPS Cells A1 A B 18 Differentiation of ES/iPS Cells: Hanging Drop Culture Plate Lid Suspended ESCs In 20ul Media After 1-2 days Embryoid Body of Defined Size 19 Early Derivation of Human Neural Progenitor Cells from 3D Neurospheres Early derivation protocols require: – Progression through EB step in serum-containing medium – End result is free-floating cell aggregates or neurospheres “In vitro differentiation of transplantable neural precursors from human embryonic stem cells” Zhang et al., 2001 Nat. Biotechnology “Neural progenitors from human embryonic stem cells” Reubinoff et al., 2001 Nat. Biotechnology 20 Presentation title in footer | 00 Month 0000 25 A General Multi-Stage Process for the ES NSC Switch Astrocytes Oligodendro cytes 21 Presentation title in footer | 00 Month 0000 Adapted from Iacovitti, L. et.al. Brain Res. 2007 Jan 5;1127(1) 19-25 Stage Specific Gene Expression of human ES Derived Primary Neural Progenitor Cells EB rosette formation Undif. hNCPs markers Tut4 Oct 4 Sox-2 Nestin BtubIII Nurr-1 Endo Ecto a-feto Ptx3 Lmxb1 AADC Mature markers for DA phenotype TH GIRK2 DAT HNF-3 Meso GATA-2 Mix-1 GAPDH Endo Epith Keratin-2 ES NSC 22 Presentation title in footer | 00 Month 0000 Neuron GAPDH Iacovetti, L 2007 Specific Antigens May Be Used to Track Glial and Neuronal Development GFAP (Glial Fibrillary Acidic Protein) is major marker. 23 Presentation title in footer | 00 Month 0000 Differentiation of ENStem-A Cells into All Neural Cell Types in vitro DAPI TUJ TUJ TUJ Hb9 24 DAPI ChAT DAPI TUJ DAT DAPI GABA TUJ Human Neural Stem Cells Are Inherently Astrocytic and Neurogenic 25 Presentation title in footer | 00 Month 0000 Moeller et al., Presented at the ISSCR, 2008. Neuronal and Glial Induction Tips • NEURONS: • Program EBs in 20ng/ml EGF + 20ng/ml FGF2 and switch to 1-5ng/ml FGF2 upon plating • Switch to 10ng/ml NT-3 + 10-20ng/ml BDNF + 0.5uM Retinoic Acid upon plating • ASTROCYTES: • Program EBs in 20ng/ml EGF and switch to 10ng/ml CNTF + 10ng/ml BMP-4 upon plating 26 16 Induction of Neurogenesis Observed in Neurospheres Alpha Internexin (C Untreated, D Treated) 27 Transfection Reagents – The Gene Delivery Tools | April 30, 2017 Moeller and Dimitrijevich, 2004, JNM. FGF2, PDGF-AA, and NT-3 May Be Used to Increase OPC Generation 28 Presentation title in footer | 00 Month 0000 Neri, M. et al. 2010. PLoS ONE 5(4). Primed hNSCs Generate Greater Numbers of Oligodendrocytes Following Directed Differentiations 29 Presentation title in footer | 00 Month 0000 Neri, M. et al. 2010. PLoS ONE 5(4). Human Oligodendrocyte Progenitors Efficiently Derived at EMD Millipore 30 3 Presentation title in footer | 00 Month 0000 “Default Differentiation” of OPCs Yields Oligodendrocytes and Neurons, But Not Astrocytes 31 3 Presentation title in footer | 00 Month 0000 Significant Expansion of Human OPCs May Be Accomplished 32 3 Presentation title in footer | 00 Month 0000 Millipore’s Oligodendrocyte Differentiation Kit Allows for Superior Numbers of Non-Immortalized Oligodendrocyte Progenitors to Be Generated Cell sorting based on surface antigens (O4, GalC) Large yield of nearly pure human oligodendrocyte Progenitors Distinct bipolar morphologies of cells GalC+/O4+ Capable of generating myelin in myelination assays FEW TO NO GFAP+ ASTROCYTES! 33 Presentation title in footer | 00 Month 0000 Human Oligodendrocytes Derived from OPCs Myelinate Neuronal Axons 34 3 Presentation title in footer | 00 Month 0000 Michael L. Moeller, MS, PhD Field Application Scientist III Bioscience Division EMD Millipore A Division of Merck KGaA Darmstadt Germany [email protected]