Induction of Neuronal and Glial Induction of Neuronal and Glial Phenotypes in Human Neural Phenotypes in Human Neural Stem CellsStem CellsMichael L. Moeller, MS, PhDMichael L. Moeller, MS, PhD
Field Application Scientist IIIField Application Scientist III
Bioscience DivisionBioscience Division
EMD MilliporeEMD Millipore
A Division of Merck KGaAA Division of Merck KGaA
Darmstadt GermanyDarmstadt Germany
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The Subventricular Zone (SVZ) Is The Source of The Subventricular Zone (SVZ) Is The Source of New Neurons For The Olfactory BulbNew Neurons For The Olfactory Bulb
www.crulrg.ulaval.ca
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““Neural Stem Cells” (NSCs) Are Multipotent Progenitor Neural Stem Cells” (NSCs) Are Multipotent Progenitor Cells Present in CNS Germinal Zones Such As The SVZCells Present in CNS Germinal Zones Such As The SVZ
NSCs
Neurons Astrocytes Oligodendrocytes
www.nih.gov www.sciencematter.files.worldpress.com www.udel.edu
Information exchange; processing Metabolic support; Insulation of neurons
of information wound healing; volumeregulation
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The Cells of the SVZ Are Organized As “Cell The Cells of the SVZ Are Organized As “Cell Nests”, Which Have Distinctive ArchitecturesNests”, Which Have Distinctive Architectures
Wood, H. 2004. Nature Reviews Neuroscience 5.
Glioblasts- Type B
Neuroblasts- Type A
Uncommitted Progenitors- Type CTransit-Amplifying Cells
Ependymal Surface of Ventricles
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Early NSCs Are FGF2-Responsive and Early NSCs Are FGF2-Responsive and Neuronal; Later NSCs Are EGF-Neuronal; Later NSCs Are EGF-Responsive and GlialResponsive and Glial
1. Temple, S. 2001. Nature 414(6859).
2. Bertrand, N. et al. 2002. Nature Reviews Neuroscience 3(7).
1.
2.
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Expand using bFGF & EGF
Multipotent NeuralProgenitors/Nestin
Post-mitotic Neurons/MAP2A/B
Astrocytes/GFAP
Differentiate with RA
Oligodendrocytes/bGalactocerebroside
NeuralNeural Progenitor IsolationProgenitor Isolation
CX
VM
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Gene and Protein Characterization of Neural Progenitor Cells
Nestin Positive Neural Progenitors
Cobblestone Morphology
V or C Myc Transgene Pos
30 hour doubling time
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Normal Male Karyotype Is Observed in ReNcell VM
Source: Dr. Carol Tang, National Neuroscience Institute, Singapore
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• 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
ReNcell human neural stem cell lines
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ReNCell VM Default DifferentiationReNCell VM Default Differentiation
A. C.
B.
Transgelin (TAGL2)- member of calponin family of cytoskeletal proteins; associated with actin polymerization; 3-fold upregulation after 4 days of differentiation.
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Multipotential Phenotypes from ReNcell VM
Neuron
βIII-tubulin (green)
Oligodendrocyte
GalC( green)
Oligodendrocyte
O1 (green)
βIII-tubulin (green) Astrocytes
GFAP-(red)
Scale = 100um
Donato et. al BMC Neuroscience 2007
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TH (Red) III Tubulin (Green)
Differentiation using Pre-Aggregation Differentiation protocolInduce 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
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ReNcell VM can differentiate into TH+ neurons
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ReNcell VM can differentiate ReNcell VM can differentiate into TH+ neurons-2into TH+ neurons-2
ββIII TubulinIII TubulinTyrosine HydroxylaseTyrosine Hydroxylase
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Two Different Ways to Culture Two Different Ways to Culture Neural Stem CellsNeural Stem Cells
ReNCell CX, p6
ReNCell VM, p6
Neurospheres in suspension culture
Monolayers in adherent culture
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Isolation of ES Cells
Culture/Characterization
Differentiation Purification DeliveryIsolation/Generation
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)
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Traditional Human ES Culture
ES Cells
Well of Tissue Culture Plate
Media
Mouse or Human Feeder Cells
Media
Knock Out Serum replacement (KOSR)
Basic Fibroblast Growth Factor (bFGF)
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Embryonic Stem Cell Feeder Free Culture
ES Cells
Well of Tissue Culture Plate
Matrigel or Other ECM Coating
Media Media
Conditioned Media – from feeder cells
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Membrane Based Co-Culture
Cells
Cell Culture Insert
Well of Tissue Culture Plate
Microporous Membrane
Media
Feeder Cells / Co-culture
Media
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Differentiation of ES/iPS Cells
A A1
B
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Differentiation of ES/iPS Cells: Hanging Drop Culture
After 1-2 days
Suspended ESCsIn 20ul Media
Embryoid Body ofDefined Size
Plate Lid
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“Neural progenitors from human embryonic stem cells” Reubinoff et al., 2001 Nat. Biotechnology
“In vitro differentiation of transplantable neural precursors from human embryonic stem cells” Zhang et al., 2001 Nat. Biotechnology
Early derivation protocols require:
– Progression through EB step in serum-containing medium
– End result is free-floating cell aggregates or neurospheres
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Early Derivation of Human Neural Early Derivation of Human Neural Progenitor Cells from 3D NeurospheresProgenitor Cells from 3D Neurospheres
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A General Multi-Stage Process for the ES A General Multi-Stage Process for the ES NSC Switch NSC Switch
Adapted from Iacovitti, L. et.al. Brain Res. 2007 Jan 5;1127(1) 19-25
Astrocytes
Oligodendro cytes
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Stage Specific Gene Expression of human ES Derived Stage Specific Gene Expression of human ES Derived Primary Neural Progenitor CellsPrimary Neural Progenitor Cells
Iacovetti, L 2007
Meso
Ecto
Endo
Mature markers for DA phenotype
Undif. hNCPs markers
EB rosette formation
Meso
Endo
Epith
Mature markers for DA phenotype
Tut4
Oct 4
Sox-2
Nestin
BtubIII
Nurr-1
Ptx3
Lmxb1
AADC
TH
GIRK2
DAT
GAPDH
GATA-2
Mix-1
a-feto
HNF-3
Keratin-2
GAPDHES NSC Neuron
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Specific Antigens May Be Used to Track Glial and Specific Antigens May Be Used to Track Glial and Neuronal DevelopmentNeuronal Development
GFAP (Glial Fibrillary Acidic Protein) is major marker.
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Differentiation of ENStem-A Cells into All Neural Cell Types in vitro
DAPITUJ
DAPIChAT
TUJHb9 GABA
DAPI
TUJTUJDAPI
DAT
TUJ
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Human Neural Stem Cells Are Inherently Human Neural Stem Cells Are Inherently Astrocytic and NeurogenicAstrocytic and Neurogenic
Moeller et al., Presented at the ISSCR, 2008.
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• 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
Neuronal and Glial Induction Tips
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Induction of Neurogenesis Observed in Neurospheres
Transfection Reagents – The Gene Delivery Tools | April 21, 2023 Moeller and Dimitrijevich, 2004, JNM.
Alpha Internexin (C Untreated, D Treated)
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FGF2, PDGF-AA, and NT-3 May Be Used FGF2, PDGF-AA, and NT-3 May Be Used to Increase OPC Generationto Increase OPC Generation
Neri, M. et al. 2010. PLoS ONE 5(4).
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Primed hNSCs Generate Greater Numbers of Primed hNSCs Generate Greater Numbers of Oligodendrocytes Following Directed DifferentiationsOligodendrocytes Following Directed Differentiations
Neri, M. et al. 2010. PLoS ONE 5(4).
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Human Oligodendrocyte Progenitors Efficiently Derived at EMD Millipore
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“Default Differentiation” of OPCs Yields Oligodendrocytes and Neurons, But Not Astrocytes
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Significant Expansion of Human OPCs May Be Accomplished
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Millipore’s Oligodendrocyte Differentiation Kit Allows for Superior Millipore’s Oligodendrocyte Differentiation Kit Allows for Superior Numbers of Non-Immortalized Oligodendrocyte Progenitors to Be Numbers of Non-Immortalized Oligodendrocyte Progenitors to Be GeneratedGenerated
Cell sorting based on surface antigens (O4, GalC)
Large yield of nearly pure human oligodendrocyteProgenitorsDistinct bipolar morphologies of cellsGalC+/O4+Capable of generating myelin in myelination assaysFEW TO NO GFAP+ ASTROCYTES!
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Human Oligodendrocytes Derived from OPCs Myelinate Neuronal Axons
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Michael L. Moeller, MS, PhDMichael L. Moeller, MS, PhD
Field Application Scientist IIIField Application Scientist III
Bioscience DivisionBioscience Division
EMD MilliporeEMD Millipore
A Division of Merck KGaAA Division of Merck KGaA
Darmstadt Germany Darmstadt Germany