Download Induction of Glial Phenotypes in Human Neural Stem Cells

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
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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
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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.
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Early NSCs Are FGF2-Responsive and
Neuronal; Later NSCs Are EGFResponsive and Glial
1.
2.
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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
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Post-mitotic Neurons/
MAP2A/B
Astrocytes/GFAP
Oligodendrocytes/
bGalactocerebroside
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Gene and Protein Characterization
of Neural Progenitor Cells
V or C Myc Transgene Pos
Cobblestone Morphology
30 hour doubling time
Nestin Positive Neural
Progenitors
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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
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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.
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Multipotential Phenotypes from ReNcell
VM

Scale = 100um
Neuron
βIII-tubulin (green)
Astrocytes
βIII-tubulin (green)
GFAP-(red)
Oligodendrocyte
O1 (green)
Oligodendrocyte
GalC( green)
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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
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ReNcell VM can differentiate
into TH+ neurons-2
βIII Tubulin
Tyrosine Hydroxylase
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Two Different Ways to Culture
Neural Stem Cells
Neurospheres in
suspension
culture
Monolayers in
adherent culture
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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)
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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
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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
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Membrane Based Co-Culture
Well of Tissue Culture Plate
Cell Culture Insert
Cells
Microporous Membrane
Media
Media
Feeder Cells / Co-culture
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Differentiation of ES/iPS Cells
A1
A
B
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Differentiation of ES/iPS Cells:
Hanging Drop Culture
Plate Lid
Suspended ESCs
In 20ul Media
After 1-2 days
Embryoid Body of
Defined Size
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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
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A General Multi-Stage Process for the ES
 NSC Switch
Astrocytes
Oligodendro
cytes
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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
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Neuron
GAPDH
Iacovetti, L 2007
Specific Antigens May Be Used to Track Glial and
Neuronal 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
DAPI
TUJ
TUJ
TUJ
Hb9
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DAPI
ChAT
DAPI
TUJ
DAT
DAPI
GABA
TUJ
Human Neural Stem Cells Are Inherently
Astrocytic and Neurogenic
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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
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Induction of Neurogenesis Observed in
Neurospheres
Alpha Internexin (C Untreated, D Treated)
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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
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Neri, M. et al. 2010. PLoS ONE 5(4).
Primed hNSCs Generate Greater Numbers of
Oligodendrocytes Following Directed Differentiations
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Neri, M. et al. 2010. PLoS ONE 5(4).
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
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!
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Human Oligodendrocytes Derived from OPCs
Myelinate Neuronal Axons
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Michael L. Moeller, MS, PhD
Field Application Scientist III
Bioscience Division
EMD Millipore
A Division of Merck KGaA
Darmstadt Germany
[email protected]