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

2 Presentation title in footer | 00 Month 0000

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

3 Presentation title in footer | 00 Month 0000

““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

4 Presentation title in footer | 00 Month 0000

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

5 Presentation title in footer | 00 Month 0000

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.

6 Presentation title in footer | 00 Month 0000

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

18

7

Gene and Protein Characterization of Neural Progenitor Cells

Nestin Positive Neural Progenitors

Cobblestone Morphology

V or C Myc Transgene Pos

30 hour doubling time

8

Normal Male Karyotype Is Observed in ReNcell VM

Source: Dr. Carol Tang, National Neuroscience Institute, Singapore

19

• 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

9

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.

10

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

11

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

22

ReNcell VM can differentiate into TH+ neurons

12

ReNcell VM can differentiate ReNcell VM can differentiate into TH+ neurons-2into TH+ neurons-2

ββIII TubulinIII TubulinTyrosine HydroxylaseTyrosine Hydroxylase

13 Presentation title in footer | 00 Month 0000

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

14

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)

15

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)

16

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

17

Membrane Based Co-Culture

Cells

Cell Culture Insert

Well of Tissue Culture Plate

Microporous Membrane

Media

Feeder Cells / Co-culture

Media

18

Differentiation of ES/iPS Cells

A A1

B

19

Differentiation of ES/iPS Cells: Hanging Drop Culture

After 1-2 days

Suspended ESCsIn 20ul Media

Embryoid Body ofDefined Size

Plate Lid

20 Presentation title in footer | 00 Month 0000

“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

25

Early Derivation of Human Neural Early Derivation of Human Neural Progenitor Cells from 3D NeurospheresProgenitor Cells from 3D Neurospheres

21 Presentation title in footer | 00 Month 0000

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

22 Presentation title in footer | 00 Month 0000

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

23 Presentation title in footer | 00 Month 0000

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.

24

Differentiation of ENStem-A Cells into All Neural Cell Types in vitro

DAPITUJ

DAPIChAT

TUJHb9 GABA

DAPI

TUJTUJDAPI

DAT

TUJ

25 Presentation title in footer | 00 Month 0000

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.

26 16

• 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

27

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)

28 Presentation title in footer | 00 Month 0000

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).

29 Presentation title in footer | 00 Month 0000

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).

30 Presentation title in footer | 00 Month 0000

30

Human Oligodendrocyte Progenitors Efficiently Derived at EMD Millipore

31 Presentation title in footer | 00 Month 0000

31

“Default Differentiation” of OPCs Yields Oligodendrocytes and Neurons, But Not Astrocytes

32

Significant Expansion of Human OPCs May Be Accomplished

Presentation title in footer | 00 Month 0000

32

33 Presentation title in footer | 00 Month 0000

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!

34

Human Oligodendrocytes Derived from OPCs Myelinate Neuronal Axons

Presentation title in footer | 00 Month 0000

34

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

michael.moeller@merckgroup.com