Laser Capture Microdissection of Neurons from Differentiated Human Neuroprogenitor Cells in Culture

Summary

Human neuroprogenitor cells (NPCs) were expanded under proliferating conditions. NPCs were differentiated into neuron-rich cultures in the presence of a combination of neurotrophins. Neuronal markers were detected by immunofluorescence staining. To isolate a pure population of neurons, NPCs were differentiated on PEN membrane slides and laser capture microdissection was performed.

Abstract

Neuroprogenitor cells (NPCs) isolated from the human fetal brain were expanded under proliferative conditions in the presence of epidermal growth factor (EGF) and fibroblast growth factor (FGF) to provide an abundant supply of cells. NPCs were differentiated in the presence of a new combination of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), dibutyryl cAMP (DBC) and retinoic acid on dishes coated with poly-L-lysine and mouse laminin to obtain neuron-rich cultures. NPCs were also differentiated in the absence of neurotrophins, DBC and retinoic acid and in the presence of ciliary neurotrophic factor (CNTF) to yield astrocyte-rich cultures. Differentiated NPCs were characterized by immunofluorescence staining for a panel of neuronal markers including NeuN, synapsin, acetylcholinesterase, synaptophysin and GAP43. Glial fibrillary acidic protein (GFAP) and STAT3, astrocyte markers, were detected in 10-15% of differentiated NPCs. To facilitate cell-type specific molecular characterization, laser capture microdissection was performed to isolate neurons cultured on polyethylene naphthalate (PEN) membrane slides. The methods described in this study provide valuable tools to advance our understanding of the molecular mechanism of neurodegeneration.

Introduction

Life-long neurogenesis is known to occur in the subventricular zone of the lateral ventricles and in the subgranular layer of the dentate gyrus of the adult mammalian brain ¹. Neuroprogenitor cells (NPCs) that originate from these regions are multipotent cells that can differentiate into neurons, astrocytes and oligodendrocytes ². NPCs have generated interest because of their potential to be transplanted in patients with various neurodegenerative disorders including Parkinson’s disease, amyotrophic lateral sclerosis, stroke and Alzheimer’s disease (AD) ³. Studies with NPCs have generally focused on this transplantation angl....

Protocol

1. Expansion of Human NPCs (Figure 1)

1. Revive the frozen stock of human NPCs from fetal brain (Lonza, Walkersville, MD, USA) and culture them in suspension in T-75 flasks as neurospheres (Figure 1A) in neurobasal medium containing proliferation supplements, EGF (10 ng/ml) and FGF (10 ng/ml).
2. After 3 days in culture, transfer the neurospheres to a 15 ml tube and centrifuge at 500 rpm for 5 min.
3. Discard the supernatant leaving behind ~100 μl medium above the cell pellet and transfer into an Eppendorf tube. A 100 μl cell pellet is enough to split into 2 T-75 flasks. If less, reduce the number of f....

Results

Expansion of NPCs (Figure 1)

When neurospheres are broken down to a single cell suspension by trituration, the pipette tip needs to touch the bottom of the tube so that there is some resistance when the suspension is pipetted up and down. The number of times of trituration will vary between individuals and needs to be decided by trial and error by examining the resulting cell suspension under a microscope. It is critical to provide sufficient density of cell suspension because th.......

Discussion

We describe in this study a neuron-rich cell culture model by differentiation of self-renewing human neuroprogenitor cells and a method to isolate a pure population of neurons by laser capture microdissection. We have used a combination of NGF, BDNF, DBC and retinoic acid for neuronal differentiation of NPCs. DBC is used to activate CREB, a transcription factor that enhances neurogenesis ¹². Retinoic acid induces cell cycle exit and reduces the glial population ¹³. The method described in this study.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Neuroprogenitor cells (NPCs)	Lonza	PT-2599
Neurocult NS-A human basal medium	Stem cell technology	5750
Neurocult NS-A Proliferation supplement	Stem cell technology	5753
Neurocult NS-A Differentiation supplement	Stem cell technology	5754
B-27 Supplement (50x)	Invitrogen	17504-044
Human epidermal growth factor	Stem cell technology	2633
Fibroblast growth factor	Sigma	F0291
Brain Derived Neurotrophic Factor	Cell signaling	3897S
Nerve Growth Factor	Invitrogen	13257-019
Dibutyryl cyclic AMP	Sigma	D-0627
poly-L-lysine	Sigma	P-5899
Mouse laminin	Sigma	L-2020
Retinoic acid	Sigma	R-2625
STAT3 antibody	Cell signaling	9132
GFAP antibody	Cell signaling	3670
GAP43 antibody	Transduction laboratories	612262
BDNF antibody	Millipore	AB1534SP
Acetyl cholinesterase antibody	Santz cruz	Sc-11409
Synaptophysin antibody	Abcam	ab18008-50
NeuN antibody	Chemicon	MAB377
Synapsin antibody	Novus	NB300-104
Anti mouse FITC	Jackson Immuno	115-095-146
	Research Laboratories
Anti Rabbit Cy3	Jackson Immuno	711-165-152
	Research Laboratories
BSA	Sigma	A1653
Triton-X 100	Acros	21568-2500
Paraformaldehye	Fisher	4042
Coverslip (Big circle cover slip)	Fisherbrand	12-545-102
Mounting medium (Prolong Gold)	Invitrogen	P36930
Pen membrane	Applied biosystems	LCM0521
Histogene LCM frozen section staining kit	Applied biosystems	KIT0401
RiboAmp RNA Amplification kit	Applied biosystems	KIT0201
Picopure RNA Isolation kit	Applied biosystems	KIT0202
CapsureMacro LCM caps	Applied biosystems	LCM0211