Progenitor-derived Oligodendrocyte Culture System from Human Fetal Brain

Summary

Primary, human fetal brain-derived, multipotential progenitor cells proliferate in vitro while maintaining the capacity to differentiate into neurons and astrocytes. This work shows that neural progenitors can be induced to differentiate through stages of the oligodendrocytic lineage by conditioning with select growth factors.

Abstract

Differentiation of human neural progenitors into neuronal and glial cell types offers a model to study and compare molecular regulation of neural cell lineage development. In vitro expansion of neural progenitors from fetal CNS tissue has been well characterized. Despite the identification and isolation of glial progenitors from adult human sub-cortical white matter and development of various culture conditions to direct differentiation of fetal neural progenitors into myelin producing oligodendrocytes, acquiring sufficient human oligodendrocytes for in vitro experimentation remains difficult. Differentiation of galactocerebroside⁺ (GalC) and O4⁺ oligodendrocyte precursor or progenitor cells (OPC) from neural precursor cells has been reported using second trimester fetal brain. However, these cells do not proliferate in the absence of support cells including astrocytes and neurons, and are lost quickly over time in culture. The need remains for a culture system to produce cells of the oligodendrocyte lineage suitable for in vitro experimentation.

Culture of primary human oligodendrocytes could, for example, be a useful model to study the pathogenesis of neurotropic infectious agents like the human polyomavirus, JCV, that in vivo infects those cells. These cultured cells could also provide models of other demyelinating diseases of the central nervous system (CNS). Primary, human fetal brain-derived, multipotential neural progenitor cells proliferate in vitro while maintaining the capacity to differentiate into neurons (progenitor-derived neurons, PDN) and astrocytes (progenitor-derived astrocytes, PDA) This study shows that neural progenitors can be induced to differentiate through many of the stages of oligodendrocytic lineage development (progenitor-derived oligodendrocytes, PDO). We culture neural progenitor cells in DMEM-F12 serum-free media supplemented with basic fibroblast growth factor (bFGF), platelet derived growth factor (PDGF-AA), Sonic hedgehog (Shh), neurotrophic factor 3 (NT-3), N-2 and triiodothyronine (T3). The cultured cells are passaged at 2.5e6 cells per 75cm flasks approximately every seven days. Using these conditions, the majority of the cells in culture maintain a morphology characterized by few processes and express markers of pre-oligodendrocyte cells, such as A2B5 and O-4. When we remove the four growth factors (GF) (bFGF, PDGF-AA, Shh, NT-3) and add conditioned media from PDN, the cells start to acquire more processes and express markers specific of oligodendrocyte differentiation, such as GalC and myelin basic protein (MBP). We performed phenotypic characterization using multicolor flow cytometry to identify unique markers of oligodendrocyte.

Protocol

Note: For routine culturing of neural progenitor and oligodendrocytic lineage cells, incubation is done at 37 °C in a humidified 5% CO₂ atmosphere. Every 2 days, the medium is replaced using 50 to 100% of fresh medium if culture is 40-70% confluent. At the time of near confluency, the cultures are passaged at 2-2.5e6/T75 flask usually on a weekly schedule.

1. Preparing the Coated Flask

1. To prepare coated flasks dilute 5 mg of poly-D-lysine (PDL) in 100 ml of deionized water (DI-water), then coat T75 flasks with 50 μg/ml of PDL at room temperature (RT) in the dark. After 1½ hr, aspirate th....

Results

It is very important to start the differentiation process from a 70%-80% confluent neural progenitor cell culture (Figure 1A). Many cells will die out after changing the culture medium from progenitor to oligo medium since it includes specific growth factors. This indicates that the growth of neural progenitor cells not committed to an oligodendrocytic phenotype will not be supported by the new medium (Figure 1B). Incubation in oligo medium + GF for one week resulted in an interm.......

Discussion

This protocol describes how to derive fetal oligodendrocytes from primary human neural progenitor cells and characterize their phenotype using both flow cytometry and immunofluorescence staining. The expansion and growth of neural progenitors from fetal CNS has been very well described^1-4. However, obtaining sufficient human oligodendrocytes for in vitro experimentation remains difficult, even though it is possible to identify and isolate glial precursors from adult human white matter^5-13

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DME/HAMS F12 1:1	Omega Scientist	DM-251	1X
Bovine Albumin	Sigma	A9418	1%
Gentamicin	Quality Biologicals	120-098-031	50 μg/ml
L-Glutamine	Quality Biologicals	118-084-061	2 mM
T3	Sigma	T2877	3 nM
N2 Components	Gibco BRL	17502	1:100
NT-3	PeproTech Inc	450-03	2 ng/ml
Shh	R&D System	1314-SH/CF	2 ng/ml
bFGF	PeproTech Inc	100-18B	20 ng/ml
PDGF-AA	PeproTech Inc	100-13A	10 ng/ml
PDL	Sigma	P6407	50 μg/m
PFA	Electron Microscopy Sciences	15712	2%
Trypsin	Quality Biologicals	118-087-721
Papain	Worthington	LK003178	20 U/ml
DNase vials	Worthington	LK003172	0.005%
EBSS	Worthington	LK003188
ProLong Gold with DAPI	Invitrogen	P36931
Table 1. Reagents.
Primary Abs(all at 1 μg/ml) Species Isotype Source Secondary Abs Flow Cytometry A2B5-Biotin Mouse IgM Gift J. Nielson Streptavidin PETR, Invitrogen, CA O4FITC Mouse IgM Gift from J. Nielson A2B5 Mouse IgM Millipore, MA gαm IgM-FITC, Invitrogen, CA O4 Mouse IgM Millipore, MA gαm IgM-FITC, Invitrogen, CA GalC Mouse IgG3 Millipore, MA gαm IgG3-PE, Southern Biotech, AL MBP Chicken IgY Millipore, MA dαck IgY-AMCA, Jackson Immu., PA Nestin Mouse IgG1 Messam et al. 200028 gαm IgG1-PECy5, Invitrogen, CA GFAP Rabbit IgG Millipore, MA gαrb IgG-PE, Jackson Immu, PA βIII tubulin Mouse IgG2 Covance, CA gαm IgG2a-PETR, Invitrogen, CA Immunocytochemistry βIII tubulin (1:1,500) Mouse IgG2a Covance, CA gαm IgG2a-FITC, Invitrogen, CA (1:1,000) GFAP (1:1,000) Rabbit IgG Millipore, MA gαrb IgG-FITC, Jackson Immu, PA (1:500) MBP (1:50) Chicken IgY Millipore, MA dαck IgY-FITC, Jackson Immu, PA (1:100) O4 (1:100) Mouse IgM Millipore, MA gαm IgM-AF546, Invitrogen, CA (1:100) GalC (1:10) Rabbit IgG Millipore, MA gαm IgM-AF750, Invitrogen, CA (1:100) Table 2. Antibodies (Abs) used for flow cytometry and immunocytochemistry assays. Antibody conjugates: PE, phyc–rythrin; PETR, phyc–rythrin Texas Red; AMCA, amino-methyl-coumarin-acetate; Cy, cyanine; FITC, fluorescein isothiocyanate. Ig: immunoglobulin. AF: Alexa Fluor; gαm: goat anti-mouse; gαrb: goat anti-rabbit; dαck: donkey anti-chicken.