1Laboratory of Molecular Medicine and Neuroscience, National Institute of Neurological Disorders and Stroke, National Institutes of Health, 2Laboratory of Neurophysiology, National Institute of Neurological Disorders and Stroke, National Institutes of Health
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Monaco, M. C. G., Maric, D., Bandeian, A., Leibovitch, E., Yang, W., Major, E. O. Progenitor-derived Oligodendrocyte Culture System from Human Fetal Brain. J. Vis. Exp. (70), e4274, doi:10.3791/4274 (2012).
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.
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
2. Starting Differentiation of Neural Progenitor Cells into Progenitor-derived Oligodendrocytes (PDO)
The protocol to isolate human CNS progenitor cells is described in a previous publication and it is not part of this protocol1. Human CNS progenitor cells were isolated from the telencephalon of an 8-week gestation fetal brain, obtained in accordance with NIH guidelines.
3. Final Step in the Differentiation of Oligodendrocytes
4. Flow Cytometry Assay
The flow cytometry assay compares the acquisition of oligodendrocyte markers during the differentiation process in relationship with those of their parental population.
5. Immunofluorescence Assay
Note: For immunofluorescence experiments, PDO are plated in oligo medium + GF or oligo medium - GF at 2.5e5 in 6 well plates coated with PDL. The cells are fixed in 2% PFA at various time points after withdrawal of growth factors . The antibody stain is performed on plastic 6 well plates instead of glass coverslips.
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 intermediate culture exhibiting a narrow, bipolar morphology (Figure 1B). The cells are kept in oligo medium + GF for 3-4 weeks (Figure 1C). Growth factor withdrawal from the culture medium resulted in progressively differentiated culture of cells with multiple processes (Figure 1D). Flow cytometry is used for quantitative representation of the data.
Figures 2A, 2B and 2C demonstrate that the majority of neural progenitors expressed the neuroepithelial stem cell marker nestin, whereas only small subpopulations expressed lineage-restrictive markers such as the astrocyte marker GFAP (Figure 2A), the neuronal marker class III β-tubulin (Figure 2B), or the oligodendrocyte marker O4 (Figure 2C). When culture medium was replaced with oligo medium + GF and cells were cultured for 1 week, decreased nestin expression and increased A2B5 expression can be observed (Figure 2D). A2B5 is a neuroglial precursor marker. In comparison, 2 weeks after medium replacement, nestin expression was decreased even further and A2B5 expression alone increased (Figure 2E) as well as the expression of another oligodendrocyte marker, O4 (Figure 2F). Two days post growth factor withdrawal, O4 expression increased as well as the expression of GalC, a late oligodendrocyte marker (Figure 2G). Six days post growth factor withdrawal, the co-expression of O4 and GalC increased (Figure 2H) and is comparable to the co-expression of GalC and MBP (Figure 2I). The multi-epitope immunostaining reveals that more than half of the cells in culture are expressing MBP (Figure 2J). Further evidence of PDO differentiation was characterized, using an immunofluorescence assay, by the temporal increase of MBP expression in these cells after growth factor withdrawal (Figure 3A-C). In summary, human fetal neural progenitor cells are able to proliferate in vitro while maintaining the capacity to differentiate into 3 major brain cell types (Figure 4).
Figure 1. Phase contrast microscopy of (A) neural progenitor cells cultured in progenitor medium; (B) neural progenitor cells grown in oligo medium + GF for a week exhibit an altered narrow, bipolar morphology; (C) differentiated progenitor-derived oligodendrocytes grown for 3 days in oligo medium after growth factor withdrawal exhibit oligodendrocyte morphology characterized by multiple processes. 20x magnification.
Figure 2. Flow cytometry analysis of neural progenitor cells co-expressing the precursor-cell marker Nestin (all vertical axes) with: (A) the astrocytic marker GFAP; (B) the neuronal marker β III tubulin; and (C) the oligodendrocytic marker O4; (D) neural progenitor cells grown for a week in oligo media + GF co-express nestin and A2B5; (E) nestin and A2B5 co-expression after two weeks of neural progenitors growth in oligo medium + GF (F) A2B5 and O4 co-expression after two weeks of neural progenitors growth in oligo medium+ GF; (G) two days after growth factor withdrawal, distinct oligodendrocyte markers of differentiation, O4 and GalC, are expressed. Six days after growth factor withdrawal (H) an increased proportion of cells are double-positive for O4 and GalC; (I) double-positive for GalC and MPB; and (J) double-positive for O4 and MBP. A-J are representative of four independent experiments. Click here to view larger figure.
Figure 3. Indirect immunofluorescence staining of progenitor-derived oligodendrocytes for MBP (A) 2 days, (B) 5 days, and (C, D) 9 days after growth factor withdrawal. (D) Represents the phase image of the 9 days culture in oligo medium - GF and (E) is an enlargement of the white box of the same image. (A, B) 20x magnification; (C, D) 32x magnification.
Figure 4. Schematic representation of our cell culture model. Primary, human fetal brain-derived, multipotential neural progenitor cells proliferate in vitro while maintaining their plasticity. Using different culture conditions, neural progenitor cells can differentiate into neurons (PDN), astrocytes (PDA) and oligodendrocyte (PDO), as shown by specific differentiation markers.
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 described1-4. 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 matter5-13. There have been different attempts in the development of various culture conditions to direct differentiation of fetal neural progenitors into myelin producing oligodendrocytes14-21. This protocol further describes nestin positive neural progenitor cells expressing the O4 marker (Figure 2) when grown for 3 weeks in a serum-free medium supplemented with select growth factors (PDGF-AA, bFGF, Shh, and NT-3), that are essential for the proliferation and survival of oligodendrocyte precursors22-24 . Removal of these growth factors in the O4+ cells resulted in further differentiation and expression of myelin components (galactocerebroside and MB). In addition, the expression of oligodendrocyte differentiation markers coincides with morphological changes from cells that at first appeared narrow and bipolar (Figure 1B) to cells that become multipolar with well-developed processes (Figure 1C). The removal of growth factors to further allow the final steps of differentiation was based on studies done both in mouse and human15,25. The presence of triiodothyronine (T3) is important for the survival and differentiation of oligodendrocytes26 while we observed that the removal of the four growth factors was necessary to the expression of final markers of differentiation. This was compared with cells kept in oligo medium + GF as a control.
We are not the first to describe the differentiation of multipotent neural progenitor cells to oligodendrocyte cells in response to signals such as Shh and bFGF24. Previous reports showed that cortical oligodendrogenesis begins around 10 weeks gestational age in humans24 with the capacity of human fetal oligodendrocytes to myelinate increasing proportionally with gestational age22. Differentiation of galactocerebroside+ and O4+ oligodendrocyte precursor cells from neural progenitor cells has been reported using second trimester fetal brain21,27. However, these cells do not proliferate in the absence of support cells including astrocytes and neurons, and are lost quickly over time in culture21. Our system supports the formation of GalC+ and MBP+ cells from human fetal culture from 8 weeks gestational age. Furthermore we described that the differentiated oligodendrocytes could be cultured in vitro without the need of supporting cells as astrocytes or neurons, although the co-cultivation with neurons could lengthen the survival of the differentiated oligodendrocytes. One more benefit of this protocol is the possibility to cultivate a large number of cells expressing the O4+ marker that can be grown and passaged in culture for weeks while maintaining their phenotype. At that moment those O4+ cells can be pushed further in the final step of differentiation when the growth factors are removed from the medium. The protocol outlined in this paper addresses the need for oligodendrocyte lineages suitable for in vitro experimentation. Moreover, we believe that the identification of specific factors that induce the differentiation cascade of neural progenitors towards progenitor-derived oligodendrocyte is important for understanding the cellular and molecular mechanisms of developmental transitions. It may also serve as an important tool for studying demyelinating disorders and virus-host cell interactions relate to the pathogenesis of human neurotropic viruses such as JCV.
No conflicts of interest declared.
This research was supported by the Intramural Research Program at the National Institutes of Health, NINDS. The authors would like to thank all the members of the Laboratory of Molecular Medicine and Neuroscience, Rick Dreyfuss for helping with microscopy and Pamela C. Sieving for helping with the editing.
|DME/HAMS F12 1:1||Omega Scientist||DM-251||1X|
|Gentamicin||Quality Biologicals||120-098-031||50 μg/ml|
|L-Glutamine||Quality Biologicals||118-084-061||2 mM|
|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|
|PFA||Electron Microscopy Sciences||15712||2%|
Table 1. Reagents.
Table 2. Antibodies (Abs) used for flow cytometry and immunocytochemistry assays. Antibody conjugates: PE, phycoerythrin; PETR, phycoerythrin 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.