Прародитель полученные из олигодендроцитов системе культуры человека от мозга плода
Summary
Первичные, человеческий мозг плода полученных, мультипотентных клеток-предшественников размножаться<em> В пробирке</em>, Сохраняя при этом способность дифференцироваться в нейроны и астроциты. Эта работа показывает, что нейронная предшественников можно индуцировать дифференцировку через этапы oligodendrocytic линии на кондиционирование с некоторыми факторами роста.
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
Representative Results
Discussion
Этот протокол описывает, как получить плода олигодендроциты из первичных человеческих нервных клеток-предшественников и характеризуют их фенотипа с использованием как проточной цитометрии и иммунофлуоресцентного окрашивания. Расширение и рост нейронных предшественников из ЦНС пл?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
Это исследование было поддержано Внутренние программы исследований в Национальном институте здоровья, NINDS. Авторы хотели бы поблагодарить всех членов лаборатории молекулярной медицины и неврологии, Рик Дрейфус за помощь в микроскопии и Памела С. Рассев за помощь в редактировании.
Materials
| Name of the reagent | Company | Catalogue number | Final concentration | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 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. |
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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. |
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