High-Content Screening Differentiation and Maturation Analysis of Fetal and Adult Neural Stem Cell-Derived Oligodendrocyte Precursor Cell Cultures

摘要

We describe the production of mixed cultures of astrocytes and oligodendrocyte precursor cells derived from fetal or adult neural stem cells differentiating into mature oligodendrocytes, and in vitro modeling of noxious stimuli. The coupling with a cell-based high-content screening technique builds a reliable and robust drug screening system.

摘要

The main hurdle in developing drug screening techniques for assessing the efficacy of therapeutic strategies in complex diseases is striking a balance between in vitro simplification and recreating the complex in vivo environment, along with the main aim, shared by all screening strategies, of obtaining robust and reliable data, highly predictive for in vivo translation.

In the field of demyelinating diseases, the majority of drug screening strategies are based on immortalized cell lines or pure cultures of isolated primary oligodendrocyte precursor cells (OPCs) from newborn animals, leading to strong biases due to the lack of age-related differences and of any real pathological condition or complexity.

Here we show the setup of an in vitro system aimed at modeling the physiological differentiation/maturation of neural stem cell (NSC)-derived OPCs, easily manipulated to mimic pathological conditions typical of demyelinating diseases. Moreover, the method includes isolation from fetal and adult brains, giving a system which dynamically differentiates from OPCs to mature oligodendrocytes (OLs) in a spontaneous co-culture which also includes astrocytes. This model physiologically resembles the thyroid hormone-mediated myelination and myelin repair process, allowing the addition of pathological interferents which model disease mechanisms. We show how to mimic the two main components of demyelinating diseases (i.e., hypoxia/ischemia and inflammation), recreating their effect on developmental myelination and adult myelin repair and taking all the cell components of the system into account throughout, while focusing on differentiating OPCs.

This spontaneous mixed model, coupled with cell-based high-content screening technologies, allows the development of a robust and reliable drug screening system for therapeutic strategies aimed at combating the pathological processes involved in demyelination and at inducing remyelination.

引言

In the central nervous system (CNS), myelin forming cells (oligodendrocytes, OLs) and their precursors (oligodendrocyte precursor cells, OPCs) are responsible for developmental myelination, a process which occurs during the peri- and post-natal periods, and for myelin turnover and repair (remyelination) in adulthood¹. These cells are highly specialized, interacting anatomically and functionally with all the other glial and neuronal components, making them a fundamental part of CNS structure and function.

Demyelinating events are involved in different CNS injuries and diseases², and mainly act ....

方案

All animal protocols described herein were carried out according to European Community Council Directives (86/609/EEC) and comply with the guidelines published in the NIH Guide for the Care and Use of Laboratory Animals.

1. Solutions and reagents

1. Prepare standard medium: DMEM/F12 GlutaMAX 1x; 8 mmol/L HEPES; 100 U/100 μg Penicillin/Streptomycin (1% P/S); 1x B27; 1x N-2.
2. Prepare neurosphere medium: add 10 ng/mL bFGF; 10 ng/mL EGF to standard medium.
3. Prepare oligosphere/OPC medium: add 10 ng/mL bFGF; 10 ng/mL PDGF-AA to standard medium.
4. Prepare oligodendroctye differentiation medium: add....

结果

The first phase of the culture may vary in duration, depending on seeding density and on whether the spheres are of fetal or adult origin. Moreover, oligospheres display a reduced population doubling compared to neurospheres (Figure 1B). Moreover, spheres production from adult tissue is slower and it may take 2–3 weeks to generate oligospheres compared to fetal that may take 1–2 weeks, depending on the seeding density.

Once seeded, the entire differentiation phase.......

讨论

The complex nature of myelination/remyelination processes and demyelinating events makes the development of predictive in vitro systems extremely challenging. The most widely used in vitro drug screening systems are mostly human cell lines or primary pure OL cultures, with increasing use of more complex co-cultures or organotypic systems¹⁵. Even if such systems are coupled with high content technologies, pure OL cultures remain the method of choice when developing screening platforms

材料

本文使用的材料清单

姓名	公司	目录编号	评论
96 孔板 - 未经处理	NUNC	267313
B27 补充剂 （100x）	GIBCO	17504-044
碱性成纤维细胞生长因子 （bFGF）	GIBCO	PHG0024
BSA	Sigma-Aldrich	A2153
睫状神经因子 （CNTF）	GIBCO	PHC7015
DMEM，不含葡萄糖	GIBCO	A14430-01
DMEM/F12 GlutaMAX	GIBCO	31331-028
DNase	Sigma-Aldrich	D5025-150KU
EBSS	GIBCO	14155-048
表皮生长因子 （EGF）	GIBCO	PHG6045
HBSS	GIBCO	14170-088
HEPES	GIBCO	15630-056
透明质酸酶	Sigma-Aldrich	H3884
IFN-&γ;	Origene	TP721239
IL-17A	Origene	TP723199
IL-1&β;	Origene	TP723210
IL-6	Origene	TP723240
层粘连蛋白	GIBCO	23017-051
N-乙酰-L-半胱氨酸	Sigma-Aldrich	A9165
N2 补充剂 （50x）	GIBCO	17502-048
非酶解离缓冲液	GIBCO	13150-016
PBS	GIBCO	70011-036
青霉素/链霉素	Sigma-Aldrich	P4333
血小板衍生生长因子 （PDGF-AA）	GIBCO	PHG0035
聚-D，L-鸟碱	Sigma-Aldrich	P4957
TGF-β;1	个 Origene	TP720760
TNF-&α;	Origene	TP723451
三碘甲状腺原氨酸	Sigma-Aldrich	T2752-1G
胰蛋白酶	Sigma-Aldrich	T1426