分离和成人神经干细胞的培养从鼠标Subcallosal区

Summary

Establishing culture systems for the expansion of adult neural stem cells (aNSCs) allows for the examination and application of aNSCs for therapy. The subcallosal zone (SCZ) has recently been recognized as a novel neuroblast-forming region in adult mice. Here, methods for the isolation, expansion, and differentiation of SCZ-aNSCs are described.

Abstract

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation into three types of cells: neurons, astrocytes, and oligodendrocytes. Identifying aNSC-derived regions and characterizing the aNSC properties are critical for the potential use of aNSCs and for the elucidation of their role in neural regeneration. The subcallosal zone (SCZ), located between white matter and the hippocampus, has recently been reported to contain aNSCs and continuously give rise to neuroblasts. A low percentage of aNSCs from the SCZ is differentiated into neurons; most cells are differentiated into glial cells, such as oligodendrocytes and astrocytes. These cells are suggested to have a therapeutic potential for traumatic cortical injury. This protocol describes in detail the process to generate SCZ-aNSCs from an adult mouse brain. A brain matrix with intervals of 1 mm is used to obtain the SCZ-containing coronal slices and to precisely dissect the SCZ from the whole brain. The SCZ sections are initially subjected to a neurosphere culture. A well-developed culture system allows for the verification of their characteristics and can increase research on NSCs. A neurosphere culture system provides a useful tool for determining proliferation and collecting the genuine NSCs. A monolayer culture is also an in vitro system to assay proliferation and differentiation. Significantly, this culture system provides a more homogenous environment for NSCs than the neurosphere culture system. Thus, using a discrete brain region, these culture systems will be helpful for expanding our knowledge about aNSCs and their applications for therapeutic uses.

Introduction

NSCs have characteristics of self-renewal and multiple-lineage differentiation. To confirm these properties, a neurosphere culture system has widely been used. The neurosphere culture system was developed in the early 1990s and served as a standard stem cell culture system¹. Depending on self-renewal potency, NSCs continuously proliferate and generate a cell mass in a suspension culture. The number of cells and the size of the neurosphere are considered to be closely associated with the proliferation properties of the NSCs. Monolayer cultures are also widely used for the maintenance and differentiation of NSCs. Compared to the neurosphere culture, the monolayer culture system provides better homogenous maintenance and expansion of NSCs². These two well-developed culture systems have contributed to the characterization of aNSCs in vitro.

NSCs reside in different brain regions, such as the subventricular zone (SVZ) of the lateral ventricle and the subgranular zone (SGZ) of the hippocampus^3-5. The subcallosal zone (SCZ) of the caudal subcortical white matter is recognized as a novel neurogenic region^6-8. It was recently reported that the SCZ-aNSCs have therapeutic potential in traumatic brain injury⁹. Compared to other neurogenic regions, the SCZ resides along the subcortical white matter. In the human brain, subcortical white matter occupies a larger region than in the mouse brain¹⁰. Therefore, an understanding of the characteristics of SCZ-aNSCs using an in vitro culture system is important in order to promote the potential use of these cells for neural regeneration. Precise dissection of the desired brain region is required to rule out possible contamination by unwanted regions containing active or quiescent NSCs. For instance, aNSCs in non-neurogenic regions can be activated and produce new neural cells in injured brains or during in vitro culturing¹¹. To obtain NSCs from the SCZ, cells were collected from brain slices containing the SCZ. Then, a careful micro-dissection of the SCZ region was performed using a fine needle. To generate the neurosphere from the SCZ, micro-dissected SCZ tissue chunks were dissociated into single cells and then cultured as a suspension in the presence of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). After SCZ-aNSCs form neurospheres, they also can be maintained as neurospheres or monolayers for expansion. This procedure also demonstrates immunostaining processes with various markers for the detection of NSCs and their progeny after their expansion and differentiation in a monolayer culture. Here, a visual protocol of the SCZ-aNSC culture system is presented. This protocol contains detailed instructions for the micro-dissection of the SCZ region and for the maintenance and passaging of the cells.

Protocol

1.材料和培养基的制备为SCZ的解剖和离解，包住大脑基质，双刃刀片，并用铝箔钳子，然后通过高压消毒他们。 制备50毫升冷PBS缓冲液洗涤全鼠脑。 设置一个解剖显微镜和制备用于脑（蒸压剪刀和镊子）的解剖所需的外科手术工具和SCZ（1ml注射器，地下30针，脑基质，和细镊子）的分离。 N2培养基的制备： 制备的F-12 / DMEM（+ L谷氨酰胺，+碳酸氢钠）有2％B27，1％…

Representative Results

从未知的神经区域定义aNSCs培养体系是了解这些细胞，并用于开发大脑修复12其潜在的使用至关重要。众所周知，神经干细胞在不同发育阶段，或在不同的区域表现不同3,4。最近，有人报告说，相对于SVZ来源的细胞7-9 SCZ来源的细胞显示出在体内和体外对神经元分化差的电位。因此，能够精确地隔离每个神经区，脑切片包括该SCZ?…

Discussion

本文介绍了详细的方案，从成年小鼠SCZ产生神经干细胞，并保持他们的各种应用。有建立净化和扩大SCZ神经干细胞所需要的体外培养体系三个关键步骤。首先，重要的是要确保该SCZ区域被精确从其他潜在神经原性区域（ 图1B）解剖出。用1毫米间隔脑矩阵获得含有SCZ区域厚和精确的部分，然后被用于从其他皮层区域（ 图1A）的SCZ的显微解剖细针。当非神经干细胞从?…

Materials

Medium components
DMEM/F12	Gibco	11320-033	+L-glutamin, +Sodium bicarbonate
Pen/Strep	Invitrogen	15140-122
N2 supplement	Gibco	17502-048
B27 supplement	Gibco	17504-044
Growth factor
bFGF	R&D	233-FB
EGF	Gibco	PHG0313
Buffer
PBS (10X)	BIOSOLUTION	BP007a	1X dilution
HBSS	Gibco	14175-095
Dissociattion buffer
Dispase II	Roche	04-942-078-001
Papain	Worthington	3126
Accutase	ICT	AT-104
Tool
Fine forceps	WPI	555229F
Scissors	Storz	E3321-C
Brain matrix (1mm)	RWD	68707
Double-edged razor	DORCO	ST-300
30 gage needle	SUNGSHIM	N1300
Materials
15 ml tubes	SPL	50015
50 ml tubes	SPL	50050
35mm dish	SPL	10035	petridish
100mm dish	SPL	10090	petridish
Cover slip (18mm)	Deckglaser	111580
12 well dish	SPL	32012	non-coating
6 well dish	SPL	32006	non-coating
Coating materials
PLO	Sigma	P4957	0.01%
Laminin	Gibco	23017-015	10 mg/ml
Primary antibodies
Nestin	Millipore	MAB353	mouse (1:1000)
EGFR	Abcam	ab2430	rabbit (1:1000)
DCX	Santa Cruz	SC8066	goat (1:500)
Tuj1	Sigma	T2200	rabbit (1:2000)
GFAP	Invitrogen	13-0300	rat (1:1000)
O4	Millipore	MAB345	mouse (1:500)
BrdU	Abcam	ab6326	Rat (1:500)
Secondary antibodies
anti-mouse 488	Invitrogen	A21202	1:500
anti-mouse cy3	Jackson	715-165-151
anti-mouse 647	Jackson	715-606-150
anti-rabbit 488	Alexa	A21206
anti-rabbit cy3	Jackson	711-165-152
anti-rabbit 647	Jackson	711-605-152
anti-goat 488	Alexa	A11055
anti-goat cy3	Jackson	705-165-147
anti-goat 647	Invitrogen	A21447
anti-rat 488	Invitrogen	A21208
anti-rat cy3	Jackson	712-166-150
anti-rat 647	Jackson	712-605-153
Immunostaining materials
BSA	Millipore	82-100-6	3% BSA with 0.1% Triton X-100 in PBS
Triton X-100	usb	22686
4% PFA	Biosesang	P2031
Hoechest33342	Life Technology	H3570	Dye for staining nuclei