Isolation of Distinct Cell Populations from the Developing Cerebellum by Microdissection

Summary

Nestin-expressing progenitors are a newly identified population of neuronal progenitors in the developing cerebellum. Using the microdissection technique presented here in combination with fluorescent-activated cell sorting, this cell population can be purified with no contamination from other cerebellar regions and can be cultured for further studies.

Abstract

Microdissection is a novel technique that can isolate specific regions of a tissue and eliminate contamination from cellular sources in adjacent areas. This method was first utilized in the study of Nestin-expressing progenitors (NEPs), a newly identified population of cells in the cerebellar external germinal layer (EGL). Using microdissection in combination with fluorescent-activated cell sorting (FACS), a pure population of NEPs was collected separately from conventional granule neuron precursors in the EGL and from other contaminating Nestin-expressing cells in the cerebellum. Without microdissection, functional analyses of NEPs would not have been possible with the current methods available, such as Percoll gradient centrifugation and laser capture microdissection. This technique can also be applied for use with various tissues that contain either recognizable regions or fluorescently-labeled cells. Most importantly, a major advantage of this microdissection technique is that isolated cells are living and can be cultured for further experimentation, which is currently not possible with other described methods.

Introduction

The cerebellum is comprised of multiple cell layers, each containing distinct cell types. During development, the EGL contains proliferating granule neuron precursors (GNPs) while the molecular layer and Purkinje layer contain Bergmann glia and Purkinje neurons, respectively. Deep within the cerebellum lies the white matter, which contains neural stem cells (NSCs) and oligodendrocytes¹.

Sonic hedgehog (Shh) plays a critical role in regulating cerebellar development and in particular, it promotes the proliferation of GNPs via binding to its receptor Patched (Ptc), a negative regulator of Shh signaling^2-4. Aberrant activ....

Protocol

Use of animals in this protocol has been performed in accordance with procedures approved by the Fox Chase Cancer Center Animal Care and Use Committee.

1. Preparation of Instruments, Solutions, and Coverslips

1. Clean two sets of #5 fine forceps, one set of #7 fine curved forceps, one large surgical scissor, one microdissecting scissor, one spatula and one perforated spoon. Place the instruments in a self-sealing sterilization pouch and autoclave. Keep instruments in pouch until ready for use.
2. Prepare a 3% low melting temperature agarose solution.
   1. Add 1.5 g agarose into 50 ml of sterile Dulbecco′s Phosph....

Results

As shown in Figure 1A, cerebellar slices were prepared from P4 Math1-GFP/Nestin-CFP mice, in which the cerebellar EGL was dominated by GFP-expressing GNPs and the molecular layer was enriched by CFP-positive glial cells. To isolate NEPs that are located in the deep part of the EGL, slices were microdissected between the EGL and the molecular layer (along dashed line; Figure 1A). Dissected EGLs (Figure 1B) were then collected for enzymatic dissociation f.......

Discussion

The microdissection method described here is the first procedure able to isolate a pure population of living cells for use in molecular and functional analyses. As demonstrated by Li et al.⁹, NEPs obtained by this method can be cultured and incorporated into various experiments and because of their high purity, can also be used for microarray analysis.

It is very important to take time to become proficient with this microdissection technique. NEPs are loca.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Neurobasal media	Gibco	21103-049
PDL	Millipore	A-003-E
Ultra pure low melting temperature agarose	Invitrogen	16520-050
DPBS	Gibco	14190144