Name: isolation of distinct cell populations from the developing cerebellum by microdissection
Uploaded: 2014-09-21T15:00:00
Description: Watch this Scientific Journal Video about Isolation of Distinct Cell Populations from the Developing Cerebellum by Microdissection at JoVE.com

The authors would like to thank James Oesterling for flow cytometry assistance. This research was supported by a grant from the US National Cancer Institute (R01-CA178380, Z.Y.) and a US National Institutes Postdoctoral training grant (5T32CA009035-37, L.W.Y.).

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
<ol>
	<li>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...

<ol>
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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...

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 signaling2-4. Aberrant activation of Shh signaling generates medulloblastoma (MB), the most common malignant brain tumor in children5,6.
Ptc mutant MB transgenic mouse models are powerful tools for the study of MB and develop tumors resembling human MB7,8. Using these mice, it was discovered that GNPs are the cell of origin for hedgehog-type MB7. Furthermore, in addition to GNPs, we have recently identified a unique population of neuronal progenitors within the EGL of the developing cerebellum that can also give rise to MB. These cells express high levels of the type VI intermediate filament protein, Nestin, and are termed NEPs9. NEPs are located within the deep part of the EGL and exist transiently only during neonatal cerebellar development. They are committed to the granule cell lineage as GNPs but are distinct as they are quiescent and do not express Math1, a well-established marker for conventional GNPs10. In addition, NEPs give rise to MB more efficiently than GNPs after activation of the Shh signaling pathway9, which makes them a novel origin for MB tumorigenesis.
We previously isolated NEPs from the cerebellar EGL at postnatal day 4 (P4) by the microdissection technique described here9. Microdissection via direct microscopic visualization of the tissue allows for specific dissection of the cerebellar EGL. This is necessary as Nestin is also expressed by NSCs in the cerebellar white matter and by Bergmann glia in the molecular layer1,7,11,12 and it was crucial that these cells not be included, as they would confound analysis. Cells isolated from microdissected tissue can be used immediately for molecular analysis or they can be cultured for further applications.
To aid in specifically microdissecting the EGL and to further purify NEPs, Math1-GFP (green fluorescent protein) mice were crossed with Nestin-CFP (cyan fluorescent protein) mice. The transcription factor Math1 is specifically expressed by GNPs and GFP expression is clearly visible in the EGL9,13. Nestin-CFP mice express a nuclear form of CFP and can be easily visualized in the molecular layer9,14. Together, GFP and CFP expression create boundaries for microdissection of the EGL (see Figure 1). CFP-positive NEPs were then isolated by FACS, following enzymatic dissociation of the dissected EGLs.
Currently, the most well utilized method to isolate cells from the EGL is Percoll gradient centrifugation of whole cerebellar tissue15,16. This method, however, is unable to completely exclude Nestin+ cell populations from the molecular layer and white matter and therefore cannot be used for the study of NEPs. Laser capture microdissection, which uses the transfer of laser energy to remove cells of interest, is another method used to isolate specific cells within a heterogeneous tissue17,18 but captured cells can only be used for recovery of DNA, RNA and protein and are not able to be cultured.
This protocol provides a way to specifically isolate an alive, pure population of NEPs from the EGL. This technique can also be applied to different tissue types that have either recognizable anatomical architecture or fluorescently labeled cells/regions. Therefore, the major advantage of incorporating this novel microdissection technique into cell isolation protocols is that cells can be isolated from specific tissue regions to eliminate contaminating neighboring tissue and the cells can be collected immediately for analysis or cultured for other applications.

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		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Name of Material/ Equipment</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Neurobasal media</td>
			<td style="width:71px;">Gibco</td>
			<td style="width:119px;">21103-049</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">PDL</td>
			<td style="width:71px;">Millipore</td>
			<td style="width:119px;">A-003-E</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">ultra pure low melting temperature agarose</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:119px;">16520-050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">DPBS</td>
			<td style="width:71px;">Gibco</td>
			<td align="right" style="width:119px;">14190144</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of distinct cell populations from the developing cerebellum by microdissection

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.

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...

Watch this Scientific Journal Video about Isolation of Distinct Cell Populations from the Developing Cerebellum by Microdissection at JoVE.com

Isolation of Distinct Cell Populations from the Developing Cerebellum by Microdissection

Nestin-expressing&#160;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.

Microdissection is a novel technique that can isolate specific regions of a tissue and eliminate contamination from cellular sources in adjacent areas. ...

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