Purification of Prominin-1+ Stem Cells from Postnatal Mouse Cerebellum

Summary

Demonstrated here is an efficient and cost-effective method to purify, culture, and differentiate white matter stem cells from postnatal mouse cerebellum.

Abstract

Most cerebellar neurons arise from two embryonic stem niches: a rhombic lip niche, which generates all the cerebellar excitatory glutamatergic neurons, and a ventricular zone niche, which generates the inhibitory GABAergic Purkinje cells, which are neurons that constitute the deep cerebellar nuclei and Bergman glia. Recently, a third stem cell niche has been described that arises as a secondary germinal zone from the ventricular zone niche. The cells of this niche are defined by the cell surface marker prominin-1 and are localized to the developing white matter of the postnatal cerebellum. This niche accounts for the late born molecular layer GABAergic interneurons along with postnatally generated cerebellar astrocytes. In addition to their developmental role, this niche is gaining translational importance in regards to its involvement in neurodegeneration and tumorigenesis. The biology of these cells has been difficult to decipher because of a lack of efficient techniques for their purification. Demonstrated here are efficient methods to purify, culture, and differentiate these postnatal cerebellar stem cells.

Introduction

The cerebellum has been long recognized as a major neuronal circuit coordinating voluntary movement¹. It receives input from the wide swathes of the neuroaxis, which includes proprioceptive information from the periphery, so as to fine tune motor output and coordinate motion. More recently, it has also been implicated in regulating cognition and emotions by potentially using similar information processing networks^2,3,4.

The adult cerebellum is composed of an outer cerebellar cortex and inner white matter. Interspersed w....

Protocol

All animal experiments were performed in compliance with the NIH’s Guide for the Care and Use of Laboratory Animals (2011) and were approved by the Northwestern University IACUC (Protocol IS00011368).

1. Preparation of Solutions

1. Prepare tissue dissociation solution made from sterile phenol red-containing Dulbecco’s phosphate-buffered saline (DPBS) with papain (100 U/ml), cysteine (0.2 mg/ml) and DNase (250 U/ml).
2. To prepare DNase solution dilute 100 mg of the lyophilized powder of DNase I (one bottle) in 50 mL of H₂O. Mix well and filter the stock solution. Prepare 10 mL stock aliquots. Divi....

Results

Prominin-1-positive postnatal cerebellar stem cells formed neurospheres in neurosphere medium rich in growth factors (EGF and bFGF). These neurospheres were positive for prominin-1-staining, the marker used for isolation, and also as a stain for other stem cell markers such as Nestin and GFAP¹³ (Figure 1). The stem cell marker expression was maintained throughout culture and for up to at least eight passages²⁰. Upon withdrawal of growth factors.......

Discussion

Prominin-1-expressing cerebellar stem cells reside in the prospective white matter during the first 3 weeks of postnatal life. Their proliferation is tightly controlled by the sonic hedgehog pathway supported by Purkinje cells¹⁷. These stem cells/progenitors contribute to later-born GABAergic interneurons called basket cells and stellate cells. These interneurons reside in the molecular layer, where they synapse onto Purkinje cells and sculpt PC topography and function via GABAergic inhibition

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.05%Trypsin	Thermo Fisher Scientific	25300054	0.05%
2% B27	Gibco; Thermo Fisher Scientific	17504001
2 mM EDTA solution	Corning	46-034-CI
Anti- Prominin-1 microbeads	Miltenyi Biote	130-092-333
Bovine serum albumin	Sigma	A9418
Column MultiStand	Miltenyi Biotec	130-042-303
Culture plates ultra - low attachment	Corning	3473
Cysteine	Sigma	C7880
DNase	Sigma	D4513-1VL	250 U/ml
Dulbecco’s Phosphate Buffer Saline	Thermo Fisher Scientific	14040141
Hank's balanced salt solution-HBSS	Gibco	14025-092
Human recombinant Basic Fibroblast Growth Factor	Promega	G507A	20 ng/mL
Human recombinant Epidermal Growth Factor	Promega	G502A	20 ng/mL
Leukemia Inhibitory Factor	Sigma	L5158
l-glutamine	Gibco	25030081
Microscopy	Lieca TCS SP5 confocal microscopes
MiniMACS separator	Miltenyi Biotec	130-042-102
Mouse anti-Prominin-1	Affymetrix eBioscience	14-1331	1 in 100
Nestin	Abcam	ab27952	1 in 200
Neurobasal medium	Thermo Fisher	25030081
O4	Millopore	MAB345
Papain	Worthington	LS003126	(100 U/mL)
Platelet- Derived Growth Factor	Sigma	H8291	10 ng/mL
Poly-D-Lysine	Sigma	P6407
Rabbit anti-tubulin, b-III	Sigma	T2200	1 in 500
Rabit anti-GFAP	Dako	Z0334	1 in 500
Separation columns-MS columns	Miltenyi Biotec	130-042-201
Sterile cell strainer	Fisher Scientific	22363547	40 μm