Isolation of Stem Cells from Postnatal Mouse Cerebellum and Differentiation into Neural Cells

Abstract

Source: Edamakanti, C. R., et al. Purification of Prominin-1⁺ Stem Cells from Postnatal Mouse Cerebellum. J. Vis. Exp. (2020).

This video demonstrates a method for isolating stem cells from the postnatal mouse cerebellum and differentiating them into neural cells. Stem cells from the isolated cell suspension are purified using antibody-coated magnetic beads and are cultured to form neurospheres. The neurospheres are then maintained in a differentiation medium to generate neural cells.

Protocol

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 deoxyribonuclease (DNase, 250 U/ml).
2. To prepare the 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. Divide one stock tube into 0.5 mL single-use aliquots. Store these single-use aliquots at -80 °C.
3. Prepare magnetic separation reagents by preparing magnetic column buffer X: 0.5% bovine serum alb....

Results

Figure 1: Isolation of prominin-1 stem cells from postnatal cerebellum. (A) Cerebellar stem cells were isolated using immunomagnetic prominin-1 beads. Top panel: Purified stem cells (column-bound) formed neurospheres with extensive prolifer.......

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
2mM 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	40um