Differentiating Mouse Embryonic Stem Cells into a Networked Neuron Population

Abstract

Source: Hubbard, K. et al., Functional Evaluation of Biological Neurotoxins in Networked Cultures of Stem Cell-derived Central Nervous System Neurons. J. Vis. Exp. (2015).

This video demonstrates a technique for embryonic stem cell differentiation into a networked population of neurons with functioning synapses. It outlines the steps involved in embryonic stem cell differentiation into neurons, induction of neuronal network formation, and enrichment of the culture with networked neurons.

Protocol

1. Neuronal Differentiation

NOTE: Conduct steps 1.1 - 1.5 before noon and step 1.7 after noon. An overview of the differentiation procedures is presented in Figure 1A.

1. Transfer 350 µl (3.5 x 10⁶) of ESCs to a 10 cm low-attachment dish containing 25 ml differentiation medium. Place on an orbital shaker set to 30 - 45 rpm inside a tissue culture incubator at 37 °C with 5% CO₂. This is the first day of differentiation, termed day in vitro (DIV) -8.
   NOTE: The use of an ultra-low attachment dish increases the cost of th....

Results

Figure 1. Suspension-adapted ESNs remain mitotically stable and express markers of pluripotency. (A) Schematic of ESC maintenance and differentiation. The presence or absence of retinoic acid (RA) or leukemia in.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Knockout DMEM	Life Technologies	10829-018	Media: ESC culture medium Formulation and notes: 500 mL
100x MEM NEAA	Life Technologies	11140-050	Media: store at 4 °C for up to 1 month Formulation and notes: 6 mL
200 mM L-Alanyl-L-Glutamine	ATCC	30-2115	6 mL
ES qualified FBS	Applied Stem Cell	ASM-5007	90 mL
100x antibiotics	Sigma-Aldrich	A5955	3 mL
55 mM 2-mercaptoethanol	Life Technologies	21985-023	1.1 mL
107 Units/mL LIF	Millipore	ESG1107	60 μL
Knockout DMEM	Life Technologies	10829-018	Media: ESC differentiation medium Formulation and notes: 436.6 mL
100x MEM NEAA	Life Technologies	11140-050	Media: store at 4 °C for up to 1 month Formulation and notes: 5 mL
200 mM L-Alanyl-L-Glutamine	ATCC	30-2115	5 mL
ESC-qualified serum	Applied Stem Cell	ASM-5007	50 mL
100x antibiotics	Sigma-Aldrich	A5955	2.5 mL
55 mM 2-mercaptoethanol	Life Technologies	21985-023	0.9 mL
Dulbecco's PBS	Sigma-Aldrich	D8537	Media: NPC trypsinization medium Formulation and notes: 100 mL
0.5 M EDTA (18.3%)	Sigma-Aldrich	3690	Media: freeze in 5 mL aliquot Formulation and notes: 0.266 mL
Trypsin	Sigma-Aldrich	T8802	50 mg
Polyethyleneimine (PEI)	Sigma-Aldrich	P3143	Media: Surface coating solutions Formulation and notes: 2.5 µg/mL in H20
Poly-D-lysine (PDL)	Sigma-Aldrich	P7280	Media: use within 1 wk Formulation and notes: 100 µg/mL in H20
Laminin	Sigma-Aldrich	L2020	5 µg/mL in H20
DMEM/F12 + GlutaMAX	Life Technologies	10565-018	Media: NPC culture medium Formulation and notes: 492.5 mL
100x N2 vitamins	Life Technologies	17502-048	Media: store at 4 °C for up to 1 month Formulation and notes: 5 mL
100x antibiotics	Sigma-Aldrich	A5955	2.5 mL
Neurobasal A	Life Technologies	10888-022	Media: ESN culture medium Formulation and notes: 482.5 mL
50x B27 vitamins	Life Technologies	17504-044	Media: store at 4 °C for up 1 month Formulation and notes: 10 mL
200 mM L-Alanyl-L-Glutamine	ATCC	30-2115	5 mL
100x antibiotics	Sigma-Aldrich	A5955	2.5 mL
5-fluoro-2'-deoxyuridine (5FDU)	Sigma-Aldrich	F0503	Media: 2000x Mitotic inhibitors aliquot and store at -20 °C Formulation and notes: dd 150 mg 5FDU and 350 mg uridine to 10 mL Neurobasal A. Sterile filter, aliquot and freeze. Use 2000x in ESN culture medium.
Uridine	Sigma-Aldrich	U3003
TrypLE Express Trypsin	Life Technologies	12605-010	Media: Miscellaneous Formulation and notes: store at RT
DMSO	Sigma-Aldrich	D8418	store at RT
soybean trypsin inhibitor (STI)	Sigma-Aldrich	T6414	store at -20 °C
ethanol	Sigma-Aldrich	E7023	store at RT
ascorbic acid	Sigma-Aldrich	A4403	Prepare 100 mM stock by dissolving 100 mg ascorbic acid in 5.7 mL of 50:50 DMSO/ethanol mix.
retinoic acid (RA)	Sigma-Aldrich	R2625	Resuspend 15 mg RA in 9 mL 50:50 DMSO/ethanol. Supplement with 1 mL of 100 mM ascorbic acid stock. Aliquot and stored at -80 °C. Stable for 6 mos.
plasma cleaner	Harrick Plasma	PDC-32G
Olympus IX51 inverted microscope	Olympus	N/A
18 mm glass coverslips	Fisher	12-545-84
cell strainer (40 µm)	Fisher	08-771-1
Stovall Belly Dancer Shaker	Fisher	15-453-211
low adhesion dishes	Fisher	05-539-101	Corning 3262
bacterial dishes	VWR	25384-302	100 x 15 mm