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 the method but produces slightly larger yields than bacterial Petri dishes since aggregates can occasionally adhere to Petri dishes. If different dish sizes are preferred, medium volumes and cell numbers can be scaled accordingly.
2. After 48 hr (DIV -6), use a 25 ml pipet to transfer the differentiating cell aggregates to a 50 ml conical tube. Immediately add a fresh 25 ml of differentiation medium to the Petri dish.
3. Allow aggregates to settle over 2 - 5 min, producing a visible pellet that is 1 - 2 mm deep. Ignore single cells or small aggregates remaining in suspension. Carefully aspirate the medium and transfer the cell pellet back to the petri dish using a P1000. Place on rotary shaker in a tissue culture incubator.
4. At DIV -4, repeat step 1.2. The pellet will be 2 - 4 mm deep. Replace 30 ml differentiation medium supplemented with 6 µM all-trans retinoic acid (RA) to the petri dish. Return to the rotary shaker in the tissue culture incubator for an additional 48 hr.
5. At DIV -2, repeat step 1.3. The pellet will be 4 - 8 mm deep at this point.
6. At DIV -1, prepare plating surfaces as in Section 2.
7. At DIV 0, thaw 5 ml of pre-aliquoted and frozen NPC trypsinization medium at 37 °C for 5 - 10 min and place in a tissue culture hood. Using a 25 ml pipet, transfer differentiating aggregates to a 50 ml conical tube. Allow aggregates to settle and carefully aspirate the medium. Wash the pellet twice with 10 ml PBS, allowing the aggregates to settle between washes.
8. After the second PBS wash, add 5 ml of NPC trypsinization medium to the pellet and incubate at 37 °C for 5 min. Gently flick the tube after 2.5 min.
9. Add 5 ml of 0.1% soybean trypsin inhibitor (STI) to inactivate trypsin and mix by inverting. Gently triturate 10 - 15 times with a 10 ml serological pipet until a relatively homogenous cell suspension is produced.
10. Slowly transfer the cell suspension to a 40 µm or 70 µm cell strainer placed on the top of a 50 ml conical tube. Once all the suspension has been filtered, add 1 ml of N2 medium to wash the remaining cells through the filter and pellet the dissociated cell suspension for 6 min at 200 x g.
11. Aspirate medium without disturbing the pellet. Wash cells twice with 10 ml N2 medium, pelleting cells for 5 min at 200 x g and triturating between washes with a P1000. Prior to the second wash, count cells using a hemocytometer.
12. Resuspend cells in N2 medium at 1 x 10⁷ cells per ml and plate ESNs at a cell density of 150,000 - 200,000 cells/cm².
13. Transfer newly plated ESNs to a humidified tissue culture incubator at 37 °C and 5% CO₂ and maintain as in Section 3.

2. Preparing Culture Surfaces for Plating Neural Precursors at DIV 0

1. Prepare tissue culture-treated dishes at least 1 day prior to plating. Add sufficient polyethyleneimine (PEI; 25 µg/ml in sterile H₂O) or poly-D-lysine (PDL; 100 µg/ml in sterile H₂O) to cover tissue-culture-treated plastic dishes and incubate O/N at 37 °C.
2. On the morning of plating, wash dishes twice with double-distilled H₂O and once with PBS. After the final wash, add sufficient N2 medium to cover the dish (e.g., 1 ml per well of a 12-well dish or 4 ml per 6 cm dish).
3. Prepare 18 mm glass coverslips at least one day prior to neuron plating. Clean the coverslips by plasma-cleaning for 4 min.
4. Immediately transfer cleaned coverslips to an ethanol-washed parafilm in the bottom of a large sterile dish and add 400 µl of PEI or PDL solution, prepared as in step 2.1. Incubate O/N at 37 °C in a tissue culture incubator.
5. In the morning, wash coverslips three times with water and add 5 µg/ml laminin in PBS for 1 - 3 hr at 37 °C. Prior to dissociating neurons, aspirate the laminin and immediately transfer the coverslip to a well of a 12-well dish containing 1 ml of NPC medium, being sure to keep the treated side facing up.
6. Store dishes and coverslips at 37 °C until NPCs are ready to plate.

3. Maintenance of Neurons

1. At DIV 1, aspirate medium and replace with N2 culture medium.
2. At DIV 2 and 4, aspirate medium and replace with B27 culture medium.
3. At DIV 8, aspirate medium and replace with B27 culture medium containing mitotic inhibitors to eliminate contaminating non-neuronal cells.
4. At DIV 12, replace with B27 culture medium.
5. Do not remove DIV 12⁺ ESNs from 5% CO₂ until ready to use.

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