Replating Differentiated Neurons Derived from Human Pluripotent Stem Cells

Abstract

Source: Calabrese, B. et al., Post-differentiation Replating of Human Pluripotent Stem Cell-derived Neurons for High-content Screening of Neuritogenesis and Synapse Maturation. J. Vis. Exp. (2019)

The video showcases a method to detach and replant human pluripotent stem cell-derived neurons using a mild proteolytic enzyme. After filtration and centrifugation, the neurons are replated on a coated multi-well plate for incubation, promoting attachment and maturation.

Protocol

1. Differentiation Period Prior to Replating

1. Differentiate neurons on 10 cm dishes, using a protocol of choice, until neurons have formed a thick network with their processes and express not only early neuronal markers such as MAP2 or TuJ1, but also late markers such as NeuN.
2. Change half the medium of choice every 4 days during the neuronal differentiation process.
   NOTE: More extensive or frequent medium changes dilute essential trophic factorsaq and could disfavor maturation.
   1. For the iPSC-derived WT126 neurons, use the following post-differentiation culturing media: 5 mL 100x N2 (neural) suppl....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
L-Ascorbic Acid	Sigma	A4403	Add 1ml of 200mM stock to 1L of N2B27 media
dibutyryl-cAMP	Sigma	D0627	Add 1 µM
Human BDNF	Peprotech	450-02	10 ng/ml final concentration
B27 (50X)	Thermofisher Scientific	17504044	Add 20 ml to 1L N2B27 media
DMEM/F12 with Glutamax	Thermofisher Scientific	31331093	Add N2 and distribute in 50 mL conicals; parafilm wrap lids
Human GDNF	Peprotech	450-10	10 ng/ml final concentration
Glutamax	Thermofisher Scientific	35050038	Add 10 ml to 1L N2B27 media; glutamine supplement
Mouse Laminin	Sigma	P3655-10mg	Add 100 µl to 50 mL N2B27
MEM Nonessential Amino Acids	Thermofisher Scientific	11140035	Add 5ml to 1L N2B27 media
N2 (100X) Supplement	Life Technologies	17502048	Add 5ml to 500mL media
Neurobasal A Media	Thermofisher Scientific	10888022	Combine with DMEM/F12 to generate N2B27 media for CVB wt cells; neural basal A media
Neurobasal Media	Thermofisher Scientific	21103049	for WT126 cells; neural basal media
SM1 Supplement	StemCell Technologies	5711	Add 1:50 to media
sodium bicarbonate	Thermofisher Scientific	25080-094	Add 10ml to 1L N2B27 media
Plate Preparation
10cm Tissue Culture Dishes	Fisher Scientific	08772-E	Plastic TC-treated dishes
6-well Tissue Culture Dishes	Thomas Scientific	1194Y80	NEST plates
Mouse Laminin	Life Technologies	23017-015	Add 1:400 on plastic
Poly-Ornithine	v	P3655-10mg	Add 1:1000 on plastic
UltraPure Distilled Water	Life Technologies	10977-015	To dilute Poly-L-Ornithine
Replating Reagents
100mM Cell Strainer	Corning	431752	Sterile, individually wrapped
384-well plate, uncoated	PerkinElmer	6007550	Coat with PLO and Laminin
DPBS	Life Technologies	14190144	Dulbecco's phosphate-buffered saline
Poly-D-Lysine-Precoated 384-well Plates	PerkinElmer	6057500	Rinse before coating with laminin