Generation of Neuronal Stem Cells Early Neural Progenitor Cells (NSCs eNPCs) from Human Induced Pluripotent Stem Cells (hi PSCs)

Researches in neuropsychiatry and neurodevelopmental disorders have been hampered, because it is difficult to obtain neuronal cells from affected patients that can be cultured and expanded in vitro. iPSCs have revolutionized such researches, as they can differentiation into neurons in vitro, potentially enabling personalized medicine by overcoming the problems of allogenic recognition. Besides, the potential to provide important molecular insights into pathogenic mechanisms, iPSC-based cell technologies can also be used for drug discovery in specific differentiated cell types.

We developed a method for efficient differentiation of human iPSCs into neurons that does not involve generation of EBs. Using the adherent culture system that does not require Noggin, we generate NSCs/eNPCs in a scalable manner.

Figure 1. Schematic diagram of differentiation into NSCs/eNPCs from hiPSC.

Materials and Equipment

Reagents Preparation

1) NP selection medium (DMEM/F12 medium, 0.5% N2, 1 mM L-glutamine, 1% nonessential amino acids, 50 U/mL penicillin G, 50 mg/mL streptomycin, 0.1 mM 2-mercaptoethanol)
2) NP expansion medium (DMEM/F12 medium, 1% N2, 20 ng/mL rhFGFβ, 1 mM L-glutamine, 1% nonessential amino acids, 50 U/mL penicillin G, 50 mg/mL streptomycin, 0.1 mM 2-mercaptoethanol)

Derivation of NSCs/eNPCs from iPSCs

1) Wash iPSC colonies with 2 mL PBS at room temperature twice.
2) Add 2 mL of pre-warmed NP selection medium and incubate under standard conditions.
3) Change medium every other day. At day 5, replace NP selection medium with NP expansion medium and culture for 7 d.
4) After 3-5 d in NP expansion medium, 50-70% of colonies will present cellular aggregates.
Note: If there is paucity of neural structures after 7 d culture in NP expansion medium, continue to culture the cells for 5-7 more days.

Generation of NLSs

1) Aspirate the medium, wash the cells twice with 1 mL DMEM/F12 and add 2 mL of pre-warmed NP expansion medium.
2) With the plate under the microscope (4X objective), use a bent 20-gauge needle to dissect cellular aggregates into pieces of approximately 40-50 μm diameter.
3) Collect the NP expansion medium containing the dissected cell aggregates and transfer into an ultra-low attachment 6-well plate. Culture in standard incubator conditions overnight.
4) On the following day, cell aggregates should round up into floating spherical cellular aggregates denoted NLSs.
5) Collect NLSs in suspension from the plate and transfer into a 15 mL conical tube. Allow NLSs to settle to the bottom of the conical tube for 10 min.
6) Replace half of the culture medium with fresh medium. Transfer the NLSs back to the 6-well ultra-low attachment plate.
Note: To distribute NLSs uniformly on low attachment plates, resuspend NLSs by pipetting up and down 3-4 times with a 5 mL pipette. As the NLSs tend to sink to the bottom rapidly, transfer into 2 wells at a time, resuspending each time.
7) Continue culturing until the NLSs reach a diameter of approximately 300 μm.

Purification of NLSs

1) Transfer the NLSs floating in the NP expansion medium into a 15 mL falcon tube and allow them to settle for 10 min.
2) Aspirate the supernatant and resuspend the NLSs into 12 mL of NP expansion medium.
3) Distribute the NLS suspension (2 mL/well) in a matrigel-coated 6-well plate.
4) Incubate in standard conditions.
5) Following overnight incubation, NLSs tend to adhere to the bottom of the culture plate and flatten. At this stage, neural rosettes can be observed.
6) Place the culture plate under an inverted microscope. Dissect and collect the neural rosettes following the same procedure described in steps Generation of NLSs 2) and 3).
7) On the following day, neural rosettes should round up into NLS. Culture NLSs for 2-3 d until the NLSs reach a size of approximately 300 μm.
8) Change half the culture medium every other day as described in step Generation of NLSs 5) and 6).

Generation of Monolayer Cultures of NSCs/eNPCs

1) Collect the NLSs into NP expansion medium and transfer into a matrigel-coated 6-well plate.
2) Incubate in standard conditions.
3) After a few hours, NLSs adhere to the surface of the culture plate and NSCs migrate away. At this stage, heterogeneous cultures will be generated, consisting of NSCs and eNPCs.
4) Fibroblast-like cells may still be observed and should be removed under the inverted microscope by scraping them with a pipette tip.

References

1. D'Aiuto L. et al. Large-scale generation of human iPSC-derived neural stem cells/early neural progenitor cells and their neuronal differentiation. Organogenesis, 2015, 10(4): 365-377.
2. Frega M. et al. Rapid neuronal differentiation of induced pluripotent stem cells for measuring network activity on micro-electrode arrays. J Vis Exp. 2017, 119: 54900.

For research use only. Not for any other purpose.