Induction of Cortical Neurons from hiPSCs

Table of Contents

• Introduction
• Materials
• Media & Buffers
• Protocol
• References

Introduction

If you're looking to speed up the process of generating early-born cortical neurons from human induced pluripotent stem cells (hiPSCs), you've come to the right place. This protocol, adapted from the fantastic work by Qi and colleagues, outlines a highly efficient method to get your cells ready for transplantation in just 8 days. By day 16, any cells left in your culture dish will even start showing functional electrophysiological properties. Let's dive in and get those neurons growing!

Materials

Human induced pluripotent stem cells (hiPSCs) Conditioned hESC Medium KSR Medium N2B7 Medium FGF2 or TCB Small Molecule FGF2 Replacements ROCK inhibitor Y-27632 (HB2297)

SB 431542 (HB3555) XAV 939 (HB0660) PD 0325901 (HB2240) LDN 193189 (HB5624) SU 5402 (HB3133) DAPT (HB3345)

Media & Buffers

Conditioned hESC Medium Cocktail

Component	Concentration
ROCK inhibitor Y-27632 (HB2297)	10 μM
FGF2 or TCB-32 or TCB-541 or TCB-621	10 ng/ml 2μM 1μM 500nM

Differentiation Medium Cocktail (Day 0-6)

Component	Concentration
LDN 193189 (HB5624)	10 μM
SB 431542 (HB3555)	10 μM
XAV 939 (HB0660)	10 μM

Differentiation Medium Cocktail (Day 2-8)

Component	Concentration
PD 0325901 (HB2240)	1 μM
SU 5402 (HB3133)	5 μM
DAPT (HB3345)	10 μM

Protocol

1. Plate the hiPSCs in conditioned hESC medium exactly 24 hours prior to initiating the differentiation timeline. Ensure the medium is supplemented with FGF2.
   
   Note: FGF2 can be replaced with stable, cost-effective small molecule FGFR1 agonists TCB-32, TCB-541, or TCB-621 to enable weekend-free feeding and significantly reduce media costs.
2. On Day 0, commence the differentiation phase by exchanging the culture medium with KSR medium supplemented with the Day 0-6 cocktail.
3. Beginning on Day 4, progressively transition the culture from KSR medium to N2B7 medium. Adjust the ratio steadily so that the cells are maintained in 100% N2B7 medium by Day 8.
4. Incorporate the Day 2-8 cocktail components into the feeding medium starting on Day 2 of the protocol.
5. By Day 8, the derived early-born cortical neurons are mature enough to be harvested and transplanted into postnatal cortex models. For in vitro applications, maintain the cultures until Day 16 to achieve functional electrophysiological properties.

References

Qi et al. (2017) Combined small-molecule inhibition accelerates the derivation of functional cortical neurons from human pluripotent stem cells. Nat. Biotechnol. 35 154. PMID: 28112759