iPSC Differentiation Protocols

01

DA Neurons

Dopaminergic Neuron Differentiation Protocol

This protocol generates midbrain floor-plate dopaminergic neurons from human iPSCs via dual SMAD inhibition followed by sonic hedgehog (SHH) and WNT pathway activation. The resulting neurons express TH, DAT, GIRK2, FOXA2, and LMX1A and exhibit spontaneous dopamine release detectable by HPLC. This protocol was optimized in collaboration with the EDDU at McGill.

35–42

Days to Maturity

>85%

TH+ Purity

9

iPSC Lines Validated

4

Phases

PHASE 1 Floor-Plate Induction (Days 0–10)

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1

Seed iPSCs on Matrigel

Dissociate iPSC colonies using Accutase (37°C, 5 min). Seed at 200,000 cells/cm² on Matrigel-coated plates in mTeSR Plus + 10 μM Y-27632 (ROCK inhibitor). Allow 24 h for attachment.

AccutasemTeSR PlusY-27632Matrigel

⏲ Day 0

2

Initiate Dual SMAD Inhibition

Switch to KSR medium containing SB431542 (10 μM, TGF-β inhibitor) and LDN193189 (100 nM, BMP inhibitor). Feed daily. This drives neuroectoderm specification.

SB431542LDN193189KSR Medium

⏲ Days 1–5

3

SHH + CHIR Patterning

Add SHH C25II (100 ng/mL) and Purmorphamine (2 μM) for ventralization. Add CHIR99021 (3 μM, WNT agonist) from Day 3 for midbrain caudalization. Begin N2 medium transition from Day 5 (25% increments every 2 days).

SHH C25IIPurmorphamineCHIR99021N2 Medium

⏲ Days 1–10

PHASE 2 Precursor Expansion (Days 11–20)

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4

Passage Floor-Plate Progenitors

At Day 11, dissociate with Accutase and replate at 400,000 cells/cm² on poly-L-ornithine/laminin/fibronectin-coated plates. Culture in N2B27 + CHIR (until Day 13) + FGF8b (100 ng/mL). Confirm FOXA2+/LMX1A+ by ICC at Day 11 (>80% expected).

PLO/Lam/FNN2B27FGF8b

⏲ Day 11

5

Neurotrophic Factor Addition

From Day 12, switch to maturation medium: Neurobasal + B27 + BDNF (20 ng/mL) + GDNF (20 ng/mL) + TGF-β3 (1 ng/mL) + dbcAMP (0.5 mM) + ascorbic acid (200 μM) + DAPT (10 μM). Feed every other day.

BDNFGDNFTGF-β3dbcAMPDAPT

⏲ Days 12–20

PHASE 3 Terminal Differentiation (Days 20–35)

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6

Final Replating & Maturation

Dissociate at Day 20 and plate at final density (100,000 cells/cm²) on PLO/laminin. Continue maturation medium (BDNF + GDNF + TGF-β3 + dbcAMP + AA). Remove DAPT after Day 25. Feed every 2–3 days with half-medium changes.

PLO/LamininMaturation Medium

⏲ Days 20–35

7

QC Checkpoint — Marker Validation

At Day 35, perform ICC panel: TH, MAP2, FOXA2, GIRK2, DAT. Expected: >85% TH+/MAP2+, >70% FOXA2+/LMX1A+. Confirm dopamine release by HPLC (18–21 ng/mL baseline). MEA should show spontaneous firing at 3–5 Hz.

ICC PanelHPLCMEA

⏲ Day 35 QC

PHASE 4 Long-Term Culture & Experiments (Days 35+)

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8

Experimental Window

Mature DA neurons can be maintained for >100 days for long-term disease modeling. From Day 35 onward, apply experimental paradigms: α-synuclein PFF exposure (5 μg/mL, 14 days for Lewy body-like inclusions), IFN-γ dual-hit (10 ng/mL), or drug screening compounds. Neurons are viable for MEA, calcium imaging, and high-content imaging readouts throughout this window.

α-Syn PFFsIFN-γDrug Compounds

⏲ Day 35+ experimental

02

Cortical

Cortical Excitatory Neuron Differentiation

This protocol generates dorsal forebrain glutamatergic neurons via dual SMAD inhibition without SHH ventralization, allowing default dorsal telencephalic fate. Neurons express TBR1, CTIP2, SATB2, and VGLUT1 and form functional glutamatergic synapses detectable by MEA and calcium imaging.

42–56

Days to Maturity

>80%

TBR1+ Purity

6.2 Hz

Mean Firing Rate

3

Phases

PHASE 1 Neural Induction (Days 0–12)

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1

Seed & Initiate Dual SMAD Inhibition

Seed iPSCs at 250,000 cells/cm² on Matrigel in mTeSR Plus + Y-27632. Switch to KSR + SB431542 (10 μM) + LDN193189 (100 nM) at Day 1. Unlike the DA protocol, no SHH or CHIR is added — the cells default to dorsal telencephalic identity.

SB431542LDN193189KSR Medium

⏲ Days 0–12

2

N2 Medium Transition

Begin gradual transition to N2 medium from Day 5: 75% KSR / 25% N2 at Day 5, 50/50 at Day 7, 25/75 at Day 9, 100% N2 at Day 11. Continue dual SMAD inhibitors throughout. At Day 12, confirm PAX6+/OTX2+ neural rosettes (>90% expected).

N2 MediumPAX6 ICC

⏲ Days 5–12

PHASE 2 Progenitor Expansion (Days 12–25)

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3

Passage Neural Progenitors

Dissociate rosettes with Accutase and plate at 300,000 cells/cm² on PLO/laminin. Culture in N2B27 + FGF2 (20 ng/mL) for progenitor expansion until Day 18. Remove FGF2 to initiate terminal differentiation.

PLO/LamininN2B27FGF2

⏲ Days 12–25

PHASE 3 Maturation & Validation (Days 25–56)

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4

Maturation with Neurotrophins

Final replate at 80,000 cells/cm². Mature in Neurobasal + B27 + BDNF (10 ng/mL) + NT-3 (10 ng/mL) + ascorbic acid (200 μM). Half-medium changes every 3 days. Cortical layer markers emerge sequentially: deep-layer (TBR1, CTIP2) by Day 35, upper-layer (SATB2, BRN2) by Day 45.

BDNFNT-3Ascorbic Acid

⏲ Days 25–56

5

QC Checkpoint

At Day 42+: ICC for TBR1 (>80%), MAP2 (>90%), VGLUT1, GABA (<15% — confirms glutamatergic enrichment). MEA recordings show network bursting at 6.2 Hz mean firing rate with synchronized calcium oscillations.

ICC PanelMEACalcium Imaging

⏲ Day 42+ QC

03

NGN2+

NGN2-Driven Rapid Neuronal Induction

This protocol uses doxycycline-inducible NGN2 overexpression to rapidly convert iPSCs into excitatory, glutamatergic-like neurons within 10–14 days. This approach bypasses the weeks-long small-molecule differentiation required for DA or cortical neurons, making it ideal for high-throughput drug screening and rapid functional assays. The resulting neurons exhibit robust electrophysiological activity and are compatible with MEA, calcium imaging, and high-content imaging platforms.

10–14

Days to Maturity

>95%

MAP2+ Purity

8.4 Hz

Mean Firing Rate

2

Phases

PHASE 1 Transgene Induction (Days 0–4)

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1

Prepare NGN2-iPSC Line

Use iPSC lines carrying a Tet-On NGN2 cassette (integrated at AAVS1 safe harbor via CRISPR or lentiviral). Verify doxycycline responsiveness and transgene copy number by qPCR prior to differentiation. Bank validated clones at passage 25–35.

Tet-On NGN2 iPSCsAAVS1 Safe Harbor

⏲ Pre-differentiation

2

Seed & Induce with Doxycycline

Seed iPSCs at 50,000 cells/cm² on Matrigel. At Day 0, switch to N2 medium + doxycycline (2 μg/mL) to activate NGN2 expression. Morphological transformation begins within 24 h — cells elongate and extend neurites by Day 2.

DoxycyclineN2 MediumMatrigel

⏲ Days 0–4

3

Puromycin Selection

From Day 1, add puromycin (1 μg/mL) to select for cells expressing the NGN2 transgene (linked to a puromycin resistance cassette). This step eliminates non-transduced cells and ensures >95% purity. Remove puromycin at Day 4.

Puromycin

⏲ Days 1–4

PHASE 2 Maturation & Validation (Days 4–14)

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4

Replate on Co-Culture or Final Substrate

At Day 4, dissociate with Accutase and plate at final density (75,000 cells/cm²) on PLO/laminin. Optionally co-plate with mouse glia (1:1 ratio) to enhance synaptic maturation. Switch to Neurobasal + B27 + BDNF (10 ng/mL) + NT-3 (10 ng/mL) + laminin (1 μg/mL). Maintain doxycycline until Day 7, then withdraw.

PLO/LamininBDNFNT-3Mouse Glia

⏲ Day 4

5

QC Checkpoint — Rapid Validation

At Day 10–14: ICC confirms MAP2+ (>95%), SYN1+ (synapsin), VGLUT1+. MEA recordings at Day 10 show robust spontaneous activity (8.4 Hz mean firing rate). Calcium imaging demonstrates synchronized transients. Neurons are ready for experimental use — drug screening, electrophysiology, or high-content assays.

ICC PanelMEACalcium Imaging

⏲ Day 10–14 QC

04

Cardio

Cardiomyocyte Differentiation from iPSCs

This protocol generates beating cardiomyocytes from human iPSCs via biphasic WNT signaling modulation: initial WNT activation with CHIR99021 drives mesoderm specification, followed by WNT inhibition with IWP2 to induce cardiac commitment. After metabolic selection with lactate, the resulting cardiomyocytes express cTnT, NKX2-5, MLC2v, and α-actinin with spontaneous beating activity suitable for cardiotoxicity screening and safety pharmacology.

15–20

Days to Beating

>90%

cTnT+ Purity

60–80

BPM Baseline

3

Phases

PHASE 1 Mesoderm Induction (Days 0–4)

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1

Prepare Monolayer & WNT Activation

Grow iPSCs to 85–90% confluency in mTeSR Plus on Matrigel. At Day 0, switch to RPMI + B27 minus insulin + CHIR99021 (8–12 μM; titre per line — critical optimization step). This activates canonical WNT signaling to induce mesodermal commitment. Brachyury (T)+ cells appear within 24 h.

CHIR99021RPMI/B27-insMatrigel

⏲ Day 0 (48 h pulse)

2

Remove CHIR & Rest

At 48 h, replace with RPMI/B27-ins (no CHIR). Rest cells for 24 h. Significant cell death is normal (30–50%); surviving cells are committed mesoderm. Do not passage.

RPMI/B27-ins

⏲ Day 2–3

PHASE 2 Cardiac Specification (Days 3–10)

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3

WNT Inhibition with IWP2

At Day 3, add IWP2 (5 μM) in RPMI/B27-ins for 48 h. This inhibits WNT secretion (Porcupine inhibitor) and drives cardiac mesoderm to committed cardiac progenitors. NKX2-5 expression begins at Day 5–6.

IWP2RPMI/B27-ins

⏲ Days 3–5

4

Cardiac Commitment & First Beating

Remove IWP2 at Day 5. Switch to RPMI + B27 with insulin from Day 7. Spontaneous beating typically appears between Day 8–10. If no beating by Day 12, the run is likely suboptimal — check CHIR concentration for the line.

RPMI/B27+ins

⏲ Days 5–10 (beating onset)

PHASE 3 Purification & Maturation (Days 10–30)

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5

Metabolic Selection with Lactate

At Day 10–12, switch to glucose-free RPMI + 4 mM sodium lactate for 4–6 days. Cardiomyocytes survive via lactate oxidation; non-cardiomyocytes (which depend on glycolysis) are eliminated. Expect >90% cTnT+ purity post-selection. Perform 2–3 rounds if needed.

Glucose-Free RPMISodium Lactate

⏲ Days 10–16

6

Maturation & Functional Validation

Return to RPMI/B27+ins. For enhanced maturation, replate on Matrigel-coated wells at Day 18–20 at 200,000 cells/cm². By Day 25–30: ICC for cTnT (>90%), α-actinin (sarcomere organization), MLC2v (ventricular identity). MEA: FPDc 350–450 ms, beat rate 60–80 BPM. Calcium transients with CTD90 ~400 ms. Ready for cardiotoxicity and safety pharmacology assays.

cTnT ICCMEACalcium ImagingImpedance

⏲ Day 25–30 QC

Dopaminergic Neuron Differentiation Protocol

PHASE 1 Floor-Plate Induction (Days 0–10)

PHASE 2 Precursor Expansion (Days 11–20)

PHASE 3 Terminal Differentiation (Days 20–35)

PHASE 4 Long-Term Culture & Experiments (Days 35+)

Cortical Excitatory Neuron Differentiation

PHASE 1 Neural Induction (Days 0–12)

PHASE 2 Progenitor Expansion (Days 12–25)

PHASE 3 Maturation & Validation (Days 25–56)

NGN2-Driven Rapid Neuronal Induction

PHASE 1 Transgene Induction (Days 0–4)

PHASE 2 Maturation & Validation (Days 4–14)

Cardiomyocyte Differentiation from iPSCs

PHASE 1 Mesoderm Induction (Days 0–4)

PHASE 2 Cardiac Specification (Days 3–10)

PHASE 3 Purification & Maturation (Days 10–30)

Protocol Comparison Overview