iPSC & Cell Models — Armin Bayati, PhD

01

iPSC Culture

iPSC Maintenance, Expansion & Differentiation

My iPSC culturing, maintenance, and differentiation training was done in association with the EDDU at McGill, generating some of the most homogenous cultures in the world. iPSCs from 9 different donors were used for modeling Parkinson's disease pathology. I have generated and banked over 20 different iPSC cell lines.

Differentiation spans ectodermal lineages (dopaminergic, cortical/excitatory neurons, astrocytes, microglia-like cells) and endodermal lineages (hepatic, lung/epithelial, gut, beta islet), in both adherent and suspension culture formats. Neuronal maturation requires 4–6 weeks to achieve full dopaminergic identity (DAT, GIRK2) or cortical maturity (VGLUT1, GABA).

iPSC differentiation protocol diagram — Schematic of iPSC differentiation pathways to dopaminergic, cortical, and other neuronal lineages, showing protocol timeline and key developmental stages.

Dopaminergic Neuron Characterization & QC

Following differentiation, I characterize neuronal identity via antibody profiling (ICC), qPCR, and flow cytometry. Electrical activity is confirmed using microelectrode arrays (MEAs) for spontaneous firing patterns and waveform analysis. I have also performed HPLC-based quantification of dopamine and metabolites from culture media to link biochemical outputs to functional phenotypes.

iPSC Differentiation Lineage Map

Pluripotent stem cells directed through ectodermal, mesodermal, and endodermal germ layers into disease-relevant cell types for modeling and screening.

Ectodermal

Mesodermal

Endodermal

Application

ICC characterization panel: DAT, GIRK2, Neurofilament 160kD, TH with Hoechst, MAP2, and composite overlays in iPSC-derived DA neurons — Immunocytochemistry panel confirming dopaminergic identity via DAT, GIRK2, Neurofilament 160kD, and TH with DAPI, MAP2, and composite overlays. From Bayati et al., Nature Neuroscience 2024.

RT-qPCR — Dopaminergic Neuron Gene Expression

RT-qPCR quantification confirms cell-type identity and treatment response at the transcriptional level. Expression is normalized to housekeeping genes (GAPDH, ACTB) and presented as fold change relative to undifferentiated or untreated controls. Error bars represent SEM from n=3 biological replicates.

RT-qPCR: Dopaminergic Marker Expression

Fold change in DA neuron markers at DIV 42 vs undifferentiated iPSCs (GAPDH-normalized)

ΔΔCt method. n=3 biological replicates per line. *p<0.05, **p<0.01 vs iPSC. Housekeeping: GAPDH, ACTB.

RT-qPCR Expression Heatmap

Relative gene expression across conditions (log₂ fold change)

Color intensity: red = upregulated, blue = downregulated relative to control. Values are log₂(fold change).

Electrophysiology & Calcium Imaging

I confirm neuronal identity and functional maturity using microelectrode array (MEA) recordings and calcium imaging. MEA captures spontaneous electrical activity, burst frequency, and network synchrony across neuronal subtypes. Calcium imaging with Fluo-4 or GCaMP reporters reveals spontaneous and evoked calcium transients in real time.

MEA raster plot with spike waveforms across dopaminergic and cortical neurons — Microelectrode array (MEA) raster plot showing spontaneous spike waveforms across dopaminergic and cortical neurons at DIV 42.

Fig. — MEA: Firing Rate Across Neuronal Subtypes

Mean firing rate and burst frequency in iPSC-derived DA, cortical, and excitatory neurons at DIV 42

Axion Maestro MEA. 5-min recording. n=6 wells per condition.

Calcium imaging stills — Fluo-4 fluorescence in DA neurons showing spontaneous transients — Calcium imaging fluorescence traces (Fluo-4) revealing spontaneous and KCl-evoked transients in iPSC-derived dopaminergic neurons.

Fig. — Calcium Imaging: Spontaneous & Evoked Transients

ΔF/F₀ traces from iPSC-derived DA neurons — spontaneous activity and KCl-evoked depolarization

Fluo-4 AM, confocal time-lapse. KCl (50 mM) applied at t=60s. 3 representative ROIs.

Fig. — HPLC Dopamine Quantification

Dopamine and DOPAC levels in iPSC-derived DA neurons ± L-DOPA treatment across PD and non-PD lines

iPSC-derived DA neurons from PD (SNCA Trip, GBA-N370S) and non-PD control lines. Conditioned media collected at DIV 42.

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Methods

Detailed Methodology & Techniques

Comprehensive descriptions of key experimental techniques, assay platforms, and analytical methods referenced throughout this page.

Cortical Neuron Differentiation

I differentiate iPSCs into cortical excitatory neurons using dual-SMAD inhibition followed by Wnt modulation, yielding VGLUT1+/TBR1+ glutamatergic neurons by DIV 35–42. These cultures serve as critical controls for dopaminergic-selective vulnerability studies and are used in network-level MEA recordings to assess excitatory synaptic transmission.

Cortical neurons are validated by immunocytochemistry for CTIP2, SATB2, BRN2 (layer markers) and VGLUT1, PSD-95 (synaptic markers). Under identical dual-hit conditions (PFF + IFN-γ), cortical neurons do not form Lewy body–like inclusions — highlighting cell-type–specific vulnerability.

Fig. — Cortical Neuron Marker Expression

ICC quantification of cortical markers at DIV 28 vs DIV 42 (n=4 wells per timepoint)

Quantified by automated high-content imaging (Opera Phenix). Error bars represent SEM.

Endodermal Lineages

Beyond neuronal lineages, I differentiate iPSCs into endodermal derivatives including hepatocyte-like cells, lung epithelial cells, intestinal organoids, and pancreatic beta islet–like cells. Protocols use Activin A–driven definitive endoderm induction followed by lineage-specific growth factor cocktails.

Hepatic differentiation yields albumin-secreting, CYP3A4-active hepatocyte-like cells suitable for drug metabolism and toxicity screening. Lung epithelial cultures express ACE2 and TMPRSS2, enabling SARS-CoV-2 infection modeling.

Fig. — Endodermal Lineage Marker Profiles

Marker expression across hepatic, lung, and gut/islet differentiation at endpoint

Flow cytometry and qPCR. n=3 independent differentiations per lineage.

02

Expression

Mammalian Expression Systems

I operate CHO and HEK293 expression platforms for recombinant protein production, lentiviral generation, and translational initiatives. This includes suspension CHO workflows (transfection → pool evaluation → clone → expression characterization → pre-banking assessment), screening funnel design, expression benchmarking, and 5 L single-use bioreactor operation.

I design and optimize expression vectors including promoter selection, signal peptide configuration, and construct architecture. Process characterization includes density-dependent productivity studies, time-course expression kinetics, and nutrient-sensitive viability shifts.

03

Disease Modeling

Lewy Body Formation: Modeling Parkinson's Pathogenesis

I developed a dual-hit model of Parkinson's disease in iPSC-derived dopaminergic neurons, published as a first-author paper in Nature Neuroscience (2024). Sequential exposure to α-synuclein preformed fibrils (PFFs) followed by proinflammatory cytokines (IFN-γ) drives formation of Lewy body–like inclusions — 5–10 µm membrane-bound perinuclear structures containing fibrils, damaged lysosomes, mitochondria, and filamentous material. These inclusions form in 15–20% of DA neurons over 14 days, are ThS-positive (amyloid-containing), and are phospho-α-synuclein–enriched — closely resembling patient-tissue Lewy bodies by EM.

Dual-Hit Neurodegeneration Model

A two-insult paradigm combining α-synuclein pre-formed fibrils (PFFs) with interferon-γ to recapitulate inflammatory Lewy body pathology in iPSC-derived dopaminergic neurons.

1

HIT 1 — PROTEOPATHIC SEED

α-Synuclein Pre-Formed Fibrils (PFFs)

Sonicated recombinant fibrils that seed endogenous α-synuclein aggregation via macropinocytic uptake. Fibrils bypass early endosomes and accumulate in lysosomes within 2 minutes, triggering lysosomal dysfunction and Lewy-like inclusion formation.

2

HIT 2 — NEUROINFLAMMATORY INSULT

Interferon-γ (IFN-γ)

Pro-inflammatory cytokine that activates JAK/STAT signaling, upregulates MHC-I, and disrupts autophagy flux. When combined with PFFs, IFN-γ dramatically accelerates α-synuclein pathology, collapses the autophagy-lysosomal pathway, and drives neuronal death.

Day 0

PFF Addition

Sonicated fibrils added to mature DA neurons

Day 1

IFN-γ Addition

10 ng/mL inflammatory insult

Day 7

Early Pathology

pS129⁺ puncta visible; lysosomal stress begins

Day 14

Inclusion Growth

Thioflavin S⁺ amyloid cores; autophagy collapse

Day 21

Mature Pathology

Lewy body–like inclusions; neuronal death

Day 21+

Drug Window

PAH / Parkin agonist rescue experiments

Multi-Readout Validation Endpoints

Pathology

Inclusion Quantification

pS129, ThioS, ubiquitin ICC + confocal

Lysosomal

ALP Integrity

LAMP1, LAMP2, TFEB, Galectin-3 WB

Autophagy

Flux Monitoring

LC3B, p62/SQSTM1, HDAC6 WB

Stress

ISR & Senescence

ATF4, ATF3, SA-β-gal, ROS

Viability

Cell Death Markers

LDH, Caspase-3/7, CellTiter-Glo

Proteomics

TMT Mass Spec

Subcellular fractionation + pathway analysis

Prion-like α-Synuclein Propagation Pathway

Pre-formed fibrils (PFFs) enter neurons via macropinocytosis, traffic through the endolysosomal system, and propagate to neighboring cells via exosomal release — a key therapeutic intervention axis.

01 · Binding

Membrane Engagement

Cell Surface

Exogenous α-synuclein PFFs bind to cell-surface heparan sulfate proteoglycans (HSPGs) and lipid rafts. Spike–ACE2 receptor engagement uses clathrin-mediated endocytosis.

t = 0 min

02 · Entry

Macropinocytosis

Plasma Membrane

PFFs are internalized via macropinocytic ruffles, bypassing classical clathrin and caveolar routes. Confirmed by EIPA and Latrunculin B inhibition.

✕ EIPA ✕ LatB

t = 0–2 min

03 · Trafficking

Lysosomal Accumulation

Endolysosomal System

PFFs bypass early endosomes and reach LAMP1⁺ lysosomes within 2 min. Nanogold EM tracing confirms rapid transit. Fibrils resist degradation and accumulate, causing lysosomal dysfunction.

t = 2 min → accumulation

04 · Seeding

Lewy-like Inclusions

Cytoplasm

Fibril seeds recruit endogenous α-synuclein, forming pS129⁺ Lewy-like inclusions. Thioflavin S⁺ amyloid cores form over 14–21 days. Dual-hit (PFF + IFN-γ) dramatically accelerates pathology.

t = 7–21 days

05 · Spread

Exosomal Propagation

MVB → Extracellular

CD63⁺ multivesicular bodies (MVBs) package fibrils into exosomes for cell-to-cell transmission. EM gold-labeled exosomes confirmed transfer to naïve neurons — the prion-like spread mechanism.

Continuous propagation

< 2 min

PFF → Lysosome transit

Bypassed

Early endosome route

14–21 d

Inclusion maturation

CD63⁺

Exosome-mediated spread

Critically, cortical neurons do not form inclusions under the same conditions, reflecting the selective vulnerability of DA neurons. IFN-γ specifically downregulates LAMP1, LAMP2, TFEB, and NRF2 in DA neurons, impairing the autophagy-lysosomal pathway. LAMP2 knockdown or GBA knockout alone is sufficient to trigger inclusion formation without immune challenge, establishing lysosomal dysfunction as the central vulnerability. Co-culture with activated microglia accelerates pathology, while astrocyte co-culture is protective.

Schematic of Parkinson's disease dual-hit model: microglia activation, cytokine release, PFF entry via endocytosis, lysosomal dysfunction, and Lewy body-like inclusion formation — Schematic of the dual-hit Parkinson's disease model: microglia activation, cytokine release, α-synuclein PFF uptake via macropinocytosis, lysosomal dysfunction, and formation of membrane-bound Lewy body–like inclusions.

Dual-hit pathological cascade: Activated microglia release proinflammatory cytokines (IFN-γ, TNF, IL-1β) that compromise lysosomal function in DA neurons. Exogenous α-synuclein PFFs enter via macropinocytosis, accumulate in dysfunctional lysosomes, seed endogenous α-synuclein, and drive formation of 5–10 µm membrane-bound Lewy body–like inclusions containing fibrils, damaged mitochondria, and disrupted organelles. From Bayati et al., Nature Neuroscience 2024.

Dual-Hit Treatment & Lewy Body EM

Confocal: PFF-only vs PFF + IFN-γ dual-hit comparison

1

EM: Lewy body–like inclusion at low and high magnification

2

EM: Compact/mature inclusion resembling patient tissue Lewy body

3

Cortical neurons: MAP2 neuronal network with DAPI nuclear stain

4

DA neuron with TH, PFF-gold, and pSyn-488 perinuclear inclusion

5

Syn-HA and phospho-synuclein colocalization in iPSC neurons

6

TH and pSyn in KO vs WT vs SNCA triplication DA neurons

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04

Co-Culture

Neuronal Co-Cultures & Immune Models

I co-cultured iPSC-derived dopaminergic neurons with activated microglia-like cells and human astrocytes. Microglia secreting IFN-γ, IL-1β, and TNF accelerated α-synuclein inclusions, while astrocyte co-culture reduced accumulation. I also established immune-mediated killing/immune-target co-culture assays using PBMCs, T-cells, and B-cells, quantifying immune-driven target-cell loss and cytokine signaling via flow cytometry and immunoassay readouts.

Neuronal Co-Culture Imaging

Neuron–microglia co-culture: accelerated α-syn inclusion formation

1

Neuron–astrocyte co-culture: reduced α-syn accumulation

2

Side-by-side comparison across co-culture conditions

3

iPSC-Derived Neurons & Cell Models

iPSC Maintenance, Expansion & Differentiation

Detailed Methodology & Techniques

Mammalian Expression Systems

Lewy Body Formation: Modeling Parkinson's Pathogenesis

Neuronal Co-Cultures & Immune Models

Cell Model Pipeline