iPSC-Derived Neurons & Astrocytes

iPSC → Midbrain Dopaminergic Neurons

I follow a Kriks/Studer-style dual-SMAD inhibition protocol with floor-plate patterning to generate authentic A9-like midbrain dopaminergic (mDA) neurons. iPSCs are committed to neuroectoderm by combined inhibition of TGFβ and BMP signaling, ventralized with sonic hedgehog and CHIR-mediated Wnt activation, then matured in BDNF/GDNF/cAMP/ascorbic-acid neurotrophic medium. These neurons are the workhorse of my Parkinson’s and PARK2 work.

1. iPSC QC and expansion

   Confirm karyotype, mycoplasma, and pluripotency (OCT4/NANOG/SSEA4) before each batch.

2. Neural induction (D0–5)

   KSR-based induction with SB431542 (10 µM) + LDN-193189 (100 nM).

3. Floor-plate patterning (D1–7)

   SHH C25II 100 ng/mL, FGF8a 100 ng/mL, Purmorphamine 2 µM, CHIR99021 3 µM.

4. Progenitor expansion (D11–25)

   N2 + B27 + BDNF (20), GDNF (20), AA (200), dbcAMP (500), DAPT (10 µM).

5. Replate & mature (D25+)

   PO/laminin/fibronectin in BrainPhys + maturation cocktail; assay at D40–60 for TH+/DAT+/GIRK2+.

iPSC → Spinal Motor Neurons

I generate spinal motor neurons (sMN) using a Maury/Wichterle-inspired patterning strategy. After dual-SMAD inhibition, iPSCs are caudalized with retinoic acid (RA) and ventralized with smoothened agonism (SAG/Purmorphamine) to specify the OLIG2+ motor-neuron progenitor domain. Notch inhibition with DAPT then drives terminal differentiation into ChAT+/ISL1+/HB9+ motor neurons used for ALS modeling, neuromuscular drug screens, and SOD1/C9ORF72/TDP-43 disease lines.

1. Neuralize (D0–6)

   iPSCs in N2/B27 + LDN-193189 100 nM + SB431542 10 µM.

2. Caudalize and ventralize (D6–12)

   Add retinoic acid (1 µM) and SAG (0.5 µM) or Purmorphamine (1 µM).

3. Specify motor-neuron progenitors (D12–18)

   Quantify OLIG2+/NKX6.1+ progenitors. Maintain RA + SAG.

4. Notch inhibition for postmitotic conversion (D18–24)

   Add DAPT 10 µM; switch to BDNF (10), GDNF (10), CNTF (10 ng/mL).

5. Mature and assay (D24+)

   Single-cell replate on laminin; functional MN assays (whole-cell patch, MEA) by D35–40.

iPSC → Cortical Glutamatergic Neurons

For cortical disease modeling (frontotemporal dementia, autism-spectrum disorders, schizophrenia genetics), I use a Shi/Livesey-style protocol that drives iPSCs through dorsal forebrain identity by dual-SMAD inhibition without ventralizing morphogens. The resulting FOXG1+/TBR1+/CTIP2+/SATB2+ neurons span deep- and upper-layer cortical fates and develop functional glutamatergic synapses by ~D60.

iPSC → NGN2+ Induced Neurons (i₃N)

For rapid, scalable neuron production I use the Zhang/Südhof transcription-factor-induction protocol: doxycycline-inducible NGN2 drives iPSCs to homogeneous excitatory glutamatergic neurons within 7–14 days. NGN2-induced neurons (i₃Ns) are ideal for high-throughput screens, isogenic comparisons, and 96/384-well disease assays where the long mDA timeline is impractical.

iPSC → Astrocytes

Co-culture with astrocytes is essential for synaptic maturation of iPSC-derived neurons and for modeling neuroinflammation. I generate astrocytes via a Krencik/Zhang-type extended differentiation: NPCs are expanded for 60–90 days under glial-promoting cytokines (CNTF/BMP4/LIF) until they convert to GFAP+/S100β+/AQP4+ astrocytes. Reactive states are induced with TNFα + IL-1α + C1q (A1) or HBEGF (A2) for inflammation studies.

α-Synuclein PFF Seeding & PARK2 Loss-of-Function

Mature mDA neurons are seeded with sonicated α-synuclein preformed fibrils (PFFs) at 1 µg/mL for 24 h. After 7–14 days, neurons accumulate phospho-S129 α-synuclein inclusions, lose TH+ neurites, and undergo death. Isogenic PARK2 KO lines exhibit baseline mitophagy defects and shortened neurites; the small-molecule Parkin agonist FB231 rescues mitochondrial-lysosomal colocalization and reduces pSyn (see Genomics & NGS).

Isogenic CRISPR-Cas9 Lines for Mechanism

I generate isogenic PARK2/Parkin, SNCA, LRRK2, and GBA KO lines by Cas9-RNP delivery. Single-cell sorted clones are sequenced (TIDE/Sanger + amplicon NGS), karyotyped, and Western-validated before differentiation. Genotype–phenotype effects are read out as bulk and single-cell RNA-seq alongside imaging and biochemistry.

Identity Confirmation — mDA Neurons

Each batch of iPSC-derived midbrain dopaminergic neurons is validated at the protein level by flow cytometry / immunofluorescence and at the transcript level by RT-qPCR (normalized to GAPDH, ACTB; compared to undifferentiated iPSC controls).