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Figure 1. Schematic for the Embryoid Body Protocol

Forebrain-type neural precursors can be generated in 18 - 19 days from hPSC-derived NPCs after selecting neural rosettes from replated embryoid bodies. For the maturation of precursors to forebrain-type neurons, see the PIS. hPSC = human pluripotent stem cell; NPCs = neural progenitor cells; PIS = product information sheet

Figure 2. Schematic for the Monolayer Protocol

Forebrain-type neural precursors can be generated from NPC monolayers derived from embryonic and induced pluripotent stem cells after three single-cell passages. For the maturation of precursors to forebrain-type neurons, see the PIS. NPC = neural progenitor cell; PIS = product information sheet

Figure 3. Forebrain-Type Neurons Are Generated After Culture in STEMdiff™ Forebrain Neuron Differentiation and Maturation Kits

NPCs generated from hPSCs in mTeSR 1™ using the STEMdiff™ SMADi Neural Induction Kit EB protocol were differentiated and matured to forebrain-type neurons using the STEMdiff™ Forebrain Neuron Differentiation and Maturation Kits. (A) Forebrain-type neurons were formed after iPS cell-derived NPCs were cultured with the STEMdiff™ Forebrain Neuron Differentiation Kit for 7 days and STEMdiff™ Forebrain Neuron Maturation Kit for 14 days. The resulting cultures contain a highly pure population of (B) class III β-tubulin-positive neurons (green), with (C) fewer than 10% astrocytes (GFAP-positive cells, red). (D) Nuclei are labeled with DAPI (blue). NPCs = neural progenitor cells; hPSC = human pluripotent stem cell; EB = embryoid body; iPS = induced pluripotent stem

Figure 4. Downstream Differentiation of Neural Progenitor Cells to Neurons Is Possible Using the STEMdiff™ Differentiation and Maturation Kits

(A) NPCs generated from STiPS-R038 hPSCs in mTeSR™1 using the STEMdiff™ SMADi Neural Induction Kit EB protocol were differentiated and matured to cortical neurons using STEMdiff™ Forebrain Neuron Differentiation Kit for 7 days and STEMdiff™ Forebrain Neuron Maturation Kit for 14 days. The resulting cultures contain a highly pure population of (B) class III β-tubulin-positive neurons (green) with less than 10% GFAP-positive astrocytes (not shown). (C) The generated neurons are also positive for FOXG1 expression (red), indicating a forebrain-type identity. (D) Nuclei are labeled with Hoechst (blue). NPCs = neural progenitor cells; hPSC = human pluripotent stem cell

Figure 5. A Mixed Population of Forebrain-Type Cortical Neurons Is Generated Using the STEMdiff™ Differentiation and Maturation Kits

Forebrain-type neurons generated from iPSC-derived NPCs (line AIW002-02) were cultured using the STEMdiff™ Forebrain Neuron Differentiation Kit for 7 days and subsequently matured for the following 6 weeks using STEMdiff™ Forebrain Neuron Maturation Kit. The resulting cultures contain a mixed population of neurons expressing VGLUT1, a glutamatergic marker of excitatory neurons (green), as well as MAP2-positive neurons, indicating mature neurons (magenta). Nuclei are labeled with Hoechst (blue). Data courtesy of Cecilia Rocha, The Neuro's Early Drug Discovery Unit (EDDU), McGill University. iPSC = induced pluripotent stem cell; NPCs = neural progenitor cells

Figure 6. PSC-Derived Astrocytes and Neurons Can Be Co-Cultured to Model Cell-Cell Interactions In Vitro

NPCs generated from the H1 cell line were differentiated to astrocytes using STEMdiff™ Astrocyte Differentiation and Maturation Kits. H9 cell-derived NPCs were differentiated to forebrain-type neurons using STEMdiff™ Forebrain Neuron Differentiation and Maturation Kits. For co-culture, matured astrocytes were seeded onto forebrain neurons that had been in STEMdiff™ Forebrain Neuron Maturation Medium for at least one week. Co-cultures were then switched to STEMdiff™ Forebrain Neuron Maturation Medium the following day and for the remaining co-culture. (A) Neurons cultured alone, following the co-culture feeding schedule, are labeled with DCX (green). (B) DCX-positive neurons (green) and astrocytes (GFAP, red) can be co-cultured for at least 1 - 2 weeks prior to analysis.For a detailed co-culture protocol, please see the Methods Library. NPCs = neural progenitor cells

Figure 7. PSC-Derived Neurons Survive and Mature when Co-Cultured with PSC-Derived Astrocytes

NPCs generated from the STiPS-R038 cell line were differentiated to astrocytes using STEMdiff™ Astrocyte Differentiation and Maturation Kits. STiPS-M001 cell-derived NPCs were differentiated to forebrain-type neurons using STEMdiff™ Forebrain Neuron Differentiation and Maturation Kits. After co-culture for one week, neurons (A) had significantly increased neurite outgrowth as measured on MAP2-positive neurons with the NeuriteTracer plugin for ImageJ (M Pool et al. J Neurosci Methods, 2008) and (B) were more numerous than neurons cultured alone using the same feeding schedule. *, p < 0.05; **, p < 0.01. NPCs = neural progenitor cells