Differentiation of Human Induced Pluripotent Stem Cells (iPSCs) into an Effective Model of Forebrain Neural Progenitor Cells and Mature Neurons

Abstract

Induced Pluripotent Stem Cells (iPSCs) are pluripotent stem cells that can be generated from somatic cells, and provide a way to model the development of neural tissues in vitro. One particularly interesting application of iPSCs is the development of neurons analogous to those found in the human forebrain. Forebrain neurons play a central role in cognition and sensory processing, and deficits in forebrain neuronal activity contributes to a host of conditions, including epilepsy, Alzheimer's disease, and schizophrenia. Here, we present our protocol for differentiating iPSCs into forebrain neural progenitor cells (NPCs) and neurons, whereby neural rosettes are generated from stem cells without dissociation and NPCs purified from rosettes based on their adhesion, resulting in a more rapid generation of pure NPC cultures. Neural progenitor cells can be maintained as long-term cultures, or differentiated into forebrain neurons. This protocol provides a simplified and fast methodology of generating forebrain NPCs and neurons, and enables researchers to generate effective in vitro models to study forebrain disease and neurodevelopment. This protocol can also be easily adapted to generate other neural lineages.

Keywords: Cortical; Forebrain; NPC; NSC; Neuron; iPSC.

Figure 1.. Sample ICC staining for high-quality iPSC cultures.

In order for differentiation to proceed effectively, ensure that you begin differentiation with high-quality iPSC cultures. IPSCs should uniformly express pluripotency markers SSEA, OCT4, TRA-1-60, and NANOG (A) when assessed using ICC. Additional pluripotency markers DNMT3b, EST2, and ZFP42 can also be assessed using a qPCR assay (B). IPSCs should be free of karyotypic abnormalities (C), possess the ability to differentiate into all three germ lineages and express characteristic markers of each lineage (D), and test negative for mycoplasma contamination (E). Scale bars represent 100 μm.

Figure 2.. Morphology of iPSCs differentiating into forebrain NPCs.

A. Day 0: Showing a single iPSC colony of appropriate size immediately prior to addition of Neural Induction Media 1. B. Day 2: The iPSC colony, which has been treated with Neural Induction Media 1 for 2 days, begins to change cellular morphology and some cells extend processes. C. Day 5: Increased expansion of the colony with some differentiation of outer cells. D. Day 12: Appearance of rosettes in the colony become visible. NSCs are present in high confluence in the middle of these structures. It is at this point that colonies are detached and re-plated on non-adherent plates at D13 for two days. E. D13 immediately after plating on adherent plates. This image shows floating rosette colonies that will continue to proliferate and differentiate in a floating mass. Non-rosette cells either remained on the dish at D12 after chemical release or float as single cells on the non-adherent plates shown in (E). F. At D15, rosette clusters expand in size and are moved to adherent plates. Cell aggregates here are 3-dimensional, but are attached to the plate. Note the purity of the clusters at this point (F). Scale bars represent 130 µm.

Figure 3.. Sample ICC staining for high-quality NPC cultures.

In order for differentiation to proceed effectively, ensure that iPSC cultures uniformly express Nestin, SOX1, and PAX6. NPC cultures should have no expression of the pluripotent marker OCT4 (DAPI shown in blue in merge of PAX6 and OCT4; all cells express PAX6, i.e., 100% purity of the culture). Scale bars represent 100 μm.

Figure 4.. Example morphology for forebrain NPCs differentiating into neurons.

Morphology of a forebrain NPC culture differentiating into neurons. Images taken at D0 (A), D5 (B), D15 (C), D30 (D). Scale bars represent 130 µm.

Figure 5.. Sample ICC staining for high-quality forebrain neuronal.

Representative ICC of forebrain neuronal culture following 30 days of differentiation (D30) from NPCs, demonstrating the relative abundance of glutamatergic, GABAergic, and astrocytic markers in the population. These cultures are approximately 65% glutamatergic, 30% GABAergic, and 5%-10% astroglial. Scale bars represent 50 μm.

Figure 6.. Electrophysiological properties of high-quality forebrain neurons.

A. Differential image contrast of a glass microelectrode recording from a single neuron in the whole-cell configuration. Scale bars represent 20 μm. B. A hyperpolarizing pulse showing a depolarizing sag followed by multiple rebound action potentials. C. Left: Representative traces of voltage clamp recordings showing inward Na⁺ currents; Right: Sodium current traces disappear after tetrodotoxin (TTX) 1 μM application. D. Representative current-clamp recording from a spontaneously active neuron with resting membrane potential -40 mV. E. Representative recording showing action potentials fired by forebrain neurons during a current ramp protocol. F. Representative voltage-clamp recording from a neuron with spontaneous synaptic input. All electrophysiological data was obtained from D14 neurons.