Directed differentiation of forebrain GABA interneurons from human pluripotent stem cells

Abstract

Forebrain γ-aminobutyric acid (GABA) interneurons have crucial roles in high-order brain function via modulating network activities and plasticity, and they are implicated in many psychiatric disorders. Availability of enriched functional human forebrain GABA interneurons, especially those from people affected by GABA interneuron deficit disease, will be instrumental to the investigation of disease pathogenesis and development of therapeutics. We describe a protocol for directed differentiation of forebrain GABA interneurons from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) in a chemically defined system. In this protocol, human PSCs are first induced to primitive neuroepithelial cells over 10 d, and then patterned to NKX2.1-expressing medial ganglionic eminence progenitors by simple treatment with sonic hedgehog or its agonist purmorphamine over the next 2 weeks. These progenitors generate a nearly pure population of forebrain GABA interneurons by the sixth week. This simple and efficient protocol does not require transgenic modification or cell sorting, and it has been replicated with multiple human ESC and iPSC lines.

Figure 1

Timeline of forebrain GABA interneuron generation. Cells are differentiated under a chemically defined system. Differentiation of human PSC to forebrain GABA interneurons involves 37 steps and three stages, including induction of neuroepithelial cells, patterning of MGE progenitors and differentiation to GABA neurons. Pur is the major factor for MGE progenitor patterning. NE, neuroepithelium; NS, neuroepithelial spheres.

Figure 2

Step-by-step illustration of neuroepithelial differentiation. (a) Under a chemically defined system, hPSCs form EBs in suspension culture for the first 7 d. (b) The EBs are then plated on day 7 at the density as shown. (c) By day 10, each EB will develop into a colony that contains neuroepithelial cells in the form of rosettes. (d) At day 16, the rosette-containing colonies are detached and grown in suspension to form neuroepithelial spheres (NS).

Figure 3

Characteristics of MGE progenitors and forebrain GABA interneurons. (a) At day 25, nearly all the cells express the forebrain marker FOXG1 (green), and the MGE marker NKX2.1 (red). Hoechst is shown in blue. Quantification is on the right; n = 5 different differentiation cultures. We counted 800–1,200 cells per replicate. Data are presented as means ± s.e.m. (b) At day 35, MGE cells (NKX2.1⁺; red) begin to express GABA (green), indicating that the GABA neurons are coming from the ventral forebrain area. (c) At day 45, nearly all the neurons express GABA (green). Quantification of the EB method and monolayer method is on the right; n = 5 different differentiation cultures. We counted 800–1,200 cells per replicate. Data are presented as means ± s.e.m. Scale bars, 50 μm. βIII-T, βIII-tubulin.

Figure 4

Human PSC-derived forebrain GABAergic interneurons are functional in vitro. (a) Electrophysiological characteristics of hPSC-derived GABAergic neurons, which were differentiated for 10 weeks in vitro. n = 12; data are means ± s.e.m. (b) Experimental setup showing the whole-cell recording on a neuron. (c) Inward and outward currents were triggered upon −50 to 50-mV voltage steps. (d) Action potentials were induced by injection of current as low as 40 pA into neurons. (e,f) Spontaneous synaptic currents were recorded (e), and the GABA receptor activity was blocked by application of bicuculine (f).

Figure 5

Maturation of GABA interneuron subtypes. (a) CB⁺ and (b) SST⁺ GABA neurons are seen from day 35, and their populations increase over time. (c) PV⁺ GABA neurons appear later. They are observed at around day 65, and their proportion increases over time. (d) nNOS⁺ neurons can be also observed after day 65. (e) Quantification of GABA interneuron subtypes. n = 5 different differentiation cultures. Nuclei are stained with Hoechst (blue). We counted 800–1,200 cells per culture. Data are presented as means ± s.e.m. Scale bar, 50 μm.

Figure 6

Pasteur pipette technique for triturating MGE-like neurospheres. Flame the end of a cotton-filtered Pasteur pipette to round the edges and narrow the opening to about 0.2–0.5 mm in diameter. Flame the narrow part of the shaft to introduce a 15–20° smooth curve. Rinse the pipette wall with the culture medium three times. Pipette neuroepithelial clusters using the treated Pasteur pipette by pulling the cells in and push them out one or two times. The narrow opening and bend in the pipette will help shear the clusters into smaller pieces of a roughly uniform size. Do not triturate more than three times.