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. 2024 Jul 15;13(14):1194.
doi: 10.3390/cells13141194.

Human-Induced Pluripotent Stem Cell (iPSC)-Derived GABAergic Neuron Differentiation in Bipolar Disorder

Daniel J Schill  1 , Durga Attili  1 , Cynthia J DeLong  1 , Melvin G McInnis  2 , Craig N Johnson  1 , Geoffrey G Murphy  3 , K Sue O'Shea  1   2
Affiliations

Human-Induced Pluripotent Stem Cell (iPSC)-Derived GABAergic Neuron Differentiation in Bipolar Disorder

Daniel J Schill et al. Cells. .

Abstract

Bipolar disorder (BP) is a recurring psychiatric condition characterized by alternating episodes of low energy (depressions) followed by manias (high energy). Cortical network activity produced by GABAergic interneurons may be critical in maintaining the balance in excitatory/inhibitory activity in the brain during development. Initially, GABAergic signaling is excitatory; with maturation, these cells undergo a functional switch that converts GABAA channels from depolarizing (excitatory) to hyperpolarizing (inhibitory), which is controlled by the intracellular concentration of two chloride transporters. The earliest, NKCC1, promotes chloride entry into the cell and depolarization, while the second (KCC2) stimulates movement of chloride from the neuron, hyperpolarizing it. Perturbations in the timing or expression of NKCC1/KCC2 may affect essential morphogenetic events including cell proliferation, migration, synaptogenesis and plasticity, and thereby the structure and function of the cortex. We derived induced pluripotent stem cells (iPSC) from BP patients and undiagnosed control (C) individuals, then modified a differentiation protocol to form GABAergic interneurons, harvesting cells at sequential stages of differentiation. qRT-PCR and RNA sequencing indicated that after six weeks of differentiation, controls transiently expressed high levels of NKCC1. Using multi-electrode array (MEA) analysis, we observed that BP neurons exhibit increased firing, network bursting and decreased synchrony compared to C. Understanding GABA signaling in differentiation may identify novel approaches and new targets for treatment of neuropsychiatric disorders such as BP.

Keywords: GABA switch; KCC2; NKCC1; SLC12A2; SLC12A5; gamma-aminobutyric acid; neuron; patient; skin biopsy; somatostatin; stem cell.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Generation and validation of CRISPR-corrected isogenic bipolar disorder cell lines. (A) Sanger sequencing representing CRISPR-mediated A to G conversion of rs1006737 in a representative BP line. (B) Karyotype analysis of a representative BP-C line. (C) Representative immunocytochemistry images of the expression of pluripotency genes OCT4, NANOG, TRA-160 and SSEA-1 for each CRISPR-corrected bipolar disorder cell line, 20×. FITC-conjugated secondary antibodies.
Figure 2
Figure 2
GABAergic neuron differentiation. (A) Protocol for the differentiation of GABAergic neurons from iPSC. (B) qRT-PCR analysis of the expression of the GABAergic marker GAD67, astrocyte-enriched transcript S100-β and marker of glutamatergic neurons vGlut1 in control, bipolar and bipolar-corrected neurons after 6 weeks of differentiation. Expressions of the GABAergic neuron subtype marker somatostatin, of CACNA1C and NKCC1/KCC2 mRNA in developing GABAergic neurons after 6 weeks of differentiation for control, bipolar and bipolar-corrected. * = p ≤ 0.05, Student’s t-test. Data from 6 independent differentiations were analyzed. (C) Immunocytochemical localization of the GABAergic marker, GAD65/67 and the neuronal protein, Map2, in representative control, bipolar and bipolar-corrected 6-week GABAergic neurons, 20X. (D) Representative scanning electron microscope (SEM) images of control, bipolar, and BP-C following 6 weeks of differentiation into GABAergic neurons, illustrating well-developed fields of processes and scattered cell bodies in BP-corrected cultures.
Figure 3
Figure 3
Bulk RNA sequencing analysis of GABAergic neurons. (A) Principal component analysis (PCA) plot of bipolar (BP), control and CRISPR-treated bipolar GABAergic neuron samples at 6 weeks post-differentiation. Two independent differentiations per line were employed. Titles of the axes indicate the proportion of variation each principal component accounts for. (B) Volcano plots compare differentially expressed genes between control vs. bipolar, highlighting select genes from the top 250 differentially expressed genes. Genes more highly expressed in BP are shown in red, and those more highly expressed in controls are shown in blue. Corresponding plots for control vs. bipolar-corrected and bipolar vs. bipolar-corrected are also shown. (C) Heatmap of the top 25 differentially expressed genes from pairwise comparisons between control, bipolar and bipolar-corrected groups. The values represent log2-fold changes relative to the average control sample.
Figure 4
Figure 4
Multi-electrode array analysis of GABAergic neurons over 6 weeks of differentiation. Weighted mean firing rate, network bursting and synchrony were assessed from d0–d50 in vitro. Initially, bipolar neurons exhibited greater firing rates and network bursting compared to control neurons, while firing synchrony was higher in control neurons throughout the culture period. Activity of control, bipolar and BP-corrected GABAergic neurons over time are represented as line graphs (A) or bar graphs (B). * = p < 0.05, ** = p < 0.01, *** = p < 0.005, Student’s t-tests. Data from 7 independent replicates were analyzed.
Figure 5
Figure 5
Live cell calcium imagining of GABAergic neurons. Six-week GABAergic neurons were subjected to 20 Hz stimulation and Ca++ handling was assessed using Fluo-5 imaging. (A) Representative images of control neurons pre- and post-stimulation are represented as line (B) and bar graphs (C), 20X. Control, bipolar and bipolar-corrected lines exhibited similar Ca++ handling patterns. Data from 11 independent differentiations are included.
Figure 6
Figure 6
Expression of NKCC1 and KCC2 in bipolar disorder GABAergic neurons derived from BP iPSC. qRT-PCR analysis of the relative expression units of NKCC1 (A), KCC2 (B) and ratio of NKCC1 over KCC2 (C) in control, bipolar and bipolar-corrected GABAergic neurons over development. Data from 11 independent differentiations were analyzed and significance assessed using Student’s t-test and ANOVA.
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