A Rapid Pipeline to Model Rare Neurodevelopmental Disorders with Simultaneous CRISPR/Cas9 Gene Editing

Abstract

The development of targeted therapeutics for rare neurodevelopmental disorders (NDDs) faces significant challenges due to the scarcity of subjects and the difficulty of obtaining human neural cells. Here, we illustrate a rapid, simple protocol by which patient derived cells can be reprogrammed to induced pluripotent stem cells (iPSCs) using an episomal vector and differentiated into neurons. Using this platform enables patient somatic cells to be converted to physiologically active neurons in less than two months with minimal labor. This platform includes a method to combine somatic cell reprogramming with CRISPR/Cas9 gene editing at single cell resolution, which enables the concurrent development of clonal knockout or knock-in models that can be used as isogenic control lines. This platform reduces the logistical barrier for using iPSC technology, allows for the development of appropriate control lines for use in rare neurodevelopmental disease research, and establishes a fundamental component to targeted therapeutics and precision medicine. Stem Cells Translational Medicine 2017;6:886-896.

Keywords: Clinical utility; Dopamine; Gene editing; Glutamate; Induced pluripotent stem cells; Neurodevelopment.

Figure 1

iPSC and forebrain neuron generation timeline. Patient fibroblasts are transfected with iPSC episomal vectors using electroporation to form colonies of iPSCs, which are purified through successive passages until pure iPSC colonies are achieved. Organoids are generated from iPSCs, and are either maintained in an aggregate state or plated as NPCs as different media are utilized to guide the differentiation of the cells into forebrain neurons. Once putative forebrain neurons have been generated, they are validated using immunocytochemistry and electrophysiology. Scale bar indicates 30 μm. Abbreviations: EBs, embryoid bodies; FBS, Fetal Bovine Serum; DMEM, Dulbecco modified Eagle's medium; iPSC, induced pluripotent stem cell; NPC, neural progenitor cells.

Figure 2

Induction of iPSCs from fibroblasts. (A): Schematic illustrating the steps of differentiation, media used and time course. Days are measured with respect to the end of selection. (B): Brightfield images showing different timepoints in the process of induction from fibroblasts to iPSCs. Scale bar indicates 30 μm. (C): Staining of iPSC colonies demonstrates all cells express the pluripotent markers TRA1‐60, Nanog, SSEA, and OCT4. Scale bar indicates 30 μm. Abbreviations: FBS, Fetal Bovine Serum; DMEM, Dulbecco modified Eagle's medium; iPSC, induced pluripotent stem cell.

Figure 3

Direct method of differentiating iPSCs into forebrain neurons. (A): Schematic illustrating the steps of differentiation, media used, and time course. Days are measured with respect to dissociation of iPSCs. (B): Brightfield images of organoids at 1 week and 4 weeks after dissociation of iPSC colonies. Image of an EB attached to a plate immediately before dissociation, and the resulting culture 5 days after replating cells. Scale bar indicates 30 μm. (C): Staining of attached organoids and dissociated cells 5 days after plating reveals cell in both conditions to uniformly express both MAP2 and TUJ1. Scale bar indicates 30 μm. (D): Top: Staining of putative forebrain neurons 4 weeks after plating from EBs shows all cells express SYT1 and TUJ1, and that both GABAergic and glutamatergic neurons are present. Bottom: Punctate staining is present for GABAα1, GluR1, and SYT1. Scale bars indicate 30 μm. Abbreviations: EB, embryoid body; FD, final differentiation; iPSC, induced pluripotent stem cell; NI, neural induction; NM, neuron maturation; NP, neural progenitor.

Figure 4

Electrophysiological characterization of forebrain neurons derived from induced pluripotent stem cells (iPSCs). (A): Sample phase images of forebrain neurons in culture at D30, D60, and D90 postdifferentiation. Scale bars indicate 25 μm. (B): Representative recordings of AP in current‐clamp mode, induced by somatic current injection (ΔI = 20 pA, from membrane potential of −70 mV) from forebrain neurons at D30, D60, and D90 postdifferentiation. (C, D): AP amplitude and AP half‐width measures in forebrain neurons over development. Stars denote statistical significance of change in AP parameters as a function of time spent differentiating cells (* ≤0 .05, ** ≤ 0.01). (E): Experimental voltage pulse‐step protocol (top) and representative voltage‐clamp recording traces (from a holding potential Vhold = −60 mV), from forebrain neurons at D30 (including expanded view of Na currents [dashed boxes, insert]), D60, and D90, postdifferentiation. (F): Average Na and K currents recorded from iPSC‐NPC1 at D30, D60, and D90 postdifferentiation, plotted as a function of step voltage amplitudes. Stars indicate significance of change in average currents as a function of time spent in differentiation (* ≤ 0.05, *** ≤ 0.001). (G, H): Representative voltage clamp traces of forebrain neurons at D90 postdifferentiating in the presence of the sodium channel block TTX and the potassium channel blocker TEA. (I): Membrane voltage at rest, determined immediately after establishing the whole‐cell configuration, without current injection. (J): Membrane capacitance determined from the compensatory circuit in voltage‐clamp. (K): Membrane resistance, while in the GOhm range, decreases during development. (L): Representative trace of miniature EPSCs from a forebrain neuron held at −60 mV. (M): Representative traces of macroscopic currents elicited by puffs of agonist‐containing solution targeting AMPA receptors, NMDA receptors, and GABA_A receptors. Abbreviations: AMPA, α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid; AP, action potentials; NMDA, N‐methyl‐D‐aspartate; GABA, γ‐aminobutyric acid.

Figure 5

Simultaneous CRIPSR/CAS9 genome editing and iPSC induction. (A): Schematic illustrating the transfection, selection, and induction process. (B): Bright field and fluorescent images of an iPSC colony 7 days and 25 following transfection. Successfully induced colonies are initially RFP+, but become RFP− due the episomal nature of the vector. Scale bars represent 30 μm. (C): Details of the CRIPSR/CAS9 and iPSC induction episomal vectors used. (D): Gel showing untransfected, homozygous KO, and heterozygous generated iPSC lines for the gene GRIN2B. (E): Expression of the GRIN2B gene as assessed via quantitative polymerase chain reaction in forebrain neurons derived from the iPSC lines shown in (D) 28 days after the initiation of differentiation. Expression levels normalized to GAPDH expression. (F): Sanger sequencing results from the GRIN2B locus of the polymerase chain reaction products shown in (D). (G): Representative chromatogram plots illustrating the deletion found in one allele of the GRIN2B knockout. All chromatogram plots can be found in the supplement. Abbreviations: iPSC, induced pluripotent stem cell; KO, knockout; RFP, red fluorescent protein.