A cellular model for sporadic ALS using patient-derived induced pluripotent stem cells

Abstract

Development of therapeutics for genetically complex neurodegenerative diseases such as sporadic amyotrophic lateral sclerosis (ALS) has largely been hampered by lack of relevant disease models. Reprogramming of sporadic ALS patients' fibroblasts into induced pluripotent stem cells (iPSC) and differentiation into affected neurons that show a disease phenotype could provide a cellular model for disease mechanism studies and drug discovery. Here we report the reprogramming to pluripotency of fibroblasts from a large cohort of healthy controls and ALS patients and their differentiation into motor neurons. We demonstrate that motor neurons derived from three sALS patients show de novo TDP-43 aggregation and that the aggregates recapitulate pathology in postmortem tissue from one of the same patients from which the iPSC were derived. We configured a high-content chemical screen using the TDP-43 aggregate endpoint both in lower motor neurons and upper motor neuron like cells and identified FDA-approved small molecule modulators including Digoxin demonstrating the feasibility of patient-derived iPSC-based disease modeling for drug screening.

Keywords: ALS; Digoxin; Drug screening; FTLD; HTS; SOD1; TARDBP, TAR DNA binding protein; TDP-43; amyotrophic lateral sclerosis; fronto-temporal lobar degeneration; high-throughput screening; iPS; iPSC; iPSC-CN; iPSC-MN; induced pluripotent stem cell-derived cortical neurons; induced pluripotent stem cell-derived motor neurons; induced pluripotent stem cells; superoxide dismutase 1.

Figure 1. Patient-specific iPSC and iPSC-derived motor neurons

Representative images of: (A) iPSC colonies from one control and one ALS patient in phase-contrast. (B) iPSC colonies stained with antibodies for pluripotency markers NANOG and TRA-1-60. (C) iPSC-MN cultures stained with nuclear marker DAPI (blue) and antibodies to motor neuron markers ISLET1 (red) and HB9 (green). (D) iPSC-MN cultures stained with antibody to axonal marker Neurofilament (SMI31). iPSC and iPSC-MN shown are from healthy control IPRN.0013 and fALS patient IPRN.0028. (Scale bars a-b: 200 µm, c-d: 75 µm). (E) (i-ii), Representative trace showing electrical activity of iPS-MN from a healthy individual (Scale bars: 100 mV; 100 ms. 8 neurons recorded from fired robust and repetitive action potentials (bursts) to depolarizing current steps. (Iii) Example trace showing action potential elicited by the rebound depolarization following 300 pA hyperpolarizing step for 100 ms (Scales bars: 100 mV; 100 ms) and example trace of spontaneous bursts of action potentials and spontaneous post-synaptic potentials from iPS derived motor neurons (50 mV; 3 Sec). (Insets) Examples of EPSP (scales: 3 mV; 250 ms), and IPSP (scales: 30 mV, 100 ms). (F) (i) Sporadic ALS iPSC-MN responses −10, 0, and 10 pA current steps for 300 msec (scale: 25 mV). (ii) APs elicited by rebound depolarization following hyperpolarizing current step (300 pA; 300 msec). (iii) Spontaneous activity of sALS iPSC-MN (scale bars; 50 mV; 500 msec) (insets example of sEPSP and sIPSP (scale bars: 5 mV; 200 msec))( n = 7).

Figure 2. TDP-43 localization in patient iPSC-derived motor neurons

(A) iPSC-MN stained for TDP-43 shows TDP-43 is ubiquitously expressed and nuclear in all cells including in ISLET1-positive neurons from control (left panel) and ALS (right panel) patients. ISLET1-positive cells from sporadic ALS iPSC-MN have nuclear staining as well as nuclear aggregates that stain with higher intensity for TDP-43 (arrowheads, right panel) but aggregates are not present in healthy controls (left panel). Fibroblasts from healthy control and sALS patients do not show nuclear aggregates. Scale bar: 30 µm. (B) Sporadic ALS patient-derived iPSC-MN cells stained with nuclear envelop marker LAMIN-A (green) and TDP-43 (red) shows TDP-43 aggregates are inside the nuclear envelope; scale bar: 20µm. (C) Quantification of healthy control IPRN.0013 and sALS IPRN.0048 clone 1 iPSC-MN cultures shows TDP-43 aggregation is present in higher fraction of ISLET1 or HB9-positive cells compared to negative cells. (30.7% of ISLET1/HB9-positive motor neurons as compared to 16.2% of ISLET1/HB9-negative cells in sALS iPSC-MN cultures. Bars are standard deviation 9% and 8% respectively; P<2.2e-16 Welch t-test; data from three differentiations, N= 458 and 466 images for control and ALS respectively, with each image containing on average 400 cells to account for more than180,000 cells counted per group). (D) Phospho-serine 409/410 TDP-43 antibody labels TDP-43 aggregates in patient iPSC-MN and co-localizes with aggregates stained with pan-TDP-43 antibody, from left to right: DAPI, Pan-TDP-43, Phospho-TDP-43 antibody, and merged images (scale bar = 60 µm). iPSC-MN cultures shown were derived from healthy control, IPRN.0013, and sALS patient, IPRN.0048.

Figure 3. TDP-43 aggregates are present in iPSC-cortical neurons and in patient postmortem CNS

(A) Healthy control IPRN.0013 and (B) ALS patient IPRN.0048 iPSC-CN cultures stained with MAP2 (green) and TDP-43 (red) show intranuclear TDP-43 aggregates are present in ALS patient cells (B) but not in healthy control cells (A). (C & E), Control and (D & F) ALS iPSC-CN stained for corticospinal neuron marker CTIP2 (green in C & D) and TDP-43 (red in C & D). (C–F), CTIP2 positive neurons have TDP-43 aggregates (arrows in D & F). (G–J), Intranuclear TDP-43 aggregates in patient (IPRN.0360) postmortem CNS stained with TDP-43 (brown) and Luxol fast blue. (G & I), Spinal cord anterior horn motor neurons have intranuclear TDP-43 aggregates (arrow) and cytoplasmic mislocalization (arrowhead). (H & J), frontal lobe neurons have intranuclear TDP-43 aggregates (arrow). (K) 11% of CTIP2-positive and 3% of CTIP2-negative cells had TDP-43 aggregates. Error bars are standard deviation (4 and 1 respectively; total cells counted = 1548). Scale bars (A–F)= 20 µm, (G–J)10 µm.

Figure 4. sALS patient iPSC-CN treated with cardiac glycosides

(A–C), sALS patient iPSC-CN control DMSO-treated for 48 hours and stained for TDP-43. (D–F), iPSC-CN treated with Digoxin at 3 µM, Lanatoside C at 1.5 µM, and Proscillardin at 1.5 µM for 48 hours and stained for TDP-43. (E–F), Dose response curves for the three compounds on iPSC-CN show dose dependent reduction in percent cells containing TDP-43 aggregates. (G–I), total cell count for compound-treated iPSC-CN shows that cell number does not decrease with increasing dose and compounds are not cytotoxic.