High-Content Assays for Hepatotoxicity Using Induced Pluripotent Stem Cell–Derived Cells

Development of predictive in vitro assays for early toxicity evaluation is extremely important for improving the drug development process and reducing drug attrition rates during clinical development. High-content imaging-based in vitro toxicity assays are emerging as efficient tools for safety and efficacy testing to improve drug development efficiency. In this report we have used an induced pluripotent stem cell (iPSC)–derived hepatocyte cell model having a primary tissue-like phenotype, unlimited availability, and the potential to compare cells from different individuals. We examined a number of assays and phenotypic markers and developed automated screening methods for assessing multiparameter readouts of general and mechanism-specific hepatotoxicity. Endpoints assessed were cell viability, nuclear shape, average and integrated cell area, mitochondrial membrane potential, phospholipid accumulation, cytoskeleton integrity, and apoptosis. We assayed compounds with known mechanisms of toxicity and also evaluated a diverse hepatotoxicity library of 240 compounds. We conclude that high-content automated screening assays using iPSC-derived hepatocytes are feasible, provide information about mechanisms of toxicity, and can facilitate the safety assessment of drugs and chemicals

Higher Vulnerability and Stress Sensitivity of Neuronal Precursor Cells Carrying an Alpha-Synuclein Gene Triplication

Parkinson disease (PD) is a multi-factorial neurodegenerative disorder with loss of dopaminergic neurons in the substantia nigra and characteristic intracellular inclusions, called Lewy bodies. Genetic predisposition, such as point mutations and copy number variants of the SNCA gene locus can cause very similar PD-like neurodegeneration. The impact of altered α-synuclein protein expression on integrity and developmental potential of neuronal stem cells is largely unexplored, but may have wide ranging implications for PD manifestation and disease progression. Here, we investigated if induced pluripotent stem cell-derived neuronal precursor cells (NPCs) from a patient with Parkinson’s disease carrying a genomic triplication of the SNCA gene (SNCA-Tri). Our goal was to determine if these cells these neuronal precursor cells already display pathological changes and impaired cellular function that would likely predispose them when differentiated to neurodegeneration. To achieve this aim, we assessed viability and cellular physiology in human SNCA-Tri NPCs both under normal and environmentally stressed conditions to model in vitro gene-environment interactions which may play a role in the initiation and progression of PD. Human SNCA-Tri NPCs displayed overall normal cellular and mitochondrial morphology, but showed substantial changes in growth, viability, cellular energy metabolism and stress resistance especially when challenged by starvation or toxicant challenge. Knockdown of α-synuclein in the SNCA-Tri NPCs by stably expressed short hairpin RNA (shRNA) resulted in reversal of the observed phenotypic changes. These data show for the first time that genetic alterations such as the SNCA gene triplication set the stage for decreased developmental fitness, accelerated aging, and increased neuronal cell loss. The observation of this “stem cell pathology” could have a great impact on both quality and quantity of neuronal networks and could provide a powerful new tool for development of neuroprotective strategies for PD.Facultad de Ciencias MédicasInstituto de Investigaciones Bioquímicas de La Plat

High-Content Assays for Hepatotoxicity Using Induced Pluripotent Stem Cell–Derived Cells

Development of predictive in vitro assays for early toxicity evaluation is extremely important for improving the drug development process and reducing drug attrition rates during clinical development. High-content imaging-based in vitro toxicity assays are emerging as efficient tools for safety and efficacy testing to improve drug development efficiency. In this report we have used an induced pluripotent stem cell (iPSC)–derived hepatocyte cell model having a primary tissue-like phenotype, unlimited availability, and the potential to compare cells from different individuals. We examined a number of assays and phenotypic markers and developed automated screening methods for assessing multiparameter readouts of general and mechanism-specific hepatotoxicity. Endpoints assessed were cell viability, nuclear shape, average and integrated cell area, mitochondrial membrane potential, phospholipid accumulation, cytoskeleton integrity, and apoptosis. We assayed compounds with known mechanisms of toxicity and also evaluated a diverse hepatotoxicity library of 240 compounds. We conclude that high-content automated screening assays using iPSC-derived hepatocytes are feasible, provide information about mechanisms of toxicity, and can facilitate the safety assessment of drugs and chemicals

Higher vulnerability and stress sensitivity of neuronal precursor cells carrying an alpha-synuclein gene triplication.

Parkinson disease (PD) is a multi-factorial neurodegenerative disorder with loss of dopaminergic neurons in the substantia nigra and characteristic intracellular inclusions, called Lewy bodies. Genetic predisposition, such as point mutations and copy number variants of the SNCA gene locus can cause very similar PD-like neurodegeneration. The impact of altered α-synuclein protein expression on integrity and developmental potential of neuronal stem cells is largely unexplored, but may have wide ranging implications for PD manifestation and disease progression. Here, we investigated if induced pluripotent stem cell-derived neuronal precursor cells (NPCs) from a patient with Parkinson's disease carrying a genomic triplication of the SNCA gene (SNCA-Tri). Our goal was to determine if these cells these neuronal precursor cells already display pathological changes and impaired cellular function that would likely predispose them when differentiated to neurodegeneration. To achieve this aim, we assessed viability and cellular physiology in human SNCA-Tri NPCs both under normal and environmentally stressed conditions to model in vitro gene-environment interactions which may play a role in the initiation and progression of PD. Human SNCA-Tri NPCs displayed overall normal cellular and mitochondrial morphology, but showed substantial changes in growth, viability, cellular energy metabolism and stress resistance especially when challenged by starvation or toxicant challenge. Knockdown of α-synuclein in the SNCA-Tri NPCs by stably expressed short hairpin RNA (shRNA) resulted in reversal of the observed phenotypic changes. These data show for the first time that genetic alterations such as the SNCA gene triplication set the stage for decreased developmental fitness, accelerated aging, and increased neuronal cell loss. The observation of this "stem cell pathology" could have a great impact on both quality and quantity of neuronal networks and could provide a powerful new tool for development of neuroprotective strategies for PD

Mitochondrial integrity, MPT opening, and apoptosis.

<p><b>A) Mitochondrial calcein loading</b> by fluorescent plate reader HTS of in NPCs grown in 96 well micro plates. Relative fluorescent signal intensities (RFU) for calcein acquired after 30 min loading with Calcein AM and CoCl₂ were normalized to mitochondrial content (Mitotracker) and to cell number by Hoechst 33342 (H33342). 1 µM ionomycin was added directly before HTS analysis as negative control (Iono) (n = 8, mean ± SD, Ctrl/SNCA-Tri: 3.4/4.9, *<i>p</i> = 0.039). <b>B)</b><b>MPT-induced mitochondrial calcein loss</b> in Ctrl and SNCA-Tri NPCs after mitochondrial calcein–AM loading. Representative fluorescence microscopy images of Ctrl and SNCA-Tri NPCs loaded with calcein (green), Mitotracker (red) and CoCl₂ were assayed 1 hr. after treatment with 4 µM staurosporine under NG conditions. MPT opening results in entry of CoCl₂ into mitochondria and loss of calcein signal (nuclear counter stain: Hoechst 33342; scale bar: 100 µm). <b>Inserts:</b> Higher magnification images obtained by conventional fluorescence microscopy (Scale bar: 10 µm). <b>C) HCI automated fluorescence microscopy analysis</b> of MPT in NPCs treated with 4 µM staurosporine as under B). Images (see B) were analyzed using MetaXpress image processing software. Depicted are data of cellular calcein signal intensities normalized to mitochondrial content (Norm. RFU Calcein/RFU Mitotracker) from two replicate wells with four image sites/well per treatment condition (n = 16, mean ± SD, Ctrl/SNCA-Tri, HG: 834/457, HG+R: 1425/1011, NG: 864/574, HG+Iono: 187/190, *<i>p</i>≤0.01). <b>D)</b><b>Kinetic evaluation of MPT opening</b> and loss of mitochondrial calcein signal after induction of MTP using fluorescence plate reader based HTS analysis. NPCs treated and prepared as under B) were loaded with 4 µM stauropsporine and changes in calcein signal normalized to cell number and mitochondrial content (Δ Norm. RFU) were recorded every 1 min for 20 min (n = 8, mean ± SD, Ctrl/SNCA-Tri, HG: −0.06/−0.12, HG+R: −0.17/−0.28, HG+Iono: −0.03/−0.04, *<i>p</i>≤0.01). <b>E)</b><b>Cytochrome c immuno-cytochemistry</b> in Ctrl and SNCA-tri NPCs challenged with 200 µM paraquat (PQ) 15 min. before fixation. Shown are permeabilized cells probed with cytochrome c antibody, detected by an Alexa-488 nm labeled secondary antibody (green). Cells were counter stained with Hoechst 33342 (blue) (Scale bar: 100 µm, insert: 10 µm). <b>F)</b><b>Immunoblot analysis of cytochrome c levels</b> in sub-cellular fractions containing either cellular organelles (containing bound cytochrome c) or cytosolic proteins (with soluble cytochrome c) from NPC cell lysates (Ctrl and SNCA-Tri) treated with paraquat (PQ) as under E). Cytochrome c (14 kDa) and GAPDH (40 kDa) specific antibodies were detected by a secondary IR-dye conjugate.</p

Mitochondrial membrane potential (MMP) and energy balance.

<p><b>A) Fluorescence microscopy</b> of MMP in live NPCs from patient (SNCA-Tri) and control (Ctrl) loaded with 100 nM TMRM in normal growth medium (HG), medium plus 20 µM Rotenone (HG+R) or with 1 µM of the ionophore CCCP (HG+CCCP) as negative control (Scale bar: 10 µm). <b>B)</b><b>Plate reader based high throughput screen (HTS) of MMP</b> in live NPCs loaded with 20 µM JC-10 for 45 min. Cells were also treated with medium w/o glucose (NG). Shown are log ratios of reduced (Ex./Em. 540 nm/590 nm) to oxidized JC-10 (Ex./Em. 488 nm/520 nm) normalized to Hoechst 33342 (Log Norm. JC-10 Ratio) after 60 min. (n = 8, mean ± SEM, Ctrl/SNCA-Tri/SNCA-Tri KD for HG+R: 202/29/194 (xE04), *<i>p</i>≤0.05; for NG: 92/30/118 (xE03) **<i>p</i>≤0.006). <b>C) Plate reader based HTS for MMP loss</b> in live NPCs prepared and analyzed as under B). Fluorescence measurements were acquired as under B) every 5 min for 10 cycles and loss of MMP with time graphed as ΔRFU/min. (n = 8, mean ± SEM, Ctrl/SNCA-Tri/SNCA-Tri KD: HG: −0.02/−0.06/−0.01, *p≤0.05; HG+R: −0.17/−0.70/−0.22 ***p<0.001, NG: −0.08/−0.33/−0.04, *p≤0.05). <b>D)</b><b>Luminescence plate reader based HTS</b><b>of ATP levels</b> in Ctrl, SNCA-Tri and SNCA-Tri KD NPCs under the above growth conditions (HG, HG+R, NG) assayed by a coupled luciferin/luciferase assay. Depicted are ATP contents in cells treated with 20 µM rotenone (R) for 18 hrs. (n = 8, mean ± SD nMATP/ug protein in: Ctrl/SNCA-Tri/SNCA-Tri KD: HG: 1.66/0.75/1.37, **<i>p</i> = 0.003; NG: 0.69/0.45/0.51, *<i>p</i> = 0.04). <b>E and F) Mitochondrial metabolic activity</b> studied by Seahorse XF24 analysis. <b>E)</b> Oxygen Consumption Rate (OCR) and <b>F)</b> Extracellular Acidification Rate (ECAR). Shown are relative OCR compared to basal values as a function of the sequential addition of mitochondrial inhibitors Oligomycin (1 µM), CCCP (1.5 µM) and Rotenone (Rot, 5 µM) + Antimycin A (Ant, 1 µM). Significant changes compared to basal OCR rates (*p<0.05) and differences between lines treated with and without 6-OHDA (250 µM) for 1 hr are indicated by # (#p<0.05, mean ± SEM, n≥17; from five independent experiments).</p