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

NPC characterization.

<p><b>A) Phase contrast microscopy</b> of α-synuclein gene triplication (SNCA-Tri), control (Ctrl) and α-synuclein knockdown (SNCA-Tri KD) iPSC-derived NPC lines (Scale bar: 50 µm) shows normal cell morphology. <b>B)</b><b>Mitochondrial and nuclear morphology</b> of NPCs visualized by fluorescence microscopy using Mitotracker Red CMX Ros (red) and Hoechst 33342 (blue) (Scale bar: 10 µm). <b>C)</b><b>Stem cell marker expression</b>. Immuno-cytochemistry on fixed NPCs detecting cytoplasmic Nestin expression pattern with secondary Alexa 588 conjugated antibody (orange) by fluorescence microscopy (Scale bar: 100 µm). Insert: Immuno-cytochemistry for the nuclear stem cell marker SOX1, detected by a secondary Alexa-488 conjugated antibody (green) (Scale bar: 20 µm). Nuclear counter stain by Hoechst 33342 (blue). <b>D) Representative α-synuclein protein expression</b> patterns (left) by immunoblot of protein lysates from a control line (Ctrl), the SNCA-Tri NPC line and the corresponding α-synuclein knock down line (SNCA-Tri KD) with β-actin serving as loading control. Right: Quantification of β-actin normalized α-synuclein expression levels (n = 4, mean ± SEM, Ctrl/SNCA-Tri/SNCA-Tri KD: 12.4/5.9/8.3, ***<i>p≤</i>0.001, t-test; from two independent experiments). <b>E) ICC of α-synuclein protein expression in adherent NPCs</b> detected by a polyclonal α-syn antibody and visualized by Alexa-488 conjugated secondary antibody (green). DAPI nuclear counterstain (blue); (Scale bar 20 µm). Insert: Higher magnification image (Scale bar: 10 µm). <b>F)</b><b>Colocalization of subcellular</b><b>α-synuclein distribution with mitochondria</b> in adherent NPCs labeled with Mitotracker Red CMX Ros (red) and probed for α-syn as under E) (Scale bar: 5 µm).</p