Journal: bioRxiv

Article Title: Small molecule screen employing patient-derived iPS hepatocytes identifies LRRK2 as a novel therapeutic target for Alpha1 Antitrypsin Deficiency

doi: 10.1101/2021.09.17.460732

Figure Lengend Snippet: The effectiveness of the small molecules is determined by the reduction of ATZ polymer (right column). The viability of the small molecules is evaluated by the DAPI + object counting (middle column) and the viability assay by Presto Blue (right column). The color of the dot represents each A1ATD patient iPSC line. Each dot represents one batch of hiPSC-hepatocytes. Statistical analysis involved a Kolmogorov-Smirnov test for normality, followed by Kruskal-Wallis test, followed by a Dunn’s multiple comparison test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: The A1ATD and healthy control iPSC-hepatocytes used in the screen were commercially sourced (Definigen), thawed in Def-HEP thawing media (Definigen), and plated at a density of 20,000 cells per well.

Techniques: Viability Assay

Journal: bioRxiv

Article Title: Small molecule screen employing patient-derived iPS hepatocytes identifies LRRK2 as a novel therapeutic target for Alpha1 Antitrypsin Deficiency

doi: 10.1101/2021.09.17.460732

Figure Lengend Snippet: ( A ) LRRK2 −/− PiZ mice were generated by crossing PiZ/C57B6 (expressing human ATZ polymer in liver) mice and LRRK2 −/− /C57B6 mice. ( B ) Representative images showing ATZ polymer levels in the livers of LRRK2 −/− x PiZ (right), PiZ (middle) and healthy controls (left). Scale bar, 200μm. ( C ) Semi-quantitative analysis of samples from (B) shows reduced polymer in LRRK2 −/− x PiZ (green bars) compared to PiZ (pink bars) mice. N=3 animals. ( D ) Representative images showing fibrosis levels (Picorsirius red staining) in the livers of LRRK2 −/− x PiZ (right), PiZ (middle) and healthy controls (left). Scale bar, 200μm. ( E ) Semi-quantitative analysis of samples in (D) shows reduced fibrosis in LRRK2 −/− x PiZ (green bars) compared to PiZ (pink bars) mice. N= 4 animals. Statistical analysis by ordinary ANOVA test followed by Dunnett’s multiple comparison test. ( F ) A panel of LRRK2 inhibitors demonstrate dose-dependent polymer reduction (pink curve, inhibition) and viability (black curve, cytotoxicity) following 48 hours treatment of Tetracycline-inducible (Tet-ON) CHO-K1 cells. N=3. Nonlinear regression by four-parameter curve fit. ( G ) Representative confocal images showing polymeric ATZ expression (green) in patient iPSC-hepatocytes treated with same panel of LRRK2 inhibitors as indicated. Scale bar, 150μm. ( H ) Quantitative assessment of confocal images from (G) demonstrates significant reduction of polymer (2C1) (left) following 48h treatment without reducing cell number (viability) (right). Each dot represents a biological replicate. Statistical analysis by ordinary ANOVA test followed by Dunnett’s multiple comparison test against the control. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: The A1ATD and healthy control iPSC-hepatocytes used in the screen were commercially sourced (Definigen), thawed in Def-HEP thawing media (Definigen), and plated at a density of 20,000 cells per well.

Techniques: Generated, Expressing, Staining, Inhibition

Journal: bioRxiv

Article Title: Small molecule screen employing patient-derived iPS hepatocytes identifies LRRK2 as a novel therapeutic target for Alpha1 Antitrypsin Deficiency

doi: 10.1101/2021.09.17.460732

Figure Lengend Snippet: ( A ) Representative images demonstrating ATZ polymer reduction in patient iPSC-Hepatocytes (middle row, green) with preserved total AAT (top row, red) after 24 hour treatment with CZC-25146 (middle column) compared to vehicle (left column) and health donor (right column) controls. Scale bar, 100μm ( B ) Quantification of polymer load (top graph), number of cells (middle graph) and cell viability (bottom graph) following increasing doses of CZC-25146 (x axis) administered for 24 hours across each of three different patient iPSC lines. 48 hour treatment of patient iPSC-Hepatocytes with CZC-25146 at high (purple bars) and lower (green bars) doses reduces intrahepatic polymeric ATZ ( C ), nuclear NFkB ( D ) and increases AAT secretion ( E ) compared to untreated (grey bars) or vehicle controls (pink bars). N=10 replicates. Age-matched healthy patient iPSC-derived hepatocytes were used as controls. Statistical analysis by Kruskal-Wallis test, followed by a Dunn’s multiple comparison test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: The A1ATD and healthy control iPSC-hepatocytes used in the screen were commercially sourced (Definigen), thawed in Def-HEP thawing media (Definigen), and plated at a density of 20,000 cells per well.

Techniques: Derivative Assay

Journal: bioRxiv

Article Title: Small molecule screen employing patient-derived iPS hepatocytes identifies LRRK2 as a novel therapeutic target for Alpha1 Antitrypsin Deficiency

doi: 10.1101/2021.09.17.460732

Figure Lengend Snippet: ( A ) Representative images of LC3-GFP fibroblasts, following treatment with CZC-25146 (CZC, middle), vehicle control (left) or positive control Rapamcyin (right). Images taken 2 hours after exposure. Scale bar, 50μm ( B ) Time course analysis following CZC (pink) or Rapamycin (green) treatment shows significant upregulation of LC3 expression from 2-10 hours compared to vehicle controls (grey). ( C ) RT-qPCR shows dose responsive increased expression of genes involved in the autophagy pathway following addition of CZC to patient iPSC-hepatocytes at Low (pink) = 14.3μM or High (green) = 28.6μM concentrations. N=4 replicates. ( D ) Representative images showing the effect of treatment with CZC alone (left middle), CZC + autophagy inhibitor, NH4Cl (right middle), NH4Cl alone (far right) or vehicle control (far left) on ATZ polymer (2C1 expression, green) in patient iPSC-hepatocytes. Scale bar, 100μm. ( E ) Semi-quantitative analysis of images from (D). ( F ) Representative macroscopic (top) or zoomed in (bottom) images of LC3 staining in livers of PiZ (far left), healthy mice (far right), CZC-25146 treated (right middle) or vehicle control (left middle). Quantification of images from (F) separately analysed by each liver lobe ( G ) or pooled ( H ). Quantification was conducted using ImageJ. N=5 mice per group. Statistical analysis by Ordinary ANOVA test, followed by Dunnet’s multiple comparisons test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: The A1ATD and healthy control iPSC-hepatocytes used in the screen were commercially sourced (Definigen), thawed in Def-HEP thawing media (Definigen), and plated at a density of 20,000 cells per well.

Techniques: Positive Control, Expressing, Quantitative RT-PCR, Staining

Journal: STAR Protocols

Article Title: Protocol for Differentiation of Human iPSCs into Pulmonary Neuroendocrine Cells

doi: 10.1016/j.xpro.2020.100068

Figure Lengend Snippet: Generation of Lung Progenitors during iPNEC Differentiation from Human iPSCs (A–D) Cultures stained for (A) lung endoderm marker NKX2.1 (green), (B) distal progenitor marker SOX9 (green), (C) proximal airway progenitor marker SOX2 (green) and (D) tight junction marker ZO1 (green) at day 17 of differentiation. Nuclei are counterstained with DAPI (blue). Scale bars, 200 μm.

Article Snippet: Healthy human lymphoblastoid cell-derived iPSCs (iPSC 2). Lymphoblastoid cell line reprogrammed by Ichida Lab USC , National Institute of Neurological Disorders and Stroke (NINDS) Biorepository, Coriell Institute , ND03719 (original lymphoblastoid cell line).

Techniques: Staining, Marker