Journal: Cell metabolism

Article Title: Generation of human fatty livers using custom-engineered induced pluripotent stem cells with modifiable SIRT1 metabolism

doi: 10.1016/j.cmet.2019.06.017

Figure Lengend Snippet: (A) Immunohistochemical staining micrographs of SIRT1 show cytoplasmic and nuclear decrease of SIRT1 expression in NASH human livers (n=4) compared to Normal human livers (n=3). Western blot analysis and quantification of SIRT1 normalized to β-Actin in Normal (n=3) and NASH (n=3) human livers (P=0.400, Mann-Whitney test). Quantitative gene expression analysis of SIRT1 expression normalized to actb in Normal (n=4) and NASH (n=4) human livers (P=0.200, Mann-Whitney test). (B) Quantitative gene expression analysis of puromycin selection cassette gene normalized to actb on human fibroblasts transduced with lentiviral vector for –iRFP (hFib-iRFP) or –iKD-SIRT1 (hFibiKD-SIRT1) and non transduced human fibroblasts (hFib) as control (*P=0.0338, *P=0.0130, Kruskal-Wallis test and Dunnett’s multiple comparisons). Quantitative gene expression analysis SIRT1 normalized to actb in hFib-iRFP and hFib-iKD-SIRT1 in the presence or absence of doxycycline (*P=0.0422, Kruskal-Wallis test and Dunnett’s multiple comparisons). Western blot analysis and quantification of SIRT1 normalized to GAPDH on hFib-iRFP (n=4) and hFib-iKD-SIRT1 (n=4) with and without doxycycline treatment (*P=0.0395, Kruskal-Wallis test and Dunnett’s multiple comparisons). (C) Immunofluorescence micrographs of SIRT1 in hFF-iKD-SIRT1 with and without doxycycline treatment, human fetal hepatocytes and human adult hepatocytes were used as controls. Light Red fluorescence and bright light micrographs were used to analyze hFib-iRFP with and without doxycycline treatment. hFib-iRFP (n=4) hFib-iKD-SIRT1 (n=4), human Fetal hepatocytes (n=4), Adult primary hepatocytes untreated (n=3). (D) Light Red fluorescence micrographs of hiPS-iRFP colony after doxycycline exposure for 48h. DAPI was used as counterstaining. Quantitative gene expression analysis of SIRT1 expression normalized to actb show knockdown of SIRT1 in hiPS-iKD-SIRT1-#17 but not in hiPS-IRFP-#3 after exposure to docycycline (*P = 0.0360, *P=0.0140, Kruskal-Wallis test and Dunnett’s multiple comparisons). Western blot analysis and quantification of SIRT1 normalized to GAPDH in hiPS-IRFP-#3 (n=4) and hiPS-iKDSIRT1-# 17 (n=4) with and without doxycycline exposure for 48h (*P=0.0222, Kruskal-Wallis test and Dunnett’s multiple comparisons). (E) Immunofluorescence micrographs of pluripotency markers Nanog, Oct4, TRA-1–60 and SSEA-4 in hiPS-IRFP-#3 and hiPS-iKD-SIRT1-#17. Quantitative gene expression analysis of pluripotency markers c-myc, Lin28 and Oct3/4 normalized to actb shows that hiPS-IRFP and hiPS-iKD-SIRT1 express bona fide pluripotency markers comprable to human Embryonic Stem (hES) cells. hiPS-IRFP#3 with (n=4) and without (n=4) doxycycline, hiPS-iKDSIRT1# 17 with (n=4), and without DOX (n=4), human embryonic stem cells (n=3) were included as controls. hiPS-IRFP-#3 and hiPS-iKD-SIRT1-#17 both carry a normal female karyotype by G-banding analysis.

Article Snippet: At day 10, the liver tissues were fixed in 4% paraformaldehyde for 12 h and 70% ethanol overnight at 4C, and then embedded in paraffin. (See ) Liver organoids formation. iHeps (representing 62.5%), Human Umbilical Vein Endothelial Cells (Lonza, Walkersville, MD) (representing 12.5%), human mesenchymal stromal cells (hMSCs) (ATCC, Manassas, VA) (representing 6.25%) and human fibroblast (representing 6.25%) and Clonal Human Macrophage Primary Cell derived from Human Peripheral Blood (Celprogen, Torrance CA) (representing 12.5%) were plated at a density of 2 × 10 4 cells per cm 2 on low attachment Petri dishes on differentiation medium.

Techniques: Immunohistochemical staining, Staining, Expressing, Western Blot, MANN-WHITNEY, Gene Expression, Selection, Transduction, Plasmid Preparation, Control, Immunofluorescence, Fluorescence, Knockdown

Journal: Cell metabolism

Article Title: Generation of human fatty livers using custom-engineered induced pluripotent stem cells with modifiable SIRT1 metabolism

doi: 10.1016/j.cmet.2019.06.017

Figure Lengend Snippet: (A) Photograph of the organ perfusion and culture organ system constituted by the organ culture chamber, perfusion pump, cell infusion pump and bubble trap. Photograph of recellularized liver matrix with iHeps, human microvascular endothelial cells, mesenchymal cells and fibroblast. Also shown are haematoxylin and eosin staining of engineered human liver tissue–iRFP and –iKD-SIRT1 in the presence of doxycycline. Human normal and fatty livers were used as controls. Asterisks (*) indicate large vacuoles of triglyceride fat with compression and displacement of the nuclei to the periphery of affected hepatocytes consistent with macrovesicular steatosis. (B) Quantitative gene expression analysis of pro-inflammatory marker CD80 and anti-inflammatory marker CD163 (*P=0.0286, Mann-Whitney test) (n=4) in co-cultured iHeps-iKD-SIRT1 or iHeps-iRFP with human primary macrophages in the presence or absence of doxycycline and free fatty acids (FFA). (C) Histological analysis of engineered human fatty liver tissue –iKD-SIRT1 (doxycycline and free fatty acids treated) in the presence or absence of human primary macrophages (n=5). Oil Red O staining show macrovesicular steatosis. Also shown are immunohistochemistry analysis of CD68, NFκB p65, MCP-1 and IL-6 showing increased parenchymal inflammation with addition of human macrophages and the absence of SIRT1 as demonstrated by immunohistochemistry quantification (*P= 0.0116, **P=0.0059, Kruskal-Wallis test and Dunnett’s multiple comparisons). Human fatty livers (n=3) were included as controls. (D) Quantitative gene expression analysis of FGF21 and Selenoprotein-P (From left to right: FGF21, *P=0.0178; Selenoprotein-P, *P=0.0318, *P=0.0213, Kruskal-Wallis test and Dunnett’s multiple comparisons). Hematoxylin and eosin (H&E) staining of engineered human fatty liver tissue –iKD-SIRT1 (free fatty acids treated) in the presence (n=7) or absence of Doxycycline (n=6) in comparison to human normal livers and human NASH livers (n=5). Immunohistochemistry analysis and quantification of FGF21 and Selenoprotein-P in human fatty liver tissues –iKD-SIRT1 −/+ doxycycline compared to human normal and NASH livers. Immunohistochemistry analysis of zonation markers Glutamine synthetase and E-cadherin in human fatty liver tissue –iKD-SIRT1 −/+ doxycycline compared to human normal and NASH liver. Immunohistochemistry analysis and quantification of Ki-67 exhibited a significant higher percentage of positive hepatocytes in human fatty liver tissue –iKD-SIRT1 −/+ doxycycline and human NASH liver compared to human normal liver. (From left to right: *P= 0.0410, *P=0.0247, Kruskal-Wallis test and Dunnett’s multiple comparisons). (E) Human NASH livers and human iPS-derived fatty liver tissues-iKD-SIRT1 with and without doxycycline were scored by Brunt scoring for histologic nonalcoholic fatty liver disease.

Article Snippet: At day 10, the liver tissues were fixed in 4% paraformaldehyde for 12 h and 70% ethanol overnight at 4C, and then embedded in paraffin. (See ) Liver organoids formation. iHeps (representing 62.5%), Human Umbilical Vein Endothelial Cells (Lonza, Walkersville, MD) (representing 12.5%), human mesenchymal stromal cells (hMSCs) (ATCC, Manassas, VA) (representing 6.25%) and human fibroblast (representing 6.25%) and Clonal Human Macrophage Primary Cell derived from Human Peripheral Blood (Celprogen, Torrance CA) (representing 12.5%) were plated at a density of 2 × 10 4 cells per cm 2 on low attachment Petri dishes on differentiation medium.

Techniques: Organ Culture, Staining, Gene Expression, Marker, MANN-WHITNEY, Cell Culture, Immunohistochemistry, Comparison, Derivative Assay

Journal: Cells

Article Title: L-Arginine Ameliorates Defective Autophagy in GM2 Gangliosidoses by mTOR Modulation

doi: 10.3390/cells10113122

Figure Lengend Snippet: Model structures of HexA (PDB:2GJX) and HexB (PDB:1NOU) sub-unit proteins, highlighting the location of pathogenic mutations. Also shown autophagy in fibroblasts obtained in culture from patients with GM2 gangliosidosis (Tay–Sachs and Sandhoff diseases). ( A ). HexA point mutations: different colours depict amino acid substitutions identified in the cognate structures identified in different mutations studied. ( B ). Frameshift mutations in the alpha subunits found in two patients with Tay–Sachs disease are shown in yellow and orange; premature stop codons are marked by an asterisk. ( C ). The surface of hexosaminidase A with the critical active site region required for hydrolysis of GM2 ganglioside (CRH_GM2). The propeptide is shown in grey and the mature protein chain is depicted in white. ( D ). Enzymatic activity of HexA in fibroblast homogenates. ( E ). Morphological changes in fibroblasts from Tay–Sachs patients compared with control cells. ( F ). Cell growth determined in healthy and Tay–Sachs fibroblasts. ( G ). Expression of autophagy proteins in control and Tay–Sachs fibroblasts: LC3-I (top panels, top band), LC3-II (top panels, bottom band). ( H ). Immunofluorescence staining with anti-p62 antibody. ( I ). Impaired autophagic flux in Tay–Sachs fibroblasts. Determination of LC3-II in the presence and absence of bafilomycin A1 in control (CTL) and fibroblasts from Tay–Sachs patients; bafilomycin A1 was used at a final concentration of 100 nM with 12 h exposure. Total cellular extracts were analysed by immunoblotting with antibodies against LC3. The data are the mean ± SD for experiments conducted on two different control cell lines. Data represent the mean ± SD of three separate experiments. *** p < 0.001, ** p < 0.005, * p < 0.05 between cells from control subjects and patients with Tay–Sachs disease. a p < 0.05; aa p < 0.01; aaa p < 0.001.

Article Snippet: Control fibroblasts were commercial primary dermal fibroblast from Juvenile and Infant donors (Primacyt Cell Culture Technology GmbH, Schwerin, Germany).

Techniques: Activity Assay, Control, Expressing, Immunofluorescence, Staining, Concentration Assay, Western Blot

Journal: Cells

Article Title: L-Arginine Ameliorates Defective Autophagy in GM2 Gangliosidoses by mTOR Modulation

doi: 10.3390/cells10113122

Figure Lengend Snippet: Expression of mTOR and AKT protein were determined in cultured control and Tay–Sachs disease fibroblasts. Data represent the mean ± SD of three separate experiments.* p < 0.05; ** p < 0.01; *** p < 0.001 between transfected and non-transfected cells.

Article Snippet: Control fibroblasts were commercial primary dermal fibroblast from Juvenile and Infant donors (Primacyt Cell Culture Technology GmbH, Schwerin, Germany).

Techniques: Expressing, Cell Culture, Control, Transfection

Journal: Cells

Article Title: L-Arginine Ameliorates Defective Autophagy in GM2 Gangliosidoses by mTOR Modulation

doi: 10.3390/cells10113122

Figure Lengend Snippet: ( A ). Expression of LC3, p62, CatB, CatD, mTOR and AKT proteins determined in human control and Sandhoff disease fibroblasts. ( B , C ). Immunofluorescence of CatB in control and pathological cells with quantification in Sandhoff disease fibroblasts. ( B , D ). Characteristic ultrastructure with altered autophagosome abundance quantified in Sandhoff disease fibroblasts. ( E ). Expression of LC3, p62, CatB and mTOR proteins in the brain and spinal cord obtained from wild type and hexb −/− mutant mice with GM2 gangliosidosis (Sandhoff disease). Densitometry results are presented as means ± SEM, n = 10 mice. * p < 0.05; ** p < 0.01; *** p < 0.001 between control and diseased fibroblasts and wild type and hexB −/− mutant mice.

Article Snippet: Control fibroblasts were commercial primary dermal fibroblast from Juvenile and Infant donors (Primacyt Cell Culture Technology GmbH, Schwerin, Germany).

Techniques: Expressing, Control, Immunofluorescence, Mutagenesis

Journal: Cells

Article Title: L-Arginine Ameliorates Defective Autophagy in GM2 Gangliosidoses by mTOR Modulation

doi: 10.3390/cells10113122

Figure Lengend Snippet: ( A ). Expression of mTOR and AKT determined in control and representative Tay–Sachs fibroblasts after L-arginine treatment. ( B ). Protein synthesis was quantified in extracts of control and Tay–Sachs fibroblasts treated with L-arginine using puromycin labeling followed by immunoblotting. ( C ). Representative image of Tay–Sachs treated fibroblasts after transfection of the mCherry-GFP-LC3 plasmid and quantification of autophagic puncta. For control cells, the data are the mean ± SD for experiments conducted on two different control cell lines. GAPDH was used as a loading control. Data represent the mean ± SD of three separate experiments. *** p < 0.001 between control and Tay–Sachs fibroblasts; aa p < 0.01; aaa p < 0.001 between non-treated and treated cells.

Article Snippet: Control fibroblasts were commercial primary dermal fibroblast from Juvenile and Infant donors (Primacyt Cell Culture Technology GmbH, Schwerin, Germany).

Techniques: Expressing, Control, Labeling, Western Blot, Transfection, Plasmid Preparation

Journal: Cells

Article Title: L-Arginine Ameliorates Defective Autophagy in GM2 Gangliosidoses by mTOR Modulation

doi: 10.3390/cells10113122

Figure Lengend Snippet: ( A , B ). Immunofluorescence of CatB and HexA in control and Sandhoff disease fibroblasts and quantification after L-arginine treatment. ( C ). Expression of CatB and HexA protein were determined in control and representative Tay–Sachs fibroblast cultures after L-arginine treatment in vivo. ( D ). Expression of mTOR, CatB, and ASS1 (arginosuccinate synthetase) proteins was determined in peripheral blood mononuclear cells obtained from a patient with juvenile Tay–Sachs disease and a patient with juvenile Sandoff disease after oral L-arginine treatment. Data represent the mean ± SD of three separate experiments.* p < 0.05; ** p < 0.01; *** p < 0.001 between control and Tay–Sachs patients; a p < 0.05; aa p < 0.01; aaa p < 0.001 between non-treated and treated cells.

Article Snippet: Control fibroblasts were commercial primary dermal fibroblast from Juvenile and Infant donors (Primacyt Cell Culture Technology GmbH, Schwerin, Germany).

Techniques: Immunofluorescence, Control, Expressing, In Vivo