Journal: Advanced healthcare materials

Article Title: Designing Inherently Photodegradable Cell-Adhesive Hydrogels for 3D Cell Culture.

doi: 10.1002/adhm.202100632

Figure Lengend Snippet: Figure 2. A) Live/dead staining of normal human dermal fibroblasts (NHDF) with calcein-AM (green, live cells) and propidium iodide (red, dead cells) embedded in GelMA-free PEGMA/PEGDMA (Gel-N0) and GelMA-containing (Gel-N10) hydrogels on day 1, 7, 14 postencapsulation, followed by imaging with confocal microscopy (Leica TCS SPE, scale bar: 200 µm). B-i) NHDF embedded in Gel-N10 hydrogels on day 1 postencapsulation. Comparison of HepG2 cells encapsulated in (ii) Gel-N10 and (iii) Gel-N0 hydrogels (scale bar: 100 µm). C) Proliferation behavior of encapsulated HepG2 cells in Gel-N10 and Gel-N0 hydrogels over a period of 7 d assessed by PrestoBlue assay (n = 3). D) Toxicity evaluation of Gel-N10 photodegradation products by MTT proliferation assay with HeLa cells after 72 h exposure (n = 3). The concentration of nondiluted degradation products was 53 mg mL−1. Data are presented as mean ± SD and statistically evaluated by (C) Student’s t-test and (D) one-way ANOVA. *, **, *** represent p < 0.05, 0.01, and 0.001, respectively.

Article Snippet: Normal human dermal fibroblasts (NHDF), human liver cancer cells (HepG2), and HeLa cells were purchased from PromoCell GmbH (Heidelberg, Germany).

Techniques: Staining, Imaging, Confocal Microscopy, Comparison, Prestoblue Assay, Proliferation Assay, Concentration Assay

Journal: Nutrients

Article Title: Essential Oils, Pituranthos chloranthus and Teucrium ramosissimum , Chemosensitize Resistant Human Uterine Sarcoma MES-SA/Dx5 Cells to Doxorubicin by Inducing Apoptosis and Targeting P-Glycoprotein

doi: 10.3390/nu13051719

Figure Lengend Snippet: Effects of Pituranthos chloranthus (PC) and Teucrium ramosissimum Desf. (TR) extracts on normal primary human uterine fibroblast cells (HUF) and primary murine Bone Marrow-Derived Macrophages (BMDM) viability. After treatment of primary HUF and murine BMDM with increasing concentrations (0–100 µg/mL) of PC and TR for 72 h, the percentage of viable cells was assessed using the (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. ( A ) Dose–response curves of PC-treated HUF (left panel) and TR-treated HUF (right panel). ( B ) Dose–response curves of PC-treated BMDM (left panel) and TR-treated BMDM (right panel). Data are expressed as a mean percentage of control growth ± Standard Deviation (SD) of two representative experiments ( n = 6 replicates per concentration).

Article Snippet: Primary human uterine fibroblast normal cells (HUF) were obtained from the ATCC.

Techniques: Derivative Assay, MTT Assay, Control, Standard Deviation, Concentration Assay

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: Rheumatology (Oxford, England)

Article Title: Distinct landscapes of fibroblast subtypes in arteries of patients with giant cell arteritis

doi: 10.1093/rheumatology/keaf143

Figure Lengend Snippet: The expression of fibroblast phenotypic markers in temporal artery biopsies (TAB). ( A ) Representative picture of the immunohistochemical (IHC) staining of CD90, fibroblast activation protein α (FAP), podoplanin (PDPN), CD248 and α-smooth muscle actin (α-SMA) in giant cell arteritis (GCA)-affected TAB. ( B ) IHC staining in control TAB. ( C ) Scoring of fibroblast markers in adventitia, media, and intima (percentage of positive cells per layer). NS, not significant; * P < 0.05, ** P < 0.01. ( D ) Triple immunofluorescence staining of CD90/FAP/PDPN and CD90/α-SMA/CD248 in GCA-affected TAB. PDPN, red; CD90, green; FAP, yellow; CD248, red; α-SMA, yellow; colocalization of CD90 and PDPN is shown in yellow, colocalization of CD90 and FAP is shown in magenta, colocalization of CD90 and αSMA is shown in magenta, colocalization of CD90 and CD248 is shown in cyan. Arrows indicate double positive areas. A, adventitia; M, media; I, intima. For GCA-TAB, intima includes media–intima (MI) and inner intima (II)

Article Snippet: Human aortic adventitia fibroblasts (HAoAF, C-12380) were obtained from PromoCell, Heidelberg, Germany.

Techniques: Expressing, Immunohistochemical staining, Immunohistochemistry, Activation Assay, Control, Immunofluorescence, Staining

Journal: Rheumatology (Oxford, England)

Article Title: Distinct landscapes of fibroblast subtypes in arteries of patients with giant cell arteritis

doi: 10.1093/rheumatology/keaf143

Figure Lengend Snippet: Fibroblast distribution patterns in aorta tissues. ( A ) Expression of fibroblast markers (CD90, fibroblast activation protein [FAP], podoplanin [PDPN], CD248, α-smooth muscle actin [α-SMA]) and cytokines related to remodelling (TGF-β, fibroblast growth factor 21 [FGF21], platelet-derived growth factor B [PDGFB]) in GCA-affected aorta tissues. Adv, adventitia; Med, media; Int, intima. ( B ) Scoring of fibroblast markers and cytokines related to remodelling in adventitia and media. NS, not significant; * P < 0.05, ** P < 0.01. ( C ) Triple immunofluorescence staining of CD90/FAP/PDPN and CD90/α-SMA/CD248 in GCA-affected aorta tissues (structurally disrupted media). CD90, green; PDPN, red; CD24, red; FAP, yellow; α-SMA, yellow. Colocalization of CD90+CD248, CD90+PDPN is shown in cyan; colocalization of CD90+FAP, CD90+α-SMA is shown in magenta. Arrows indicate double positive areas. ( D ) Masson trichrome staining and collagen scoring in aorta tissues. NS: not significant; * P < 0.05

Article Snippet: Human aortic adventitia fibroblasts (HAoAF, C-12380) were obtained from PromoCell, Heidelberg, Germany.

Techniques: Expressing, Activation Assay, Derivative Assay, Immunofluorescence, Staining

Journal: Rheumatology (Oxford, England)

Article Title: Distinct landscapes of fibroblast subtypes in arteries of patients with giant cell arteritis

doi: 10.1093/rheumatology/keaf143

Figure Lengend Snippet: The relationship between fibroblast activating protein (FAP) and human aortic fibroblast proliferation. ( A , B ) Triple immunofluorescent staining of CD90/FAP/Ki67 in GCA-affected temporal artery ( A ) and aorta (structurally disrupted media) ( B ). Ki67, red; CD90, green; FAP, yellow; colocalization of CD90/FAP, cyan; arrows indicate CD90 + FAP + Ki67 + cells. A, adventitia; M, media; I, intima; GCA, giant cell arteritis. ( C ) TGF-β1 5 ng/ml 48 h promoted mRNA expression of FAP and proliferation in human aortic adventitial fibroblasts (HAoAF). Proliferation rate was calculated as Ki67 positive nuclei divided by 4′,6-diamidino-2-phenylindole positive nuclei; Ki67, red. ( D ) HAoAF were transfected with small interfering RNA (siRNA) targeting FAP, and were treated with TGF-β1 6 h later. After 24 h, cells were collected for qPCR; 72 h later, cells were collected for Ki67 staining. FAP knockdown decreased the effect of TGF-β1 on cell proliferation. FAP, red; Ki67, green. NB si: non-binding small interfering RNA; FAP siRNA: small interfering RNA targeting FAP. NS: not significant; * P < 0.05, ** P < 0.01

Article Snippet: Human aortic adventitia fibroblasts (HAoAF, C-12380) were obtained from PromoCell, Heidelberg, Germany.

Techniques: Staining, Expressing, Transfection, Small Interfering RNA, Knockdown, Binding Assay