Journal: Stem Cells International

Article Title: BMP-2 Enhances Osteogenic Differentiation of Human Adipose-Derived and Dental Pulp Stem Cells in 2D and 3D In Vitro Models

doi: 10.1155/2022/4910399

Figure Lengend Snippet: Alizarin red staining of MG-63 cells, hADSCs and hDPSCs cultured in 2D for 4 weeks with (a–c) proliferation medium or with (d–f) commercial MesenCult or (g–i) BMP2-supplemented osteogenic differentiation media. Cell nuclei were stained with hematoxylin. Calcium deposits are showed in red (arrows). Scale bar = 200 μ m.

Article Snippet: Human adipose-derived stem cells (hADSCs) and human dental pulp stem cells (hDPSCs) were purchased from Lonza (Basel, Switzerland), and MG-63 cells were from the American Type Culture Collection (ATCC® CRL-1427™; Sigma-Aldrich; Madrid, Spain).

Techniques: Staining, Cell Culture

Journal: Stem Cells International

Article Title: BMP-2 Enhances Osteogenic Differentiation of Human Adipose-Derived and Dental Pulp Stem Cells in 2D and 3D In Vitro Models

doi: 10.1155/2022/4910399

Figure Lengend Snippet: Relative levels of gene expression of 2D-cultured MG-63 cells, hADSCs and hDPSCs, for 2 and 3 weeks with proliferation medium or commercial MesenCult osteogenic differentiation medium of (a) RUNX2, (b) BGLAP, (c) IBSP, (d) SPP1, (e) CSF1, (f) CSF2, and (g) MT-CO1. Samples cultured with proliferation media were taken as relative control. Mean ± SD are shown, with statistical significance at ∗ p ≤ 0.05 with respect to the control group.

Article Snippet: Human adipose-derived stem cells (hADSCs) and human dental pulp stem cells (hDPSCs) were purchased from Lonza (Basel, Switzerland), and MG-63 cells were from the American Type Culture Collection (ATCC® CRL-1427™; Sigma-Aldrich; Madrid, Spain).

Techniques: Expressing, Cell Culture

Journal: Stem Cells International

Article Title: BMP-2 Enhances Osteogenic Differentiation of Human Adipose-Derived and Dental Pulp Stem Cells in 2D and 3D In Vitro Models

doi: 10.1155/2022/4910399

Figure Lengend Snippet: Relative levels of gene expression of 2D-cultured MG-63 cells, hADSCs and hDPSCs, for 2 and 3 weeks with proliferation medium or BMP2-supplemented osteogenic differentiation medium of (a) RUNX2, (b) BGLAP, (c) IBSP, (d) SPP1, (e) CSF1, (f) CSF2, and (g) MT-CO1. Samples cultured with proliferation media were taken as relative control. Mean ± SD are shown, with statistical significance at ∗ p ≤ 0.05 with respect to the control group.

Article Snippet: Human adipose-derived stem cells (hADSCs) and human dental pulp stem cells (hDPSCs) were purchased from Lonza (Basel, Switzerland), and MG-63 cells were from the American Type Culture Collection (ATCC® CRL-1427™; Sigma-Aldrich; Madrid, Spain).

Techniques: Expressing, Cell Culture

Journal: PLoS ONE

Article Title: Targeted Genomic Integration of a Selectable Floxed Dual Fluorescence Reporter in Human Embryonic Stem Cells

doi: 10.1371/journal.pone.0046971

Figure Lengend Snippet: ( A ) pAAVS1ZFN vector diagram showing two reverse orientation human PGK promoters driving the left and right AAVS1 zinc finger nucleases (ZFNs). ( B ) pZDonor-mTmG-2a-Puro vector diagram and the AAVS1 genomic integration site on chromosome 19 in the first intron of the PPP1R12C gene. The PGKNeoKan cassette enables selection of cells with stable transgene integration and the CAG-mTmG-2a-Puro selectable “stoplight” cassette allows for visual and antibiotic selection of cells that have been recombined by Cre recombinase. Yellow and blue colored bars represent ∼800 bp stretches of sequence homologous to either side of the AAVS1 target locus. ( C ) In coordination with the AAVS1 ZFNs, the mTmG-2a-Puro transgene was integrated into the AAVS1 site by homologous recombination. Cre mediated recombination of LoxP sites removes the tdTomato expression cassette and results in eGFP-2a-Puro expression. Abbreviations: pA = bovine growth hormone polyadenylation signal, mT = myristoylated tdTomato, mG = myristoylated eGFP, NeoKan = neomycin/kanamicin resistance, Puro = puromycin resistance, loxP = Cre recombinase locus of chromosomal crossover, Ex = exon.

Article Snippet: 5×10 5 undifferentiated RUES2 hESCs were dispersed to single cells using Versene (Gibco) and electroporated with 5 µg of pAAVS1ZFN and 15 µg of pZDonor mTmG-2a-Puro (Lonza, Kit 1 (Cat#: VAPH-5012), program A-23).

Techniques: Plasmid Preparation, Zinc-Fingers, Selection, Sequencing, Homologous Recombination, Expressing

Journal: PLoS ONE

Article Title: Targeted Genomic Integration of a Selectable Floxed Dual Fluorescence Reporter in Human Embryonic Stem Cells

doi: 10.1371/journal.pone.0046971

Figure Lengend Snippet: Photomicrographs of an undifferentiated mTmG-2a-Puro RUES2 colony ( A ) under brightfield, tdTomato (red) and eGFP (green) fluorescent illumination. Note that the transgenic hESCs are tdTomato + and eGFP - . Scale bar is 75 µm. ( B ) eGFP and tdTomato flow cytometry of wild type RUES2 hESCs (WT) and RUES2 mTmG-2a-Puro undifferentiated hESCs after G418 selection (mTmG-2a-Puro). G418 selection produces >94% purity of mTmG-2a-Puro cells as measured by flow cytometry for tdTomato fluorescent expression (see the panel on the right). ( C ) (i) Schematic showing the NheI, NdeI and KpnI cut sites within the transgene. The orange bar represents the binding site for the neomycin probe. The blue bar represents the binding site for the 3′ genomic probe. Non-locus-specific Southern blot detection of the neomycin transgene detects only a single copy of the mTmG-2a-Puro construct (ii), while Southern blot analysis of the AAVS1 locus shows heterozygous targeting of the mTmG-2a-Puro transgene to the AAVS1 locus (iii). ( D ) G-banded karyotyping of mTmG-2a-Puro RUES2 undifferentiated cells demonstrating a normal (46, XX) karyotype.

Article Snippet: 5×10 5 undifferentiated RUES2 hESCs were dispersed to single cells using Versene (Gibco) and electroporated with 5 µg of pAAVS1ZFN and 15 µg of pZDonor mTmG-2a-Puro (Lonza, Kit 1 (Cat#: VAPH-5012), program A-23).

Techniques: Transgenic Assay, Flow Cytometry, Selection, Expressing, Binding Assay, Southern Blot, Construct

Journal: PLoS ONE

Article Title: Targeted Genomic Integration of a Selectable Floxed Dual Fluorescence Reporter in Human Embryonic Stem Cells

doi: 10.1371/journal.pone.0046971

Figure Lengend Snippet: ( A ) Flow cytometry showing changing numbers of tdTomato + and eGFP + RUES2 mTmG-2a-Puro cells at various timepoints after transduction with an EF1α-Cre lentivirus. ( B ) Quantitation of the fluorescence switch shown in panel A (n = 3 biological replicates). ( C ) Scatter plot of undifferentiated mTmG-2a-Puro cells treated with EF1α-Cre lentivirus and then selected with puromycin for 48 hours. Note that the resultant cultures were >99% eGFP + by flow cytometry (i). Comparison of untreated (red) and EF1α-Cre treated, puromycin selected (blue) undifferentiated mTmG-2a-Puro cells (ii). Quantitation of flow cytometry data from three independent experiments in which cells were transduced with EF1α-Cre and puromycin selected (iii). ( D ) Phase contrast and fluorescent photomicrographs of undifferentiated mTmG-2a-Puro cells (i), mTmG-2a-Puro cells treated with EF1α-Cre lentivirus (ii), and mTmG-2a-Puro cells treated with EF1α-Cre and selected with puromycin (iii). Scale bars are 50 µm. Error bars represent +/− one standard error of the mean. ( E ) mTmG-2a-Puro cells transduced with EF1α-Cre lentivirus were immunostained with an anti-Cre recombinase antibody. Note that Cre (magenta) co-localizes only with eGFP + cell nuclei. Scale bar is 10 µm.

Article Snippet: 5×10 5 undifferentiated RUES2 hESCs were dispersed to single cells using Versene (Gibco) and electroporated with 5 µg of pAAVS1ZFN and 15 µg of pZDonor mTmG-2a-Puro (Lonza, Kit 1 (Cat#: VAPH-5012), program A-23).

Techniques: Flow Cytometry, Transduction, Quantitation Assay, Fluorescence

Journal: PLoS ONE

Article Title: Targeted Genomic Integration of a Selectable Floxed Dual Fluorescence Reporter in Human Embryonic Stem Cells

doi: 10.1371/journal.pone.0046971

Figure Lengend Snippet: ( A ) Flow cytometry for cardiac troponin T (cTnT) indicating the percentage of cardiomyocytes present after mTmG-2a-Puro hESCs were subjected to a directed cardiac differentiation protocol. ( B ) Mean percentage of cTnT + cells resulting from three independent differentiation runs. ( C ) The percentage of cells that immunostained for eGFP, tdTomato, and/or the cardiomyocyte marker α-actinin was determined before and after puromycin selection (n = 230–600 cells per condition for three independent differentiation runs). ( D ) Magnification at 10x (i and ii) and 60x (iii and iv) showing fluorescent photomicrographs of cardiac differentiated mTmG-2a-Puro cells before (i and iii) and after (ii and iv) puromycin selection. Scale bars are 200 µm in (i and ii) and 50 µm in (iii and iv). Error bars represent one standard error of the mean.

Article Snippet: 5×10 5 undifferentiated RUES2 hESCs were dispersed to single cells using Versene (Gibco) and electroporated with 5 µg of pAAVS1ZFN and 15 µg of pZDonor mTmG-2a-Puro (Lonza, Kit 1 (Cat#: VAPH-5012), program A-23).

Techniques: Flow Cytometry, Marker, Selection