Journal: Tissue Engineering. Part C, Methods

Article Title: Isolation and Characterization of Alveolar Epithelial Type II Cells Derived from Mouse Embryonic Stem Cells

doi: 10.1089/ten.tec.2013.0415

Figure Lengend Snippet: 3′hprt.mSPCP-NEOR.Rosa26 targeting vector. Schematic diagram of 3′hprt.mSPCP-NEOR.Rosa26 targeting vector (showing relevant positional information, not scaled proportionally based on sequence lengths). The NEOR transgene controlled by mATIIC-specific SPC promoter (mSPCP-NEOR) was cloned into 3′hprt targeting vector backbone, containing the puromycinR gene (PURO), the K14Agouti transgene (Ag), and an LoxP site (arrow). One 5.0 kb DNA fragment homologous to Rosa 26 gene was included in the vector for site-specific targeting by homologous recombination. To generate Rosa 26-targeted mESCs, NdeI was used to linearize the vector before transfection of mESCs. The primer A is located within Rosa 26 gene region upstream of the NdeI site and the primer B within the NEOR gene downstream of the NdeI site in the targeting vector. Thus, PCR can be performed by using primers A and B to identify Rosa 26-targeted mESC clones. mESC, mouse embryonic stem cell; ATIIC, alveolar epithelial progenitor type II cell; mATIIC, mouse primary ATIIC; NEOR, neomycinR; SPC, surfactant protein C.

Article Snippet: Approximately 1×10 6 mESCs were re-suspended in 100 μL of supplemented mESC Nucleofector Solution (VAPH-1001; Lonza), mixed with 5 μg of the linearized targeting vector, and then transfected by using Nucleofector II as previously described.

Techniques: Plasmid Preparation, Sequencing, Clone Assay, Homologous Recombination, Transfection

Journal: Tissue Engineering. Part C, Methods

Article Title: Isolation and Characterization of Alveolar Epithelial Type II Cells Derived from Mouse Embryonic Stem Cells

doi: 10.1089/ten.tec.2013.0415

Figure Lengend Snippet: In vitro differentiation of mESCs into ATIICs. (A) Diagram of differentiation protocols. Dissociated mESCs were cultured in either Matrigel-coated (Gel) or Collagen IV-coated (C4) plates in hDM, mDM, or SR medium for the first 5 days. From day 6, the differentiating cultures were either maintained in the original medium (hDM*, mDM*, or SR* procedure) or transferred into a serum-free medium supplemented with FGF2 (hDM-Sf*, mDM-Sf*, or SR-Sf* procedure) for an additional 2, 4, or 6 days. (B) Expression of SPC RNA in differentiating cultures of mESCs. Total RNA samples were isolated from differentiating cultures of mESCs on days 7, 9, and 11, respectively, for analysis of SPC expression by RT-PCR. The 18S expression in each sample was shown in the lower blot of each panel as a control to demonstrate that changes in the amount of SPC-specific RT-PCR product was due to corresponding changes in SPC RNA expression. hDM, hESC differentiation medium; mDM, mESC differentiation medium.

Article Snippet: Approximately 1×10 6 mESCs were re-suspended in 100 μL of supplemented mESC Nucleofector Solution (VAPH-1001; Lonza), mixed with 5 μg of the linearized targeting vector, and then transfected by using Nucleofector II as previously described.

Techniques: In Vitro, Cell Culture, Expressing, Isolation, Reverse Transcription Polymerase Chain Reaction, Control, RNA Expression

Journal: Tissue Engineering. Part C, Methods

Article Title: Isolation and Characterization of Alveolar Epithelial Type II Cells Derived from Mouse Embryonic Stem Cells

doi: 10.1089/ten.tec.2013.0415

Figure Lengend Snippet: Biological functions of mESC-derived ATIICs. (A) QRT-PCR was performed to analyze the expression levels of ATIC markers, AQP5 and T1α, as well as the expression levels of ATIIC-specific SPC in the differentiating cultures of mESC-ATIICs on days 0, 2, 4, 6, and 8 (n=6). (B) To examine the proliferation capacity of mESC-ATIICs, G418-selected mESC-ATIICs from day 11 were cultured on Matrigel-coated six-well plates in MEF-conditioned SR medium, with or without 30 ng/mL of rhKGF, for 4 days. The number of cells/well was counted on days 2 and 4 (n=6). ##p<0.01, #p<0.05 versus the groups on day 0. **p<0.01 versus the cultures in the absence of rhKGF. (C) Surfactant lipid secretion from cultured mESC-ATIICs in response to secretagogue (n=6). Secretion of 3H-labeled PC was shown as percent of total 3H-labeled PC. **p<0.001 versus the cultures in the absence of secretagogue. (D) Surfactant protein secretion from cultured mESC-ATIICs in response to the treatment of Bt2cAMP+Dex. Media collected from cultured mESC-ATIICs and mATIICs were analyzed by immunoblotting for the secretion of SPB and SPC. AQP5, Aquaporin-5; ATIC, alveolar epithelial type I cell; Dex, dexamethasone; MEF, mouse embryonic fibroblast; PC, phosphatidylcholine.

Article Snippet: Approximately 1×10 6 mESCs were re-suspended in 100 μL of supplemented mESC Nucleofector Solution (VAPH-1001; Lonza), mixed with 5 μg of the linearized targeting vector, and then transfected by using Nucleofector II as previously described.

Techniques: Derivative Assay, Quantitative RT-PCR, Expressing, Cell Culture, Labeling, Western Blot

Journal: Tissue Engineering. Part C, Methods

Article Title: Isolation and Characterization of Alveolar Epithelial Type II Cells Derived from Mouse Embryonic Stem Cells

doi: 10.1089/ten.tec.2013.0415

Figure Lengend Snippet: Identification of Rosa 26-targeted mESC clones. (A) PCR analysis using primer A and B was performed to screen Rosa 26-targeted mESC clones and demonstrated a 1.35 kb Rosa 26-targeted transgene fragment in mESC clones 32, 33, 34, and 39. (B) Southern blot analysis of integration sites of targeting vector. A 425 bp NEOR gene DNA fragment was used as a hybridization probe to analyze BamHI-, HindIII-, and NheI-digested genomic DNA samples of Rosa 26-targeted mESC clones (32, 33, and 34). The probe can recognize a 6.4 kb-targeted BamHI fragment, a 7.2 kb-targeted HindIII fragment, a 5.7 kb-targeted NheI fragment, and a randomly inserted targeting vector (Fig. 1). Southern blot demonstrated that mESC clones 32, 33, and 34 contain the 6.4 kb-targeted BamHI fragment, the 7.2 kb-targeted HindIII fragment, and the 5.7 kb-targeted NheI fragment, indicating a site-specific targeting. Although the randomly inserted targeting vector was also demonstrated in mESC clones 33 and 34, the result showed that mESC clone 32 only has a single copy of targeting vector in Rosa 26 gene locus.

Article Snippet: Approximately 1×10 6 mESCs were re-suspended in 100 μL of supplemented mESC Nucleofector Solution (VAPH-1001; Lonza), mixed with 5 μg of the linearized targeting vector, and then transfected by using Nucleofector II as previously described.

Techniques: Clone Assay, Southern Blot, Plasmid Preparation, Hybridization

Journal: Tissue Engineering. Part C, Methods

Article Title: Isolation and Characterization of Alveolar Epithelial Type II Cells Derived from Mouse Embryonic Stem Cells

doi: 10.1089/ten.tec.2013.0415

Figure Lengend Snippet: Relative ATIIC Content in the Differentiating Cultures of mESCs

Article Snippet: Approximately 1×10 6 mESCs were re-suspended in 100 μL of supplemented mESC Nucleofector Solution (VAPH-1001; Lonza), mixed with 5 μg of the linearized targeting vector, and then transfected by using Nucleofector II as previously described.

Techniques:

Journal: Tissue Engineering. Part C, Methods

Article Title: Isolation and Characterization of Alveolar Epithelial Type II Cells Derived from Mouse Embryonic Stem Cells

doi: 10.1089/ten.tec.2013.0415

Figure Lengend Snippet: Surfactant protein expression and lamellar body formation in mESC-ATIICs. (A) The G418-selected mESC-ATIICs on days 7, 9, and 11, as well as control mATIICs, were immunostained by rabbit anti-human SPA, proSPB, and proSPC antibodies (Green) with Draq5 counterstaining (Scale bar=25 μm). (B) Transmission electron micrographs showed well-developed lamellar bodies with normal concentric, tightly packed multi-lamellar lipid membranes in selected mESC-ATIICs on day 11 (b, at 4000 magnification) as observed in normal mATIICs (a, at 3000 magnification). (c) Magnified view (25,000 magnification) of * region in (a). (d) Magnified view (25,000 magnification) of ** region in (b). SPB, surfactant protein B.

Article Snippet: Approximately 1×10 6 mESCs were re-suspended in 100 μL of supplemented mESC Nucleofector Solution (VAPH-1001; Lonza), mixed with 5 μg of the linearized targeting vector, and then transfected by using Nucleofector II as previously described.

Techniques: Expressing, Control, Transmission Assay