Bmi1<\/em>CreER\/+<\/sup> Heart immunohistochemistry was performed as previously described 7]. Specific yellow fluorescent protein (YFP) detection with anti-GFP antibody was confirmed Adult cardiomyocytes were fixed in 0.25 % glutaraldehyde (Sigma; 5 min), washed with Hearts were collected from Bmi1<\/em>-YFP mice five days after TM induction, perfused with PBS to remove blood cells, and Purified YFP Cardiomyocytes were isolated from hearts of TM-induced adult Bmi1<\/em>-YFP mice. The heart was removed rapidly and retrograde-perfused under constant pressure For Fluo-4\u00c2\u00a0AM labeling, we prepared a stock solution of 1 mM Fluo-4\u00c2\u00a0AM (Invitrogen) Hearts from one-day-old Wistar rats were minced to 1 mm Explants were prepared from hearts of eight-week-old TM-induced Bmi1<\/em>-YFP mice, as described 34], and cultured in IMDM containing 20 % embryonic stem cell-screened FBS (Hyclone, To evaluate spontaneous endothelial\/smooth muscle differentiation potential of Bmi1<\/em>-CPC, cells were obtained from eight-week-old TM-induced Bmi1<\/em>-YFP mouse hearts and seeded on gelatin-coated plates as above. After seven to ten To generate bone marrow (BM) chimeras, eight to ten week-old C57BL\/6 Bmi1<\/em>-YFP mice were TM-induced, lethally irradiated (one dose each of 4.75 and 4.5 Gy, RNA was extracted from hearts of eight-week-old TM-induced Bmi1<\/em>-YFP mice, or from the indicated subpopulations (Bmi1<\/em>-CPC and SCA-1 CPC) purified using the sorting strategy described above, with a Cells-to-CT We used genomic PCR to detect recombined and Rosa26-<\/em>YFP alleles, with primers 5?-AAAGTCGCTCTGAGTTGTTAT, 5?-AAGACCGCGAAGAGTTTGTC and 5?-AGCTC Statistical analysis was performed with Prism 5.0 (GraphPad Software). Significance Transgenic mice and tamoxifen administration Bmi1CreER\/+ ;Rosa26YFP\/+ (Bmi1-YFP) mice were generated by crossing the Bmi1CreER\/+ strain with Rosa26YFP\/+ reporter mice. Male and female Bmi1CreER\/+ ;Rosa26YFP\/+ double heterozygous mice received tamoxifen (TM; Sigma, Madrid, Spain) injections between postnatal days 30 (P30) and P60. TM was dissolved in corn oil (Sigma) to a final concentration of 20 […]<\/p>\n","protected":false},"author":1,"featured_media":0,"comment_status":"closed","ping_status":"closed","sticky":false,"template":"","format":"standard","meta":{"footnotes":""},"categories":[],"tags":[],"class_list":["post-27001","post","type-post","status-publish","format-standard","hentry"],"_links":{"self":[{"href":"http:\/\/healthmedicinet.com\/news\/wp-json\/wp\/v2\/posts\/27001","targetHints":{"allow":["GET"]}}],"collection":[{"href":"http:\/\/healthmedicinet.com\/news\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"http:\/\/healthmedicinet.com\/news\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"http:\/\/healthmedicinet.com\/news\/wp-json\/wp\/v2\/users\/1"}],"replies":[{"embeddable":true,"href":"http:\/\/healthmedicinet.com\/news\/wp-json\/wp\/v2\/comments?post=27001"}],"version-history":[{"count":0,"href":"http:\/\/healthmedicinet.com\/news\/wp-json\/wp\/v2\/posts\/27001\/revisions"}],"wp:attachment":[{"href":"http:\/\/healthmedicinet.com\/news\/wp-json\/wp\/v2\/media?parent=27001"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"http:\/\/healthmedicinet.com\/news\/wp-json\/wp\/v2\/categories?post=27001"},{"taxonomy":"post_tag","embeddable":true,"href":"http:\/\/healthmedicinet.com\/news\/wp-json\/wp\/v2\/tags?post=27001"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}
\n ;Rosa26<\/em>YFP\/+<\/sup>
\n (Bmi1<\/em>-YFP) mice were generated by crossing the Bmi1<\/em>CreER\/+<\/sup>
\n strain with Rosa26<\/em>YFP\/+<\/sup>
\n reporter mice. Male and female Bmi1<\/em>CreER\/+<\/sup>
\n ;Rosa26<\/em>YFP\/+<\/sup>
\n double heterozygous mice received tamoxifen (TM; Sigma, Madrid, Spain) injections
\n between postnatal days 30 (P30) and P60. TM was dissolved in corn oil (Sigma) to a
\n final concentration of 20 mg\/ml and mice received TM (i.p.) every 24 h on three consecutive
\n days (9 mg per 40 g body weight). When indicated, Bmi1<\/em>CreER\/+;<\/sup>Rosa26<\/em>tomato\/+<\/sup>
\n (Bmi1<\/em>-tomato), Myh6<\/em>MerCreMer\/+<\/sup>
\n ;Rosa26<\/em>YFP\/+<\/sup>
\n (Myh6<\/em>-YFP) and Bmi1<\/em>CreER\/+;<\/sup>Rosa26<\/em>LacZ\/+<\/sup>
\n (Bmi1<\/em>-LacZ) (Jackson Laboratory, Sacramento, California, United States) were used and TM-induced
\n as above. All animal procedures conformed to EU Directive 86\/609\/EEC and Recommendation
\n 2007\/526\/EC regarding the protection of animals used for experimental and other scientific
\n purposes. The ethics committees of the Fundaci\u00c3\u00b3n Centro Nacional de Investigaciones
\n Cardiovasculares (CNIC) and Centro Nacional de Biotecnolog\u00c3\u00ada (CNB) approved animal
\n studies.\n <\/p>\nImmunodetection analysis<\/h4>\n
\n by control immunofluorescence analysis of heart sections of TM-injected Rosa26<\/em>YFP\/+<\/sup>
\n mice and of non-induced Bmi1<\/em>-YFP mice (Bmi1<\/em>-YFP
NI<\/sup>
\n ); no signal was observed for either (Fig.\u00c2\u00a02a). For immunodetection, sections were fixed in 2 % paraformaldehyde (PFA) and rinsed
\n in PBS or PHEM buffer (25 mM Hepes, 10 mM EGTA, 60 mM PIPES, 2 mM MgCl
2<\/sub>
\n ; all from Sigma). Slides were rinsed in blocking buffer (BB; 0.5 % porcine skin gelatin,
\n 0.1 % bovine serum albumin; BSA; Sigma), incubated in 150 mM glycine (Merck, Madrid,
\n Spain) (10 min, room temperature (RT)), followed by sodium borohydride (Sigma; 10
\n min) and finally in PBS with 0.1 % Triton X-100 (Sigma). Preparations were incubated
\n with primary antibodies (see Additional file 1: Table S1) (1\u00e2\u20ac\u201c3 h, RT), washed and incubated with the appropriate secondary antibody
\n (1 h). Slides were incubated with Sytox Green and mounted in ProLong antifade reagent
\n (both from Invitrogen, Madrid, Spain). Images were captured with a Leica SP5, Zeiss
\n LSM 700 or LSM 780 coupled to a two-photon Spectra-Physics Mai Tai laser scanning
\n confocal microscope and were assembled with ImageJ software (NIH). Processing, including
\n assignment of pseudo-colors and changes in brightness, was applied uniformly to the
\n entire image exclusively to equalize the appearance of multiple panels in a single
\n figure. Immunocytochemistry was performed as above.\n <\/p>\nLacZ staining<\/h4>\n
\n PBS twice (5 min), then incubated with wash buffer (0.1 M Na
2<\/sub>
\n HPO
4<\/sub>
\n :2H
2<\/sub>
\n O, 0.1 M NaH
2<\/sub>
\n PO
4<\/sub>
\n :H
2<\/sub>
\n 0, 2 mM MgCl
2<\/sub>
\n , 0.11 % sodium deoxycholate, 0.2 % Igepal, 20 mM Tris\u00e2\u20ac\u201cHCl pH\u00c2\u00a07.3) (3 min). Cells
\n were incubated overnight with staining buffer (1 mg\/ml X-Gal, 5 mM K
4<\/sub>
\n Fe(CN)
6<\/sub>
\n , 5 mM K
3<\/sub>
\n Fe(CN)
6<\/sub>
\n , followed by three washes with PBS (5 min).\n <\/p>\nCell isolation, culture and flow cytometry<\/h4>\n
\n processed by enzymatic digestion using 0.1 % collagenase IV (Sigma) and 10 ?g\/ml DNAse
\n (Roche, Madrid, Spain) (40 min, 37 \u00c2\u00b0C). The resulting single cell suspension was passed
\n through a 40 ?m filter to remove debris. YFP
+<\/sup>
\n cells were separated from total heart mass with a BD FacsAria II Special Order System
\n cell sorter fitted with a 488 nm laser to excite YFP (collected in the 525\/50 channel).
\n To discriminate YFP
+<\/sup>
\n from autoflorescent cells, a 488 nm laser was used to excite cells, followed by collection
\n in the 585 channel (phycoerythrin). For flow cytometry analysis, cardiac cells from
\n hearts of TM-induced Bmi1<\/em>-YFP mice were incubated with the following primary and secondary antibodies as indicated:
\n APC (allophycocyanin)-conjugated rat anti-c-KIT, APC-rat anti-SCA-1\/Ly6a, biotin-rat
\n anti-SCA-1\/Ly6a, biotin-rat anti-CD45, biotin-rat anti-CD31 (all at 1:100; all from
\n BD Pharmingen, Madrid, Spain), and streptavidin-Alexa Fluor 405 conjugate (1:500;
\n Invitrogen). Labeled cells were examined with a BD Facs Canto II flow cytometer and
\n data analyzed using Facs DIVA Software.\n <\/p>\n
+<\/sup>
\n cells were cultured in Iscove\u00e2\u20ac\u2122s modified Dulbecco\u00e2\u20ac\u2122s medium (IMDM, Invitrogen) containing
\n 10 % fetal bovine serum (ESCell FBS, Gibco, Madrid, Spain), 100 U\/ml penicillin, 100
\n mg\/ml streptomycin and 2 mM L-glutamine (all from Invitrogen), 10
3<\/sup>
\n units ESGRO Supplement (Millipore, Madrid, Spain), 10 ng\/ml EGF (epidermal growth
\n factor; Sigma) and 20 ng\/ml FGF (fibroblast growth factor; Peprotech, Rocky Hill,
\n New Jersey, United States) (37 \u00c2\u00b0C, 3 % O
2<\/sub>
\n , 5 % CO
2<\/sub>
\n ). The SCA-1
+<\/sup>
\n population was prepared by incubating the Lin
?<\/sup>
\n primary cell suspension with rat anti-SCA-1\/Ly6a biotin antibody (1:100: Abcam, Cambridge,
\n United Kingdom), followed by isolation with Mouse Anti-Rat Kappa Microbeads (Miltenyi
\n Biotec). In all cases, when preparing SCA-1
+<\/sup>
\n cells, the CD45
+<\/sup>
\n fraction was removed by indirect sorting using the MACS system and AUTOMACS technology
\n (Miltenyi Biotec, Teterow, Germany). Cells were cultured in the same medium as YFP
+<\/sup>
\n cells. Flow cytometry analysis of adult cardiomyocytes was performed in a BD LSR Fortessa
\n TM using a neutral density filter 1.0. R1 mouse embryonic stem cells (ES), a gift
\n from Dr. Miguel Torres (CNIC, Spain), were cultured on mitomycin C (Sigma)-inactivated
\n murine embryonic fibroblasts as feeder cells in DMEM\/Glutamax (Invitrogen) supplemented
\n with 20 % ES-qualified FBS (Invitrogen), 10
3<\/sup>
\n U\/ml LIF (Millipore), 50 ?M ?-mercaptoethanol (Merck) and 1 % non-essential amino
\n acids (Thermo, Madrid, Spain).\n <\/p>\nIsolation and biochemical properties of adult mouse cardiomyocytes<\/h4>\n
\n (60 mmHg; 37 \u00c2\u00b0C, 8 min) in Ca
2+<\/sup>
\n -free buffer containing 113 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO
4<\/sub>
\n , 5.5 mM glucose, 0.6 mM KH
2<\/sub>
\n PO
4<\/sub>
\n , 0.6 mM Na
2<\/sub>
\n HPO
4<\/sub>
\n , 12 mM NaHCO
3<\/sub>
\n , 10 mM KHCO
3<\/sub>
\n , 10 mM Hepes, 10 mM 2,3-butanedione monoxime, and 30 mM taurine. Digestion was initiated
\n by adding a mixture of recombinant enzymes (0.2 mg\/ml Liberase Blendzyme (Roche),
\n 0.14 mg\/ml trypsin (Invitrogen), and 12.5 ?M CaCl
2<\/sub>
\n to the perfusion solution). When the heart became swollen (10 min), it was removed
\n and gently teased into small pieces with fine forceps in the same enzyme solution.
\n Heart tissue was further dissociated mechanically using 2, 1.5, and 1 mm-diameter
\n pipettes, until all large heart tissue pieces were dispersed. The digestion buffer
\n was neutralized with stopping buffer containing 10 % FBS and 12.5 ?M CaCl
2<\/sub>
\n . Cardiomyocytes were pelleted by gravity (20 min), the supernatant aspirated and
\n cells resuspended in the perfusion solution containing 5 % FBS and 12.5 ?M CaCl
2<\/sub>
\n . The calcium concentration was increased by gradually adding CaCl
2<\/sub>
\n from 62 ?M to 1 mM final concentration. Cardiomyocytes were plated in culture dishes
\n precoated with 0.5 mg\/ml mouse laminin (BD Biosciences) in PBS (1\u00e2\u20ac\u201c2 h, RT). Plating
\n medium was Medium 199 Hank\u00e2\u20ac\u2122s (Invitrogen), 0.25 % BSA (Sigma), 22 mM NaHCO
3<\/sub>
\n , 0.05 % FBS (Sigma), 0.001 % ITS (insulin-transferrin-selenium (Gibco), 10 mM 2,3-butanedione
\n monoxime and 25 ?M blebbistatin. After 2 h, cardiomyocytes were fixed with 2 % PFA
\n or were used for the in vitro<\/em> calcium transient studies. To detect YFP
+<\/sup>
\n cardiomyocytes (YFP
+<\/sup>
\n CM), we used a confocal microscope LSM 780 upright scanning system (Zeiss) equipped
\n with a W 20X Plan-APOCHROMAT dipping objective (numerical aperture (NA)?=?1.0). YFP
+<\/sup>
\n CM were detected using the 514 nm laser to excite YFP (acquired in the 535 channel).
\n A transmitted light detector (T-PMT) was used to screen cardiac cell morphology. We
\n captured and stored YFP cardiomyocyte images based on cell coordinates before Fluo-4
\n labeling.\n <\/p>\n
\n in DMSO with an equal volume of 20 % Pluronic F-127 DMSO (1:1 ratio); the working
\n concentration was 1 ?M. Fluo-4\u00c2\u00a0AM was added to DMEM supplemented with 100 U\/ml penicillin,
\n 100 mg\/ml streptomycin and 2 mM L-glutamine; cells were incubated in the dark (20\u00e2\u20ac\u201c30
\n min). We washed the cells and added fresh DMEM without phenol red (Sigma) and images
\n were acquired by confocal microscopy as for Ca
2+<\/sup>
\n fluorescence. Fluo-4 was excited with the 488 nm line of an argon laser and 505 nm
\n signal emissions were collected. Images were captured in a time series (xyt, pixel
\n dwell 1.58 ?s) and 2D images (512?\u00c3\u2014?512 lines) were obtained and stored for offline
\n analysis.\n <\/p>\nPrimary culture of neonatal rat cardiomyocytes<\/h4>\n
2<\/sup>
\n and digested with 0.05 % trypsin (Invitrogen) in Hank\u00e2\u20ac\u2122s balanced salt solution (Sigma)(37
\n \u00c2\u00b0C, 40 min). The fragments were digested with 0.1 % collagenase (class II, Worthington
\n Biochemical, Lakewood, New Jersey, United States). Single-cell suspensions were prepared
\n by mechanical pipetting. Cells were passed through a 40 ?m filter and preplated in
\n DMEM (Invitrogen) supplemented with 10 % FBS, 100 U\/ml penicillin, 100 mg\/ml streptomycin
\n and 2 mM L-glutamine (2 h, 37 \u00c2\u00b0C). Newborn rat cardiomyocytes (NBRC) were collected
\n and seeded on coverslips precoated with gelatin (Invitrogen) and fibronectin (BD Biosciences)
\n in a final medium containing DMEM, M-199 (Gibco) at 4:1, 10 % horse serum (Sigma),
\n 5 % FBS, 100 U\/ml penicillin, 100 mg\/ml streptomycin, 2 mM L-glutamine and 1 ?g\/ml
\n cytosine ?-D-arabinofuranoside (Sigma). Neonatal mouse cardiomyocytes were isolated
\n as indicated for adult mice, using M-199 medium supplemented with 0.05 % FBS, 0.001
\n % ITS and 25 ?M blebbistatin.\n <\/p>\nExplant cultures<\/h4>\n
\n GE Healthcare Life Sciences, Madrid, Spain), 100 U\/ml penicillin, 100 mg\/ml streptomycin
\n and 2 mM L-glutamine (37 \u00c2\u00b0C, 5 % CO
2<\/sub>
\n ).\n <\/p>\nB-CPC differentiation potential<\/h4>\n
\n days, plates were fixed with 2 % PFA in PHEM buffer (15 min, RT) and processed for
\n immunocytochemistry. For cardiomyocyte differentiation, B-CPC cardiospheres were plated
\n on a monolayer of NBRC or adult transgenic mouse GFP
+<\/sup>
\n CM derived from the beta-actin<\/em> GFP mouse strain (Jackson Laboratory). For B-CPC\/adult mouse CM co-culture, we used
\n M-199 medium, 0.05 % FBS, 0.001 % ITS and 25 ?M blebbistatin. After four to five days
\n co-culture, cells were fixed with 2 % PFA in PHEM buffer (15 min, RT) and processed
\n for immunocytochemistry.\n <\/p>\nBone marrow transplant<\/h4>\n
\n separated by 24 h) and then transplanted (i.v.) with 10
7<\/sup>
\n whole BM cells isolated age-matched C57BL\/6 Act<\/em>-RFP mice 35]. At two months post-TM induction (54 days after BM transplant), after confirmation
\n of full chimerism, transplanted Bmi1<\/em>-YFP mouse hearts were digested and analyzed as above.\n <\/p>\nRT-qPCR and genomic PCR analysis<\/h4>\n
\n kit (Ambion, Thermo, Madrid, Spain). RNA from ES cells was prepared as previously
\n described 36]. Complementary DNA was obtained by reverse transcription with the High Capacity cDNA
\n Reverse Transcription Kit (Applied Biosystems, Madrid, Spain). cDNAs were analyzed
\n by real time PCR using the Power SYBR Green PCR Master Mix (Applied Biosystems). Amplification,
\n detection and data analysis were carried out with an ABI PRISM 7900HT Sequence Detection
\n System. The crossing threshold values for individual mRNAs were normalized to GusB<\/em> expression for mRNA. Changes in mRNA expression were denoted as the x-<\/em>fold change relative to the control. (See Additional file 2: Table S2 for primers used).\n <\/p>\n
\n CTCGCCCTTGCTCACCATG 17]. PCR conditions were 96 \u00c2\u00b0C for 2 min to separate strands, followed by 34 amplification
\n cycles (96 \u00c2\u00b0C for 30 s, 56 \u00c2\u00b0C for 30 s, 72 \u00c2\u00b0C for 30 s) and a 5 min elongation step
\n at 72 \u00c2\u00b0C. The specific PCR product (320 bp) derived from the floxed allele is detected
\n in all transgenic Bmi1<\/em>-YFP and Myh6<\/em> -YFP mice, but the diagnostic fragment (550 bp) associated with the floxed-out allele
\n is only detectable in the Myh6<\/em> -YFP CM-enriched fraction post-TM induction 37].\n <\/p>\nStatistical analysis<\/h4>\n
\n between groups was evaluated in all experiments as detailed in the figures. A value
\n of P??0.05 was considered significant. All replicates considered are biological replicates.\n <\/p>\n","protected":false},"excerpt":{"rendered":"