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 mg/ml and mice received TM (i.p.) every 24 h on three consecutive
days (9 mg per 40 g body weight). When indicated, Bmi1CreER/+;Rosa26tomato/+
(Bmi1-tomato), Myh6MerCreMer/+
;Rosa26YFP/+
(Myh6-YFP) and Bmi1CreER/+;Rosa26LacZ/+
(Bmi1-LacZ) (Jackson Laboratory, Sacramento, California, United States) were used and TM-induced
as above. All animal procedures conformed to EU Directive 86/609/EEC and Recommendation
2007/526/EC regarding the protection of animals used for experimental and other scientific
purposes. The ethics committees of the Fundación Centro Nacional de Investigaciones
Cardiovasculares (CNIC) and Centro Nacional de BiotecnologÃa (CNB) approved animal
studies.
Immunodetection analysis
Heart immunohistochemistry was performed as previously described 7]. Specific yellow fluorescent protein (YFP) detection with anti-GFP antibody was confirmed
by control immunofluorescence analysis of heart sections of TM-injected Rosa26YFP/+
mice and of non-induced Bmi1-YFP mice (Bmi1-YFP
NI
); no signal was observed for either (Fig. 2a). For immunodetection, sections were fixed in 2 % paraformaldehyde (PFA) and rinsed
in PBS or PHEM buffer (25 mM Hepes, 10 mM EGTA, 60 mM PIPES, 2 mM MgCl
2
; all from Sigma). Slides were rinsed in blocking buffer (BB; 0.5 % porcine skin gelatin,
0.1 % bovine serum albumin; BSA; Sigma), incubated in 150 mM glycine (Merck, Madrid,
Spain) (10 min, room temperature (RT)), followed by sodium borohydride (Sigma; 10
min) and finally in PBS with 0.1 % Triton X-100 (Sigma). Preparations were incubated
with primary antibodies (see Additional file 1: Table S1) (1–3 h, RT), washed and incubated with the appropriate secondary antibody
(1 h). Slides were incubated with Sytox Green and mounted in ProLong antifade reagent
(both from Invitrogen, Madrid, Spain). Images were captured with a Leica SP5, Zeiss
LSM 700 or LSM 780 coupled to a two-photon Spectra-Physics Mai Tai laser scanning
confocal microscope and were assembled with ImageJ software (NIH). Processing, including
assignment of pseudo-colors and changes in brightness, was applied uniformly to the
entire image exclusively to equalize the appearance of multiple panels in a single
figure. Immunocytochemistry was performed as above.
LacZ staining
Adult cardiomyocytes were fixed in 0.25 % glutaraldehyde (Sigma; 5 min), washed with
PBS twice (5 min), then incubated with wash buffer (0.1 M Na
2
HPO
4
:2H
2
O, 0.1 M NaH
2
PO
4
:H
2
0, 2 mM MgCl
2
, 0.11 % sodium deoxycholate, 0.2 % Igepal, 20 mM Tris–HCl pH 7.3) (3 min). Cells
were incubated overnight with staining buffer (1 mg/ml X-Gal, 5 mM K
4
Fe(CN)
6
, 5 mM K
3
Fe(CN)
6
, followed by three washes with PBS (5 min).
Cell isolation, culture and flow cytometry
Hearts were collected from Bmi1-YFP mice five days after TM induction, perfused with PBS to remove blood cells, and
processed by enzymatic digestion using 0.1 % collagenase IV (Sigma) and 10 ?g/ml DNAse
(Roche, Madrid, Spain) (40 min, 37 °C). The resulting single cell suspension was passed
through a 40 ?m filter to remove debris. YFP
+
cells were separated from total heart mass with a BD FacsAria II Special Order System
cell sorter fitted with a 488 nm laser to excite YFP (collected in the 525/50 channel).
To discriminate YFP
+
from autoflorescent cells, a 488 nm laser was used to excite cells, followed by collection
in the 585 channel (phycoerythrin). For flow cytometry analysis, cardiac cells from
hearts of TM-induced Bmi1-YFP mice were incubated with the following primary and secondary antibodies as indicated:
APC (allophycocyanin)-conjugated rat anti-c-KIT, APC-rat anti-SCA-1/Ly6a, biotin-rat
anti-SCA-1/Ly6a, biotin-rat anti-CD45, biotin-rat anti-CD31 (all at 1:100; all from
BD Pharmingen, Madrid, Spain), and streptavidin-Alexa Fluor 405 conjugate (1:500;
Invitrogen). Labeled cells were examined with a BD Facs Canto II flow cytometer and
data analyzed using Facs DIVA Software.
Purified YFP
+
cells were cultured in Iscove’s modified Dulbecco’s medium (IMDM, Invitrogen) containing
10 % fetal bovine serum (ESCell FBS, Gibco, Madrid, Spain), 100 U/ml penicillin, 100
mg/ml streptomycin and 2 mM L-glutamine (all from Invitrogen), 10
3
units ESGRO Supplement (Millipore, Madrid, Spain), 10 ng/ml EGF (epidermal growth
factor; Sigma) and 20 ng/ml FGF (fibroblast growth factor; Peprotech, Rocky Hill,
New Jersey, United States) (37 °C, 3 % O
2
, 5 % CO
2
). The SCA-1
+
population was prepared by incubating the Lin
?
primary cell suspension with rat anti-SCA-1/Ly6a biotin antibody (1:100: Abcam, Cambridge,
United Kingdom), followed by isolation with Mouse Anti-Rat Kappa Microbeads (Miltenyi
Biotec). In all cases, when preparing SCA-1
+
cells, the CD45
+
fraction was removed by indirect sorting using the MACS system and AUTOMACS technology
(Miltenyi Biotec, Teterow, Germany). Cells were cultured in the same medium as YFP
+
cells. Flow cytometry analysis of adult cardiomyocytes was performed in a BD LSR Fortessa
TM using a neutral density filter 1.0. R1 mouse embryonic stem cells (ES), a gift
from Dr. Miguel Torres (CNIC, Spain), were cultured on mitomycin C (Sigma)-inactivated
murine embryonic fibroblasts as feeder cells in DMEM/Glutamax (Invitrogen) supplemented
with 20 % ES-qualified FBS (Invitrogen), 10
3
U/ml LIF (Millipore), 50 ?M ?-mercaptoethanol (Merck) and 1 % non-essential amino
acids (Thermo, Madrid, Spain).
Isolation and biochemical properties of adult mouse cardiomyocytes
Cardiomyocytes were isolated from hearts of TM-induced adult Bmi1-YFP mice. The heart was removed rapidly and retrograde-perfused under constant pressure
(60 mmHg; 37 °C, 8 min) in Ca
2+
-free buffer containing 113 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO
4
, 5.5 mM glucose, 0.6 mM KH
2
PO
4
, 0.6 mM Na
2
HPO
4
, 12 mM NaHCO
3
, 10 mM KHCO
3
, 10 mM Hepes, 10 mM 2,3-butanedione monoxime, and 30 mM taurine. Digestion was initiated
by adding a mixture of recombinant enzymes (0.2 mg/ml Liberase Blendzyme (Roche),
0.14 mg/ml trypsin (Invitrogen), and 12.5 ?M CaCl
2
to the perfusion solution). When the heart became swollen (10 min), it was removed
and gently teased into small pieces with fine forceps in the same enzyme solution.
Heart tissue was further dissociated mechanically using 2, 1.5, and 1 mm-diameter
pipettes, until all large heart tissue pieces were dispersed. The digestion buffer
was neutralized with stopping buffer containing 10 % FBS and 12.5 ?M CaCl
2
. Cardiomyocytes were pelleted by gravity (20 min), the supernatant aspirated and
cells resuspended in the perfusion solution containing 5 % FBS and 12.5 ?M CaCl
2
. The calcium concentration was increased by gradually adding CaCl
2
from 62 ?M to 1 mM final concentration. Cardiomyocytes were plated in culture dishes
precoated with 0.5 mg/ml mouse laminin (BD Biosciences) in PBS (1–2 h, RT). Plating
medium was Medium 199 Hank’s (Invitrogen), 0.25 % BSA (Sigma), 22 mM NaHCO
3
, 0.05 % FBS (Sigma), 0.001 % ITS (insulin-transferrin-selenium (Gibco), 10 mM 2,3-butanedione
monoxime and 25 ?M blebbistatin. After 2 h, cardiomyocytes were fixed with 2 % PFA
or were used for the in vitro calcium transient studies. To detect YFP
+
cardiomyocytes (YFP
+
CM), we used a confocal microscope LSM 780 upright scanning system (Zeiss) equipped
with a W 20X Plan-APOCHROMAT dipping objective (numerical aperture (NA)?=?1.0). YFP
+
CM were detected using the 514 nm laser to excite YFP (acquired in the 535 channel).
A transmitted light detector (T-PMT) was used to screen cardiac cell morphology. We
captured and stored YFP cardiomyocyte images based on cell coordinates before Fluo-4
labeling.
For Fluo-4Â AM labeling, we prepared a stock solution of 1 mM Fluo-4Â AM (Invitrogen)
in DMSO with an equal volume of 20 % Pluronic F-127 DMSO (1:1 ratio); the working
concentration was 1 ?M. Fluo-4Â AM was added to DMEM supplemented with 100 U/ml penicillin,
100 mg/ml streptomycin and 2 mM L-glutamine; cells were incubated in the dark (20–30
min). We washed the cells and added fresh DMEM without phenol red (Sigma) and images
were acquired by confocal microscopy as for Ca
2+
fluorescence. Fluo-4 was excited with the 488 nm line of an argon laser and 505 nm
signal emissions were collected. Images were captured in a time series (xyt, pixel
dwell 1.58 ?s) and 2D images (512?×?512 lines) were obtained and stored for offline
analysis.
Primary culture of neonatal rat cardiomyocytes
Hearts from one-day-old Wistar rats were minced to 1 mm
2
and digested with 0.05 % trypsin (Invitrogen) in Hank’s balanced salt solution (Sigma)(37
°C, 40 min). The fragments were digested with 0.1 % collagenase (class II, Worthington
Biochemical, Lakewood, New Jersey, United States). Single-cell suspensions were prepared
by mechanical pipetting. Cells were passed through a 40 ?m filter and preplated in
DMEM (Invitrogen) supplemented with 10 % FBS, 100 U/ml penicillin, 100 mg/ml streptomycin
and 2 mM L-glutamine (2 h, 37 °C). Newborn rat cardiomyocytes (NBRC) were collected
and seeded on coverslips precoated with gelatin (Invitrogen) and fibronectin (BD Biosciences)
in a final medium containing DMEM, M-199 (Gibco) at 4:1, 10 % horse serum (Sigma),
5 % FBS, 100 U/ml penicillin, 100 mg/ml streptomycin, 2 mM L-glutamine and 1 ?g/ml
cytosine ?-D-arabinofuranoside (Sigma). Neonatal mouse cardiomyocytes were isolated
as indicated for adult mice, using M-199 medium supplemented with 0.05 % FBS, 0.001
% ITS and 25 ?M blebbistatin.
Explant cultures
Explants were prepared from hearts of eight-week-old TM-induced Bmi1-YFP mice, as described 34], and cultured in IMDM containing 20 % embryonic stem cell-screened FBS (Hyclone,
GE Healthcare Life Sciences, Madrid, Spain), 100 U/ml penicillin, 100 mg/ml streptomycin
and 2 mM L-glutamine (37 °C, 5 % CO
2
).
B-CPC differentiation potential
To evaluate spontaneous endothelial/smooth muscle differentiation potential of Bmi1-CPC, cells were obtained from eight-week-old TM-induced Bmi1-YFP mouse hearts and seeded on gelatin-coated plates as above. After seven to ten
days, plates were fixed with 2 % PFA in PHEM buffer (15 min, RT) and processed for
immunocytochemistry. For cardiomyocyte differentiation, B-CPC cardiospheres were plated
on a monolayer of NBRC or adult transgenic mouse GFP
+
CM derived from the beta-actin GFP mouse strain (Jackson Laboratory). For B-CPC/adult mouse CM co-culture, we used
M-199 medium, 0.05 % FBS, 0.001 % ITS and 25 ?M blebbistatin. After four to five days
co-culture, cells were fixed with 2 % PFA in PHEM buffer (15 min, RT) and processed
for immunocytochemistry.
Bone marrow transplant
To generate bone marrow (BM) chimeras, eight to ten week-old C57BL/6 Bmi1-YFP mice were TM-induced, lethally irradiated (one dose each of 4.75 and 4.5 Gy,
separated by 24 h) and then transplanted (i.v.) with 10
7
whole BM cells isolated age-matched C57BL/6 Act-RFP mice 35]. At two months post-TM induction (54 days after BM transplant), after confirmation
of full chimerism, transplanted Bmi1-YFP mouse hearts were digested and analyzed as above.
RT-qPCR and genomic PCR analysis
RNA was extracted from hearts of eight-week-old TM-induced Bmi1-YFP mice, or from the indicated subpopulations (Bmi1-CPC and SCA-1 CPC) purified using the sorting strategy described above, with a Cells-to-CT
kit (Ambion, Thermo, Madrid, Spain). RNA from ES cells was prepared as previously
described 36]. Complementary DNA was obtained by reverse transcription with the High Capacity cDNA
Reverse Transcription Kit (Applied Biosystems, Madrid, Spain). cDNAs were analyzed
by real time PCR using the Power SYBR Green PCR Master Mix (Applied Biosystems). Amplification,
detection and data analysis were carried out with an ABI PRISM 7900HT Sequence Detection
System. The crossing threshold values for individual mRNAs were normalized to GusB expression for mRNA. Changes in mRNA expression were denoted as the x-fold change relative to the control. (See Additional file 2: Table S2 for primers used).
We used genomic PCR to detect recombined and Rosa26-YFP alleles, with primers 5?-AAAGTCGCTCTGAGTTGTTAT, 5?-AAGACCGCGAAGAGTTTGTC and 5?-AGCTC
CTCGCCCTTGCTCACCATG 17]. PCR conditions were 96 °C for 2 min to separate strands, followed by 34 amplification
cycles (96 °C for 30 s, 56 °C for 30 s, 72 °C for 30 s) and a 5 min elongation step
at 72 °C. The specific PCR product (320 bp) derived from the floxed allele is detected
in all transgenic Bmi1-YFP and Myh6 -YFP mice, but the diagnostic fragment (550 bp) associated with the floxed-out allele
is only detectable in the Myh6 -YFP CM-enriched fraction post-TM induction 37].
Statistical analysis
Statistical analysis was performed with Prism 5.0 (GraphPad Software). Significance
between groups was evaluated in all experiments as detailed in the figures. A value
of P??0.05 was considered significant. All replicates considered are biological replicates.