Materials and Reagents

Bacterial dishes (100 x 15 mm) (Corning, catalog number: 351006 )
Standard culture Petri dishes (100 x 15 mm) (Corning, catalog number: 70165-101 )
6 multiwell-plates (Corning, catalog number: 353224 )
50 ml tubes (Falcon^®, catalog number: 352098 )
P19 cells (Izsler-Istituto Zooproﬁlattico Sperimentale, Lombardy and Emilia Romagna “Bruno Ubertini”, Brescia, Italy) (Izsler, catalog number: BS-TCL 206 , Passage 0)
Minimum Essential Medium alpha (α-MEM) (Sigma-Aldrich, catalog number: M8042 )
Penicillin-streptomycin 10,000 U/ml (Thermo Fisher Scientific, Gibco^TM, catalog number: 15140-122 )
L-glutamine 2 nM (Thermo Fisher Scientific, Gibco^TM, catalog number: 25030-081 )
Dimethyl sulfoxide (Sigma-Aldrich, catalog number: D4540-100 ml )
Fetal Bovine Serum (FBS) (Sigma-Aldrich, catalog number: F6178-50 ml )
Trypsin-EDTA (0.5%), no phenol red (Thermo Fisher Scientific, Gibco^TM, catalog number: 15400-054 )
Optimem^® (Thermo Fisher Scientific, Gibco^TM, catalog number: 31985-070 )
siPORT^® NeoFX transfection agent (Thermo Fisher Scientific, Invitrogen™, catalog number: AM4511 )
Pre-miRNA 1 (ID: 000385) (Thermo Fisher Scientific, Applied Biosystems^®, catalog number: AM17100 ), Pre-miRNA 133 (ID: 000458) (Thermo Fisher Scientific, Applied Biosystems^®, catalog number: AM17100), Pre-miRNA 499 (ID: 001045) (Thermo Fisher Scientific, Applied Biosystems^®, catalog number: AM17100) molecules and scramble miRNA (negative control) (Thermo Fisher Scientific, Ambion™, catalog number: AM17110 )
Culture medium (see Recipes)
Standard differentiation medium (see Recipes)
Mix A (see Recipes)
Mix B (see Recipes)

Equipment

Pipetus^® Pipettes
Laminar flow-hood (EuroClone S.p.A., model: S@feflow 1.8 )
Humidified cell culture incubator set at 37 °C, 5% CO₂ (Panasonic Corporation, Sanyo, model: MCO-18AC )
Inverted bright light microscope equipped with a phase-contrast filter (ZEISS, model: Observer Z1 )

Procedure

Expand the P19 cells form passage 0 to passage 5, in a standard Petri dish and feed the cells with culture medium. When the cells reach 80-90% confluence, harvest the P19 cells using 5% trypsin/EDTA for 5 minutes at 37 °C, 5% CO₂ (Figure 1A).
On day 1, transfer 4 x 10⁵ cells to a 100 mm bacterial dish in 8 ml of standard differentiation medium and incubate the cells for 24 h at 37 °C, 5% CO₂.
On day 2, add 5 ml of fresh standard differentiation medium (total volume medium: 13 ml, fresh differentiation medium must be prepared just before the use).
On day 3, the morphologic changes of P19 cells start to be visible: some cell clusters sediment at the bottom of the culture dish, become EB and begin to differentiate into CMC. Some isolated cells may remain in suspension: these cells are not committed toward the cardiac lineage and will not adhere at the bottom of the culture dish since bacterial dish are used. These undifferentiated P19 will be discarded when exchanging the cell culture medium.
Replace 5 ml of fresh standard differentiation medium: with a 10 ml pipette gently aspirate 5 ml of medium, avoiding to aspirate also the EB; then, with a new 10 ml pipette, add 5 ml of fresh differentiation medium.
On day 4, the EB are ready for transfection (Figure 1B). Prepare mix A and mix B in two separate 15 ml conical tubes.
Gently add mix B to mix A, invert the tube 3 times and incubate at RT for 10 min.
Using a 25 ml pipette, collect and transfer the EB contained in one bacterial dish to a 50 ml tube and let the EB to sediment.
Aliquot 1 ml of transfection mix into each well of a 6-multiwell-plate; add only standard differentiation medium in those wells that will be used as control.
EB previously collected in the 50 ml tube will now be settled. Carefully aspirate the medium and re-suspend the EB in 14.4 ml of fresh standard differentiation medium.
Transfer 2.4 ml of re-suspended EB to each well and gently rock the multiwell-plate using a 1 ml pipette tip. Now shake back and forth the plate to obtain homogeneous distribution of the cells. Every time you pick an aliquot of the mixture, pipette up and down to re-suspend the EB. Incubate over-night at 37 °C, 5% CO₂.
On day 5, EB will have adhered to the bottom of the dish (Figure 1C). You can now exchange the medium with fresh differentiation medium to allow the expansion of EB. This step represents “day 1” of the cardiac induction protocol. A Real-Time PCR can be performed in order to quantify the miRNA transfection efficiency.
On day 5 of the induction protocol, the first contracting EB should appear and will be visible at the microscope (Video 1).

Video 1. Beating embryoid bodies
Early cardiac differentiation can be documented by PCR and/or immunocytochemistry starting from day 7 of the induction protocol. More advanced cardiac differentiation will appear starting from day 14 of the induction protocol (Figure 1D) (Pisano et al., 2015 http://onlinelibrary.wiley.com/doi/10.1002/stem.1928/pdf, Figures 2, 3 and 6).

Figure 1. P19 cells and EB. A. Images of undifferentiated P19 maintained in culture medium; B. EB formation during the cardiac differentiation protocol, day 4; C. When EB are transferred from a bacterial dish to a Petri dish they adhere to the plate; D. After 7 days of cardiac differentiation the attached EB will have expanded and are beating.

Notes

Manipulation of miRNA molecules requires the use of RNAse-free plastic and reagents. Moreover, if the flow-hood used for miRNA transfection is utilized also for other experiments, we suggest to pre-treat the surfaces with 10% NaClO.
During the transfection protocol you must work carefully and aseptically under the flow-hood since Optimem^® medium does not contain antibiotics.

Recipes

Culture medium
Store the medium at 4 °C; when the phenol red contained in the α-MEM turns to pink (or to yellow) discard the medium and use a fresh culture medium.
α-MEM 500 ml
FBS 55 ml
L-glutamine 5.5 ml
Penicillin-streptomycin 5.5 ml
Standard differentiation medium (prepare fresh before use)
Culture medium
DMSO 0.5%
Mix A (prepare fresh before use)
Dilute the pre-miRNA solution using Optimem^®
We use pre-miRNA1 and pre-miRNA499 at a concentration of 10 nM, and pre-miRNA133 and scramble miRNA at 5 nM.
Incubate the mix at room temperature (RT) for 10 min
For one 6-multiwell dish we use 3 ml of Mix A
Mix B (prepare fresh before use)
Dilute siPORT^® 1:50 using Optimem^®
Incubate at RT for 10 min
For one 6-multiwell dish we use 3 ml of Mix B

Acknowledgments

The development of this protocol was supported by the Fondazione IRCCS Policlinico San Matteo Pavia, Italy; the Fondazione Cariplo (2007-5984) and the Ministero Italiano della Sanità (GR-2008-114278). We want to thank Laurene Kelly for help with editing the manuscript.

References

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