D Jacob

E Mimoun

L De Sarlo

M Weitz

J Dalibard

F Gerbier

Production of Sodium Bose-Einstein condensates in an optical dimple trap

Keywords: numbers: 67.85.Hj, 37.10.De, 37.10.Jk

We report on the realization of a sodium Bose-Einstein condensate (BEC) in a combined red-detuned optical dipole trap, formed by two beams crossing in a horizontal plane and a third, tightly focused dimple trap propagating vertically. We produce a BEC in three main steps: loading of the crossed dipole trap from laser-cooled atoms, an intermediate evaporative cooling stage which results in efficient loading of the auxiliary dimple trap, and a final evaporative cooling stage in the dimple trap. Our protocol is implemented in a compact setup and allows us to reach quantum degeneracy even with relatively modest initial atom numbers and available laser power.

Introduction

The preparation of degenerate atomic quantum gases is interesting from both a fundamental and an applied point of view. On the one hand, the unprecedented level of control on these systems allows one to study quantum many-body phenomena in the absence of perturbing effects unavoidable in solid-state systems [START_REF] Bloch | Many-body physics with ultracold gases[END_REF]. On the other hand, degenerate gases are a promising starting point to reliably produce highly entangled states, which could pave the way for a new generation of atom-based quantum sensors (see [START_REF] Bouyer | Heisenberg-limited spectroscopy with degenerate Bose-Einstein gases[END_REF][START_REF] Gross | Nonlinear atom interferometer surpasses classical precision limit[END_REF] and references therein).

In view of the sensitivity of these strongly correlated states to the perturbations caused by magnetic fields fluctuations, experimental schemes in which evaporative cooling is performed without the use of external magnetic fields are particularly interesting. These so-called "all-optical evaporation" schemes rely on far off-resonant optical dipole traps. They have been developed by several groups to produce Bose-Einstein condensates (BEC) of various atomic species, in particular alkali atoms (Rb [START_REF] Barrett | All-optical formation of an atomic Bose-Einstein condensate[END_REF][START_REF] Kinoshita | All-optical Bose-Einstein condensation using a compressible crossed dipole trap[END_REF][START_REF] Cennini | Bose-Einstein condensation in a CO2-laser optical dipole trap[END_REF], Li [START_REF] Granade | All-optical production of a degenerate Fermi gas[END_REF], Cs [START_REF] Weber | Bose-Einstein condensation of Cesium[END_REF] and Na [START_REF] Dumke | All-optical generation and photoassociative probing of Sodium Bose-Einstein condensates[END_REF]). In such all-optical setups, the trapping potential is almost independent of the internal state, opening the route to the study of spinor condensates [START_REF] Stenger | Spin domains in ground-state Bose-Einstein condensates[END_REF].

Experiments relying on all-optical setups are based on a common experimental scheme: Laser-cooled atoms are first loaded into an optical dipole trap and then evaporatively cooled by lowering the trapping laser intensity and thus the trap depth. In this paper we discuss how to optimize these two steps for producing an all-optical BEC of sodium atoms, starting with relatively modest atom numbers and laser powers.

The first issue, dealing with the transfer from the magneto-optical trap (MOT) to the optical dipole trap, has extensively been studied (see e.g. [START_REF] Kuppens | Loading an optical dipole trap[END_REF]). Laser cooling forces and light-assisted losses can be strongly modified by the presence of the dipole trap potential. The size and the depth of the trapping potential have to be adapted to the size, density and temperature of the MOT. A convenient configuration is a laser trap consisting of two crossed gaussian beams [START_REF] Adams | Evaporative cooling in a crossed dipole trap[END_REF], as in our experiment. The loading of the trap then occurs in two steps: Atoms are first captured in both arms of the trap, and then start filling the crossing region by "free evaporation" once the near-resonant cooling beams have been turned-off [START_REF] Barrett | All-optical formation of an atomic Bose-Einstein condensate[END_REF]. We will describe our procedure to optimize the atom number N in the crossing region and the temperature T at the end of the free evaporation to get a high phase-space density D and a large collision rate γ coll . We recall that D = N ( ω/k B T ) 3 and γ coll ∝ N/ω 3 T for a Boltzmann gas in a harmonic trap, with the Planck constant, k B the Boltzmann constant, and ω the average trapping frequency. For a given beam size, we find that the optimal trap depth is different for loading and free evaporation, and propose that trap-induced light-shifts on the cooling transition are the physical mechanism behind this observation.

The second issue is related to the efficiency of evaporative cooling. In these respect, optical traps differ in several aspects from magnetic traps. In magnetic traps, evaporative cooling takes place in the so-called runaway regime, where the elastic collision rate γ coll and evaporation efficiency stay constant or even increase with time [START_REF] Ketterle | Evaporative Cooling of Trapped Atoms[END_REF]. In optical traps, this regime is not easily reachable because the trap depth and trap confinement both increase with the trapping laser power. In practice, decreasing the trap depth to force evaporation results in a looser confinement, so that the collision rate can decrease even if the phase-space density increases. Solutions involving modification of the trapping potential have been demonstrated to resolve this issue. For example a dynamical change of the beam size using a zoom lens allows one to maintain constant confinement while reducing the trap depth [START_REF] Kinoshita | All-optical Bose-Einstein condensation using a compressible crossed dipole trap[END_REF], thus preserving a high collision rate during evaporation. Runaway evaporation in an optical trap can also be obtained, by using an additional expelling potential independent of the trapping laser (gravity or "pulling" laser) in order to decouple trap confinement and potential depth [START_REF] Hung | Accelerating evaporative cooling of atoms into Bose-Einstein condensation in optical traps[END_REF][START_REF] Clément | All-optical runaway evaporation to Bose-Einstein condensation[END_REF]. A third solution, based on the addition of a tighter "dimple" potential [START_REF] Weber | Bose-Einstein condensation of Cesium[END_REF][START_REF] Lin | Rapid production of 87 Rb Bose-Einstein condensates in a combined magnetic and optical potential[END_REF], has been realized and characterized theoretically [START_REF] Ma | The evaporative cooling of a gas of Caesium atoms in the hydrodynamic regime[END_REF][START_REF] Ma | Optimized evaporative cooling using a dimple potential: an efficient route to Bose-Einstein condensation[END_REF][START_REF] Comparat | Optimized production of large Bose-Einstein condensates[END_REF]. This solution, which is the one investigated in this paper, leads to a two-step evaporation sequence: After the loading of a larger trap, atoms are first transferred by cooling into the "dimple" trap, then further cooled down in this trap. The major advantages of this technique are its relative technical simplicity (as compared, for instance, to a "zoom-lens" method), the increase of phase-space density during the transfer, and the high efficiency of the second evaporation step due to the high confinement in the "dimple" trap. Here we describe the first application of this technique to 23 Na. Starting with 3 × 10 5 trapped atoms, a Bose-Einstein condensate of ∼ 10 4 23 Na atoms is produced after ∼ 2 s evaporation time.

The paper is organized as follows. In section 2, we give an overview of our experimental setup. In section 3, we investigate the loading of a dipole trap from a MOT of sodium atoms and study how the compression of the trap after the atom capture improves the initial conditions for evaporative cooling. We then present in section 4 how evaporative cooling works in presence of the auxiliary dimple trap, detailing its filling dynamics, and the last evaporative cooling stage to reach Bose-Einstein condensation.

Experimental Setup

Laser cooling

Our experiment starts with a sodium magneto-optical trap (MOT) capturing approximately 10 7 atoms in 10 s from a vapor whose pressure is modulated using light-induced atomic desorption [START_REF] Mimoun | Fast production of ultracold Sodium gases using light-induced desorption and optical trapping[END_REF]. After the MOT is formed, a far off-resonant dipole trap is switched on (see subsection 2.2). The detunings and powers of both the cooling (tuned to 3S 1/2 , F = 2 → 3P 3/2 , F ′ = 3 transition) and repumping (tuned to 3S 1/2 , F = 1 → 3P 3/2 , F ′ = 2 transition) lasers are modified in order to optimize the trap loading. During a first "dark MOT" phase [START_REF] Ketterle | High densities of cold atoms in a dark spontaneous-force optical trap[END_REF], we lower the power of the repumping laser in about 100 ms, from I rep = 300 µW.cm -2 to I rep = 10 µW.cm -2 per beam while keeping the magnetic gradient on. This reduces the loss rate due to light-induced collisions by limiting the population of the excited states [START_REF] Kuppens | Loading an optical dipole trap[END_REF]. We keep the cooling laser intensity at the same value as for MOT loading, I cool = 0.9 mW.cm -2 per beam, which corresponds to one sixth of the saturation intensity (I sat = 6.3 mW.cm -2 ). During this "dark MOT" phase, both the spatial density in the dipole trap and the temperature increase. We then apply a 30 ms-long "cold MOT" phase, where the cooling beam detuning is shifted from δ cool ≈ -Γ to δ cool ≈ -3.8Γ (Γ/2π ≈ 10 MHz is the natural linewidth). The temperature of the atoms after this cooling sequence is around 50 µK.

Trapping lasers configuration

The far off-resonant dipole trap results from the combination of three beams, two forming a crossed dipole trap (CDT) in the horizontal x -y plane and a tightly focused one propagating vertically along the z axis (see figure 1(a)), which we refer to as "dimple trap" (dT). The CDT is derived from a 40 W fiber laser (IPG Photonics) at 1070 nm. This trap is formed by folding the beam onto itself at an angle θ ≃ 45 the laser power using a motorized rotating waveplate (OWIS GmbH) followed by a Glan-Taylor polarizer (bandwidth ∼ 10 Hz), and a control input on the current in the laser pump diodes (bandwidth ∼ 50 kHz). The waveplate is used for coarse reduction of laser power by changing the amount of light transmitted by the polarizer, whereas the current control is used at the end of the evaporation ramp (low laser powers) and for fast servo-control of the intensity to reduce fluctuations. Combining both servo loops, we can control the laser power from its maximal value (P CDT ≈ 36 W) down to ≈ 100 mW. We can switch off the trapping potential to an extinction level greater than 90 % in less than 10 µs using the laser current input. We use motorized mirrors (Agilis, Newport Corporation) for alignment. Special care is taken to ensure the orthogonality of the polarization of both arms, realized by the insertion of a λ/2 waveplate that is positioned with a precision 0.5 • . A misalignement of only 1 • results in a measurable heating of the sample [START_REF] Mimoun | Fast production of ultracold Sodium gases using light-induced desorption and optical trapping[END_REF].

The auxiliary dimple trap is produced using a 500 mW laser (Mephisto-S, InnoLight GmbH) at 1064 nm. As sketched on figure 1(a), the beam propagates vertically, and crosses the CDT with a waist w dT ≈ 8 µm. The laser beam is transmitted through a single-mode optical fiber, and focused to a waist size w dT using a custom-made microscope objective (CVI Melles Griot, NA 0.3). An acousto-optic modulator placed before the fiber allows us to control the intensity and to quickly switch off the dT-beam.

To fix the notations that will be used in the following, we give here the expressions of the dipole trap potentials. The expression of the CDT potential is given by

V CDT (x, y, z) = - V 0 CDT 2 e -2(x 2 +z 2 )/w(y) 2 (w(y)/w CDT ) 2 + e -2(u 2 +z 2 )/w(v) 2 (w(v)/w CDT ) 2 , (1)
with w(y) = w CDT 1 + y 2 /y 2 R and with y R the Rayleigh length y R = πw 2 CDT /λ ≈ 5.2 mm. We have also introduced the rotated coordinates: (u, v) = (x cos(θ) + y sin(θ), -x sin(θ) + y cos(θ)). The expression of the dT potential is given by

V dT (x, y, z) = -V 0 dT e -2(x 2 +y 2 )/w 2 dT , (2)
neglecting the confinement of the dT along the z-axis, always negligible compared to the vertical confinement of the CDT. Typical trapping frequencies and potential depths are given in table 1.

In figure 1(c), we give schematically the temporal evolution of the powers of the two lasers during the experimental sequence. This time evolution is optimized for loading and evaporation, as explained in sections 3 and 4.

Imaging

We monitor the time evolution of the trapped cloud using both fluorescence and absorption imaging [START_REF] Kaiser | Spinor Condensates and Light Scattering from Bose-Einstein Condensates[END_REF]. Fluorescence light is captured by the same high numerical aperture microscope used to focus the dT. The photons are collected on a low-noise charge-coupled device camera (PIXIS, Princeton Instruments). In figure 1(b), we show a typical fluorescence image. We typically observe the atoms after a time-of-flight t ToF = 0.1 ms during a short pulse (t mol = 50 µs) performed with the six cooling and repumping beams.

Absorption images are recorded with a vertically propagating resonant probe beam. It is better suited for the analysis of the central denser part of the trapped cloud but not very precise for the arms of the CDT. Indeed, the regions corresponding to the CDT arms display low optical densities (< 0.1) only slightly above the noise level (∼ 0.04, limited by residual fringes on the background). Atom counting in the arms of the CDT is thus more accurate using fluorescence images.

Loading and free evaporation in the Crossed Dipole Trap

Dipole trap loading dynamics

We can distinguish two stages in the dynamics of the trap loading. At first, during the MOT/CDT overlap period, atoms are captured mainly in the arms of the CDT without a notable enhancement of the density in the crossing region. In the second phase that follows the extinction of the MOT beams, which we call "free evaporation", the hottest atoms leave the arms and the remaining ones fill the crossing region through thermalization. The quantity of interest is the number of atoms in the central region N C , which corresponds approximately to the number of atoms with an energy comprised between -V 0 CDT and -V 0 CDT /2 (as defined in equation ( 1)). We show in figure 2 the potential V CDT in the z = 0 plane, truncated at three different energy levels. One can see that atoms having energies lower than V 0 CDT /2 explore only the central region, as expected. This dense part is the relevant component that matters for further evaporative cooling. Although both trapping lasers (CDT and dT) are turned on simultaneously, the CDT is much deeper than the dimple trap, the latter playing a negligible role during this initial stage. In this section, we discuss auxiliary experiments where the dT is absent.

In order to understand the loading dynamics during the first stage, we give a brief overview on the relevant mechanisms (see [START_REF] Kuppens | Loading an optical dipole trap[END_REF] for a detailed analysis). The loading rate of atoms in the CDT is proportional to the probability of an atom to be trapped by the dipole potential and to the atomic flux in the CDT/MOT overlap region. The first term corresponds to the damping of the velocity of an atom when it crosses one arm of the CDT, leading to a reduction of its total energy below the CDT potential depth. The second term is proportional to the spatial density and the average velocity of the atoms in the MOT, and thus depends on the temperature of the atoms. The relevant parameters for optimizing the loading rate, namely the atomic density and the temperature, can be adjusted by the "dark MOT" and "cold MOT" phases (see subsection 2.1). The presence of the dipole potential changes locally the cooling properties, due to the light shifts induced by the CDT laser beams. During this phase, atom accumulation in the trap crossing region is limited by light-assisted inelastic collisions, such as radiative escape.

In the second stage, after the MOT light is extinguished, the trapped atoms thermalize and the sample cools down by evaporative cooling (at a fixed potential depth). Atoms concentrate in the crossing region and the phase-space density increases substantially as compared to the MOT [START_REF] Barrett | All-optical formation of an atomic Bose-Einstein condensate[END_REF].

We have experimentally tested CDT configurations with different beam sizes w CDT (from 30 µm to 50 µm). A larger beam size helps to trap more atoms during the capture stage due to higher overlap volume. However, at a given available power, larger beams imply a weakening of the trap stiffness, which in return penalizes the thermalization after capture. The data presented in this paper are taken with a beam waist w CDT ≈ 42 µm. We obtain very similar results for w CDT ≈ 35 µm, but with different optimal powers at each stage. In the next subsection, we will concentrate on the optimization of the laser power to find the optimal trap depth for filling the center region.

Optimization of CDT loading

In order to characterize the filling dynamics of the crossing region, we define the filling factor α = N C /N as the fraction of atoms in this region relatively to the total number of atoms in the dipole trap. Images as that in figure 1(b) are processed with a multicomponent fitting routine that extracts the temperature, the density, the total atom number N and N C . Details about the fitting procedure are presented in Appendix A. The results of the optimization of the CDT power are presented in figure 3, where we plot the evolution of atom number N C and filling factor α with time. We fit the function α(t) = a(1 -e -t/τ ) + b to our data.

We look first at a situation in which "free evaporation" occurs at constant CDT power, keeping the same power during the free evaporation phase as during the capture stage. We report in figure 3 the evolution of N C and α with time for three different powers (P CDT = 7.9, 13.7 and 36 W). The values of the loading time τ and the asymptotic value α ∞ = a + b of the filling factor obtained from the fit are shown in figure 4. We find an optimal power P CDT = 13.7 W that maximizes both the number of atoms N C and the stationary filling fraction α ∞ .

In a second set of experiments, the CDT is kept at constant P CDT = 13.7 W during the "cold MOT" phase, and ramped up in 50 ms to another value just after switching off the resonant lasers. As shown in figure 4, ramping up the power to the maximum available power results in quicker loading of the central region (≃ 2 sec) and better filling ratio (α ∞ ≃ 0.6), the best values being apparently limited by the available laser power. A slower, linear power ramp to P CDT = 36 W in 2 sec (also shown in figure 3), leads to a slightly better loading ratio (α ∞ ≃ 0.7), and also a slightly lower temperature, which altogether results in a higher number of atoms (about twice as many atoms in the center of the CDT as compared to the loading at constant P CDT = 13.7 W). This particular ramp provides the best starting point we could achieve for the evaporative cooling stage.

The results of the two series of experiments show the existence of an optimal power P opt CDT = 13.7 W for the loading of the atoms during the period in which the MOT and the CDT are simultaneously present. We interpret this observation in the following way. The CDT laser exerts different light shifts on the various hyperfine states in the ground (3s) and excited (3p) manifolds. These differential light shifts can perturb the laser cooling dynamics in the CDT region and thus degrade the capture efficiency. For instance, if we take the |g = |F = 2, m F = 2 → |e 3 = |F ′ = 3, m F = 3 transition and a π-polarized CDT laser, we obtain that near the trap bottom, the laser detuning changes according to

δ 33 = ω L -ω 33 + α 33 I, (3)
with ω L the cooling laser frequency, ω 33 the "bare" transition frequency, I = 2P CDT /πw 2 CDT the intensity of the CDT laser ‡. For sodium, we find α 33 /2π ≈ 27 Hz.cm 2 /W. For our optimal cooling beam detuning δ cool = ω L -ω 33 ≈ -3.8Γ (see section 2.1) we obtain that the detuning on the cooling transition vanishes when I ≈ |δ cool |/α 33 ≈ 1.4 × 10 6 W.cm -2 . Experimentally, the optimum P opt CDT = 13.7 W corresponds to I opt = 4.7 × 10 5 W.cm -2 , close to the value calculated above, and a change of detuning from -3.8Γ to δ 33 ≈ -2.5Γ. We reached a very similar optimum in another set of experiments with w ′ CDT = 35 µm, where we found an optimum power P ′opt CDT = 10 W corresponding to I ′opt = 5.2×10 5 W.cm -2 and a comparable final detuning δ 33 ≈ -2.4Γ.

One could think that tuning the cooling beam frequency further than -3.8Γ on the red side of the |g → |e 3 transition could help to mitigate the effect, thus increasing the optimal power and ultimately the number of atoms captured. However, two separate effects work against this strategy. First, this compensation is efficient only near the trap bottom and not across the whole trapping region. Second, it brings the MOT-beams closer to resonance with neighbouring transitions that can shift in opposite ways. For example the |g = |F = 2, m F = 2 → |e 2 = |F ′ = 2, m F = 2 transition has an intensity dependance δ 22 = ω L -ω 22 -α 22 I, with ω 22 the corresponding frequency and α 22 /2π ≈ 16 Hz.cm 2 /W. The latter effect is limiting for 23 Na, which has an hyperfine structure splitting ω 33 -ω 22 much smaller than heavier alkalis ( 87 Rb and 133 Cs). ‡ For the calculation we use the data from the NIST atomic spectra database [START_REF] Yu | NIST atomic spectra database (version 4.0)[END_REF] and consider the 3s → 3p, 3p → 3d, 4d transitions (see also [START_REF] Viering | Spatially resolved single atom detection of neutral atoms[END_REF]).

Two-stage evaporation

Evaporation in the crossed dipole trap alone

As pointed out in the introduction, lowering the laser intensity to reduce the trap depth for evaporation is inevitably accompanied by a reduction of trap stiffness (near the trap bottom, the trap frequency ω is proportional to √ P /w), unlike in magnetic traps where the depth and confinement are independent. The resulting decrease in density and collision rate can make the cooling due to evaporation stop at low laser power, and this is precisely what is observed in our experiment. For a harmonic trap the classical phasespace density is given by D = N ( ω/k B T ) 3 , where ω stands for the mean trapping frequency. In a simple model where the evaporation parameter η = V CDT 0 /k B T is assumed constant and where losses are neglected, the gain in phase-space density when the trap depth is lowered from V CDT 0 to V CDT 0 /r (r > 1 is the reduction factor) is given by [START_REF] O'hara | Scaling laws for evaporative cooling in time-dependent optical traps[END_REF]

D = D 0 r β , β = 3 2 η 2 -7η + 11 η 2 -6η + 7 . ( 4
)
The starting point in our experiment (about 3 × 10 5 atoms at T ≃ 100 µK) corresponds to η ≈ 10 and a phase-space density D 0 ∼ 10 -4 . According to equation ( 4), evaporating with a reduction factor r = 200 leads to a final phase-space density of ∼ 0.2. In our experiment, the laser power is ramped down during evaporation according to

V CDT (t) = V 0 CDT (1 + t/τ evap ) -αevap , (5)
where the parameters τ evap = 30 ms and α evap = 1.2 were optimized empirically. Even after optimization we have not been able to achieve a final phase-space density greater than ∼ 10 -2 in the CDT alone †. We also observe that the collision rate after ∼ 1 s is lower than 10 s -1 , and that evaporative cooling stops near this point. Such a collision rate is too low to maintain efficient thermalization and sustain the cooling process.

Evaporation in the dimple trap

The decrease of evaporation efficiency presented in the previous subsection is caused by the relation between trap depth and confinement strength inherent to optical traps.

To circumvent this problem, one needs to break this relation. To this aim, we have added a tight "dimple" to the initial trap [START_REF] Mimoun | Fast production of ultracold Sodium gases using light-induced desorption and optical trapping[END_REF][START_REF] Weber | Bose-Einstein condensation of Cesium[END_REF][START_REF] Stamper-Kurn | Reversible formation of a Bose-Einstein condensate[END_REF][START_REF] Garrett | Growth dynamics of a Bose-Einstein condensate in a dimple trap without cooling[END_REF]. We turn on the auxiliary dT together with the main CDT, but keep it at constant power P dT = 200 mW during the CDT ramp §. With the "dimple" addition, the atoms feel a more and more confining potential as they cool, in stark contrast to the situation in the CDT alone. In our experiment, we take advantage of the dissipative nature of evaporative cooling to fill such a "dimple" trap (dT). As the atoms in the CDT are evaporatively cooled, they get progressively trapped in the stiffer potential which results in a substantial increase in spatial density [START_REF] Weber | Bose-Einstein condensation of Cesium[END_REF][START_REF] Ma | Optimized evaporative cooling using a dimple potential: an efficient route to Bose-Einstein condensation[END_REF]. Since the temperature remains the same, this translates into a huge boost in the phase-space density. This is markedly different from an adiabatic trap compression which increases spatial density but leaves the phase-space density unchanged [START_REF] Ketterle | Atom cooling by time-dependent potentials[END_REF]. After 1 s evaporation in the CDT, the dT power is reduced to provide the final stage of evaporative cooling (see figure 1(c)). The plots in figure 5 summarize the evaporation dynamics. We show atom number N, temperature T , dimple trap filling α d = N d /N where N d is the number of atoms present in the dT, and phase-space density D during the ramp, and compare it to the evaporation without dimple trap * . From figure 5(c), we observe that almost all atoms accumulate rapidly (within a few 100 ms) in the dT. At this stage, the atoms are essentially trapped by the dT in the x -y plane and by the weaker CDT in the z-direction. We therefore call this stage "evaporative filling". At the end of it, we obtain a cold sodium gas with high phase-space density and collision rate (γ coll ≈ 2 × 10 3 s -1 ), well suited to start a second evaporative cooling stage.

The difference in trapping frequencies between the cases with and without dimple trap leads to an increase of about 100 in phase-space density at t = 1 s. We quantify the evaporation efficiency κ evap leading from the starting point (N 0 ,D 0 ) to (N 1 ,D 1 ), using the the definition given in [START_REF] Ketterle | Evaporative Cooling of Trapped Atoms[END_REF],

κ evap = - ln (D 1 /D 0 ) ln (N 1 /N 0 ) . (6)
Typical values in magnetic traps are κ evap ∼ 1 -2. In our experiment, we get much better evaporation efficiencies using the dimple trap (κ dT evap ≃ 3.5) than evaporation in the CDT alone (κ CDT evap ≃ 1.6) ♯. We pursue the evaporation by reducing the dT depth, with an exponential ramp from P dT = 200 mW to P dT = 2 mW in 1.5 sec, with a time constant τ dT = 0.6 s. This results in a phase-space density increase and a crossing of the BEC threshold after ∼ 1 sec ramping, with ≃ 2 × 10 4 atoms at T ≃ 1 µK. At the end of this ramp, we obtain an almost pure BEC with ≃ 10 4 atoms.

Finally, we note that the dimple trap is used here in a quite different way compared to the experiment reported in [START_REF] Stamper-Kurn | Reversible formation of a Bose-Einstein condensate[END_REF][START_REF] Garrett | Growth dynamics of a Bose-Einstein condensate in a dimple trap without cooling[END_REF]. In these works, the authors studied an adiabatic process, in which the gain in phase-space density is obtained isentropically by modifying the trap potential shape [START_REF] Pinkse | Adiabatically changing the phase-space density of a trapped Bose gas[END_REF]. In the present work, the entropy is reduced by evaporative cooling as the transfer between the CDT and the dT proceeds. * The measurement of α d is done in the following way: The dT laser is switched off 2.8 ms after the CDT laser, and we let the cloud expand during time-of-flight t ToF = 0.2 ms before taking an absorption image. At this time, atoms released from the CDT have expanded in the x -y plane more than those released from the dT, which is appropriate for counting each component. ♯ We have found experimentally that turning on the dimple trap at a later stage during the evaporation ramp still results in a boost in phase-space density. However the cooling is not as efficient, so that the final phase-space density and the evaporation efficiency are both slightly worse.

Conclusion and prospects

We have demonstrated a method to reach Bose-Einstein condensation of 23 Na in an all-optical experimental setup. We have shown the importance of adapting the trapping potential to the magneto-optical trap cooling dynamics for optimizing the capture in the arms of the crossed trap. In the free evaporation step that follows, an increase in trap depth leads to a fast transfer of the atoms from the arms to the central region, providing thus a dense sample. We have also described the implementation of a two-step evaporation stage using a tightly focused "dimple" trap. "Evaporative filling" of the dimple trap occurs at almost the same atom number and temperature as in the crossed dipole trap alone. As a result, the phase-space density increases as (ω dT /ω CDT ) 3 , where ω dT is the dT average frequency and ω CDT the CDT average frequency at low power. Experimentally this corresponds to a large gain in phase-space density, of ∼ 100. After a final evaporation stage in the dimple trap, we are able to obtain almost pure BECs containing ∼ 10 4 atoms.

The efficient "evaporative filling" of the dimple trap suggests to generalize the scheme by adding a second, even smaller dimple trap to shorten the time to reach Bose-Einstein condensation. Such a scheme with imbricated evaporative cooling steps (like the layers of an "atomic matryoshka") can be taken into consideration if the aim is the production of Bose-Einstein condensates with small atom number, confined in a microscopic potential [START_REF] Kolomeisky | Ground-state properties of artificial bosonic atoms, Bose interaction blockade, and the single-atom pipette[END_REF][START_REF] Chuu | Direct observation of sub-poissonian number statistics in a degenerate Bose gas[END_REF][START_REF] Dudarev | Quantum many-body culling: Production of a definite number of ground-state atoms in a Bose-Einstein condensate[END_REF]. 1, 2 model the arms so that σ 1y ≫ σ 1x and σ 2y ≫ σ 2x . The third component models the denser crossing region. The second arm propagates with an angle θ: (x 2 , y 2 ) = (x 1 cos(θ) + y 1 sin(θ), -x 1 sin(θ) + y 1 cos(θ)). Each arm is supposed to be radially symmetric, the size in the z direction is therefore taken equal to the radial size in the (x, y) plane. From the sizes and the calibration of the total fluorescence counts on the CCD with the atom number measured from an absorption image, we infer the atom number in each component N 1 , N 2 and N 3 .

In order to evaluate how well equation (A.1) can fit the density profile, we perform the fit on a computed density profile of an atomic cloud at thermal equilibrium in a crossed dipole trap potential U(r) = V CDT (r) (see equation ( 1)), for P CDT = 13.7 W and w CDT = 42 µm. For a classical gas, the phase-space density f (r, p) is given by

f (r, p) = 1 Z e -p 2 /2M +U (r) k B T Θ(-p 2 /2M -U(r)) (A.2)
with Z the partition function chosen such that f (r, p) d 3 rd 3 p (2π ) 3 = 1, and Θ the Heavyside step function. An integration of f (r, p) along the imaging direction z yields a 2D-profile n sim 2D (x, y) n sim 2D (x, y) = dz f (r, p)

d 3 p (2π ) 3 = dz e -U (r)/k B T Γ inc U(r) k B T , 3 2 , (A.3)
where Γ inc is the incomplete gamma function. We also calculate the density of states ρ(ǫ) (with -V 0 CDT < ǫ < 0) This can be used to determine the number of atoms N C that have an energy between -V 0 CDT and -V 0 CDT /2,

ρ(ǫ) = d 3 rd 3 p (2π ) 3 δ ǫ -p 2 /2M -U(r) = 1 (2π) 2
N C = N -V 0 CDT ǫ -V 0 CDT /2 = n 0 Λ 3 dB V 0 CDT /2 0 dǫ ρ(ǫ)e -ǫ/k B T , (A.5)
with n 0 the density in the center of the trap, and Λ dB = h/ √ 2πmk B T the thermal de Broglie wavelength. We take N C as an estimate of the number of atoms in the central region. Equations (A.4) and (A.5) are evaluated numerically using Monte-Carlo integration.

We apply to the simulated profiles n sim 2D the same fitting routine as used for the experimental images. In figure A1, we compare the fit output with the parameters used for the simulation. As one can see, the number of atoms in the center is found to be very close to N C . This validates our method to estimate of the loading ratio α = N C /N tot with the result from the fit N 3 /(N 1 + N 2 + N 3 ). Note however that the procedure systematically over-estimates the temperature in the arms by ∼ 30 %. This is due to the gaussian shape of the trap that causes a radial density profile wider than the profile created by a harmonic trap with the same curvature. We checked that for a truncated harmonic trap, the fitted temperature is equal to the temperature T obtained for the simulated profile (equation (A.3)).

ǫ = V 0 CDT /4 ǫ = V 0 CDT /2 ǫ = V 0 CDT × 3/4 V CDT (x, y, 0) V 0 CDT /2 x [µm] x [µm] x [µm] y [µm] y [µm] y [µm] (a) (b) (c)

Figure 1 .

 1 Figure 1. (a) Sketch of the laser geometry showing the crossed dipole trap (CDT) propagating in the horizontal plane and the dimple trap (dT) propagating vertically. (b) Fluorescence image of the atoms trapped in the CDT taken after short time-offlight . The thermal equilibrium state in such a potential has a characteristic spatial structure: two elongated "arms" and a denser crossing region. (c) Evolution of the powers of the crossed dipole trap and the dimple trap during the sequence. The first step corresponds to the loading of the CDT from a "cold-MOT" phase, followed by a compression that helps to fill the central trapping region. The next step consists in evaporatively cooling the CDT and results in the filling of the dimple trap. The last step is evaporative cooling in the dimple trap, which leads to Bose-Einstein condensation.

Figure 2 .

 2 Figure 2. CDT potential in the z = 0 plane, truncated at an energy ǫ = V 0 CDT /4 (a), ǫ = V 0 CDT /2 (b), and ǫ = V 0 CDT × 3/4 (c).

Figure 3 .

 3 Figure 3. Evolution of the atom number in the center of the CDT (a) and the loading ratio α = N C /N (b) in four different loading situations: low power (circles), highest power (diamonds), ∼ 1/3 of maximum power (crosses), ramping up in 2 s after loading at low power (stars). The loading ratio α(t) is fitted with the function a(1 -e -t/τ ) + b. The results of the fit τ and α ∞ = a + b are indicated in (b).

Figure 4 .

 4 Figure 4. (a) Filling time τ and (b) center filling fraction α ∞ for CDT in two different situations: the solid curve shows the results of the experiments where the CDT laser is hold at any time at the same power.The dashed curve denotes the compression experiments where the power starts at P CDT = 13.7 W in the "cold MOT" phase and is ramped up in 50 ms to the final value indicated after switching off the molasses beams. The error-bars correspond to 90% confidence bounds on the fit coefficients τ and α ∞ . For P CDT = 13.7 W, both curves should intersect as the experimental sequence is the same. The observed difference indicates systematic variations between different experimental runs, probably due to dipole trap pointing fluctuations and total atom number variations. The vertical dashed line corresponds to the optimal power P CDT = 13.7 W for the "cold MOT" phase.

Figure 5 .

 5 Figure 5. Evaporative cooling trajectories in the combined trap (CDT and dT) (circles) and in the CDT alone (stars). We show the time evolution of atom number N (a), temperature T (b), dimple filling α d (c) and phase-space density D (d).

Figure A1 .

 A1 Figure A1. Test of the multicomponent fitting routine used to analyze CDT images. For (a), (b) and (c), black diamonds show the result from the fit. In (a), the center atom number is calculated from the density of states and shown as solid line. For the solid lines in (b) and (c), n 0 and D are taken from the formulas at thermal equilibrium. In (d), we compare the temperatures extracted from the size of the arms and the size of the central component to the one used for compute the distribution.

Table 1 .

 1 • in the horizontal plane. At the crossing point, both arms have a waist w CDT ≈ 42 µm. We control Dipole Trap ω x /2π (kHz) ω y /2π (kHz) ω z /2π (kHz) V 0 /k B (mK) Trapping frequencies and trap depths at P CDT ≈ 36 W and P dT ≈ 200 mW for the crossed dipole trap (CDT) and the dimple trap (dT), respectively.

	CDT	2.5	4.5	5.1	1.2
	dT	3.7	3.7	0.021	0.10

† The measured phase-space density is lower than the prediction of equation (4). This should be attributed to the crudeness of the model underlying this equation. In particular, three-body losses, which are important at the densities present in the CDT, are not accounted for. § Keeping the dT power high causes no modification for the CDT loading and compression.

Acknowledgments

We wish to thank Lingxuan Shao and Wilbert Kruithof for experimental assistance. D.J. acknowledges financial support by DGA, Contract No. 2008/450. L.D.S. acknowledges financial support from the EU IEF grant No. 236240. This work was supported by IFRAF, by the European Union (MIDAS STREP project), and DARPA (OLE project). Laboratoire Kastler Brossel is a Unité Mixte de Recherche (UMR n • 8552) of CNRS, ENS, and UPMC.

Appendix A. Analysis of CDT images

A figure of merit for the loading in the CDT is the atom number in the crossing region of the trap as mainly these atoms will participate to evaporative cooling. We will take the ratio between the atoms in this region and the arms as an indicator of the loading efficiency. We fit the atomic density profiles with a sum of three gaussians, two of them fitting the arms region and the last one fitting the center region,

with G(A, x, y, σ x , σ y ) = Ae -1 2 (x/σx) 2 -1 2 (y/σy) 2 . Here A is the amplitude, σ x and σ y the sizes of the distribution along the directions x and y. The first two components


Adrien Acquistapace

Thierry Bru

Pierre-François Lesault

Florence Figeac

Amélie E Coudert

Olivier Le Coz

Christo Christov

Xavier Baudin

Fréderic Auber

René Yiou

Pierre-Fran Ois Lesault

Am lie E Coudert

Fr Deric Auber

Jean-Luc Dubois-Rand

Anne-Marie Rodriguez

Marie Rodriguez
email: marie.rodriguez@inserm.fr

Human mesenchymal stem cells reprogram adult cardiomyocytes toward a progenitor-like state through partial cell fusion and mitochondria transfer

Keywords: Animals, Cell Differentiation, genetics, physiology, Cell Fusion, Cell Line, Cells, Cultured, Fibroblasts, cytology, metabolism, Humans, Immunohistochemistry, In Situ Hybridization, Male, Mesenchymal Stem Cells, cytology, metabolism, Mice, Mitochondria, metabolism, Myocytes, Cardiac, cytology, metabolism, Nuclear Reprogramming, genetics, physiology, Reverse Transcriptase Polymerase Chain Reaction mesenchymal stem cells, cardiomyocytes, cell fusion, nuclear reprogramming

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Heart degenerative diseases such as myocardial infarction and heart failure are among the main causes of death in western countries.

Such mortality has its roots in the poor ability of the myocardium to restore cardiomyocyte loss upon injury, which explains why rebuilding the damaged heart remains a critical challenge. One of the most promising approaches to repair this organ is cell therapy, particularly that based on multipotent adult stem cells because such cells might benefit from immune priviledge , and can be

[1 -3 ] [3 4 ]
expanded on a large scale . However, although many studies suggested the benefit of stem cell grafting, the mechanisms by in vitro [1 -3 ] which such cells contribute to cardiac wound healing remain a matter of debate.

The functional benefit of stem cells may result from several processes, including transdifferentiation into cardiac and endothelial cells [ or secretion of paracrine factors with various effects such as protection against apoptosis and induction of angiogenesis . Another 5 -7 ] [START_REF] Mirotsou | Paracrine mechanisms of stem cell reparative and regenerative actions in the heart[END_REF] intriguing possibility is that permanent cell fusion between stem and resident cardiac cells may promote replacement of the dead myocardium by generating new cardiomyocytes in response to injury. Indeed, several and studies involving the Cre/loxP in vivo in vitro system or transgenic rodents expressing GFP or -galactosidase reporter genes showed that both adult and embryonic stem cells could fuse β with somatic cells such as neurons, hepatocytes, and cardiomyocytes . Permanent cell fusion results in the formation of binucleated [9 -11 ] heterokaryons, which in some cases undergo nuclear fusion to give rise to mononucleated hyperploid synkaryons. In heart tissue, permanent cell fusion has been detected after stem cell delivery in various settings, including the healthy neonatal heart , myocardial [START_REF] Ishikawa | Purified human hematopoietic stem cells contribute to the generation of cardiomyocytes through cell fusion[END_REF] infarction , dystrophin-deficient cardiomyopathy , and monocrotaline-induced hypertension . However, despite [13 -15 ] mdx [START_REF] Payne | Regeneration of dystrophin-expressing myocytes in the mdx heart by skeletal muscle stem cells[END_REF] [17 ]

evidence of hybrid cells with a cardiac phenotype , such a phenomenon was described as being extremely rare and led critics to [13 -17 ] cast doubts on its biological significance , , .

[6 18 19 ] Recently, 2 studies mimicking the cardiac environment by co-culturing stem cells and cardiomyocytes suggested that hybrid in vitro cells may be generated by a mechanism other than permanent cell fusion, one that consists of partial cell fusion through transient direct cell-to-cell communication and intercellular exchange of various compounds , . This new transient cell fusion pathway, discovered [20 21 ] in 2004, is based on the formation of membrane thin channels, referred to as tunneling nanotubes, that mediate membrane continuity between connected cells, sometimes over long distances . These nanotubular structures have been found to connect a broad range of [START_REF] Rustom | Nanotubular highways for intercellular organelle transport[END_REF] cultured mammalian cells and to permit transfer of several components such as multi-protein complexes , organelles and [START_REF] Gerdes | Intercellular transfer mediated by tunneling nanotubes[END_REF] [24 ] [25 -27 ] pathogens . Specially, stem cells and cardiomyocytes have been reported to exchange cytoplasm macromolecules and [28 -30 ] in vitro organelles through intercellular structures resembling nanotubes , . However, whether these cellular transfers play a role in cell fate [ 20 21 ] change and tissue repair remains to be formally demonstrated.

Thus, the phenomenon of cell fusion in heart tissue appears to be poorly understood. In particular, the respective physiological importance of partial versus permanent cell fusion processes is unclear, and little is known about the cellular and molecular mechanisms underlying spontaneous generation of hybrid cells, as well as the characterization of their phenotypes.

Here, we hypothesized that stem-cell mediated fusion with terminally differentiated cells constitutes a regenerative mechanism by which the nuclei of somatic cells are reprogrammed, thus resulting in selective survival and proliferative advantages. To promote cell fusion events, we developed a species mismatch co-culture approach using mouse terminally differentiated cardiomyocytes and human multipotent adipose-derived stem cells (hMADS cells) as a model of adult stem cells. Using a Cre/loxP system, we found that hMADS [START_REF] Rodriguez | Transplantation of a multipotent cell population from human adipose tissue induces dystrophin expression in the immunocompetent mdx mouse[END_REF] cells could reprogram post-mitotic murine cardiomyocytes into proliferating cardiac progenitor-like cells through spontaneous cell fusion.

In addition, we showed that successful somatic reprogramming of cardiomyocytes required both intercellular structures composed of f-actin and microtubules and transfer of functional stem-cell mitochondria into cardiomyocytes.

MATERIALS AND METHODS

Cell isolation and culture

For hMADS cell isolation, adipose tissues were obtained from young donors, with written informed consent of the parents, as surgical scraps from surgical specimens obtained from various surgeries, as approved by the Regional Ethical Committee ( Comit de Protection " é

des Personnes Ile de France IX ). HMADS cells were isolated as previously described . Human bone-marrow mesenchymal stem cells

" [ 3 ]
(hBMSCs) were a generous gift from Dr H l ne Rouard (Etablissement Fran ais du Sang, Cr teil, France). hBMSCs were isolated from é è ç é iliac-crest bone-marrow aspirates from patients undergoing standard bone-marrow transplantation (Henri Mondor Hospital, Cr teil, é France) after receiving their informed consent .

[31 ]

The human lung fibroblast MRC5 cell line was a gift from Dr. Jorge Boczkowski (INSERM U955, Cr teil). HMADS cells, hBMSCs For mitochondrial DNA depletion, hMADS cells were cultivated in DMEM supplemented with 10 FBS and 0.5 g/ml ethidium % μ bromide (Sigma-Aldrich), 50 g/ml uridine and 100 g/ml pyruvate for at least 1 month .

μ μ [32 -34 ]

Isolation of adult mouse ventricular myocytes

Ventricular cardiomyocytes were isolated from hearts of 2-to 5-month old male mice after intracardiac perfusion of enzymatic solution containing 12.5 M CaCl (Sigma-Aldrich), 0.1 mg/ml liberase blendzyme IV (Roche diagnostics), 0.14 mg/mL trypsine μ 2 (Sigma-Aldrich), as described . Cardiomyocytes were obtained from several mouse lines, including C57BL/6J mice (Janvier), [START_REF] Mitra | A uniform enzymatic method for dissociation of myocytes from hearts and stomachs of vertebrates[END_REF] GCAG-GFP transgenic mice (gift of Dr Fabrice Chr tien, Pasteur Institute, Paris, France) and Rosa26R transgenic mice (gift of Dr.

[36 ] é

Shaharagim Tajbakhsh, Pasteur Institute, Paris, France).

Co-culture with adult mouse cardiomyocytes

Freshly isolated mouse cardiomyocytes were co-cultured with hMADS cells, hBMSCs or MRC5 cells in DMEM supplemented with 10 FBS in a 1:1 ratio, each cell type seeded at 3,500 cells/cm .

% 2
In experiments preventing physical contact between the 2 cell types, cardiomyocytes were seeded on cell-culture inserts containing a polycarbonate membrane with a 0.4-m pore (Millicell, Millipore) placed in 35-mm dishes plated with hMADS cells. μ To assess passive human mitochondria transfer, cells were indirectly co-cultured by use of polycarbonate cell-culture inserts with an 8m pore (Millicell, Millipore). For co-culture with hMADS cells, culture medium was supplemented with 50 g/ml uridine and 100 μ ρ 0 μ μ g/ml pyruvate to permit survival of stem cells devoid of functional mitochondria.

Detection of cell fusion

For detection of fused cells, hMADS cells were infected with a human serotype V adenovirus expressing Cre under a cytomegalovirus promoter (gift of Dr Athanassia Sotiropoulos, Cochin Institute, Paris, France) at a multiplicity of infection of 50 at 72 hr before co-culture with Rosa26R cardiomyocytes. Rosa26R mice carried the lacZ gene that encodes for -galactosidase under the control of an internal stop β codon flanked by loxP sites . When Cre-expressing cells fuse with Rosa26R cells, Cre recombinase contacts the genome of Rosa26R [START_REF] Soriano | Generalized lacZ expression with the ROSA26 Cre reporter strain[END_REF] cells and excises its floxed stop cassette, thus resulting in expression of LacZ in the fused cells. Consequently, fused cells and their progeny can be detected by Xgal staining (substrate of -galactosidase) or by immunocytochemistry with antibodies against β β -galactosidase.

Xgal staining

For Xgal staining, co-cultured cells were fixed with 2 paraformaldehyde (PFA) before being placed in phosphate buffer containing % 10 mM K Fe(CN) , 10 mM K Fe(CN) and 1 mg/ml Xgal (Qiagen) at 37 C for 24 hr. DNA probes (Roche Diagnostics and Invitrogen) previously labeled by NICK translation (Roche Diagnostics) with Cy3 and biotin, respectively. Mouse biotin-labeled DNA was detected with a streptavidin-fluorescein conjugate (Sigma-Aldrich). In experiments combining GATA-4 immunocytochemistry and FISH, co-cultured cells were stained with GATA-4 and then labeled with all human or all mouse centromere probes conjugated to FITC (Kreatech Diagnostics). After hybridization, nuclei were counterstained with Hoechst and examined by confocal microscopy (Zeiss LSM 510 Meta).

Electronic microscopy

Co-culture derived cells were fixed overnight in 3 glutaraldehyde in 0.1 M sodium phosphate buffer, pH 7.4, post-fixed for 1.5 h

- %
with 1 osmium tetraoxide, dehydrated by successive ethanol washes (50 , 70 , 80 , 95 , 100 , 100 ), and impregnated with epoxy

% % % % % % %
resin. After polymerization, 70-to 80-nm sections were cut by use of a Reichert Ultracut E ultramicrotome, stained with 2 uranyl acetate % plus Reynold s lead citrate, and visualized under a Philips CM120 transmission electron microscope.

'

Inhibition of f-actin or microtubule polymerization

After proper cell adhesion (~6 hr), co-cultured cells were treated for 12 hr with 1 M nocodazole to inhibit -tubulin polymerization or μ α 0.5 M cytochalasin D or 0.25 M latrunculin A, two inhibitors of f-actin polymerization. Inhibitory treatments were repeated 3 days later.

μ μ

Stem-cell mitochondria transfer

Prior to co-culturing, hMADS cells were labeled with MitoTracker Green FM (200 nM, Invitrogen). Images were obtained by time-lapse video microscopy (Leica DMI6000, Metamorph 7 acquisition software, Universal Imaging) or fluorescence microscopy (Zeiss Axioplan 2 Imaging, Axiovision LE 4.6 software).

RESULTS

Fusion of hMADS cells and adult cardiomyocytes leads to formation of hybrid cells with cardiac progenitor-like features

To investigate whether hMADS cells fused with fully differentiated cardiomyocytes, we used a Cre/loxP system that allows lacZ gene expression only in fused cells. Cre-expressing hMADS cells were co-cultured with cardiomyocytes from Rosa26R transgenic mice. From 2 days after co-culture initiation, we detected colonies of small rounded cells that were lacZ-positive and whose morphology differed from that of both hMADS cells and adult cardiomyocytes (

). Xgal staining was not due to endogenous -galactosidase activity because Fig. 1A β co-cultures with non-genetically modified hMADS cells and Rosa26R mouse cardiomyocytes were devoid of blue cells (

). In Fig. 1B addition, immunocytochemistry revealed that colony-derived cells co-expressed -galactosidase with the early cardiac transcription factor β GATA-4, which suggests that fused cells belonged to the cardiac lineage (

). Again, the presence of false-positive -galactosidase Fig. 1C β cells in co-cultures was ruled out by immunocytochemistry of co-cultures without induction of the Cre/loxP system ( ). Fig. 1D To further characterize the phenotype of fused cells, we used immunostaining for GATA-4 with a series of markers directed against cardiac, smooth muscle or fibroblastic cells. GATA-4 cells expressed early cardiac cell markers such as the transcription factors Nkx2.5

+ and MEF-2C (

). In contrast, later cardiac differentiation markers such as desmin and cardiac troponin I (cTnI) were expressed Fig. 2A,B only in a small proportion (<5 ) of GATA-4 cells ( ), and -sarcomeric actinin was never detected ( ). Furthermore, % + Fig. 2C,D α Fig. 2E GATA-4 colony-derived cells were negatively stained for -smooth muscle actin and P4HB, which confirms that these cells did not

+ α
originate from smooth muscle or fibroblastic lineages, respectively ( ). Of note, hMADS cells that were cultured alone showed Fig. 2F,G negative staining for all these antibodies, except P4HB. Finally, hybrid GATA-4 cells expressed Ki67, a cell-cycling and proliferation + marker ( and ). Counting of such hybrids from day 2 to 5 of co-culture allowed for estimating their Fig. 2H Supporting information Fig. 1A population doubling time at about 24 hr. MTT assay performed from day 8 to 14 of co-culture depleted in human stem cells after 5 days of oubain exposure confirmed the proliferative potential of cardiac hybrid cells. Nevertheless, it also indicated that such potential was limited because cell growth decreased with time to become undetectable after 2 weeks of co-culture (

). Supporting information Fig. 1B,C According to these results, cell fusion between stem cells and post-mitotic cardiomyocytes led to the generation of hybrid cells exhibiting proliferative potential, the expression of early cardiac markers and absence of an organized contractile cytoskeleton. These features were reminiscent of a cardiac progenitor cell type. Therefore, hybrid cells are hereafter designated cardiac progenitor-like cells.

Cardiac progenitor-like cells originate from somatic reprogramming of cardiomyocytes, not from stem-cell transdifferentiation

To determine the parental origin of cardiac genes expressed in hybrid cells, we performed real-time PCR analysis with species-specific primers on cells co-cultured from day 0 to 7. As compared with mouse cardiomyocytes alone, co-cultured cells exhibited transcriptional activation of mouse genes involved in early cardiac commitment and proliferation, including GATA-4, MEF-2C, p300 (a histone acetyltransferase implicated in cardiac precursor cell induction ) and Ki67, whereas expression of later cardiac mouse genes such as [START_REF] Yanazume | Cardiac p300 is involved in myocyte growth with decompensated heart failure[END_REF] desmin and cardiac troponin T (cTnT) appeared unaffected ( ). In contrast, co-culture cells and hMADS cells cultivated alone did Fig. 3A not differ in human gene expression ( ). Fig. 3B Real-time PCR results suggesting a mouse phenotype of cardiac progenitor-like cells were further confirmed by immunostaining for GATA-4 with both human/mouse and human -specific Lamin A/C antibodies and by co-culturing GFP transgenic mouse cardiomyocytes.

+

As expected, progenitor-like GATA-4 cells expressed only mouse Lamin A/C, as shown by the negative stain with human-specific + antibody (

). In agreement with this finding, cardiac progenitor-like cells were GFP when coculture was performed with mouse Fig. 3C +

GFP cardiomyocytes (

).

+

Fig. 3D

The mouse phenotype of hybrid cells highlighted that cellular fusion between stem cells and cardiomyocytes did not trigger cardiomyogenic conversion of stem cells but, rather, promoted somatic reprogramming of cardiomyocytes toward a progenitor-like state.

To show that somatic reprogramming resulted from cell fusion, co-cultures were performed with (i) a cell-culture insert to impede physical interactions between the 2 cell types without affecting the diffusion of soluble factors or (ii) living cardiomyocytes and hMADS cells previously killed with PFA to inhibit cellular fusion . Indeed, PFA pre-fixation prevented fusion of live sperm cells or neural stem [START_REF] Yoon | Bone marrow-derived side population cells are capable of functional cardiomyogenic differentiation[END_REF] cells with fixed sea urchin eggs or endothelial cells, respectively, but did not disrupt receptor-mediated recognition and association of the co-cultivated cell types , . Reprogramming did not occur in either condition, which indicates that soluble signaling molecules or [ 41 42 ] cell-to-cell interaction through a receptor had little or no effect on the generation of mouse cardiac progenitor-like cells ( and not Fig. 3E,F shown).

Finally, we addressed the frequency of cardiomyocyte reprogramming by counting the number of immunostained cardiac gal β + /GATA-4 hybrid cells resulting from co-cultures rendered mitotically inactive by mitomycin C treatment. We detected a mean of 10 + hybrid cells per 100 000 cardiomyocytes initially seeded. However, because propidium iodide staining and flow cytometry revealed that more than 90 of cardiomyocytes were suffering before co-culture (

), the rate of cardiomyocytes efficiently % Supporting information Fig. 2 reprogrammed might be about 0.1 when considering the fraction of healthy cardiomyocytes at the start of co-culture.

%

In addition, we tried to improve reprogramming efficiency by increasing cardiomyocyte survival because living in vitro cardiomyocytes were found to have limited lifespan (< 24 hr) under our co-culture conditions. We performed co-cultures on laminin-coated dishes in the presence of medium containing insulin-transferrin-selenium (ITS) and 2,3butanedione monoxime (BDM) as previously reported

. Nevertheless, under these conditions, the yield of reprogrammed cardiomyocytes seemed not affected.

[43 ]

Human bone-marrow derived stem cells (hBMSC) but not human fibroblasts can reprogram mature cardiomyocytes

To determine whether reprogramming of cardiomyocytes was specific to hMADS cells, we created co-cultures with hBMSCs as another model of multipotent stem cells and with human fibroblasts (MRC5 cell line) as an example of proliferative cells devoid of plasticity potential. Real-time PCR of BMSC co-cultures revealed results similar to those with hMADS cells: increased transcription of mouse but not human genes involved in proliferation and early cardiac commitment such as Ki67, GATA-4, MEF-2C and p300, as well as unchanged expression of both mouse and human later cardiac differentiation genes such as desmin and cardiac troponin T ( ). In Fig. 3G,H contrast, co-culture with MRC5 fibroblasts resulted in different mouse and human gene expression patterns. Even fibroblasts promoted enhanced transcription of mouse GATA-4; they failed to enhance that of mouse p300 and MEF-2C, which indicates that reprogramming of mouse cardiomyocytes was incomplete (

). In On the other hand, coculture with human fibroblasts exhibited increased transcription Fig. 3I of human GATA-4 gene, which suggests that these cells initiate a process of cardiomyogenic conversion (

). Accordingly, somatic Fig. 3J reprogramming seems to be a feature of multipotent stem cells that is not shared by fibroblastic cells.

Cardiomyocyte reprogramming is mainly mediated by a partial cell-fusion process

To gain additional insights into the mechanisms underlying somatic reprogramming, we investigated whether hybrid cells originated from permanent or partial cell fusion processes. The occurrence of permanent cell fusion was tested by FISH nuclear staining with species-specific DNA probes. Rare synkaryons (1:200 hybrid cells) whose nuclei exhibited both human and mouse DNA were detected by this method ( ), whereas colony-derived GATA-4 cells contained only mouse DNA ( ). Such observations argued Fig. 4A + Fig. 4B D against permanent cell fusion as the principle mechanism of the formation of cardiac progenitor-like hybrid cells. Further supporting this hypothesis, live cell imaging and electron microscopy revealed partial cell fusion events between co-cultured hMADS cells and cardiomyocytes. Live cell imaging microscopy revealed intercellular structures resembling tunneling nanotubes that ensure membrane continuity between stem and cardiac cells (

). Electron microscopy revealed 2 other types of heterocellular interactions: one Fig. 5A,B connecting distant cells and consisting in long slender protrusions differing from nanotubes in that they adhered to the substratum (

), and the other consisting in focal, broad, tight cell-to-cell contact between neighboring cells ( ). Membrane disruptions Fig. 5C,D Fig.

5F suggestive of partial cell fusion seemed to occur focally in both cases (

). Cytoskeletal composition of intercellular structures Fig. 5E,G connecting hMADS cells and cardiomyocytes was then analysed by staining f-actin and microtubules with phalloidin and anti--tubulin α antibody, respectively; most of the intercellular communications between the 2 cultivated cell types contained both microtubule and f-actin filaments (

). Fig. 6A,B To assess the involvement of such connections in somatic reprogramming, co-cultured cells were transiently treated to not alter cell viability with cytochalasin D or latrunculin A, 2 inhibitors of f-actin polymerization, or with nocodazole, which inhibits -tubulin α polymerization. The expression of mouse and human cardiac genes was analyzed in day-7 co-cultures by real-time PCR. As compared with untreated co-cultures, nocodazole-, cytochalasin D-or latrucunlin A-treated co-cultures showed decreased transcriptional expression of mouse early cardiac GATA-4 gene with relative abundances of 0.7, 0.6 and 0.82, respectively. This effect was associated with increased expression of mouse later cardiac genes, with relative abundances of 3, 3.8 and 2 for desmin and 2.7, 3.1 and 2.8 for cardiac troponin T,

respectively (

). This cardiac gene expression pattern reminiscent of that of fully differentiated cardiomyocytes, these results Fig. 6C indicated that transient disruption of f-actin filaments or microtubules prevented cardiomyocyte reprogramming back to a progenitor-like state. In addition, we observed an increased level of transcripts encoding for human GATA-4 and desmin, whereas the expression of human MEF-2C remained quite similar to that in untreated co-cultures (

). These data indicated that transient disruption of f-actin Fig. 6D or microtubule networks favored transdifferentiation of hMADS cells into a cardiac lineage. The phenotype of transdifferentiated hMADS cells was further characterized by immunohistochemistry. We detected the protein expression of GATA-4 but no desmin, cardiac troponin I or MEF2C, which supports that cardiomyogenic conversion of hMADS cells was incomplete ( and data not Supporting information Fig. 3 shown). Interestingly, the effect of nocodazole on mouse and human cardiac gene transcription was comparable to that of cytochalasin D and latrunculin A (no significant statistical differences between drugs)(

). Thus, f-actin and microtubules may act in concert and Fig. 6C,D hence, somatic reprogramming might be mainly mediated by intercellular structures containing both f-actin and -tubulin filaments. α

Transfer of functional stem cell mitochondria into cardiomyocytes is essential for somatic reprogramming

Because the transfer of functional mitochondria from stem cells was suggested to rescue cardiomyocytes in co-culture , we [START_REF] Plotnikov | Cell-to-cell cross-talk between mesenchymal stem cells and cardiomyocytes in co-culture[END_REF] analysed whether mitochondria of hMADS cells labelled with mitotracker before co-culture were transferred to mouse cardiomyocytes. As soon as 6 hr after co-culture initiation, live cell imaging revealed some mouse cardiomyocytes containing fluorescent human mitochondria ( ). Passive transfer of human mitochondria toward cardiomyocytes was tested in indirect co-cultures in which hMADS cells Fig. 7A previously labeled with mitotracker dye were separated from cardiomyocytes by an 8-m pore cell-culture insert. Under these conditions, μcardiomyocytes were unable to capture mitotracker dye from hMADS cells, which supports that stem-cell mitochondria were mainly transferred through intercellular structures and not from extracellular medium (data not shown). Accordingly, human mitochondria were detected inside intercellular structures connecting hMADS cells to cardiomyocytes ( ). Fig. 7B To determine whether transferred human mitochondria persisted in mouse progenitor-like cells derived from cell fusion, day-7 co-cultures of Rosa26R mouse cardiomyocytes and hMADS cells expressing Cre recombinase underwent immunocytochemistry for GATA-4, -gal and human mitochondria. Human mitochondria were revealed inside the cytoplasm of mouse-reprogrammed GATA-4 /

β + β
-gal cells ( ). As expected, the human mitochondria antibody stained human but not mouse cells ( ).

+ Fig. 7C Fig. 7D
To assess the role of stem-cell mitochondria transfer in cardiomyocyte reprogramming, hMADS cells were pre-treated for a long time (>1 month) with low doses of ethidium bromide. Such a treatment allows mutations and depletion of mitochondrial DNA without altering nuclear function, as previously reported for other cell types . After 1 month of ethidium bromide exposure, more than 95 of [32 -34 ] % treated cells, commonly designated as hMADS cells, exhibited greatly decreased mitochondrial DNA as compared with untreated ρ 0 hMADS cells ( ). To investigate their cell fusion ability, hMADS cells were genetically modified to express Cre recombinase Fig. 7E ρ 0 before co-culture with Rosa26R mouse cardiomyocytes. The proportion of cardiac GATA-4 / -gal progenitor-like cells was 70 to 90

+ β + % %
lower in treated ( ) than untreated co-cultures ( ), indicating that functional stem cell mitochondria transfer is essential for Fig. 7F Fig. 7G successful achievement of somatic reprogramming. The detection of a low amount of fused cardiac progenitor-like cells was likely due to the presence in the hMADS population of a few human cells whose mitochondria were still unaltered. The requirement of functional ρ 0 stem-cell mitochondria for reprogramming was confirmed by real-time PCR analysis. As compared with untreated co-cultures, hMADSρ 0 co-cultures showed significantly decreased mouse early but also late cardiac differentiation transcripts, with relative abundances of 0.43 for GATA-4, 0.22 for MEF-2C, 0.02 for desmin and 0.07 for cTnT ( , left panel). The relative transcription level of mouse Fig. 7H GATA-4 and MEF-2C genes should reflect the presence of a few cardiac hybrid cells previously identified by immunocytochemistry, whereas the marked decrease of mouse desmin and cTnT transcripts strongly suggests that in the absence of functional stem-cell mitochondria, adult cardiomyocytes died, which explains why they were unable to be reprogrammed. Again, concomitant with somatic reprogramming inhibition, we observed enhanced transcription of human GATA-4 gene, with a relative abundance of 2.71 as compared with that in untreated co-cultures, which suggests the entry of hMADS cells into the cardiomyogenic lineage ( , right panel).

Fig. 7H

DISCUSSION

To further characterize stem cell fusion as tissue repair mechanism, our study provides the first evidence that hMADS cells can rescue post-mitotic cardiomyocytes via cell fusion and reprogramming to a rejuvenated progenitor-like state. Interestingly, hBMSCs but in vitro not human fibroblasts exhibited similar reprogramming ability, which suggests that this feature is restricted to some types of multipotent stem cells. Another important consideration concerns the frequency with which somatic reprogramming occurs. According to our results, the rate of this phenomenon is rare and might be underestimated because of the poor viability of cardiomyocytes after dissociation from the heart and their low survival potential on culture. Consequently, future efforts to optimize reprogramming efficiency might focus on improving methods for dissociation from the heart and culture of mouse cardiomyocytes.

Additionally, our study reveals information on the phenotype of hybrid cardiac cells, which was until now largely unexplored. We found that fused cells are in fact an intermediate state between stem and fully differentiated cells because they look like a type of cardiac progenitor in terms of their proliferative potential, expression of early cardiac transcription factors and lack of contractile proteins.

Therefore, whether such cells resemble or differ from cardiac progenitors or cardiac stem cells reported to reside in heart tissue , [15 45remains a question that requires further knowledge of cardiac hybrid cells. In particular, whether co-culture derived hybrid cells can 47 ] differentiate into beating cardiomyocytes needs to be determined to show whether they are truly cardiac progenitors. Although this phenomenon was never observed in our co-culture conditions, we cannot exclude that such a differentiation could occur in experimental settings more faithfully reproducing the heart environment. In strengthening this hypothesis, recent unpublished data from our lab suggest that cardiac hybrid cells generated by co-culture differentiate into mature cardiomyocytes when engrafted into mouse infarcted in vivo myocardium.

Besides assessing the hybrid phenotype, our study also provides insights into mechanisms underlying cell fusion processes leading to cardiomyocyte reprogramming. Despite the occurrence of permanent cell fusion during co-culture, this process fails to fully explain the generation of hybrid cells in terms of its frequency, which is extremely rare, and the fact that we never detected colonies of synkaryons expressing GATA-4. Rather, our observations favor a role of partial cell fusion in the emergence of cardiac hybrid cells. Evidence of partial cell fusion events was reported from co-culture of neonatal cardiomyocytes and endothelial progenitor cells or bone-marrow in vitro derived stem cells , . These studies revealed thin membranous connections between stem and cardiac cells, but did not consider

- [ 21 48
] their cytoskeletal composition. Here, we report the possibility of partial cell fusion by transient thin channels resembling tunneling nanotubes or by close cell-to-cell membrane apposition accompanied by focal membrane disruption. Our results also highlight the involvement of both f-actin and microtubule filaments in this process. These 2 cytoskeletal components may act in concert inside the same intercellular connections. Interestingly, intercellular channels containing both f-actin and microtubules have been described to transfer mitochondria between macrophages and, according to our observations, may also participate in conveyance of stem-cell in vitro [START_REF] Onfelt | Structurally distinct membrane nanotubes between human macrophages support long-distance vesicular traffic or surfing of bacteria[END_REF] mitochondria toward cardiomyocytes.

Despite previous evidence of cell-to-cell connections and organelle transfer between stem and cardiac cells , , the formation of [20 21 ] cardiac progenitor-like cells has never been reported in co-culture studies. The fact is probably ascribable to differences in developmental stage of cardiomyocytes used for co-culture. Indeed, other investigators used neonatal cardiac cells capable of division , , , [7 49 50 ] whereas we used non-replicative adult cardiomyocytes. Finally, one of the most exciting outcomes of our study relates to the role of stem-cell mitochondria in cardiomyocyte reprogramming. Here, we provide the first evidence to our knowledge that human stem-cell mitochondria transferred toward mature cardiomyocytes persisted over time in mouse cardiac progenitor-like cells derived from cell fusion.

Importantly, we found that depletion of stem-cell mitochondrial DNA did not alter the cell fusion ability of stem cells but, rather, led to a dramatic decrease in somatic reprogramming frequency. Thus, delivery of stem-cell intact mitochondria to distressed cardiomyocytes whose mitochondria are rapidly altered after isolation may prolong the survival of such cells and maintain their nuclear nuclear integrity, which is a pre-requisite for reprogramming.

In support of our findings, Spees and collaborators recently described that transfer of stem-cell mitochondria can rescue aerobic [START_REF] Spees | Mitochondrial transfer between cells can rescue aerobic respiration[END_REF] respiration in mammalian cells with non-functional mitochondria. However, whether stem-cell mitochondria may affect somatic reprogramming by favoring cardiomyocyte transcriptional machinery remodeling through conveyance of key factors from stem cells or by modulating transcription factor activities into cardiomyocytes through mitochondria-to-nucleus retrograde signaling is yet unclear .

[51 ]

Whatever the fine mechanisms by which stem-cell mitochondria sustain somatic reprogramming, these findings may reconcile current controversies about mechanisms underlying the cardiac benefit of stem cells by proposing an alternative regenerative pathway to other well-accepted candidates including transdifferentiation, permanent cell fusion and paracrine effects. In particular, transfer of stem-cell mitochondria may well explain why heart functional improvements are observed despite the fact that few of the donor cells are engrafted at long term. Nevertheless, determining whether partial cell fusion, mitochondria transfer and cardiomyocyte reprogramming take place in is currently difficult. However, several recent studies have strengthened the hypothesis that such processes occur in living tissues. For vivo instance, the existence of tunneling nanotubes has been confirmed in cornea following injury , and heart regeneration in zebrafish has [START_REF] Chinnery | Cutting edge: Membrane nanotubes in vivo: a feature of MHC class II cells in the mouse cornea +[END_REF] been shown to be mediated by cardiomyocyte dedifferentiation and proliferation .

[53 ]

Future research into partial cell fusion will contribute to a better understanding of the regenerative properties of stem cells and lead to the development of more efficient cell therapies to treat heart degenerative diseases.

CONCLUSIONS

Our study provides the first evidence that adult stem cells can reprogram cardiomyocytes back to a more immature state through partial cell fusion processes and transfer of functional mitochondria. The timeline of cardiomyocyte reprogramming is summarized in . In contrast, permanent cell fusion and the formation of synkaryons were rare in our co-culture conditions supporting information Fig. 4 and therefore do not seem to be the principle mechanisms of hybrid cardiac cell generation.

élow

  and MRC5 cells were cultured in Dulbecco s modified Eagle s medium (DMEM), 1 g/l glucose containing 10 heat inactivated fetal' ' %bovine serum (FBS) (Dominique Dutscher), 100 U/ml penicillin, 100 g/ml streptomycin, and 10 mM HEPES (Invitrogen) in a 5 CO μ % 2 atmosphere at 37 C. As described , hMADS cells had the following phenotype: CD44 , CD49b , CD105 , CD90 , CD13 , Stro-1 , , CD117 , Flk-1 , Gly-A , CD133 , HLA-DR and HLA-I .Generation of hMADS cells devoid of functional mitochondria (hMADS cells)ρ 0

Figure 1 Figure 2 Figure 3 Figure 4 Figure 5

 12345 Figure 1 Formation of GATA-4 hybrid cells after fusion of hMADS with cardiomyocytes + (A) Detection of hybrid cells (blue) by Xgal staining on day 2 of co-culture of Rosa26R cardiomyocytes with Cre-human multipotent adipose-derived stem cells (hMADS) cells (arrowhead). (B) Negative Xgal staining on co-culture with non-transduced hMADS cells (arrowhead). (C D) Co-immunostaining for -galactosidase ( -gal, white) and GATA-4 (red) on day 7 of co-culture of Rosa26R -β β cardiomyocytes and (C) Cre-or (D) non-transduced hMADS cells. Arrow: dead cardiomyocyte bodies. Nuclei were counterstained with Hoechst 33342 (blue). (A D) Scale bars, 50 m.μ

Figure 6 Figure 7

 67 Figure 6 Involvement of f-actin and microtubule filaments in cardiomyocyte reprogramming (A) Six hours after co-culture initiation, thin membrane channels interconnecting stem (arrow) to cardiac (arrowhead) cells contained both f-actin (rhodamine-phalloidin staining, red) and microtubules (FITC-conjugated -tubulin, green). (B) Close membrane apposition between α stem and cardiac cells showing concentration of microtubule (green) and f-actin (red) filaments at the contact site. Scale bars (A, B), 10 m. μ (C D) Impaired somatic reprogramming by inhibitors of f-actin (cytochalasin D 0.5 M, latrunculin A 0.25 M) or microtubule -μ μ polymerization (nocodazole 1 M) assessed by real-time PCR at 7 days of co-culture. No statistical differences between inhibitory effects of μ nocodazole, cytochalasin D or latrunculin A were observed. Relative expression was compared to day-7 untreated co-cultures. Data represent the mean SD of at least 3 independent experiments <0.05; <0.01; <0.001. ± *P

Table 1 ±

 1 Between-group comparisons for biochemical data involved the two-tailed Student s test. < 0.05 was considered statistically significant.

	3	6	4	6	°Immunocytochemistry
	Cells were fixed with 4 PFA or methanol-acetone (50:50) and stained with antibodies (Abs) against GATA-4 (goat polyclonal Ab %	[
	pAb , 1:20, R&D Systems, or rabbit pAb, 1:50, Santa Cruz Biotechnology), -galactosidase (chicken pAb, 1:500, Abcam), Ki67 (rabbit ] β
	pAb, 1:500, Abcam), Nkx2.5 (rabbit pAb, 1:50, Santa Cruz Biotechnology), myocyte enhancer factor 2C (MEF-2C; goat pAb, 1:50, Santa
	Cruz Biotechnology), desmin (rabbit pAb, 1:100, Abcam), cardiac troponin I (cTnI, rabbit pAb, 1:100; Abcam), -sarcomeric actinin α
	(mouse monoclonal Ab mAb , 1:500, Sigma-Aldrich), -smooth actin (rabbit pAb, 1:100, Abcam), prolyl 4 hydroxylase subunit beta [ ] α
	(P4HB; mouse mAb, 1:100, Novus Biologicals), GFP (rabbit pAb, 1:100, Gene Tex), Lamin A/C (rabbit pAb, 1:100, Cell Signaling
	Technology), human Lamin A/C (mouse mAb, 1:100, Novocastra), or human mitochondria (mouse mAb, 1:800, Abcam). Donkey
	secondary anti-goat, -rabbit, -chicken and -mouse antibodies (FITC-, Cy3-or Cy5-conjugated, 1:100) were purchased from Jackson
	ImmunoResearch Laboratories. Nuclei were stained with Hoechst 33342 (Sigma-Aldrich). For co-staining with phalloidin-rhodamine (5 μ
	g/ml, Sigma-Aldrich) and FITC-conjugated -tubulin (mouse mAb, 1:100, Abcam), hMADS cells were fixed with 4 PFA, then with cold α %
	acetone. For fluorescence analysis, we used a Zeiss Axioplan 2 Imaging microscope.
	Real-time PCR
	Total RNA was extracted from cells at co-culture days 0, 1, 2, 4, and 7 by use of the Qiagen RNeasy Mini Kit (Qiagen), then
	reverse-transcribed with the Superscript First-Strand Synthesis System (Invitrogen) and Oligo(dT) . Quantitative RT-PCR reactions were 20
	performed in triplicate on a 7900 real-time PCR detection system (Applied Biosystems) with use of Platinium SYBR Green qPCR
	SuperMix (Invitrogen). PCR conditions were 50 C for 2 min, 95 C for 2 min, 45 cycles at 95 C for 15 s, and 60 C for 45 s, with GAPDH

°°°°u sed as the reference gene. Primer sequences are described in . Results are reported as mean SD. supporting information ' t P hybridization In situ FISH experiments were performed as previously described . Co-cultured cells were hybridized with human and mouse COT-1

[START_REF] Brown | Dynamic repositioning of genes in the nucleus of lymphocytes preparing for cell division[END_REF]

[START_REF] Oh | Cardiac progenitor cells from adult myocardium: homing, differentiation, and fusion after infarction[END_REF]

[START_REF] Oh | Cardiac progenitor cells from adult myocardium: homing, differentiation, and fusion after infarction[END_REF]

[START_REF] Oh | Cardiac progenitor cells from adult myocardium: homing, differentiation, and fusion after infarction[END_REF]

[START_REF] Oh | Cardiac progenitor cells from adult myocardium: homing, differentiation, and fusion after infarction[END_REF]

[START_REF] Oh | Cardiac progenitor cells from adult myocardium: homing, differentiation, and fusion after infarction[END_REF]

[START_REF] Oh | Cardiac progenitor cells from adult myocardium: homing, differentiation, and fusion after infarction[END_REF]

[START_REF] Oh | Cardiac progenitor cells from adult myocardium: homing, differentiation, and fusion after infarction[END_REF]

Proc Natl Acad Sci U S A . 2005 ; 102 : 3766 -3771

[START_REF] Oh | Cardiac progenitor cells from adult myocardium: homing, differentiation, and fusion after infarction[END_REF]

[START_REF] Oh | Cardiac progenitor cells from adult myocardium: homing, differentiation, and fusion after infarction[END_REF]

[START_REF] Oh | Cardiac progenitor cells from adult myocardium: homing, differentiation, and fusion after infarction[END_REF]

[START_REF] Oh | Cardiac progenitor cells from adult myocardium: homing, differentiation, and fusion after infarction[END_REF]

[START_REF] Oh | Cardiac progenitor cells from adult myocardium: homing, differentiation, and fusion after infarction[END_REF]

Acknowledgements:

We are grateful to M. Goossens, A. d Anglemont de Tassigny, T Piolot and X Decrouy for their helpful advice regarding FISH, ' cardiomyocyte dissociation, time-lapse videomicroscopy and confocal microscopy experiments, respectively. This work was financially supported by INSERM, the Association Fran aise contre les Myopathies and the Association pour la Recherche et l Etude des Maladies

Author contributions: A.A. conception and design, collection and assembly of data, data analysis and interpretation, manuscript writing; T.B. conception and design, collection and assembly of data, data analysis and interpretation, manuscript writing; P.F.L. collection and assembly of data, data analysis and interpretation; F.F. collection and assembly of data, data analysis and interpretation; A.E.C. collection and assembly of data, data analysis and interpretation; O.L.C. collection and assembly of data, data analysis and interpretation; C.C. collection and assembly of data; X.B. collection and assembly of data; F.A. provision of study material or patients; R.Y. conception and design, final approval of manuscript; J.L.D. financial support, conception and design, final approval of manuscript; A.M.R. conception and design, collection and assembly of data, data analysis and interpretation, manuscript writing.