Ildar Khalidov

Dimitri Van De Ville

Jalal Fadili

Michael Unser

Activelets and sparsity: A new way to detect brain activation from fMRI data

Keywords: fMRI, wavelets, exponential-spline wavelets, sparse approximations

FMRI time course processing is traditionally performed using linear regression followed by statistical hypothesis testing. While this analysis method is robust against noise, it relies strongly on the signal model. In this paper, we propose a non-parametric framework that is based on two main ideas. First, we introduce a problem-specific type of wavelet basis, for which we coin the term "activelets". The design of these wavelets is inspired by the form of the canonical hemodynamic response function. Second, we take advantage of sparsity-pursuing search techniques to find the most compact representation for the BOLD signal under investigation. The non-linear optimization allows to overcome the sensitivity-specificity trade-off that limits most standard techniques. Remarkably, the activelet framework does not require the knowledge of stimulus onset times; this property can be exploited to answer to new questions in neuroscience.

INTRODUCTION

Functional magnetic resonance imaging (fMRI) has become a predominant modality for imaging human brain function. The blood-oxygenation-level-dependent (BOLD) signal, which originates from an overcompensation in oxygenated hemoglobine following neuronal activity by the neurovascular system, can be measured and localized using fast acquisition of T2* MRI volumes. The analysis of these large spatio-temporal datasets is difficult due to noise, measurement artifacts, and the variability of the BOLD response.

The classical approach puts forward a linear model to explain a voxel's time course. The modeled BOLD response is the convolution of the hemodynamic response function (HRF) with the stimulus function, which can be a train of boxcar functions (block-based paradigm) or Dirac delta-functions (event-related paradigm). A widely used model for the HRF is the sum of two gamma-functions. Finally, voxels are declared as activated based on the results of statistical hypothesis testing. [START_REF] Frackowiak | Human Brain Function[END_REF] The variability of the BOLD response is particularly important in event-related fMRI (efMRI). It has been shown that (1) the inter-subject differences in BOLD response are stronger than spatial ones inside one subject;

(2) the intra-subject variability is stronger in space than in time; and (3) task-related responses have more coherent shapes than other activations, such as cardiac and respiratory ones. [START_REF] Menz | Variability of the BOLD response over time: An examination of within-session differences[END_REF] Several extensions have been proposed to the classical approach to deal with variations in BOLD response. Friston et al. include the orthonormalized versions of first partial derivatives of the HRF model with respect to parameters in the regression procedure. [START_REF] Friston | Event-related fMRI: Characterizing differential responses[END_REF] Adding more regressors reduces the number of degrees of freedom and requires looking for an optimal trade-off between sensitivity (ability to identify the active voxels) and specificity (ability to identify non-active voxels). Friman et al. use canonical correlation analysis to find basis functions having this optimal trade-off. [START_REF] Friman | Adaptive analysis of fMRI data[END_REF] To get ultimate adaptivity, one could build up the model for the HRF directly from the data. The FIR basis approach constructs the HRF as an FIR filter using statistical significance as an optimization criterion. Finally, we mention PCA and ICA methods that consider the problem as blind deconvolution of multiple sources. This strategy provides a data-dependent decomposition based on an information-theoretic criterion.

Further author information: (ildar.khalidov, dimitri.vandeville, michael.unser)@epfl.ch jalal.fadili@greyc.ensicaen.fr

In this paper, we propose a new dictionary of building blocks (activelets) that approximate well a class of gamma-function HRF models, instead of having a single HRF model. Recently, a family of exponential-spline wavelets has been introduced. [START_REF] Khalidov | From differential equations to the construction of new wavelet-like bases[END_REF] Their properties are essentially transpositions of polynomial-related properties of traditional wavelets to the exponential-polynomial (gamma functions) setting. This makes exponential-spline wavelets natural candidates that can be tuned for our purpose.

Another important ingredient of our approach is the usage of sparsity-driven algorithms to recover (in a data-dependent way) the underlying activations. To get the minimum-budget building atoms of the eventrelated activation response, our problem is formulated as finding the sparsest solution to an underdetermined linear system of equations, which is the solution with the smallest number of nonzero elements. Therefore, we would like to solve a sparsity-promoting optimization problem, where a quadratic data fidelity term is optimized under a sparsity-budget constraint as measured by the l 1 norm. Solving such problems is the area of an active research work.

We validated this approach as follows. An initial experiment consisted into approximating arbitrary shifts of a single gamma function. For the noiseless case, we found that the exponential-spline wavelet basis allowed a very good approximation with as few as 4 coefficients. Interestingly, traditional B-spline wavelets required more than 30 coefficients to obtain the same approximation quality. To further demonstrate the feasibility of our approach, we evaluated its performance using a stochastic signal simulator. Three synthetic activations were generated as HRFs with random onset and parameters (time-to-peak and decay) and corrupted with additive white noise. The sparsity algorithms nicely detected all the activations within the presence of noise, even at SNRs at 15dB. Note that the activations were detected without any prior knowledge on the stimulus function, as opposed to model-based approaches. In addition, our method appears to cope well with the variability of BOLD responses.

This paper is organized as follows. In Section 2, we use the gamma-function model for the fMRI time series to find an appropriate wavelet (activelet) basis that could be used for the analysis. The activelet dictionary is used to formulate the sparse optimisation problem in Section 3. We outline our algorithm in Section 4 and show the validation results in Section 5. Finally, in Section 6, we discuss current challenges and the potential usage of the activelet method in neuroscience.

ACTIVELETS REPRESENTATION

Consider a number of neuronal events at the time points t k , which can be modeled by a train of Dirac deltafunctions as

s(t) = N k=1 δ(t -t k ).
In efMRI, the timings t k are known by the stimulation pattern. If we further assume the canonical HRF model with the linear-shift-invariant assumption, we can then expect the induced BOLD signal x(t) to be

x(t) = s * h(t), (1)
where h(t) is the canonical HRF that is described by a difference of gamma functions:

h(t) = g(t; τ 1 , l 1 ) - 1 6 g(t; τ 2 , l 2 ), g(t; τ l ) = l τ t τ -1 + e -lt Γ(τ ) . ( 2
)
The parameters of the Gamma functions are chosen by specifying the delay to peak (τ /l) and the dispersion (τ /l 2 ). The standard parameters are: delay=6s, dispersion=1s for the initial peak, and delay=16s, dispersion=1s for the undershoot. [START_REF] Frackowiak | Human Brain Function[END_REF] Therefore, x(t) can be rewritten as

N k=1 h(t -t k ). The measured (noisy) data corresponds to y[k] = x(k) + n[k], (3)
where n[k] represents the additive gaussian noise.

In traditional fMRI analysis, the relevant question is whether the voxel is active or not with respect to the known stimulus function. This question is answered by measuring the strength of the signal (1)

in the data y[k].

A regression is fitted to the data; the resulting signal weight is subject to statistical testing for each voxel, using its corresponding t-value. In the case of efMRI, this method can be very sensitive to HRF variations: inaccurate estimation of the parameters τ 1 and τ 2 could lead to a false negative response. At the same time, τ 1 and τ 2 are fixed for the whole volume. In order to account for the variability of h(t), state-of-the-art data analysis tools, such as SPM, include the partial derivatives dx/dt and dx/dτ 1 in the regression. This makes the model more flexible; note, however, that each added regressor increases the risk of fitting non-stimuli related components in the signal, potentially giving way to false positive responses. Selection of regressors always boils down to setting a trade-off between sensitivity and specificity.

In this paper, we choose a different approach, which does not rely on the prior knowledge of the timings t k . We note that the gamma-functions in (2) are piecewise exponential polynomials. A convenient tool to compactly represent such signals is the exponential-spline wavelet (ESW) transform. [START_REF] Khalidov | From differential equations to the construction of new wavelet-like bases[END_REF] This transform is governed by a vector parameter α; similar to traditional wavelets, it provides a stable representation of

L 2 (R) = ⊕ ∞ i=-∞ W i in terms of orthogonal wavelet spaces W i = span{ψ i (t -2 i k)} k∈Z .
As compared to the traditional case, the wavelets become scale-dependent. Nevertheless, the ESW transform can still be performed by means of fast Mallat's filterbank algorithm with scale-dependent filters.

Qualitatively, the ESW transform shows the same properties regarding exponential polynomials as the traditional wavelet transform has for polynomial functions. In particular, for piecewise exponential polynomials, it has the energy-compaction property, leading to sparse representation in the ESW domain. All these favourable properties of exponential-spline wavelets make them a natural tool for fMRI time-course analysis.

One degree of freedom is the choice of the ESW parameter α. We have optimized α by minimizing the reconstruction error of the canonical HRF from K wavelet coefficients averaged over an interval of values for τ 1 and τ 2 . With the optimal choice of α = {-0.6, -0.6, -0.6, -0.6}, we are able to reconstruct the HRF from as little as K = 4 coefficients with an average SNR of 30dB. We coin the term activelets the corresponding HRFadapted exponential-spline wavelets. In comparison, to obtain the same quality using regular B-spline wavelets, which corresponds to setting α = {0, 0, 0, 0}, we need about 30 coefficients.

The activelet transform gives us a representation that concentrates the energy of an fMRI time course on few coefficients. In the next section, we describe a way detect BOLD activity in the noisy signal by means of a sparse optimization problem.

OPTIMIZATION PROBLEM

We observe the noisy measurements y[k] of the signal x(t) as in (3), and we would like to find the sparsest set of wavelet coefficients β 0 that fits y[k] best after reconstruction. Mathematically, we can state this as a constrained optimisation problem min

β β 0 subject to y -Φβ 2 ≤ , (4)
where Φ is the inverse ESW transform matrix, is a constant of the same order as the noise norm, and the 0 quasi-norm of a vector is the number of its non-zero components. Unfortunately, the functional to be optimized here is not convex, which makes the problem (4) NP-hard, demanding exorbitant computational efforts in general. This is why authors in 6 advocated the convexified 1 form:

min β β 1 subject to y -Φβ 2 ≤ , (5)
Problem ( 5) can be also stated in the augmented lagrangian form

β 0 = arg min β y -Φβ 2 2 + λ β 1 , ( 6
)
where λ is a regularisation parameter. The best choice of λ is determined by the actual noise level, which is estimated using e.g. the MAD. [START_REF] Chen | Atomic decomposition by basis pursuit[END_REF] Table 1. Mean and standard deviation of random parameters in the synthetic experiment.

Parameter Mean Standard deviation a 1 , a 2 , a 3 1 0.2 τ 1 6 1.5 τ 2 16 1.5

Note that the problems ( 6) and ( 4) are equivalent under an appropriate correspondence of parameters; that is, for a given 0 ≤ λ < +∞ there exists > 0 such that the two problems share the same solution, and vice versa. Formulation ( 6) is a (perturbed) linear programming problem known as BPDN. [START_REF] Chen | Atomic decomposition by basis pursuit[END_REF] It has been shown in [6, Theorem 3.1] that if the noiseless ideal signal x is sufficiently sparse with β 0 ≤ (µ -1 Φ + 1)/4 * , then β 0 is the unique maximally sparse representation of x, and stable recovery is possible by minimizing (5). As far as the solution to ( 6) is concerned, Tropp [8, Theorem 8 and Corollary 9] proved that if x is sufficiently sparse in Φ with β 0 < µ -1 Φ /2, and if the regularization parameter λ is chosen higher than the noise level, then the support of the solution to ( 6) is either contained or identical to the support of β 0 . In summary, if the signal has a sufficiently sparse representation, then, by minimizing either ( 5) or ( 6), we obtain a solution that (i) is the unique sparsest solution, (ii) is recovered with the appropriate sparsity pattern, and (iii) is stable to noise.

The optimisation in ( 6) can be cast as a (perturbed) linear program, and solved using general purpose solvers such as simplex and interior point methods which are slow for large-scale problems. There has also been interest in the statistical community in fitting regression models while imposing 1 -norm constraints on the regression coefficients. In, [START_REF] Tibshirani | Regression shrinkage and selection via the Lasso[END_REF][START_REF] Efron | Least angle regression[END_REF][START_REF] Osborne | A new approach to variable selection in least squares problems[END_REF] the Lasso, LARS and Homotopy methods were proposed to solve (6) in the overdetermined case for all λ ∈ [0, Φ T y ∞ ]. In this paper, we propose an approximate path-following algorithm which identifies groups of atoms at each iteration using a stagewise iterative-thresholding (IT) variant of LARS, where the sequence {λ k } k≥0 is not data-adapted, but allowed to be strictly decreasing. [START_REF] Fadili | Sparse representation-based image deconvolution by iterative thresholding[END_REF] It can be shown that the computational complexity of IT-LARS is O (KV ). Here, K is the number of non-zero coefficients in the solution, and V is the cost of the ESW decomposition.

We now have all the tools needed for the activelet method, which is outlined in the next section.

THE ACTIVELET APPROACH

The algorithm takes the fMRI time course y[k] as input, which is fed into the sparse-solution search algorithm that uses the activelet decomposition routine to perform fast dictionary search. We use the undecimated wavelet (activelet) transform in order to gain shift-invariance. In addition, large-scale B-splines are incorporated in the dictionary to fit the fMRI baseline signal.

In case when the the voxel's time course encodes brain activity, the activelet part of the representation becomes non-zero but still remains sparse. The full reconstruction contains BOLD signal both due to activation and baseline. Contrarily, for a pure noisy voxel, coefficients of the activelet decomposition are kept to zero by the sparsity-pursuing algorithm and the fit reduces essentially to the baseline component.

SYNTHETIC EXPERIMENT

As a proof of concept, we have validated our approach on a synthetic dataset with 3 stimuli occurring at nonregularly spaced time moments. The variability is of HRF has been modelled by randomizing the amplitudes a 1 , a 2 , a 3 and the canonical HRF parameters τ 1 , τ 2 of the 3 responses. The means and standard deviations of these variables in our experiment are shown in Table 1. The data was corrupted by gaussian white noise with standard deviation σ = 0.15.

In Figure 1, we show a typical activelet fit to a noisy dataset. We observe good visual quality of the reconstructed signal and a significant gain (+6 dB on average) in the SNR, despite the strong noise level in the original data. Importantly, as seen in Figure 2, a simulated pure-noise time course does not have a sparse activelet representation, and the algorithm detects no activations. We repeat the same experiment using traditional Bspline wavelets (cf. Fig. 3 and Fig. 4). important degradation of results stresses the importance of a properly designed, problem-adapted wavelet basis. In particular, sparse optimisation with traditional wavelets cannot distinguish pure noise from the activity-related signal.

CONCLUSION AND OUTLOOK

We have presented a novel approach for detecting activity in fMRI. The fMRI time course is decomposed in the newly designed activelet basis that concentrates activity-related energy on few wavelet coefficients; these coefficients are then identified by means of a sparse search algorithm.

The activelet representation and the sparse search algorithm are the two main ingredients for our method. From the sensitivity-specificity viewpoint, activelets alone are nothing but a very flexible model; used with a least-squares fit, they would "detect" the whole signal. At the same time, we have shown that sparse optimisation with traditional wavelets can not fit the signal properly.

Undecimated wavelet transform has a disadvantage of having highly correlated elements in the dictionary. This has a double negative impact on the algorithm: on one hand, the computation time amounts up to 5 sec/voxel; on the other hand, the noise could eventually well fitted by a linear combination of correlated elements. A potential improvement to the algorithm would consist in taking advantage of the knowledge of the dictionary structure and avoiding selection of correlated elements. Interestingly, unlike traditional fMRI time-course processing methods, our algorithm does not use the knowledge of the stimulus times t k . This opens a new horizon to the usage of fMRI in neuroscientific applications, such as mental chronometry, where t k could be estimated from the time course itself.

Figure 1 .

 1 Figure 1. Activelet fit to a noisy fMRI time-course. Thin line: original signal. Dashed line: noisy measurements. Thick line: reconstruction from sparse activelet representation.

Figure 2 .

 2 Figure 2. Activelet fit (thick line) to pure noisy data (dashed line). No activations are detected.

Figure 3 .

 3 Figure 3. Wavelet fit to a noisy fMRI time-course. Thin line: original signal. Dashed line: noisy measurements. Thick line: reconstruction from sparse wavelet representation.

Figure 4 .

 4 Figure 4. B-spline wavelet fit (thick line) to pure noisy data (dashed line). Noise is partially fitted by the traditional wavelets.

ACKNOWLEDGMENTS

This work was supported by the Center for Biomedical Imaging (CIBM) of the Geneva -Lausanne Universities and the EPFL, the foundations Leenaards and Louis-Jeantet, as well as by the Swiss National Science Foundation under grant 200020-109415.


Icia Santos-Zas

Elisa Negroni

Kamel Mamchaoui

Carlos S Mosteiro

Rosalia Gallego

Gillian S Butler-Browne

Yolanda Pazos

PhD. Vincent Mouly
email: vincent.mouly@upmc.fr

PhD. Jesus P Camiña

Obestatin increases the regenerative capacity of human myoblasts transplanted intra-muscularly in an immunodeficient mouse model: proliferation, migration and differentiation

Although cell-based therapy is considered as promising method aiming at treating different muscular disorders, little clinical benefit has been reported. One of major hurdles limiting the efficiency of myoblast transfer therapy is the poor survival of the transplanted cells. Any intervention upon the donor cells focused at enhancing in vivo survival, proliferation and expansion is essential to improve the effectiveness of such therapies in regenerative medicine.

In the present work, we investigated the potential role of obestatin, an autocrine peptide factor regulating skeletal muscle growth and repair, to improve the outcome of myoblast-based therapy by xenotransplanting primary human myoblasts into immunodeficient mice. The data proved that short in vivo obestatin treatment of primary human myoblasts not only enhances the efficiency of engraftment but also facilitates an even distribution of myoblasts in the host muscle. Moreover, this treatment leads to a hypertrophic response of the human-derived regenerating myofibers. Taken together, the activation of the obestatin/GPR39 pathway resulted in an overall improvement of the efficacy of cell engraftment within host's skeletal muscle. These data suggest considerable potential for future therapeutic applications and highlight the importance of combinatorial therapies.

INTRODUCTION

Cell-based therapy is considered to be one of the innovative methods aiming at treating different muscular disorders [START_REF] Negroni | Cellular Therapies for Muscular Dystrophies: Frustrations and Clinical Successes[END_REF][START_REF] Guiraud | The Pathogenesis and Therapy of Muscular Dystrophies[END_REF]. Cell therapy is based on the delivery of precursor cells, autologous or heterologous, that will contribute to the regeneration of muscle fibers and tissue repair. This type of restorative therapy has the potential to establish genetically corrected myofibers and reverse the pathological phenotype. The fundamental requirements for the ideal cell candidate are related their myogenic potential, their homing capacities to sites of degeneration, and their motility in the target tissue. Furthermore, their isolation and amplification must be transferable under clinical conditions, preferably in an autologous context. Several cell therapy strategies have been tested using different types of cells with a myogenic potential and derived from muscle (satellite cells/myoblasts or muscle derived stem cells), vessels (pericytes and their progeny mesoangioblasts), bone marrow, blood, or embryonic tissues, including, recently, induced pluripotent stem cells (reviewed in [START_REF] Negroni | Cellular Therapies for Muscular Dystrophies: Frustrations and Clinical Successes[END_REF][START_REF] Maffioletti | Stem cell transplantation for muscular dystrophy: the challenge of immune response[END_REF][START_REF] Benedetti | Repair or replace? Exploiting novel gene and cell therapy strategies for muscular dystrophies[END_REF]. However, the data obtained from these studies resulted in suboptimal, although promising, effectiveness in restoring functional skeletal muscle tissue. First, the availability of an adequate number of stem cells to transplant constitutes an important challenge. This includes issues related to the harvesting from donors or from the same patient, genetic correction in case of autologous transplant, large-scale amplification in clinical conditions, maintenance of a myogenic potential prior to transplantation, and their compatibility with the host immune system. Once implanted, the lack of efficiency and survival of the transplanted cells remains a major hurdle for clinical application. Moreover, for diseases targeting a large number of muscles, their complexity and distribution throughout the body adds to the difficulties of delivering cells to muscles with restricted accessibility [START_REF] Negroni | Cellular Therapies for Muscular Dystrophies: Frustrations and Clinical Successes[END_REF][START_REF] Guiraud | The Pathogenesis and Therapy of Muscular Dystrophies[END_REF][START_REF] Maffioletti | Stem cell transplantation for muscular dystrophy: the challenge of immune response[END_REF][START_REF] Bareja | Satellite cell therapy-from mice to men[END_REF][START_REF] Bentzinger | The emerging biology of muscle stem cells: implications for cell-based therapies[END_REF].

Myoblasts were the first candidate cell to be tested for cell therapy. Myoblasts are wellcharacterized physiological progenitors, for which isolation and expansion is feasible in vitro.

Several studies have been successfully carried out in mice, showing that injected myoblasts are able to participate to muscle regeneration [START_REF] Watt | Incorporation of donor muscle precursor cells into an area of muscle regeneration in the host mouse[END_REF] and restore the missing protein dystrophin in the mdx mouse, a widely used animal model of Duchenne muscular dystrophy (DMD) [START_REF] Partridge | Conversion of mdx myofibres from dystrophin-negative to -positive by injection of normal myoblasts[END_REF][START_REF] Morgan | Normal myogenic cells from newborn mice restore normal histology to degenerating muscles of the mdx mouse[END_REF][START_REF] Kinoshita | Utilization of myoblasts from transgenic mice to evaluate the efficacy of myoblast transplantation[END_REF][START_REF] Huard | Utilization of an antibody specific for human dystrophin to follow myoblast transplantation in nude mice[END_REF][START_REF] Huard | High efficiency of muscle regeneration after human myoblast clone transplantation in SCID mice[END_REF][START_REF] Asselin | Quantification of normal dystrophin mRNA following myoblast transplantation in mdx mice[END_REF].

However, the results of the first clinical trials in DMD patients were rather disappointing [START_REF] Huard | Myoblast transplantation produced dystrophin-positive muscle fibres in a 16-year-old patient with Duchenne muscular dystrophy[END_REF][START_REF] Gussoni | Normal dystrophin transcripts detected in Duchenne muscular dystrophy patients after myoblast transplantation[END_REF][START_REF] Tremblay | Results of a triple blind clinical study of myoblast transplantations without immunosuppressive treatment in young boys with Duchenne muscular dystrophy[END_REF]. Among the main reasons, the poor survival of cells, their lack of dispersion from the injection site and immunological rejection by the host have been proposed as potentially hampering any clinical benefit. Several studies have indicated that the vast majority of injected murine myoblasts die during the first hours following transplantation [START_REF] Beauchamp | Dynamics of myoblast transplantation reveal a discrete minority of precursors with stem cell-like properties as the myogenic source[END_REF][START_REF] Skuk | Resetting the problem of cell death following muscle-derived cell transplantation: detection, dynamics and mechanisms[END_REF]. The reasons for this early death are poorly understood and may be due to apoptosis, necrosis, anoıkis or nonspecific immune destruction. Furthermore, the lack of cell survival was also observed in immunodeficient or immunosuppressed animals, illustrating that it is independent of any event related to the host's adaptive immune response [START_REF] Guérette | Prevention by anti-LFA-1 of acute myoblast death following transplantation[END_REF]. It was demonstrated that muscle damaging or irradiation improved the migration capacities of myoblasts [START_REF] Morgan | Normal myogenic cells from newborn mice restore normal histology to degenerating muscles of the mdx mouse[END_REF][START_REF] Morgan | Long-term persistence and migration of myogenic cells injected into pre-irradiated muscles of mdx mice[END_REF], but such protocol is not clinically applicable. In fact, grafted myoblasts do migrate, but in a manner most closely associated with their physiological function to support myofiber regeneration rather than in one ideally suited to facilitate their transplantation as a potential therapy [START_REF] Skuk | Transplanted myoblasts can migrate several millimeters to fuse with damaged myofibers in nonhuman primate skeletal muscle[END_REF][START_REF] Skuk | Electroporation as a method to induce myofiber regeneration and increase the engraftment of myogenic cells in skeletal muscles of primates[END_REF]. It is well known that myoblasts migrate in response to signals deriving both from damaged fibers and infiltrating cells [START_REF] Goetsch | In vitro myoblast motility models: investigating migration dynamics for the study of skeletal muscle repair[END_REF]. In clinical settings, cell repartition was improved by innovative systems of injection [START_REF] Skuk | Dystrophin expression in muscles of duchenne muscular dystrophy patients after high-density injections of normal myogenic cells[END_REF][START_REF] Skuk | First test of a "high-density injection" protocol for myogenic cell transplantation throughout large volumes of muscles in a Duchenne muscular dystrophy patient: eighteen monthsfollow-up[END_REF]. This protocol remains applicable to small and accessible muscles (e.g. thenar eminence, extensor digitorum or even biceps brachii) in DMD patients, or in the context of autologous cell transplantation for localized tissue repair in less extended diseases such as oculopharyngeal muscular dystrophy [START_REF] Périé | Autologous myoblast transplantation for oculopharyngeal muscular dystrophy: a phase I/IIa clinical study[END_REF].

Several research groups have developed studies to improve the comprehension of the biology of these satellite cells such as their heterogeneity or their niche [START_REF] Yin | Satellite Cells and the Muscle Stem Cell Niche[END_REF]. Recent studies involving purification of satellite cells or sub-populations and implantation without any in vitro amplification [START_REF] Cerletti | Highly efficient, functional engraftment of skeletal muscle stem cells in dystrophic muscles[END_REF][START_REF] Sacco | Self-renewal and expansion of single transplanted muscle stem cells[END_REF][START_REF] Tanaka | Syndecan-4expressing muscle progenitor cells in the SP engraft as satellite cells during muscle regeneration[END_REF] or implantation of one single intact myofiber [START_REF] Collins | Stem cell function, self-renewal, and behavioral heterogeneity of cells from the adult muscle satellite cell niche[END_REF] in mice have shown that cells nearer to satellite cell state or even still within their niche can robustly contribute to muscle repair. Particularly, the implantation of single fibers was by far the most efficient procedure in terms of efficiency of participation in host's regeneration. In fact transplantation in dystrophic mouse muscles of a single muscle fiber that contained as few as seven satellite cells led to an increasing number of new satellite cells that in turn generated more than 100 new muscle fibers [START_REF] Collins | Stem cell function, self-renewal, and behavioral heterogeneity of cells from the adult muscle satellite cell niche[END_REF]. Although reasons for this increased efficiency are not yet fully understood, this is a much more efficient way to generate new muscle fibers than transplantation of cultured satellite cells, in which normally the number of donor-derived new fibers that are generated is several orders of magnitude less than the number of injected cells. These results open new avenues for research on cell transplantation, based on comparisons between cultured and uncultured cells. Despite the promising value of this approach, this is not yet applicable in clinical situations, where a massive amplification of donor cell is still required, particularly in autologous trials.

In spite of progress made, significant gaps remain in the knowledge of areas such as survival, proliferation and migration of transplanted human myoblasts. The development of xenotransplantation strategies in immunodeficient preclinical mouse models allowed to analyze human myoblast behavior and the regulation of key biological events occurring in the host muscle to design rational strategies to improve grafting. Among them, recent studies demonstrated that a significant portion of grafted human myoblasts survives and engrafts host muscle, and although a cell death peak is noted, it seems far less than for murine myoblasts [START_REF] Riederer | Slowing down differentiation of engrafted human myoblasts into immunodeficient mice correlates with increased proliferation and migration[END_REF]. Interestingly, in situ proliferation of human myoblasts was observed in the first days posttransplantation that was able to compensate for the loss of cells occurring after injection. Furthermore, the migration of grafted human myoblasts was limited to the same time window after transplantation, after which proliferation is downregulated and grafted myoblasts start to differentiate in situ, suggesting that proliferation, migration, and differentiation are tightly linked and that modifying one of these three processes has an impact on the others. Thus, insights into the molecular and cellular mechanisms regulating myoblast activation, proliferation and/or differentiation should allow to optimize conditions to improve the regenerative potential of amplified myoblasts. p38 was recently identified as a determining switch to promote differentiation of satellite cells [START_REF] Charville | Ex Vivo Expansion and In Vivo Self-Renewal of Human Muscle Stem Cells[END_REF]. Thus, reversible pharmacological inhibition of p38 in cultured human satellite cells resulted in a gene expression program consistent with the promotion of human satellite cells self-renewal, and it allowed for expansion of a population ex vivo with enhanced self-renewal and engraftment potential. Recently, it was also proposed that compounds stimulating the expansion of the satellite cell pool, such as recombinant Wnt7a, improve motility and engraftment leading to significantly improved muscle function [START_REF] Bentzinger | Wnt7a stimulates myogenic stem cell motility and engraftment resulting in improved muscle strength[END_REF]. These results highlight the need to develop strategies to reduce early cell death and promote proliferation so as to limit the number of cells required for successful engraftment.

Autocrine factors that can regulate the activation and proper differentiation of myoblasts would be particularly useful to improve the outcome of myoblast-based therapies. In this context, we have recently reported that obestatin, a 23-amino acid peptide derived from a polypeptide called preproghrelin, is involved in muscle regeneration, exerting an autocrine function to control the myogenic differentiation program [START_REF] Gurriarán-Rodríguez | The obestatin/GPR39 system is up-regulated by muscle injury and functions as an autocrine regenerative system[END_REF]. Obestatin regulates multiple steps of myogenesis: myoblast proliferation, cell cycle exit, differentiation and recruitment to fuse and form multinucleated hypertrophic myotubes [START_REF] Gurriarán-Rodríguez | Action of obestatin in skeletal muscle repair: stem cell expansion, muscle growth, and microenvironment remodeling[END_REF]. This action is coordinated by the interplay between G-protein-dependent and ß-arrestin-dependent mechanisms [START_REF] Santos-Zas | β-Arrestin scaffolds and signaling elements essential for the obestatin/GPR39 system that determine the myogenic program in human myoblast cells[END_REF]. The obestatin-associated mitogenic action is determined by G-protein-dependent activation defining the intricate pathways related to the ERK1/2 and JunD axis; and, the transactivation of EGFR through the βarrestin signal complex determines the cell cycle exit, the development and the progression of differentiation through a kinase hierarchy determined by the Akt, CAMKII, c-Jun and p38 axes.

Added to the functional features, the chemical/biochemical properties and the existence of an easily available 23-amino acid peptide with high dispersal profile in skeletal muscle [START_REF] Gurriarán-Rodríguez | Action of obestatin in skeletal muscle repair: stem cell expansion, muscle growth, and microenvironment remodeling[END_REF], undoubtedly facilitate its use as a therapeutic agent.

In this work, we investigated the potential of obestatin to improve the outcome of myoblastbased therapy by xenotransplanting primary human myoblasts into immunodeficient mice. Our results showed that a short in vivo treatment of human myoblasts with obestatin not only enhances the efficiency of engraftment but also facilitates an even distribution of myoblasts in the host muscle. Moreover, this treatment leads to a hypertrophic response of the humanderived regenerating myofibers. Our observations suggest obestatin as a pharmacological mean to improve the outcome of myoblast-based therapy, which might be beneficial for future regenerative therapies of skeletal muscle.

RESULTS

Functional validation of the obestatin/GPR39 system in human primary myoblasts. The expression of the obestatin/GPR39 system was assessed in human primary myoblasts during myogenesis. Upon reaching confluence, serum and additives were withdrawn to induce the differentiation from proliferative myoblasts into terminally differentiated myotubes. As shown in Figure S1a, obestatin is not expressed in primary myoblasts but is upregulated in differentiated myotubes (6-day differentiation period). However, GPR39 was detected in both myoblasts and myotubes (Fig. S1a). In human primary myoblasts, obestatin (10 nM, 5 min) activated MAPKs [ERK1/2, c-Jun/JunD, and p38], Akt, and CAMKII signaling pathways (Fig. S1b). These kinase hierarchies are decoded by the myogenic effectors that execute different phases of the differentiation process in response to the obestatin/GPR39 system [START_REF] Santos-Zas | β-Arrestin scaffolds and signaling elements essential for the obestatin/GPR39 system that determine the myogenic program in human myoblast cells[END_REF]. In the context of obestatin-induced activity, obestatin increased mitogenic event in a dose-effect relationship with maximal effect at 10 nM obestatin in GM (proliferating conditions; Fig. S1c). To assess the activity of obestatin as a promoter of the differentiation process, primary myoblasts were switched to DM supplemented with obestatin at 10 nM for 6 days. Immunoblot analyses revealed enhanced protein level of MHC when compared with control cells (DM for 6 days; Fig. S1d). The myotube areas were larger in the obestatin-treated cells (DM + 10 nM obestatin; 3401 ± 33 µm 2 ) as compared to control conditions (DM; 168 ± 36 µm 2 ) at 6 days after differentiation. To quantify myoblast fusion, we calculated the fusion index by expressing the number of nuclei within MHC + myotubes with ≥3 nuclei as a percentage of the total nuclei. The fusion index was significantly higher in the obestatin-treated cells compared with untreated control cells (47.0 ± 0.8 and 33.4 ± 1.2%, respectively; Fig. S1c).

Nuclear number analysis revealed that the percentage of myotubes with >30 nuclei was significantly higher in obestatin-treated cells compared with control cells (Fig. S1c). Thus, obestatin regulated multiple steps of myogenesis, including myoblast proliferation, differentiation and recruitment to fuse and form multinucleated hypertrophic myotubes.

Mitogenic effect in vivo of obestatin in regenerating muscle.

To test the obestatin effect on human cells within the murine recipient muscle, we transplanted 5×10 5 myoblasts cells into freeze damaged TA muscle followed by obestatin delivered via intramuscular injection every 24 hours up to 5 consecutive days (Fig. 1a). The expression of ki67, (a protein expressed in all cell-cycle phases except G 0 ), was analyzed on cross sections of TA muscles grafted with human cells, in parallel with the detection of human Lamin A/C. At 24 hours posttransplantation, the number of human Ki67 + cells (Lamin A/C + cells) in grafted TAs was not significantly different whether they had been treated with obestatin or not (17.6 ± 3.5 and 19.3 ± 2.9 ki67 + cells, respectively; Fig. 1b). 5 days post-transplantation there was a 38% decline in the number of human Ki67 + cells in the untreated animals. By contrast, the analysis of Ki67 expression in obestatin-treated mice showed an 80% increase in the number of human Ki67 + cells compared untreated recipients (21.4±2.2 and 11.9±0.7 ki67 + cells, respectively; Fig. 1b), thus maintaining the levels of proliferating cells observed at 24 hours post-transplantation. An analysis of phospho-histone-3 (S10) (pH3) expression, a marker of the late G2-and M-phase of the cell cycle, demonstrated an 114% increase in pH3 + human cells in the obestatin-treated group as compared to the control group at 24 hours post-transplantation (10.4 ± 1.0 and 4.9 ± 0.9 pH3 + cells, respectively; Fig. 1c). Despite the decline in the relative numbers of pH3 + cells at 5 days post-transplantation, the number of human pH3 + cells was double in the obestatintreated group than in the control group (5.6 ± 1.1 and 2.8 ± 0.6 pH3 + cells, respectively; Fig. 1c). Calculation of the apoptotic cells from TUNEL labeling showed that obestatin treatment significantly attenuated the number of apoptotic nuclei at 5 days of transplantation (15.0 ± 2.4 and 1.0 ± 0.4 cells, respectively; Fig. 1d). Furthermore, the expression of cleaved caspase-3 (activated caspase-3) decreased in human cells in the obestatin-treated group as compared to the control group at 5 days post-transplantation (Fig. 1e). Thus, the increased expression of Ki67 and pH3 proteins, together with reduced rates in apoptosis, supports that the proliferation of muscle precursors is stimulated by the obestatin treatment decreasing, at least in part, the loss of injected cells.

Obestatin treatment increases cell migration in vitro and in vivo.

We next investigated whether the activation of obestatin/GPR39 system could modulate human cell migration in vivo, while colonizing the recipient´s tissue. In order to do this, we measured the area occupied by the human cells on transverse sections along the entire length of the muscle at the different time points described above. At 24 hours post-transplantation, the area occupied by the human cells did not show significant differences between control and obestatin-treated muscles (422.5 ± 38.9 and 369.8 ± 124.0 µm 2 , respectively; Fig. 2a). At 5 days posttransplantation, the area occupied by human cells within regenerating muscle treated with obestatin was increased by 132% as compared to untreated muscle (1245 ± 239.6 and 536.3 ± 110.2 µm 2 , respectively; Fig. 2a). Importantly, 21 days post-transplantation (i.e.16 days after the last obestatin administration), the area occupied by human cells in obestatin-treated muscles was 59% greater compared to untreated group (1833 ± 241.2 and 1146 ± 100.7 µm 2 , respectively; Fig. 2a). Similar results were obtained when relative areas, i.e. total area/human cell number, or the cell number per unit area were compared (Fig. 2a). These results ruled out the possibility that the increased area might be related to the proliferation response.

The effect of obestatin on cell motility was further tested in scratch assays. To exclude a bias arising from different rates of proliferation, we treated the human primary cells with mitomycin-C (15 µg/mL; 2-hours), a DNA replication inhibitor. Addition of obestatin (10 nM) to the culture medium increased the migration of human cells by ∼28.7 and ∼44.6% when compared to untreated control at 24-and 48-hours, respectively (Fig. 2b). To identify potential effectors of obestatin/GPR39 signaling, we investigated the requirement of Disheveled 2 protein (Dvl2).

Despite primary cells provided a valuable complement to in vivo experiments, their in vitro

culture requirements limited the efficacy of siRNA transfection [siRNA-mediated knockdown of Dvl2 (si-Dvl2); 8.7 ± 2% reduction relative to scrambled siRNA (si-control)]. To achieve maximum efficacy, siRNA experiments were carried out in the hTERT/cdk4 immortalized human myoblasts (KM155C25 clone 48; see Material and Methods for details), a reliable and stable immortalized cell line derived from primary cells that retain the characteristics of non-senescent primary line, i.e. in vitro and in vivo differentiation, with extended proliferative lifespan [START_REF] Mamchaoui | Immortalized pathological human Obestatin stimulates human myoblast engraftment-Revised version 1: MTJ-17-1113 30 myoblasts, towards a universal tool for the study of neuromuscular disorders[END_REF].

siRNA-mediated knockdown of Dvl2 [60.4 ± 0.5% reduction relative to scrambled si-control] significantly prevented obestatin-mediated migration in scratch assays when compared with the si-control (Fig. 2c). The small GTPases, Rac1 and Rho, have also been proposed to be involved in obestatin-mediated cell migration. Indeed, obestatin significantly increased the activation of Rho (160% relative to si-control; Fig. 2d) while it had no significant effect on active Rac1 (Fig. 2D). si-Dvl2 significantly reduced by 73% the activation of Rho in obestatin-stimulated cells when compared with the si-control (Fig. 2d). We conclude that obestatin/GPR39 increase the migration of human cells in the host muscle through activation of Dvl2 and the small GTPase Rho.

Obestatin promotes differentiation of muscle precursors in vivo.

We analyzed the expression of myogenin, a key myogenic regulatory factor, which is also a marker of early skeletal muscle cell differentiation [START_REF] Bigot | Replicative aging down-regulates the myogenic regulatory factors in human myoblasts[END_REF] as well as of MHC, a marker of terminal differentiation [START_REF] Ph | Regulation of myogenic differentiation in the developing limb bud[END_REF]. As determined by immunostaining for myogenin on muscle sections, obestatin treatment tends to increase the number of myogenin + cells although this is not significant when compared to controls at 5 days post-transplantation (20.4 ± 1.0 and 15.9 ± 2.9 % myogenin + cells, respectively; Fig. 3a). When the expression of MHC was evaluated in the injected human muscle cells at the same time point, the treated-to-control ratios showed a 33% decrease in MHC + cells (47.0 ± 2.0 and 70.0 ± 1.0 % MHC + cells, respectively; Fig. 3b). However, the cross-section areas of fibers with centrally located human Lamin A/C + nuclei (i.e. regenerating fibers) were 289.8% larger in obestatin-treated mice (752.0 ± 63.0 µm 2 ) than in control mice (193.0 ± 14.0 µm 2 ) at 5 days post-transplantation (Fig. 3b). Furthermore, the number of human myonuclei per regenerating myofiber was significantly increased by 105% in the obestatintreated group (2.1 ± 0.07 cells) when compared with the control muscles (1.0 ± 0.01 cells; Fig. 3b). Thus, myogenin and MHC expression excluded rapid differentiation of the transplanted human cells and correlates with activation of the proliferating stage. Despite the fact that there is fewer fibers with human nuclei, obestatin treatment was accompanied by an increase in fiber hypertrophy and in myonuclear content.

Obestatin increase the regenerative capacity of human myoblasts in vivo.

To assess the overall efficiency of in vivo engraftment of human cells in a regenerated muscle in the presence of obestatin/GPR39 activation, TA muscles were immunostained for human Lamin A/C and mouse laminin at 21 days post-transplantation. The number of myofibers containing human nuclei was increased by 112% in obestatin-treated compared to control muscles (193.7 ± 19.2 and 91.0 ± 20.0, respectively; Fig. 4a). Furthermore, the total number of human cells engrafted into mouse myofibers was 135% greater in the obestatin-treated muscles than in the control group (386.7 ± 52.5 and 164.3 ± 46.6 human grafted-cells, respectively; Fig. 4a). The participation of the injected cells in muscle regeneration was further quantified by counting the number of human spectrin + fibers per muscle section at 21 days post-transplantation. As shown in Figure 4B, obestatin increased by 1.8-fold the number of human spectrin + fibers. Related to the total number of fibers per muscle section, 15 ± 3 % spectrin + fibers were observed in obestatin-treated TA versus 8 ± 1 % in the control group. The analysis of cross-sectional areas showed that the treated-to-control ratio of all fibers (spectrin + and spectrin -fibers) was 16% larger in obestatin-treated mice (1605.0 ± 46.0 µm 2 ) than in control mice (1389.0 ± 40.0 µm 2 ) at 21 days post-transplantation, respectively (Fig. 4b). The cross-sectional areas of spectrin + fibers were 98% larger in obestatin-treated mice (2629.8 ± 172.0 µm 2 ) than in control mice (1327.9 ± 92.0 µm 2 ) with a shift of the muscle fiber size distribution towards major caliber fibers (Fig. 4c).

However, cross-sectional areas of spectrin -fibers were similar in obestatin-treated mice (1440.9 ± 49.0 µm 2 ) compared to control mice (1397.9 ± 49.0 µm 2 ). Overall, these data demonstrate that obestatin globally enhanced the regenerative capacity of the injected cells, as shown by the increase in both the number of myofibers containing human nuclei and/or expressing human protein and the significantly increased size of the human-derived regenerated myofibers.

Once the differentiation process is triggered, myoblasts have a dual fate: while most commit to myogenic differentiation, there is a population that withdraws from the cell cycle and reexpresses markers of quiescent satellite cells, including Pax7. We investigated whether the obestatin/GPR39 system may also regulate this dual fate. To identify progenitor cells of donor origin, muscle sections were stained with antibodies against human Lamin A/C and Pax7 (Fig. 5). In representative sections, the number of Pax7 + cells per 100 fibers, which include cells of both human and mouse origin (total Pax7 + cells), was 50 ± 3 in control muscles, and the number of human Pax7 + /Lamin A/C + cells per 100 fibers was 8 ± 1, which accounts for 16 ± 2% of total Pax7 + cells in the absence of treatment. By contrast, the numbers of total Pax7 + and Pax7 + / Lamin A/C + cells per 100 fibers were 107 ± 5 and 69 ± 8 in obestatin-treated muscles, respectively. This accounts for 65 ± 10% of total Pax7 + cells. The numbers of Pax7 + / Lamin A/C -cells per 100 fibers (i.e. host satellite cells) were 42 ± 3 and 38 ± 11 in control and obestatin-treated muscles, respectively. To further determine whether donor-derived Pax7 + cells were in the satellite cell position, triple labeling of human Lamin A/C, Pax7, and laminin was performed. Donor-derived Pax7 + cells were located both in the satellite cell position (underneath the basal lamina of myofibers; Fig. 5) and outside the basal lamina. There were 1.0 ± 0.3 in control group and 14.8 ± 1.2 in obestatin group human Pax7 + / Lamin A/C + cells per 100 fibers located inside the basal lamina. These data indicate that 21.4 ± 1.7 % of Pax7 + donor cells are in the position of satellite cells, the remainder being undifferentiated interstitial myoblasts located outside the basal lamina. We further quantified the number of human cells that had not been incorporated into the muscle fibers but remained located in an interstitial position outside the basal lamina of the fibers This number was significantly larger for control compared to obestatin-treated muscles (39 ± 3% and 18 ± 4% relative to total number of human cells, respectively).

Discussion

Although myoblast transfer therapy has appeared as promising from the results obtained in murine models, the results of clinical trials for DMD patients to date are disappointing, since little clinical benefit have been reported [START_REF] Negroni | Cellular Therapies for Muscular Dystrophies: Frustrations and Clinical Successes[END_REF][START_REF] Skuk | Cell therapy in muscular dystrophies: many promises in mice and dogs, few facts in patients[END_REF]. The poor survival of the transplanted cells remains a major hurdle limiting its efficiency. Previous results have also shown that a reduced proliferation of the transplanted human myoblasts is correlated with their early differentiation within the host tissue [START_REF] Riederer | Slowing down differentiation of engrafted human myoblasts into immunodeficient mice correlates with increased proliferation and migration[END_REF]. An analysis of the proliferation of human myoblast cells transplanted into regenerating muscles of immunodeficient mice using Ki67 or pH3 as markers of dividing cells, showed a limited in vivo amplification of the injected cells after engraftment. This reduced proliferation and early differentiation within the host tissue, was correlated to a limited migration of the transplanted human myoblasts within the host tissue. Any intervention upon the donor cells and/or the recipient's microenvironment focused at enhancing in vivo survival, proliferation and expansion before differentiation would be essential to improve the effectiveness of cell-based therapies in regenerative medicine. Obestatin, an autocrine factor regulating the function of myoblast pool and improving the ultimate efficiency of the engraftment, fulfill some of these requirements. Our results demonstrate that: 1) stimulation of proliferation of human myoblasts through the activation of the obestatin/GPR39 pathway delays myogenic differentiation, thus increasing the number of cells which can then colonize a much larger area within the recipient's muscle; 2) obestatin treatment results in increased cell migration through activation of Dvl2 and the small GTPase Rho; 3) obestatin-stimulated myoblasts maintain their ability to differentiate, since a higher number of myofibers containing human nuclei and/or expressing human proteins is found. Moreover, obestatin treatment leads to a hypertrophic response in the human-derived regenerating myofibers; and, 4) obestatin signaling enhances the capacity to reconstitute a satellite cell pool from transplanted cells, advocating their potential for long-term treatment of muscle diseases. Combined together, these effects of the activation of the obestatin/GPR39 pathway result in an overall improvement of the efficacy of cell engraftment within host's skeletal muscle.

It was already shown that transplantation of these myogenic precursors in the presence of serum, which maintain the proliferative state of myoblasts in vitro, improved the outcome of myoblast transplantation [START_REF] Riederer | Slowing down differentiation of engrafted human myoblasts into immunodeficient mice correlates with increased proliferation and migration[END_REF]. In agreement with these observations, we show that an exogenous induction of myoblast proliferation via obestatin stimulation has similar consequences on their participation to host's tissue, with the advantage of using a single molecule instead of serum which is clinically irrelevant. The fact that more cells are cycling and expressing markers of proliferation at 5 days post-transplantation clearly indicates that obestatin extends the phase of proliferation of implanted cells. Despite this extended proliferation, it should be noted that their myogenic potential is not compromised, since they can still be committed to differentiate after obestatin treatment as demonstrated by the number of myogenin + human cells observed at day 5 post-engraftment. These observations support the hypothesis that self-renewal is a mechanism regulated by the obestatin/GPR39 system where myoblasts-derived myoblasts give rise to both differentiated cells and functional myoblasts.

Further evidence for self-renewal has come from studies of myoblast transplantation into adult muscle, where neonatal myoblasts/satellite cell-derived myoblasts gave rise to both differentiated myonuclei and functional satellite cells [START_REF] Blaveri | Patterns of repair of dystrophic mouse muscle: studies on isolated fibers[END_REF][START_REF] Heslop | Transplanted primary neonatal myoblasts can give rise to functional satellite cells as identified using the Myf5nlacZ/+ mouse[END_REF]. Observations on the dynamic expression of Pax7, MyoD, and myogenin in ex vivo cultured single myofibers are crucial to guiding satellite cells/myoblasts from an active to a quiescent state [START_REF] Day | Nestin-GFP reporter expression defines the quiescent state of skeletal muscle satellite cells[END_REF][START_REF] Halevy | Pattern of Pax7 expression during myogenesis in the posthatch chicken establishes a model for satellite cell differentiation and renewal[END_REF][START_REF] Zammit | Muscle satellite cells adopt divergent fates: a mechanism for self-renewal?[END_REF]. While most of proliferating Pax7 + /MyoD + myoblasts differentiate into Pax7 -/MyoD + /myogenin + cells, some Pax7 + /MyoD + myoblasts, repress MyoD expression preserving Pax7 expression and ultimately withdraw from the cell cycle. Future studies are needed to identify the regulatory mechanisms governing this process linked to obestatin/GPR39signaling.

In addition to efficient proliferation of the transplanted human cells, the efficiency to reconstitute the host skeletal muscle involves also extensive migration within the recipient's muscle. Previous studies have shown that myoblasts have a very limited migration and remain confined at the injection site [START_REF] Riederer | Slowing down differentiation of engrafted human myoblasts into immunodeficient mice correlates with increased proliferation and migration[END_REF]. Indeed, control engrafted cells only occupy a small volume of the muscle at 21 days post-injection. By contrast, obestatin clearly extended the dispersion and consequently the area occupied in the recipient's muscle during the same time. In agreement with these observations, our experiments with primary or immortalized myoblasts support that the enhanced tissue dispersion of obestatin-stimulated cells is due to an increased intrinsic migratory capacity at early time points after myoblast transplantation and does not involve induction of proliferation. Importantly, blocking Dvl2 signaling by siRNA transfection inhibited obestatin-induced cell migration, indicating that Dvl2 activation participates in the regulation of the myoblast migration. In our study, specific downregulation of Dvl2 signaling in immortalized myoblasts suppresses obestatin-induced activation of small GTPases Rho. These findings suggest a molecular pathway linking obestatin/GPR39 signaling with Dvl2/Rho in governing cytoskeletal architecture and cell motility [START_REF] Wallingford | The developmental biology of Dishevelled: an enigmatic protein governing cell fate and cell polarity[END_REF][START_REF] Gao | Dishevelled: The hub of Wnt signaling[END_REF].

At late stage of muscle regeneration, obestatin treatment increased the number of spectrinexpressing myofibers in the host muscle. Furthermore, the efficiency of the engraftment was ameliorated by the increased cross-sectional area of the newly formed muscle fiber that resulted from human cell fusion. This increase in both fusion and fiber size might be interpreted as a result of mitotic expansion of human cells, so that more cells can differentiate and participate to the repair of the tissue. However, we have already demonstrated that the muscle hypertrophic response to obestatin is not associated with the addition of new myonuclei via proliferation and further fusion [START_REF] Gurriarán-Rodríguez | The obestatin/GPR39 system is up-regulated by muscle injury and functions as an autocrine regenerative system[END_REF][START_REF] Gurriarán-Rodríguez | Action of obestatin in skeletal muscle repair: stem cell expansion, muscle growth, and microenvironment remodeling[END_REF][START_REF] Santos-Zas | β-Arrestin scaffolds and signaling elements essential for the obestatin/GPR39 system that determine the myogenic program in human myoblast cells[END_REF]. It should be considered that hypertrophy is strongly associated to Akt activation, a pathway that is activated by obestatin during the differentiation program and skeletal muscle regeneration [START_REF] Gurriarán-Rodríguez | The obestatin/GPR39 system is up-regulated by muscle injury and functions as an autocrine regenerative system[END_REF][START_REF] Gurriarán-Rodríguez | Action of obestatin in skeletal muscle repair: stem cell expansion, muscle growth, and microenvironment remodeling[END_REF][START_REF] Santos-Zas | β-Arrestin scaffolds and signaling elements essential for the obestatin/GPR39 system that determine the myogenic program in human myoblast cells[END_REF]. This pathway controls mechanisms of protein synthesis at several stages (e.g., translation capacity, translation efficiency) through the increase of specific mRNAs translation, which results in myotubes and fiber enlargement [START_REF] Glass | Skeletal muscle hypertrophy and atrophy signaling pathways[END_REF][START_REF] Lee | mTOR pathway as a target in tissue hypertrophy[END_REF][START_REF] Egerman | Signaling pathways controlling skeletal muscle mass[END_REF]. Therefore, obestatin action is a highly coordinated process in which various signaling pathways converge to activate specific gene expression programs associated to each stage of myofiber formation: proliferation, differentiation, fusion, and hypertrophic response.

On the whole, obestatin treatment increased the cross-sectional area of the human-derived regenerating myofibers with no significant effect found on mouse regenerating myofibers. Furthermore, obestatin did not modify host satellite cell population. Because of the hypertrophic effect of obestatin in murine skeletal muscle and its role in the expansion of satellite cells [START_REF] Gurriarán-Rodríguez | Action of obestatin in skeletal muscle repair: stem cell expansion, muscle growth, and microenvironment remodeling[END_REF], these data seem disconcerting since the whole regenerating muscle is being treated. However, these results are consistent with the existence of a species-specific activity associated to obestatin because mice were treated with human obestatin. In fact, mouse and human obestatin exhibit clear conformational differences beyond their differences in primary structure [START_REF] Alén | The NMR structure of human obestatin in membrane-like environments: insights into the structure-bioactivity relationship of obestatin[END_REF]. The mouse analogue adopts a distinct three-dimensional structure, which cannot activate human GPR39 [START_REF] Alén | The NMR structure of human obestatin in membrane-like environments: insights into the structure-bioactivity relationship of obestatin[END_REF]. These structural differences are responsible for the different bioactivities observed. This species-specific activity allowed us to demonstrate the usefulness of exogenous induction of myoblast proliferation in vivo to improve the efficacy of engraftment. However, this property also provide indirect evidence that, if used clinically, host satellite cells can become activated by human-specific obestatin and contribute to muscle regeneration as grafted donor cells. In particular, endogenous satellite cells might outcompete the transplanted cells and give rise to regenerated sections of myofibers that contain, for example, no dystrophin within DMD patient muscles. Thus, the next important goal is to enhance donor cell response in relation to that obtained from host satellite cell. It is possible that an ex vivo treatment of human myogenic cells with obestatin is sufficient to enhance donor cell engraftment and tissue dispersion. Future experiments will investigate both how to regulate in a specific, proportionate manner the donor cells through the obestatin/GPR39 system, and the utility of modulating this system in vivo to augment the efficacy of engraftment muscle.

The ideal cell approach for muscle therapy must be capable of repopulating the precursor cell pool, providing an enduring source of regenerative cells for future muscle repair after injury.

Previous studies have shown that murine as well as human myoblasts were able to give rise to satellite cells after transplantation in mice [START_REF] Gross | Muscle precursor cells injected into irradiated mdx mouse muscle persist after serial injury[END_REF][START_REF] Heslop | Transplanted primary neonatal myoblasts can give rise to functional satellite cells as identified using the Myf5nlacZl+ mouse[END_REF][START_REF] Brimah | Human muscle precursor cell regeneration in the mouse host is enhanced by growth factors[END_REF][START_REF] Cousins | Regeneration of skeletal muscle from transplanted immortalised myoblasts is oligoclonal[END_REF][START_REF] Ehrhardt | Human muscle precursor cells give rise to functional satellite cells in vivo[END_REF][START_REF] Negroni | In vivo myogenic potential of human CD133+ muscle-derived stem cells: a quantitative study[END_REF][START_REF] Xu | In vivo Fluorescence Imaging of Muscle Cell Regeneration by Transplanted EGFP-labeled Myoblasts[END_REF]. This is further supported by the present work in which transplanted myoblasts generate stem cell-like populations that reside in the satellite cell location, providing evidence for human stem cell self-renewal. Strikingly, this capacity was notably enhanced after obestatin treatment, increasing the number of human Pax7 + cells into the satellite cell compartment. Therefore, obestatin not only allows reconstitution of mature muscle fibers but also replenishes the muscle precursor pool by engrafted donor cells, thus seeding a reserve pool of cells that may be recruited for subsequent rounds of muscle repair. This observation supports the postulated hypothesis that the obestatin/GPR39 pathway can regulate cell assignment: dividing myoblast can either enter terminal differentiation or remain in a precursor state via self-renewal, involving withdrawal from the terminal myogenic program to maintain the satellite cell pool. This population of reserve cells retains the ability to reenter cell cycle and differentiate.

Overall, the present data validate the concept that any intervention upon the donor cells aiming at improving proliferation and migration should be done before differentiation of implanted cells. Furthermore, our work identifies a clinically relevant pharmacological mean to significantly improve the outcome of cell-based therapy for muscle diseases, highlighting the importance of combinatorial therapies. The proper activation and differentiation of transplanted cells via obestatin/GPR39 offer unique opportunities for muscle regenerative medicine.

Material and Methods

Materials.

Human obestatin was obtained from California Peptide Research (Napa, CA, USA).

Antibodies used are listed in Table S1. All other chemical reagents were from Sigma Chemical Co. (St. Louis, MO, US).

Animals. Rag2 -/-Il2rb -/-immunodeficient mice aged 2-3 months were used as recipients for human myoblast transplantation [START_REF] Vosshenrich | Roles for common cytokine receptor gamma-chain-dependent cytokines in the Obestatin stimulates human myoblast engraftment-Revised version[END_REF]. Experimental procedures were performed in accordance with the legal regulations in France and with European Union ethical guidelines for animal research.

Primary human myoblasts.

Human myoblasts were isolated from the semitendinosus muscle of a 25-year-old male in accordance with the French legislation on ethical rules, as described previously (61). The initial muscle biopsy was obtained anonymously through MyoBank, affiliated to EuroBiobank, in accordance with European recommendations and French legislation (approval reference AC-2013-1868), washed through two changes of DMEM medium (Lonza, Pontevedra, SP) and carefully microdissected under aseptic conditions to exclude fat and connective tissue. Cleaned muscle pieces were then cut into ~400 µm 3 cubes and plated onto FBS-washed 60 mm 2 dish for 1 hour. After microexplants attachment, growth medium [GM: Medium 99:DMEM (1:4, v: v; Lonza, Pontevedra, SP) supplemented with 20% FBS (v/v), 25 µg/µL fetuin, 5 ng/mL hEGF, 0.5 ng/mL bFGF and 50 µg/mL gentamycin (Invitrogen; Thermo Fisher Scientific, CO, USA)] was added. All cultures were performed at 37°C in a humid atmosphere containing 5% CO 2 . Mononucleated cells migrating out of the explants were removed from the dish by using a 1.5% trypsin-0.04% ethylenediaminetetraacetic acid (EDTA) solution in PBS and then plated at a density of 2x103 cells/cm 2 . The myogenic cell population was enriched using a CD56 magnetic bead sorting system (MACS; Miltenyi Biotec, Paris, France) according to the manufacturer's instructions. Briefly, cells were labeled with anti-CD56 (a specific marker of myoblasts) microbeads, and then separated in a MACS column placed in a magnetic field. Purification was checked by immunochemistry using a desmin marker (62). All of the primary cell preparations used in this study, C25 cells, had a myogenic purity of at least ≥ 90%. The mean population doubling (MPD) was determined at each passage as described previously [START_REF] Mamchaoui | Immortalized pathological human Obestatin stimulates human myoblast engraftment-Revised version 1: MTJ-17-1113 30 myoblasts, towards a universal tool for the study of neuromuscular disorders[END_REF]. Experiments were performed with C25 cells between 10 and 15 mean population doubling [START_REF] Mamchaoui | Immortalized pathological human Obestatin stimulates human myoblast engraftment-Revised version 1: MTJ-17-1113 30 myoblasts, towards a universal tool for the study of neuromuscular disorders[END_REF]. Cell counting was carried out by means immunofluorescence using desmin as a marker for myoblasts and TOPRO ® -3 (Thermo Fisher Scientific; Topro, fluorescent nuclear and chromosome counterstain) for nuclear staining. For differentiation into myotubes, C25 cells were cultured on Matrigel-covered glass coverslips in GM. At 80% confluence, GM was switched to differentiation medium [DM: DMEM or DMEM supplemented with obestatin 10 nM] for 5 days unless otherwise stated.

Cell preparation and myoblast transplantation.

Mice were anesthetized by an intraperitoneal injection of 80 mg/kg of ketamine hydrochloride and 10 mg/kg xylasine (Sigma-Aldrich, St Louis, MO, USA) as described previously (63). Surgical procedures were performed under aseptic conditions and in accordance with the legal regulations in France and with European Union ethical guidelines for animal research. Primary myoblasts (C25 cells), grafted in vivo in this study had achieved 10-15 MPD, and were thus far from senescence [START_REF] Mamchaoui | Immortalized pathological human Obestatin stimulates human myoblast engraftment-Revised version 1: MTJ-17-1113 30 myoblasts, towards a universal tool for the study of neuromuscular disorders[END_REF]64). Cells were trypsinized, centrifuged, and resuspended in DMEM. Before cell implantation, right TA muscles were subjected to three consecutive cycles of freeze/thawing by applying on the muscle surface exposed by opening the skin, a liquid nitrogen cooled metallic rod as described previously [START_REF] Riederer | Slowing down differentiation of engrafted human myoblasts into immunodeficient mice correlates with increased proliferation and migration[END_REF]. Animals were assigned to one of two-matched experimental groups (n=10 per group): (i) obestatin-treated group and (ii) control group. Cell suspensions containing 5×10 5 myoblasts in DMEM or DMEM+obestatin (300 nmol/Kg body weight) were injected in a single injection site in the mid belly of the TA immediately after cryodamage. The skin was then closed using fine sutures. Obestatin treatment was prolonged 24-hours after cell transplantation injecting 20 µl of obestatin solution in DMEM (300 nmol/kg body weight) into the target muscles each 24 hours during 5 days following the protocol previously described [START_REF] Gurriarán-Rodríguez | Action of obestatin in skeletal muscle repair: stem cell expansion, muscle growth, and microenvironment remodeling[END_REF]. Control group was treated with 20 µl of DMEM under same conditions. Injections were performed inserting the needle of a 0.3 mL/29 gauge syringe (Becton, Dickinson and Company, Franklin Lakes, NJ) for 1 mm in the distal part of the muscle. At 24 hours, 5 and 21 days after engraftment, mice were sacrificed (n=5 for each time point) and the TAs were dissected. In all experiments, the TAs were mounted in tragacanth gum (Sigma-Aldrich), and frozen in isopentane precooled in liquid nitrogen.

Cell culture and differentiation of human immortalized myoblasts. Immortal human myoblasts were derived from the C25 primary culture as previously described [START_REF] Mamchaoui | Immortalized pathological human Obestatin stimulates human myoblast engraftment-Revised version 1: MTJ-17-1113 30 myoblasts, towards a universal tool for the study of neuromuscular disorders[END_REF].

Immortalized human myoblasts, KM155C25 Clone 48 (KM155C25 cells), were cultivated in GM containing Medium 199:DMEM (1:4, v/v; Lonza, Pontevedra, SP) supplemented with 20 % FBS (v/v), 25 µg/µL fetuin, 5 ng/mL hEGF, 0.5 ng/mL bFGF, 0.2 µg/mL dexamethasone (Sigma-Aldrich; MO, USA) and 50 µg/mL gentamycin (Invitrogen) as previously described [START_REF] Santos-Zas | β-Arrestin scaffolds and signaling elements essential for the obestatin/GPR39 system that determine the myogenic program in human myoblast cells[END_REF][START_REF] Mamchaoui | Immortalized pathological human Obestatin stimulates human myoblast engraftment-Revised version 1: MTJ-17-1113 30 myoblasts, towards a universal tool for the study of neuromuscular disorders[END_REF]. To confirm that immortalized cell line maintained their myogenic signature, the expression of several myogenic markers [desmin, neural cell adhesion molecule (N-CAM) and MyoD] was assessed in proliferating primary and immortalized cell line. In addition, the ability to differentiate into myotubes, using immunostaining with MF20 antibody, which recognizes all skeletal-muscle myosin heavy chains (MHC), was investigated. Differentiation into myotubes was initiated at 90 % confluence by switching to differentiation medium [DM; DMEM supplemented with obestatin and 50 µg/mL gentamycin (Invitrogen)] for 6 days. Immortalized cells retain the characteristics of the unmodified parental population (C25 cells) [START_REF] Mamchaoui | Immortalized pathological human Obestatin stimulates human myoblast engraftment-Revised version 1: MTJ-17-1113 30 myoblasts, towards a universal tool for the study of neuromuscular disorders[END_REF].

GST pull downs: Rac1/Rho activity assays. Active Ras-related C3 botulinum toxin substrate 1 (Rac1) and Ras homolog family member (Rho) pull-down and detection kits (Thermo Fisher Scientific, CO, USA) were used to perform Rac1 and Rho activation assays. In both cases, KM155C25 cells were washed in cold PBS and then lysed in equal volumes of 1X Lysis/Binding/Wash buffer (25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1% NP-40 and 5% glycerol) with protease and phosphatase inhibitor mixtures (Sigma-Aldrich, Missouri, USA). Lysates were added into prewashed agarose glutathione beads and incubated for 90 min at 4 °C with gentle rocking. GTP-bound Rac1 and GTP-bound Rho were affinity precipitated from cell lysates (300-500 µg of protein) using an immobilized GST fusion construct containing the p21-binding domain (PBD) of human p21-activated protein kinase 1 (Pak1) that binds to Rac1-GTP but not to Rac1-GDP and using an immobilized GST-fusion protein of the Rhotekinbinding domain (RBD) along with glutathione agarose resin to specifically pull down active Rho. The complexes were then subjected to immunoblot analysis using Rac1-specific antibody.

Total cellular lysates were also separated by SDS-PAGE, and immunoblot blot analysis with anti-Rac1 and anti-Rho antibodies was done as a control for protein loading.

Small interfering RNA (siRNA) silencing assays. Chemically synthesized doublestranded siRNA duplexes targeting Dishevelled 2 (Dvl2) were selected from ON TARGETplus SMARTpool siRNA from Dharmacon (Thermo Fisher Scientific; Dvl2, GCUCAAAGCAGGC-CUGAUC, GGGAGACGAAGGUGAUUUA, CGCUAAACAUG-GAGAAGUA, CCACAAU-GUCUCUCAAUAU). An ON-TARGETplus nontargeting siRNA was used as a control. KM155C25 cells were transfected in suspension with Lipofectamine 2000 (Invitrogen). In short, siRNA is put in each well prior to transfection and combined with diluted Lipofectamine 2000 to form complexes in each well. Cells are added directly to the Lipofectamine 2000-siRNA complexes and transfection occurs while cells are attaching to the well.

In vitro migration/wound healing assays. Cells were seeded on an Ibidi single-culture insert (Ibidi GmbH, Munich, DE) in a 35 mm µ-dish, consists of two reservoirs separated by a 500 µm thick wall. Such inserts create a linear gap by adhering to the treated dish bottom and preventing cell growth in a predefined region. For the myoblasts migration assay an equal number of cells (3 x 10 4 ) were seeded in GM into the two reservoirs of the same insert and incubated at 37°C and 5% CO 2 until their attachment. After 12 hours, cells were treated for 2 hours with mytomicin (15 µg/mL; Sigma-Aldrich) to prevent proliferation. Subsequently, the insert was gently removed creating a gap of ∼500 µm and the plate was filled with GM supplemented with different obestatin concentrations. The progress of migration was photographed immediately, 8 and 24 hours after removing the reservoirs, near the crossing point. Unless indicated otherwise, obestatin was used at a concentration of 10 nM in scratch assays. The wound was calculated by tracing along the border of the scratch using the ImageJ64 analysis software and using the following equation: percentage of wound closure = [(wound area (0h)-wound area (xh))/wound area (0h)] x 100 [START_REF] Gurriarán-Rodríguez | The obestatin/GPR39 system is up-regulated by muscle injury and functions as an autocrine regenerative system[END_REF].

Histology and immunofluorescence analysis. Muscle samples were prepared as described previously [START_REF] Gurriarán-Rodríguez | Action of obestatin in skeletal muscle repair: stem cell expansion, muscle growth, and microenvironment remodeling[END_REF]. Immunostaining analyses of grafted TA muscles were performed using mouse monoclonal antibodies specific for human spectrin and human Lamin A/C. These antibodies were used to visualize fibers expressing human proteins (anti-spectrin) and to detect human nuclei (anti-Lamin A/C). To evaluate the proliferation and differentiation of human cells during regeneration, double immunofluorescence analyses were performed combining antibodies directed against human Lamin A/C with the following antibodies: anti-Ki67 (a pan marker for cells within the cell cycle), anti-phosphohistone H3 (pH3; a marker of the M phase of cell cycle), and anti-myogenin (a marker of the early phase of myoblast differentiation). To evaluate the apoptosis of human cells, immunofluorescence analyses were performed using anti-Lamin A/C antibody and a terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) assay (TACS® TdT In Situ Apoptosis Detection Kit-Fluorescein; R&D Systems, Minneapolis, MN). The nuclei of cells were further counter-stained with DAPI. The number of TUNEL-and Lamin A/C-positive nuclei was counted in ten images from non-overlapping areas of each group. Furthermore, apoptosis was evaluated by immunofluorescence analyses combining antibodies directed against human Lamin A/C with anti-cleaved caspases-3. Staining for MHC, a global marker for early and full differentiation of myoblasts, was performed using the antibody anti-Mf20 (a pan MHC antibody recognizing all isoforms expressed in differentiated skeletal muscle) together with an anti-human Lamin A/C. In addition, we used the anti-Pax7 antibody to detect satellite cells in the muscle tissue together with anti-human Lamin A/C and anti-laminin antibodies. General characteristics of all antibodies are summarized in Table S1. DAPI was used to counterstain nuclei (Life Technologies). The digital images of the cell cultures were acquired with a Leica TCS-SP8 spectral confocal microscope (Leica Microsystems, Heidelberg, DE). Muscles were analyzed as previously described [START_REF] Negroni | In vivo myogenic potential of human CD133+ muscle-derived stem cells: a quantitative study[END_REF]. Briefly, TA muscles were entirely cut into 5 µm sections. For every 450 µm along the complete length of the muscle, 10 sections corresponding to a 50 µm length were used for quantitative analyses.

The number of spectrin-positive profiles in each section examined was counted and the maximum value was determined for each TA investigated.

  To quantify mononucleated interstitial Lamin A/C positive cells, three of the 10 sections analyzed, each separated by at least 15 µm, were assessed and the mean number of Lamin A/C positive cells was calculated. Finally, the mean value of Lamin A/C positive cells was calculated for all of the slides examined covering the entire length of the TA muscle. Immunoblot analysis. The cell samples were directly lysed in ice-cold RIPA buffer [50 mmol/L Tris-HCl (pH 7.2), 150 mmol/L NaCl, 1 mmol/L EDTA, 1% (v/v) NP-40, 0.25% (w/v) Na-deoxycholate, protease inhibitor cocktail (Sigma Chemical), phosphatase inhibitor cocktail (Sigma-Aldrich)]. The lysates were clarified by centrifugation (14,000 ×g for 15 minutes at 4 °C) and the protein concentration was quantified using the QuantiProTM BCA assay kit (Sigma Chemical). For immunoblotting, equal amounts of protein were fractionated by SDS-PAGE and transferred onto nitrocellulose membranes. Immunoreactive bands were detected by enhanced chemiluminescence (Pierce ECL Western Blotting Substrate; Thermo Fisher Scientific, Pierce, Rockford, IL). Statistical analysis. All values are presented as mean ± SEM. Statistical analyses were performed using Graph Pad Prism (version 5.0b; Graph Pad Software, San Diego, CA). Statistical significance was assessed by one-way ANOVA with the Bonferroni post-test or Student's unpaired t-test, with P < 0.05 being considered significant (*). generation, differentiation, and maturation of NK cell precursors and peripheral NK cells in vivo. J Immunol 174: 1213-1221 61. Edom F, Mouly V, Barbet JP, Fiszman MY, Butler-Browne GS (1994) Clones of human satellite cells can express in vitro both fast and slow myosin heavy chains. Dev Biol 164: 219-229 62. Kaufman SJ, Foster RF (1988) Replicating myoblasts express a muscle-specific phenotype. Proc Natl Acad Sci USA 85: 9606-9610 63. Silva-Barbosa SD, Butler-Browne GS, Di Santo JP, Mouly V (2005) Comparative analysis of genetically engineered immunodeficient mouse strains as recipients for human myoblast transplantation. Cell Transplant 14: 457-467 64. Decary S, Mouly V, Hamida CB, Sautet A, Barbet JP, Butler-Browne GS (1997) Replicative potential and telomere length in human skeletal muscle: implications for satellite cell-mediated gene therapy. Hum Gene Ther 8: 1429-1438

Figure 1

 1 Figure 1 Obestatin activates human myoblast proliferation in regenerating

Figure 2

 2 Figure 2 Obestatin treatment increases cell migration in vitro and in vivo. (a)

Figure 3

 3 Figure 3 Obestatin promotes differentiation of muscle precursors in vivo.

Figure 4

 4 Figure 4 Obestatin increase engraftment capacity of human myoblasts in vivo.

Figure 5

 5 Figure 5 Obestatin regulates self-renewal and expansion of transplanted human

Figure S1

 S1 Figure S1 Validation of obestatin/GPR39 functionality in human primary

Figure 1 Obestatin

 1 Figure 1

Figure 2 Obestatin

 2 Figure 2

Figure 3 Obestatin

 3 Figure 3

Figure 4

 4 Figure 4

ACKNOWLEDGMENTS

This work was supported by grants from Instituto de Salud Carlos III, European Regional Development Fund (ISCIII and Fondos FEDER; MINECO, Spain; PI15/015037) and Association Française contre les Myopathies (AFM-Téléthon). The work of JP Camiña and Y Pazos are funded by the SERGAS through a research-staff stabilization contract. An EMBO Fellowship supported the work of I Santos-Zas. Marta Picado Barreiro from IDIS (Santiago de Compostela, Spain) is greatly acknowledged for assistance with microscope experiments, as well as the platform for immortalization of human cells from the Myology Institute (Paris, France).


Aurélien Manchon

Stefania Pizzini

Jan Vogel

Vojtech Uhlir

Lucien Lombard

Clarisse Ducruet

Stéphane Auffret

Bernard Rodmacq

Bernard Dieny

Michael Hochstrasser

A Manchon Spintec

V Uhl

G Panaccione

X-Ray Analysis of Oxygen-induced Perpendicular Magnetic Anisotropy in Pt/Co/AlOx trilayer

Keywords: numbers: 72.25.-b, 73.43.Jn, 73.40.Rw, 73.43.Qt

The discovery of perpendicular magnetic anisotropy (PMA) in Pd/Co multilayers [1] has opened an exciting field of research questioning the fundamental origins of such PMA and the role of interfacial orbital hybridization [2,3]. Although the mechanism of interfacial PMA is complex (see Ref. 2), it has been clearly shown that spin-orbit coupling plays a key role in PMA. Daalderop et al. [2] showed theoretically an oscillating behavior of the anisotropy energy as a function of the valence band filling of Co. Schematically, hybridization of the 3d levels of cobalt with the d levels of Ni, Pd or Pt [3] can modify this filling and induce an important anisotropy in the d orbitals at the interface, thus creating an important PMA in thin X/Co multilayers [4,11] (X=Ni,Pt,Pd).

It has recently been shown that strong PMA can be obtained in optimally oxydized Pt/Co/MOx trilayers [5,6] (M=Al, Cr, Ta, Mg, Ru), starting from unoxidized films with inplane magnetization. Recent work of Lacour et al. [7] on similar Pt/Co/Al trilayers showed that Co magnetization in their samples already lies out-of-plane even without oxidation. Consequently, one can expect a strong enhancement of PMA due to the presence of Co-O bondings at the interface. X-ray studies achieved by Telling et al. [12,13] on Co/AlOx/Co MTJs showed that a maximum of Co magnetic moment, corresponding to a maximum in TMR, can be reached by optimally oxidizing the tunnel barrier. Designing high TMR-MTJ using oxygen-induced PMA [6] requires a deep understanding of this phenomenon.

In this article, we investigate the role of interfacial oxidation in the anisotropy crossover induced by annealing of a Pt/Co/AlOx sandwich. The macroscopic magnetic properties are analyzed in parallel with the microscopic structural properties of the samples obtained via X-ray Photoelectron Spectroscopy (XPS), X-ray Absorption Spectroscopy (XAS) and X-ray Magnetic Circular Dichroïsm (XMCD).

Pt(3 nm)/Co(0.6 nm/Al(1.6 nm) trilayers were deposited on a thermally oxidized silicon wafer by conventional dc magnetron sputtering with a base pressure of 5 × 10 -8 mbar. The sample was oxidized by using oxygen rf plasma with a partial pressure of 3 × 10 -3 mbar and a power of 10 W during 20s. One sample was left as-deposited and a second one was annealed at 350 • C for 30 minutes. The samples are highly stable so that no capping layer had to be deposited on top of AlOx.

The macroscopic magnetic properties of these samples were studied by extraordinary Hall effect (EHE) in a standard four-probes Hall geometry. The Hall resistance R H has the form

R H = R 0 H + 4πR EHE M z ,
where H is the magnetic field (applied perpendicularly to the plane of the sample) and M z is the out-of-plane component of the sample magnetization. R 0 is the ordinary Hall resistance and R EHE is the extraordinary Hall resistance [14]. Fig. 1 shows the hysteresis loops obtained by EHE at room temperature for as-deposited and annealed samples.

The EHE measurement of the as-deposited sample gives no hysteresis and zero remanence.

Magnetization saturates at high field (-H-¿1.4 kOe), where M z no longer depends on H and the residual slope comes from the ordinary Hall contribution. This demonstrates that the magnetization of the Co layer possesses no effective out-of-plane component at remanence. In fact, EHE measurements with in-plane external field as well as anisotropic magnetoresistance measurements (not shown) demonstrate that the magnetization of the Co layer lies out-of-plane forming a domain structure. Then, one needs to apply a large external field (H=1.4 kOe) to saturate it in the out-of-plane direction. Note that this field is much lower than 4πMs (≈ 12 kOe) which is consistent with the existence of PMA at the Co/Pt interface. After annealing, the sample shows a square hysteresis loop with sharp magnetization reversal and a coercivity of 100 Oe (see bottom inset of Fig. 1). These features indicate that the perpendicular anisotropy strongly increases in the annealed sample. The perpendicular magnetic anisotropy, estimated from both SQUID and EHE with in-plane external field measurements, is enhanced during the thermal annealing from H an ≈ 0.4 kOe to H an ≈ 5.2 kOe. SQUID measurements applying an out-of-plane external field (top inset of Fig. 1) show that an enhancement of 14% of the Co magnetic moment is obtained after annealing. Note that the magnetic moment of our ultrathin Co (≈950 emu.cm -3 ) is lower than for bulk Co (≈1400 emu.cm -3 ). The large increase of the EHE magnitude (200%) in the annealed sample is then mostly attributed to the enhancement of R EHE . This point will be adressed elsewhere.

To understand the microscopic origin of the anisotropy crossover associated with annealing, we performed soft X-ray Photoelecton Spectroscopy (XPS) of Co 2p levels, as well as X-ray Absorption Spectroscopy (XAS) and X-ray Magnetic Circular Dichroism (XMCD) measurements at the Co L 2,3 edges. The aim of these element-specific measurements is to correlate the changes in macroscopic magnetic properties induced by annealing with changes in average chemical composition at the Co/Al interface. For the Co thickness used here (0.6 nm), XAS and XMCD give information on the composition and magnetic moments averaged over the Co layer, while XPS will provide information on the electronic structure of Co at the Co/AlOx interface mainly.

Measurements were carried out at the Advanced Photoelectric-effect Experiments (APE) beamline of the ELETTRA synchroton in Trieste (Italy), using the experimental setup given in the inset of Fig. 3(a). Co 2p XPS measurements were performed setting the incident photon energy at hν = 1130 eV, with an incident angle of θ = 60 • . Fig. 2 shows the spectra obtained for as-deposited and annealed samples. The spectrum of pure CoO is given for reference in the top of the same figure. In this spectrum, one can distinguish two main peaks, corresponding to CoO 2p 1/2 and CoO 2p 3/2 core levels (lying at 796.3 eV and 781.1 eV respectively) and two satellite peaks (denoted S and lying at 803.3 eV and 786.7 eV resp.) which arise from the charge transfer between O 2p and Co 3d. The main peaks are shifted towards larger binding energies with respect to Co metal peaks, due to oxygen environment.

The spectrum of the as-deposited sample is similar to that of pure cobalt (see Ref. 15), with Co 2p 1/2 and Co 2p 3/2 lying at the binding energies of 792.5 eV and 778.1eV respectively.

The spectrum of the annealed sample clearly shows contributions from both Co and CoO spectra. Gaussian fits (not shown) indicate that the contribution of CoO to the XPS spectra of the annealed sample is larger than 80%, showing that most of the Co atoms located near the Co/Al interface are bonded to oxygen atoms. Furthermore, the ratio between the estimated areas of the CoO 2p 1/2 peak (796.3 eV) and its satellite (803.3 eV) gives 0.97, which means that the cobalt oxide is a monoxide [16].

In order to confirm the previous studies, Co L 2,3 absorption spectra were obtained by measuring the drain current of the sample via the sample holder. To probe the out-of-plane Co remanent magnetization (no out-of-plane magnetic field could be applied), the sample surface was set perpendicular to the incident X-ray beam (see inset of Fig. 3(a) with θ = 0 • ). Fig. 3 shows the Co L 2,3 absorption spectrum measured for the as-deposited and annealed samples, for left (dashed line) and right (dotted line) circular polarization. The spectrum of the as-deposited sample (Fig. 3(a)) shows no dichroism (solid line) in this configuration, indicating that the Co magnetic moment, averaged over the whole Co thickness, has no net component in the direction perpendicular to the surface. This is in agreement with macroscopic measurements. An important dichroism signal is found for the annealed sample, which indicates that it possesses a net, remanent out-of-plane magnetic moment consistent with the EHE measurements. XMCD measurements therefore confirm the increase of the perpendicular magnetic anisotropy associated to annealing.

The spectral shape of the Co L 2,3 absorption spectra is typical of metallic Co (see Ref. 15 for example), both before and after annealing (see the derivatives of the XAS spectra in the inset of Fig. 3(b)). This indicates that the Co chemical environment, averaged over the whole Co layer, does not change significantly upon annealing. As we have seen, the more surface-sensitive XPS measurements (the collected intensity is exponentially decreasing with a penetration depth of the order of 5-6 Å) indicate, on the other hand, a large oxidation of the Co layer. As a consequence, the comparison of the two spectroscopic techniques implies that the Co-O environment is specific to the topmost Co/AlOx interface.

We therefore deduce that the enhancement of perpendicular magnetic anisotropy induced

  These results are not in contradiction with those of Ref.5, 6, but are simply explained by the larger role of the PMA at the Pt/Co interface induced by the presence of a Ta buffer layer and a thicker Pt underlayer. This is a well-known phenomenon as described for instance by Ref. 8. The studies presented in Ref. 5, 6 and in the present work can be put in parallel with the theoretical work of Oleinik et al. [9] and Belaschenko et al. [10] on Co/AlOx/Co and Co/STO/Co magnetic tunnel junctions (MTJ), which consider the role of interfacial oxygen in the enhancement of the magnetic moment of cobalt. The charge transfer which occurs between Co and O enhances the asymmetry of the valence band of Co, increasing the interfacial spin polarization. This charge transfer reduces the energy of Co d orbitals pointing towards O, creating a splitting between in-plane (d xy , d x 2 -y 2 ) and out-of-plane (d xz , d yz , d z 2 ) d orbitals (parameter ∆ in Ref. 11) increasing the crystalline field effect [11].

  by thermal annealing is related to the appearance of a significant density of interfacial Co-O bonds at the Co/AlOx interface. This is the result of temperature-induced diffusion of excess oxygen in the amorphous AlOx structure towards the Co/Al interface. In conclusion, combining bulk and interfacial-sensitive spectroscopy techniques (XAS and XPS), we have shown that interfacial oxidation of Co in Pt/Co/AlOx trilayers is at the origin of strong perpendicular magnetic anisotropy induced by annealing. Thermal annealing favours the oxygen diffusion towards the Co/AlOx interface, without penetration of the O atoms within the Co layer. These studies support the seminal role of oxygeninduced splitting of the Co 3d bands in PMA, as presented at the beginning of this article. The role of the Co/Pt interface have been disregarded in this study because the potential thermally-induced Co-Pt mixing at the Pt/Co interface is expected to reduce the perpendicular magnetic anisotropy, not to enhance it. Ab-initio calculations, aiming at clarifying the role of Co-O hybridization in magnetic anisotropy energy, are presently underway.

Figure 1 :

 1 Figure 1: Hall resistance as a function of the applied field for as-deposited (open squares) and annealed (filled squares) samples. The field is applied perpendicular to the plane of the trilayer. Top inset: hysteresis cycles of the samples obtained by SQUID measurements. Bottom inset: zoom of the Hall resistance loops at low field for as-deposited and annealed samples.

Figure 2 :

 2 Figure 2: Co 2p XPS spectra for the as-deposited sample (bottom curve), annealed sample (middle curve) and pure CoO (top curve).

  Photon Energy (eV)

Figure 3 :

 3 Figure 3: Co L 2,3 X-ray absorption spectra measured for the as-deposited sample (a) and the annealed sample (b), with no applied magnetic field (H=0) and perpendicular incident beam (θ = 0 • ). The solid curves represent the XMCD signal, obtained from the difference between left (dashed line) and right (dotted line) circularly polarized light spectra. Inset of (a): experimental set-up. Inset of (b): derivative of the XAS spectra of as-deposited (dotted line) and annealed (solid line) samples.

The authors are grateful for the experimental support and assistance of B. Pang, L. Ranno, O. Fruchart and P. Torelli.