Gianluca Aloi
email: aloi@dimes.unical.it

Marco Di Felice
email: difelice@cs.unibo.it

Valeria Loscrì
email: valeria.loscri@inria.fr

Pasquale Pace
email: ppace@dimes.unical.it

Giuseppe Ruggeri
email: giuseppe.ruggeri@unirc.it

SPONTANEOUS SMARTPHONE NETWORKS AS USER-CENTRIC SOLUTION FOR THE FUTURE INTERNET

In this paper we focus on a special case of User-Centric Networks (UCNs), named Spontaneous Smartphones-based Networks (SSNs), where the role of the end-user devices is played by smartphones that are, "evolutionary" and more active in supporting communication services. SSNs present key features like spontaneity in the creation of the network and redefinition of the devices' role in order to make them continuously adaptive to both network and users requirements. This work is devoted to identify the potential advantages of SSNs, by also providing a clear definition of the challenges and the issues that need to be faced in order to make this emerging paradigm effective and practically deployable.

INTRODUCTION

Nowadays, Spontaneous Smartphone Network (SSN) is emerging as a potential new communication paradigm, characterized by the fact that the access network has a strong self-organizing nature and it is primarily made up of users' owned devices. These latter can also act as routers by actively cooperating to forward data on multi-hop paths. SSNs can be considered a special case of User-Centric Networks (UCNs) made up only by users' devices.

This new approach of network deployment undoubtedly offers great advantages in terms of (i) lower costs required to set up the network access, (ii) reduced or no maintenance at all for network management, and (iii) possibility to set-up a network even on scenarios where the infrastructure is poorly available (i.e., disaster scenarios, rural areas, least developed countries, etc.). Despite the enormous potentials of SSN the multi-hop communication between smartphones, or similar devices (e.g., phablet, tablet), is still not an affirmed paradigm and self-organized SSNs are a challenge [START_REF] Iera | Making a meshrouter/gateway from a smartphone: Is that a practical solution?[END_REF].

Usually, the network creation and management requires a massive intervention from the users that, however, would prefer being agnostic about technological issues. This requirement constitutes a unique issue of SSNs compared to traditional self-organizing systems and generic multi-hop ad hoc networks.

Hence, a framework able to limit the human intervention, with the aim of making the network management as much spontaneous as possible, is mandatory for the success of the SSN paradigm.

In this work, we present and discuss results of a novel framework, named STEM-Net [START_REF] Aloi | STEM-Net: an evolutionary network architecture for smart and sustainable cities[END_REF] and specifically adapted to SSNs, in order to provide two main enabling features for users' devices: (i) network self-configuration and, (ii) evolvability, i.e., the capacity of continuously adapting to the needs of both user and network. Through STEM-Net, a smartphone can switch between different roles. It can produce/receive data, it can forward the traffic of other terminals through multi-hop communications or it can provide the access to global network resources to other terminals, but above all, it can autonomously assume the most suitable role without any user intervention.

The main contributions of the paper are: (i) to specify advantages and issues of SSNs by identifying strategic scenarios and use cases on which SSNs can be utilized, (ii) to review the enabling technologies of SSNs, by also pointing out limitations of existing software frameworks in terms of selforganizing deployments, and, finally, (iii) to describe how the application of the STEM-Net framework can solve such issues, by also presenting a proof of concept implementation on real smartphones.

USE CASES AND SCENARIOS

In this section, we propose few communication scenarios (depicted in Figure 1) well suited for SSNs to motivate the need of a new and flexible system architecture for network creation and management.

Pervasive wireless Internet access:

SSNs can be used to deploy a fully pervasive and infrastructure free Internet access by naturally extending the coverage of wide areas. This goal could be easily achieved because of the huge amount and density of smartphones located in all daily life environments.

As a result, a pervasive scenario can be implemented by adding self-configuration capabilities to smartphones, making them able to cooperate with the aim of sharing the access to global resources.

Emergency and post-disaster recovery:

When natural catastrophes disrupt traditional network infrastructures, SSNs can help both survivals and rescue teams. The survivors, by using their mobile phones and the scarce communication resources still available, can share updates, post photos, and upload videos on social media sites. We are confident that in the long run emergency services, spontaneous smartphones-based networks will complement the safety networks (prospectively based on LTE) in place for officials only such as police and fire brigade.

Large crowd gathering places:

When special events (e.g., concerts, trade fairs, Olympic games) involve huge numbers of people with risks of overloading the communication infrastructure, SSNs can be used to offload mobile data traffic from cellular networks. As an example, cooperative smartphones can wisely forward user's data "hop-by-hop" to the nearest Wi-Fi access point so that the amount of offloaded data can further increase.

ENABLING TECHNOLOGIES

In this Section, we provide a quick review of existing hardware and software technologies that might support the implementation of SSNs on today's smartphones.

COMMUNICATION TECHNOLOGIES

Communication among smartphones can be supported by a plethora of short/medium range communication interfaces and technologies; in the following, we review the main features of existent or emerging wireless communication technologies available on today's smartphones, in order to discover the more suitable (if any) for the deployment of SSNs.

Bluetooth offers a short communication range and a rate that ranges from 1.2Mb/s in the early releases (version 1.2) to a maximum of 24Mb/s in the latest releases (version 4). Bluetooth requires a significant involvement by the user, since the interface should be activated, the recipient device should be discovered and the content to be sent should be manually specified. The poor communication performance, the annoying procedures and the users' reluctance hindered the diffusion of Bluetoothbased SSNs.

Near Field Communication (NFC) technologies have been successfully proposed to support content sharing between smartphones. The very short communication range (in the order of few cm) provides a natural protection against malware diffusion. However, such strength is also a limitation, since the utilization of NFC-based SSNs is limited to few specific scenarios such as "Delay Tolerant" content sharing, in addition to the need of involving the user supervision and management.

Recent smartphones are equipped with IEEE 802.11 interface (Wi-Fi) supporting high transmission rate up to 600Mb/s (IEEE 802.11n). However, most of existing Mobile Operating Systems (MOSs) do not allow to configure the Wi-Fi interface in Ad Hoc mode, unless breaking some safety procedures and creating a super-user account, which is quite far to be practical for common users. Hence direct communication between smartphones are practically not feasible.

Wi-Fi Direct has been recently standardized to allow communication between enabled devices without any infrastructure. The devices activate a negotiation procedure at their first connection to determine which one shall act as an access point, while the other devices can connect to it by using, de facto, the Wi-Fi Infrastructured mode. However, despite the benefits in terms of increased security compared to the Ad Hoc mode, Wi-Fi Direct causes an unfair workload distribution since the smartphones acting as access points will consume more resources. Furthermore, Wi-Fi Direct does not supports multi-hop communication and, consequently, communications are limited to terminals within reciprocal communication range.

A further opportunity to support SSNs is given by LTE and also HSDPA/HSUPA. Even if these technologies are mainly conceived to provide the devices connectivity toward a network infrastructure, it is expected that LTE in 3GPP Release 12 will support the so called Device to Device (D2D) communications mostly based on local, opportunistic and single hop data exchange.

In Table 1, we briefly report the main characteristics of the wireless technologies discussed so far.

SOFTWARE SUPPORT

While the characteristics of communication at the lower layers (MAC/PHY) depend on the wireless technology in use, the network functionalities are provided by the MOS. Two main functionalities are fundamental to implement a SSN: the possibility to turn a smartphone into a network gateway, and the routing capabilities. Nowadays, most of the MOSs (i.e., Android, IOS, Windows Phone) make available "tethering" modules, which enable a smartphone to provide mobile Internet connectivity through its short-range wireless interfaces (e.g., Wi-Fi, Bluetooth). However, the enabling/disabling of tethering modules must be performed manually by the user. Routing capabilities on Wi-Fi networks are not provided by most popular MOSs for security reasons, even if the NIC (Network Interface Card) usually supports Ad Hoc communication mode. Existing software can be categorized into two approaches [START_REF] Nishiyama | Relay by Smartphone: Realizing Multi-hop Device-to-Device Communications[END_REF], respectively DTN-based [START_REF] Ntareme | Delay tolerant network on smartphones: applications for communication challenged areas[END_REF] or MANET-based [START_REF] Zhuang | Managing Ad Hoc Networks of Smartphone[END_REF] [START_REF] Mitra | Emergency Response in Smartphone-based Mobile Ad Hoc Networks[END_REF]. The first approach allows facing the problem of intermittent connectivity caused by end-user mobility, although it poses severe challenges in terms of performance and battery consumption [START_REF] Ntareme | Delay tolerant network on smartphones: applications for communication challenged areas[END_REF]. The second approach relies on traditional routing protocols used over generic MANET; we cite experimental studies of multi-hop SSNs using OLSR [START_REF] Aloi | STEM-Net: an evolutionary network architecture for smart and sustainable cities[END_REF] and AODV [START_REF] Zhuang | Managing Ad Hoc Networks of Smartphone[END_REF] protocols. Since most of the MOSs do not allow intercepting IP packets or modifying the routing tables at the kernel layer, these implementations run in user space [START_REF] Zhuang | Managing Ad Hoc Networks of Smartphone[END_REF], thus introducing additional overhead in terms of power consumption and data transfer. At the same time, since the routing policy is performed on the basis of the route length only, energy consumption can be unbalanced or lead to suboptimal SSN performance.

CHALLENGES IN THE IMPLEMENTATION OF SSNS

From all the argumentations reported above we argue that the main challenge to the implementation of SSNs is constituted by the lack of adequate software support for network creation and management. In this paper we focus on three limitations of existing software architectures:

1. No autonomous device configuration capabilities. In most of today's smartphone, configuration and set-up of network functionalities must be carried out by users manually, like pairing operations with other devices/networks, network formation, adjustment of transmitting parameters. This approach is not scalable and then it is not suitable for scalable deployments (e.g., the pervasive access scenario) or dynamic environments (e.g., the emergency scenario).

2.

No cooperative network management capabilities. Although computational and communicational capabilities of smartphones are continuously improving, their performance are still not comparable with those of dedicated network equipments. For instance, in [START_REF] Iera | Making a meshrouter/gateway from a smartphone: Is that a practical solution?[END_REF], the authors have compared the performance of a SO-HO Wi-Fi network router to a smartphone provided with mesh routing capabilities, and have found that in this latter case power consumption constitutes a severe concern, since look-up operations are highly demanding for the smartphone's CPU. Vice versa, energy efficiency of the single device can be improved when smartphones cooperate to share the effort of network management, by dynamically deciding the role to act (e.g., router/gateway) on the basis of their actual resources.

3.

No network self-organization capabilities. SSNs are intrinsically dynamic environments, due to the end-users' mobility and to the variable traffic loads produced by the mobile applications. Self-organizing principles are required to manage the network and guarantee continuity of service in front of dynamic and unforeseen events. Moreover, in several scenarios (e.g., the post-disaster recovery), the goal of the SSN becomes to maximize the operativeness of the network, considered as a single entity, rather than the performance of a single component. Achieving such distributed intelligence requires cooperation, autonomous sensing, and decision making capabilities, which of course cannot be managed in case of human control.

STEM-NET: A FRAMEWORK TO SUPPORT THE IMPLEMENTATION OF SSNS

In this section we describe the STEM-Net framework, originally introduced in [START_REF] Aloi | STEM-Net: an evolutionary network architecture for smart and sustainable cities[END_REF] for a Smart Cities environment, and we show how STEM-Net paradigm can overcome the previous challenges becoming a viable and effective solution also in the SSN context.

The logical architecture of a generic end-user smartphone is illustrated in Figure 2. We refer to the "stemness" property of a smartphone as the ability to perform a protocol reconfiguration achieved throughout the combination of built-in features and algorithmic solutions. From here on, we use the term Stem-Phone to indicate this novel family of software enhanced smartphones. We further note that in our view, Stem-Phones may be a limited fraction of the existing smartphones but they can still provide useful services to the neighboring legacy phones.

Each Stem-Phone, participating to the SSN setup, can play a given set of roles according to the network capabilities/functionalities supported by the specific device. The basic set of roles of each Stem-Phone include the ability (i) to produce/receive data, (ii) to forward the traffic of other terminals and (iii) to act as a gateway providing access to global network resources to other terminals. The set of roles played by each Stem-Phone can vary in accordance of its own built-in characteristics (i.e., hardware features or physical constraints). For example, the gateway role could require the simultaneous use of different communication technologies to connect other nodes (e.g., Wi-Fi Direct, Bluetooth) and to access to global network resources (e.g., LTE, HSDPA). In addition, a Stem-Phone could also play new roles that can be dynamically configured and "learnt" from other Stem-Phones by relying on cooperation with them. This might be the case, for instance, of a Stem-Phone that upgrades its software, downloaded from its neighbors, in order to gain the ability to serve as an access point.

Moreover, each role is mapped to a specific network configuration and it is foreseen the possibility for a Stem-Phone to change its configuration over time for self-optimization purposes. This might be the case, for instance, of a Stem-Phone configured as a Wi-Fi router that dynamically adjusts its transmitting power level based on measured interference conditions.

STEM-NET FOR SSNS: THE GATEWAY ELECTION CASE

In this section we show how the STEM-Net framework can be usefully applied to handle autonomous configuration tasks by focusing on a basic SSN issue: the dynamic gateway election.

Let us consider a set of smartphones that cooperate to build a SSN. Without loss of generality, we suppose that the multi-hop communication is supported by following the approach proposed in [START_REF] Iera | Making a meshrouter/gateway from a smartphone: Is that a practical solution?[END_REF]. Wi-Fi cards are configured in Ad Hoc Mode and the OLSR protocol is deployed. Some of the smartphones should be elected to play the role of Gateway that consists in forwarding the packets coming from the SSN on the Wi-Fi interface toward the Internet backbone by using the 3G interface. This role requires the fulfillment of several requirements: i) acting as gateway implies an high power consumption, therefore the gateway should be chosen among those Stem-Phones with a sufficient residual energy, ii) the connection between the gateway and the cellular backbone should offer an adequate throughput to sustain the traffic generated in the SSN, iii) since the throughput on Wi-Fi multi-hop communication roughly decreases with the number of hops, the gateway should be located in a central position with respect to the other terminals in the SSN and finally, iv) the Wi-Fi network nearby the gateway should be as uncongested as possible.

To address this problem, a simple but effective spontaneous gateway election procedure has been proposed in [START_REF] Di Felice | Smartphones Like Stem Cells: Cooperation and Evolution for Emergency Communication in Post-Disaster Scenarios[END_REF]. Since we only aim at showing how this election procedure is functional to SSNs, we briefly summarize our strategy in the following by referring the reader to the work in [START_REF] Di Felice | Smartphones Like Stem Cells: Cooperation and Evolution for Emergency Communication in Post-Disaster Scenarios[END_REF] for further details. Once a SSN is created, a gateway is randomly selected; after this initialization phase, the gateway role is passed to the most suitable node by following a "stimulus-response" model. Each node monitors the following parameters: i) its own residual energy, ii) the congestion level experienced at the Wi-Fi interface, and iii) the average distance from other nodes in the SSN. The gateway 𝑔 also computes the congestion experienced at the Cellular interface. All the monitored parameters are periodically exchanged between the nodes. The exact computation of the relevant parameters are given in Table 2.

The node acting as gateway constantly evaluates its attitude to keep its role and quantifies it through a specific metric called Stimulus Metric (SM) [START_REF] Di Felice | Smartphones Like Stem Cells: Cooperation and Evolution for Emergency Communication in Post-Disaster Scenarios[END_REF]:

𝑆𝑀(𝑔) = 𝐺(𝑟𝑒𝑠𝑖𝑑𝑢𝑎𝑙 𝑒𝑛𝑒𝑟𝑔𝑦, 𝑐𝑜𝑛𝑔𝑒𝑠𝑡𝑖𝑜𝑛 𝑜𝑛 𝑐𝑒𝑙𝑙𝑢𝑙𝑎𝑟 𝑖𝑛𝑡𝑒𝑟𝑓𝑎𝑐𝑒)
Here, G represents a suitable function that is given in Table 2. Periodically, the gateway in charge broadcasts its SM value by starting a "gateway handover procedure"; upon receiving a SM message, each Stem-Phone 𝑖 evaluates its capability to take over the gateway role and summarizes it in a new metric called Threshold Metric (TM) defined as follows [START_REF] Di Felice | Smartphones Like Stem Cells: Cooperation and Evolution for Emergency Communication in Post-Disaster Scenarios[END_REF]:

𝑇𝑀 𝑖 = 𝐹 (𝑟𝑒𝑠𝑖𝑑𝑢𝑎𝑙 𝑒𝑛𝑒𝑟𝑔𝑦, 𝑐𝑜𝑛𝑔𝑒𝑠𝑡𝑖𝑜𝑛 𝑜𝑛 𝑊𝑖𝐹𝑖 𝑖𝑛𝑡𝑒𝑟𝑓𝑎𝑐𝑒, 𝑎𝑣𝑒𝑟𝑎𝑔𝑒 𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑓𝑟𝑜𝑚 𝑛𝑒𝑖𝑔ℎ𝑏𝑜𝑢𝑟𝑠)
Here, 𝐹 is a suitable function that is given in Table 2. If the stimulus 𝑆𝑀(𝑔) perceived by a node 𝑖 exceeds its threshold 𝑇𝑀(𝑖) , the node 𝑖 assumes the role of gateway and announces its decision to the SSN.

Table 2. Parameters estimation and Stimulus-Threshold computation.

We would like to remark here that the described gateway election procedure can be easily supported by the proposed Stem-Phone architecture (Fig. 2

A PROOF OF CONCEPT IMPLEMENTATION

The effectiveness of the gateway election has been already shown in [START_REF] Di Felice | Smartphones Like Stem Cells: Cooperation and Evolution for Emergency Communication in Post-Disaster Scenarios[END_REF]. Here, we are interested in providing some brief guidelines on the feasible implementation of the election process on commonly available smartphones. As hardware platform we used a Samsung Galaxy S model, which can be considered as representative of a wide range of user devices on the market.

On top of the hardware platforms we have developed a software suite, which implements the gateway election procedure as described in the former section. The software suite includes two classes of programs that implement: i) the basic communication functions, and ii) the components of the Stem-Phone architecture. The basic communication over multi-hop paths is supported by using the OLSR daemon (www.olsr.org).

The components of the Stem-Phone architecture have been implemented as follows:

• The Context Manager consists of a series of self-developed scripts that also leverage some routines offered by the Android operating system. Specifically Table 2 summarizes how RP is computed.

• Cooperation Manager has been developed by extending the OLSR protocol with two custom message types: i) STEM HELLO MESSAGE to disseminate RP computed by each Stem-Phone, ii) STEM GATEWAY MESSAGE to start a gateway election procedure.

• The Knowledge Database is implemented through a set of files in ~/proc/Stem/ directory. Specifically a file named ~/proc/Stem/context.local stores the information relevant for the node itself, while the file ~/proc/Stem/context.extra is filled with the information received by surrounding nodes.

• The Control & Decision Brain functionalities have been supported by implementing a dedicated C program which, starting from the information stored in the Knowledge Database, computes and compares Stimulus and Threshold according to the formerly described algorithm.

The realized prototypes have been tested in the deployment shown in Figure 3 where three Stem-Phones constitute a small SSN. One of the three Phones (Phone A) is unable to directly transmit data to the 3G network. This condition has been forced by instructing the Android OS to disable data communication through the 3G interface. Hence, Phone A should necessarily forward its data to one of the other two Phones (B or C) that instead have their 3G connection active. Data produced by Phone A towards Internet consists of a constant bit rate flow of 350kb/s, generated by using a custom software. To control the residual charge available to Phones B and C, their battery has been replaced with a wooden one and the phones have been powered through a tunable power supplier. Commonly available phones estimate the State of Charge (SOC) of their battery by probing the voltage provided by this latter. Thus, in our set-up we forced the phones to change their estimation of the residual charge by varying the voltage value throughout a tunable power supplier. In our test campaign the voltage provided to Phone C has been kept constant to 3.7 V to emulate a residual battery charge of about 50% of the nominal value; on the contrary, the voltage provided to Phone B has been varied in [3.2V-4.2V] to emulate the residual amount of charge showed in figure 4.

Three different approaches for the gateway election have been evaluated:

1. Phone B is manually selected. This approach is equivalent to tethering operation, which is the only solution commercially available today.

2. OLSR is installed on all the phones. In particular, Phone B and Phone C are configured as potential gateways and the choice of which one to select is left to the routing protocol. This case represents what can be obtained through the framework presented in [START_REF] Iera | Making a meshrouter/gateway from a smartphone: Is that a practical solution?[END_REF] or by using similar MANET-based approaches.

3. The STEM-Net architecture is deployed on the Phones, through the software modules formerly described in Section 4.1.

Figure 4 reports the throughput achieved by Phone A when varying the gateway selection method and the residual charge at phones B and C. The worst performance is achieved when the gateway is manually chosen. In this case, as soon as the residual charge at Phone B approaches zero, this latter switches off its communication interfaces and stops forwarding data coming from Phone A, hence the communication is interrupted.

The performance improves when the choice of the gateway is carried out by using OLSR. In this case Phone B is selected as gateway as long as its available charge goes to zero. At this instant, the communication path is broken and OLSR looks for a new available gateway. After about 10 seconds Phone C is selected as new gateway. This behavior constitutes an improvement compared to the first case (manual selection) nonetheless it presents some drawbacks: (i) it leads to an uneven power consumption between the available gateways, (ii) the search for a new gateway is started only after Phone B became inoperative and following this late reaction a service disruption of about (10s) is experienced, (iii) the owner of Phone B may be annoyed to have his/her phone inoperative due to the lack of charge.

In the STEM-Net framework the gateway election procedure is carried out considering also the residual charge, so the role of the gateway is always assumed by the node having the maximum residual charge.

As soon as the residual charge of Phone B falls below that of Phone C, the role of gateway is passed from the former to the latter. Since the change of role is done while Phone B is still operative, the handover procedures are much faster compared to the OLSR case. Finally the STEM-Net approach does not starve Phone B, hence it provides a better experience to the end-user. This simple experiment confirms the viability and effectiveness of the STEM-Net framework on commonly available devices and it paves the way for additional experimentations for upcoming SSNs.

LESSONS LEARNED AND OPEN ISSUES

Based on the experimental results, we can conclude that STEM-Net represents a viable solution to face the challenges on SSNs implementation introduced in Section 3.3. Indeed, cooperative network management policies can be implemented in a straight-forward way through the concepts of "roles" and node mutation (point two of Section 3.3), while network adaptiveness and distributed selforganization capabilities (point three of Section 3.3) can be achieved through the proper modeling of stimulus/threshold functions, that are intrinsically scenario-dependent. However, several practical issues regarding autonomous device configuration capabilities (point one of Section 3.3), have emerged from the test-bed implementation, based on which we can argue that a completely user-agnostic SSN deployment model might not be 100% feasible on today's smartphones. Skipping the technical details, most of such issues derive by the poor support provided by the MOS at the APIs level, so that several low-level network functionalities (e.g., multi-hop routing) cannot be implemented without breaking some safety mechanisms of the original software equipment, which is clearly not a solution.

Finally, we conclude the paper by highlighting the existence of additional open issues that need to be addressed for practical deployments of SSNs. Security is a major concern while designing and developing spontaneous network based on mobile devices [START_REF] Gilaberte | A Secure Protocol for Spontaneous Wireless Ad Hoc Networks Creation[END_REF]. A secure self-configured protocol is required for user authentication, validation and data transfer. Moreover, novel and robust reputation mechanisms are required to identify user misbehavior within different communities [START_REF] Sofia | Moving towards a socially-driven internet architectural design[END_REF]. Connected to the security issues, novel cooperation models and utility functions have to be designed to encourage users to share their resources with the certainty of obtaining future benefits [START_REF] Pace | Managing and Deploying Pervasive Wireless Internet Access through Attractive Connection Sharing and Reselling Mechanisms[END_REF]. In this context, smart mechanisms based on local pricing strategies could be used to control and prevent network congestion by prolonging the overall lifetime of the SSN, considered again as a single entity. We plan to address these open issues as future works.

CONCLUSION

In this paper we have investigated the potential of Smartphone Spontaneous Networks (SSNs), by analyzing the main issues and challenges from the perspective of minimizing users' involvements for network set-up and maintenance. We believe that the implementation of an actual network "spontaneity" model, based on "software" intelligence local at each smartphone, could favor the diffusion of SSNs. To this purpose, we have introduced the STEM-Net framework, and presented results of a small-scale test-bed in which gateway nodes are dynamically selected. The interface driver offers an estimation of total transmitted 𝑇𝑋 !"## , received 𝑅𝑋 !"## , dropped 𝐷𝑅𝑂𝑃 !"## and corrupted 𝐸𝑅𝑅 !"## packets:

𝐶 !"## = !"#$ !"## ! !"" !"## !" !"## ! !" !"## 𝐶 !"!!" 𝑖 :

Congestion on the Wi-Fi interface of node i

The interface driver offers an estimation of total transmitted 𝑇𝑋 !"#" , received 𝑅𝑋 !"#" , dropped 𝐷𝑅𝑂𝑃 !"#" and corrupted 𝐸𝑅𝑅 !"#" packets:

𝐶 !"#" = !"#$ !"#" ! !"" !"#" !" !"#" ! !" !"#"

𝐷 𝑖 :

Average distance from neighbors of node i

The OLSR routing tables include the hop distance from other nodes in the SSN. 𝐷 𝑖 is the average of those hop distances.

Section B

Stimulus and Threshold computation

Figure 1 .

 1 Figure 1. Use cases and communication scenario.

Figure 2 .

 2 Figure 2. The generic architecture of a Stem-Phone.

  ); in particular, the Context Manager estimates the most Relevant Parameters (RP) (residual energy, congestion on Wi-Fi and Cellular interfaces, average distance), the Cooperation Manager takes care of the RP exchanges between smartphones, the Knowledge Database stores the RP both concerning the smartphone itself and neighboring ones. The Policy Manager translates the user preferences such as his/her reluctance to share the residual charge and their acceptable performance. Finally the Control & Decision Brain computes the metrics: 𝑆𝑀(𝑔), 𝑇𝑀(𝑖) and takes decisions.

Figure 3 .

 3 Figure 3. The test-bed set up.

Figure 4 .

 4 Figure 4. Throughput experienced by Phone A using different residual charge for both Phones B and C.

Figure 1 .

 1 Figure 1. Use cases and communication scenario.

Figure 2 .

 2 Figure 2. The generic architecture of a Stem-Phone.

Figure 3 .

 3 Figure 3. The test-bed set up.

Figure 4 .

 4 Figure 4. Throughput experienced by Phone A using different residual charge for both Phones B and C.

1 Table 2 .

 12 Stimulus at node g 𝑆𝑀(𝑔) = 𝛼(1 -𝐸(𝑔)) + 𝛽(𝐶 !"## (𝑔)) where the smoothing factors 𝛼 and 𝛽 are chosen to respect the relation 𝛼 + 𝛽 = 1 𝐹(. . . ) Threshold at node i 𝑇𝑀 𝑖 = 1smoothing factors 𝛾 , 𝛿 , and 𝜑 are chosen to respect the relation 𝛾 + 𝛿 + 𝜑 = Parameters estimation and Stimulus-Threshold computation.

Table 1 . Enabling technologies for SSNs.

 1

AUTHORS' BIOGRAPHIES

Gianluca Aloi [S'99-M'02] (aloi@dimes.unical.it) received his M.S. degree in Computer Science in 1999 and his Ph.D. degree in Systems Engineering and Computer Science in 2003. He joined the University of Calabria in 2004 where, currently, he is Assistant Professor at the Department of Informatics, Modeling, Electronics and Systems (DIMES). His research interests include spontaneous and reconfigurable wireless networks, cognitive networks, resource provisioning and sharing, software defined radio systems, localization systems and satellite communications. Giuseppe Ruggeri. (giuseppe.ruggeri@unirc.it) received the master degree in electronics engineering in 1998 From University of Catania ,Italy. In 2002 he received the Ph.D. in electronics, computer science and telecommunications engineering from University of Palermo (Italy). He is currently Assistant Professor at the Department of ICT, Infrastructures, and Sustainable Energy (DIIES) at the University "Mediterranea" of Reggio Calabria (Italy). His research interests focus on wireless networking, Self Organizing Networks, and Information Centric Networking (ICN).

Marco


Valérie Bellamy

Valérie Vanneaux

Alain Bel

Hany Nemetalla

Emmanuelle Solène

Yohan Boitard

Pierre Farouz

Marie-Cécile Joanne

Estelle Perier

Chantal Robidel

Mandet

Emmanuelle Boitard

Yohan Farouz

Pierre Joanne

Marie-Cécile Perier

Estelle Robidel

Chantal Mandet

Albert Hagège

Patrick Bruneval

Jérôme Larghero

Onnik Agbulut
email: onnik.agbulut@upmc.fr

Philippe Menasché
email: philippe.menasche@egp.aphp.fr

Long-term functional benefits of human embryonic stem cell-derived cardiac progenitors embedded into a fibrin scaffold

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

At least, two important lessons can be drawn from the multiple cardiac stem cell therapy reported so far. The first is that regardless of whether the transplanted cells directly generate a new myocardial tissue or, more likely, act by harnessing endogenous repair pathways to induce host-derived regeneration, 1 the most effective seem those committed to a cardiac lineage. [START_REF] Crisostomo | Embryonic stem cells attenuate myocardial dysfunction and inflammation after surgical global ischemia via paracrine actions[END_REF][START_REF] Li | Direct comparison of different stem cell types and subpopulations reveals superior paracrine potency and myocardial repair efficacy with cardiosphere-derived cells[END_REF] In this setting, along with mesenchymal stem cells exposed to a cocktail of cardiopoietic factors prior to transplantation [START_REF] Bartunek | Cardiopoietic stem cell therapy in heart failure: the C-CURE (Cardiopoietic stem Cell therapy in heart failURE) multicenter randomized trial with lineage-specified biologics[END_REF] and cardiospheres, 5 pluripotent stem cells are attractive because of their intrinsic capacity to be differentiated in any lineage, including the cardiomyogenic one, in response to appropriate cues. [START_REF] Pal | Embryonic stem (ES) cell-derived cardiomyocytes: a good candidate for cell therapy applications[END_REF] The second important lesson is that conventional needle-based intramyocardial injections have several limitations, including random distribution of cells, stress-induced cell damage [START_REF] Aguado | Improving viability of stem cells during syringe needle flow through the design of hydrogel cell carriers[END_REF] and, possibly, induction of arrhythmias. [START_REF] Fukushima | Direct intramyocardial but not intracoronary injection of bone marrow cells induces ventricular arrhythmias in a rat chronic ischemic heart failure model[END_REF][START_REF] Terajima | Autologous skeletal myoblast sheet therapy for porcine myocardial infarction without increasing risk of arrhythmia[END_REF] Since cardiac surgery provides the unique opportunity of a direct control over the heart, several studies have assessed the effects of replacing multiple intramyocardial injections by the epicardial delivery of a cellularized patch and, overall, their results support the superiority of the latter approach with regard to cell survival, [START_REF] Sekine | Cardiac cell sheet transplantation improves damaged heart function via superior cell survival in comparison with dissociated cell injection[END_REF][START_REF] Visage | Mesenchymal stem cell delivery into rat infarcted myocardium using a porous polysaccharide-based scaffold: a quantitative comparison with endocardial injection[END_REF][START_REF] Sekiya | Muscle-derived stem cell sheets support pump function and prevent cardiac arrhythmias in a model of chronic myocardial infarction[END_REF][START_REF] Araña | Epicardial delivery of collagen patches with adiposederived stem cells in rat and minipig models of chronic myocardial infarction[END_REF] reduction of fibrosis, [START_REF] Sekiya | Muscle-derived stem cell sheets support pump function and prevent cardiac arrhythmias in a model of chronic myocardial infarction[END_REF][START_REF] Araña | Epicardial delivery of collagen patches with adiposederived stem cells in rat and minipig models of chronic myocardial infarction[END_REF][START_REF] Hamdi | Cell delivery : Intramyocardial injections or epicardial deposition ? A head-to-head comparison[END_REF] increase in angiogenesis [START_REF] Sekiya | Muscle-derived stem cell sheets support pump function and prevent cardiac arrhythmias in a model of chronic myocardial infarction[END_REF][START_REF] Araña | Epicardial delivery of collagen patches with adiposederived stem cells in rat and minipig models of chronic myocardial infarction[END_REF][START_REF] Hamdi | Cell delivery : Intramyocardial injections or epicardial deposition ? A head-to-head comparison[END_REF] and improvement of function. [START_REF] Sekine | Cardiac cell sheet transplantation improves damaged heart function via superior cell survival in comparison with dissociated cell injection[END_REF][START_REF] Sekiya | Muscle-derived stem cell sheets support pump function and prevent cardiac arrhythmias in a model of chronic myocardial infarction[END_REF][START_REF] Araña | Epicardial delivery of collagen patches with adiposederived stem cells in rat and minipig models of chronic myocardial infarction[END_REF][START_REF] Hamdi | Cell delivery : Intramyocardial injections or epicardial deposition ? A head-to-head comparison[END_REF][START_REF] Hamdi | Epicardial adipose stem cell sheets results in greater post-infarction survival than intramyocardial injections[END_REF] Additional advantages of the patch-based approach include strengthening of the infarcted wall which may contribute to limit adverse remodelling [START_REF] Gaballa | Grafting an acellular 3dimensional collagen scaffold onto a non-transmural infarcted myocardium induces neoangiogenesis and reduces cardiac remodeling[END_REF] and provision of a template for cells to proliferate and secrete their own matrix to which they can anchor. [START_REF] Hamdi | Epicardial adipose stem cell sheets results in greater post-infarction survival than intramyocardial injections[END_REF] Among the diverse materials that can be used, fibrin is attractive because of its multiple advantages which include biocompatibility, suitability for cell adhesion, [START_REF] Barsotti | Fibrin as a scaffold for cardiac tissue engineering[END_REF] possibility to act as a reservoir for controlled-release growth factors [START_REF] Rajangam | Fibrinogen and fibrin based micro and nano scaffolds incorporated with drugs, proteins, cells and genes for therapeutic biomedical applications[END_REF] and a long-standing safety record when used as an hemostatic sealant in surgery. [START_REF] Rousou | Use of fibrin sealants in cardiovascular surgery: a systematic review[END_REF] The present study was therefore designed to leverage these data by combining the use of human embryonic stem cell (ESC)-derived cardiac progenitors with their incorporation into a fibrin scaffold and to assess the outcome of such a composite construct in a rat model of myocardial infarction subjected to a serial and extended (4 months) functional follow-up.

Methods

Animals

Seven-hundred thousand SSEA-1-positive progenitors were first added to 150 µL of a solution made of fibrinogen (20 mg/mL) and α-MEM medium (Macopharma, Tourcoing, France) in an agarosecoated Petri dish. Four units of thrombin were then added as evenly distributed 25-µL droplets of an equivalent volume of culture medium supplemented with thrombin to induce polymerization of the gel, which occurred in less than 10 minutes (at 37°C). Both fibrinogen and thrombin were components of the Evicel® kit (Ethicon Biosurgery, Omrix Biopharmaceuticals-Ethicon Biosurgery, Rhode Saint Genèse, Belgium). Previous studies in the laboratory have shown that this fibrinogen-thrombin ratio is appropriate to generate mechanically robust patches with good handling and suturability characteristics and well-preserved cell viability.

Assessment of left ventricular function

Left ventricular function was assessed by echocardiography at baseline and every month thereafter until sacrifice at 4 months, as described previously [START_REF] Hamdi | Cell delivery : Intramyocardial injections or epicardial deposition ? A head-to-head comparison[END_REF] (more details are available in the Supplementary Material online section).

Histological and immunohistochemical analyses

Four months following the sham or transplantation procedures, rats were euthanized and sacrificed.

Explanted hearts were processed for the determination of fibrosis and angiogenesis. In separate experiments, tracking of cell fate was performed one day and one week after transplantation using immunostaining against lamin A/C (all procedures detailed in the Supplementary Material online section) and completed, at 4 months only, by the search for human-specific Alu sequences by RT-PCR.

Relative quantification of gene expression by real-time PCR

Total RNA was extracted from heart tissue samples using TRIzol® lysis reagent (Life Technologies, Saint-Aubin, France) and TissueLyser II system (Qiagen France SAS, Courtaboeuf, France) following the manufacturers' instructions. Real-time PCR was performed using LightCycler 480® (Roche Diagnostics, France) as described in the Supplementary Material online section.

Assessment of a heart failure index

In 20 randomly selected hearts (n=7, 7 and 6 for sham-operated, control acellular fibrin patches and cell-treated patches, respectively), a whole set of functional and PCR data were available. This cohort was therefore used for generating a 5-component heart failure score including the absolute change in LV end-systolic volume (ESV) between 4 months and baseline, and the qPCR-based expression of natriuretic peptides A and B, myosin heavy chain 7 and periostin.

Statistics

Data are summarized using mean (IC95%) or mean (SEM) values. Survival between groups was compared using the log-rank test and Kaplan Meier curves were plotted. Within each group, LV function variables measured at baseline and at 4 months were compared using paired Student tests.

Across groups, the changes of LV function variables between repeated assessment (baseline, 1, 2, 3 and 4 months) were compared using a linear mixed model analysis. The mean percentages of fibrosis and angiogenesis were compared between groups using analysis of variance. Pairwise comparisons were done and p-values were adjusted for multiple testing (Tukey adjustment). The "heart failure index" was constructed by standardizing the 5 components on the overall population (= computing zscore for each variable) and then averaged them. Comparisons of this index between groups were made using analysis of variance (Tukey adjustment for multiple testing). For all analyses, a two-tailed p-value <0.05 was considered statistically significant.

Analyses were conducted using SAS 9.4 (Statistical Analysis System, Cary, NC, USA).

RESULTS

Characteristics of the transplanted SSEA1 + cell population.

Following immunomagnetic sorting, the purity rate of the SSEA-1-positive cell population was 92.5% ± 2.6%. The transcriptomic profiling of this population, as assessed by Affymetrix microarray analysis, showed the down-regulation of pluripotency-associated genes like SOX2, SOX21, NANOG, HESX1, LEFTY, ZIC3 (Figure 1A), as compared with undifferentiated I6 ESC and this pattern was mirrored by the upregulation of cardiac mesoderm-committed genes like HAND1, ISL1, EOMES, HAPLN1, BMP4, PKP2 (Figure 1B). Based on that same transcriptomic analysis, the SSEA-1negative cell fraction was also characterized by a switch-off of most pluripotency genes (Figure 1A). Four hours after sorting, a time point selected to match the timing by which the patch would be effectively delivered onto the heart, less than 10% of the SSEA-1-positive scaffold-embedded cells were found apoptotic, as shown by TUNEL staining and caspase-3 expression (data not shown), while markers suggestive of a cardiomyogenic lineage like Isl-1 were identified (Figure 2, upper panel).

Moreover, the ability of the SSEA-1-positive cells to keep proliferating into the fibrin scaffold was demonstrated by Ki67 staining (Figure 2, lower panel).

Left ventricular function assessment.

After exclusion of rats with preserved LV functions (LVEF ≥ 55%), baseline LVEF did not differ between the three groups: 47.46% [95%CI:45.28%;49.63%], 47.68% [45.96%;49.40%], and 47.37% [44.97%;49.77%], in sham-operated (n=18), control (n=19) and treated (n=20) hearts, respectively .

Over the 4-month follow-up period, the death rate did not significantly differ among the three groups (12, 13 and 14 deaths in the sham, control and treated groups, respectively; Figure 3A) which let a total of 41 surviving animals (13 sham-operated rats, 12 controls and 16 treated rats) meeting the preset inclusion criterion (LVEF ≤55%) available for the last 4-month echocardiographic assessment.

Over this 4-month follow-up period, the patterns of changes remained fairly consistent within each group (Figure 3B). Function remained stable in the sham group in which the LVEF, at 4 months, was 48.45% (44.71%;52.18%); it slightly increased in the control acellular fibrin group (50.36% [47.29%;53.42%]) and to a still greater extent in the cell-treated group (53.19% [50.10%;56.29%], p=0.002 vs sham). When data were expressed as the absolute difference between the baseline value and the 4-month final study point, LVEF was found to have increased by 0.09%±0.99% (mean±SEM) in sham-operated hearts, 2.36%±0.80% in control cell-free fibrin-treated hearts (p=0.014 vs baseline), and 5.68%±1.29% in cell+fibrin-treated hearts (p=0.0005 vs baseline).

Left ventricular end-diastolic volumes (LVEDV) were likewise similar at baseline between the three groups:

344.81μL (95%CI:323.89;391.37) in sham, control and treated hearts, respectively. However, the patterns of changes were then markedly different (Figure 3C). Volumes steadily increased to a similar extent in the sham and control groups (absolute difference between 4 months and baseline: 44.68 μL±20.83μL; p=0.053 and 50.27μL±25.95μL, p=0.07 respectively) whereas the LV dilatation was much less pronounced in the treated group (absolute difference between 4 months and baseline: 4.64μL±24.07 μL, p=0.85). Patterns of changes in LV end-systolic volumes (LVESV) still more clearly delineated between-group differences (Figure 3D) as they increased in the sham and control groups (absolute difference between 4-month and baseline values: 28.08μL±14.65μL and 15.41μL±13.36μL, respectively), whereas they decreased by 16.76μL±15.55μL in hearts receiving the cell-loaded fibrin patch (p=0.013 vs sham; p=0.052 vs controls).

The functional efficacy of epicardially delivered SSEA-1-positive progenitors was partially supported by real-time PCR analysis, which demonstrated a reduced expression of the stress-induced betamyosin heavy chain genes, atrial natriuretic factor, brain natriuretic peptide and periostin in the treated group as compared to sham (data not shown). When these PCR data were combined with LVESV changes to generate a 5-component heart failure index (Figure 4), the highest score was seen in the sham-operated group (2.24±1.29). It was lower in control hearts (-0.74±0.84) and further decreased in the treated group (-1.75±0.84, p=0.04 vs sham).

Histological and immunohistochemical analyses.

The extent of fibrosis did not significantly differ between the three groups (Figure 5A). Conversely, angiogenesis in the border zone was significantly greater in the cell+fibrin group (p=0.006 vs sham, Figure 5B). At the 4-month time point, there was no teratoma in 6 hearts treated with the cell-loaded fibrin patch which were specifically examined for this complication. Lamin A/C-positive human cells were detected by immunostaining one day after transplantation in the patch implantation area (Supplemental Figure 1) but could no longer be found after one week. Based on this result, we reasoned that the probability for cells to persist at 4 months was extremely small and therefore used PCR rather than immunostaining to sensitize their tracking in 6 cells+fibrin-treated hearts, taking 7 sham-operated hearts and 7 acellular fibrin patch-grafted hearts as controls. Three primers were used for probing different regions of the human-specific Alu DNAsequence but, as expected, there was no detectable signal with either primer at this late time point in any heart tissue sample (comprising both the area of patch implantation and the nongrafted underlying myocardium).

DISCUSSION

The major finding of the present study is that a fibrin patch, in which ESC-derived cardiac progenitors have been embedded, improves pump function and limits adverse ventricular remodelling in a rat model of myocardial infarction and that, given the life expectancy of rats, these effects seem to be sustained over a reasonably long period of time.

Cell scaffolding.

. After having tested several materials, including scaffold-free cell sheets, [START_REF] Hamdi | Cell delivery : Intramyocardial injections or epicardial deposition ? A head-to-head comparison[END_REF][START_REF] Hamdi | Epicardial adipose stem cell sheets results in greater post-infarction survival than intramyocardial injections[END_REF][START_REF] Hamdi | Efficacy of Epicardially-delivered Adipose Stroma Cell Sheets in Dilated Cardiomyopathy[END_REF] we selected fibrin as the cell matrix. In addition to its multiple above mentioned advantages, fibrin is attractive because of the tunability of its mechanical properties through an adjustment of the fibrinogen to thrombin ratio.

Thus, in the present experiments, the selected ratio (20mg/mL and 4 Units/mL, respectively) allowed to achieve both an elasticity modulus (in the range of 6kPa) matching that reported appropriate for cells of the mesodermal lineage [START_REF] Jacot | Substrate stiffness affects the functional maturation of neonatal rat ventricular myocytes[END_REF] and a fiber network density allowing ingrowth of host-derived vessels (Figure 5). The choice of transplanting 700,000 progenitor cells was based on preliminary screening experiments but we acknowledge that upscaling this number might have increased cell engraftment, at least early after patch delivery, as suggested by our observation of numerous donor cells inside the scaffold in a separate experiment where it was loaded with two million cells (Figure 6).This observation matches the finding that fibrin can increase the amount of cells retained into the myocardium 24 hours after an intramyocardial injection. 28

Interpretation of outcomes.

The present results demonstrate that hearts receiving the cell-free fibrin patch tended to have slightly better functional outcomes than the sham-operated animals, which has been previously reported with the use of injected fibrin alone [START_REF] Christman | Fibrin glue alone and skeletal myoblasts in a fibrin scaffold preserve cardiac function after myocardial infarction[END_REF] and which likely reflects an angiogenic effect. , [START_REF] Liu | Autologous stem cell transplantation for myocardial repair[END_REF][START_REF] Ye | Thymosin β4 increases the potency of transplanted mesenchymal stem cells for myocardial repair[END_REF] However, embedding SSEA-1 + cells in the fibrin matrix further improved contractile function and stabilization of remodelling as cells+fibrin-treated hearts consistently yielded higher values of LVEF (the increase of which between 4 months and baseline was more than two-fold higher than in the acellular fibrin group) but more importantly lower values of LVEDV and LVESV compared with their cell-free counterparts. Although focus on EF could suggest that LV function was only moderately impaired at baseline (after infarction), analysis of LV volumes at the same time point shows that they were dramatically enlarged compared with healthy hearts, [START_REF] Kang | Human vasculogenic cells form functional blood vessels and mitigate adverse remodeling after ischemia reperfusion injury in rats[END_REF][START_REF] Sabatini | Effects of injectable anesthetic combinations on left ventricular function and cardiac morphology in Sprague-Dawley rats[END_REF] which makes the 4-month attenuation of remodelling yielded by the cells+fibrin patch functionally relevant, particularly if one keeps in mind that LVESV has been reported to be a strong predictive factor of outcome in patients following a myocardial infarction. [START_REF] Norris | Prognosis after recovery from myocardial infarction: the relative importance of cardiac dilatation and coronary stenoses[END_REF] In keeping with these data, the cell-scaffold product also achieved the lowest value of the heart failure index generated by combining genomic and functional end points associated with heart failure, 35 cardiomyopathy 36 and adverse LV remodelling. [START_REF] Stansfield | Periostin is a novel factor in cardiac remodeling after experimental and clinical unloading of the failing heart[END_REF] Overall, these results are consistent with those previously reported with the use of fibrin used either in an injectable form mixed with skeletal myoblasts [START_REF] Christman | Injectable fibrin scaffold improves cell transplant survival, reduces infarct expansion, and induces neovasculature formation in ischemic myocardium[END_REF] or bone marrow-derived cells [START_REF] Nakamuta | Cell therapy attenuates cardiac dysfunction post myocardial infarction: effect of timing, routes of injection and a fibrin scaffold[END_REF][START_REF] Ryu | Implantation of bone marrow mononuclear cells using injectable fibrin matrix enhances neovascularization in infarcted myocardium[END_REF] or as an epicardial patch loaded with mouse ESC-derived BMP-2-treated cardiac progenitors [START_REF] Vallée | Embryonic stem cell-based cardiopatches improve cardiac function in infarcted rats[END_REF] or human hESC-derived vascular cells 41 in rat and porcine myocardial infarct models, respectively. Compared with the latter two studies, the present one differs by (1) the phenotype of the cells (in Vallée's study, [START_REF] Vallée | Embryonic stem cell-based cardiopatches improve cardiac function in infarcted rats[END_REF] ESC-derived cardiac progenitors were of mouse origin while in Xiong's study, [START_REF] Xiong | A fibrin patch-based enhanced delivery of human embryonic stem cell-derived vascular cell transplantation in a porcine model of postinfarction left ventricular remodeling[END_REF] the derivatives of the human ESC were not cardiomyocytes), (2) the extension of follow-up to 4 months (versus 50 days and 4 weeks in Vallée's and Xiong's studies, respectively), and (3) the fact that the tested cell-scaffold construct was manufactured, processed and quality-controlled under similar conditions as those used for the product planned for a human use. It is even possible that the beneficial effects of the fibrin material could have been optimized by seeding cells at the surface of the fibrin biomatrix instead of incorporating them into it. [START_REF] Gerard | Fibrin gel improves the survival of transplanted myoblasts[END_REF] The mechanism of the incremental benefit afforded by the cell-loaded fibrin patch remains to be investigated. A de novo muscularization of the infarct tissue by the grafted cells can be excluded. In the present study, two methods were used for tracking donor cell fate: lamin A/C protein expression and human Alu gene expression. As early as one week after patch implantation, lamin A/C-positive cells could no longer be detected and, expectedly, were not identified either after 4 months despite the use of a highly sensitive PCR-based tracking method. [START_REF] Schneider | Quantification of human Alu sequences by real-time PCR -an improved method to measure therapeutic efficacy of antimetastatic drugs in human xenotransplants[END_REF] With the caveat of a shorter follow-up, a higher survival rate has been reported with the use of a fibrin patch loaded with human hESC-derived vascular cells, [START_REF] Xiong | A fibrin patch-based enhanced delivery of human embryonic stem cell-derived vascular cell transplantation in a porcine model of postinfarction left ventricular remodeling[END_REF] which might be related to differences in cell phenotype, processing, or timing of delivery. Whatsoever, the contrast, in the present experiments, between the absence of a long-term cell engraftment and a sustained functional benefit, similar to what has been reported with other cell types, [START_REF] Levit | Cellular encapsulation enhances cardiac repair[END_REF] strongly supports a cell-induced paracrine signalling initiating a cascade of self-sustained events. This would be consistent with an attenuation of fibrosis and the increase in angiogenesis seen in the treated group. Our failure to identify cells expressing both markers of proliferation (Ki67) and cardiac lineage (troponin I) does not necessarily rule out an additional increase in the contractile cell pool (by mobilization of endogenous stem/progenitors or new divisions of mature cardiomyocytes) which could have been missed because of the late postprocedural sampling time (4 months). This paracrine hypothesis is relevant to the patch-based delivery strategy as shown by the ability of a cardiomyocyte-containing alginate scaffold to improve postinfarction heart function even though the grafted cells remain insulated from the host myocardium and cannot therefore couple with them. [START_REF] Wendel | Functional consequences of a tissueengineered myocardial patch for cardiac repair in a rat infarct model[END_REF] Reliance on paracrine effects has practical implications in that it leads to focus on the enhancement of early retention of grafted cells (rather than on their sustained survival), within a time frame matching the release of cell-derived biomolecules which could then paracrinally foster innate repair pathways before clearance of the donor cells . [START_REF] Levit | Cellular encapsulation enhances cardiac repair[END_REF] We acknowledge that we did not dissect out the molecular events underpinning the postulated paracrine activation of endogenous reparative responses. Indeed, an increasing role is attributed to exosomes as possible mediators of the cells' paracrine effects [START_REF] Chen | Cardiac progenitor-derived exosomes protect ischemic myocardium from acute ischemia/reperfusion injury[END_REF][START_REF] Ibrahim | Exosomes as critical agents of cardiac regeneration triggered by cell therapy[END_REF] and deciphering the nature of their content, particularly the miRNA profile, should help in mechanistically identifying the various pathways targeted by the cell-released biomolecules while providing clues for developing cell mimetics more suitable for large-scale clinical applications.

An important observation was the lack of teratoma in the immunodeficient rats after 4 months. This likely reflects the efficiency of the selection procedure aimed at discarding the cells unresponsive to the cardioinstructive cues. In this study, the population of progenitors was purified by immunomagnetic sorting against SSEA-1 (also called Sialyl-Lewis X ), a carbohydrate moiety expressed early during the differentiation of pluripotent ESC [START_REF] Mummery | Differentiation of human embryonic stem cells and induced pluripotent stem cells to cardiomyocytes: a methods overview[END_REF] which is consistent with the fact that cell surface proteins incur changes in their glycosylation patterns when they transition from pluripotency to differentiation. The transcriptomic analysis of the purified SSEA-1-positive population has confirmed the down-regulation of pluripotency genes (primarily NANOG and SOX-2) which may still be expressed at various degrees in the residual contaminating SSEA-1-negative cells but probably not to the extent that a teratoma can develop since injection of this sole fraction in immunodeficient mice still failed to induce teratoma formation. [START_REF] Menasché | Towards a clinical use of human embryonic stem cell-derived cardiac progenitors: a translational experience[END_REF] The disappearance of the cells over time may also be considered as an additional flail-safe mechanism.

Limitations.

We acknowledge several limitations of this study. First, the follow-up was not extended beyond 4 months. However, such a duration is yet significant given the rat's life expectancy and the stability of functional data, as evidenced by the serial echocardiograms, provides an encouraging signal regarding the maintenance of the functional benefits over time. A second limitation is that cells may not migrate into the myocardium. This would be an issue if the grafted cells were supposed to generate a de novo myocardial tissue but, indeed, is not if they act primarily by releasing a blend of factors which can be released from the patch towards the underlying myocardium to harness endogenous repair pathways.

Third, we did not document the potential occurrence of arrhythmic events. However, (i) the survival rate of the treated rats was not different from that of sham-operated animals and although we were not able to determine the causes of death in each group, this indirectly argues against a substantially greater occurrence of cell-induced life-threatening arrhythmias, and (ii) patches have been reported to be less arrhythmogenic than injections. ,9 Fourth, our "customized" heart failure score should be interpreted cautiously because it has not been externally validated and was constructed as a post-hoc composite of variables; of note, however, this index was not designed to predict survival, like the heart failure scores commonly used in the clinics, [START_REF] Levy | The Seattle Heart Failure Model: prediction of survival in heart failure[END_REF] but rather to assess whether the combination of end points generated by different methods (echocardiography and genomics) but all relevant to heart dysfunction, would confirm the benefits of the cells+scaffold approach, which actually turned out to Nuclei were counterstained by hematoxyline (30 sec) and slides were mounted with Eukitt (Kindler, Freiburg, Germany). The detection of human cells entailed the use of a monoclonal anti-human lamin A/C antibody (dilution 1/300; Novocastra, A.Menarini, Rungis, France). Furthermore, six hearts were specifically examined for the search of a teratoma. Photographs were taken using a Leica DM2000 microscope (Leica, Wetzlar, Germany) coupled to a Quicam CCD camera (Qimaging Corp., Surrey, BC, Canada). Results were distinguished from autofluorescence by checking all three filters. Data were collected using QCapture software, QCam Driver DLL version 1.73.0 (Qimaging Corp.) and quantified using Metamorph Offline Version 6.3r.2 (Molecular Devices, Sunnyvale, CA).

Relative quantification of gene expression by qPCR

Total RNA was extracted from heart tissue samples using TRIzol® lysis reagent (Life Technologies, Saint-Aubin, France) and TissueLyser II system (Qiagen France SAS, Courtaboeuf, France) following the manufacturers' instructions. Extracted RNA was spectrophotometrically quantified using NanoDrop 2000 (Thermo Fisher Scientific, Saint Herblain, France). From 1000 ng of extracted RNA, the first-strand cDNA was then synthesized using the RevertAid First Strand cDNA Synthesis Kit

Figure 1 .

 1 Figure 1. In vitro characterization of human embryonic stem cells (ESC)-derived SSEA-1-positive progenitors. Expression level of selected pluripotency-associated (A) and cardiac lineage commitment-associated (B) genes, as assessed by a transcriptome analysis using Affymetrix microarrays, in SSEA-1-positive progenitors, SSEA-1-negative cells and undifferentiated pluripotent I6 ESC taken as the reference (100%). Data are reported as means±SEM. Heatmaps of the transcriptome have been previously published.[START_REF] Menasché | Towards a clinical use of human embryonic stem cell-derived cardiac progenitors: a translational experience[END_REF]

Figure 2 .

 2 Figure 2. Immunolabeling of SSEA-1-positive progenitors in the fibrin patch. The upper panel shows that the fibrin-embedded SSEA-1-positive progenitors express the cardiac transcription factor Isl-1 (green fluorescence) and the proliferation-associated protein Ki67 (green fluorescence, lower

Figure 3 .

 3 Figure 3. Four-month follow-up of SSEA-1-positive progenitor-grafted hearts in comparison with sham and control groups. (A) Kaplan-Meier survival curves during the 4-month follow-up. Percentages of surviving animals, in sham (n=13 out of 25), control (n=12 out of 25) and treated (n=16 out of 30) groups. No significant difference was observed in the survival rate between the different groups. Significance was analyzed with the log-rank test. (B) Changes in left ventricular ejection fraction (LVEF) over the 4-month follow-up period. Values are presented as percentages. * p=0.014 vs baseline; ** p=0.0005 vs baseline; p=0.002 vs sham. (C) Absolute changes in left ventricular enddiastolic volume (LVEDV) over the 4-month follow-up period; * p=0.053 vs baseline. (D) Absolute changes in left ventricular end-systolic volume (LVESV) over the 4-month follow-up period; * p=0.013 vs sham; p=0.052 vs controls. Data are reported as means±SEM.

Figure 4 .

 4 Figure 4. Box-blot representation of the heart failure index. The absolute change in LVESV between 4 months and baseline and the qPCR-based expression of natriuretic peptides A and B, myosin heavy chain 7 and periostin were used to construct a 5-component heart failure index. The score yielded by the control and treated groups was calculated relative to that of the total 20-heart cohort. The boxes represent the lower to upper quartile range, the error bars represent the entire range, the horizontal bar represents the median value and dot points represent mean values. Note the significantly lower heart failure index in the treated group vs sham.

Figure 5 .

 5 Figure 5. Box-blot representation of fibrosis (A) and number of capillaries (B) in sham, control and treated hearts. Four months after treatment, 6-10 sections per heart were stained by Sirius red (fibrosis) or immunostained with a rat-specific endothelial cell antibody (to label capillaries; 7 sections per heart). The area of fibrosis and number of capillaries were quantified using Metamorph software (Universal Imaging Corporation, USA). Fibrosis was expressed as the ratio between the area of scar tissue to the left ventricular area. The number of capillaries was expressed as per mm 2 of the infarct border zone. The boxes represent the lower to upper quartile range, the error bars represent the entire range, the horizontal bar represents the median value and dot points represent mean values.

(

  Thermo Fisher Scientific) with anchored-oligo(dT)18 primer and according to the manufacturer's instructions using Light Cycler 480 system (Roche Diagnostics, Meylan, France). The reaction was carried out in duplicate for each sample in a 6-µl reaction volume containing 3 µl of SYBR Green Master Mix, 500 nM each of the forward and reverse primers and 3 µl of diluted (1:25) cDNA. The -25thermal profile for SYBR Green qPCR was 95°C for 8 min, followed by 40 cycles at 95°C for 15 s, 60°C for 15 s and 72°C for 30 s. To exclude PCR products amplified from genomic DNA, primers were designed to span one exon-exon junction. Primer sequences used in this study are available on request. The gene expression stability of B2m, Actb, Hprt1 and Nubp1 were calculated using geNorm software and after analysis only Nubp1 and Actb were used as the references transcripts. Data were collected and analyzed using the LightCycler® 480 software release 1.5.0 (Roche).

Fig 5 - 30 -

 530 Fig 1

FUNDINGS

This work was supported by public funds from INSERM, University Paris Descartes and the LabEx REVIVE; and by charity funds from the Fondation de France, the Fondation Coeur et Artères, the Fondation de l'Avenir (ET2-658) and the Association Française contre les Myopathies, ANR-12-RPIB-0015. P.J. was supported by a fellowship from the Fondation Lefoulon-Delalande and S.B. from the Conseil Régional d'Ile de France (DIM Biothérapies).

Eighty female Nude rats (Charles River) weighing an average of 200-250 g were used in this study.

All procedures were approved by our institutional Ethics Committee and complied with the European legislation (European Commission Directive 86/609/EEC) on animal care.

Experimental protocol

Myocardial infarction was created as described previously [START_REF] Hamdi | Cell delivery : Intramyocardial injections or epicardial deposition ? A head-to-head comparison[END_REF] . Five to 7 weeks after myocardial infarction, rats underwent a baseline echocardiographic assessment of left ventricular (LV) function and all those with an ejection fraction (EF) ≥55% were excluded. The remaining rats were reoperated on by a median sternotomy and randomly allocated to receive a cell-free fibrin patch (n=25) or a fibrin patch loaded with 700,000 ESC-derived SSEA-1 + progenitors (n=30). The patch was delivered onto the clearly visible akinetic area and secured to the epicardium by three 6/0 polypropylene sutures.

Twenty-five additional rats assigned to a sham group only underwent the open-chest placement of similar sutures, without any other procedure. No medical treatment for heart failure was given. Three additional rats receiving a progenitor cell-loaded fibrin patch were used for an early posttransplantation (1, 2 and 7 days) histological assessment.

Preparation of the SSEA-1 + progenitors

The progenitors were derived from the I6 ESC line (generously provided by J. Itskovitz and M. Amit, Technion Institute, Haïfa, Israël) according to a a relatively simple growth factor-based protocol attempting to recapitulate signalling events involved in embryonic cardiogenesis [START_REF] Leschik | Cardiac commitment of primate embryonic stem cells[END_REF][START_REF] Verma | Steering signal transduction pathway towards cardiac lineage from human pluripotent stem cells: a review[END_REF][START_REF] Vallier | Activin/Nodal and FGF pathways cooperate to maintain pluripotency of human embryonic stem cells[END_REF] (detailed in the Supplementary Material online section). This protocol allowed to reproducibly generate a population of SSEA-1-positive early cardiac progenitors, primarily expressing Isl-1, a transcription factor for both the first and second heart fields [START_REF] Moretti | Multipotent embryonic isl1+ progenitor cells lead to cardiac, smooth muscle, and endothelial cell diversification[END_REF] but also additional genes characteristic of different developmental stages of cardiogenesis. [START_REF] Menasché | Towards a clinical use of human embryonic stem cell-derived cardiac progenitors: a translational experience[END_REF] The cardiomyocyte-oriented fate of this population has previously been confirmed in vivo in a nonhuman primate model of myocardial infarction. [START_REF] Blin | A purified population of multipotent cardiovascular progenitors derived from primate pluripotent stem cells engrafts in post-myocardial infarcted non-human primates[END_REF] Preparation of the fibrin patch be the case. Finally, no mechanistic study was done to specifically determine the causes of cell loss which might have been facilitated by two model-intrinsic feature : (1) an immune response related to the xenogenic setting of the transplantation and which cannot be excluded, even in the nude rat, and

(2) the stress that human cells have to withstand when they are implanted in a fast-beating rodent heart..

In conclusion, the present data support the functional efficacy of fibrin scaffolds harbouring ESCderived cardiac progenitors and provide additional evidence that a long-term cell engraftment may not be a prerequisite for a sustained benefit. As such, they may serve as a platform for the further development of cell-, cell-mimetics-or biomaterial-based strategies aimed at fostering endogenous reparative responses.

CONFLICT OF INTEREST

None of the Authors has a conflict of interest to disclose.

Supplementary Materials

Preparation of the SSEA-1 + progenitors

The progenitors were derived from the human embryonic stem cell (ESC) line I6 generously provided by J. Itskovitz and M. Amit (Technion Institute, Haïfa, Israël). Following amplification onto irradiated human fibroblasts in Nutristem ™ (Biological Industries, Kibbutz Beit-Haemek, Israel), cells were committed towards a mesodermal-cardiac lineage by reverting the medium to α-Minimum Essential Medium Eagle (MEM) solution (Macopharma, Tourcoing, France) enriched with 2% insulinfree B27 supplement (Invitrogen, Life Technologies, Carlsbad, CA), Bone Morphogenetic Protein (BMP)-2 (Dibodermine alpha, 10 ng/mL, marketed as InductOs® [Wyeth pharmaceuticals, now part of Pfizer, New York, NY]) and a fibroblast growth factor receptor (FGFR)-specific tyrosine kinase inhibitor, SU-5402 (1µM; Merck, Whitehouse Station, NJ). This specification phase lasted for 4 days, at the completion of which cells were immunomagnetically sorted after labeling with a microbead-coupled -23anti-SSEA-1 antibody (Miltenyi, Teterow, Germany). The collected SSEA-1-positive cell yield was checked for its purity (percentage of SSEA-1-positive cells, as assessed by flow cytometry) and a full transcriptomic analysis of the parent undifferentiated I6 ESC, their SSEA-1-positive progeny as well as the SSEA-1-negative fraction was performed using the AffymetrixGeneAtlas TM system coupled to the new Human Genome U219 array (BIOAlternatives, Gencay, France). Heatmaps representative of this transcriptomic profiling have been previously published [START_REF] Menasché | Towards a clinical use of human embryonic stem cell-derived cardiac progenitors: a translational experience[END_REF] and only the most salient data are presented in Figure 1A andB.

Assessment of Left ventricular function

Left ventricular function was assessed by echocardiography at baseline and every month thereafter until the sacrifice at the 4-month study point as described previously. [START_REF] Hamdi | Cell delivery : Intramyocardial injections or epicardial deposition ? A head-to-head comparison[END_REF] Briefly, transthoracic echocardiography (Sequoia 516, Siemens, Seine-St.Denis, France) was performed with a 15 MHz transducer in animals sedated with 2 % isoflurane. Parasternal long and short axis views were obtained with assessment of LV end-diastolic (EDV) and end-systolic (EDS) volumes using the biplane arealength method [(8/3π) x (LVarea 2 /LVlength)]. Ejection fraction was then calculated as (LVEDV-LVESV)/(LVESV)x100. All measurements were made in triplicate and averaged by an investigator blinded to the treatment group.

Histological and immunohistochemical analyses

Four months following the sham or transplantation procedures, rats were euthanized and sacrificed.

Explanted hearts were rinsed in PBS, immediately fixed with Shandon Cryomatrix (Thermo Scientific, Illkirch, France) and frozen in -196 °C nitrogen. Blocks were stored at -80 °C until they were sliced in 7 µm-cryosections using a cryostat (CM 1850, Leica, Wetzlar, Germany). Vascularisation was assessed in 23 hearts (n=8, 7 and 8 for sham-operated, control acellular fibrin patches and cell-treated patches, respectively), in both the border zone of the infarct and in remote normal myocardium by immunostaining against rat endothelial cell antigen (RECA, Serotec, Oxford, UK). After exposure to the RECA antibody (diluted 1:30 in PBS-NHS 7 %) for 30 min, sections were incubated 30 min with a biotin-labelled anti-mouse antibody (Vector Laboratories Inc., Burlingame, CA 94010, USA) and preformed avidin-biotin peroxidase complexes (ABC/PO-solution, Vectastain ABC, Vector Labs