S3 begins within a region/country by exploiting place-based expertise and industrial skills within the regional innovation eco-system. The paper refers to emerging research which indicates that some regions suffer from insufficient innovation eco-system complexity, followed by sub-optimal innovation performances and path lock-in. This indicates that regional innovation eco-systems could be further strengthened through transnational learning and collaboration. Several major forms of collaboration are identified. The paper suggests that macro-regional and trans-European smart specialisation strategies could be based on multi-level approaches to experimentally extend and strengthen regional innovation eco-systems. In order to achieve robust and long-lasting outcomes, these experiments could apply some existing S3 tools. Here, an important issue is the transition from temporary programmes, projects and networks to new institutional frameworks for co-evolution and collaboration between smart specialised regions. The next important step is to exploit the European diversity identified through regional RIS3 strategies. The long-term challenge is the strengthening of emergent European and macro-regional systems of innovation, and thus supporting the regions.