**1. Introduction**

#### **1.1. The adipose-derived stem cell — A pluripotent adult stem cell?**

In 2001, the journal Tissue Engineering published an article describing the isolation of a population of putative multipotent stem cells from adipose tissue termed Processed Lipoas‐ pirate Cells or PLA cells [1]. Based on isolation methods designed for the harvest of adherent, fibroblastic cells from the adipose stroma capable of adipogenic differentiation in vitro [2], this work by Zuk et al. described the differentiation of their PLA cells toward multiple mesodermal lineages, including fat, bone and cartilage. This ground-breaking article has since been followed by over 3500 studies published and available through PubMed, describing the differentiation capacity of ASCs in a variety of in vitro and in vivo model systems. Early works continued the characterization of PLA cells – now termed ASCs for Adipose-derived Stem Cells - identifying a unique CD "signature" for these cells [3]-[8] and studying their mesoder‐ mal differentiation capacity at a molecular and biochemical level [8]. Subsequent studies have since confirmed the ASC's mesodermal differentiation capacity in vitro reporting osteogenic, adipogenic, chondrogenic and skeletal myogenic capacities [9]-[20]. These works have since been expanded into in vivo translational models using a variety of animal systems for bone formation [21]-[25], cartilage [26]-[28], fat [29]-[32] and skeletal muscle [33]-[35]. In addition, recent years have presented some exciting results, expanding ASC potential to add smooth muscle [36], [37] and cardiac myogenesis [38], [39] to the growing list of ASC capacities.

With these increased capacities, it became natural to ask if the ASC possessed pluripotent potential and initial in vitro studies appeared to answer this question, reporting ectodermal [8], and endodermal differentiation [40], [41]. However, the true test of these germ line potentials still lies in the in vivo model. Consistent with the in vitro studies, numerous in vivo

model systems have reported possible ectodermal and endodermal potentials, describing the repair of nervous and epithelial tissues [42], [43], together with hepatic and pancreatic regeneration [44]-[46]. With these in vivo results, combined with earlier in vitro analysis, it becomes easier to conclude that the ASC is an adult pluripotent stem cell population.

#### **1.2. ASC-mediated tissue regeneration: Secretion of soluble factors**

Despite the in vivo translational studies above suggesting that ASCs are capable of enhancing tissue healing and regeneration, many of these studies cannot confirm the direct differentiation of the ASC into a specific cell type. For example, while bone regeneration is observed upon implantation of ASCs, very few studies report the presence of the ASC within the newly formed bone. Whether this is an oversight by the research team or an indication that the ASC does not directly form part of the new tissue is unclear. It is entirely possible that the ASC does not directly differentiate into the desired regenerating tissue, but simply directs tissue formation "from the sidelines". Tissue development and healing is incredibly complex and the role of paracrine signaling is still not entirely understood. Therefore, it is possible that ASCs may be intimately involved in tissue regeneration and health through their ability to mediate the host's regenerative capacity using paracrine signaling.

Two arguments can be made in support of this theory. First, in many translational models, it does not appear that the ASC has any difficulty in surviving within the transplantation region for extended periods of time. In addition, the range of tissues capable of engrafting ASCs appears to be quite broad. Initial studies by Nolta and researchers show that systemic admin‐ istration of human ASCs is followed by multi-organ engraftment in nude mice [47]. In support of this, human ASCs administered via tail vein migrate and home efficiently to multiple tissues (epithelial and endothelial) in irradiated mice [48], [49]. The specific migration of ASCs to injured tissues has also been shown by the Longaker group, who confirm the presence of ASCs specifically in parietal bone defects and their persistence as the defect heals [50]. Second, stem cells like bone marrow MSCs and ASCs are known to secrete numerous factors and cytokines, including VEGF, HGF, NGF, BDNF and multiple interleukins [49], [51]. In fact, Salgado's article calls these factors the "secretome" of ASCs. This secretome may have powerful paracrine effects on the health, repair and function of a tissue and has resulted in an exciting, new theory that proposes the ASC as a mediator of tissue regeneration through the secretion of specific soluble factors. In this regard, the ASC could be used in an incredibly broad range of applications. However, the most popular are reviewed below.
