In the present study, we compared the differentiation potential and reparative properties of ADSCs derived from pericardial fat in relation to subcutaneous fat from the groin. Our data suggest that although both ADSCs are phenotypically identical, periADSCs constitute a vigorous potential toward myogenic differentiation by endogenously expressing transcriptional factors for cardiogenesis. Notably, transplantation of periADSCs into the injured myocardium resulted in significant functional improvement and structural repair by robust myogenesis and vasculogenesis. Therefore, pericardial fat-derived ADSCs represent attractive donor cells for the treatment of ischemic heart disease.
In recent years, increasing evidence demonstrated that multipotent stem cells derived from mature adipose tissues are able to differentiate into osteocytes, chondrocytes, adipose cells, cardiomyocytes, and neural cells . Because the adipose tissues are abundant and clinically easy to obtain, they have been considered to be an ideal source for obtaining donor cells in regenerative medicine [11, 12]. In those ongoing preclinical and clinical studies, the ADSCs were mainly isolated from subcutaneous liposuction , although visceral adipose tissues also can serve as a source for generating ADSCs. Given that the biology of subcutaneous adipose tissue differs from that of visceral fat in terms of insulin resistance, lipometabolism, and secretion patterns, the subpopulation of the stromal fraction may also vary on where tissue stromal cells are located [14, 15]. For instance, cardiac MSCs have been shown to present cardiovascular-associated features and were more efficient for cardiac repair . However, location-related diversity of ADSCs in terms of differentiation potential and regenerative capacity has not been well documented.
In the present study, we first compared the phonotypic properties of two types of ADSCs from different sources and found that the ADSCs of subcutaneous and visceral origin display the same morphology and surface epitopes that were undistinguishable with flow cytometry (Table 1). Both periADSCs and ingADSCs express mesenchymal markers, as previously reported [6, 17], with only minor heterogeneity of CD106 expression. CD106 is also known as vascular cell adhesion molecule 1 (VCAM-1) and identifies a subpopulation of MSCs with unique immunomodulatory properties . Whether the more sub-fractions of CD106 positive cells in the periADSCs attributed to the in vivo regenerative activity needs to be further characterized.
The striking finding in this study is that periADSCs constitutively express of some key transcriptional factors important for cardiomyogenesis, including GATA-4, Isl-1, Nkx-2.5, and MEF-2c, whereas ingADSCs were fairly absent, indicating that the periADSCs hold a potent intrinsic capability towards cardiac differentiation. This notion was further supported by our inductive experiment, showing the significant formation of cardiospherical structure and the vigorous generation of cTnT-expressing cardiac progenitors in comparison to ingADSCs. Although subcutaneous ADSCs have been well demonstrated to display cardiac potential [1, 2], the more efficient cardiac differentiation of periADSCs may be related to their developmental origin . The epicardial and pericardial MSCs developmentally initiated from the proepicardial cluster of cells located dorsal and adjacent to the heart tube and migrated onto the surface layer to form the second heart field  and retained postnatally the ability to form cardiac colony-forming-unit fibroblasts (cCFU-Fs) . Thus, the ADSCs isolated from the pericardial adipose tissue are supposed to constitute some biologic properties similar to cardiac progenitors that contributed to the homeostasis of the heart .
The diversity of ADSCs from different locations has been previously described, but their myogenic activity has not been compared . The unique feature of myogenic potency of periADSCs prompts us to investigate further the regenerative capacity relevant to the use of ADSCs for cardiovascular regeneration. In this context, we injected the same amounts of two types of ADSCs into the middle of the infarcted area and analyzed the follow-up structural and functional repair.
Our results demonstrate that, in direct contrast to ingADSCs, periADSCs were able to superiorly induce significant myogenesis and angiogenesis restrictively at the site of injection, but did not alter the infarct size (data not shown) or collagen deposition (Figure 5B). The structural and functional benefits after the transplantation of periADSCs did not result from the direct conversion of the ADSCs into the cardiac lineage, as nearly all prelabeled eGFP-positive periADSCs disappeared 28 days after transplantation (Figure 5A), likely because of apoptosis-mediated cell loss . Regardless of the minimal engraftment in the host myocardium, periADSCs exclusively triggered significant formation of cardiomyocytes (Figure 4), whereas ingADSCs failed to do so, suggesting a unique property of periADSCs on the myogenesis via an indirect action on endogenous cardiac stem cells. The mechanistic interlink showing how periADSCs communicated with endogenous stem cells is not clear, but may relate to apoptotic body-mediated exchange of genetic materials (that is, transcriptional factors) via horizontal gene transfer  and/or via indirect paracrine-related proangiogenic and immunomodulatory effects, as reported previously by Naftali-Shani et al.
. Therefore, those data underscore the definition of paracrine factors and the underlying mechanism by which the endogenous regeneration was evoked.
We are aware of several limitations in the present experiments. First, the differences in adipogenesis and osteogenesis of two types of ADSCs were compared only in in vitro conditions and must be characterized further in the in vivo condition as to cardiac transplantation. Second, cardiac transplantation was made of a single injection in the infarcted area, and in the future, multiple spots of injection in the border zone may be needed to maximize the functional benefits after myocardial injury. Also, the comparison of cardiac function of two types of ADSCs was analyzed only at the end point (28 days after transplantation), whereas the starting baseline data is still missing. Therefore, further study must assess cardiac function at various time points to demonstrate the kinetics of functional recovery after ADSC-based therapy.
Finally, de novo formation of cardiomyocytes must be experimentally proved.