In the present study, we successfully identified for the first time MSCs in adipose retobulbar tissue of patients with GO (GO-MSCs) using standard protocols for adipose tissue-derived stem cell isolation and expansion. We use the term mesenchymal stem/stromal cells (MSCs) because we could demonstrate that orbital fat-derived MSCs fulfill the minimal criteria for defining multipotent MSCs together with immunoregulatory function, in agreement with MSC phenotype definition of the International Society for Cellular Therapy.
18 Importantly, we could demonstrate for the first time that GO-Fs, an accepted in vitro cell model of GO since their first isolation by Bahn and coworkers,
11 exhibit the same morphologic features, as well as a surface marker set and multilineage differentiation potential similar to GO-MSCs (
Figs. 1,
2,
3). Interestingly, the GO-MSCs and -Fs expressed the same surface marker as orbital fat-derived stem cells obtained from fat tissue of the intraorbital cavity during blepharoplastic surgery.
19 In addition, the observed reduction of the surface markers CD31 and CD146 during the early passages (
Fig. 2A,
Supplementary Fig. S1) was found to be a characteristic of mesenchymal progenitor cells derived from orbital adipose tissue.
20 Taken together, the observed surface marker profile supports the notion that GO-MSCs and -Fs isolated from the retrobulbar tissue during decompression procedure of GO patients represent orbital fat-derived stem cell populations. Indeed, GO-MSCs and GO-Fs shared the most important characteristic of orbital adipose tissue-derived stem cells, namely, the potential to differentiate into adipocytes, chondrocytes, and osteoblasts, and in addition into myogenic and neuronal precursors.
13 These findings demonstrate that our isolation procedure effectively yields the generation of MSCs from retrobulbar fat tissue. In contrast, GO-EthC fibroblasts showed no multilineage differentiation capacity and therefore could not be considered as containing MSC populations although isolated with a MSC protocol. Similarly, fibroblasts isolated from the eyelid skin during blepharoplastic surgeries did not differentiate toward the adipogenic or osteogenic lineage, and they showed less pronounced signs of chondrogenic differentiation even though they expressed a similar set of surface markers as the orbital fat-derived stem cells.
14 Several working groups previously reported differentiation of GO-Fs into adipocytes or myocytes.
19–21 However, we found that GO-Fs were also able to differentiate along the osteocytic, chondrocytic, and neuronal lineages (
Fig. 3), hence shared characteristics of classical adipose tissue-derived stem cells that reside in the vascular stroma of adipose tissue as well as in the bone marrow.
22 Because adipose cells are mesodermal in origin, the differentiation of adipogenic stem cells into neural tissue of ectodermal origin seemed to be very unlikely. However, they can take on a neuronal morphology when exposed to different induction agents.
23 While MSCs, adipose-derived stem cells, and most tissue fibroblasts are of mesenchymal origin, the GO-Fs have been hypothesized to be derived from the neuroectodermal origin.
24 Although ocular and orbital components have been shown to be derived from a combination of mesodermal and neural crest cells,
25 the origin of the retrobulbar connective/fat tissue and derived cells remains unclear. Interestingly, it has been reported that in the cephalic region a subset of adipocytes arises from the neural crest.
26 Further, a subpopulation of adipogenic progenitors derived from the neural crest has been identified in adipose stromal cells.
27 Because our in vitro data provide the first experimental evidence that the GO-F population contains progenitors with neuronal differentiation potential, it appears possible that GO-Fs contain neural crest–derived progenitors. However, we found that GO-Fs differentiate and showed multilineage potency, indicating that mesenchymal multipotent progenitor cells were effectively present in the GO-F population. In comparison to GO-Fs, the GO-MSCs showed better cell differentiation ability in terms of adipogenesis, osteogenesis, and myogenesis. These data may suggest that GO-Fs, in contrast to GO-MSCs, represent a more heterogeneous population of fibroblastoid cells containing more mature differentiated cells and a minority of MSC-like multipotent progenitor cells. Further studies are needed to fully elucidate the functional heterogeneity of orbital progenitor cells.