Late-passage ASCs fail to form 3D spheroids
In the presence of HA gel, ASC cultures at early passage (0-2) were observed to produce uniform-sized spheroids, while cultures at 4-6 passages produced irregularly-shaped structures filled with cell aggregates. (Figure.1B). When cultured in monolayers, passage 8 ASCs were either spindle-shaped or large, flat, and irregularly shaped, which are characteristics of cellular senescence. Further, late-passage ASCs were unable to form 3D spheroids in the presence of HA gel.
ECFCs were identified by positive expression of characteristic markers including CD31, VEGFR2, eNOS, and CD105, and negative expression of CD133, and CD45 . ECFCs are named for their ability to form colonies of cells. Primary ECFCs were cultured for 28 days until colonies were observed and could be selected for further expansion (Figure.2A). Flow cytometry analysis revealed that ECFCs and ASCs had similar size and granularity. Compared with primary ASCs, ECFCs are positive for the mesenchymal marker CD105 and endothelial marker CD31, and negative for the hematopoietic marker CD45 and stem cell marker CD133 (Figure.2B). Thus, it was confirmed that the isolated primary cells were ECFCs.
3D co-culture of late-passage ASC and ECFCs
To generate a co-culture, ECFCs were seeded on a monolayer culture of ASCs (passage 6). In the presence of ECFCs, passage 8 ASCs were observed to maintain their spindle-shaped morphology (Figure.3A). To determine the most efficient protocol for generation of 3D co-cultures, the seeding density of ASCs was optimized (1x106-6x106) together with serum supplementation (1 or 2.5% FBS). Medium containing 2% FBS provided improved cell mobility compared with 1% FBS, as indicated by the large cell aggregates. Further, an ASC seeding density of 4x106 in cultures supplemented with 2% FBS promoted the formation of tubular-like structures in the HA gel (Figure.3B). We speculate that this structure may represent the neovascular properties of endothelial cells.
Trophic functionality of 3D co-culture spheroids
The secretory functions of 3D co-cultures were assessed by ELISA. Immunological analysis revealed that secretions of VEGF, HGF, and EGF were general higher under co-culture conditions than when mixed conditioned media were employed. The ASC+ECFC co-culture promoted the secretion of HGF/EGF when compared with the conditioned medium mixture from both 2D and 3D mono culture conditions. PDGF secretion did not differ between the groups. The 3D co-culture model significantly enhanced the secretion of VEGF and HGF compared with the other experimental groups (Figure.3C).
Viability and characterization of 3D co-cultured cells
Cell viability of the 3D co-culture spheroid was assessed by flow cytometry using the 7-AAD stain. Over time, cell death increased after 24 hours (22.1±4.3%), 48 hours (45.8±7.1%) and 72 hours (90.5±9.0%) incubation (Figure. 4A). Twenty-four hours incubation was therefore considered as the most favorable culture period for achieving reduced cell death. 3D co-culture spheroids incubated in HA gel for 24 hours were enzymatically suspended into single cells for characterization by flow cytometry analysis. Like the mixed cultures, the 3D co-culture maintained expression of the mesenchymal marker CD105. In comparison, there was an increase in the CD31 (approx. 2.8 fold and CD133 (approx. 3 fold) positive populations when cells were co-cultured in 3D (Figure. 4B). These results indicated that the ASCs were undergoing transdifferentiation.
Chronic injury assessment of radiation-ulcer mice
Overall, ulcers created in the irradiated mouse tissues showed impaired healing compared with those in the normal tissue. Eighteen days post-injury, the vehicle-treated (HA) ulcers in the irradiated tissue remained unhealed (Fig. 5B). Seven days post-injury, ASC/ECFC -treated ulcers showed improved re-epithelialization and wound healing (wound area: 40.1±3.0%) compared with ulcers treated with only ASCs (63.3±6.7%) or ECFCs (65.8±7.2%). The late-passage ASC-treated ulcers healed similarly to vehicle-treated ulcers (76.16±5.2%) (p>0.05). Fourteen days post-injury, the ASC/ECFC -treated ulcers had healed more (77% wound closure) than those in mice in the other treatment groups, which showed significantly impaired healing.
Histological evaluation of the ulcer wound demonstrated the formation of scar tissue in the central area (Fig. 5C). Despite normal dermal thickness, ASC/ECFC -treated mice had a thicker subcutaneous adipose layer in the wound marginal area. ASC/ECFC -treated mice exhibited more collagen deposition in the wound center.
Microscopic differences in the ulcer vasculature was noted between ASC/ECFC -treated ulcers and other treatment groups. To investigate further, vascularization of the wound tissue was examined by immunocytochemistry of α-SMA-stained capillaries (Figure 6A). ASC/ECFC -treated ulcers had approximately 30% more α-SMA positive cells than the other three treatment groups. Adipogenesis was evaluated by quantification of perilipin staining. There was a 4-fold increase in the adipose content of the ASC/ECFC ulcers compared with the other groups. However, ulcers treated with only passage 8 ASCs or ECFCs alone did not differ from the vehicle-control.
To trace transplanted ASCs, DiI labeling was performed. Eighteen days post-transplantation, cells in the deep subcutaneous layer of ASC/ECFC ulcers were arranged in circular patterns, a proportion of which was positive for both α-SMA and DiI (Figure 6B). Double-positive cells indicated that the engrafted ASCs were developing into blood vessels in the host tissue.