Immunogenicity of allogeneic adipose-derived stem cells in a rat spinal fusion model

Abstract

Adipose-derived stem cells (ASCs) express a nonimmunogenic profile as shown by in vitro studies that demonstrate a lack of T cell proliferation to allogeneic ASCs as well as ASC-mediated suppression of mixed lymphocyte reactions. To determine whether these observations would translate in vivo, immune monitoring studies were carried out in conjunction with a rat spinal fusion study. ASCs derived from Fischer or ACI strain rats were loaded onto scaffolds and implanted in Fischer recipients that had undergone the following treatments: (1) No treatment; (2) Scaffold only; (3) Syngeneic ASCs+Scaffold; or (4) Allogeneic ASCs+Scaffold. Half of each group was sacrificed at 4 weeks postimplantation, and the remaining animals were sacrificed at 8 weeks. As determined in a separate study, allogeneic and syngeneic ASCs were equally efficacious in accelerating spinal fusion compared to No treatment and Scaffold only control groups. To determine whether donor ASCs induced an immune response in recipient rats, lymph nodes were harvested for T cell proliferation studies and serum was collected to assess antibody responses. Although T cell priming was not detected to donor alloantigens in recipients at either time point, significant antibody responses were detected to ACI ASCs in animals implanted with syngeneic or allogeneic ASCs. Antibodies were of the IgG isotype, noncytotoxic in the presence of complement, and reactive to fetal bovine serum. These results support the use of allogeneic ASCs for spinal fusion.

FIG. 1.

Immunologic characteristics of ACI rat strain ASCs. Passage 5 ASCs were evaluated by flow cytometry (A) for cell surface markers indicated on each histogram. The immunogenicity experiment in (B) shows the T cell proliferative response of Fischer strain LNCs (5 × 10⁵ cells/well) to increasing numbers of syngeneic Fischer spleen cells and allogeneic ACI spleen cells and ASCs. Results are shown as the mean ± standard deviation of triplicate culture wells. Significant differences from the background response to syngeneic Fischer spleen cells are denoted with an asterisk (p < 0.05). The suppression experiment in (C) shows the effect of adding increasing numbers of ASCs to one-way MLR cultures consisting of Fischer responder cells (5 × 10⁵ cells/well) and ACI spleen cells (2 × 10⁵ cells/well). Results are shown as the mean ± standard deviation of triplicate culture wells. Significant differences from the MLR response (no ASCs added) are denoted with an asterisk (p < 0.05).

FIG. 2.

Group and individual rat T cell proliferative responses to ACI spleen cells. Lymph node cells (LNCs) were removed from recipient rats at 4 or 8 weeks after implantation with no carrier or cells (No Trt), scaffold only (Scaf), syngeneic Fischer ASCs plus scaffold (Syn+Scaf), or allogeneic ACI ASCs plus scaffold (Allo+Scaf). LNCs (3 × 10⁵ cells/well) were stimulated with ACI or Fischer spleen cells (2 × 10⁵cells/well), and cultures were harvested at 3 or 7 days as indicated. Data are reported as ΔCPM that reflect that background proliferative responses to Fischer spleen cells have been subtracted from the proliferative responses to ACI spleen cells. The mean group response ± standard error of the mean is shown in (A). Individual rat responses from the Syn+Scaf and Allo+Scaf groups are shown in (B). All responses are from cultures harvested at 7 days. The significance of differences between groups are shown by brackets. NS, not significant.

FIG. 3.

Group responses to stimulation with Con A. Groups corresponding to each bar in the graph are described in Figure 2A and in the Figure 2 legend. LNCs (3 × 10⁵ cells/well) obtained from individual rats at 4 or 8 weeks were stimulated with Con A and harvested at 3 days. The response of the cells cultured in medium has been subtracted from the Con A–stimulated response (ΔCPM). The mean group response ± standard error of the mean is shown. There were no significant differences between treatment groups at either time point.

FIG. 4.

Group responses of recipient Fischer rat LNCs to control stimuli. Groups are described in Figure 2 and the Figure 2 legend. LNCs (3 × 10⁵ cells/well) obtained from individual rats at 4 or 8 weeks were cultured in medium alone (A) or with syngeneic Fischer strain spleen cells (2 × 10⁵ cells/well, B). The cultures were harvested at 3 or 7 days as indicated. Proliferative responses are designated as CPM because background responses were not subtracted. The mean group response ± standard error of the mean is shown. Significant differences from the No Trt background response are denoted with an asterisk (p < 0.05).

FIG. 5.

Serum antibody binding to ACI strain rat ASCs. Mean group serum antibody responses of recipient Fischer rats at 4 and 8 weeks postimplantation are shown in (A). Groups are described in Figure 2. The mean group MFI response ± standard error of the mean is shown. Significant differences from the No Trt background response are denoted with an asterisk (p < 0.05). Individual rat serum antibody responses to ACI strain rat ASCs are shown in (B). Antibody responses from individual rats are shown for the Syn+Scaf and Allo+Scaf groups at 4 and 8 weeks postimplantation. The significance of differences between groups is shown by brackets. NS, not significant.

FIG. 6.

Characteristics of the humoral response. The isotype and titer of the antibody response to ACI ASCs at 4 and 8 weeks is shown in (A) for No Trt and Allo+Scaf–treated rats. Rat sera were diluted as indicated, allowed to bind to ASCs and labeled with FITC-conjugated goat anti-IgM or IgG secondary antibodies before evaluation by flow cytometry. Significant differences from No Trt controls are denoted by an asterisk (p < 0.05). Sera from the same groups of rats were evaluated for cytotoxicity against ASC target cells (B). After allowing sera to bind ASCs, guinea pig complement was added, and cell viability was determined by propidium iodide staining and flow cytometry. As controls, ASCs were incubated with complement only (“None”) or with anti-CD90 mAb (positive control). The specificity of rat sera for FBS is shown in (C). Rat sera from all four groups in the spinal fusion study were serially diluted and evaluated for binding to FBS by ELISA. In all panels, the number of rat sera tested per group is shown in parentheses, and group means are shown ± standard error.