ESC Lineage Differentiation and the Effect of IFNγ on MHC-I Expression
As early evasion of immune detection by transplanted ESCs has been reported to be reliant on an absence of MHC-I expression [3
], we speculated that similar immune mechanisms may govern the fate of ESC-derived VE-CAD+
endothelial progenitors in our in vivo syngeneic models. To investigate this possibility, ESCs were cultured using defined serum-free media [7
]. FACS characterization of undifferentiated ESCs revealed negligible expression of MHC-I, Bracyhury, Flk-1, or VE-CAD (, and data not shown).
Figure 1 Embryonic stem cell (ESC) lineage differentiation and the effect of IFNγ on MHC-I expression. (A): ESCs were cultured in the presence of BMP-4 for 3.25 days. After this time, we detected a discrete population of Bry+Flk-1+ (Aa) cells that was (more ...)
A two-step culture-differentiation process was used to derive Bry+
cells (hemangioblasts) [7
] and VE-CAD+
endothelial progenitor cells as previously described [7
]. By FACS, we observed that following this differentiation period Bry+
cells represented 27.8% ± 2.5% of all cells in culture (n
= 7; ). To derive endothelial progenitor cells, Bry+
cells were isolated by FACS-sorting and returned to culture for further 7 days and with supplemental VEGF. Following this, we observed that VE-CAD+
cells constituted 30.9% ± 2.9% of all cells in culture (n
= 7) (). Immunohistochemistry revealed that in addition to being VE-CAD+
positive, these cells also expressed both CD31 () and vWF (data not shown). All cell populations remained MHC-I negative throughout differentiation ().
MHC-I expression was induced using IFNγ during our culture-differentiation process. As anticipated, IFNγ treatment significantly increased MHC-I expression in both Bry+
(0.2% ± 0.17% to 41.3% ± 4.89%, both n
= 7, p < .0001) () and VE-CAD+
cells (0.18% ± 0.09% to 87.3% ± 5.24%, both n
= 7, p < .0001) (). IFNγ treatment did not induce MHC-II expression in ESC-derived vascular progenitor cell populations (Supporting Information, Fig. 1
). In addition, IFNγ treatment and induced MHC-I expression did not interfere with cell differentiation, with IFNγ-treated Bry+
cells representing 25.8% ± 7.1% of all cells in culture (P
= n.s., n
= 7 compared with no IFNγ treatment) (). VE-CAD+
cells constituted 26.8% ± 8.3% of all cells in culture (P
= n.s., n
= 7 compared with no IFNγ treatment) (). Treatment with IFNγ did not change CD31 and VCAM-1 expression. However, ICAM-1 expression increased in the presence of IFNγ as compared with no IFNγ treatment (42.35% ± 5.43% and 5.50% ± 0.85%, respectively, n
= 5; Supporting Information, Figs. 2
Given that IFNγ may increase susceptibility to apoptosis [21
], we verified that this treatment did not alter progenitor cell survival, proliferation, or differentiation. Bry+
cells were returned to culture for 7 days according to our endothelial progenitor differentiation protocol. These cells survived, proliferated, and exhibited unremarkable differentiation characteristics, as shown by FACS ( and data not shown) and immunohistochemistry staining ( and data not shown). Both VE-CAD+
cells were subjected to a Matrigel angiogenesis assay, revealing identical tube-forming ability and thereby further confirming that IFNγ treatment does not affect the endothelial properties of ESC-derived VE-CAD+
cells (Supporting Information, Fig. 4
MHC-I Expression by ESC-Derived Endothelial Progenitor Cells Promotes Neovascularization in Matrigel Plugs
VE-CAD+MHC-I+ and VE-CAD+MHC-I− cells, mixed with liquid Matrigel, were injected subcutaneously into syngeneic recipient mice (129/ola) to form Matrigel plugs. Fluorescence stereomicroscopic analysis at day 14 postimplantation revealed significantly more vessel formation in VE-CAD+MHC-I+ cell Matrigel plugs than in VE-CAD+MHC-I− cell plugs (). Fluorescence quantitation revealed significantly higher fluorescence in VE-CAD+MHC-I+ cell plugs than in VE-CAD+MHC-I− cell plugs (, ***, p < .001, five plugs per test group). Immunostaining for DsRed/CD31 and DsRed/α-SMA on plugs derived from the VE-CAD+MHC-I+ cell group demonstrated that ESC-derived VE-CAD+ cells had functionally integrated into the new blood vessels (). As a whole, these results suggest that ESC-derived VE-CAD+ cells are able to promote and functionally participate in neovascularization in a Matrigel plug model, and highlight the importance of MHC-I expression in this process.
Figure 2 Matrigel plug neovascularization assay and embryonic stem cell (ESC)-derived cell integration into new vessels. Fluorescence stereo-microscopic imaging revealed that while VE-CAD+MHC-I− cells in Matrigel exhibited new vessel formation (A), significantly (more ...)
MHC-I Expression is Critical for ESC-Derived Endothelial Progenitor Survival in a Syngeneic Murine Ischemic Hindlimb Model
To further elucidate the function of MHC-I and the mechanisms of interaction between transplanted cells and their syngeneic host (129/ola mice), we proceeded to investigate the fate of ESC-derived vascular progenitors with and without MHC-I expression in a syngeneic ischemic model. In control and PBS-injected mice, our ischemic murine hindlimb model was characterized by an initial inflammatory response in the ischemic musculature, in association with a degree of tissue degeneration/necrosis. Surprisingly, 2 weeks after femoral artery ligation, transplantation of ESC-derived VE-CAD+MHC-I− cells was associated with markedly increased tissue degeneration in tibialis muscle, while transplantation of VE-CAD+MHC-I+ cells was associated with only a minor increase in tissue degeneration when compared with PBS-injected control hindlimbs (). Consistent with this, compared with PBS-injected controls, immunohistochemistry revealed increased macrophage and T-cell infiltration in the VE-CAD+MHC-I− cell-injected tibialis muscle (), with significantly less inflammatory cell infiltration observed in muscle injected with VE-CAD+MHC-I+ cells ().
Figure 3 Morphology, immunohistochemistry, Y-chromosome FISH, and Fluorescence-activated cell sorting (FACS) analysis following syngeneic transplantation. Tibialis muscle from female recipients was harvested 2 weeks after femoral artery ligation and male VE-CAD (more ...)
As our experimental protocol utilized male donor ESC-derived cells and recipient female mice, Y-chromosome expression was used to ascertain the fate of transplanted cells. Thus, Y-chromosome FISH of ischemic tissues in the MHC-I− group, but not MHC-I+ group, revealed a widespread atypical staining pattern, consistent with ESC-derived VE-CAD+MHC-I− cells undergoing active degeneration/necrosis (). Indeed, we detected very few Y-chromosome positive cells with a normal morphologic and FISH staining pattern, suggesting extensive rejection of transplanted VE-CAD+MHC-I− cells. Conversely, FISH staining performed on muscle injected with VE-CAD+MHC-I+ cells revealed typical staining, similar to that of normal male muscle, suggesting that MHC-I expression may modulate donor cell integrity and survival in this syngeneic transplantation model ().
FACS analysis for DsRed was used to identify ESC-derived cells from total cells extracted from ischemic hindlimb muscle at 2 weeks post-transplantation. We identified that the survival of transplanted VE-CAD+MHC-I+ cells (), or their progeny, was significantly greater than that of VE-CAD+MHC-I− cells (DsRed+ cells in tibialis muscle, 5.42% ± 0.61% vs. 0.97% ± 0.38%, respectively, both n = 7, p < .005; (). In addition to ESC-derived endothelial progenitor (VE-CAD+) cells, we also transplanted earlier stage ESC-derived hemangioblast Bry+Flk-1+MHC-I+ () and Bry+Flk-1+MHC-I− cells (). Similar to VE-CAD+ cells, survival of transplanted Bry+Flk-1+MHC-I+ cells was significantly greater than that of Bry+Flk-1+MHC-I− cells (DsRed+ cells in tibialis muscle, 5.62% ± 0.69% vs. 0.68% ± 0.09%, respectively, both n = 7, p < .005) ().
NK Cell Attack Plays a Major Role in the Rejection of ESC-Derived VE-CAD1MHC-I− Cells and MHC-I Expression Promotes ESC-Derived Progenitor Cell Resistance to NK Cell Attack
We elected to further explore the pathobiology of the processes underlying the apparent rejection of ESC-derived MHC-I− cells. Initially, to exclude the possibility of poor graft viability or senescence, sorted VE-CAD+MHC-I− cells (as prepared for transplantation) were returned to culture for up to 3 weeks. These cells grew, proliferated, and were passaged normally throughout this additional culture period (data not shown). We next examined the nature of the host inflammatory cell infiltrate after donor cell transplantation. Immunohistochemistry revealed an abundance of infiltrating NK cells in VE-CAD+MHC-I− cell-injected tibialis muscle, whereas very few NK cells were observed in muscle injected with VE-CAD+MHC-I+ cells (7.32% ± 0.58% vs. 0.70% ± 0.27% of total cells respectively, both n = 7, p < .0001) ().
Figure 4 MHC-I expression by embryonic stem cell-derived cells and subsequent NK cell attack plays a major fate-determining role following syngeneic transplantation. (A): Immunohistochemistry staining using anti-NKp46/NCR1 antibody to identify NK cells 2 weeks (more ...)
We also employed an in vitro 51Chromium release assay to examine the susceptibility of various cell populations to syngeneic NK cell attack, including ESC-derived VE-CAD+MHC-I− progenitors, IFN-induced VE-CAD+MHC-I+ progenitors, MHC-I− control cells (YAC-1 cells, H-2Db− ), and MHC-I+ control cells (Concanavalin A blasts, H-2Db+). These studies revealed that MHC-I− cells, including ESC-derived VE-CAD+MHC-I− cells, were highly susceptible to NK cell-mediated killing. On the other hand, MHC-I+ control cells and VE-CAD+MHC-I+ progenitors evaded or were resistant to NK cell attack ().
To confirm the importance of NK cell attack in vivo, a blocking antibody against NK cells was administered to abrogate NK cell activity at the time of donor cell transplantation. Two weeks after transplantation, survival of ESC-derived VE-CAD+MHC-I− cells was significantly greater in NK antibody-treated mice than in PBS-treated animals (5.37% ± 0.41% vs. 0.97% ± 0.38% of total single cells in tibialis muscle, p < .001; ).
We also investigated the survival of ESC-derived hemangioblast Bry+
cells in the setting of absent NK cell function by using NSG mice, which lack functional NK cells [22
]. In agreement with our initial findings, following transplantation into the ischemic hindlimbs of NSG mice the survival of ESC-derived Bry+
hemangioblasts was similar to that of ESC-derived Bry+
cells (5.32 ± 0.76 vs. Five.51 ± 0.68 respectively, both n
= 6, p
= .61; Supporting Information, Figs. 5A and 5B
Collectively, these in vitro and in vivo studies led us to conclude that NK cell attack is an important factor in the immune-rejection of transplanted ESC-derived vascular progenitor cells. IFNc-induced MHC-I expression promotes ESC-derived progenitor cell resistance to NK cell attack in vitro and enhances cell survival in vivo after syngeneic transplantation.
Continued Survival, Proliferation, and Differentiation of MHC-I1 ESC-Derived Progenitors After Syngeneic Transplantation
Given the improved early survival of MHC-I+ ESC-derived progenitor cells after syngeneic transplantation, we proceeded to investigate the intermediate-term survival and differentiation of these cells. The percentage of ESC-derived cells in total single cells extracted from tibialis muscle was assessed by FACS for DsRed expression at 4 weeks after MHC-I+ cell transplantation. At 2 weeks compared with 4 weeks post-transplantation, the numbers of Bry+Flk-1+MHC-I+ cells and VE-CAD+MHC-I+ cells (and/or their progeny) were unchanged (Bry+Flk-1+MHC-I+ cells: 5.62% ± 0.69% vs. 6.08% ± 0.28%, both n = 7, p = .41; VE-CAD+MHC-I+ cells: 5.42% ± 0.6132% vs. 5.58 ± 0.33, n = 7, p = .82; ). Although fluctuating levels of infiltrating inflammatory and local cells may have influenced these results (altered total number of cells), this data appears to suggest stable engraftment of transplanted MHC-I+ ESC-derived cells.
Figure 5 Continued survival, proliferation, and differentiation of MHC-I+ ESC-derived progenitors after syngeneic transplantation. (A): The percentage of transplanted cells (and/or their progeny) in total single cells extracted from tibialis muscles at 2 and 4 (more ...)
We performed double immunofluorescence staining on ischemic tibialis muscle transplanted with VE-CAD+
cells. Using DsRed to identify the ESC-origin of transplanted cells, we readily detected ESC-derived cells that were double positive for CD31 and DsRed (), as well as cells double positive DsRed and vWF (Supporting Information, Fig. 6
), indicating that MHC-I+
ESC-derived endothelial progenitor cells can contribute functionally to the endothelial vascular layer in vivo. We also identified ESC-derived cells (and/or their progeny) that expressed α-SMA or vascular smooth muscle cell (VSMC)-specific myosin heavy chain (SM-MHC) ( and Supporting Information, Fig. 6
). Particularly, as SM-MHC expression is indicative of a mature VSMC phenotpye, and consistent with our Matrigel plug experiments, these data suggest that transplanted ESC-derived VE-CAD+
cells can give rise to VSMCs.
To confirm these results, we further investigated the in vivo fate of transplanted ESC-derived MHC-I+ cells using Y-chromosome FISH to locate donor cells that exhibited either endothelial (costaining for CD31) or smooth muscle (costaining for α-SMA) characteristics. Similar to the immunohistochemistry results, we identified frequent Y-chromosome+CD31+ and also Y-chromosome+α-SMA+ double positive cells () in VE-CAD+MHC-I+ cell-transplanted muscle. We also identified frequent Y-chromosome+CD31+ and Y-chromosome+ α-SMA+ double positive cells in the Bry+Flk-1+MHC-I+ cell-transplanted group (). Importantly, Bry+Flk-1+MHC-I+ cells were negative for both CD31 and a-SMA prior to transplantation (data not shown).
Finally, FACS analysis was used to identify CD31 and VSMC-specific marker Sm22 positive donor DsRed+
cells in tibialis muscle at 2 and 4 weeks after transplantation. Consistent with our results above, FACS analysis revealed a significant number of donor-derived endothelial (CD31+
) and smooth muscle (Sm22+
) cells at both time-points ( and Supporting Information, Fig. 7
). Interestingly, we observed that transplantation of Bry+
hemangioblasts, as compared with VE-CAD+
endothelial progenitor cells, gave significantly higher numbers of Sm22+
cells at both the 2 and 4 week time-points. Conversely, transplantation of VE-CAD+
endothelial progenitor cells, rather than Bry+
hemangio-blasts, was associated with greater numbers of CD31+
cells at both time-points ( and Supporting Information, Fig. 7
As a whole, these results suggest that transplanted Bry+Flk-1+MHC-I+ and/or VE-CAD+MHC-I+ ESC-derived cells are able to engraft in a syngeneic model of hindlimb ischemia, and that a significant proportion of these cells are able to survive over the intermediate term. Both cell populations appeared capable, in vivo, of giving rise to both smooth muscle and endothelial progeny. However, the respective ESC-derived progenitor populations exhibited a clear bias toward an in vivo fate that was consistent with their anticipated in vitro phenotype.