Recently, several elegant studies have shown that IPCCs are generated in vitro from both human and murine ASCs 
, suggesting that ASC-derived IPCCs may circumvent worldwide shortage of donor islets and eventually provide a cure for human type 1 diabetes. In this study, we found that ASC-derived IPCCs have the intrinsic survival disadvantage over freshly isolated islets in syngeneic recipients. Nevertheless, IPCC allografts exhibited certain immune privilege and enjoyed long-term survival in diabetic NOD mice in the presence of CD28/CD154 and Fas blockade. Therefore, long-term survival of ASC-derived IPCCs in syngeneic recipients requires only blocking Fas and TNF death pathways whereas blocking both death pathways and CD28/CD154 costimulation is needed for long-term IPCC allograft survival in diabetic NOD mice. Thus, our studies suggest that ASC-derived IPCCs may provide an effective cell replacement therapy in human type 1 diabetes.
Using IPCCs that differentiated in vitro from murine ASCs based on a recent protocol 
, we first compared their survival to that of freshly-isolated intact islets in syngeneic recipients. Surprisingly, we found that overwhelming majority of IPCC grafts did not survive for long-term in syngeneic WT or Rag1-/- mice whereas nearly all of syngeneic islet grafts survived for long-term (>100 days). Our findings suggest that freshly isolated islets have a survival advantage over in vitro-generated IPCCs upon transplantation. Therefore, IPCC syngrafts die out in vivo even in the absence of alloimmune responses, although exact mechanisms responsible for their shortened survival in syngeneic hosts remain to be defined. Our results are consistent with a previous study showing that IPCC grafts, derived from murine embryonic stem cells, have survived for only a average of two to three weeks in syngeneic Rag-/- hosts 
, implying that IPCC grafts generated from stem cells are incapable of surviving for long-term even in the absence of alloimmune-based rejection. However, our findings differ from a recent study showing that human ASC-derived IPCC grafts have survived beyond 60 days in 50% of xenogeneic recipient mice without immunosuppression 
. It is unclear how IPCC xenografts survived for such a long time without immunosuppression.
We then investigated how to prevent IPCC graft dysfunction in the absence of alloimmunity or any immune-based rejection, which is an important step prior to in-depth IPCC allotransplantation. Since Fas or TNF signaling mediates apoptosis in most cell types, including β cells 
, we asked whether blocking their signaling pathways would enhance IPCC graft survival or restore their function in syngeneic recipient mice. Indeed, blocking either Fas or TNF signaling significantly prolonged IPCC graft survival and inhibited IPCC cell apoptosis in syngeneic recipients while blocking both pathways induced long-term IPCC syngraft survival in overwhelming majority, if not all, of recipient mice. Our findings suggest that IPCC graft dysfunction due to passive cell death, which is mediated by Fas and TNF signal pathways but independent of alloimmune-based rejection, must also be overcome upon IPCC allotransplantation.
We next examined IPCC allograft survival in alloimmune-competent WT recipient mice. We found that IPCC allografts derived from both Fas-replete and Fas-deficient ASCs were acutely rejected in allogeneic WT recipient mice, suggesting that ASC-derived IPCCs are highly immunogenic and can evoke acute allograft rejection. Blocking CD40-CD154 costimulation induced Fas-deficient IPCC allograft survival for long-term in 63% of recipient mice. Given that the same percentage of Fas-deficient IPCC grafts survived in syngeneic recipients without CD154 costimulatory blockade, we conclude that blocking CD40-CD154 costimulation is sufficient to suppress IPCC allograft rejection mediated by alloimmune responses. Furthermore, simultaneously blocking CD154, Fas and TNF-receptor signaling induced long-term IPCC allograft survival in overwhelming majority, if not all, of recipient mice. Taken together, both death signaling of β cells and alloreactivity of recipients need to be suppressed in order to achieve long-term IPCC allograft survival.
In this study, we determined whether costimulatory blockade promotes long-term IPCC allograft survival in NOD mice that are closely relevant to human type 1 diabetes. Costimulatory blockade has been shown to suppress alloimmune responses and induce long-term allograft survival 
. Previous studies have also shown that blocking both CD28 and CD154 costimulatory pathways prevents autoimmune diabetes induced by transfer of transgenic T cells in NOD.SCID mice 
but fails to induce long-term islet allograft survival in NOD recipients 
, suggesting that CD28/CD154 blockade is sufficient to suppress autoimmunity but insufficient to simultaneously suppress both alloimmunity and autoimmunity in NOD recipients. In contrast, another study has shown that ICOS/CD154 blockade is capable of inducing islet allograft tolerance and preventing autoimmune diabetes in NOD mice 
. Here we found that CD28/CD154 blockade induces long-term Fas-/- IPCC, but not Fas-/- islet, allograft survival in diabetic NOD recipients. We utilized Fas-deficient IPCCs because Fas-replete IPCC grafts died out over time even in syngeneic recipients due to Fas-mediated cell death. Our data have further confirmed that CD28/CD154 blockade does not induce long-term islet allograft survival in diabetic NOD recipients. On the other hand, we have demonstrated for the first time that blocking CD28 and CD154 costimulatory signaling is capable of inducing long-term survival of ASC-derived IPCC allografts in diabetic NOD mice, although a recent study has shown that blocking CD28/CD154/LFA-1 enhances engraftment of allogeneic ESC-derived endothelial cells in non-diabetic environment 
It is unclear whether CD154 costimulatory blockade can be translated to the clinic in the future, although it prolongs IPCC allograft survival. Clinical trials using anti-CD154 Ab were halted due to its thromboembolic side effects 
. The progress in its clinical application, hence, has been hampered. Recently, Abs against its counter receptor, CD40, have been sought as an alternative to blocking CD40/CD154 costimulatory pathway. These promising Abs have been shown to potently suppress allograft rejection in both mice and non-human primates 
. However, it remains to be defined whether they will also cause the same side effects in the future clinical trials.
Our finding indicates that it is easier to suppress the rejection of ASC-derived IPCC allografts than freshly isolated islet allografts in NOD recipient mice once passive cell death is blocked. This is consistent with previous studies showing that ESC-derived IPCCs or tissues exhibit a certain degree of immune privilege 
. Therefore, ASC-derived IPCCs enjoy at least two advantages over freshly isolated islets: relatively immune privilege and unlimited sources while displaying an intrinsic survival disadvantage that is unrelated to alloimmune-based rejection. A recent study has demonstrated that transplantation of IPCCs derived from human umbilical cord mesenchymal stem cells alleviates hyperglycemia in diabetic NOD mice without any immunosuppressive treatment 
, although it was not observed how long IPCC xenografts survived and how their rejection could be suppressed. Taken together, previous and our current studies indicate that clinical application of ASC-derived IPCCs for treating type 1 diabetes is no longer elusive.