Immunobiology of SCNT Derived Embryonic Stem Cells: Pivotal Role of mtDNA in Rejection of Transplanted Stem Cells

Abstract

Background: For regenerative therapies, somatic cell nucleus transfer (SCNT) can be used to transfer the nucleus of a somatic cell into the cytoplasm of an enucleated egg. The resulting SCNT-embryo has the capability to develop to the blastocyst stage. Subsequently, embryonic stem cells (ESCs) can be derived from the inner cell mass of these blastocysts. These ESCs are genetically identical with the donor nucleus, with the exception of mitochondrial DNA (mtDNA) from the receiving egg. This study aimed to investigate the role of mtDNA in ESC transplantation and its immunological recognition. Methods: SCNT-ESCs were generated by transferring donor cells from Balb/C background into enucleated oocytes from BDF1 background. 1×106 of these SCNT-ESCs or SCNT-ESC-derived cardiomyocytes (CMs) were injected into Balb/C (mitochondria-incompatible) or BDF1 (nucleus-incompatible) mice (n=10). After 5 days, cellular and humoral responses were investigated by ELISPOT, donorspecific antibodies, histopathology, and confocal immunofluorescence microscopy. Immunodeficient SCIDbeige mice were used as controls (n=6). In vivo bioluminescence imaging was used to visualize cell survival longitudinal. The amount of mitochondria in undifferentiated ESCs was compared to ESC-derived CMs using the TMRM assay (Figure 1).[Figure 1]Results: Using ELISPOT assays for Th1, Th2, and Th17-cells, we found that foreign mitochondria caused a similar immune response compared to ESCs with foreign nuclear DNA, both being significant higher than in SCIDbeige (p< 0.001). Similar results were observed for the humoral response. Re-injection of ESCs resulted in an adaptive immune response, highlighting the role of T-cell mediated rejection as shown by ELISPOT assays and in vivo bioluminescence imaging. Antigenicity of ESCs increased with their differentiation into CMs, correlating with increased MHCI-expression and amount of mitochondrial antigens (Figure 1). Conclusion: mtDNA is coding for 13 proteins, which surprisingly resulted in an adaptive immune response and donorspecific antibody production after transplantation. Upon ESC differentiation into CMs we observed an increase of mitochondrial antigens resulting in a greater immunological recognition of transplanted cells. In summary, SCNT-derivates are not sufficient immune privileged and undergo immune rejection after transplantation.