Pluripotent parthenogenetic stem cells (pSCs) can be derived by pharmacological activation of unfertilized oocytes. Homozygosity of the major histocompatibility complex (MHC) in pSCs makes them an attractive cell source for applications in allogeneic tissue repair. This was recently demonstrated for pSC-based tissue-engineered heart repair. A detailed analysis of immunological properties of pSC-derived cardiomyocytes and engineered heart muscle (EHM) thereof is, however, lacking. The aim of this study was to determine baseline and cytokine-inducible MHC class I and MHC class II as well as programmed death ligand-1 (PDL-1) and co-stimulatory protein (CD40, CD80, CD86) expression in pSC-derived cardiomyocytes and pSC-EHM in vitro and in vivo. Cardiomyocytes from an MHC-homologous (H2d/d) pSC-line were enriched to ~90% by making use of a recently developed cardiomyocyte-specific genetic selection protocol. MHC class I and MHC class II expression in cardiomyocytes could only be observed after stimulation with interferon gamma (IFN-γ). PDL-1 was markedly upregulated under IFN-γ. CD40, CD80, and CD86 were expressed at low levels and not upregulated by IFN-γ. EHM constructed from H2d/d cardiomyocytes expressed similarly low levels of MHC class I, MHC class II, and costimulatory molecules under basal conditions. However, in EHM only MHC class I, but not MHC class II, molecules were upregulated after IFN-γ-stimulation. We next employed a cocultivation system with MHC-matched and MHC-mismatched splenocytes and T-cells to analyze the immune stimulatory properties of EHMs. Despite MHC-mismatched conditions, EHM did not induce splenocyte or T-cell proliferation in vitro. To evaluate the immunogenicity of pSC-derived cardiomyocytes in vivo, we implanted pSC-derived embryoid bodies after elimination of non-cardiomyocytes (cardiac bodies) under the kidney capsules of MHC-matched and -mismatched mice. Spontaneous beating of cardiac bodies could be observed for 28 days in the matched and for 7 days in the mismatched conditions. Teratomas formed after 28 days only in the MHC-matched conditions. Immunohistochemistry revealed single clusters of CD3-positive cells in the border zone of the implant in the mismatched conditions with few CD3-positive cells infiltrating the implant. Taken together, MHC-matched pSC-cardiomyocyte allografts show little immune cell activation, offering an explanation for the observed long-term retention of pSC-EHM allografts in the absence of immunosuppression.
Here I declare that my doctoral thesis entitled "Immunological properties of parthenogenetic stem cell derived cardiomyocytes and their application in cardiac tissue engineering." has been written independently with no other sources and aids than quoted.Furthermore, I confirm that this thesis has not yet been submitted as part of another examination process neither in identical nor in similar form.
Background: Pluripotent parthenogenetic stem cells (PSCs) can be directed towards a cardiac fate and utilized in tissue engineered heart repair. In vivo applications of tissue engineered allografts are compromised by expression of mismatching major histocompatibility complex proteins (MHC; encoded in the murine H2 locus). Here we investigated whether PSC-derived cardiomyocytes (CM) express MHC-I. Methods: Mouse PSCs (A3-line from B6D2F1 strain with haploidentical H2K d -locus) expressing a CM-specific neomycin-resistance and GFP were differentiated and purified for CM by addition of G418 (85% purity by FACS for actinin). To simulate heart muscle biology in vitro, we made use of engineered heart muscle (EHM) constructed from PSC-derived CM (75%), growth-inhibited murine embryonic fibroblasts (MEF (25%); NMRI mice), and collagen type I. MHC class-I H2K d (MHC-I) expression was assessed on CM and Non myocytes before EHM assembly and from enzymatically digested EHMs (cultured for 10 days) by FACS. Interferon gamma (IFNγ) was added for 48 h to stimulate MHC-I expression. As a reference, we investigated MHC-I expression in CM from neonatal mice and adult mouse hearts by FACS and by immunofluorescence staining. Results: EHM showed a positive ionotropic response to beta-adrenergic stimulation which could be reduced by muscarinergic stimulation. A3-CM, in contrast to Non myocytes, showed negligible expression of MHC-I (1±0.5% vs. 60±10% positive cells; n=3). EHM culture did not change MHC-I expression in CM. IFNγ treatment resulted in a marked increase of MHC-I-expression in CM monolayer culture (40±6%; n=3) and in EHM (30±8%; n=3). For comparison, 30% (n=2) neonatal CM expressed MHC-I while MHC-I was not detectable in adult CM. Conclusion: PSC-derived CM show a similarly low expression of MHC-I as adult CM and respond with MHC-I upregulation to IFNγ stimulation. This suggests a mature immunological response in PSC-CM with important implications for in vivo applications, i.e., MHC-I matching will likely be a prerequisite for successful allografting of PSC-EHM.
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