Etablierung einer Methode zur Herstellung von adulten pluripotenten Stammzellen

Abstract

The irreversible loss of cardiomyocytes after myocardial infarction can lead to chronic heart failure. A stem cell-based cell therapy could be a promising approach for organ regeneration. In the present thesis the direct generation of adult pluripotent stem cells from adult cells was investigated. In the first part of this work mesenchymal stem cells (MSCs) of mice and humans were compared to pluripotent embryonic stem cells (ESCs). We analysed pluripotency-specific transcription factors on both mRNA and protein level. We found no comparable expression of pluripotency markers in MSCs and ESCs. However, there was a weak and variable expression of few pluriotency markers, such as Oct4. Furthermore, MSCs showed no capacity of differentiation into cardiac lineage. This indicates that MSCs do not possess pluripotency. Furthermore, the ability of reprogramming of MSCs using ESC-cytoplasm was examined. A method for isolation of the ESC-cytoplasm fragment was established. ESC-cytoplasm fragments could be isolated with a purity of 95%. After fusion of MSCs with ESC-fragments, the derived cells were tested for their pluripotency at the mRNA and protein level. The new derived cells showed no increase of pluripotency. In the second part of this work, unipotent adult spermatogonial stem cells (SSCs) derived from the mouse testis have been investigated under different culture conditions with respect to their pluripotency. We found that adult SSCs responded to culture conditions and could convert into pluripotent stem cells in culture in the presence of leukemia inhibitory factor (LIF) and mouse embryonic fibroblasts (MEFs). The obtained cells showed an ESC-like morphology and strong alkaline phosphatase activity. Furthermore, we found a continuous expression of Oct4, Nanog, Utf1, and Rex1, Esg1 comparable with ESCs at the mRNA level. Additionally, we detected protein expression of Oct4, Nanog, Sox2 and SSEA1 comparable with ESCs. These results suggest that initially unipotent adult SSCs respond to culture conditions and acquire ESC properties under LIF and MEFs culture conditions. We therefore name these cultured cells multipotent adult germline stem cells (maGSCs). Our data show that MSCs have no pluripotency and can not be reprogrammed with single fusion of ESC-cytoplasm. However, adult SSCs respond to culture conditions and acquire pluripotent properties. The successful isolation of pluripotent stem cells from adult SSCs in this work opens new perspectives in the stem cell-based regenerative medicine.