In English

Optimizing culturing conditions using factorial experimental design for culturing of SSEA4+/CD34- cells isolated from human cardiac tissue

märta jansson
Göteborg : Chalmers tekniska högskola, 2016. 92 s.
[Examensarbete på avancerad nivå]

Cardiac disease is one of the major causes of death worldwide and current treatment aims at reducing further loss of viable tissue and treating symptoms rather than replenish lost cardiomyocytes in response to cardiac pathology. Therefore, finding an alternative method for treating cardiac disease is of essence. Recently cardiac progenitor cells were found residing in the adult human heart capable of cardiac regeneration. Isolation and in vitro cardiomyocyte differentiation of such cells would provide a potential platform for cardiac regeneration. The SSEA4+/CD34- cardiac progenitor cell population have previously shown signs of cardiac commitment, but no stable culture system have yet been suggested. The major aim of this Master’s Thesis project was therefore to establish a stable culture system for primary isolated SSEA4+/CD34- cells present in the adult human heart. Real time quantitative PCR was used to confirm the cardiac commitment of the SSEA4+/CD34- population, based on expression of cardiac-specific genes and to determine the medical relevance of such cells in comparison to differentiated induced pluripotent stem cells. It was also determined that short-term culturing of all isolated cells before sorting the population of interest using fluorescence-activated cell sorting, rather promoted endothelial commitment amongst cultured cells. Initial differentiation of SSEA4+/CD34- cells using the GiWi protocol published by Lian et. al. did not yield cardiomyocytes, but rather CD31 mRNA specific for endothelial cells was slightly increased while cardiac specific mRNA was maintained. Factorial experimental design was used to determine the relevance of each medium component to maintain primary isolated SSEA4+/CD34- cells. Statistical analysis in MODDE 10.1 showed the model to be unfitted and of low significance, both when using primary isolated cells and pre-ordered iCell CPCs, making all obtained results uncertain. A great variance could also be seen between samples, which could be limited by reducing model size through limiting the number of factors included in the model for future experiments. Thereby the relevance of each factor can be determined with certainty, allowing implementation of a stable culture system for primary isolated SSEA4+/CD34- cells, which potentially could be beneficial for future cardiac regeneration strategies.

Nyckelord: Cardiac regeneration, cardiac progenitor cells, tissue dissociation,fluorescence-activated cell sorting, real time quantitative PCR, cardiomyocyte differentiation, factorial experimental design, mammalian cell culture, induced pluripotent stem cells

Publikationen registrerades 2017-01-19. Den ändrades senast 2017-01-19

CPL ID: 247463

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