In vitro Expansion humaner mesenchymaler Stammzellen in 2D und 3D Kultursystemen und Analyse genetischer und epigenetischer Folgen der Langzeitkultivierung

  • In vitro expansion of human mesenchymal stem cells in 2D and 3D culture systems and analysis of genetic and epigenetic sequels of long-term culture

Schellenberg, Anne; Wagner, Wolfgang (Thesis advisor); Zenke, Martin (Thesis advisor); Elling, Lothar (Thesis advisor)

Aachen (2016, 2017)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2016

Abstract

MSCs are attractive therapeutic agents for regenerative medicine due to their differentiation potential and immunomodulatory properties. Most clinical applications demand high cell numbers, which requires marked in vitro expansion of MSCs prior to transplantation. However, lifespan of MSCs in vitro is limited - after a certain number of cell divisions they reach replicative senescence, which is accompanied by morphological and functional changes. In this study, the analysis of the composition of subpopulations during culture expansion demonstrates that the number of fibroblastoid colony forming units (CFU-f) and clonogenic subsets with the highest differentiation capacity decrease rapidly already at early passages. The data obtained in the present study indicate that monitoring the CFU-f frequency over subsequent passages might be used as an indicator for remaining population doublings before entering the senescent state. Furthermore, genetic and epigenetic stability in long-term culture has to be taken into account for clinical use of these cell preparations. Analysis of genetic sequels of long-term culture using Karyotyping and SNP-microarrays revealed no relevant chromosomal aberrations. Subsequently, DNA-methylation profiles showed that highly consistent senescence-associated modifications occur at specific CpG sites upon long-term culture. These DNA-methylation changes correlated with repressive histone marks of previously published data sets, such as trimethylation of H3K9, H3K27 and EZH2 targets. Furthermore, sequels of MSC-culture on elastic PDMS substrates were analyzed. It could be confirmed that the adipogenic and osteogenic differentiation potential increases on soft and rigid substrates, respectively. However this disposition was erased when cells were reseeded on TCP. In line with these findings, global gene expression and DNAm profiles showed only few differences in MSCs cultivated on various substrate elasticities. As a next step, the impact of surface topography of PVDF fibers and pore architecture in 3D non-wovens on MSC fate was evaluated. In these scaffolds cells align along the fibres and form cell sheets that span over non-woven pores. Cell proliferation is significantly enhanced in scaffolds with round fiber shape and small pore size. Furthermore, long-term proliferation over a time course of 15 days and efficient adipogenic differentiation could be demonstrated for PVDF scaffolds. Taken together, the results of this study provide further insight in the effect of long-term culture and substrate properties on MCS fate.