PhD Project: Development of Physiologic 3D Cultivation Approaches for Primary Human Mesenchymal Stem Cells
Julia Moldaschl - Doctoral Candidate DocSchool BioMatInt
The project focuses on the investigation of physiological cultivation approaches for primary human mesenchymal stem cells (MSC) with particular emphasis on 3D and hypoxic oxygen culture conditions. The application of both parameters pursues the goal of mimicking the cell’s natural environment resembling the in vivo situation better and therefore providing higher relevant data derived from in vitro models – especially in the context of safe and effective translation of cell-based therapies (CBT) into clinical applications.
An essential part of this work will be the participation in the Crazy 8 Initiative project “Tracking Ewing Sarcoma (EwS) Origin by Developmental and Trans-Species Genomics”. EwS is a malignant pediatric bone tumor lacking appropriate preclinical models recapitulating the human disease. While the genetic cause is already resolved, the knowledge about EwS histogenesis is still poor. However, juvenile mesoderm- or neural-crest-derived mesenchymal stem cells (MSC) have been proposed as candidate cell types of origin. Thus, the declared primary aim is to decipher the histogenesis of EwS in order to drive the development of models for preclinical drug testing.
The thesis will contribute particularly to the achievement of the Crazy 8 Initiative project’s first subgoal, namely the establishment of the first time- and lineage-resolved single-cell reference atlas of hMSC development based on single-cell transcriptome analyses. Therefore, the optimization of expansion and differentiation processes of pediatric bone marrow-derived MSCs towards adipocytes, chondrocytes, and osteoblasts will be conducted in scaffold-free physiological 3D cell culture systems that allow MSCs to form three-dimensional aggregates (spheroids) by themselves. This will be performed before and after lentiviral oncogene induction and under normoxia vs. hypoxia since bone marrow is a hypoxic microenvironment and a subgroup of tumor cells in EwS reveal a hypoxic gene expression signature. In addition, whole-mount staining protocols will be established in order to confirm the identity of the differentiated cells.
Over the last decades, the awareness of the positive effects of physiological cultivation systems on cells increased, resulting in a continuous shift from 2D to 3D cell culture systems. In detail, MSCs cultured as spheroids have shown to provide enhanced angiogenic, anti-inflammatory, and immunomodulatory effects as well as improved stemness and survival rates after transplantation.
This project will cover both scaffold-free 3D models arranging the cells in spheroids by using micropatterned multi-well plates and scaffold-based cultures focusing on hydrogels.
