Developing quality control metrics for RNA sequencing of aging and robust stem cells
Mesenchymal Stromal Cells (MSC) are the most common cell type used for cell-based treatments of human diseases. MSCs are heterogeneous and exhibit significant cell-to-cell variation in regenerative properties. A challenge faced by MSC therapies is the inability to create uniform cell populations to produce consistent patient outcomes. A previous study determined that CD264, a decoy receptor for tumor factor-related apoptosis-inducing ligand, the expression has a significant negative correlation with colony-forming efficiency. This finding signifies CD264 as a biomarker for cellular aging. After using CD264 to sort into aging and robust MSCs, RNA Sequencing (RNA-Seq) can be used for differential gene expression. This thesis will analyze quality control metrics for aging MSCs with the following hypothesis. Among quality control metrics performed on live MSCs, counts of CFUs per run provide the largest average ratio between CD264-/+ populations; therefore, CFU counts are the best quality control metric used to determine which MSC sorts produced unique populations before performing RNA-Seq. For experimentation, only CFU counts with a CD264-/+ ratio greater than two for each donor were used. This ratio proved that the CD264-/+ populations are distinct from each other and can be further analyzed using RNA-Seq. The statistical analysis of live-cell quality control metrics proved that statistical significance exists between the average ratios of CFU count with both forward and side scatter. The CFU findings determined two distinct populations for aging and robust cells while the quality control metric analysis determined that compared to traditional aging cell quality control metrics, CFU counts provide the largest ratio between CD264-/+ populations. Using the best quality control metric for RNA-Seq provides optimal conditions to identify differential gene expressions in aging vs. robust stem cells. Results from RNA-Seq can identify new quality controls for aging MSCs and identify new targets for MSC rejuvenation.