Effect of Passage Number on Human Induced Pluripotent Stem Cell Derived Neurons Culture Contaminants with Non-Neuronal Cell Types

Abstract

Background: The study of live human neurons has been hindered due to the complexity and potential irreversible damage to the patient during biopsy. However, reprogramming of adult human somatic cells into induced pluripotent stem cells (iPSCs) has proved to be a novel method in the study of the pathophysiology of disease and therapeutic targets of the human nervous system. There are several approaches, and the optimum time (i.e., passage number) to generate highly pure cultures is being studied. Therefore, our laboratory has investigated the effect of passage number on culture contaminants with non-neuronal cell types. 
 
 Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood from three cell lines and reprogrammed into iPSCs. Each cell line consisted of three samples that were analyzed after low (5-10), middle (20-26), and high (30-38) number of passages. Cells were maintained in an induction medium for eight days. On day nine, cells were dissociated and replated in a maintenance medium. On day 33, total RNA was extracted from cells. Normalized values for non-neuronal cell marker genes were compared using paired Student’s t-tests and two-way ANOVA, with the cell line and passage number as independent variables. P-values less than 0.05 were considered significant. 
 
 Results: Our results showed that lower passage number was associated with decreased astrocyte and chondrocyte marker expression. High passage number was associated with decreased oligodendrocyte and glial precursor marker expression. Of the fibroblast markers evaluated, there were similar trends of expression between all three groups. There was no significant difference in microglial cell marker gene expression between all three groups. 
 
 Conclusion and Potential Impact: Low gene expression suggests a purer culture. According to these results, as passage number increases, there is more contaminants with oligodendrocytes and glial precursor cells. Conversely, with low passage numbers, there are more contaminants with astrocytes and chondrocytes. Future studies will identify the impact of these non-neuronal contaminants and implications on research.