Submission
| Poster Title: | Passage Number Impacts the Heterogeneity of Human Induced Pluripotent Stem Cell-Derived Neuron Cultures |
| Authors: | Dominique Ketsoglou, Erica Cantor, Guanglong Jiang, Xi Wu, Santosh Phillips, Fei Shen, Bryan P. Schneider |
Abstract
Background/Significance/Rationale:
The reprogramming of adult human somatic cells into induced pluripotent stem cells (iPSCs) with subsequent sensory neuronal differentiation has proved to be a reliable method for the study of the pathophysiology of diseases of the human peripheral nervous system (PNS). The impact of iPSC culture conditions, such as passage number, on the generation of pure, mature neuronal cultures has not been definitively established. Therefore, we set out to determine the effect of iPSC passage number on maturity and presence of contaminating cell types in iPSC-derived sensory neuron (iPSC-dSN) cultures.
Methods:
Peripheral blood mononuclear cells were isolated from whole blood of three individual donors and reprogrammed into iPSCs. The three iPSC lines were passaged until each of three target passage numbers were reached: low (5-10), middle (20-26), and high (30-38). Neuronal differentiation was then induced for eight days and maintained for an additional 24 days. On day 33, total RNA was extracted from the cells. Normalized expression values for marker genes of potential contaminating cell types and pluripotency were compared.
Results/Findings:
Lower passage number was associated with decreased expression of pluripotency and astrocyte markers, and increased expression of myelinating glial cell markers. No significant differences in the expression of markers for other common contaminating cell types were observed.
Conclusions/Discussion:
Lower passage numbers optimally replicated a pure, PNS phenotype, with lower expression of marker genes for common contaminating cell types, and higher expression of marker genes for key components of the PNS.
Translational/Human Health Impact:
The study of primary human neurons is hindered due to the potential for irreversible damage to a patient during biopsy. Optimizing this model from pure iPSC-dSNs cultures will improve the studies of neurological diseases and drug induced neurotoxicities. This work is also poised to provide insight into novel therapeutic interventions to improve patient outcomes.
