Evaluation of inter-batch differences in stem-cell derived neurons

Abstract

Differentiated cells retain the genetic information of the donor but the extent to which phenotypic differences between donors or batches of differentiated cells are explained by variation introduced during the differentiation process is not fully understood. In this study, we evaluated four separate batches of commercially available neurons originating from the same iPSCs to investigate whether the differentiation process used in manufacturing iPSCs to neurons affected genome-wide gene expression, modified cytosines, or neuronal sensitivity to drugs. No significant changes in gene expression, as measured by RNA-Seq, or cytosine modification levels, as measured by the Illumina 450K arrays, were observed between batches relative to changes over time. As expected, neurotoxic chemotherapeutics affected neuronal outgrowth, but no inter-batch differences were observed in sensitivity to paclitaxel, vincristine and cisplatin. As a testament to the utility of the model for studies of neuropathy, we observed that genes involved in neuropathy had relatively higher expression levels in these samples across different time points. Our results suggest that the process used to differentiate iPSCs into neurons is consistent, resulting in minimal intra-individual variability across batches. Therefore, this model is reasonable for studies of human neuropathy, druggable targets to prevent neuropathy, and other neurological diseases.

Human Induced Pluripotent Stem Cell Derived Sensory Neurons are Sensitive to the Neurotoxic Effects of Paclitaxel

“…33 In recent years, studies of chemotherapy neurotoxicity have been performed in commercially available neurons from iPSCs (e.g., iCell Neurons and Peri.4U Neurons). [9][10][11][12][13][14][15][16]19,40 In these cases, cells are typically tested shortly after plating and the outgrowth of neurites is measured using high content imaging. Although neurite outgrowth is a common measurement of the neurodevelopmental effects of chemicals, 41,42 the mature neurite networks investigated in this study more accurately reflect the conditions during exposure to paclitaxel in humans.…”

“…There are several advantages of the current iPSC-SN model to study chemotherapy-induced neurotoxicity compared with similar studies in iCell neurons and Peri.4U neurons. [9][10][11][12][13][14][15][16]19,40 The iPSC-SNs are derived from a well-characterized and widely used iPSC line, 25 with a known genetic background that can be edited for mechanistic investigation of specific genes and for the introduction of human polymorphisms that are associated with chemotherapy-induced peripheral neuropathy. Other iPSC-derived sensory neurons have proven useful for the study of peripheral nerve injury, mechanotransduction, and neurodegeneration.…”

Stretch Injury of Human Induced Pluripotent Stem Cell Derived Neurons in a 96 Well Format

“…hiPSCNs offer unique insight into the pathology of human disease by enabling human in vitro models with cells such as cardiomyocytes and neurons that are difficult to ethically obtain by biopsy from human subjects. Batch-to-batch variability in cells produced in academic labs has historically presented significant challenges, but the recent availability of hiPSCNs from commercial vendors with little batch-to-batch variability 6 has broadened access to this technology and improved reproducibility across labs. iCell neurons from Cellular Dynamics are a mixed population of GABAergic and glutamatergic hiPSCNs expressing multiple ligand gated and voltage gated ion channels 7 .…”

Mechanosensitivity of Human Oligodendrocytes

“…There are several possible explanations for heterogeneity of behavior of hiPSC lines and their derivatives. Key sources of heterogeneity include the genetic background of the hiPSC donors and batch-to-batch variations among cells obtained from the same hiPSC donor during manual steps of multi-month differentiation procedures (Rouhani et al, 2014 ; Morrison et al, 2016 ; Carcamo-Orive et al, 2017 ). Age and sex of cell donors can influence stem cell fate and may be explanatory variables in the behavior of derived cells (Siegel et al, 2013 ).…”