Reprogramming method does not impact the neuronal differentiation potential of 16p11.2 deletion patient iPSCs
bioRxiv (Cold Spring Harbor Laboratory)(2023)
Abstract
SUMMARY A major impediment to the actualization of the induced pluripotent stem cell (iPSC)-based personalized medicine revolution is the lack of widely accepted standard operating procedures (SOPs) across different groups and institutions. The various methods employed can include choice of starting materials, reprogramming agents, and culture conditions, with each of these factors hypothesized to influence the reprogramming efficiency and transcriptional identity of iPSCs. As such, we systematically compared iPSC reprogramming procedures using cells derived from the somatic cells of three patients with 16p11.2 deletion syndrome (16p11.2del) and found remarkable similarity among the different methods. FACS analysis revealed that regardless of somatic cell type (fibroblast, lymphocyte, erythroblast), route of reprogramming factor introduction (mRNA, Sendai virus, episome), donor sex, or facility (Rutgers, NYSCF), 16p11.2del patient iPSCs were viable as high purity cultures expressing pluripotency marker proteins. This observation was supported at the transcript level by qPCR analysis, which demonstrated the ability for the iPSCs to differentiate into all three embryonic germ cell lineages after 12 days in culture as embryoid bodies. NGN2-mediated differentiation of these iPSCs produced functional neurons that formed active synaptic networks as revealed by multi-electrode array (MEA) recordings. Importantly, no group-wise comparisons among the reprogramming methods yielded consistent statistically significant differences, indicating that these procedures are equally capable of producing pluripotent stem cells that can efficiently differentiate into mature, functional neurons. This work highlights the utility of these reprogramming methods and supports the use of differentially reprogrammed iPSCs for direct comparative studies of human neurodevelopment.
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Key words
neuronal differentiation potential,deletion patient
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