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. 2022 Sep 23;12(1):15869.
doi: 10.1038/s41598-022-19018-6.

Passage number affects differentiation of sensory neurons from human induced pluripotent stem cells

Erica L Cantor  1 , Fei Shen  1 , Guanglong Jiang  2 , Zhiyong Tan  3 , Geneva M Cunningham  2 , Xi Wu  1 , Santosh Philips  4 , Bryan P Schneider  5
Affiliations

Passage number affects differentiation of sensory neurons from human induced pluripotent stem cells

Erica L Cantor et al. Sci Rep. .

Abstract

Induced pluripotent stem cells (iPSCs) are a valuable resource for neurological disease-modeling and drug discovery due to their ability to differentiate into neurons reflecting the genetics of the patient from which they are derived. iPSC-derived cultures, however, are highly variable due to heterogeneity in culture conditions. We investigated the effect of passage number on iPSC differentiation to optimize the generation of sensory neurons (iPSC-dSNs). Three iPSC lines reprogrammed from the peripheral blood of three donors were differentiated into iPSC-dSNs at passage numbers within each of the following ranges: low (5-10), intermediate (20-26), and high (30-38). Morphology and pluripotency of the parent iPSCs were assessed prior to differentiation. iPSC-dSNs were evaluated based on electrophysiological properties and expression of key neuronal markers. All iPSC lines displayed similar morphology and were similarly pluripotent across passage numbers. However, the expression levels of neuronal markers and sodium channel function analyses indicated that iPSC-dSNs differentiated from low passage numbers better recapitulated the sensory neuron phenotype than those differentiated from intermediate or high passage numbers. Our results demonstrate that lower passage numbers may be better suited for differentiation into peripheral sensory neurons.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Morphology and pluripotency of the three iPSC lines (06401-2sb, CMC226, rsAA-12) at different passage numbers (low, intermediate, and high). (A) Phase-contrast images of three iPSC cell lines at different passage numbers. Scale bar = 1000 microns. (B) The average proportion of pluripotent cells in iPSCs at different passage numbers based on measurements of the expression of Sox2, Oct3/4, and Nanog using flow cytometry. (C) The expression level of SOX2, OCT3/4, and NANOG in iPSCs at different passage numbers based on RNA-seq. LP = low-passage, IP = intermediate-passage, HP = high-passage.
Figure 2
Figure 2
The morphology and expression of neuronal markers peripherin and βIII-tubulin in iPSC-dSNs differentiated from iPSCs at each of low, intermediate, and high passage numbers. (A) Representative immunofluorescent images of neurons stained for neuronal markers peripherin and βIII-tubulin. Scale bar = 200 microns. (B) Average expression levels of TUBB3 in iPSC-dSNs derived from iPSCs with different passage numbers by RNA-seq. (C) Average expression levels of PRPH in iPSC-dSNs derived from iPSCs with different passage numbers by RNA-seq. (D) Representative phase-contrast images of iPSC-dSNs derived from iPSCs with different passage numbers. Scale bar = 200 microns. LP = low-passage, IP = intermediate-passage, HP = high-passage; *FDR = 0.005.
Figure 3
Figure 3
Comparison of average expression levels of neuronal marker genes at day 33 post-induction in three iPSC-dSN lines induced at three different iPSC passage numbers. LP = low-passage, IP = intermediate-passage, HP = high-passage; * FDR < 0.05.
Figure 4
Figure 4
Electrophysiological assessment of iPSC-dSNs differentiated from iPSCs at different passage numbers. (A) Average cell size, (B) average membrane capacitance, and (C) average sodium current amplitude and (D) density from whole-cell patch-clamp recordings. Experiments were performed using 16, 14, and 18 iPSC-dSNs of low-, intermediate-, and high-passage, respectively. LP = low-passage, IP = intermediate-passage, HP = high-passage; *p-value < 0.05.
Figure 5
Figure 5
Timeline of reprogramming iPSCs from peripheral blood and subsequent neuronal differentiation.
See this image and copyright information in PMC

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