Characteristic analyses of a neural differentiation model from iPSC-derived neuron according to morphology, physiology, and global gene expression pattern.
- Authors
- Kang, Sai; Chen, Xiaoxia; Gong, Siyi; Yu, Panpan; Yau, SukYu; Su, Zhenghui; Zhou, Libing; Yu, Jiandong; Pan, Guangjin; Shi, Lingling
- Year
- 2017
- Journal
- Scientific reports
- PMID
- 28947763
- DOI
- 10.1038/s41598-017-12452-x
- PMCID
- PMC5612987
Induced pluripotent stem cells (iPSCs) can differentiate into neural progenitor cells (NPC) under proper conditions. NPC can be used as a model and is a useful tool for disease mechanism exploration and drug screening. However, the characteristics of the cells in various stages from NPC to functional neurons have not been fully described. This study investigated the characteristics of iPSC-derived NPCs during differentiation. Morphological characteristics of the NPCs, including soma area, neurite length, and the number of neurite branches, were examined on selected differentiation days. Physiological functions were assessed by recordings of sodium current, spontaneous excitatory postsynaptic current (sEPSC), and spontaneous inhibitory postsynaptic current (sIPSC). Furthermore, gene expression patterns were assessed with RNA-seq. We found that NPCs derived from iPSCs can be differentiated into glutamatergic and gabaergic neurons. Cell growth peaked during differentiation day 7-12, as the soma area decreased after day 12, growth cone and the number of branches peaked at day 9 and decreased afterwards; whereas a functional synapse formed after day 23. RNA-seq analysis found that a differential expression pattern emerged by day 7. Overall, the study provides a framework for the differentiation process of hiPSC-derived NPCs.
In vitro neural development model. Neural progenitor cells (NPCs) were differentiated from hiPSCs, which were then further induced to differentiate into neurons (A–H). The majority of cells differentiated from hiPSCs stained positive for NESTIN, indicating the cells were NPCs (E). NPCs derived from hiPSC maintained differentiation potential. HiPSC derived NPCs can diffentiated into both neural and glial lineage as stained by neuron marker TUJ-1, astrocyte marker GFAP (I–L).
The area of soma increased during development. The soma area of the cultured neurons increased significantly when measured on D9, D12 and D15, compared with D3 (n = 60, ***P < 0.05).
Both the length and the number of branches of neuritis increased during NPC maturation. (A) Cells expressing RFP were used for morphological study. The length of neuritis and the number of branches were measured and counted. (B–D) The number of primary and secondary branches of neurites reached a peak on D9. The number of primary branches showed a decreasing trend after D9. (E–G) The length of neurites reached a peak on D12 (n = 10 for D9, n = 20 for D3, D6, D12 and D15 measurement, ***P < 0.001, **P < 0.01, *P < 0.05).
Development pattern of the growth cone. (A) The growth cones are categorized into three subtypes: blunt-ended (collapsed with no visible filopodia or lamellipodia), filopodial (growth cones with numerous filopodia and a small or absent lamellipodial veil), or lamellipodial (well spread growth cones with elaborate lamellipodia). (B) Area of growth cone reached a peak on D9. (C) The percentage of the lamellipodial type growth cone increased before D9 and then decreased (n = 60, ***P < 0.001, *P < 0.05).
Sodium channels were expressed in early days of differentiation, but became functional only in later days. (A) RFP expressing cell visualized under microscope. (B) Sodium current traces recorded from cultured cells on different days during differentiation. (C) The depolarization that was required to elicit sodium current decreased. (D) The amplitude of sodium current increased. (E) The resting membrane potential of cultured cells decreased. (F) The capacitance of the cultured cells increased. (G) The membrane resistance of the cells decreased.
Frequency of sEPSCs and sIPSC progressively increased. (A) Example sEPSC traces recorded from a cultured cell. (B) The amplitude of the EPSCs increased. (C) The frequency of EPSC increased. (D) The charges transferred between cultured cells increased. (E) Example sIPSC traces recorded from a cultured cell. (F) The amplitude of sIPSC increased. (G) The frequency of sIPSC increased. (H) The charges transferred between cultured cells increased.
The enriched gene modules were divided into three groups by trend in expression. Squares with colors indicate a P value of <0.01. Four modules (red, No. 11, 12, 13, 15) belong to the increasing gene expression module, 2 modules (yellow, No. 2, 3) belong to the decreasing cluster, and the expression level of 2 modules (purple and green, No 1, 14) correlate with disorders.
WNT signal pathway gene expression. WNT2B, WNT5A, and WNT7A gene expression were quantified by qRT-PCR. The results were consistent with the findings of RNA-Seq.
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