Generation of a TPH2-EGFP reporter cell line for purification and monitoring of human serotonin neurons in vitro and in vivo

Xu, Ting; Duan, Jinjin; Li, Yingqi; Wang, Guanhao; Li, Shuanqing; Li, You; Lu, Weiyue; Yan, Xiaojun; Ren, Yixuan; Guo, Fei; Cao, Lining; Lu, Jianfeng

doi:10.1016/j.stemcr.2022.08.012

Cited by 3 publications

(3 citation statements)

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“…To facilitate the examination of the electrophysiological properties of SNs, a TPH2 EGFP reporter system was constructed into the TPH2 locus to indicate TPH2‐positive SNs with EGFP as we described previously. [ 23 ] The whole‐cell patch‐clamp recording was applied. The voltage clamp was used to generate stepped membrane voltages, and the current flowing through the membrane was measured.…”

Section: Resultsmentioning

confidence: 99%

“…Generation of FKO-TPH2EGFP Reporter Cell Line: FKO-TPH2 EGFP reporter cell line was generated on the background of an FKO2 hPSC line by CRISPR/Cas9 mediated gene editing as it was previously described. [23] Differentiation of hPSCs toward SNs: hPSCs were differentiated into SNs with some modifications were previously described. [5] Briefly, hPSCs were dissociated by TrypLE into single cells and passaged at the density of 5 × 10 4 cells cm −2 onto Matrigel-coated plates with mTeSR1 medium and 1 μM Y27632.…”

Section: Methodsmentioning

confidence: 99%

“…FKO‐TPH2 EGFP reporter cell line was generated on the background of an FKO2 hPSC line by CRISPR/Cas9 mediated gene editing as it was previously described. [ 23 ]…”

Section: Methodsmentioning

confidence: 99%

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Uncovering the role of FOXA2 in the Development of Human Serotonin Neurons

Xu,

Cao,

Duan

et al. 2023

Advanced Science

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Directed differentiation of serotonin neurons (SNs) from human pluripotent stem cells (hPSCs) provides a valuable tool for uncovering the mechanism of human SN development and the associated neuropsychiatric disorders. Previous studies report that FOXA2 is expressed by serotonergic progenitors (SNPs) and functioned as a serotonergic fate determinant in mouse. However, in the routine differentiation experiments, it is accidentally found that less SNs and more non‐neuronal cells are obtained from SNP stage with higher percentage of FOXA2‐positive cells. This phenomenon prompted them to question the role of FOXA2 as an intrinsic fate determinant for human SN differentiation. Herein, by direct differentiation of engineered hPSCs into SNs, it is found that the SNs are not derived from FOXA2‐lineage cells; FOXA2‐knockout hPSCs can still differentiate into mature and functional SNs with typical serotonergic identity; FOXA2 overexpression suppresses the SN differentiation, indicating that FOXA2 is not intrinsically required for human SN differentiation. Furthermore, repressing FOXA2 expression by retinoic acid (RA) and dynamically modulating Sonic Hedgehog (SHH) signaling pathway promotes human SN differentiation. This study uncovers the role of FOXA2 in human SN development and improves the differentiation efficiency of hPSCs into SNs by repressing FOXA2 expression.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Uncovering the role of FOXA2 in the Development of Human Serotonin Neurons

Xu,

Cao,

Duan

et al. 2023

Advanced Science

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Advances in human pluripotent stem cell reporter systems

Puspita,

Juwono,

Shim

2024

iScience

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Transcriptomic profiling across human serotonin neuron differentiation via the FEV reporter system

Li,

Duan,

et al. 2024

Stem Cell Res Ther

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Background The detailed transcriptomic profiles during human serotonin neuron (SN) differentiation remain elusive. The establishment of a reporter system based on SN terminal selector holds promise to produce highly-purified cells with an early serotonergic fate and help elucidate the molecular events during human SN development process. Methods A fifth Ewing variant (FEV)-EGFP reporter system was established by CRISPR/Cas9 technology to indicate SN since postmitotic stage. FACS was performed to purify SN from the heterogeneous cell populations. RNA-sequencing analysis was performed for cells at four key stages of differentiation (pluripotent stem cells, serotonergic neural progenitors, purified postmitotic SN and purifed mature SN) to explore the transcriptomic dynamics during SN differentiation. Results We found that human serotonergic fate specification may commence as early as day 21 of differentiation from human pluripotent stem cells. Furthermore, the transcriptional factors ZIC1, HOXA2 and MSX2 were identified as the hub genes responsible for orchestrating serotonergic fate determination. Conclusions For the first time, we exposed the developmental transcriptomic profiles of human SN via FEV reporter system, which will further our understanding for the development process of human SN. Graphical Abstract

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Generation of a TPH2-EGFP reporter cell line for purification and monitoring of human serotonin neurons in vitro and in vivo

Cited by 3 publications

References 28 publications

Uncovering the role of FOXA2 in the Development of Human Serotonin Neurons

Uncovering the role of FOXA2 in the Development of Human Serotonin Neurons

Advances in human pluripotent stem cell reporter systems

Transcriptomic profiling across human serotonin neuron differentiation via the FEV reporter system

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