Multi-omics approach reveals posttranscriptionally regulated genes are essential for human pluripotent stem cells

Iwasaki, Mio; Kawahara, Yuka; Okubo, Chikako; Yamakawa, Tatsuya; Nakamura, Michiko; Tabata, Tsuyoshi; Nishi, Yoshimi; Narita, Megumi; Ohta, Akira; Saito, Hirohide; Yamamoto, Takuya; Nakagawa, Masato; Yamanaka, Shinya; Takahashi, Kazutoshi

doi:10.1016/j.isci.2022.104289

“…Interestingly, Nestin and β-3-tubulin were expressed in undifferentiated iPSCs, despite being commonly used as ectodermal and neuronal differentiation markers. This result is consistent with a previous report of their expression in some iPS cell lines (Kuang et al, 2019 ) and with published RNA-seq and proteomic data for the 201B7 cell line (Iwasaki et al, 2022 ). The detection of a broader range of endogenous protein expression in live cells will require further improvements in the brightness of fluorescent proteins or in the sensitivity of detectors.…”

Section: Discussionsupporting

confidence: 91%

Endogenous tagging using split mNeonGreen in human iPSCs for live imaging studies

Husser,

Pham,

Law

et al. 2023

Preprint

0

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Endogenous tags have become invaluable tools to visualize and study native proteins in live cells. However, generating human cell lines carrying endogenous tags is difficult due to the low efficiency of homology-directed repair. Recently, an engineered split mNeonGreen protein was used to generate a large-scale endogenous tag library in HEK293 cells. Using split mNeonGreen for large-scale endogenous tagging in human iPSCs would open the door to studying protein function in healthy cells and across differentiated cell types. We engineered an iPS cell line to express the large fragment of the split mNeonGreen protein (mNG21-10) and showed that it enables fast and efficient endogenous tagging of proteins with the short fragment (mNG211). We also demonstrate that neural network-based image restoration enables live imaging studies of highly dynamic cellular processes such as cytokinesis in iPSCs. This work represents the first step towards a genome-wide endogenous tag library in human stem cells.

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“…We also expected some of these genes to be expressed at varying levels in iPSCs. The expression levels for these genes are shown on a graph of RNA-seq data from Iwasaki et al, 2022 ; Figure 2—figure supplement 1A . While ACTB and TUBA1B should be highly expressed, there is a shift to lower expression levels for KRT18, RHOA, and H2BC11, while SOX2, CDH1, NES, TUBB3, ANLN, and RACGAP1 are all weakly expressed.…”

Section: Resultsmentioning

confidence: 99%

“…Genes targeted for tagging with mNG2 11 are indicated by triangles (green for tagged proteins that were detected and blue for tagged proteins that were not detected in induced pluripotent stem cells, iPSCs). The RNA-seq data was obtained from Iwasaki et al, 2022 ; GEO accession number GSE199820. ( B–T ) Flow cytometry plots show 17 different cell populations with smNG2 fluorescence after being edited with the mNG2 11 tag.…”

Section: Resultsmentioning

confidence: 99%

Endogenous tagging using split mNeonGreen in human iPSCs for live imaging studies

Husser,

Pham,

Law

et al. 2024

eLife

0

View full text Add to dashboard Cite

Endogenous tags have become invaluable tools to visualize and study native proteins in live cells. However, generating human cell lines carrying endogenous tags is difficult due to the low efficiency of homology-directed repair. Recently, an engineered split mNeonGreen protein was used to generate a large-scale endogenous tag library in HEK293 cells. Using split mNeonGreen for large-scale endogenous tagging in human iPSCs would open the door to studying protein function in healthy cells and across differentiated cell types. We engineered an iPS cell line to express the large fragment of the split mNeonGreen protein (mNG21-10) and showed that it enables fast and efficient endogenous tagging of proteins with the short fragment (mNG211). We also demonstrate that neural network-based image restoration enables live imaging studies of highly dynamic cellular processes such as cytokinesis in iPSCs. This work represents the first step towards a genome-wide endogenous tag library in human stem cells.

show abstract

“…We also expected some of these genes to be expressed at varying levels in iPSCs. The expression levels for these genes are shown on a graph of RNA-seq data from (Iwasaki et al 2022 Fig. S3A).…”

Section: Resultsmentioning

confidence: 99%

“…Genes targeted for tagging with mNG2 11 are indicated by triangles (green for tagged proteins that were detected and blue for tagged proteins that were not detected in iPSCs). The RNA-seq data was obtained from Iwasaki et al (2022; GEO accession number GSE199820). B-T) Flow cytometry plots show 17 different cell populations with smNG2 fluorescence after being edited with the mNG2 11 tag.…”

Section: Figure S1mentioning

confidence: 99%

Endogenous tagging using split mNeonGreen in human iPSCs for live imaging studies

Husser

¹

,

Pham

²

,

Law

³

et al. 2023

Preprint

0

View full text Add to dashboard Cite

Endogenous tags have become invaluable tools to visualize and study native proteins in live cells. However, generating human cell lines carrying endogenous tags is difficult due to the low efficiency of homology-directed repair. Recently, an engineered split mNeonGreen protein was used to generate a large-scale endogenous tag library in HEK293 cells. Using split mNeonGreen for large-scale endogenous tagging in human iPSCs would open the door to studying protein function in healthy cells and across differentiated cell types. We engineered an iPS cell line to express the large fragment of the split mNeonGreen protein (mNG21-10) and showed that it enables fast and efficient endogenous tagging of proteins with the short fragment (mNG211). We also demonstrate that neural network-based image restoration enables live imaging studies of highly dynamic cellular processes such as cytokinesis in iPSCs. This work represents the first step towards a genome-wide endogenous tag library in human stem cells.

show abstract

Multi-omics approach reveals posttranscriptionally regulated genes are essential for human pluripotent stem cells

Cited by 10 publications

References 52 publications

Endogenous tagging using split mNeonGreen in human iPSCs for live imaging studies

Endogenous tagging using split mNeonGreen in human iPSCs for live imaging studies

Endogenous tagging using split mNeonGreen in human iPSCs for live imaging studies

Endogenous tagging using split mNeonGreen in human iPSCs for live imaging studies

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