Molecular characterization of a toxicological tipping point during human stem cell differentiation

Saili, Katerine S.; Antonijevic, Todor; Zurlinden, Todd J.; Shah, Imran; Deisenroth, Chad; Knudsen, Thomas B.

doi:10.1016/j.reprotox.2019.10.001

Cited by 20 publications

(15 citation statements)

References 69 publications

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“…SB431542 is a small molecule inhibitor of TGFβ signaling through ALK4, ALK5, and ALK7 that blocks primitive streak induction in mESCs 46 . Both SB431542 and atRA 23,47 block hPSC definitive endoderm differentiation and were shown here to disrupt LPM differentiation in a CRBN-and SALL4-independent fashion. In contrast, thalidomide, lenalidomide, and pomalidomide inhibited hiPSC mesendoderm differentiation via CRBN-mediated SALL4 degradation.…”

Section: Discussionmentioning

confidence: 64%

Thalidomide Inhibits Human iPSC Mesendoderm Differentiation by Modulating CRBN-dependent Degradation of SALL4

Belair¹,

Lü²,

Waller³

et al. 2020

Sci Rep

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exposure to thalidomide during a critical window of development results in limb defects in humans and non-human primates while mice and rats are refractory to these effects. Thalidomide-induced teratogenicity is dependent on its binding to cereblon (CRBN), the substrate receptor of the Cul4A-DDB1-CRBN-RBX1 E3 ubiquitin ligase complex. Thalidomide binding to CRBN elicits subsequent ubiquitination and proteasomal degradation of CRBN neosubstrates including SALL4, a transcription factor of which polymorphisms phenocopy thalidomide-induced limb defects in humans. Herein, thalidomide-induced degradation of SALL4 was examined in human induced pluripotent stem cells (hiPSCs) that were differentiated either to lateral plate mesoderm (LPM)-like cells, the developmental ontology of the limb bud, or definitive endoderm. Thalidomide and its immunomodulatory drug (iMiD) analogs, lenalidomide, and pomalidomide, dose-dependently inhibited hipSc mesendoderm differentiation. Thalidomide-and IMiD-induced SALL4 degradation can be abrogated by CRBN V388I mutation or SALL4 G416A mutation in hiPSCs. Genetically modified hiPSCs expressing CRBN E377V/ V388I mutant or SALL4 G416A mutant were insensitive to the inhibitory effects of thalidomide, lenalidomide, and pomalidomide on LPM differentiation while retaining sensitivity to another known limb teratogen, all-trans retinoic acid (atRA). Finally, disruption of LPM differentiation by atRA or thalidomide perturbed subsequent chondrogenic differentiation in vitro. the data here show that thalidomide, lenalidomide, and pomalidomide affect stem cell mesendoderm differentiation through CRBN-mediated degradation of SALL4 and highlight the utility of the LPM differentiation model for studying the teratogenicity of new cRBn modulating agents.

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Section: Discussionmentioning

confidence: 64%

Thalidomide Inhibits Human iPSC Mesendoderm Differentiation by Modulating CRBN-dependent Degradation of SALL4

Belair¹,

Lü²,

Waller³

et al. 2020

Sci Rep

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“…Our clutch analysis suggests that this threshold might be strongly dependent on genetic polymorphisms affecting enzymatic activity or gene expression parameters. In support, a threshold or toxicological tipping point for RA signaling was recently described in a cell-based model (Saili et al, 2019). The clutch analysis also showed that the network response, i.e.…”

Section: Discussionmentioning

confidence: 74%

Retinoic acid fluctuation activates an uneven, direction-dependent network-wide robustness response in early embryogenesis

Parihar

Bendelac-Kapon

Gur

et al. 2020

Preprint

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Retinoic acid (RA) is a central developmental signal whose perturbation results in teratogenic outcomes. We performed transient physiological RA signaling disturbances during embryogenesis followed by kinetic RNAseq and high-throughput qPCR analysis of the recovery. Unbiased comparative pattern analysis identified the RA network as a key differentially regulated module aimed at achieving RA signaling robustness. We analyzed this module using a principal curve-based approach determining clutch-wise variability, and organized the results into a robustness efficiency matrix comparing the shifts in RA feedback regulation and hox gene expression. We found that feedback autoregulation was sensitive to the direction of the RA perturbation: RA knock-down exhibited an upper response limit, whereas RA addition did not activate a feedback response below a threshold. These results suggest an asymmetric capacity for robust feedback control of RA signal during early development based on genetic polymorphisms, likely a significant contributor to the manifestation of developmental defects.

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“…To explore whether DNA damage does indeed redirect differentiation in a p53-dependent manner, we examined changes in expression in key pluripotency and lineage-specific markers ( Saili et al., 2019 ) in each condition ( Figure 4 F). MMS-treated wild-type cells expressed lower levels of DE markers and higher levels of most mesoderm markers, along with small changes in ectoderm markers.…”

Section: Resultsmentioning

confidence: 99%

A p53-Dependent Checkpoint Induced upon DNA Damage Alters Cell Fate during hiPSC Differentiation

et al. 2020

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Summary The ability of human induced pluripotent stem cells (hiPSCs) to differentiate in vitro to each of the three germ layer lineages has made them an important model of early human development and a tool for tissue engineering. However, the factors that disturb the intricate transcriptional choreography of differentiation remain incompletely understood. Here, we uncover a critical time window during which DNA damage significantly reduces the efficiency and fidelity with which hiPSCs differentiate to definitive endoderm. DNA damage prevents the normal reduction of p53 levels as cells pass through the epithelial-to-mesenchymal transition, diverting the transcriptional program toward mesoderm without induction of an apoptotic response. In contrast, TP53 -deficient cells differentiate to endoderm with high efficiency after DNA damage, suggesting that p53 enforces a “differentiation checkpoint” in early endoderm differentiation that alters cell fate in response to DNA damage.

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Molecular characterization of a toxicological tipping point during human stem cell differentiation

Cited by 20 publications

References 69 publications

Thalidomide Inhibits Human iPSC Mesendoderm Differentiation by Modulating CRBN-dependent Degradation of SALL4

Thalidomide Inhibits Human iPSC Mesendoderm Differentiation by Modulating CRBN-dependent Degradation of SALL4

Retinoic acid fluctuation activates an uneven, direction-dependent network-wide robustness response in early embryogenesis

A p53-Dependent Checkpoint Induced upon DNA Damage Alters Cell Fate during hiPSC Differentiation

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