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Updated: Jun 8, 2026
A Simple Method to Identify Kinases That Regulate Embryonic Stem Cell Pluripotency by High-throughput Inhibitor Screening
Published on: May 12, 2017
High-content screening of feeder-free human embryonic stem cells to identify pro-survival small molecules
Paul D Andrews1, Melissa Becroft, Anders Aspegren
1University of Dundee, Scotland, UK. p.d.andrews@dundee.ac.uk
Abstract:
The propensity of human embryonic stem cells to die upon enzymatic disaggregation or low-density plating is an obstacle to their isolation and routine use in drug discovery and basic research. Equally, the very low rate of establishment of implanted cells hinders cell therapy. In the present study we have developed a high-content assay for human embryonic stem cell survival and used this to screen a range of libraries of 'lead-like' small molecules and known bioactives. From this we identified 18 confirmed hits with four structural classes being represented by multiple compounds: a series of 5-(acyl/alkyl-amino)indazoles, compounds with a 4-(acylamino)pyridine core, simple N⁶,N⁶-dialkyladenines and compounds with a 5-(acylamino)indolinone core. In vitro kinase profiling indicated that the ROCK (Rho-associated kinase)/PRK2 (protein kinase C-related kinase 2) protein kinases are of pivotal importance for cell survival and identified previously unreported compound classes that inhibited this important biological activity. An evaluation using an extensive panel of protein kinases showed that six of our hit compounds exhibited better selectivity for ROCK inhibition than the routinely used commercially available ROCK inhibitor Y-27632. In this screen we also identified the K(+)-ATP channel opener pinacidil and show that it probably promotes cell survival, by 'off-target' inhibition of ROCK/PRK2. We have therefore identified novel pro-survival compounds of greater specificity, equivalent potency and reduced toxicity relative to the routinely employed ROCK inhibitor Y-27632.
Insights
Researchers identified new small molecules that improve human embryonic stem cell survival, crucial for drug discovery and cell therapy. These compounds offer greater specificity and reduced toxicity compared to existing methods.
Area of Science:
- Stem cell biology
- Pharmacology
- Biochemistry
Background:
- Human embryonic stem cells (hESCs) exhibit poor survival during enzymatic disaggregation and low-density plating, hindering isolation and research applications.
- Low engraftment rates of implanted cells impede the progress of cell-based therapies.
Purpose of the Study:
- To develop a high-content assay for screening compounds that enhance hESC survival.
- To identify novel small molecules and bioactive compounds that promote hESC survival and overcome current therapeutic limitations.
Main Methods:
- A high-content assay was developed to screen libraries of 'lead-like' small molecules and known bioactives for hESC survival.
- In vitro kinase profiling was performed to identify the molecular targets of the identified compounds.
- Hit compounds were evaluated for their selectivity and potency against ROCK (Rho-associated kinase) inhibition compared to Y-27632.
Main Results:
- Eighteen confirmed hits were identified, falling into four main structural classes: 5-(acyl/alkyl-amino)indazoles, 4-(acylamino)pyridine, N⁶,N⁶-dialkyladenines, and 5-(acylamino)indolinone.
- ROCK/PRK2 (protein kinase C-related kinase 2) were identified as pivotal kinases for hESC survival.
- Six hit compounds demonstrated superior ROCK inhibition selectivity and potency compared to the standard ROCK inhibitor Y-27632.
- The K(+)-ATP channel opener pinacidil was identified as a novel ROCK/PRK2 inhibitor promoting cell survival.
Conclusions:
- Novel pro-survival compounds for hESCs have been identified with improved specificity, potency, and reduced toxicity over Y-27632.
- Targeting ROCK/PRK2 kinases represents a promising strategy for enhancing hESC survival in vitro and in vivo.
- These findings pave the way for improved stem cell isolation, drug discovery, and cell therapy applications.
