A Chemical Biology Study of Human Pluripotent Stem Cells Unveils HSPA8 as a Key Regulator of Pluripotency

Abstract

SummaryChemical biology methods such as high-throughput screening (HTS) and affinity-based target identification can be used to probe biological systems on a biomacromolecule level, providing valuable insights into the molecular mechanisms of those systems. Here, by establishing a human embryonal carcinoma cell-based HTS platform, we screened 171,077 small molecules for regulators of pluripotency and identified a small molecule, Displurigen, that potently disrupts hESC pluripotency by targeting heat shock 70-kDa protein 8 (HSPA8), the constitutively expressed member of the 70-kDa heat shock protein family, as elucidated using affinity-based target identification techniques and confirmed by loss-of-function and gain-of-function assays. We demonstrated that HSPA8 maintains pluripotency by binding to the master pluripotency regulator OCT4 and facilitating its DNA-binding activity.

Investigating core genetic-and-epigenetic cell cycle networks for stemness and carcinogenic mechanisms, and cancer drug design using big database mining and genome-wide next-generation sequencing data

“…Of these, HSPA8 has been reported as a constitutively expressed gene involved in M phase regulation. 53 Therefore, we suggest that M phase-specific genes, which may be the constitutively expressed, could play an important role in promoting properly timed cell cycle exit.…”

A small molecule-based strategy for endothelial differentiation and three-dimensional morphogenesis from human embryonic stem cells

“…A delicate state of balance exists between the pluripotency regulatory network and the differentiation regulatory pathways, and is required for the maintenance of pluripotency [ 19 , 20 ]. Disrupting this balanced state, by altering the expressions or activities of different components important for the maintenance of this balance, will trigger differentiation in different ways and toward different directions; for example, inhibition of heat shock 70 kDa protein 8 (HSPA8) by a small molecule leads to un-directional differentiation [21] , whereas small molecule inhibition of TRPM6/TRPM7 magnesium channel activity specifically enhances mesoderm and definitive endoderm differentiations [22] . We thus hypothesized that by applying a stimulus that undermines this delicate balance in a certain way during the initial phase of differentiation, hESCs may be poised to differentiate in such a way that a self-organizing organoid structure can be generated.…”

Mesendogen, a novel inhibitor of TRPM6, promotes mesoderm and definitive endoderm differentiation of human embryonic stem cells through alteration of magnesium homeostasis

“…We previously conducted a high-throughput chemical screening and found 29 bioactive small molecules that potently disrupt hESC pluripotency ( Geng et al, 2015 ). Using this compound collection we conducted a small scale chemical screen (Materials and Methods) to search for small molecules that specifically induce mesoderm and DE differentiations, and identified compound 6528694 (N-({[2-chloro-5-(trifluoromethyl) phenyl]amino}carbonothioyl)-4-isopropylbenzamide; Fig.…”

“…Previously a high-throughput chemical screening was conducted at the High-Throughput Screening Facility (HTSF) at the University of Illinois at Urbana−Champaign ( http://www.scs.illinois.edu/htsf/index.html ). A total of 171,077 compounds from the Marvel Library, the HTSF House Library, the ChemBridge MicroFormat Library, and the National Cancer Institute (NCI) library were screened, giving rise to 29 bioactive small molecules which potently disrupt hESC pluripotency ( Geng et al, 2015 ). Using this collection of 29 bioactive compounds, we conducted the small scale screen for mesoderm and DE inducers.…”

“…For this screen, hESCs were maintained in the pluripotent state in mTeSR1 medium ( Ludwig et al, 2006 ). Small molecules were added at 1–10 μM, depending on their respective potencies and toxicities shown at the validation stage of the previous high-throughput screen ( Geng et al, 2015 ), and incubated for 5–7 days to induce differentiation. Differentiated cultures were then assayed for mesoderm, DE, and neural lineage-specific marker expressions using Western blotting.…”