Revealing a core signaling regulatory mechanism for pluripotent stem cell survival and self-renewal by small molecules

Abstract

Using a high-throughput chemical screen, we identified two small molecules that enhance the survival of human embryonic stem cells (hESCs). By characterizing their mechanisms of action, we discovered an essential role of E-cadherin signaling for ESC survival. Specifically, we showed that the primary cause of hESC death following enzymatic dissociation comes from an irreparable disruption of E-cadherin signaling, which then leads to a fatal perturbation of integrin signaling. Furthermore, we found that stability of E-cadherin and the resulting survival of ESCs were controlled by specific growth factor signaling. Finally, we generated mESC-like hESCs by culturing them in mESC conditions. And these converted hESCs rely more on E-cadherin signaling and significantly less on integrin signaling. Our data suggest that differential usage of cell adhesion systems by ESCs to maintain self-renewal may explain their profound differences in terms of morphology, growth factor requirement, and sensitivity to enzymatic cell dissociation.human embryonic stem cell survival | cell-cell adhesion | cell-ECM adhesion C onventional murine and human embryonic stem cells (hESCs) derived from blastocysts can be propagated indefinitely and have the ability to generate all cell types (1-3). They express pluripotency transcription factors, including the ones that can reprogram somatic cells back to pluripotent states: Oct4, Sox2, Nanog, and Klf4. However, murine and human ESCs respond very differently to several key signaling pathways in selfrenewal or differentiation. For example, murine ESCs (mESCs) self-renew under leukemia inhibitory factor (LIF) and bone morphogenic protein (BMP) (4, 5), whereas human ESCs (hESCs) appear dependent on FGF, and TGFβ/Activin/Nodal pathway activity for self-renewal (6-11). These studies clearly suggest that there exist two distinct self-renewal mechanisms. In addition, hESCs grow in vitro as large flattened 2D colonies, whereas mESCs display characteristic small-domed 3D appearances of compact colonies. Moreover, unlike mESCs, hESCs are very vulnerable to single-cell dissociation. Massive cell death occurs after complete single-cell dissociation, which has been a significant hurdle for rapid expansion and genetic manipulation of hESCs. To address this critical challenge and understand the molecular mechanisms that govern hESC survival, we used a high-throughput chemical screening approach and identified two small molecules with distinct mechanisms of action that significantly increase hESC survival after single-cell dissociation. In depth characterizations of compounds' mechanism of action revealed that hESC survival and self-renewal is regulated by the interplay between two cell adhesion systems: cell-cell adhesion and cell-ECM adhesion. Our studies also uncovered a common mechanism that underlies and integrates two seemingly distinct self-renewal states represented by conventional murine and human ESCs.

LacdiNAc (GalNAcβ1-4GlcNAc) Contributes to Self-Renewal of Mouse Embryonic Stem Cells by Regulating Leukemia Inhibitory Factor/STAT3 Signaling

“…By contrast, mESCs are derived from ICM and are maintained in the naïve state. Recent reports indicate that hESCs (primed state) can be induced to revert to mESC-like cells (naïve state) under the specific culture conditions that are required for maintenance of mESCs; after reversion, these naïve hESCs can be maintained under culture conditions containing LIF [25,53]. Thus, these reports have reinforced the view that hESCs and mESCs are at developmentally different stages.…”

Low-molecular-weight inhibitors of cell differentiation enable efficient growth of mouse iPS cells under feeder-free conditions

“…In combination with CHIR99021, PD0325901 affects the self-renewality of ES and iPS cells to prevent cell differentiation (Lin et al 2009). Thiazovivin acts as a Rho-associated Kinase inhibitor, which can improve the survival of ES and iPS cells from single cell dissociation (Lin et al 2009;Xu et al 2010). The iPS cell colonies were more rounded with clearer boundaries than those obtained under condition 2 (Fig.…”

The evolving biology of small molecules: controlling cell fate and identity

“…We ultimately found two: thiazovivin (Tzv; a 2,4-disubstituted thiazole) and pyrintegrin (Ptn; a 2,4-disubstituted pyrimidine). Both compounds enhance survival following dissociation more than 30-fold, without adversely affecting pluripotency over long-term culture in chemically defined medium [26].…”

“…More recently, another study generated rat ESCs using four small molecules, including PD0325901, A-83-01, CHIR99021 and Y27632 [89]. Y27632 is a Rho-associated protein kinase inhibitor that can stabilize E-cadherin [26]. These derived cells successfully contributed to the generation of transgenic rats via germline transmission, proving their occupancy of the naive ground state.…”