Progress toward the goal of tissue rejuvenation via stem cells and tissue engineering (“RepleniSENS”) is badly hampered by the surprising fragility of human embryonic stem cells (hESC) relative to mouse ESC (mESC). Unlike their murine counterparts, hESC undergo extensive cell death following enzymatic single-cell dissociation; as a result, researchers are forced to rely on laborious mechanical microdissection, or on narrowly-control enzymatic dissociation that ensures that hESC remain above a minimum cluster size. These requirements make their expansion extremely tedious and inefficient. The reasons for the intolerance of hESC to full dissociation — and the development of means to ameliorate it — are therefore of considerable biomedical as well as scientific interest. This month, researchers at the Scripps Research Institute led by Dr. Sheng Ding report that they have at once apparently provided the detailed molecular basis for this frustrating anomaly, and its abrogation using either modified culture protocols or either of two small molecules.
Xu et al(1) began with a high-throughput screen to “identify small molecules that promoted hESC survival after trypsin dissociation … while maintaining pluripotency”. Of 50 000 synthetic candidate molecules, two emerged — named Thiazovivin (Tzv) and Tyrintegin (Ptn) — that enhanced the survival of several hESC lines on matrigel plating >30 fold without altering proliferation rates, while maintaining full pluripotency, including the ability to form all 3 germ layers in teratomas in nude mice.
The researchers’ attention was drawn to the fact that the treatments caused substantial increases in hESC adhesion to extracellular matrix (ECM) protein substrates for integrin binding (laminin or matrigel), but not in adhesion to gelatin.This led to a series of experiments confirming the involvement of integrin activity and integrin-mediated growth and survival signaling in the improved survival of treated cells bound to ECM proteins.
But while either Tzv or Ptn enhanced attachment to ECM substitutes, Tzv alone could additionally support the formation and survival of free-floating hESC aggregates. This was traced to Tzv-induced increases in cell-cell adhesion via increased E-cadherin levels. It was found that E-cadherin was cleaved by trypsin dissociation, and while cells did synthesize E-cadherin, the newly-synthesized molecules were unstable and rapidly degraded. Tzv subsequently restored its levels by stabilizing the molecule and inhibiting its endocytosis rather than by increasing its transcription. Pulldown studies revealed that Tzv — and not Ptn — inhibits Rho-associated kinase (ROCK); inhibiting Rho-ROCK signaling by Tzv or any of several other agents enhanced the level of E-cahedrin and promoted cell survival, supporting either cell attachment to ECM or alternatively cell-cell adhesion and formation of cell aggregates. By contrast, while Ptn could also inhibit Rho activity and stabilize E-cadherin, it could only do so in the presence of ECM. Consistent with this, plating on matrigel itself inhibited Rho activity, while plating on gelatin did not.
Collectively, these findings revealed the culpability of increased Rho-ROCK signaling in the high fragility of hESC following enzymatic dissociation, and its prevention by ECM or their novel small molecules. Turning their attention to mESC, the Scripps researchers found that trypsin dissociation degraded far less E-cadherin than in hESC, and led to no significant increase in Rho or ROCK activity. Their higher stability was traced to the different culture conditions used to maintain mESC, and by modifying the standard mESC culture medium they were able to support the survival of hESC after trypsin dissociation, most markedly in the absence of ECM; this improved stability was once again associated with an increase in E-cadherin stability.
The modified medium allowed hESC to under leukaemia inhibitory factor (LIF), when self-renewal of conventionally-cultured hESC requires fibroblast growth factor (FGF) instead. This, combined with the known dependence of conventional hESC but not mESC on integrin signaling for self-renewal, led them to further probe integrins’ role in the process. Attachment and proliferation of conventional hESC was found to be dependent on both integrin and E-cadherin signaling, but modified hESC could persist when integrin (but not E-cadherin) signaling was blocked. This was found to be associated with increased expression of E-cadherin and lowered levels of an intermediate of integrin inhibition of Rho; this was consistent with a higher reliance of mESC on E-cadherin and reduced dependency on integrins, and with their earlier work showing the involvement of integrins in the improved survival of ECM-bound conventional hESC when treated with their two small molecules.
The researchers speculate that the differences in cytokine and adhesion-molecule signaling requirements between conventional hESC and mESC may indicate that conventional hESC culture maintains them at a later developmental phase than mESC, and that modified hESC may thus also be retained at an earlier stage of pluripotency. But clearly the significance of these findings extends well beyond any insight that they might give to developmental biology. Ding’s group have given us two powerful tools for more facile culture and expansion of hESC, removing a significant impediment to progress in the field. Injected into an area that already enjoys a high level of government and industry investment, these tools bring us closer to realizing the promise of cell therapies and tissue engineering for the treatment of a range of age-related and traumatic diseases and disorders, as well as for the rejuvenation of aging tissues.
This intensive funding has fueled rapid advancement in pluripotent stem cell research such as the new report, and the early entry of the more straightforward cell- and tissue-based therapies into clinical trials. The close relationship between the availability of resources and the rate of scientific progress underscores the importance of increasing investment in more neglected areas of rejuvenation engineering such as novel lysosomal hydrolases (LysoSENS) for the clearance of recalcitrant intracellular wastes, allotopic expression of mitochondrially-encoded proteins (MitoSENS) for the obviation of age-related mitochondrial DNA mutations, and the targeted ablation of accumulating death-resistant cells (ApoptoSENS) for the rejuvenation of the immune system and other key goals. SENS Foundation continues to pursue critical-path biomedical research within the broader suite of biomedical therapies against the morbidity and mortality of aging.
1: Xu Y, Zhu X, Hahm HS, Wei W, Hao E, Hayek A, Ding S. Revealing a core signaling regulatory mechanism for pluripotent stem cell survival and self-renewal by small molecules. Proc Natl Acad Sci U S A. 2010 Apr 20. [Epub ahead of print] PubMed PMID: 20406903.