This study shows that the colony morphology characteristic of pluripotent hES and hiPS cells is maintained through a pertussis toxin-sensitive mechanism downstream of Gi
-coupled GPCRs. Wild-type colonies were discrete and radial with flat, monolayer organization. Absence of this characteristic morphology and organization usually suggests a lack or loss of pluripotency in human pluripotent cell cultures 
signaling may specifically mediate the morphology and organization of pluripotent stem cell colonies by outwardly regulating the density of cells within the colony. We refer to this as the “outward model” of pluripotent stem cell organization ().
Model of organization of pluripotent stem cell colonies.
Human pluripotent stem cells organize as a flat monolayer of cells that tightly adhere to one another. The Rho-Rock-myosin signaling axis is required to maintain this strong cell-cell adhesion 
. Inhibition of Rock renders hES cell colonies more resistant to enzymatic disassociation 
and survival as a single-cell suspension 
. Although the Rho-Rock-myosin pathway is known to induce changes in hES cell colony morphology 
, activation of this pathway is known to be independent of Gi
signaling and is insensitive to pertussis toxin in somatic cells 
. GPCRs coupled to the G proteins Gq
, and G13
activate Rho in a pertussis toxin-independent manner 
, so the Rho-Rock-myosin pathway will need to be closely examined as we extend our studies to these G protein pathways. The exact molecular mechanism for Gi
-induced morphology change is unknown and there are likely other ways that the GPCR superfamily could activate changes to cellular morphology. We anticipate that there will be multiple GPCR pathways that induce morphology change that work in parallel with the Gi
signaling pathway. The morphology change that we observe with pertussis toxin is also likely to alter the expression and function of adhesion molecules such as E-cadherin, a key regulator of ES cell adhesion 
. Unfortunately, we do not at this time have a complete annotation of all cell surface adhesion molecules involved in this process. These genes are often expressed at low levels where DNA microarray data is not accurate. We are currently embarking on deep sequencing of hES and hiPS cells to gain a more robust list of the cell surface molecules that can be probed for involvement in human pluripotent cell morphology.
Our findings indicate that Gi signaling orchestrates the organization of human pluripotent stem cell colonies as an interconnected monolayer of cells. Gi signaling may maintain pluripotent stem cell colony morphology by outwardly organizing cells much like the springs anchoring the stretched fabric of a trampoline (). The remarkable wound healing exhibited by wild-type pluripotent colonies suggests that colonies can detect the wounded area and actively reform to maintain colony integrity. Disrupting Gi signaling with pertussis toxin blocks the outward forces so that the colonies cannot re-enter the denuded area of the scratch assay, thereby inducing a morphological and organizational change.
Inhibition of Gi
signaling with pertussis toxin converted the monolayer colony characteristic of wild-type pluripotent cells into a multilayered colony. The monolayer colony is a hallmark of human pluripotent cells, first described for hES cells 
and later for hiPS cells 
. These results suggest that a Gi
-coupled receptor actively participates in maintaining the morphology of human pluripotent colonies. Characteristic colony morphology serves as an important initial screen during the production of hiPS cells 
. Insights into how the morphology and organization of pluripotent colonies are regulated will thus be important to understanding the molecular mechanisms of somatic cell reprogramming. We hypothesize that similar Gi
-coupled GPCRs are involved in pluripotent colony formation and maintenance during iPS cell induction. These mediators of iPS cells formation may provide clues to identify factors important for the production of hiPS cells free of genetic modification.
signaling may also play a role in the long observed difference between mouse and human pluripotent colonies. In contrast to monolayer human ES colonies, mouse ES cells form multilayered colonies that are clearly pluripotent as they satisfy the most stringent criteria for pluripotency including tetraploid complementation 
. In view of the significant differences in colony morphology, some investigators have wondered whether human ES cells are truly as pluripotent as mouse ES cells. Interestingly, mouse ES cells treated with pertussus toxin exhibit a phenotype that is similar to that in hES and hiPS cells but attenuated (data not shown). The mouse cell phenotype may be harder to elicit because they are already retracted in their basal state. This suggests that mouse and human pluripotent stem cell colonies share a common Gi
-mediated mechanism for colony organization. The difference in wild-type colony morphology between mouse and human pluripotent cells may reflect differential amounts of Gi
-coupled GPCR signaling in pluripotent colonies. This hypothesis can be tested in future studies by directly stimulating Gi
-coupled GPCRs in mouse and human ES cells using engineered receptors such as RASSLs (receptor activated solely by a synthetic ligand) 
By analyzing a large tissue and cell line meta-dataset, we identified GPCRs, including Gi-coupled receptors, with evidence of expression in human pluripotent stem cells. Such an analysis has several drawbacks over more quantitative measures (e.g., transcript sequencing, protein detection), but nevertheless provides a preliminary candidate list for identifying mediators of the Gi sensitivity of hES and hiPS cells. Manipulating the processes by which pluripotent colonies form and organize may also be critical for therapeutic applications. As hiPS cell technology matures as a viable source for cell-based therapeutics and regenerative medicine, controlling the multi-cellular morphological and organizational characteristics of pluripotent stem cells ex vivo will become increasingly important. GPCRs and their downstream signaling pathways are attractive targets for study in human pluripotent stem cells.