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Cell Cycle and Stem Cells: An intimate Relationship Delineated

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The importance of the cell cycle to human embryonic stem cells (hESCs) is demonstrated by a wealth of data emanating from multiple labs across the world. The unique pattern of the cell cycle in hESCs is signified by a large S phase and a short but vitally important G1 phase (Coronado et al and Savatier et al) which lengthens as a cell moves towards its final somatic state, indicating that differentiation affects cell-cycle regulation and that the short G1 phase is highly linked to the pluripotent state (Calder et al and Coronado et al). Moreover, research has also shown that pluripotent cells are more receptive to differentiation signals when in G1 compared to other phases of the cell cycle (Sela et al).   Siim Pauklin and Ludovic Vallier from the Cambridge Stem Cell Institute, University of Cambridge, UK have now utilised new tools in an attempt to delineate the mechanisms that integrate pluripotency, differentiation and the cell cycle in hESCs, in a study published in Cell with an excellent accompanying summary article.

The exciting results generated within this study relied on the use of the FUCCI reporter system which, in short, allows for Red Fluorescent Protein (RFP) expression during G1, Green Fluorescent Protein (GFP) expression during S, G2, and M phases and overlapping expression at the G1/S transition which manifests itself as a yellow colour. Transfection of this system into hESCs allows for the red-through-yellow-to-green transition to track progression through the cell cycle.

To assess the effect of cell cycle state on cell fate, the researchers sorted cells by cell cycle stage and placed them under specific conditions to drive homogenous populations of endoderm, mesoderm and neuroectoderm cells. This demonstrated that early G1-hESCs were tightly linked to the initiation of endoderm differentiation and lost their capacity to express neuroectoderm markers, while late G1-hESCs were linked to neuroectoderm differentiation. Analysis of the differentiation of hESCs to terminally differentiated endodermal cells; pancreatic and hepatic cells, also found that early G1-hESCs were able to differentiate more efficiently into insulin-expressing cells and hepatocyte-like cells when compared to unsorted cells or cells sorted in late G1, altogether demonstrating that the cell cycle stage of hESCs can influence their differentiation capacity.

Analysis of Activin-Nodal-Smad2/3 mediated signaling, associated with endoderm differentiation, found that Smad2/3 bound specifically to endoderm genes in early G1 and its activity was also maximal at this stage only. CyclinD1, 2 and 3 and their associated partners CDK4/6 were then assessed as possible controlling factors due to their central role in co-ordinating G1 progression. Germ layer specific differentiation of hESCs was associated with a distinct profile of Cyclin D expression, with endoderm differentiation linked to low Cyclin D1, 2 and 3 expression, which is known to be associated with active Activin-Nodal signaling. Single knockdowns by short hairpin RNA in hESCs led to moderate increases in differentiation markers, while double knockdowns demonstrated increased endodermal and decreased pluripotency and neuroectoderm marker expression, with conditional triple knockouts leading to endodermal differentiation.

These findings were confirmed by reciprocal over-expression studies, which also demonstrated decreased Smad2/3 transcriptional activity in hESCs/endoderm cells with cyclin D over-expression which also decreased the ability of early G1-hESCs to undergo endodermal differentiation, with less Smad2/3 chromatin occupation. Smad2 was shown to be nuclear-localized and induce endodermal markers in late G1, and was also shown to interact with CyclinD1/2/3 through co-immunoprecipitation assays, which may control nuclear transport. Furthermore, inhibition of CDK4/6 induced endoderm and mesoderm markers in hESCs and enabled an increased capacity of hESCs to differentiate into mesoderm/endoderm rather than ectoderm under specific differentiation conditions. This was associated with increased Smad2/3 chromatin occupancy, and inhibition in late G1 phase was also associated with increased Smad2/3-dependent transcription and a derepression of endoderm differentiation. Post-translational modifications can affect nuclear shuttling and phosphorylation of Smad2/3 differed during the cell cycle and, in late G1, when excluded from chromatin, the linker region was phosphorylated, while in early G1 when chromatin associated, the MH2 region was phosphorylated. Further mutation studies of the phosphorylation sites confirmed these findings.

Finally, the researchers used the ability of CDK4/6 inhibition to induce endoderm markers and differentiation to develop an efficient differentiation protocol for endodermal tissues. Combinatorial analysis demonstrated that ActivinA could be replaced by CDK4/6 inhibition alongside BMP4 and FGF2 in ESCs and iPSCs; boosting endoderm differentiation and enhancing hepatic and pancreatic marker expression. This enhancement was also observed in induced pluripotent stem cells refractory to differentiation allowing for the generation of pancreatic and hepatic cells, which was not observed using normal techniques.

In summary, the researchers demonstrate that early G1 is associated with a differentiation tendency towards an endodermal fate, linked to the binding of Smad2/3 to chromatin to activate transcription and boost differentiation. However, in late G1 Cyclin D/CDK4/6 expression binds to Smad2/3, inhibiting nuclear shuttling, repressing endodermal differentiation and boosting neuroectodermal differentiation. While this elegant study has revealed some of the machinery linking the cell cycle and differentiation propensities of hESCs using the elegant FUCCI system, it has also allowed some refinement of differentiation protocols for the generation of therapeutically relevant cells such as pancreatic and hepatic cells.

 

References

Calder, A. et al.
Lengthened G1 phase indicates differentiation status in human embryonic stem cells.
Stem Cells Dev., 22 (2013), pp. 279–295

Coronado, D. et al.
A short G1 phase is an intrinsic determinant of naïve embryonic stem cell pluripotency.
Stem Cell Res. (Amst.), 10 (2013), pp. 118–131

Savatier, P. et al.
Withdrawal of differentiation inhibitory activity/leukemia inhibitory factor up-regulates D-type cyclins and cyclin-dependent kinase inhibitors in mouse embryonic stem cells.
Oncogene, 12 (1996), pp. 309–322

Sela, Y et al.
Human embryonic stem cells exhibit increased propensity to differentiate during the G1 phase prior to phosphorylation of retinoblastoma protein.
Stem Cells 30 (2012), pp. 1097–1108

 

Stem Cell Correspondent Stuart P Atkinson reports on those studies appearing in current journals that are destined to make an impact on stem cell research and clinical studies.