Molecules that replace, sustain pluripotency factors make “non-permissive” mouse strains yield stable embryonic stem cells
Embryos from nonobese diabetic mice don't yield stable embryonic stem cells, which make the mice unsuitable for several sorts of experiments. New research reveals not only how to generate these stem cells but also how to toggle between different states of pluripotency.
Rudolf Jaenisch of the Massachusetts Institute of Technology in Cambridge says the work, reported in Cell Stem Cell, grew out of several strands of research. In 2007, scientists led by Ron McKay at the National Institutes of Health in Bethesda, Maryland, and Roger Pedersen at the University of Cambridge, UK, had found that mouse embryos could generate a type of embryonic stem (ES) cell that very much resembled human ES cells in terms of gene expression and requirement of growth factors. Because these stem cells came from the epiblast, or the outer layer of early ball-shaped embryos, these cells are called epiSCs2,3. Unlike mouse ES cells, which are generated from the inner cell mass of ball-shaped embryos, epiSCs do not, if mixed with a mouse embryo, contribute widely to tissues in the resulting mouse pups1.
Another strand of the research comes from work published this year by Austin Smith at the University of Cambridge, which showed that epiSCs could be converted into regular ES cells with the insertion of the gene for pluripotency factor Klf4 (ref. 4). If the gene was removed, the cells reverted back to epiSCs.
That got Jaenisch wondering if the epiSCs that can be generated from nonobese diabetic mice could also be converted into typical ES cells. His lab began to explore the issue by trying to reprogram fibroblasts from these mice, using the four standard reprogramming genes: cMyc, Klf4, Oct4 ) and Sox2. The cells reprogrammed, but only if genes for either cMyc or Klf4 remained active.
Then came the pursuit of small molecules that could replace these pluripotency factors. Researchers led by Peter Schultz of The Scripps Research Institute in La Jolla, California, developed a high-throughput screen that could test greater numbers and diversity of molecules than previous screens5. (The assay, which relies on a gene for the glowing luciferase protein tied to the Nanog promoter, can be performed in 1536-well plates, which are the same size as standard 96-well plates.)
This identified a promiscuous kinase inhibitor called kenpaullone that was capable of replacing Klf4. Other small molecule combinations that had previously been shown to prevent ES cell differentiation or allow derivation of rat ES cells could also work to generate stable ES cells from the nonobese diabetic mice. If these small molecules were withdrawn, however, the cells became unstable.
Jaenisch says his work shows both that a cells' genetic background controls how cells become pluripotent and that external factors, like culture conditions, can compensate for nonpermissive backgrounds. That, in turn, could lead to the generation of ES cells from more species, a research tool that could not only help livestock science but also create better ways to study human disease. Interestingly, work published last month by researchers at the University of Washington in Seattle also showed the ability of human ES cells to apparently toggle between different pluripotent states, depending on the concentration of a histone deacetylase inhibitor called butyrate, which affects the way cells control whether genes are active or silent6.
But Jaenisch says he's not sure whether this work will lead to techniques to convert human ES cells to a more mouse-like state. “Mouse cells are much easier to work with than human cells, but I'm not sure that I can transfer them so easily.”
Hanna, J. et al. Metastable pluripotent states in NOD-mouse-derived ESCs. Cell Stem Cell 10.1016/j.stem.2009.04.015 (published online 7 May 2009).
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Tesar, P.G. et al. New cell lines from mouse epiblast share defining features with human embryonic stem cells. Nature 448, 196–199 (2007).
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Baker, M. New types of embryonic stem cells generated by stabilizing pluripotency. Nat Rep Stem Cells (2009). https://doi.org/10.1038/stemcells.2009.74