Embryonic stem cells: variability in differentiation potential underscores the need for a multitude of available lines

by Lisa Girard, PhD
HSCI Science Editor

We tend to think of embryonic stem cells as being all the same for obvious reasons. Embryonic stem cell lines are all similarly derived from the inner cell mass (ICM) of blastocyst-stage embryos and share a number of common features including:

• the ability to divide and produce more stem cells
• the ability to differentiate into cell types from all three germ layers
• the sharing, on a molecular level, of a number of markers that have been correlated with pluripotency.

However, recent work from a number of labs suggests that the homogenicity assumed by virtue of their isolation from a common source is an over-generalization. Instead, embryonic stem cell lines retain a significant degree of heterogeneity that can appear as a propensity to differentiate into particular cell types. Understanding these biases enriches our understanding of embryonic stem cell specification and the biological underpinnings of pluripotency. Relevant to the progress of stem cell research, this heterogeneity underscores the need to have available a number of well-characterized cell lines representing those biased toward each of the array of cell types desired. Such resources are key to providing researchers and clinicians with optimal material for studies, screens, and therapies.

A range of studies has explored genetic variation in stem cell lines and its consequences through a number of approaches. Gene expression profiling of embryonic stem cells using DNA microarrays (Ramalho-Santos et al., 2002) to compare genes expressed in embryonic stem cells, hematopoietic stem cells, and neural stem cells, provided initial clues for what would later be borne out experimentally. While the researchers identified 216 genes that were expressed in all three types of stem cells, they found a significant degree of overlap between the embryonic stem cell program and the neural stem cell program, supporting the model, at least in frogs and mice, that barring additional cell signaling events the neural program is default.

In addition to heterogeneous gene expression patterns, experiments also identified a significant degree of variation between embryonic stem cell lines among other parameters including proliferation rate, hematopoietic differentiation, and teratoma formation (Burridge et al., 2007). In order to delineate which factors affect differentiation potential due to inherent genetic variation, versus cellular location of origin within the ICM, researchers studied differentiation potentials between isogenic lines in culture using pairs of isogenic, monozygotic twin, and single inner cell mass-derived clonal embryonic stem cell lines (Lauss et al., 2005). They measured levels of the leukemia inhibitory factor receptor (lifr) gene in these stem cell lines, a protein that predisposes cells toward cardiomyocyte fate, and found that its levels varied even among the supposedly isogenic lines. Furthermore, levels of lifr varied across the ICM, suggesting these levels may account for different differentiation predispositions. The tendency of the cells to develop into cardiomyocytes correlated with the levels of lifr gene implying a molecular basis for this propensity, as well as provided evidence for genetic variability across the ICM.

Changes in cell culture conditions have also been explored as a source of differentiation bias. Using four different human embryonic stem cell lines cultured under identical conditions, researchers compared differentiation variability. They found that the four lines differed significantly with respect to embryoid body formation and bias toward cardiomyocyte differentiation (Burridge et al., 2007). Additional studies of human embryonic stem cell lines significantly expanded these conclusions (Osafune et al., 2008), comparing the differentiation potential of 17 embryonic stem cell lines created at Harvard University. These experiments examined the expression levels of marker genes characteristic of the different germ layers. While the expression levels at undifferentiated time points were very similar across all the lines, during differentiation time points marker expression levels varied and different lines were found to have different likelihoods of developing into particular cell types.

In order to be able to optimally utilize stem cells in cell-based therapies and regenerative approaches a consistent means by which to direct targeted differentiation into specific cell types must be determined. The inherent genetic variability between stem cell lines has represented a challenge for researchers pursuing this goal. Many sources for the variability in differentiation propensity have been proposed. The variation in ICM marker gene levels (see Lauss et al., 2005) depicts the ICM from which embryonic stem cells are derived as a heterogeneous group of cells. These studies, taken with those finding different levels of germ cell-specific markers (Osafune et al., 2008) and dependence on culture conditions (Burridge et al., 2007) not surprisingly yielding embryonic stem cells with different expression profiles and non-uniform differentiation biases. Further, these differences are not shown to correlate with chromosomal abnormalities (common in embryonic stem cells) or epigenetic differences. Key to these studies is an understanding that underlines the importance of having many lines in existence in order to optimally populate the stem cell researcher's toolbox. It will be critical to both in vitro and in vivo future work to determine if a comparable degree of variability exists among stem cell lines generated from approaches other than from blastocysts ICM isolation such as somatic cell nuclear transfer and induced pluripotent stem cells. If researchers are not able to use material that can generate consistent results, then the basis for creating reproducible, therapeutically valuable material for regenerative, cell-based approaches and screens is hindered.

References

• Burridge, P.W., Anderson, D., Priddle, H., Barbadillo Munoz, M.D., Chamberlain, S., Allegrucci, C., Young, L.E., Denning, C. (2007) Improved human embryonic stem cell embryoid body homogeneity and cardiomyocyte differentiation from a novel V-96 plate aggregation system highlights interline variability. Stem Cells. 25, 929-38.
• Lauss, M., Stary, M., Tischler, J., Egger, G., Puz, S., Bader-Allmer, A., Seiser, C., Weitzer, G. (2005) Single inner cell masses yield embryonic stem cell lines differing in lifr expression and their developmental potential. Biochem Biophys Res Commun. 331, 1577-86.
• Osafune, K., Caron, L., Borowiak, M., Martinez, R.J., Fitz-Gerald, C.S., Sato, Y., Cowan, C.A., Chien, K.R., Melton, D.A. (2008) Marked differences in differentiation propensity among human embryonic stem cell lines. Nat Biotechnol 26, 313-5.
• Ramalho-Santos, M., Yoon, S., Matsuzaki, Y., Mulligan, R.C., Melton, D.A. (2002) “Stemness": transcriptional profiling of embryonic and adult stem cells. Science 298, 597-600.