When induced pluripotent stem (iPS) cells came on the scene four years ago, they created considerable excitement within the stem cell research community. Unlike their embryonic stem (ES) cell counterparts, iPS cells can be generated from virtually any cell in the body by using a cocktail of three to four genes. The approach makes it possible to generate patient-specific stem cell populations and bypasses the myriad of ethical issues associated with ES cells. But there have been questions for these transformed pluripotent cells; the main one aimed at determining how close they truly are to ES cells.

“They are definitely molecularly similar,” says William Lowry—assistant professor in the Department of Molecular, Cell, and Developmental biology at the University of California, Los Angeles—who carried out one of the first extensive studies comparing the gene expression profiles between human iPS and ES cells [1]. “The long-term question is whether they are molecularly or functionally identical. That’s a difficult question to answer one way or the other based on how many lines you analyze and how you prepare the cells.”

Reprogramming quality

In their analysis, Lowry’s group found that iPS cells actually carry a unique gene expression signature that distinguishes them from ES cells, but this difference becomes less and less apparent the longer iPS cells remain in culture [1,3]. “Basically, we found consistent differences from lab to lab when comparing iPS to ES cells,” says Lowry. “And when you passage iPS cells for a long time, they seem to get closer and closer to ES cells, at least at the gene expression level.” [3] Interestingly, the method used to reprogram iPS cells (i.e., using polycistronic versus four factor vectors) also differentially impacts whether ‘younger’ iPS cells resemble ES cells.

“That is neither saying that they are identical or not,” explains Lowry. “If you look at hundreds of iPS and ES cell lines, maybe you won’t find consistent differences. But we were able to detect some differences depending on how you reprogram and how long you leave the cells in culture.”

Quality control becomes an issue because each lab generates their iPS cells using different techniques. The first generation of iPS cells used four separate viral vectors, one of which contained the oncogene c-myc. But this method is becoming less popular because the vectors randomly integrate into the genome, leading to genetic changes that could alter other functions of the cell. The second generation of iPS cells combined the four factors into one larger vector and also added a way to control expression of the vector using the tet-system. Methods to generate iPS cells are still evolving and include the use of transient or non-integrating vectors (such as adenoviruses, plasmids, and piggyBac transposons), as well as recombinant proteins. The most promising method involves the use of chemicals or small molecules that mimic the four factors, eliminating the need for vectors altogether.

The proper execution of these lab techniques certainly impacts the quality of iPS cells produced. Two other groups found iPS and ES cells indistinguishable at the gene expression and chromatin levels except for differences due to lab environments [4,5]. Although these findings appear to conflict with Lowry’s original analysis, Lowry suspects that his team’s conclusions are not reflective of biological differences between iPS and ES cells. “The different conclusions arose because of the way the analyses were done, not because of differences in the biology.”

Background noise

“Many of the differences seen in the past were likely the result of looking at noise in gene expression data caused by genetic background,” says Konrad Hochedlinger from the Harvard Stem Cell Institute/Massachusetts General Hospital in Boston, MA.

Hochedlinger’s group examined genetic background effects in a study in which they reprogrammed mouse ES cells to generate mice with different adult cell types that carry the reprogramming cassette [6]. By exposing these cells to a drug, all the reprogramming genes were turned on to generate iPS cells. Hochedlinger found these cells to be very similar to ES cells (with only very small or subtle differences) that all mapped to one particular location in the genome.

“The bottom line,” says Hochedlinger, “is that there are differences between [genetically matched] mouse ES and iPS cells, but they are very subtle—just a handful of genes are differentially expressed. But these subtle differences can cause huge developmental abnormalities.”

Interestingly, Hochedlinger observed improper silencing of a single imprinted gene cluster in a huge majority of iPS cells. Only about 10% of iPS cells from the same reprogramming batch normally expressed this gene cluster and were considered to be ‘good iPS cells,’as measured by a stringent assay called tetraploid complementation.

Tetraploid complementation investigates whether mouse pluripotent cells can generate an entire mouse, similar to a cloned animal. While mouse ES cells almost always pass this assay, most mouse iPS cells do not. While some researchers suspect this has to do with culture conditions or heterogeneity of cell populations, no one fully knows why this is the case.

Human iPS cells

The challenge for humans is finding an assay to determine if human iPS cells are functionally equivalent to human ES cells. In mice, the truest test is the tetraploid complementation assay, which generates a whole mouse from iPS cells—an experiment that is not feasible with human iPS cells. This leaves a hole for iPS researchers at the moment. So what is a good in vivo pluripotency assay?

“We don’t have one!” says Lowry. “If we did, we would have been testing it all along.” There is no ideal assay to test full pluripotency in human iPS cells other than to see if they can form teratomas. But finding a way to perform the teratoma assay quantitatively will help settle the issue of functional equivalence of human ES and iPS cells.

The verdict
“Unfortunately, the media has incorrectly portrayed that iPS cells are worse than ES cells,” says Hochedlinger. “But the truth is that this is not the case.”

Differences in gene expression between iPS and ES cells has been shown to occur only transiently, right after reprogramming. These differences may be due to heterogeneity of cell populations, analytical techniques, culture conditions, and reprogramming strategies. But the general consensus is that iPS cells become more similar to ES cells the longer they are in culture.

What matters in the long run is linking molecular differences to functional differences, similar to the experiments done by Hochedlinger in the mouse. This knowledge will be important to study human development, to create new model diseases, and to develop clinical applications. Already, hundreds of iPS cell lines have been made from people with diseases ranging from Down’s syndrome to spinal muscular atrophy, and much has been learned of what truly makes a cell pluripotent.

“There’s so much still to be understood,” says Hochedlinger, “and [the field] is still in its early days.”

References
1. Chin, M.H., M.J. Mason, W. Xie, S. Volinia, M. Singer, et al. 2009. Induced pluripotent stem cells and embryonic stem cells are distinguished by gene expression signatures. Cell Stem Cell 5: 111–123.
2. Hochedlinger, K. and K. Plath. 2009. Epigenetic reprogramming and induced pluripotency. Development 136: 509–523.
3. Chin, M.H., M. Pellegrini, K. Plath, and W.E. Lowry. 2010. Molecular analyses of human induced pluripotent stem cells and embryonic stem cells. Cell Stem Cell 7: 263–269.
4. Guenther, M.G., G.M. Frampton, F. Soldner, D. Hockemeyer, M. Mitalipova, et al. 2010. Chromatin structure and gene expression programs of human embryonic and induced pluripotent stem cells. Cell Stem Cell 7: 249–257.
5. Newman, A.M. and J.B. Cooper. 2010. Lab-specific gene expression signatures in pluripotent stem cells. Cell Stem Cell 7: 258–262.
6. Stadtfeld, M., N. Maherali, M. Borkent, and K. Hochedlinger. 2010. A reprogrammable mouse strain from gene-targeted embryonic stem cells. Nature Methods 7: 53–55.