To evaluate iPSCs as a research tool, we generated a large panel of cell lines from multiple donors, then examined aspects of their pluripotency and ability to generate terminally-differentiated motor neurons. The results of our comparisons confirm the remarkable value of iPSC lines for in vitro studies, and demonstrate that they can perform as well as standard ESC lines. When procedures were standardized, this observation held true for experiments performed in two geographically distinct laboratories. The analyses provided here serve as a quality control for this stem cell resource, while also providing sufficient data on specific aspects of variability to allow investigators to select lines that will be of particular relevance to their own research.
Our study is not the first to compare human iPSC and ESC lines, but it is the most extensive comparison of their ability to generate a specific terminally-differentiated cell thus far. Most studies have used panels of four or fewer iPSC lines17–22
, limiting the possibilities for understanding variability between cell lines or drawing general conclusions about functional similarities between iPSCs and ESCs. Similar to a comparable study9
, we find that variation exists between the differentiation efficiencies of individual iPSC lines. However, contrary to the conclusions of that study, which were that the differentiation capacity of iPSCs needs be improved in order to match that of ESCs, we found that iPSC lines could be made to differentiate on average as well as ESC lines. Whether this is due to differences in protocols for reprogramming and motor neuron differentiation between the two studies, or whether it reflects the larger sample analyzed here, remains to be determined.
While all cell lines in our test set were capable of generating motor neurons, the standard protocol for motor neuron production did reveal significant quantitative differences in the propensity of the lines for terminal differentiation. These differences were highly reproducible, suggesting that they represent intrinsic characteristics of the lines. Our initial hypothesis was that the poorly performing lines would be identified by anomalies in standard tests for stem cell quality. However, all cell lines tested expressed pluripotency markers, could form the three germ layers in vitro
, and in teratomas. Moreover, although variations in karyotype and transgene expression were observed, they were not accurate predictors of differentiation capacity. Fortunately, a solution for identifying such predictors has now been proposed by Bock et al.11
, who used our test set to search for epigenetic and transcriptional differences that correlate with differentiation potential. They used the lines we describe here, to develop a scorecard for stem cell quality that predicted our motor neuron differentiation results () with remarkable precision.
We anticipate that one of the major uses of the cell lines provided through this resource will be to model ALS. Importantly, our data demonstrate that several conditions that are necessary for reliable disease modeling are met. First, since ALS is not a developmental disease, our finding that iPSCs carrying an ALS-triggering mutation differentiated similarly to those from healthy controls is as expected. Second, although lines from different healthy donors taken together showed donor-related variation in differentiation efficiency, the pairwise comparisons did not reach significance. This increases the chances that phenotypic differences we may eventually observe between ALS cases and controls are related to disease. Nevertheless, since we found real line-to-line differences, it will be essential to confirm that any phenotypes are ALS-related by silencing the mutant SOD1 disease gene. Lastly, the remarkable concordance between the results from two different laboratories reported here suggests that, once ALS-related phenotypic differences are discovered, they will prove sufficiently reproducible to serve as a general paradigm for the field.