Thus guest post is by Irene Gallego Romero (@ee_reh_neh) on her paper Gallego Romero et al “Generation of a Panel of Induced Pluripotent Stem Cells From Chimpanzees: a Resource for Comparative Functional Genomics” bioRxived here.
Genetic divergence in protein coding regions between humans and chimpanzees cannot explain phenotypic differences between the two species, or, more broadly, between other closely related groups. Although we have known this since the early days of genetic sequencing, it has been very hard to formally test the hypothesis that follows logically – that it may be changes in gene expression and regulation that underlie the divergence in phenotypes. This is especially true in the great apes, where there are plenty of ethical and practical impediments to experimentation. For instance, our ability to carry out functional studies and really decode cellular mechanisms is restricted to tissues that can be sampled non-invasively. To date, this has mostly meant fibroblasts and immortalised lymphoblastoid cell lines. The rest of comparative work in primates tends to be done in tissue samples collected post-mortem, where experimental manipulation is not a possibility.
Together, these limitations provided the impetus for us to develop a panel of high-quality induced pluripotent stem cell (iPSC) lines from chimpanzees. The promise of this panel lies, of course, not just in insights into the pluripotent state in chimpanzees (although that is certainly a worthy subject) but in how it opens the door to a tantalizing number of previously inaccessible questions, when we combine it with any of the many protocols available for differentiating iPSCs into particular somatic cell types that have remained out of reach until now.
The amount of work that went into developing an effective reprogramming protocol is not readily apparent in our preprint, but it was exhaustive – and exhausting! We began by using retroviral vectors to deliver the four factors that are commonly used to reprogram somatic cells to pluripotency, but soon encountered two fairly sizable problems with that approach. First, these viral vectors are integrated into the host genome during the course of reprogramming, and one never knows what they’re going to disrupt. This is an issue that everyone using retro- or lentiviral vectors has to contend with, and indeed, when we began working on the project three and a half years ago they were the most reliable and established reprogramming method around, so we were prepared to take our chances and scan the resulting lines to determine insertion sites. Regardless, the thought of random insertions of pluripotency genes set us somewhat on edge!
However, for reasons that we never fully understood, those chimpanzee lines had a lot of trouble silencing the retroviral vectors and maintaining pluripotency solely through endogenous mechanisms, as we show in one of our supplemental figures. At the time, we were making human iPSC lines in tandem using exactly the same vector stocks. While the human lines would lose most exogenous vector expression after 12 to 15 passages, in chimpanzee iPSCs of the same age we would generally find that expression of at least one, if not more, exongenous genes was as high as it had been on day one. This did not bode well for the lines, or for our ability to do interesting things with them! So we scrapped the integrating approach, and began optimizing protocols all over again. Fortunately for us, Shinya Yamanaka’s group had just published a very thorough protocol on reprogramming cells using non-integrating episomal vectors, which ended up laying the foundations of the one we present in our preprint.
The lines we have generated with it are of fantastic quality, and they have passed every test we have thrown at them with flying colours. Pluripotency is being endogenously maintained, they’re karyotypically normal, and they differentiate into all three germ layers both spontaneously as embryoid bodies and teratomas when injected into mice, and when we use directed protocols to push them towards a particular fate.
We were very interested in quantifying how human and chimpanzee iPSC lines differ from each other. To this end, we collected RNA-sequencing and methylation data from the chimpanzee iPSCs and the fibroblast lines they were generated from, as well as from seven human iPSC lines from various ethnic and cellular origins and their precursors, and compared them to one another. We find large numbers of inter-species differences both before and after reprogramming, but crucially, most of them are not the same differences. Of all the genes with strong evidence for differential expression between species at the iPSC stage, only 38% are also differentially expressed before reprogramming, and the situation is quite similar with regards to methylation.
Another thing we have found very striking in the data is the very clear increase in homogeneity within (and possibly between, although our design makes that harder to effectively quantify) species at the iPSC level relative to the precursor cells, both in gene expression levels and in DNA methylation. This finding will be very interesting to keep in mind as we go forward and differentiate the iPSCs into a suite of somatic cell types and see how these measures fluctuate through differentiation.
Ultimately, however, where the biggest significance of this work lies for us is in the fact that the lines are not just for our own use. They’re available to other researchers, and this is something we have had in mind from the earliest stages of the work. There is no possible way for our lab to even begin to tackle all the questions that these lines can be used to answer. So if you want to work with our chimpanzee iPSC lines, get in touch.