Development in reverse: A better model of human induced pluripotency

What: Studying the reprogramming process in human cells is now easier and more reliable, thanks to work by a team of scientists led by Broad Institute researcher Tarjei Mikkelsen. The team designed an improved method for generating human induced pluripotent stem (iPS) cells in the lab that reduces variability. The system enables a high-resolution look at the intermediate cellular and molecular changes taking place as somatic cells are reprogrammed to become iPS cells, something much more difficult to study before this new model. It also makes it easier to perform large-scale studies of how different drugs or genetic modifications influence the reprogramming process.

Why: Since they were first reported nearly a decade ago, induced pluripotent stem cells have been used to develop drugs, to study cellular development, and to model disease, and they could one day help grow new tissues or organs for transplantation. The process by which they are created is, however, not yet fully understood.

iPS cells are created by taking defined, “differentiated” cells, such as skin or muscle cells, and inducing them in a dish to behave more like embryonic stem cells. These so-called “reprogrammed” cells are “pluripotent,” meaning they can then be coaxed into becoming many different adult cell types. Because it’s still unclear exactly what happens inside the cell during reprogramming, scientists want to study the process itself. However, the existing model for generating human iPS cells produces inconsistent, heterogeneous cultures that make it difficult to compare datasets between different experiments or labs.

“Every lab sees something slightly different,” said Mikkelsen. “There’s a lot of variation in how the cells are treated, how they’re cultured, where they come from. We still don’t have a good handle on which variables actually matter and which don’t, for producing high-quality iPS cells or tissues from those iPS cells.”

How: With the goal of improving the model system for studying reprogramming, Mikkelsen and his colleagues employed a molecular trick that has been used to extend the life of other cellular cultures. By overexpressing the gene for telomerase — an enzyme that protects the ends of DNA like the plastic tip on a shoelace — they hoped to produce a better culture of differentiated cells that can be reliably converted to iPS cells.

Their method begins with human fibroblasts (connective tissue cells), which are made to express four genes — the so-called “Yamanaka factors” – and become iPS cells. A subset of these cells reprogram well and are selected and coaxed to differentiate back into fibroblasts. Those cells can then be reprogrammed again at higher efficiency. This double-reprogramming process is known as “secondary reprogramming,” and is used widely to study induced pluripotency. By selecting cells that successfully reprogrammed in the first phase, researchers have more confidence during the second phase that they are working with cells that have the capacity to reprogram. In the secondary process, the resulting cultures are generally clonal, or genetically identical, which also reduces variability between cells.

One problem with secondary reprogramming, however, is that cells lose their ability to reprogram over time in a kind of cellular aging. With the goal of using somatic cells generated during the first phase in large-scale studies of reprogramming, Davide Cacchiarelli, a postdoctoral fellow in the Mikkelsen lab, induced the secondary somatic cells to overexpress telomerase, which prevents cellular aging. By lasting longer in culture, the cells were allowed to “catch up” to each other developmentally, producing a much more homogenous, reliable model system to study.

Taking advantage of their improved model system, the researchers characterized the dynamic molecular and epigenetic changes happening during reprogramming. With less variability among the cells, the team generated a higher-resolution look at the reprogramming process in human cells than has ever been possible before.

In the data, they also saw evidence that as cells reprogram, they undergo gene expression and epigenetic changes that are hallmarks of less- and less-developed cells, even back to markers expressed by pre-implantation embryos. This supports the hypothesis that reprogramming involves a kind of reversal of development, with cells moving backwards through a cellular trajectory that is reminiscent of the one they normally move forwards through. The team was also able to “short-circuit” the process by coaxing fibroblasts in the early stages of reprogramming to develop into a muscle cell type without first becoming iPS cells, a process known as trans-differentiation or “direct lineage conversion”.

For now, this new method offers a model system that makes it easier and more practical to study reprogramming in human cells. Mikkelsen added, “So far, our data suggest that what we find with this model will generalize to the more therapeutically and clinically relevant primary reprogramming systems that are harder to study.”

Who: In addition to Mikkelsen, the research team includes Eric Lander, Alex Meissner, George Daley, John Rinn, John Doench, Cole Trapnell, Michael Ziller, Magali Soumillon, Marcella Cesana, Rahul Karnik, Julie Donaghey, Zachary Smith, Sutheera Ratanasirintrawoot, Xiaolan Zhang, Shannan Ho Sui, Zhaoting Wu, Veronika Akopian, and Casey Gifford.

Where to find it: The paper appears in the July 16, 2015 edition of Cell.

Cacchiarelli et al., Integrative analyses of human reprogramming reveal dynamic nature of induced pluripotency, Cell (2015), DOI: 10.1016/j.cell.2015.06.016