Stem cells hold promise for regenerative medicine because they can turn into any type of tissue, but the need to harvest them from human embryos has stymied research in this field. One way around this issue is to turn back the clock on differentiated cells, reverting them to a stage before they grew into a specific type.

And because these induced pluripotent stem (iPS) cells could be made from a patient’s own adult cells, tissue grown from them would always be a genetic match.

Researchers could also use these iPS cells to study disease. Rather than taking a tissue sample from a patient with a genetic disorder—especially challenging when the affected organ is the brain—researchers could use iPS cells derived from that patient’s skin cells to grow model organs as needed.

Treatments and research using this technology have been slow to develop, however. Even though techniques that turn adult cells into these iPS cells have existed for a decade, the process is not foolproof. After reverting to their pluripotent state, these cells don’t always correctly differentiate back into adult cells.

Penn bioengineers Jennifer Phillips-Cremins, an assistant professor in the Department of Bioengineering in the School of Engineering and Applied Science, and Jonathan Beagan, a graduate student in her lab, have now discovered one of the potential reasons why: The reversion process does not always fully capture the way a cell’s genome is folded up inside its nucleus.

Phillips-Cremins’ area of research is “3-D epigenetics,” or the way that the folding of DNA might influence cell-type-specific gene expression. Classic epigenetic marks are a diverse set of modifications on top of the linear DNA sequence that provide an additional layer of information contributing to the complexity of gene expression regulation. Looking at these marks in a linear fashion does not reveal the whole picture, however, as genome folding can bring two disparate regions of the DNA into spatial and functional contact.

By applying experimental and computational techniques that the Cremins lab has developed, her group was able to identify folding patterns in iPS cells that had previously been unseen.

“Previous methods provided data analogous to an old television, with large, black-and-white pixels,” Phillips-Cremins says. “One could generate a blurry image and tell there was a person on the screen, but it would be difficult to parse finer scale facial features. We employed methods to create high-resolution maps of genome folding, so we could distinguish detailed topological features and evaluate their similarities and differences among traditional embryonic stem cells, iPS cells, and mature, differentiated cells.”

The approach Beagan used to create the high-resolution maps involves chemically gluing the DNA such that its 3-D folding patterns are preserved prior to sequencing. As a result, two distant parts of the sequence will end up in the same string of hybrid DNA when it is sequenced.

“Each of the pixels on our maps is a representation of the frequency that any two given segments in the genome interact,” Beagan says. “You can represent the contact frequency numbers as colors and the entire region of DNA as a heat map. You end up seeing intriguing patterns of high and low intensity, and from these patterns one can infer the folding configurations of the genome.” 

The researchers found that traditional embryonic stem cells and more mature neural stem cells from the brain had strikingly different genome folding patterns. Surprisingly, however, the genetic material from iPS cells did not fold in a manner that perfectly resembled traditional embryonic stem cells, but instead exhibited traces of the 3-D configurations of the brain cells from which they were derived.

Importantly, the researchers also found they could make iPS cells with more accurate folding configurations by changing the medium in which they are grown. This result provides a window of insight into possible ways that researchers and clinicians could better engineer iPS cells for experiments and treatments.