Why lab-made stem cells might fail: Errors in DNA folding

Why lab-made stem cells might fail: Errors in DNA folding

Induced pluripotent stem cells hold promise for regenerative medicine because they can, in theory, turn into any type of tissue and because they are made from a patient's own adult cells, guaranteeing compatibility. However, the technique that turns adult cells into these iPS cells is not foolproof; after reverting to their pluripotent state, these cells don't always correctly differentiate back into adult cells.

Researchers from the University of Pennsylvania have now discovered one of the reasons why: the reversion process does not always fully capture the way a cell's genome is folded up inside its nucleus. This folding configuration directly influences gene expression and therefore the functionality of the cell.

The new study shows that current techniques might not produce iPS cells that are equivalent to the pluripotent stem cells found in embryos, as some clones retain folding patterns that partially resemble those found in the adult cells from which they are made.

Classic epigenetic marks are chemical modifications on top of the DNA sequence that provide an additional layer of information on top of the long sequence of base-pair letters. 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 team was able to identify folding patterns in iPS cells that had previously been unseen.

The approach used to create the high-resolution maps involves fixing the DNA such that its 3-D folding patterns are preserved prior to sequencing. Sections of the linear genetic sequence that are separated by marked distances but are spatially adjacent when the DNA is folded are chemically glued together. As a result, two distant parts of the linear sequence will end up in the same string of hybrid DNA and will thus be detected together when the DNA is sequenced.

Analyzing these hybrid pieces provides information that allows the researchers to infer which DNA segments are adjacent to each other in the genome's folded state. Critically, the teams approach targets only specific sites in the genome, which allows high-resolution analysis across these regions to be much more easily achievable than with alternative genome-wide approaches. The maps reported by the Penn Engineering lab are the highest-resolution maps of genome folding to date in iPS cells.

The team can plot the sequencing data in heat maps, thereby providing a picture of the DNA segments that are spatially adjacent to each other in the 3-D nucleus of stem cells.

The Penn researchers targeted several locations along the genome to perform their computational analyses, comparing the iPS cells to the cells from which they were generated and to the embryonic stem cells they ideally should perfectly replicate.

They found that traditional embryonic stem cells and mature, differentiated brain cells 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.