Skip to main content
Premium Trial:

Request an Annual Quote

Stem Cell Surface Protein Panel Distinguishes Between Primed, Naïve States


NEW YORK (GenomeWeb) – Scientists at the Babraham and Karolinska institutes have identified a panel of human stem cell surface markers that can be used to distinguish between cells in naïve and primed pluripotent states.

Described in a paper published last week in Cell Stem Cell, the markers could help more precisely define stem cell states, allowing for better research and comparisons across different populations of cells and enabling study of the molecular events involved in naïve cell formation.

Human pluripotent stem cells can be broadly classified as being either naïve or primed, with each state characteristic of different aspects of cellular development. Naïve cells can be obtained from human embryos but also via various processes through which primed cells are reset to the naïve state.

However, said Peter Rugg-Gunn, a Babraham researcher and senior author on the paper, the relatively novelty and diversity of these resetting processes has raised the need for some way to characterize and standardize the stem cells they produce.

"The human naïve cell field is potentially confusing at the moment, with several reported cell types and cell culture requirements," he said. "This can make the area daunting to move into and sometimes challenging to see how one set of findings can be extended to other naïve systems. The naïve state cells are relatively new, and we are all trying to find the best ways to work with them."

He added that he and his colleagues hoped that establishing sets of cell surface markers could "make it easier to define human pluripotent states, at least at an operational level, and to compare between different cell types."

As Rugg-Gunn and his co-authors noted, previous researchers have proposed certain transcriptional and epigenetic profiles that could be used to distinguish between primed and naïve cells, but, he said, they believed that their cell surface markers could prove more straightforward and robust to measure.

He added that another significant advantage is the fact that, because the cell surface markers allow for a readout of live cells via techniques like flow cytometry, they "can be used to isolate specific cell types for downstream functional assays."

In the study, the researchers took primed human pluripotent stem cells and converted and maintained them in the naïve state using two commonly employed approaches, the 5i/L/FA and the t2i/L+PKCi method. They then used two antibody panels totaling 486 antibodies against 377 cell surface proteins to identify markers that could distinguish between primed and naïve states.

Through this analysis they identified a series of primed- and naïve-specific markers that they then winnowed down to a smaller panel consisting of the markers CD75, CD7, CD77, CD130, CD24, CD57, and CD90.

The smaller panel is somewhat less precise than the full set of markers, Rugg-Gunn said, but it has advantages in cost and ease of use.

"The downsides of a large panel are the cost and also that it is not trivial to set up a flow cytometer to accurately detect many different fluorophore-conjugated antibodies in one sample," he said. "The smaller panel gives researchers the option to avoid these drawbacks, yet retain the ability to isolate specific cell types, albeit with a bit less precision."

Even so, the panel proved quite effective at distinguishing between primed and naïve state cells, he said. "For example, if we spike in 10 percent naïve cells into a sample of primed cells, then our markers selectively recover essentially all of the naïve cells."

The study also identified differences between the stem cell resetting protocols they looked at. For instance, they found that at 10 days the 5i/L/A process produced a larger proportion of cells with protein signatures similar to that of established naïve cells than did the t2i/L+PKCi protocol.

One potential limitation of the surface marker analysis, Rugg-Gunn noted, is the fact that although they appear useful for defining cell states, it's unclear what, if any, role these proteins have in actual regulating cell pluripotency.

"Further work is needed to look at this in more detail," he said.

Regardless, though, of their role in regulating pluripotency, the surface marker panels should allow researchers to identify "emerging naïve cells at a much earlier time point in their reprogramming than was previously possible," Rugg-Gunn said, adding that this could help researchers identify ways to further improve their control of cell states as well as provide insights into the biology of pluripotency more generally.

In the Cell Stem Cell paper he and his colleagues put the panel to this purpose in an investigation of the transcriptional dynamics occurring during the formation of naïve cells, gathering information on the sequence of gene expression changes during the resetting process.

Among their findings were that the transcription factors KLF17, DPPA5, and NANOG "are induced at a relatively late stage of resetting" while genes including DPPA3 and TBX3, which are known to play roles in pluripotency, are induced early in the process. Additionally, a number of genes characteristic of naïve cells, including MEG3, XIST, and a number of zinc finger proteins, were not induced by the 10-day point.

"Through this approach, we can begin to observe the temporal sequence of molecular events that are triggered during cell resetting, thereby providing a first step toward mapping the route of PSC state transitions," the authors wrote.

The study also provides a variety of potential leads for other researchers to follow up on, Rugg-Gunn suggested. "Our dataset contains information on 377 cell surface proteins. There are interesting classes of proteins in there, including signaling receptors and adhesion molecules. We hope that [it] will be useful for other researchers to mine and then follow up on."