Creating stem cells that can give rise to any cell type in the early embryo and its supporting structures, including the placenta: some call it 'the holy grail' of stem cell research. An international team of researchers offer new criteria to determine whether a mouse stem cell line has this much-wanted ability, known as totipotency.
Replicating mouse stem cells in a totipotent state within a laboratory has been a long-sought goal of stem cell biologists around the world, says Associate Professor Vincent Pasque from the Department of Development and Regeneration and the Stem Cell Institute at KU Leuven. "In normal foetal development, totipotency is typically only found during the first few cell divisions of an embryo and is then lost rapidly as the embryo develops. Capturing stem cells in a totipotent state has been difficult. But if successfully created, such a stem cell line could be a big step forward for the study of development and regenerative medicine."
Researchers already know how to create mouse stem cell lines in a pluripotent state, which can create any cell type of an embryo - but not the supporting placental structures. Researchers can also isolate stem cells that can give rise to all placental cell types, but not other embryo cells. Therefore, the creation of stem cells with totipotency, which could make cells leading to both an embryo and the placenta, has thus far remained elusive. Vincent Pasque: "Isolating stem cells with totipotency is a major goal in stem cell biology at the moment, and the field needed better ways of assessing totipotency."
Checklist for promising candidates
Vincent Pasque and a team of researchers from KU Leuven - including PhD fellow Adrian Janiszewski - collaborated with colleagues from the Hospital for Sick Children (SickKids) in Canada and the Karolinska Institute in Sweden to provide criteria to assess whether a mouse stem cell line shows true totipotency. The team included developmental biologist Dr Janet Rossant (SickKids), Dr Eszter Posfai, a former postdoctoral fellow in the Rossant laboratory and now Assistant Professor at Princeton University, Dr Fredrik Lanner, Assistant Professor in the Department of Obstetrics at the Karolinska Institute, and Dr John Schell, who recently obtained his PhD in the Lanner laboratory. "I am so pleased that we could join the expertise in single-cell technologies, stem cell and developmental biology of KU Leuven with that of the Karolinska Institute and SickKids," says Vincent Pasque.
The international team came together as they all have expertise in embryo development and were sceptical of previously published research on stem cells with features of totipotency. They joined forces to generate new data and re-examine published data. The team determined three criteria for a mouse stem cell line to be totipotent:
- The genetic activity of the cells needs to be closer to that of an earlier embryo than to pluripotent stem cells.
- The cells should be able to readily transform into placental stem cells or into an early embryo-like structure in an artificial environment.
- Most importantly, the cells should be able to contribute to the placenta as well as the foetus when returned into the environment of an early embryo.
Ongoing search
The team tested two different mouse stem cell lines that had been reported as potentially totipotent and assessed them using their criteria. While both lines showed some gene expression differences from pluripotent stem cells, they didn't look like totipotent early embryo cells and didn't make certain functional cells in the placenta.
"Ultimately, we found the search for a totipotent stem cell is not over so it's back to the drawing boards in our respective labs to find better ways of capturing these cell types," says Dr Posfai. "Now, we have a clear set of criteria to validate any new cell lines, which will need to be considered in all future publications."
In addition to establishing the criteria, the team also developed a comprehensive analysis of gene expression and regulation at the single-cell level for embryos and different pluripotent stem cell lines. "Single-cell technologies are transforming our ability to understand embryos and stem cells," says doctoral researcher Adrian Janiszewski (KU Leuven). "We have made reference datasets and codes publicly available online as an invaluable resource to enable the community to engage in future studies."
"We can now study gene regulation from atlases of thousands of individual cells, enabling us to determine how gene regulatory networks control cell identity, embryo development, and stem cell states," Pasque adds. "The fundamental knowledge about the criteria to evaluate the totipotency of stem cells in mice could also help to create human totipotent stem cells by reprogramming from differentiated patient cells. Human totipotent stem cells are going to be very important to understand the mechanisms of embryo and placenta development. And once we understand these mechanisms better, we could then try to apply this knowledge to find better therapies in regenerative medicine."
