Finding a Sweet Spot for Cultivating Embryonic Cell Lines

Dr. Carol Ware and Dr. Julie Mathieu
Dr. Carol Ware and Dr. Julie Mathieu

For scientists who study how the human body grows from a single cell into a complex organism, the earliest stages of development are both a subject of profound intrigue and a rich source of stem cells that are useful for exploring a wide range of questions about our own biology.

One of those scientists is Professor Emeritus Carol Ware, PhD, a founding member of the Institute for Stem Cell and Regenerative Medicine (ISCRM) and the inaugural director of the Ellison Stem Cell Core. In that role, Ware helped ISCRM labs answer research questions by using different types of stem cells to model a wide range of diseases and biological processes.

Embryonic stem cells are found in the inner cell mass of the blastocyst, a structure that appears three to five days after fertilization in humans and contains cells which form the embryo. Embryonic stem cells are pluripotent, which means they can mature into any cell type in the body (except the placenta). Because embryonic stem cells are indefinitely renewable, and can be grown in labs, they are powerful tools of discovery for regenerative medicine researchers studying human development and disease.

Unlike embryonic stem cells, which can only be derived from embryonic tissue, induced pluripotent stem cells can be derived from adult cells that are abundant in skin, urine, and blood. Induced pluripotent stem cells are engineered in labs by resetting adult cells to a stem cell-like state. This gives regenerative medicine researchers an easily accessible and replenishable source of stem cells they can use to study human development, disease onset, and potential therapies. Although induced pluripotent stem cells are not yet used in clinics, this technology also means patients could one day be treated with their own cells.

In either case, Ware has always been interested in the best ways to culture stem cells. In a 2014 breakthrough, she generated one of the first human embryonic stem cell lines in a naïve state, which more closely resembles a pre-implantation stage of development. (Cells that are derived from a post-implantation stage are known as primed.) Successfully generating and stabilizing naïve cells opened up new possibilities in embryonic modeling.

However, there was still much to learn about how to cultivate these cells in ways that are most useful to the field. Ware and her team encountered one problem that is now the focus of a paper published in Stem Cell Review and Reports. Essentially, the researchers were struggling to produce self-sustaining cell lines from embryos frozen at the blastocyst stage. Ware explains, “in the study, we wanted to know why the naïve cell lines were not surviving and what we could do to promote viability.”

Ware is the first author of the paper. ISCRM faculty member Julie Mathieu, who is the current director of the Stem Cell Core is the lead investigator. ISCRM faculty member Marshall Horwitz and ISCRM Regulatory Manager Erica Jonlin are also authors. Ware and Mathieu emphasize the importance of Jonlin’s work, who managed the complex regulatory approval and consent processes required for the use of human embryos.

Beyond leading to the propagation of more cells, one mark of a useful embryonic stem cell line is a more plastic imprint pattern that allows reprogramming back to an earlier developmental stage.

“Throughout your life, your DNA is always the same,” says Ware. “Imprinting is what changes and allows cells to take on different attributes. The paper sheds light on the imprint pattern in early development, which is that early naïve cells do not reliably maintain a stable imprint pattern, whereas later naïve cells have a very stable, but reversible, imprint pattern that allows for much more thorough differentiation into various cell types.”

In other words, along a continuum of naïve and primed pluripotency stages, late naïve appears to be a sweet spot for cultivating embryonic cell lines. How exactly to derive cells in the late naïve stage was one question facing the researchers. The somewhat surprising answer has its roots in cancer studies done at ISCRM.

In previous research led by ISCRM co-founder Tony Blau, PhD, ISCRM investigators studied a molecule that inhibits a checkpoint (CHK1) that when normally active, detects and weeds out harmful cells. This inhibitor represents a potential cancer treatment because turning off this cell cycle checkpoint allows tumor cells to progress through the cell cycle allowing heightened sensitivity to genotoxic cancer drugs.

Ware and Mathieu recognized that the CHK1 inhibitor might also help late naïve cells survive but were worried that unchecked cell growth might lead to mutations. This turned out not be the case. Rather than seeing cultures of karyotypically abnormal cells, the researchers found cells that were not mutated and grow and develop normally.

Why exactly this happened is a question to explore, says Ware.  “There are a number of avenues to pursue next. One is what allowed the checkpoint inhibitor to work in this unique way at this late naïve stage. In the bigger picture, understanding the states of pluripotency, how they differ from each other, and what the pros and cons of each are will yield important insights for the future of embryo modeling.”