When expert agreement fails to instigate progress, the teratoma assay stands out as a focal point in stem cell research. Teratomas, tumors formed from a chaotic mix of various tissue types, can include structures like teeth, skin, and even limbs. The term "teratoma" was coined by Rudolf Virchow, deriving from the Greek word for "monster," reflecting their bizarre compositions. The earliest known reference to such tumors can be traced back to a clay tablet from the Chaldean Royal Library of Nineveh, dated between 600 and 900 BC, as noted in the review by Floriana Bulic-Jakus et al.

The groundbreaking work by Kazutoshi Takahashi and Shinya Yamanaka in 2006, titled "Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors," revolutionized the field. This pivotal paper has garnered over 6,000 citations, illustrating its lasting impact. Takahashi and Yamanaka's research revealed that introducing just four specific transcription factors—Oct3/4, Sox2, c-Myc, and Klf4—into differentiated fibroblast cells could revert them to a pluripotent state, enabling them to differentiate into any cell type.

Why does this discovery matter so much?

1. It unveiled crucial regulators of cellular identity.
2. Induced pluripotent stem cells (iPSCs) from patients with diseases provide valuable models for studying human conditions.
3. iPSCs can lead to personalized therapies to treat a range of ailments, from macular degeneration to heart disease.

One particular experiment described in their paper piqued my interest. The researchers injected cells from their proposed pluripotent stem cell populations into the skin of immunocompromised mice and observed the results after four weeks. As anticipated, the injected stem cells developed into visible tumors.

After removal, the tumors were chemically preserved, sliced thinly, and stained with hematoxylin and eosin, which highlighted the cellular components in vivid colors. Microscopic examination revealed that these tumors represented a chaotic mixture of various tissue types not typically found at the injection site—teratomas. The stem cells had proliferated rapidly, forming distinct tissue types, including muscle, epithelium, cartilage, and neural tissues, demonstrating their pluripotent potential.

This compelling result was noteworthy, although I noted, "gross," next to the figure. As a former scientist, I often reflect on the implications of the experiments I read about, and this one would be quite disheartening.

The transplantation method, known as the teratoma assay, was not new; it was first utilized by Leroy Stevens and CC Little, who discovered naturally occurring testicular teratomas in mice and grafted them into healthy mice to observe growth. One tumor managed to grow and differentiate across 16 generations of serial transplantation, a phenomenon attributed to the presence of pluripotent embryonic-like cells that can both differentiate and self-renew.

The 2006 study by Takahashi and Yamanaka sparked a surge of interest in iPSCs, with many research teams attempting to create human stem cells from various starter cells. Each group needed to validate the pluripotency of their generated cells, often through the teratoma assay.

However, it wasn't long before some scientists began to challenge the teratoma assay's status as the definitive measure of pluripotency. Critics argued that the assay's lengthy process and ethical implications concerning animal testing did not justify its use.

> “Many stem cell biologists worry that the current metric for characterizing these cells — the teratoma test — might be setting a low bar for defining pluripotency. ‘It is the most ridiculous assay on the planet,’ says Owen Witte, director of the Broad Stem Cell Research center at the University of California, Los Angeles.” — Elie Dolgin, Nature Medicine, 2010.

To quantify the impact, the Human Pluripotent Stem Cell Registry currently lists 1,880 cell lines, with at least three mice required for the teratoma assay for each line. This results in 5,640 mice subjected to considerable stress for one to two months before being euthanized for analysis. This figure likely underestimates the total number of mice used, as only successfully validated cell lines make it to the registry.

For human cell therapies, generating iPSCs from a patient's own cells is optimal. If the teratoma assay were required for every individualized iPSC line, it would result in a significant number of mice being sacrificed. Such an approach might be justified if the teratoma assay were the sole method available and reliably confirmed pluripotency, but it is not.

During the 2010 International Stem Cell Initiative workshop, participants scrutinized the teratoma assay but could not reach a consensus on its efficacy. Instead of eliminating the assay, they agreed on the need for improvements while also exploring alternative methods. This led to the publication of “A call to standardize teratoma assays used to define human pluripotent stem cell lines” by Müller and Loring et al. in Cell.

In 2013, Buta et al. questioned whether alternative assays were already adequate, contributing to the discussion with their article “Reconsidering pluripotency tests: do we still need teratoma assays?” in Stem Cell Research.

By 2018, at a different International Stem Cell Banking Initiative meeting, the conclusion emerged that the teratoma assay was no longer the best assessment for pluripotency and may not be necessary. The findings from that meeting are compiled in the Sullivan et al. white paper, “Quality control guidelines for clinical-grade human induced pluripotent stem cell lines,” published in Regenerative Medicine.

A quick search on PubMed reveals that the teratoma assay is still in use and has yet to be standardized, despite being published multiple times since July 2018. Are scientists disregarding the consensus, fearing publication difficulties without the teratoma assay, or do they see merit in the assay that the broader community does not? What will it take to retire this outdated practice?

> “Maybe it’s time to let the old ways die > It takes a lot to change a man > Hell, it takes a lot to try > Maybe it’s time to let the old ways die” > — Lyrics from “Maybe It’s Time,” from the 2018 A Star is Born soundtrack.

This situation exemplifies a widespread issue. Experts in various fields gather at workshops or form committees to conduct extensive research, producing reports and recommendations that often go unheeded.

Consider the ongoing challenges in combating climate change or the CDC and WHO's guidelines urging factory farms to reduce antibiotic overuse. We possess the knowledge and expert recommendations, yet the implementation of change, when it occurs, is agonizingly slow, with many individuals choosing to ignore the available evidence and advice.

This raises an intriguing question: why does this happen? I welcome your thoughts on this matter.