The tumor-generating assay that will not die

When expert consensus fails to generate change

Tumor harvest from immunodeficient mice. Photo by littlepeggy
“Teratoma is a tumor comprised of a disorganized mixture of tissues otherwise found in the adult organism. Sometimes it contains organotypic structures such as teeth, skin with its appendages (hair and sebaceous glands), and limbs. The name teratoma was derived by Rudolf Virchow from the Greek τέρατος (monster), and a suffix denoting tumor. The first reference to such a structure in human pathology appears to be on a clay tablet dating from 600 to 900 BC from the Chaldean Royal Library of Nineveh.” From a review by Floriana Bulic‐Jakus et al.

In 2006, Kazutoshi Takahashi and Shinya Yamanaka published their nobel prize winning scientific discovery, “Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors.”

This paper was a huge deal when it came out. It is still a huge deal, and has been cited over 6,000 times. Takahashi and Yamanaka discovered that just four molecular ingredients need to be added to a dish of differentiated fibroblast cells to transform them into a pluripotent state, capable of becoming any cell type. Four transcription factors — Oct3/4, Sox2, c-Myc, and Klf4 — were all that were needed to teach these old cultured cells new tricks.

Fibroblast cells. Fluorescence microscopy image by vshivkova

Why is that such a big deal?

  1. The induction of pluripotency revealed key regulators of cellular identity.
  2. Induced pluripotent cells from diseased individuals are useful models to study human disease.
  3. Induction of pluripotent stem cells can be used to deliver personalized cell therapies to alleviate a variety of human ailments, from macular degeneration to heart disease.

One experiment in the paper particularly caught my eye. Takahashi and Yamanaka injected cells from each of their putative pluripotent stem cell populations under the skin of immunocompromised mice and waited. Four weeks later, the hundred microliter drop of stem cells had grown into visible tumors, as expected.

The tumors were removed, chemically preserved, thinly sliced, and stained with hematoxylin and eosin to color the cellular components in bright red, blue, and violet. Looking under a microscope, they could see that the tumors were a disorganized mosaic of many tissue types that would not normally be present at the injection site — a type of tumor called a teratoma. The injected stem cells had rapidly proliferated and differentiated, forming pockets of identifiable tissue structures, including muscle, epithelium, cartilage, and neural tissues. By demonstrating that their putative stem cells could become tissues from all three germ layers, Takahashi and Yamanaka proved that their stem cells were pluripotent.

Histology of teratomas confirm that the injected stem cells were able to differentiate into tissue types from all three germ layers. Figure 5A from “Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors,” by Kazutoshi Takahashi and Shinya Yamanaka. Cell (2006).

It was a beautiful and highly convincing result. Next to the figure, I wrote, “gross.” As a former scientist, I tend to think about what it would be like to do the experiments I read about, and this experiment would really bum me out.

The transplantation experiment is known as the teratoma assay. Takahashi and Yamanaka were not the first to do it. That honor goes to Leroy Stevens and CC Little, who identified naturally occurring testicular teratomas in mice, then “minced and grafted” those tumors into healthy mice to test their growth. One tumor continued to grow and differentiate into all three germ layers for 16 generations of serial transplantation, a feature they attributed to pluripotent embryonic-type cells that are able to differentiate and self-renew (read their 1954 paper in full). They had found stem cells in the teratoma.

Takahashi and Yamanaka’s 2006 paper was the beginning of a boom time for induced pluripotent stem cells, with many research groups aiming to create human stem cells induced from their starter cell or individual of choice. Each of those groups had to prove that the cells they were generating were in fact pluripotent, and each of them did it by performing a battery of tests, including the teratoma assay.

It didn’t take long for some scientists to question the use of this assay as the gold standard for pluripotency. Those against the assay said it took too long and was not convincing enough to be worth the cost of all those animal lives and human hours.

“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.” Excerpt from a Nature Medicine article by Elie Dolgin, written in 2010.

To put a number to it, the Human Pluripotent Stem Cell Registry currently reports 1,880 cell lines. For each one, at least three mice were used for the teratoma assay to confirm pluripotency. That’s 5,640 mice living in a burdened state for one or two months before the tumor is ready to be harvested for tissue analysis, at which time the mouse is euthanized. That number is certainly much lower than the total number of mice used in teratoma assays, as only the fully vetted, successful cell lines are put into the registry.

Photo by electravk

For cell therapies in humans, inducing pluripotency from the patient’s own cells is ideal. Imagine if a teratoma assay had to be completed for every patient-specific induced pluripotent stem cell line. That’s a lot of mice. It would be worth it if the teratoma assay was the only assay available and did a perfect job of confirming pluripotency — I don’t want people to suffer just so that mice don’t have to get tumors — but it is not, and it doesn’t.

At the 2010 International Stem Cell Initiative workshop, the scientists present called the teratoma assay into question but could not agree on its usefulness. Rather than getting rid of the assay, the consensus was to make it better while working toward alternative assays. Soon after “A call to standardize teratoma assays used to define human pluripotent stem cell lines,” by Müller and Loring et al., was published in Cell.

In 2013, Buta et al. asked whether the alternative assays were already sufficient, publishing the article “Reconsidering pluripotency tests: do we still need teratoma assays?” in Stem Cell Research.

At the 2018 International Stem Cell Banking Initiative meeting (different from the workshop mentioned above), the consensus was that the teratoma assay is no longer the best test for assessing pluripotency and is not necessary. This and other outcomes of the meeting are summarized in the Sullivan et al. white paper, “Quality control guidelines for clinical-grade human induced pluripotent stem cell lines,” published in Regenerative Medicine.

A brief look on pubmed reveals that presently, the teratoma assay is still in use, and has not been standardized (example 1, 2, 3, 4, 5, 6, 7 — all published after July 2018). Are individual scientists not getting the message? Or are they worried that journals will not publish their cell line without the teratoma assay? Or do they find value in the teratoma assay not represented in the group consensus? What does it take to kill this monster?

“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 is an example of a common problem. We assemble leaders in the field at workshops or create committees who do a tremendous amount of research in order to produce reports and recommendations for their respective fields. And then…those recommendations get largely ignored.

Picture by stockstudioX

Consider efforts to mitigate climate change, or the CDC and WHO mandates that factory farms stop the overuse of antibiotics. We have the information. We have the expert advice on how to proceed. Yet change, if it occurs at all, happens at a snail’s pace, with many individuals ignoring the evidence and advice completely.

An interesting question, then, is why? I am curious to hear what you think.