Stemming the Unwanted

Published in Lab Times 05-2013.

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Elimination of undesirable pluripotent cells in induced stem cells preparations. Following exposure to a chemical compound, the residual undifferentiated cells are killed (red dots) while differentiated endodermal cells are spared (green).

Stem cell therapy has all the potential it takes to revolutionize regenerative medicine. But in spite of all that it has to offer, the technology is yet not infallible for therapy. In an attempt to minimize safety issues concerning the use of stem cells in transplantation, scientists in Israel led by Nissim Benvenisty make a major stride.

What does it take for a novel technology to win the support of major funding agencies like the National Institutes of Health or EMBO? A groundbreaking and a medically conducive concept. Stem cell research gets a fat billion dollars annually in the United States alone, and even more worldwide, so there’s no doubt that the health sector vouches for its value.

The “stem cell era” spurred off as early as the 80’s when scientists in Europe and the US first grew embryonic stem (ES) cells on a petri dish. A fascinating trend in stem cell research given its colossal implications in medicine, has been the wave of fervor that it spread worldwide. While scientists in the West were caught up using human ES cells for mouse regenerative therapy, in the East, Korean researchers isolated adult stem cells from umbilical cord blood and a Japanese group led by Shinya Yamanaka made the Nobel-winning discovery of induced pluripotent stem cells (iPSCs). This way, scientists across the globe recognized the gravity of stem cell technology and the latter contributions in fact, reinstated the technology when it staggered from the ethical stymie on ES cell usage.

Besides their capacity to regenerate, human stem cells in culture are an affordable and accessible model to follow developmental processes, or to test the efficacy of drugs. All that being said, it is perhaps unsurprising that a technology as promising as this comes with a hitch. Nissim Benvenisty and his group at the Hebrew University in Jerusalem, Israel have been studying the tumorigenic effect of human stem cells that challenges their use in therapy. They propose a chemical approach for dealing with this problem in their recent papers (Nat Commun 4:1992; Cell Stem Cell vol.12(2):167-79).

The magic and its price

The property of self-renewal and the ability to mature into any tissue type, termed “pluripotency”, make stem cells very attractive for research and medicine. As we age, the tissues and organs in our body become irreversibly specialized to perform a certain function and lose the plasticity for regrowth or regeneration. Stem cell therapy is like the magic dust that can reverse this process of differentiation. In culture dishes, stem cells can be directed to become any given cell type such as a nerve cell or a heart cell. These “committed” cells are now like the building blocks that can reconstitute a whole new tissue when grafted into a host. Fortunately, the pluripotent cells can be derived from the adult host (iPSCs) itself, by the process of “reprogramming”, to minimize the risk of rejection. Now, that’s like the icing on the cake.

In reality, stem cell therapy is still in its infancy and will require many pre-clinical trials before the technology can be medically translated. One of the major impediments is that the proliferative potential of stem cells is uncontrolled and often tumorigenic. Even after differentiation, stem cells in culture are associated with some residual undifferentiated cells. This extraneous cell population is notorious for teratomas, which are benign tumors, or even carcinomas and unsafe for transplantation.

For over a decade now, the Benvenisty lab has been trying to understand what makes stem cells tumorigenic and to what extent stem cells and cancer cells share biological pathways. “Our work is aimed at achieving safer cell therapies based on differentiation of human pluripotent stem cells (hPSCs). We have been identifying vulnerabilities in pluripotent stem cells and harnessing them for their selective elimination”, Prof. Benvenisty lists the goals of his lab.

Frighteningly similar

Stem cells and cancer cells may personify the good and the bad guy but strangely they are two of a kind. Besides their limitless proliferative potential, both cell types share a high propensity for mutations and genomic alterations. “Stem cells can acquire chromosomal aberrations such as copy number variations even within very few passages. If they are not regularly checked for genomic integrity, you run the risk of transplanting aneuploid cells”, remarks Uri Ben-David, a graduate student in the Benvenisty lab.

To have rigorous checks on the stem cell genome at each round of replication seems like a tedious task. As an alternative, Uri and his professor built a ‘virtual karyotyping’ interface (Nat Protoc vol.8(5):989-97). Aided by a public database of gene expression profiles of over a thousand cell lines, combined with bioinformatics tools, their protocol allows the comparative analysis of genomic integrity for any stem cell line. Using this interface, the duo established that indeed stem cells are highly prone to mutations with every passage. The nature of chromosomal aberrations is characteristic to each stem cell line and even exhibits correlation with the tumors of the corresponding tissue (Cell Stem Cell vol.9(2):97-102). “Adult stem cells like mesenchymal stem cells that form the endoderm or neural stem cells that develop into brain tissue develop cell-type specific aberrations. For instance, we saw trisomy of chromosome 21 only in mesenchymal stem cells in culture. Also, the exact same trisomy 21 is what we see in endodermal tumors suggesting common mechanisms in stem cells and cancer cells”, Uri explains.

Cracking the vulnerabilities

Even a minor fraction of undifferentiated cells in a committed stem cell line is deleterious as their tumorigenic properties can rapidly engorge the culture. Classic paradigms to selectively eliminate these undifferentiated cells rely on genetic introduction of ‘suicidal genes’ or cell sorting based on surface markers of pluripotency. “These techniques are not very efficient. Sorting for example, requires dissociation of cells in culture and stem cell differentiation protocols do not always support dissociated cells”, Prof. Benvenisty hints at the need for a better solution. “We suggest for the first time a chemical approach to deal with the risk of tumors. Future research should focus on adopting the chemical approach to various clinically-relevant differentiation protocols, and test it in preclinical trials”.

The Israeli group used two carefully laid out strategies to selectively kill the residual pluripotent cells. Their first approach (Cell Stem Cell vol.12(2):167-79) was a high throughput screen for chemical inhibitors. In collaboration with Roche, they performed a fascinating semi-automated screen of over 50,000 chemical compounds and assessed the effect of each of these “anonymous compounds” on the viability of ESCs and iPSCs. About 700 compounds turned out to be cytotoxic to pluripotent cells in their first screen. They then counter-screened for cytotoxic effects of these hits on committed cells to exclude false-positives. After further increasing the stringency of selection by statistical methods, they eventually arrived at 15 “hot” candidates, dubbed the ‘pluripotent-selective inhibitors or PlurSIns’.

Of course an ambitious lab would not leave it at that, so they set out to identify the mode of action of these drugs. In a series of experiments that followed, which Uri calls his “Aaha! moments”, they pinned down on a lipid synthesis pathway that was targeted. “We tried to figure out how our best hit, PlurSIn #1, worked. We walked piece-by-piece to understand its mechanism of action. It was like a shot in the dark. I collected some evidence from previous work that interfering with the synthesis of oleic acid in cancer cells can cause oxidative stress, protein synthesis inhibition and eventually death. We saw the same changes in our treated cultures too, so I started working backwards. The real breakthrough was when I applied oleic acid to the PlurSIn-treated stem cells and saw a complete rescue of cell death”, the grad student is excited, as he recollects his discovery. PlurSIn #1 they proved, is an inhibitor of an enzyme involved in oleic acid synthesis and apparently disrupting this pathway is detrimental to stem cell survival, and importantly, highly selective to undifferentiated cells.

In their second approach, that also forms the crux of their latest paper, the group decided to reverse manipulate the system. They compared the global gene expression patterns in differentiated versus undifferentiated cells. Among the major candidates, Claudin-6 (CLDN-6), a tight junction protein, turned out to be robustly expressed in hPSCs but absent in somatic tissues. Having found a lead, they first eliminated CLDN-6 positive cells by appending toxins to CLDN-6 antibodies. “This immunogenic method was successful, so we next tried a chemical approach. Clostridium perfringens enterotoxin or CPE is a well-known inhibitor of Claudins. Remarkably, even a short exposure of the culture to CPE abolished virtually all hPSCs. Committed cells viz. mesodermal or neural cells were spared, however, as they are Claudin-negative”, Uri summarizes their results. CPE is so potent that hPSCs that normally develop teratomas when injected into immunodeficient mice, fail to do so if they are pre-treated with the toxin.

Getting there

No treatment option is free of flaws but it is worthwhile to tap the ‘vulnerabilities’ or the weaknesses of a particular system upfront and work systematically to find ways to tackle them. Nissim Benvenisty and his lab have pioneered the analysis of tumorigenic properties of hPSCs and have discovered novel ways to limit their expansion in differentiated cultures. Clearly with these experimental insights, it looks like we are closing in on bringing stem cells to the clinic after all.

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