Increased Flexibility in Induced Pluripotent Stem Cell Derivation Might Solve Tumor Concerns

Regenerative medicine depends on stem cells for the promises that it can potentially deliver to ailing patients. Training stem cells to repair injured tissues with custom-grown tissue substitutes and to replace dead cells are some of the goals of regenerative medicine. A major player in regenerative medicine is induced pluripotent stem cells (iPSCs), which are made from a patient’s own tissues. Because iPSCs are derived from a patient’s own cells, their chance of being rejected by the patient’s own immune system is rather low. Unfortunately, Shinya Yamanaka’s formula for making iPSCs, for which he was awarded last year’s Nobel Prize, utilizes a strict recipe that uses a precise combination of genes, some of which increase the risk of cancer risk, and, therefore, restricts their full potential for clinical application.

From left: Emmanuel Nivet and Juan Carlos Belmonte. Seated: Ignacio Sancho Martinez. (Source: Salk Institute for Biological Studies)
From left: Emmanuel Nivet and Juan Carlos Belmonte. Seated: Ignacio Sancho Martinez. (Source: Salk Institute for Biological Studies)

However, the laboratory Juan Carlos Izpisua Belmonte and his colleagues at the Salk Institute have published a paper in the journal Cell Stem Cell that shows that the recipe for iPSCs is much more versatile than originally thought. For the first time, Izpisua Belmonte and his colleague have replaced a gene that was once thought to be impossible to substitute in the production of iPSCs. This creates the potential for more flexible recipes that should speed the adoption of iPSCs for stem cell-based therapies.

Pluripotent stem cells come from two main sources. Embryonic stem cells (ESCs) are derived from early human blastocyst-stage embryos. The cells of the inner cell mass are extracted and these immature cells that have never differentiated into specific cell types, and are cultured, grown, and propagated to form an embryonic stem cell line. Secondly, induced pluripotent stem cells or iPSCs, are derived from mature cells that have been reprogrammed back into an undifferentiated state. In 2006 by Yamanaka introduced four different genes into a mature cell to reprogram the cell to pluripotency. This pluripotent cell can be cultured and grown into an iPSCs line. Because of Yamanaka’s initial success in iPSC production, most stem cell researchers adopted his recipe, even though variations on his protocol have been examined and used.

Izpisua Belmonte and his colleagues used a fresh approach for the derivation of iPSCs. They played around with the Yamanka protocol and in doing do discovered that pluripotency (the stem cell’s ability to differentiate into nearly any kind of adult cell) can also be programmed into adult cells by “balancing” the genes required for differentiation. What genes? Those genes that code for “lineage transcription factors,” which are proteins that direct stem cells to differentiate first into a particular cell lineage, or type, such as a blood cell versus a skin cell, and then finally into a specific cell, such as a white blood cell.

“Prior to this series of experiments, most researchers in the field started from the premise that they were trying to impose an ’embryonic-like’ state on mature cells,” says Izpisua Belmonte, who holds the Institute’s Roger Guillemin Chair. “Accordingly, major efforts had focused on the identification of factors that are typical of naturally occurring embryonic stem cells, which would allow or further enhance reprogramming.”

Despite these efforts, there seemed to be no way to determine through genetic identity alone that cells were pluripotent. Instead, pluripotency was routinely evaluated by functional assays. In other words, if it acts like a stem cell, it must be a stem cell.

That condition led the team to their key insight. “Pluripotency does not seem to represent a discrete cellular entity but rather a functional state elicited by a balance between opposite differentiation forces,” says Izpisua Belmonte.

Once they understood this, they realized the four extra genes weren’t necessary for pluripotency. Instead, adult cells could be reprogrammed by altering the balance of “lineage specifiers,” genes that were already in the cell that specified what type of adult tissue a cell might become.

“One of the implications of our findings is that stem cell identity is actually not fixed but rather an equilibrium that can be achieved by multiple different combinations of factors that are not necessarily typical of ESCs,” says Ignacio Sancho-Martinez, one of the first authors of the paper and a postdoctoral researcher in Izpisua Belmonte’s laboratory.

Izpisua Belmonte’s laboratory showed that more than seven additional genes can facilitate reprogramming adult cells to iPSCs. Most importantly, for the first time in human cells, they were able to replace a gene from the original recipe called Oct4, which had been replaced in mouse cells, but was still thought indispensable for the reprogramming of human cells. Their ability to replace it, as well as SOX2, another gene once thought essential that had never been replaced in combination with Oct4, demonstrated that stem cell development must be viewed in an entirely new way. In point of fact, Belmonte’s group showed that genes that specify mesendodermal lineage can replace OCT4 in human iPSC generation, and ectodermal lineage specifiers are able to replace SOX2 in hiPSC generation. Simultaneous replacement of OCT4 and SOX2 allows human cell reprogramming to iPSCs

“It was generally assumed that development led to cell/tissue specification by ‘opening’ certain differentiation doors,” says Emmanuel Nivet, a post-doctoral researcher in Izpisua Belmonte’s laboratory and co-first author of the paper, along with Sancho-Martinez and Nuria Montserrat of the Center for Regenerative Medicine in Barcelona, Spain.

Instead, the successful substitution of both Oct4 and SOX2 shows the opposite. “Pluripotency is like a room with all doors open, in which differentiation is accomplished by ‘closing’ doors,” Nivet says. “Inversely, reprogramming to pluripotency is accomplished by opening doors.”

This work should help to overcome one of the major hurdles in the widespread adoption of iPSC-based therapies; namely, that the original four genes used to reprogram stem cells had been implicated in cancer. “Recent studies in cancer, many of them done by my Salk colleagues, have shown molecular similarities between the proliferation of stem cells and cancer cells, so it is not surprising that oncogenes [genes linked to cancer] would be part of the iPSC recipe,” says Izpisua Belmonte.

With this new method, which allows for a customized recipe, the team hopes to push therapeutic research forward. “Since we have shown that it is possible to replace genes thought essential for reprogramming with several different genes that have not been previously involved in tumorigenesis, it is our hope that this study will enable iPSC research to more quickly translate into the clinic,” says Izpisua Belmonte.

Other researchers on the study were Tomoaki Hishida, Sachin Kumar, Yuriko Hishida, Yun Xia and Concepcion Rodriguez Esteban of the Salk Institute; Laia Miquel and Carme Cortina of the Center of Regenerative Medicine in Barcelona, Spain.

Induced Pluripotent Stem Cells Lead Neuroscientists to the Cause of Neuron Loss in Parkinson’s Disease

Salk Institute scientists have made induced pluripotent stem cells (iPSCs) from patients with early-onset Parkinson’s disease (PD) in order to study precisely what goes wrong in the brains of PD patients. Their findings may lead to new ways to diagnose and even treat PD.

At the Salk Institute for Biological Studies in La Jolla, CA, Juan Carlos Izpisua Belmonte and his colleagues have examined the effects of mutations in a gene that encodes the leucine-rich repeat kinase 2 (LRRK2) protein on cultured neurons. LRRK2 mutations are responsible for approximately 2% of all inherited and sporadic cases of PD in North American Caucasian populations and up to 20% of all PD cases in Ashkenazi Jewish patients and approximately 40% of all PD cases in patients of North African Berber Arab ancestry. Therefore, the LRRK2 gene product plays a central role in PD pathology.

When iPSCs derived from PD patients who carry LRRK2 mutations, they were differentiated into neurons that were cultured in the laboratory. Cultured neurons from PD patients show profound disruption of the nuclear membrane and this undoes all nuclear architecture, which leads to cell death.

According to Dr. Izpisua Belmonte, “This discovery helps explain how PD, which had traditionally been associated with loss of neurons that produce dopamine and subsequent motor impairment, could lead to locomotor dysfunction and other common non-motor manifestations, such as depression and anxiety. Similarly, current clinical trials explore the possibility of neural stem cell transplantations to compensate for dopamine deficits. Our work provides the platform for similar trials by using patient-specific corrected cells. It identifies degeneration of the nucleus as a previously unknown player in PD.”

Izpisua Belmonte and his colleagues were also able to confirm that these disruptions of the nuclear membrane also occur in brain tissue from deceased PD patients. While it is still unclear if these disruptions to the nuclear membrane are the result of PD or are a cause of PD, Izpisua Belmonte’s lab used gene replacement techniques that were initially developed and perfected in work with mouse ESCs to fix the mutation in the PD patient-derived iPSCs. When they fixed the mutation, the disruptions to the nuclear membrane failed to form. Belmonte thinks that this could open the door for drug treatments of PD patients, although he did speculate as to how a pharmacological agent might mitigate abnormal nuclear architecture.

These results underscore the power of using iPSCs to model genetic diseases. As Belmonte noted, “We can model disease using these cells in ways that are not possible using traditional research methods, such as established cell lines, primary cultures and animal models.”

Another finding that nicely comports with data from clinical observations of PD patients is the tendency for patients to become progressively worse as they age. Likewise, in their cultured neurons differentiated from that were iPSCs derived from PD patients, Belmonte and his group observed progressively greater deformities in the nuclear membranes of the cells as they aged.

“This means that, over time, the LRRK2 mutation affects the nucleus of neural stem cells, hampering [sic] both their survival and their ability to produce neurons. It is the first time to our knowledge that human neural stem cells have been shown to be affected during Parkinson’s pathology due to aberrant LRRK2. Before development of these reprogramming technologies, studies on human neural stem cells were elusive because they needed to be isolated directly from the brain,” said Belmonte.

Belmonte further opined that dysfunctional neural stem cell populations that are afflicted with LRRK2 mutations might also contribute to other health issues associated with this particular form of PD, which includes depression, anxiety, and the inability to smell.

Modeling diseases with iPSCs also has an added bonus, since this model system can effectively recapitulate the effects of aging. Since unique dysfunctions result from aging, there are very few ways to model such events. However, using cultured cells made from iPSCs can bypass this problem, since the age-related pathologies will typically show up in culture.

Embryonic Stem Cell Cultures Fluctuate Between Pluripotence and Totipotence

In the journal Nature, a fascinating paper appeared from the laboratory of Samuel Pfaff at the Howard Hughes Medical Institute at the Salk Institute for Biological Studies in La Jolla, California near San Diego. In this paper, Todd Macfarlan and his colleagues show that embryonic stem cells cycle between a very primitive developmental stage and a later stage. This cycling is also due to gene expression that is linked to transposable DNA elements.

First, we need some background. The term “totipotent” means that a cell can form any structure in the embryonic or adult body. For example, when the egg undergoes fertilization, it becomes a zygote, which has the capacity to grow into the embryo and all the extraembryonic membranes (amnion, chorion, allantois, placenta, and so on). Another example is a sponge. When a small piece of the sponge is cut from it, the cells in that small piece can de-differentiate and grow into an entire new sponge. Sponge cells are, therefore, totipotent.

Secondly, there is a term “pluripotent,” which means that the cells can form all the adult cell types. Embryonic stem cells are generally thought to be pluripotent and not totipotent. Once the embryo forms the two-cell stage, these two blastomeres are totipotent. However, when the blastocyst stage forms, the inner cell mass cells become pluripotent and lose the ability to form the placenta.

Many years ago, Beddington and Robertson, (1989) implanted mouse embryonic stem cells into the outer layer of cells (trophoblast) to determine if the inner cell mass cells could form the placenta (Development 105, 733–737). The embryonic stem cells were incorporated into the placenta at a very low rate. These data led to an intriguing question: Was the low incorporation due to contamination with trophoblast cells, or could a small proportion of the embryonic stem cells actually become placenta? When gene expression studies examined embryonic stem cells, gene expression was stable in the majority of the cells, but unstable in a small minority of cells (a condition called metastable). It was not surprising that embryonic stem cells were a mixed bag of different cells, but some cells expressed genes that were normally found at earlier developmental stages or were normally expressed in cells that make placenta:
1. Niakan, K. K. et al. Sox17 promotes differentiation in mouse embryonic stem cells by directly regulating extraembryonic gene expression and indirectly antagonizing self-renewal. Genes Dev. 24, 312–326 (2010).
2. Hayashi, K., Lopes, S. M., Tang, F. & Surani, M. A. Dynamic equilibrium and heterogeneity of mouse pluripotent stem cells with distinct functional and epigenetic states. Cell Stem Cell 3, 391–401 (2008).
3. Singh, A. M., Hamazaki, T., Hankowski, K. E. & Terada, N. A heterogeneous expression pattern for Nanog in embryonic stem cells. Stem Cells 25, 2534–2542 (2007).
4. Chambers, I. et al. Nanog safeguards pluripotency and mediates germline development. Nature 450, 1230–1234 (2007)
5. Zalzman, M. et al. Zscan4 regulates telomere elongation and genomic stability in ES cells. Nature 464, 858–863 (2010).

Weird, huh?

Into the fray comes Macfarlan and company to save (or explain) the day. It turns out that our genomes are loaded with DNA from transposable elements. These DNA elements either have or had at one time, the ability to jump from one location in the genome to another. There are large numbers of these transposable elements in our genomes and almost 50% of the human genome is composed of the remains of these elements.  Current transposable elements include Long INterspersed Elements (LINEs), Short INterspersed Elements (SINEs) and SVA (SINE/VNTR/Alu) elements.  Others include elements such as Mariner, MIR, HERV-K, and others.  The significance of all this is that during development, when the embryo gets to the two-cell stages, in the mouse, particular transposable elements are expressed at very high levels (they produce 3% of the transcribed messenger RNAs, see Peaston, A. E. et al., Dev. Cell 7, 597–606 (2004); Evsikov, A. V. et al., Cytogenet. Genome Res. 105, 240–250 (2004); Kigami, D., et al., Biol. Reprod. 68, 651–654 (2003)), and after two-cell stage, the expression of these transposable elements is silenced (Svoboda, P. et al. Dev. Biol. 269, 276–285 (2004); Ribet, D. et al. J. Virol. 82, 1622–1625 (2008)).

Since these transposons are characteristic of gene expression at the two-cell stage, they can be used as a marker for embryonic stem cells that have reverted back to the two-cell stage.  MacFarlan and his co-workers made a reporter gene and placed it into embryonic stem cells that was controlled by the same sequences as the transposons that are activated at the two-cell stage.  After growing these engineered embryonic stem cells in culture, they discovered that a small minority of cells expressed this reporter gene.

Did these reporter-expressing cells have characteristics like unto those of the two-cell stage embryos?  Yes they did.  When Macfarlan and his buddies examined the genes expressed in the cells that expressed the reporter, they found that the traditional genes that are so characteristic of inner cell mass cells (Oct4, Nanog, Sox2, etc.) were not expressed and other genes normally expressed in two-cell stage embryos, such as Zscan4, were expressed.  Other features that are found in two-cell-stage embryos were also found in these cells that expressed the reporter gene. (methylation of histone 3 lysine 4 (H3K4) and acetylation of H3 and H4 for those who are interested).

Finally, the reporter-expressing cells were able to contribute to the formation of the placenta when transplanted into a mouse embryo.  This shows that these cells not only express the genes of the totipotent stage of development, but they also are totipotent.

These experiments show that most, maybe all embryonic stem cells pass through a short-lived state during which they display features that are characteristic of the totipotent two-cell stage: unlike the vast majority of the ES cells in the culture.  During this transition, they lack expression of the pluripotency-promoting proteins Oct4, Sox2 and Nanog, and have the ability to form cells of both the placenta and the fetus.

There is also a moral implication of these experiments.  In his book Challenging Nature and on the book’s web site, Lee Silver argues that embryonic stem cells are essentially embryos, and if we don’t object to using and discarding embryonic stem cells, then we should not have any problem with using and discarding embryos.  His reasons for asserting that embryonic stem cells are embryos is that in the mouse, embryonic stem cells can be inserted into the inner cell mass of an embryo that has four copies of each chromosome.  The tetraploid embryos can form the placenta, but they cannot form the embryo that is attached to the embryo.  Inserting embryonic stem cells into the inner cell mass of these embryos will rescue them from dying because the embryonic stem cells with make the embryo and the tetraploid embryos will form the placenta.  This experiment is called “tetraploid rescue” and Silver uses it to argue that embryonic cells are essentially embryos.

I find this argument unconvincing for several reasons.  First of all, these embryonic stem cells are being manipulated by being inserted into an embryo.  Granted this embryo is abnormal, but it is an embryo all the same and it provides a vital function that the embryonic stem cells cannot supply – the making of the placenta.  This manipulation helps the embryonic stem cells make the embryo, but not everything else.  In this case the embryonic stem cells are only doing part of the job and they are also receiving the structure and inductive signals from the tetraploid embryo to form the embryo proper.  This is something that embryonic stem cells do not do in culture.

Secondly, embryos undergo development, a process that we understand rather well.  This process of development has a goal toward which the embryo proceeds during development.   Embryonic stem cells are not in the process of development.  They can be induced to form particular cell types or even tissues, but this is part of the embryo or fetus and forming part of the fetus does not constitute embryonic development but only a small part of it.  Embryos do not go backwards during development.  Cells that do go backwards are usually cancer cells that grow uncontrollably and cannot move to a more differentiated state that puts the brakes on cell division.  The fact that embryonic stem cells do move developmentally backward is another indication that they are not embryos.  They do something that embryos do not do and this disqualifies them from being embryos.

Thus another argument against the humanity of the early embryo falls into the pit of very bad arguments.