Josh P. Roberts

Find out just how far we’ve come in the field.

The stem cell field—not too long ago hamstrung by crippling restrictions and embroiled in scandal—has come a long way. Pluripotent somatic cells have been found, and “stem-like” cells have been generated by what amounts to either wholesale or piecemeal gene therapy. Now it’s time to see just what they are and what they can do.

Researchers at the International Society for Stem Cell Research 11th Annual Meeting, taking place this week in Boston, will be discussing such creations, just how “stem-like” they are, and what they can tell us in terms of development, disease, drug screening and discovery, and even the future of personalized medicine. A number of them previewed their talks for GEN.

“We have finally been able to use human oocytes as a reprogramming machine” to make pluripotent, embryonic stem cell (ESC)-like cells, said Shoukhrat Mitalipov, Ph.D., of the Oregon Health and Science University (OHSU). Using a technique known as somatic cell nuclear transfer (SCNT), his group replaced the nuclear material of an unfertilized human egg with that of a skin cell to create NT-ESC lines. “The egg cytoplasm has the factors that can reset the identity of the donor cell nucleus and make it look like early embryonic.”

He explained that although this type of reprogramming had long been predicted, it took more than a decade to make it work. “We knew this works in the mouse, and we were the first to show that it works in the monkey, but translating to human was difficult.” Human SCNT embryos would divide, but never develop into viable stem cell colonies; rather, they arrest at the 4–8 cell stage.

Dr. Mitalipov hypothesized that this may have been due to a mismatch in the cell cycles of the donor nucleus and the oocyte. “It looks like the human eggs have very sensitive and unstable metaphase,” and the manipulations induce the cell’s exit from metaphase. Yet metaphase cytoplasmic factors seem to be responsible for synching up the incoming nucleus, which is harvested in interphase.

They used special techniques such as gentle enucleation and introduction of donor nucleus, and drugs including caffeine, to maintain the oocyte’s metaphase artificially during the microsurgical procedures. The cytoplasmic metaphase factors chew up the nuclear membrane and condense the chromatin back into chromosomes, now probably stripped of many somatic cell transcription factors and epigenetic factors. “Then they will be reassembled once the cell actually goes into the interphase naturally, in this case with the somatic nucleus inside of the egg. And that is where, probably, the oocyte-specific transcription factors will replace the somatic cell factors, and how reprogramming was enhanced,” he theorized.

So now that other methods of making pluripotent cells have been demonstrated, why bother with SCNT? For one thing, human induced pluripotent stem cells (iPSCs) have a high frequency of copy number alterations, and reprogramming is often not complete. Dr. Mitalipov has begun comparing his NT-ESCs to iPSCs, as well as distributing them to other labs for them to study to see if they suffer the same fate. He has another, more direct, reason for creating NT-ESC cells: Dr. Mitalipov’s lab studies mitochondrial DNA, which as part of the cytoplasmic compartment remains in the oocyte while the nuclear compartment is completely replaced. Thus, SCNT replaces mutated mitochondrial DNA in a patient cell in NT-ESCs—which iPSCs do not accomplish.

It’s The Substrate

Most of the work in cell programming and reprogramming to date has focused on transcription factors—the master controllers, regulated in development, that tell a cell what it should be. Adding exogenous genes for transcription factors—basically conducting gene therapy—is an inefficient way of reprogramming, with perhaps 1/1,000 or 1/10,000 cells becoming converted, pointed out Kenneth Zaret, Ph.D., of the University of Pennsylvania School of Medicine. He wants to know why.

There are chromatin impediments to the reprogramming process, Dr. Zarat noted, and these “are a major feature that don’t allow a cell to jump from one fate to another—they help keep cells nice and tidy where they should be.” Chromatin is formed when DNA wraps around histone molecules, and when the histones are chemically modified (methylated or acetylated, for example) they act like flags to recruit additional proteins—some of which are signals to turn a gene on, while others signal to turn it off.

His lab studies the chromatin state, and the molecules involved with chromatin that govern where transcription factors can bind versus where they’re not allowed to bind. In work published last year in Cell they showed that the canonical reprogramming factors Oct4, Sox2, Klf4, and c-Myc (collectively called OSKM) occupy pluripotent cells such as iPSCs and ESCs in very different patterns than they do in the differentiated human fibroblast genome. Ectopically expressed O, S, and K seem to act as pioneering factors by binding “closed” chromatin at distal elements of genes required for reprogramming and helping to recruit c-Myc. But there are also still megabase-sized domains of the genome refractory to OSKM binding.

“What I’m going to discuss [at ISSCR] are our latest insights into different types of repressive structures. One type prevents OSKM binding, and when knocked down, enhances the production of pluripotent cells. Another is an impediment to the production of pancreatic beta cells in normal, embryonic development. Similarly, when we knock this down, we observe an increase in pancreatic beta cells,” he said. The goal is—“because we know the mechanistic basis for it, and because these chromatin modifications, or histone marks (so to speak) are laid down by enzymes, and enzymes can be inhabitable with small molecules”—to ultimately be able to use small molecules to relieve the inhibition and allow more of the cells to be specified to one fate or another.

Give It Time

Benoit Bruneau, Ph.D., is exploring how transcriptional and chromatin-level regulation are integrated during the process of differentiation. To do this his lab at the Gladstone Institute of Cardiovascular Disease at the University of California–San Francisco uses a directed differentiation approach to drive mouse ESCs toward a cardiomyocyte lineage, and looked at cells at various intermediate stages along that developmental pathway.

Combining chromatin immunoprecipitation with massively parallel sequencing (ChIP-Seq) of both polyadenylated RNA and noncoding RNA, they queried the transcriptome along with several histone modifications of the induced cardiac precursors. More than 13,500 genes were found to be expressed during the time course, and these were clustered according to expression pattern. Among these was “a relatively small but important group of genes—essentially all of the genes that are involved in contractile function of the heart”—which were seen being regulated at the chromatin level, completely differently from another group of genes that has the same expression pattern, Dr. Bruneau explained.

“If you looked just at their RNA levels, you’d say these genes are regulated the exact same way. But at the chromatin level they have a unique signature over time. And so that implies that there are specific mechanisms that are deployed for groups of not just co-regulated genes but functionally related genes.”

The notion that certain histone modifications are associated with enhancers is of course not new. What is unique, he noted, is the fact that they have been able to establish dynamic changes that were not predicted by querying only single time points.

They also took enhancer elements that were predicted on a genome scale and computationally asked what flavor of DNA binding protein motifs are there. They found “the usual suspects, but also binding sites for transcription factors that were not known to be associated with cardiac differentiation.” This, Dr. Bruneau said, enabled them to hypothesize new interactions.

He plans to present not only relatively recent efforts in describing the epigenomic landscape of cardiac differentiation, but also a recent important foray into understanding how mesoderm lineage commitment is set up and what impact that has on cardiac differentiation. “We’ve found a mechanism by which dynamic enhancers are established, involving the function of the chromatin remodeling complex,” he said.

Be Patient

Lawrence Goldstein, Ph.D., reprograms iPSCs to create cell lines that carry “the unique genetic constitution of whatever person donated the skin cells. So if it’s a person with hereditary Alzheimer’s disease [AD], you can then make human brain cells—human neurons—in a dish that have the genetic change that caused AD, and study what’s different about those neurons from normal.”

Similarly, his lab also investigates neurons made from the skin of patients with sporadic AD. Sixty to eighty percent of the risk for developing the disease comes from genetic variation, and so it is likely that these nouveau brain cells will manifest some of the same biochemical changes that caused AD in their donors.

“Can you analyze that and figure out what’s going on?” the University of California–San Diego distinguished professor asked. If so, “that lets you do experiments on mechanism: What’s going wrong? Can you treat it with drugs? Can you look for drugs? And all the rest.”

They purified cultures by flow cytometry to greater than 90% neurons, “enabling a much more precise biochemical evaluation,” Dr. Goldstein explained. Lines made from the hereditary AD donors and from one of the two sporadic AD donors were found to have significantly higher levels of various pathological AD markers, including amyloid-β and phospho-tau; they were able to pharmacologically block the effect on phospho-tau expression.

He has also been using the same stem cell technologies to study common variants associated with sporadic AD, regardless of whether the donor has any clinical signs of disease. “I will talk about a project at this meeting to nail down what the molecular behavior is that is different in people who have a particular variant in the human population.”

Stanford University School of Medicine’s Joseph Wu, M.D., Ph.D., agrees with this philosophy. He takes issue with the pharmaceutical industry’s reliance on Chinese hamster ovary (CHO) cells—transgenic for a single heart ion channel (HERG), easy to grow, but not even able to beat—being used for cardiotoxicity drug screening: “The human heart has over 15 major channel genes,” he pointed out. “Relying on CHO cells expressing HERG channels can lead to many false positive and false negative hits.”

To circumvent the problem his lab has derived cardiomyocytes from human skin cells (hiPSC-CMs). “The cardiac cell that we obtain beats on a dish so we can measure several parameters. Does the cardiac cell beat faster or slower after exposure with drugs? Does it beat stronger or weaker? Does the drug cause arrhythmia in different patient cohorts? So we have a variety of readouts that are very similar to the human heart physiology which allows us to what’s the effect of the drugs on these cells in a dish,” not just on the HERG channel, noted Dr. Wu.

Many drugs can be safe for the majority of the population, but harmful or fatal for those with certain abnormalities or genetic backgrounds. Wu has been creating disease-specific hiPSC-CMs from patients with various hereditary cardiac disorders. By using these population-specific cells to screen drug candidates, such negative interactions may be identified and avoided while potentially rescuing otherwise valuable pharmaceuticals.

Dr. Wu sees iPSC-derived cell testing as playing a major role in the future, and “not just for the heart. People will make patient-specific models for brain diseases, for hepatic metabolism, for diabetes, and try to show that this can be useful for predicting drug response.”

With the pace of current technology, in 5–10 years CROs will do drug testing on panels derived from 1,000 people of various genetic and ethnic backgrounds, he predicted. And in 20 years, “you should be able to take a patient’s skin or blood, make these iPSC-derived cells on a dish or in 3D organoids, and use them as phenotype readouts along with patients’ genotype data for personalized medicine that is safer and more predictive.”

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