Just as one cannot step into the same river twice, one cannot find anything fixed in stem cell research, where one will find, instead, that revelations and still other revelations are ever flowing. Accordingly, much has changed since the original revelation, the almost four-decade-old discovery of embryonic stem cells in mice. Subsequent studies led to the isolation of human embryonic stem cells, which were then grown in the laboratory, as well as the development of reprogramming techniques and the generation of induced pluripotent stem cells (iPSCs). More recently, advances in biotechnology and genome editing have fostered a modern evolution of stem cell advances that shows no signs of slowing.

The evolution of stem cell technology continues despite ongoing controversies. Besides objections to research involving human embryonic stem cells, there are concerns about unproven and unapproved stem cell–based therapies. Such therapies have been linked to serious complications. The only stem cell–based products that have received FDA approval are those that consist of blood-forming stem cells (hematopoietic progenitor cells) derived from cord blood. Such products are suitable only for patients with diseases of the blood or bone marrow.

Despite warnings from reputable scientists and regulatory authorities, people eager to benefit from regenerative medicine may seek care from stem cell clinics, if not for themselves, then for their beloved pets. For example, dog owners may be attracted to an animal biotech company called Gallant, which recently launched a stem cell banking service for dogs. The company obtains a dog’s stem cells from tissues collected during the animal’s routine spay or neuter procedure. These cells, the company suggests, may benefit the animal’s health in the future.

Far removed from stem cell services that offer only anecdotal evidence of efficacy, or no evidence at all, there are bona fide advances in stem cell research. These advances may not beguile a credulous public, but they are the most reliable indicators of progress. In this article, due attention is given to some of the most significant stem cell advances: the application of CRISPR genome editing to iPSCs; the development of stem cell therapeutics for diabetes; and the creation of synthetic stem cell–derived embryos.

Changing stem cells while maintaining their state

Stem cell research is hardly indifferent to genome editing, which is undergoing revolutionary change. Given the ability of patient-derived iPSCs to model diseases and the promise of gene editing to provide cures, some researchers are marrying the two disciplines. Together, they may bring about treatments far superior to those attainable with either discipline alone.

Until recently, notes Lee Spraggon, PhD, head of cell engineering development at Synthego, there was an inertia to moving gene editing at scale into iPSCs. At present, however, momentum is building. According to Spraggon, there are clinical centers that have large repositories of iPSC lines that they are keen to edit. They could, for example, introduce disease-associated edits in iPSC lines or revert patient-derived iPSCs back to a wild-type genotype.

A leader in the field, Bruce Conklin, MD, senior investigator at the Gladstone Institute of Data Science and Biotechnology at the University of California, San Francisco, tells GEN that “human iPSCs and human embryonic stem cells were very hard to do genetics with at first. It was very difficult to make genetic modifications.” He explains that new genome engineering tools (such as zinc finger nuclease, transcription activator-like effector nuclease, and CRISPR-Cas nuclease systems) have revolutionized researchers’ ability to introduce targeted genetic modifications that can reveal disease mechanisms and therapeutic targets.

By showing that a specific genotype (modification) is sufficient to make phenotypic change, Conklin notes, proof of the relationship can be provided.  This genotype–phenotype link may allow researchers to uncover once-hidden connections. And exploiting these connections could help researchers establish new uses for iPSCs in human genetics, disease modeling, and drug discovery.

Some researchers choose to perform the editing themselves. But there are unique challenges that iPSC lines present. Working with iPSCs in a culture environment, Spraggon explains, is not like working with other cells commonly used in cell culture. With immortalized cell lines, he says, one can be “less cautious.” With stem cells, more care must be taken. Everything must be “on point.” According to Spraggon, stem cell cultures represent the gold standard cell culture technology.

Synthego offers a service that can introduce genomic edits at scale. Offering knock outs or knock ins, the service makes edits in Synthego’s internal cell lines or patient-derived iPSC lines. The company can also add epitope tags that allow proteins to be followed, for example, in an effort to understand a protein’s physiological role in the cell.

The first area of research that Spraggon thinks will benefit from a such a service is neuroscience. Indeed, Synthego has announced a partnership with the NIH for a multimillion-dollar program to better understand neurodegenerative disease. Synthego has been tasked to generate iPSCs with various genomic edits and will make many highly characterized iPSC clones for the research community. This work could be done manually, notes Spraggon. But it would take an incredibly long time.

“Our approach,” explains Mark Cookson, PhD, senior investigator in the Laboratory of Neurogenetics at the National Institute on Aging, “is to use CRISPR-Cas9 to introduce human genetic variants associated with Alzheimer’s disease and related dementias, with varying levels of pathogenic impact, into a consistent genetic background.” The laboratory is trialing several different background lines for their ability to stay genomically stable after editing, he explains, so that the resulting resource will be as qualified as possible for the community.

It was not easy for Synthego to modify its gene editing platform to accommodate iPSCs, describes Spraggon. It was particularly challenging, he asserts, to obtain consistency. Nonetheless, the company has committed to ensuring a high level of quality control to guarantee that the cells that come off the platform are the same as the cells that are put into it—except for the intended genome edits.

“Doing the editing in the cell is not that hard,” Spraggon asserts. The challenge, he says, is in maintaining the iPSCs in their pluripotent, undifferentiated state. If the cells differentiate, they are worthless.

Type 1 diabetes may be next in line

Stem cells are surging down a path toward disease treatments. Evidence that the surge is well on its way includes the recent announcement that Semma Therapeutics, a developer of stem cell technologies for treating diabetes, agreed to be acquired by the pharma giant Vertex Pharmaceuticals. Semma, which was founded by stem cell pioneer Douglas A. Melton, PhD, will become a separate operating subsidiary of Vertex following a sizable cash transaction: $950 million. After the acquisition, Melton will continue in his role as chair of Semma’s scientific advisory board.

In addition to his work at Semma, Melton is the Xander University Professor at Harvard and co-director of Harvard’s Stem Cell Institute. At Harvard, Melton participates in research that uses animal models to illuminate the developmental biology of the pancreas. Applied to human cells, the research could lead to the development of insulin-producing b-cells for diabetics. The ultimate goal is to find a cure for type 1 diabetes, the chronic condition that affects more than 1.5 million people in the United States.

Currently, diabetics require multiple daily insulin injections and blood glucose monitoring. Soon, better alternatives may be available. For example, diabetics might receive transplants of insulin-producing b-cells of the pancreatic islets. The transplant material would be generated by stem cells grown in the laboratory.

The stem cell research conducted by Melton at Harvard informs Semma’s developmental work. And both pursuits are inspired, in part, by one of Melton’s personal journeys. Years ago, when Melton’s own children were diagnosed with type 1 diabetes, he resolved to steer his laboratory’s work to developing a cure.

Melton’s academic and industry interests were both apparent in a research paper published in Nature earlier this year. The paper, entitled “Charting cellular identity during human in vitro b-cell differentiation,” was contributed by authors representing both Melton’s lab and Semma. It described how single-cell RNA sequencing was used to assay more than 100,000 cells during stem cell differentiation into hormone-producing cells. In addition, the paper detailed the differentiation steps that pancreatic progenitor cells follow.

The authors’ analysis, noted an accompanying News & Views article in Nature, showed a “deep mechanistic understanding of islet-cell differentiation from stem cells.” For their part, the research paper’s authors asserted that their work “will guide future endeavors that focus on the differentiation of pancreatic islet cells and their applications in regenerative medicine.”

Elaborating on the developmental perspective, David Altshuler, MD, PhD, executive vice president, global research and CSO of Vertex, contributed the following quote to a press release about the Semma acquisition: “The therapeutic approach pioneered by Semma has the potential to address the causal human biology of type 1 diabetes, a serious disease inadequately controlled by existing therapies. Unlike insulin injections and insulin pumps, islet cell transplantation can provide physiologic regulation of blood glucose, thereby potentially ameliorating or preventing both the hyperglycemic and hypoglycemic episodes associated with the current standards of care.” (Vertex declined an invitation to comment for this story.)

From cultured stem cells to artificial embryos

The work of Juan Carlos Izpisua Belmonte, PhD, professor in the Gene Expression Laboratory at the Salk Institute, has led to innovations in multiple areas of stem cell biology and mammalian development.

Salk Institute team
The Salk Institute team that led the stem cell–derived synthetic embryos research: Ronghui Li, PhD, Juan Carlos Izpisua Belmonte, PhD, and Cuiqing Zhong, PhD.

In 2017, he presented a proof-of-concept study showing that functional organs from one species can be grown in another. Combining gene editing and stem cell technologies, Izpisua Belmonte and colleagues were able to grow a rat pancreas, heart, and eyes in a developing mouse. They were also able to generate human cells and tissues in early-stage pig and cattle embryos. This work contributed to Izpisua Belmonte being selected as one of Time magazine’s “50 Most Influential People in Health Care” in 2018. Other work by his laboratory has focused on wound healing and aging.

Jun Wu
Jun Wu, PhD, Assistant Professor, UT Southwestern Medical Center

In collaboration with Jun Wu, PhD, assistant professor at UT Southwestern Medical Center and a former postdoc in the Belmonte laboratory, Izpisua Belmonte has established that models of early embryos can be generated from mouse stem cells. More specifically, the investigators showed that extended pluripotent stem cells from mice—cells that are treated to maintain pluripotency—can, in culture, self-organize to form blastocyst-like structures. The work is published in Cell in a paper entitled “Generation of Blastocyst-Like Structures from Mouse Embryonic and Adult Cell Cultures.” This work, which marks the first time that blastocyst-like structures have been created from a single cultured cell, may eliminate the need for sex cells to create living organisms.

“This work advances the field related to stem cell–derived synthetic embryos,” Wu tells GEN. By building artificial embryos in vitro using cultured stem cells, Wu notes, “we’ll gain novel insights into the molecular and cellular processes that occur during early development.” Studying mammalian development, he explains, is critical to develop effective stem cell differentiation protocols for regenerative medicine.

Stem cell–derived synthetic embryos are mostly used for basic studies at this stage. But, in the future, if human synthetic embryos can be generated, Wu explains, they may be used for modeling disease and advancing preimplantation and implantation. Wu sums up the role of this work in advancing the stem cell field by quoting the Nobel-prize winning physicist Richard Feynman, PhD: “What I cannot create, I do not understand.”

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