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By Nick Miller
Christopher Mayhew, PhD, appreciates the importance of converting skeptics into believers.
He finds this transformation happening more readily as time passes — certainly compared to four years ago. That is when Mayhew helped establish the Pluripotent Stem Cell Facility at Cincinnati Children’s — one of the nation’s first such core research laboratories at an academic medical center. The move was a significant investment and statement for Cincinnati Children’s.
It signaled a strong commitment to an emerging and — at the time — somewhat uncertain technology called induced pluripotent stem cells (iPSCs). Only a few years later, the capabilities of iPSCs are resulting in dramatic advances in how scientists study disease.
For Mayhew, it has been rewarding to see fellow scientists learn they can use new, human-based modeling systems that do not require a reliance on animal models and their inherent functional or translational limitations.
“There has been a small cohort of pioneers and true believers from the very beginning, but what is exciting is to see people come into the fold with new ideas, and for the projects to actually reach a level of fulfillment,’’ says Mayhew, who co-directs the facility with Jim Wells, PhD.
The technology uses gene-based biochemical solutions to transform human skin cells into cells capable of forming any tissue in the human body. Functionally based on the transformative powers of human embryonic stem cells (hESCs), which are derived from a fertilized egg, induced pluripotent stem cells were devised to accomplish the same goals, without the complications of hESCs.
For the purpose of regenerating tissues to be used for disease research modeling and eventual therapeutic purposes, iPSCs have the additional advantage of having the same genetics of the person who donated the cells. This means regenerated tissues should not risk rejection by that person’s immune system.
“For years, people have been aware that this technology is out there and that you can do some interesting and novel studies with it,” explains Mayhew. “But scientists are really starting to understand that iPSCs can be used for discovery research, and it’s not just a novelty this technology is being used to answer some very important questions, and providing new insights into the mechanisms of human disease is the next big explosion to come.”
As the technology of making iPSCs from skin cells becomes more refined, and as reputations and word-of-mouth grow, so does the amount of work at the facility and the types of projects it takes on. Mayhew estimates the lab’s project load has quadrupled from its start four years ago.
With a staff that includes Mayhew, Wells, a full-time research assistant and a part-time assistant, the lab performs an array of very specialized and complex services. They can generate and maintain iPSC cultures for a research lab studying a specific organ or disease, or they can train the laboratory’s staff to generate and maintain the cells themselves. The cells require constant and precise nurturing, Mayhew says.
It can take up to three months from the point of starting the reprogramming process to having iPSCs ready for a laboratory to start experiments. At that point, it is up the researchers receiving the iPSCs, with assistance from Mayhew and his team as needed, to turn the cells into an organ- or disease-specific model system.
In some cases, as in studies involving intestinal tissues, Mayhew’s team can generate the organoids for researchers studying that organ. The lab also hopes to be able to generate other specific tissue types for researchers as those capabilities and needs develop.
Most of the lab’s services are currently supplied to research labs at Cincinnati Children’s or the University of Cincinnati. But the facility’s growing reputation means its services are also being sought out by institutions from around the U.S. and as far away as Europe.
This means that, on any given day the lab might be generating iPSCs or training scientists who want to make heart cells. The day after, the focus might be on immune cells called macrophages, and the day after that the project may involve cells of the intestine or lung.
Some of the increasing acceptance of iPSCs comes with refinements in how they are generated from skin cells. When first described in 2006 by researchers in Japan, iPSCs were produced by introducing a set of genes that caused the cells to turn back their developmental clock to act like human embryonic stem cells.
Despite understandable excitement in the scientific community, the cells did come with a few challenges. Some genes in the original mixture (c-Myc in particular) were associated with causing cancer. This was not the only concern.
“The old method used viruses to deliver the genes that also jumped into the DNA of the recipient cells, and that can generate mutations and cancer,” says Wells. “The method we use now is an approach called episomal reprogramming, meaning the genes don’t jump into the host cell’s chromosomes.”
The new delivery method involves the use of plasmids containing the reprogramming genes, which enter the cells and float around. This process produces specific proteins that actually cause cellular reprogramming, avoiding insertion directly into the cells’ chromosomes. “You don’t have to worry about what is called insertional mutagenesis, and we have had great success with this method,” Mayhew says.
Human embryonic stem cells are still used today, although much less than several years ago. They serve primarily as a gold standard and benchmark for measuring functional capabilities, and to make sure iPSCs are doing what they are supposed to do, according to Wells.
He expects an increasing shift toward using iPSCS to continue, especially as the technology continues to improve.
Bruce Trapnell, MD, and Takuji Suzuki, MD, PhD, Division of Pulmonary Biology, use induced pluripotent stem cells (iPSCs) to study a rare genetic disorder, pulmonary alveolar proteinosis (PAP). The lungs of children with PAP fill with surfactant, a thick fatty substance normally present in a small amount to help keep the lungs from collapsing. Current treatment is a whole-lung wash, an invasive procedure performed under general anesthesia to remove the excess surfactant.
The study used iPSCs derived directly from the cells of children with the disease. The iPSCs, which contained the mutated gene that causes PAP, were then converted into macrophages, immune system cells that become dysfunctional in PAP. Researchers analyzed the cells as they grew and differentiated into macrophages, acquiring the same PAP disease characteristics observed in patients.
The researchers then used an engineered virus to deliver a correct copy of the mutated gene to the diseased macrophages. This caused PAP disease manifestations in the cells to cease, providing a proof of principle for a potential gene therapy. The research was published earlier this year in the American Journal of Respiratory and Critical Care Medicine. Additional research is needed before the findings could be used clinically, but iPSC models of PAP will be central to the researchers’ ongoing work.
Gastrointestinal disorders affect up to a fourth of the U.S. population. Many of these disorders involve a lack of spontaneous muscle contractions that help food and waste get through the body’s digestive tract. There are few treatments — especially drugs — that target these ailments.
The laboratories of Jim Wells, PhD, and Samantha Brugmann, PhD, are using patient-specific iPSCs to generate human intestinal organoids with functioning epithelium. Wells’ lab has already produced intestinal tissue, but this project aims to generate intestine with enteric nerves. The goal is to produce a system on a 3D chip with living cells and tissues to model functioning human intestine. The chip will be used to test prospective drugs for toxicity and safety.
Wells and Brugmann were selected in 2012 to be part of a 17-grant, multi-center consortium formed by the National Institutes of Health’s National Center for Advancing Translational Sciences. The consortium’s goal is to provide human tissue-based testing platforms to predict new drug safety in a faster, more cost-effective way.
Researchers have long suspected that the gene neurogenin-3 might be a key to generating human pancreatic beta cells in vitro, and that its mutation may drive a genetic form of diabetes. Beta cells are vital to regulating blood glucose and preventing the disease. But some people born with mutations in the neurogenin-3 gene do not have diabetes, although they suffer from intractable diarrhea.
This sent researchers back to their labs. Doubt set in about whether mouse studies in this instance translated well to humans. But there was no way to study beta cell development in people.
Wells and Sean McGrath, a graduate student in Developmental Biology, decided to use iPSCs to model the human condition. They produced human PSCs in a petri dish and tested to see if neurogenin-3 is important for making beta cells.
So far, testing suggests that expression variance in mutated neurogenin-3 makes it possible for just enough gene function to produce beta cells, but not enough to manufacture intestinal cells critical for food absorption and preventing diarrhea. Although additional research on the findings is needed, Wells expects the study will demonstrate that complex human diseases can be effectively studied “in a dish.” He believes the approach will increasingly replace the need to use animals in research.
Dr. Chris Mayhew with stem cell facility research assistants Amy Pitstick and Matt Kuhar.
Click image for caption.
On-screen image shows a colony of induced pluripotent stem cells.
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