Directly Reprogramming Gut Cells into Beta Cells to Treat Diabetes

Type 1 diabetes mellitus results from destruction of insulin-producing beta cells in the pancreas. Diabetics have to give themselves routine shots of insulin. The hope that stem cells offer is the production of cells that can replace the lost beta cells. “We are looking for ways to make new beta cells for these patients to one day replace daily insulin injections,” says Ben Stanger, MD, PhD, assistant professor of Medicine in the Division of Gastroenterology, Perelman School of Medicine at the University of Pennsylvania.

Some diabetics have had beta cells from cadavers transplanted into their bodies to replace the missing beta cells. Such a procedure shows that replacement therapy is, in principle possible. Therefore, transplanting islet cells to restore normal blood sugar levels in type 1 diabetics could treat and even cure disease. Unfortunately, transplantable islet cells are in short supply, and stem cell-based approaches have a long way to go before they reach the clinic. However, Stanger and his colleagues have tried a different strategy for treating type 1 diabetes. “It’s a powerful idea that if you have the right combination of transcription factors you can make any cell into any other cell. It’s cellular alchemy,” comments Stanger.

New research from Stanger and a postdoctoral fellow in his laboratory, Yi-Ju Chen that was published in Cell Reports, describes the production of new insulin-making cells in the gut of laboratory animals by introducing three new transcription factors. This experiment raises the prospect of using directly reprogrammed adult cells as a source for new beta cells.

In 2008, Stanger and others in Doug Melton’s laboratory used three beta-cell reprogramming factors (Pdx1, MafA, and Ngn3, collectively called PMN) to convert pancreatic acinar cells (the cells in the pancreas that secrete enzymes rather than hormones) into cells that had many of the features of pancreatic beta cells.

Following this report, the Stanger and his team set out to determine if other cells types could be directly reprogrammed into beta cells. “We expressed PMN in a wide spectrum of tissues in one-to-two-month-old mice,” says Stanger. “Three days later the mice died of hypoglycemia.” It was clear that Stanger and his crew were on to something. Further work showed that some of the mouse cells were making way too much extra insulin and that killed the mice.

When the dead mice were autopsied, “we saw transient expression of the three factors in crypt cells of the intestine near the pancreas,” explained Stanger.

They dubbed these beta-like, transformed cells “neoislet” cells. These neoislet cells express insulin and show outward structural features akin to beta cells. These neoislets also respond to glucose and release insulin when exposed to glucose. The cells were also able to improve hyperglycemia in diabetic mice.

Stanger and his co-workers also figured out how to turn on the expression of PMN in only the intestinal crypt cells to prevent the deadly whole-body hypoglycemia side effect that first killed the mice.

In culture, the expression of PMN in human intestinal ‘‘organoids,’ which are miniature intestinal units grown in culture, also converted intestinal epithelial cells into beta-like cells.

“Our results demonstrate that the intestine could be an accessible and abundant source of functional insulin-producing cells,” says Stanger. “Our ultimate goal is to obtain epithelial cells from diabetes patients who have had endoscopies, expand these cells, add PMN to them to make beta-like cells, and then give them back to the patient as an alternate therapy. There is a long way to go for this to be possible, including improving the functional properties of the cells, so that they more closely resemble beta cells, and figuring out alternate ways of converting intestinal cells to beta-like cells without gene therapy.”

This is hopefully a grand start to what might be a cure for type 1 diabetes.

Stem Cell-based Baldness Cure One Step Closer

Scientists might be able to offer people with less that optimal amounts of hair new hope when it comes to reversing baldness. Researchers from the University of Pennsylvania say they’ve moved closer to using stem cells to treat thinning hair — at least in mice.

This group said that the use of stem cells to regenerate missing or dying hair follicles is considered a potential way to reverse hair loss. However, the technology did not exist to generate adequate numbers of hair-follicle-generating stem cells.

But new findings indicate that this may now be achievable. “This is the first time anyone has made scalable amounts of epithelial stem cells that are capable of generating the epithelial component of hair follicles,” Dr. Xiaowei Xu, an associate professor of dermatology at Penn’s Perelman School of Medicine, said in a university news release.

According to Xu, those cells have many potential applications that extend to wound healing, cosmetics and hair regeneration.

In their new study, Xu’s team converted induced pluripotent stem cells (iPSCs) – reprogrammed adult stem cells with many of the characteristics of embryonic stem cells – into epithelial stem cells. This is the first time this has been done in either mice or people.

The epithelial stem cells were mixed with certain other cells and implanted into mice. They produced the outermost layers of the skin and hair follicles that are similar to human hair follicles. This study was published in the Jan. 28 edition of the journal Nature Communications.

This suggests that these cells might eventually help regenerate hair in people.

Xu said this achievement with iPSC-derived epithelial stem cells does not mean that a treatment for baldness is around the corner. Hair follicles contain both epithelial cells and a second type of adult cells called dermal papillae.

“When a person loses hair, they lose both types of cells,” Xu said. “We have solved one major problem — the epithelial component of the hair follicle. We need to figure out a way to also make new dermal papillae cells, and no one has figured that part out yet.”

Experts also note that studies conducted in animals often fail when tested in humans.

Reactivation of Hair Follicle Stem Cells Restarts Hair Growth

Sarah Millar and her team at the Perelman School of Medicine at the University of Pennsylvania have exploited a known property of hair follicle stem cells to restart hair growth in laboratory animals.

The Wnt signaling pathway is an important regulator of hair follicle proliferation, but does not seem to be required for hair follicle survival. Wnt signaling in cells culminated in the activation of a protein called beta-catenin, which goes to the nucleus of the cell and causes changes in gene expression.

wnt signaling

Millar and her colleagues disrupted Wnt signaling in laboratory animals by expressed an inhibitor called Dkk1 in hair follicles. Dkk1 expression prevented hair growth, and when the hair follicles were examined, they still had their stem cell populations, but these stem cells were dormant. Removal of Dkk1 resumed Wnt/beta-catenin signaling, and restored hair growth.

Dkk1 activity

Interestingly, Millar’s group found Wnt activity in non-hairy regions of the skin, such as palms, soles of feet, and so on. Therefore, in order for Wnt signaling to induce hair growth, it must occur within specific cell types.

This work also has additional applications: skin tumors often show over-active beta-catenin. Removing beta-catenin could prevent the growth of skin tumors, just as removing beta-catenin in the skin of these mice prevented proliferation of any hair follicles. However, agents that can activate beta-cateinin in hair follicles could reactivate dormant hair follicles and induce new hair growth.

Finding ways to safely reactivating the Wnt pathway in particular cells in the skin is a major focus of Millar’s research group.  Such work may lead to treatments for male pattern baldness.

Gene Therapy Makes Huge Advance in Cancer Fight

Gene therapy has been used to transform patients’ blood cells into soldiers that seek and destroy cancer. A small group of leukemia patients were given a one-time, experimental therapy several years ago and today, some remain cancer-free today. As a follow-up, at least six research groups have treated more than 120 patients with many types of blood and bone marrow cancers, with stunning results.

“It’s really exciting,” says Janis Abkowitz, blood diseases chief at the Univ. of Washington in Seattle and president of the American Society of Hematology. “You can take a cell that belongs to a patient and engineer it to be an attack cell.”

In one study, all five adults and 19 of 22 children with acute lymphocytic leukemia (ALL) showed complete remission of their cancer (i.e. no cancer could be found after treatment), although a few patients have relapsed since then.

These were gravely ill patients who were out of options. Some of them had tried multiple bone marrow transplants and up to 10 types of chemotherapy or other treatments. In the case of eight-year-old Emily Whitehead of Philipsburg, Pa., her cancer was so advanced that her doctors said Emily’s major organs would fail within days. Ms. Whitehead was the first child given the gene therapy and nearly two years later, shows no sign of cancer.

Physicians think that this has the potential to become the first gene therapy approved in the U.S. and the first for cancer worldwide. Only one gene therapy is approved in Europe, for a rare metabolic disease.

This gene therapy involves filtering patients’ blood to remove millions of T-cells, a type of white blood cell. These T-cells are then genetically engineered in the laboratory with a gene that targets cancer. These cells were subsequently returned to the patient in infusions, given over three days.

“What we are giving essentially is a living drug” – permanently altered cells that multiply in the body into an army to fight the cancer, says David Porter, a Univ. of Pennsylvania scientist who led one study.

Several drug and biotech companies are working hard to develop these therapies. The University of Pennsylvania has patented its method and licensed it to the Switzerland-based company Novartis AG. Novartis AG is building a research center on the Penn campus in Philadelphia and plans a clinical trial next year that could lead to federal approval of the treatment as soon as 2016. Hervé Hoppenot, president of Novartis Oncology, the division leading the work, said that “there is a sense of making history… a sense of doing something very unique.” Lee Greenberger, chief scientific officer of the Leukemia and Lymphoma Society, agrees: “From our vantage point, this looks like a major advance. We are seeing powerful responses… and time will tell how enduring these remissions turn out to be.”

This group has given $15 million to various researchers who are testing this strategy. Since there are nearly 49,000 new cases of leukemia, 70,000 cases of non-Hodgkin lymphoma and 22,000 cases of myeloma expected to be diagnosed in the U.S. in 2013, there are no shortage of potential subjects.

Many patients are successfully treated with chemotherapy or bone marrow or stem cell transplants, but transplants are risky and donors can’t always be found. Thus, gene therapy has been used as a fallback strategy for patients who were in danger of dying once all the other treatments failed.

The gene therapy must be made individually for each patient, since laboratory costs now are about $25,000, without a profit margin. That’s still less than many drugs to treat these diseases and far less than a transplant. The treatment can cause severe flu-like symptoms and other side effects, but these have been reversible and temporary, according to the physicians who administer the gene therapy treatment and observe the patients afterwards.

Penn doctors have treated 59 patients so far, which is the most of any center so far. Of the first 14 patients with B-cell chronic lymphocytic leukemia (CLL), four showed complete remissions, four showed partial remission, and the remaining patients did not respond to the treatment. However, some of the patients who showed partial remission continued to see their cancer shrink a year after treatment. “That’s very unique to this kind of therapy” and gives hope the treatment may still purge the cancer, says Porter.

Another 18 CLL patients were treated and half have responded so far. University of Pennsylvania doctors also treated 27 ALL patients. All five adults and 19 of the 22 children had complete remissions, which is an “extraordinarily high” success rate, according to Stephan Grupp at the Children’s Hospital of Philadelphia (CHOP). Six patients have, since then, suffered relapse of their cancer. The attending physicians are considering administering a second gene therapy treatment.

At the National Cancer Institute, James Kochenderfer and others have treated 11 patients with lymphoma and four with CLL, starting roughly two years ago. Six showed complete remission, six patients had partial remission, and one has stable disease but it is too soon to tell for the rest.

Ten other patients were given gene therapy to try to kill the leukemia or lymphoma cells that remained after bone marrow transplants. These patients received infusions of gene-treated blood cells from their transplant donors instead of using their own blood cells. One had a complete remission and three others had significant reduction of their disease.

“They’ve had every treatment known to man. To get any responses is really encouraging,” Kochenderfer says. The cancer institute is working with a Los Angeles biotech firm, Kite Pharma Inc., on its gene therapy approach.

Patients are encouraged that relatively few have relapsed.

“We’re still nervous every day because they can’t tell us what’s going to happen tomorrow,” says Tom Whitehead, eight-year-old Emily’s father.

Doug Olson, 67, a scientist for a medical device maker, shows no sign of cancer since his gene therapy in September 2010 for CLL he has had since 1996. “Within one month he was in complete remission. That was just completely unexpected,” says Porter, his doctor at Penn. Olson ran his first half-marathon in January and no longer worries about how long his remission will last. “I decided I’m cured. I’m not going to let that hang over my head anymore,” he says.

Decoding the Mechanisms Behind Stem Cell Reprogramming

Kenneth Zaret is the associate director of the Penn Institute for Regenerative Medicine and is also Professor of Cell and Developmental Biology at the University of Pennsylvania. Zaret’s laboratory has examined the process by which adult cells are reprogrammed to make induced pluripotent stem cells (IPSCs). Shinya Yamanaka won the Nobel Prize this year for the discovery of iPSCs, and iPSCs seem to offer many of the advantages of embryonic stem without the moral messiness of destroying human embryos to make them.

The production of iPSCs required genetic engineering techniques that introduce genes into cells. Introducing four genes – Oct4, Sox2, Klf4, and c-Myc – into adult cells drives them to de-differentiate at become iPSCs, but this process is very slow – it can take up to a month – and is quite inefficient – one in 1,000 cells becomes an iPSC. Also, even though iPSCs share many characteristics with embryonic stem cells, they are not exactly the same and differ in some ways.

Oct4 in Mammalian ESC Pluripotency

See this site for this figure.

This study, which was published in the journal Cell, attempted to define the reasons why iPSC production takes so long and is so inefficient. Zaret and his group examined the genomes of adult cells, 48 hours after the introduction of the four genes, and compared them to the genomes of the starting adult cells, fully reprogrammed iPSCs, cells near the end of the re-programming process (pre-iPSCs), and embryonic stem cells.

At 48 hours, Zaret and others found that of the transcription factors that had been introduced and overexpressed in the adult cells, three of them, Oct4, Klf4, and Sox2, tended to bind to the “distal enhancer elements” of genes. The phrase “distal enhancer elements” refers to regions of genes that help control when the gene is turned on. Most genes consist of a sequence of DNA known as the “coding region” that contains the sequences that are used to make the messenger RNA that will be translated into protein. However, genes also have other sequences that tell the cell when and where to make the messenger RNA. These control sequences are called “enhancers.” The Oct4, Klf4, and Sox2 proteins bind to enhancers in target genes and influence their expression. Because these proteins do this early heavy lifting, Zaret called them “pioneer factors.”

Now, there is a problem. The DNA of the cell is not an open book, but is bound up into a compact structure called chromatin. DNA tightly wound into chromatin does not allow access to proteins. Therefore, the pioneer factors for cell reprogramming are ready to bind to enhancers, but the DNA of the genome is too tightly wound to permit their binding. What is the cell to do? This is the job of the c-Myc protein, which enhances the binding of the other pluripotency factors to chromatin.


There is another problem, however, and this is the genuinely remarkable finding of Zaret’s lab: 48 hours after the initiation of reprogramming, large sections of the genome of the cell are refractory to the binding of the pioneering factors. In Zaret’s own words: “Basically, large chunks of the human genome were physically resisting these factors from entering. That provided some understanding that you’ve got to overcome the binding requirement to get these factors to their final destination.”

What caused these chunks of the genome to be off-limits to the pioneer factors? Chromatin results from the assembly of DNA with very positively-charged proteins called histones. Histones act as miniature spools around which the DNA is wound and packaged. Chemical modification of the histones can influence the tightness of the chromatin. For example, the attachment of acetate groups tends to make chromating rather loose, and gene expression can readily occur, but the attachment of methyl (CH3-) groups tends to cinch the chromatin down so tightly that little gene expression can occur.

Histone modification

In the case of the off-limits portions of the genome in adult cells undergoing reprogramming, a histone modification called “H3K9me3,” which is a short hand for saying lysine residue number 9 on histone #3 had three methyl groups attached to it, blocks the pioneer factors from accessing the DNA under its structural compaction. However, if Zaret and his workers treated cells with an inhibitor that prevents the enzymes from modifying histones in this manner, they found that the reprogramming process was significantly accelerated.

Zaret thinks that these findings might not only tell us about the roadblocks to reprogramming, but also give us clues as to a way to work around the difficulties to reprogramming. His lab has uncovered a normal mechanism by which cells protect themselves from being reprogrammed under normal circumstances. In his own words: “We went into this thinking that we were going to learn something about the mechanism of conversion to pluripotency, but at the end of the day we ended up discovering new ways that cells control gene expression by shutting down parts of their genome.”

The importance of this work is difficult to overstate. In the words of Susan Haynes, from the National Institutes of Health General Medical Sciences division, which funded Zaret’s work, “These studies provide detailed insights into how reprogramming factors interact with the chromatin of differentiated cells and start them down the path toward becoming stem cells. Dr. Zaret’s work also identified a major structural roadblock in the chromatin that the factors must overcome in order to bind DNA. This knowledge will help improve the efficiency of reprogramming, which is important for any future therapeutic applications.”