In a study led by Martin Fussenegger of ETH Zurich, stem cells extracted from the fat of a 50-year-old test subject were transformed into mature, insulin-secreting pancreatic beta cells.
Fussenegger and his colleagues isolated stem cells from the fat of a 50-year-old man and used these cells to make induced pluripotent stem cells (iPSCs). These iPSCs were then differentiated into pancreatic progenitor cells and then into insulin-secreting beta cells but means of a “genetic software” approach.
Genetic software refers to the complex synthetic network of genes required to differentiate pancreatic progenitor cells into insulin-secreting beta cells. In particular, three genes, all of which expression transcription factors, Ngn3, Pdx1, and MafA, are particularly crucial for beta cell differentiation.
Fussenegger and his team designed a a protocol that would express within these fat-based stem cells the precise concentration and combination of these transcription factors. This feature is quite important because the concentration of these factors changes during the differentiation process. For example, MafA is not present at the start of beta cells maturation, but appears on day four on the final data of maturation when its concentration rises precipitously. The concentration of Ngn3 rises and then falls and the levels of Pdx1 rise at the beginning and towards the end of maturation.
The Zurich team used ingenious genetic tools to reproduce these vicissitudes of gene expression as precisely as possible. By doing so, they were able to differentiate the iPSC-derived pancreatic progenitor cells into insulin-secreting beta cells.
The fact that Fussenegger’s team was able to use a synthetic gene network to form mature beta cells from adult stem cells is a genuine breakthrough. The genetic network approach also seems to work better than the traditional technique of adding various chemicals and growth factors to cultures cells. “It’s not only really hard to add just the right quantities of these components (growth factors) at just the right time, it’s also inefficient and impossible to scale up,” said Fussenegger.
This new process can successfully transform three out of four fat stem cells into beta cells. Also the beta cells made with this method have the same microscopic appearance of natural beta cells in that they contain internal granules full of insulin. They also secrete insulin in response to increased blood glucose concentrations. Unfortunately the amount of insulin made by these cells is lower than that made by natural beta cells.
Pancreatic islet transplants have been performed in diabetic patients, but such transplantations also require treatment with potent antirejection drugs that have potent side effects.
“With our beta cells, there would likely be no need for this action (administering antitransplantation drugs), since we can make them using endogenous cell material taken from the patient’s own body. This is why our work is of such interest in the treatment of diabetes,” said Fussenegger.
Fussenegger and his group have made these beta cells in the laboratory, but they have yet to transplant them into a diabetic patient. However, the success of this synthetic genetic software technology might also be useful in the reprogramming of adult cells into other types of cells that are useful for therapeutic purposes.
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.
Embryonic stem cell lines are made from four-five-day-old human embryos. At this stage of development, the embryo is a sphere of cells with two distinct cell populations; an outside layer of flat trophoblast cells and an inner clump of round inner cell mass (ICM) cells. The embryo consists of ~100 cells four days after fertilization, and ~150 cells five days after fertilization.
Embryonic stem cell (ESC) derivation involves the removal of the trophoblast cells (which are collectively called the trophectoderm) and the isolation of the ICM cells. There are several ways to remove the trophectoderm, but the most commonly-used technique is “immunosurgery,” which uses antibodies that bind to proteins on the surfaces of the trophectoderm, and serum to initiate destruction of the trophoblast cells. The isolated ICM cells are then cultured, and if they grow, they may produce an embryonic stem cell line.
Immunosurgery was first perfected by Davor Solter and Barbara B. Knowles on mouse embryos. They used an antiserum that was raised in rabbits when the rabbits were immunized against mouse spleen tissue. When mouse embryos were incubated with this antiserum plus serum from mice, all the cells of the mouse embryo died. However, if they used the rabbit antiserum and serum from guinea pig, then only the trophoblast cells were destroyed. For human embryonic stem cell derivation, the rabbits are immunized again human red blood cells, and this rabbit antiserum is used with guinea pig serum. The serum contains proteins called “complement,” which bind to cells that have antibodies attached to them and bore holes in those cells, thus destroying them.
When ICM cells are cultured, they are placed on a layer of mouse cells that have been treated with a chemical called mitomycin C to prevent them from dividing. These non-dividing cells act as “feeder cells” that keep the ICM cells from differentiating. Because ICM cells are grown on animal cells, they cannot be used for clinical purposes, since they will possess animal proteins can carbohydrates on their surfaces, which would be attacked by the patient’s immune system. However, several ESC lines have been derived without animal products, and it is possible to make ESC lines that would be safe or human use.
ESC derivation results in the destruction of human embryos. There is not two ways about it. Even though there are potential ways around this problem, the majority of ESC lines were made literally over the dead bodies of very young human beings. All the rationalization in the world (the embryo is too young, too small, too inchoate, too unwanted, going to die anyway, in the wrong place at the wrong time) do not undo the fact that the embryo is a very young human being, and making an ESC line from it ends his/her life.
Getting the ESC line to differentiate in what you want it to be is another problem. If any undifferentiated cells remain after differentiation, they can cause tumors. Therefore, there is a need to ensure that differentiation is efficient and complete. To this end, Doug Melton’s lab at Harvard University has published a remarkable paper in the journal Nature that uses mesenchymal stem cells from particular organs to direct the differentiation of ESC lines.
Melton’s lab, in particular Julie M. Sneddon and Malgorzata Borowiak (say that fast five times), established 16 lines of tissue-specific mesenchymal stem cells (MSCs) from embryonic, neonatal and adult mouse intestine, liver, spleen, and pancreas and human pancreas too. Then they cultured mouse ESCs on these MSC lines to determine if they could drive the ESCs to differentiate into pancreas cells. In the embryo, pancreatic precursors express several genes in a nested, hierarchical fashion. First, they express Sox17, which is a common endodermal marker, and then pancreatic progenitors all express Pdx1. Of these pancreatic progenitors, some express Ngn3 and these will become endocrine rather than exocrine cells, and othe the Ngn3-expressing cells, a few will become beta cells that make insulin.
Melton and his co-workers tried to determine if any of these genes was up-regulated in their ESC lines if that were co-cultured with their established MSC lines. They discovered that four lines – MSC1, 2, 3, & 4, all affected gene expression when co-cultured with ESCs. MSC 1 and 2 induced and increase in Sox17 expression and MSC 3 and 4 increased the expression of Ngn3 in ESCs.
These changes in gene expression were due to increased cell proliferation of cells actually expressing these genes and not due to differential survival. Also, no combination of growth factors could achieve the same results as the accompanying MSC lines. Thus there is more going on here than the MSCs just secreting the right growth factors. The MSCs must be making contact with the ESCs and inducing them to differentiate into a particular cell type.
Next Melton and his colleagues determined if this interaction with MSCs caused the ESCs to lose their ability to self-renew. The answer was a clear “no.” Even though these ESC lines were expressing genes characteristic of endodermal or pancreatic tissue, they did not lose their ability to differentiate into pancreatic tissue when appropriately induced to do so, and they also id not lose their ability to self-renew and grow competently in culture.
In a more stringent test, these ESCs that had been grown on tissue-specific MSCs were implanted into mice. As Melton points out in the paper, the “most efficient published protocols for in vitro differentiation of pluripotent cells to beta-cells yield only a small percentage (typically 0-15%) of insulin-positive cells, and these do not secrete insulin in a glucose-responsive manner.” Could the MSC-conditioned ESCs do any better?
Before implantation, the ESCs were differentiated into endodermal progenitors (Sox17-expressing cells), and co-cultured with MSCs for at least 3-7 passages. Then they were differentiated into beta cells and transplanted into mice. There were a few important controls that were used; Just saline, implantations of MSCs alone, and ESCs that had been differentiated into beta cells, but had never been passaged on MSCs. Finally, human pancreatic islets were used as a positive control.
The results were interesting to say the least. The saline and MSC alone implantations showed no insulin production with or without glucose. Likewise the human pancreatic islets made insulin in a glucose-dependent manner (no surprise there). The ESC-derived beta cells that had never been passaged on MSCs made insulin, and even showed some ability to respond to glucose and make more insulin after glucose ingestion. However, the beta cells derived from ESCs that had been passaged on MSCs made insulin in a glucose-dependent manner. The experiment produced a wide range of variability since the number of transplanted cells differed between each trial, but the implanted beta cells derived from ESCs passaged on tissue-specific MSCs definitely performed the best, and even did as well or better than the implanted human beta cells in some cases.
Melton notes at the end of his paper that this technique worked rather well for coaxing ESCs to form pancreatic derivatives, but it could very well be applicable to other systems as well. Also, it could probably work with induced pluripotent stem cells, which have many (though not all) of the characteristics of ESCs and can be made without killing human embryos. Thus another technique for increasing ESC differentiation seems to be on the table.