Stem Cells Analyze the Cause and Treatment of Diabetes


A research group that is part of the New York Stem Cell Foundation or NYSCF has generated patient-specific beta cells (the cells that secrete insulin in the pancreas), and these cultured beta cells accurately recapitulate the features of maturity-onset diabetes of the young or MODY.

In this research, NYSCF scientists and researchers from the Naomi Berrie Diabetes Center of Columbia University used skin cells from MODY patients to produce induced pluripotent stem cells (iPSCs) that were differentiated in the culture dish to form insulin-secreting pancreatic beta cells (if that sounds like a lot of work that’s because it is).

Other laboratories have succeeded in generating beta cells from embryonic stem cells and iPSCs, but questions remain as to whether or not these cells accurately recapitulate genetically-acquired forms of diabetes mellitus.

Senior co-author of this study, Dieter Egli, a senior research fellow at NYSCF, said: “We focused on MODY, a form of diabetes that affects approximately one in 10,000 people. While patients and other models have yielded important clinical insights into this disease, we were particularly interested in its molecular aspects – how specific genes can affect responses to glucose by the beta cell.”

MODY is a genetically inherited form of diabetes mellitus, and the most common form of MODY, type 2, results from mutations in the glucokinase or GCK gene. Glucokinase is a liver-specific enzyme and it adds a phosphate group to sugar so that the sugar can be broken down to energy by means of a series of reactions known as “glycolysis.” Glucokinase catalyzes the first step of glycolysis in the liver and in pancreatic beta cells. Mutations in GCK increase the sugar concentration in order for GCK to properly metabolize sugar, and this increases blood sugar levels and increases the risk for vascular complications.

The steps of the enzymatic pathway glycolysis, which is used by cells to degrade sugar to energy.
The steps of the enzymatic pathway glycolysis, which is used by cells to degrade sugar to energy.

MODY patients are usually misdiagnosed as type 1 or type 2 diabetics, but proper diagnosis can greatly alter the treatment of this disease. Correctly diagnosing MODY can also alert family members that they too might be carriers or even susceptible to this disease.

NYSCF researchers worked with skin cells from two patients from the Berrie Center who had type 2 MODY. After reprogramming these skin cells to become iPSCs, they differentiated the cells into beta cells, These cells had the impaired GCK activity, but in order to compare them to something, the NYSCF group also made iPSCs with a genetically engineered version of GCK that was impaired in the same way as the GCK gene in these two patients, and another cell line with normal versions of the GCK gene. They used these iPSCs to make cultured beta cells.

“Our ability to create insulin-producing cells from skin cells, and then to manipulate the GCK gene in these cells using recently developed molecular methods, made it possible to definitely test several critical aspects of the utility of stem cells for the study of human disease,” said Haiqing Hua, lead author of this paper and a postdoctoral fellow in the Division of Molecular Genetics.

The beta cells made from these iPSCs were transplanted into mice and these mice were given an oral glucose tolerance test. An oral glucose tolerance test is used to diagnose diabetes mellitus. The patient fasts for 12 hours and then is given a concentrated glucose concentration (4 grams per kilogram body weight), which the patient drinks and then the blood glucose level is examined at 30-minute intervals. The blood glucose levels of diabetic patients will rise and only go down very sluggishly whereas the blood glucose levels of a nondiabetic patient will rise and then decrease as their pancreatic beta cells start to make insulin. Insulin signals cells to take up glucose and utilize it, which lowers the blood glucose levels. A reading of over 200 milligrams per deciliters

When mice with the transplanted beta cells made from iPSCs were given oral glucose tolerance tests, the beta cells from MODY patients   showed decreased sensitivity to glucose.  In other words, even in the presence of high blood sugar levels, the beta cells made from iPSCs that came from MODY patients secreted little insulin.  However, high levels of amino acids, which are the precursors of proteins, also induces insulin secretion, and in this case, beta cells from MODY patients secreted sufficient quantities of insulin.

When the iPSCs made from cells taken from MODY patients were subjected to genetic engineering techniques that repaired the defect in the GCK gene, these iPSCs differentiated into beta cells that responded normally to high blood glucose levels and secreted insulin when the blood glucose levels rose.

By making beta cells from MODY patients and then correcting the genetic defect in them and returning them to normal glucose sensitivity, NYSCF scientists showed that this type of diagnosis could lead to cures for MODY patients.

“These studies provide a critical proof-of-principle that genetic characteristics of patient-specific insulin-producing cells can be recapitulated through use of stem cell techniques and advanced molecular biological manipulation.  This opens up strategies for the development of new approaches to the understanding, treatment, and, ultimately, prevention of more common types of diabetes,” said Rudolph Leibel of the Columbia University Medical Center.

Plastic Bags Coated With Plasma For Culturing Stem Cells


Clinical products that are used to treat patients must be manufactured under a set of standards known as “Good Manufacturing Practice” or GMP. Drugs, catheters, stents, implants, pacemakers and so on must all be manufactured in a facility that strictly adheres to GMP standards and produces products that are consistently safe for patient use. Products made to GMP standards are free of contaminating microorganisms, free of molecules that cause robust rejection by the immune system, and known to be safe for use in a human patient.

Producing stem cells for regenerative medicine represent a tough case for several reasons. Most of the laboratory products sold off the shelf for tissue culture have some animal products in them, which disqualifies them for clinical use, since culture media with animal products can contain animal viruses or animal antigens that will cause patient’s immune systems to reject them. Growing stem cells under animal-free conditions is tedious, expensive, and the results are not always consistently reproducible. While some laboratories have made remarkable strides in growing cells under animal-free conditions, doing so in a manner that meets GMP standards is even more exacting.

New work by Kristina Lachman and Michael Thomas and their colleagues from the Fraunhofer Institute for Surface Engineering and Thin Films in Braunschweig, Germany, has shown that plastic bags coated with a plasma can provide excellent vessels for stem cell culture and can be manufactured in a manner that meets GMP standards.

The term “plasma” in physics refers to the state of a gas when a strong enough electric current is passed through it so that the gas mainly consists of ions. In such a state, the gas is no longer a gas and has properties unlike a solid, liquid or gas, but is considered a distinct state of matter. When a plasma is used to coat the inside surface of a plastic bag, it modifies the surface of the internal surface of the bag so that different types of cells can grow on it. The plasma also acts as a disinfectant while it transforms the surface of the bag so that cells are able to grow on it and even want to grow on it.

plasma_image

“Our goal was to realize a closed system in which cells grow undisturbed and without the risk of contamination. Coating the bags with plasma enables us to use them as a GMP laboratory,” said Henk Garritsen from Braunschweig Municipal Hospital.

To date, stem cells cultured from the patient’s own body have been grown in plastic culture dishes, spinner bottles, and bioreactors. However, systems like these, though initially sterile, must be opened in order to refill the culture medium or extract the cells. Every time the culture is opened there is a risk of contamination and the cells are rendered unusable.

Enter Werner Lindenmaier and Kurt Dittmar from the Helmholtz Center for Infection and Research who were already working on bags for stem cell cultivation. By collaborating with the experts at Fraunhofer who knew how to coat plastics with plasma, these scientists embarked on a very fruitful venture that culminated in experiments that showed that stem cells could robustly grow on plasma-coated films. Then a joint venture sponsored by the German Federal Ministry of Economics and Technology investigated the feasibility of plastic bags coated with plasma as a closed system for stem cells cultivation and growth.

To coat the bags with plasma, they are first filled with a non-reactive gas and then hit with low-voltage electrical currents. This generates a plasma in the bag and this plasma is a “luminous, ionized gas that chemically alters and at the same time disinfects the surface of the plastic,” said Lachmann.

The bags were coated in a pilot plant at Fraunhofer IST and then tested at the Helmholtz Center and the Braunschweig Municipal Hospital to test the diverse types of coatings used on the bags for their ability to support the growth of stem cells. Dittmat noted that, “We work with stem cells for bones, cartilage, fat, and nerves – the coating can be optimized for each of these cell types.”

The pilot plant at Fraunhofer IST has designed an automated system for making these stem cell culture bags. This automated system can make bags that are wholly reproducible in their composition and properties.

“We use medically approved bags for the coating,” said Thomas. “Nevertheless, the plasma treatment must be demonstrated to be innocuous before being approved for clinical use.”