New York Stem Cell Foundation Invents Robotic Platform for Making Induced Pluripotent Stem Cells


Induced pluripotent stem cells (iPSCs) are made from mature adult cells through a combination of genetic engineering and cell culture techniques. Because they are made from cells isolated from specific patients, they are patient-specific cells that can be used for drug testing, model experimental systems, and potentially cells for regenerative therapies.

Induced-pluripotent-stem-cell-picture.jpg

Unfortunately, iPSCs are made in different laboratories that use different reagents and different protocols and with workers with different skill levels. Consequently, laboratory-made iPSC lines show a very wide range of variation that are not due to genetic differences in the cells from which they were made. Additionally, the production of iPSCs is labor intensive and expensive and there is a deep need to standardize the whole process. What are stem-cell scientists to do?

New York Stem Cell Foundation (NYSCF) has announced the development of a robot-driven apparatus that automates and completely standardizes the production of iPSCs. This modular, robotic platform for iPSC reprogramming enables automated, high-throughput conversion of cells isolated from skin biopsies into iPSCs and differentiated cells derived from them with minimal manual intervention. In a paper in the journal Nature Methods, NYSCF scientists in collaboration with bioengineers demonstrates that automated reprogramming of mature cells with this robotic platform (that uses pooled selection of pluripotent cells) results in high-quality, stable iPSC lines. These lines show less line-to-line variation than either manually produced iPSC lines or iPSC lines produced through automation followed by single-colony subcloning.

“The capacity to test drugs on thousands of patients in a dish will change how we cure disease. We
will be more informed about how drug candidates will behave in patients before the clinical trial phase accelerating the discovery process. This technology will enable us to bring precision medicine
treatments and personalized pharmaceuticals to more patients,”noted Dr. Thomas Singer, Senior
Vice President, F. Hoffmann-La Roche Ltd, Pharmaceuticals Division.
“This technology may help us predict how drug candidates behave in patients before the most complex and expensive phase of drug development: clinical trials. This insight could speed up new biomedical R&D and open the door to a larger number of high impact precision therapies,”said Freda Lewis-Hall, M.D., DFAPA, Chief Medical Officer and Executive Vice President, Pfizer Inc.
“Our goal is to understand and treat diseases. This is not an artisanal pursuit. Researchers need to
look at genetically diverse populations at scale, which means creating large numbers of standardized, human pluripotent stem cells. The NYSCF Global Stem Cell Array’s massive parallel processing
capabilities make this research possible,” said NYSCF Research Institute CEO and Founder Susan
L. Solomon, an author of the paper.

This robotic platform can potentially enable the application of iPSCs to population-scale biomedical problems including the study of complex genetic diseases and the development of personalized clinical treatments.

Human Stem Cells Elucidate the Mechanisms of Beta-Cell Failure in Diabetes


Wolfram syndrome is a rare form of diabetes characterized by high blood sugar levels that result from insufficient levels of the hormone insulin.  The chronically high blood sugar levels cause degeneration of the optic nerve, leading to progressive vision loss (optic atrophy).  Wolfram syndrome patients often also have abnormal pituitary glands that release abnormally low levels of the hormone vasopressin (also known as antidiuretic hormone or ADH), which causes hearing loss caused by changes in the inner ear (sensorineural deafness), urinary tract problems, reduced amounts of the sex hormone testosterone in males (hypogonadism), or neurological or psychiatric disorders.

Diabetes mellitus is typically the first symptom of Wolfram syndrome, usually diagnosed around age 6. Nearly everyone with Wolfram syndrome who develops diabetes mellitus requires insulin replacement therapy. Optic atrophy is often the next symptom to appear, usually around age 11. The first signs of optic atrophy are loss of color vision and peripheral (side) vision. Over time, the vision problems get worse, and people with optic atrophy are usually blind within approximately 8 years after signs of optic atrophy first begin.

Mutations in the WFS1 gene cause more than 90 percent of the cases of Wolfram syndrome type 1.  The WFS1 gene encodes a protein called wolframin that regulates the amount of calcium in cells.  A proper calcium balance is important for a whole host of cellular processes, including cell-to-cell communication, the tensing (contraction) of muscles, and protein processing.  Wolframin protein is found in many different tissues, such as the pancreas, brain, heart, bones, muscles, lung, liver, and kidneys.  Inside cells, wolframin is in the membrane of a cell structure called the endoplasmic reticulum that is involved in protein production, processing, and transport. Wolframin is particularly important in the pancreas, where it helps process proinsulin into mature hormone insulin, the hormone that helps control blood sugar levels.

WFS1 gene mutations lead to the production of a sub-functional versions of wolframin.  As a result, calcium levels within cells are not properly regulated and the endoplasmic reticulum does not work correctly.  When the endoplasmic reticulum does not have enough functional wolframin, the cell triggers its own cell death (apoptosis).  In the pancreas, the cells that make insulin (beta cells) die off, which causes diabetes mellitus.  The gradual loss of cells along the optic nerve eventually leads to blindness, and the death of cells in other body systems likely causes the various signs and symptoms of Wolfram syndrome type 1.

A certain mutation in the CISD2 gene also causes Wolfram syndrome type 2. The CISD2 gene provides instructions for making a protein that is in the outer membrane of cell structures called mitochondria,the energy-producing centers of cells.  Even though the function of the CISD2 protein is unknown, CISD2 mutations produce nonfunctional CISD2 protein that causes mitochondria to eventually break down. This accelerates the onset of cell death.  Cells with high energy demands such as nerve cells in the brain, eye, or gastrointestinal tract are most susceptible to cell death due to reduced energy, and people with mutations in the CISD2 gene have ulcers and bleeding problems in addition to the usual Wolfram syndrome features.

Some people with Wolfram syndrome do not have an identified mutation in either the WFS1 or CISD2 gene. The cause of the condition in these individuals is unknown.

Now that you have a proper introduction to Wolfram syndrome, scientists from the New York Stem Cell Foundation and Columbia University Medical Center have produce induced pluripotent stem cells (iPSCs) from skin samples provided by Wolfram syndrome patients.  All of the patients who volunteered for this study were recruited from the Naomi Berrie Diabetes Center and had childhood onset diabetes and required treatment with injected insulin, and all had vision loss.  Control cell lines that did not have mutations in WFS1 were obtained from Coriell Research for Medical Research.

These skin samples contained cells known as fibroblasts and these were reprogrammed into induced pluripotent stem cells.  In order to show that these cells were truly iPSCs, this group implanted them underneath the kidney capsule of immuno-compromised mice, and they formed the teratoma tumors so characteristic of these cells.

When these iPSCs were differentiated into insulin-secreting pancreatic beta cells, Linshan Shang and her colleagues discovered that the beta cells made from cells that did not come from Wolfram syndrome patients secreted normal levels of insulin.  However, those beta cells made from iPSCs derived from Wolfram patients failed to secrete normal quantities of insulin either in culture or when transplanted into the bodies of laboratory animals.  Further investigations of these cells showed these beta cells showed elevated levels of stress in the endoplasmic reticulum as a result of an accumulation of unfolded proteins.

What on earth is endoplasmic reticulum protein-folding stress?  First some cell biology.  When the cell needs to make a protein that will be secreted, embedded in a membrane or vesicle. that protein begins its life on ribosomes (protein synthesis factories of the cell) in the cytoplasm, but later those ribosomes are dragged to a cellular structure called the endoplasmic reticulum.  While on the surface of the endoplasmic reticulum, the ribosome completes the synthesis of the protein and extrudes the protein into the interior of the endoplasmic reticulum or embeds the protein into the endoplasmic reticulum membrane.  From there, the protein is trafficked in a vesicle to another subcellular structure called the Golgi apparatus, were it undergoes further modification, and from the Golgi apparatus, the protein goes to the membrane, secretory vesicle or other places.

If the proteins in the endoplasmic reticulum cannot fold properly, they clump and build up inside the endoplasmic reticulum, and this induces the ERAD or Endoplasmic Reticulum-Associated Protein Degradation response.  The players in the ERAD response are shown below.  As you can see, this response is rather complicated, but if it fails to properly clear the morass of unfolded proteins in the endoplasmic reticulum, then the cell will undergo programmed cell death.

ERAD Response

However, this research team did not stop there.  When they treated the cultured beta cells made from cells taken from Wolfram syndrome patients with a chemical called 4-phenyl butyric acid, the stress on the cells was relieved and the cells survived.  This experiment shows that relieving this unfolded protein stress is a potential target for clinical intervention.

“These cells represent an important mechanism that causes beta-cell failure in diabetes.  This human iPS cell model represents a significant step forward in enabling the study of this debilitating disease and the development of new treatments,” said Dieter Egli, the principal investigator of the study, and senior research fellow at the New York Stem Cell Foundation.

Because all forms of diabetes mellitus ultimately result from an inability of the pancreatic beta cells to provide sufficient quantities of insulin in response to a rise in blood sugar concentrations, this Wolfram patient stem cell model enables an analysis of a more specific pathway that leads to beta-cell failure in more prevalent forms of diabetes.  Furthermore, this strategy enables the testing of strategies to restore beta-cell function that may be applicable to all types of diabetes.

Susan L. Solomon of the New York Stem Cell Foundation, said, “Using stem cell technology, we were able to study a devastating condition to better understand what causes the diabetes syndromes as well as discover possible new drug targets.”

Rudolph L. Leibel, a professor of diabetes research and co-author of this study, said, “This report highlights again the utility of close examination of rare disorders as a path to elucidating more common ones.  Our ability to create functional insulin-producing cells using stem cell techniques on skin cells from patients with Wolfram’s syndrome has helped to uncover the role of ER stress in the pathogenesis of diabetes.  The use of drugs that reduce such stress may prove useful in the prevention and treatment of diabetes.”

The ERAD response seems to play a role in the survival of insulin-producing beta cells in both type 1 and type 2 diabetes.  The ERAD response opposes the stress of the immune assault in type 1 diabetes and the metabolic stress of high blood glucose levels in both types of diabetes.  When the ERAD response fails, cell death ensues and this reduces the number of insulin-producing cells.

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.

A New Automated Protocol to Prepare and Purify Induced Pluripotent Stem Cell Lines


Induced pluripotent stem cells (iPSCs) come from adult cells and not embryos. By genetically engineering adult cells to express a cadre of genes that are normally found in early embryonic cells, scientists can de-differentiate the adult cells into cells that resemble embryonic stem cells in many (although not all) ways.

Generating iPSCs from human adult cells is tedious and not terribly efficient, but there are ways to increase the efficiency of iPSC generation (see here). Additionally, iPSCs can show a substantial tendency to form tumors, but this tendency is cell line-specific (see here and here). Furthermore, there are ways to screen iPSC lines for tumorgenicity.

Because iPSCs are directly from the patient’s cells, the chances of rejection by the immune system are less likely (see here). Therefore, many stem cells scientists believe that iPSCs may represent one of the best future possibilities for regenerative medicine. However, a hurdle in iPSC development is the ability to generate and evaluation iPSC lines in a rapid, but reliable manner. Once adult cells are induced to become iPSCs, the iPSC cultures are a mixed bag of iPSCs, undifferentiated adult cells that failed to make the transition to iPSCs, and partially reprogrammed cells. Selecting the iPSCs by merely eye-balling the cells through the microscope is tricky and fraught with errors. If the scientist wants to select iPSCs for toxicity studies and not partially differentiated cells, selecting the wrong cells for the experiment can be fatal to the experiment itself.

Scientists from the New York Stem Cell Foundation (NYSCF) Research Institute have developed a protocol for iPSC generation and evaluation is automated and efficient, and may bring us closer to the goal of using iPSCs in the clinic some day. This protocol is the culmination of three and a half years of work. This protocol uses a technology called “fluorescence activated cell sorting” or FACS to identify fully reprogrammed cells. FACS sorts the cells according to their expression of two specific cell surface molecules and the absence of another cell surface molecule. This negative selection for a cell surface molecule found in partially reprogrammed cells but not iPSCs is a very powerful technique for purifying iPSCs.

David Kahler, the NYSCF director of laboratory automation, said, “To date, this protocol has enabled our group to derive (and characterize over) 228 individual iPS cell lines, representing one of the largest collections derived in a single lab.” Kahler continued: “This standardized method means that these iPS cells can be compared to one another, an essential step for the use in drug screens and the development of cell therapies.”

This particular cell selection technique provides the basis for a new technology developed by NYSCF, the Global Stem Cell Array, which is a fully automated, robotic platform to generate cell lines in parallel.

Underway at the NYSCF Laboratory, the Array reprograms thousands of adult cells from kin and blood samples taken from healthy donors and diseased patients into iPSC lines. Sorting and characterizing cells at an early stage of reprogramming allows efficient development of iPSC clones and derivation of adult cell types.

“We are excited about the promise this protocol holds to the field. As stem cells move towards the clinic, Kahler’s work is a critical step to ensure safe, effective treatments for everyone.” said Susan L. Solomon, who is the Chief Executive Officer of NYSCF.