Using Patient Stem Cells to Make “Heart-Disease-on-a-Chip”


Personalized medicine is a new but quickly advancing innovation in medicine that tailors the diagnosis and treatment of a particular patient according to their specific genetic and physiological idiosyncrasies. As an example, if a patient has high blood pressure, which treatment would work the best? If the patient is an African-American, it is unlikely that a group of drugs called ACE inhibitor or another group called ARBs would work terribly well because African-American patients tend to lack sufficient quantities of the targets of these two type of drugs for them to work properly. Therefore, diuretics or beta blockers are better drugs to lower the blood pressure of such patients.

Other examples include the enzymes that chemically modify drugs are they circulate throughout our bodies. Some patients have excessive amounts of a liver enzyme called CYP2D6, and this enzyme modifies the painkiller codeine.  Codeine, you see, is not given in an active form.  It only becomes active after the liver enzyme CYP2D6 modifies it.  People with large amounts of CYP2D6, which includes about 10% of Arabs, over-activate codeine, which causes side effects like profound sedation and stomach cramps (codeine or hydrocodone is a form of morphine).  Therefore, before the patient is prescribed codeine, which is present in several different types of prescription painkillers (e.g., Norco, Lortab, Tusnel-HC, Canges-HC, Drocon-CS, Excof-SF, TriVent-HC, etc.), it would be immensely useful to know if your patient had this condition in order to cut their codeine dose or prescribe an altogether different pain-killer.

Now that you have, hopefully been convinced that personalized medicine can potentially save lives, I hope to tell you about a new advance that brings stem cells into the personalized medicine arena.  Kevin Kit Parker and William Pu have used stem cell and “organ-on-a-chip” technologies to grow functioning heart tissue that carries an inherited cardiovascular disease.  This research appears to be a big step forward for personalized medicine.

Parker and Pu modeled a cardiovascular disease called Barth Syndrome, which is caused by mutations on a gene that resides on the X chromosome called TAZ, which encodes the Tafazzin protein.  Barth Syndrome is also affects heart and skeletal muscle function.  Skin biopsies were taken from two male patients who suffer from Barth Syndrome.  These cells were de-differentiated into induced pluripotent stem cells (iPSCs) that were further differentiated into heart muscle cells.  To differentiate the iPSCs into heart muscle cells , the cells were grown on small slides known as chips lined with human extracellular matrix proteins that mimicked the environment of the human heart.  The cells were tricked into thinking that they were in a heart and they differentiated into heart tissue.  Not surprisingly, the heart tissue made on a chip contracted very weakly compared to normal heart tissue.

To confirm that they were not barking up the wrong tree, Parker and Pu used normal cells that had been genetically engineered to possess mutations in the TAZ gene.  When these engineered cells were used to make heart tissue on a chip, they too contracted very weakly.  This told Parker and Pu that they were definitely on the right track.

“You don’t relay understand the meaning of a single cell’s genetic mutation until you build a huge function,” said Parker, who has spent over a decade working on “organs-on-a-chip” technology.  “In the case of the cells grown out of patients with Barth Syndrome, we saw much weaker contractions and irregular tissue assembly.  Being able to model the disease from a single cell all the way up to heart tissue, I think that’s a big advance.”.

The TAZ mutation disrupts the activity of the powerhouse of the cell, a small structure called the mitochondrion.  Even though the TAZ mutation did not affect the over all energy supply of the cells, it seems to affect the way the heart muscle constructs itself so that it can properly contract.

Since mitochondria use a process known as oxidative phosphorylation to make the lion share of their chemical energy in the form of the molecule ATP (adenosine triphosphate), mitochondria also generate toxic byproducts called reactive oxygen species or ROS.  Cells have mechanisms to squelch ROS, but these mechanisms can be overwhelmed if cells make excessive quantities of ROS.  Heart muscle that contains the TAZ mutation seems to make excessive quantities of ROS, and this affects the integrity of the heart muscle.

Can drugs that quench ROS be used to retreat patients with Barth Syndrome?  It is difficult to say, but this chip-on-a-dish is surely an excellent model system to determine if such an approach can work.  Already, Pu and his colleagues are testing drugs to treat this disorder by testing those drugs on heart tissue grown on chips.

“We tried to thread multiple needles at once and it certainly paid off,” said Parker.  “I feel that the technology that we’ve got arms industry and university-based researchers with the tools they need to go after this disease.”.

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.