Large Screening and Analyses of Established Induced Pluripotent Stem Cell Lines Finds Rogue Lines


Induced pluripotent stem cells (iPSCs) have come a long way since the first lines were made by Shinya Yamanaka and his colleagues in 2006. Initial successes of iPSCs in animal models generated a good deal of hope that iPSCs might find a place in the annals of regenerative medicine. However, since that time, further work has created doubts about the safety of these cells, since some, though admittedly not all, iPSC lines show some genetic abnormalities. However, as screening techniques have become better and have increased in sensitivity, the possibility of accurately ascertaining the quality of iPSC lines draws closer and closer.

A new paper that appeared in the June 9 edition of the journal Stem Cell Reports by Carolyn Lutzko and others from a multi-institutional research group known as the Progenitor Cell Biology Consortium, have used these new screening technologies to screen large numbers of established iPSC lines. The results were somewhat sobering; about 30 percent of iPSC lines analyzed from 10 research institutions were genetically unstable and not safe for clinical use.

This work comprehensively characterized of a large collection of iPSC lines. The technology to produce safe and effective iPSCs exists. Nevertheless, this does not mean that all iPSC lines were produced safely and effectively. In this paper, Lutzko and her colleagues discovered that some iPSC lines that were made with inferior protocols. Some iPSC lines were contaminated with bacteria or carried mutations associated with cancer.

“It was very surprising to us the high number of unstable cell lines identified in the study, which highlights the importance of setting safety standards for stem cell therapies,” said Carolyn Lutzko, PhD, senior author and director of translational development in the Translational Core Laboratories at Cincinnati Children’s Hospital Medical Center. “A good number of the cell lines we studied met quality standards, although the unexpected number of lines that did not meet these standards could not be used for clinical therapies.”

In this paper, Lutzko and her collaborators compared 58 different iPSC lines that had been submitted by various research institutions. The cells were generated with a variety of genes, methods and cells of origin that ranged from skin fibroblasts to infant cord blood cells. All iPSC lines were analyzed for genetic stability, degree of pluripotency, and several other scientific criteria.

In order for an iPSC line to be considered for clinical work, they must exhibit a high degree of genetic stability. Genetically unstable iPSC lines run the risk of form derivatives that can become cancerous, show poor survival, or differentiate into unwanted cell types upon transplantation. It also is essential that iPSC lines exhibit the ability to continuously renew and expand without losing pluripotency or introducing new genetic mutations.

All iPSC lines were also compared to human embryonic stem cell lines in order to compare them to an outside standard.

How did these 58 iPSC lines fare in this rather exacting gauntlet of tests? It depended on several factors. First of all the cell of origin was very important. Skin fibroblasts tended to make rather low-quality iPSC lines, on the average, but cord blood stem cells usually made rather high-quality iPSC lines. Additionally, the specific reprogramming method employed also made a difference. Some of the iPSC lines included in the test were reprogrammed by means of viruses that integrate into the genome of the host cell (24%). Others were reprogrammed with plasmids (64%), which do not integrate into the host cell genome and are lost soon after reprogramming and growth occurs. Others were reprogrammed with modified RNAs (7%), and a few others (5%) were reprogrammed with other types of viruses that do not integrate into the genome of the host cell (Sendai virus). In all cases, the iPSC lines were made by introducing genes into a mature cell that drove that cell to de-differentiate and grow. Slightly different cocktails of genes were used, but the results were largely the same – the induction of pluripotency.  On the average, non-integrating methods of introducing reprogramming genes into cells resulted in higher-quality iPSC lines, with a few notable exceptions.

Pluripotency for each iPSC line was tested by means of implanting undifferentiated iPSCs into nude mice and observing the cells form differentiated tumors called “teratomas.” Teratomas contain tissues derived from all three primary germ layers; endoderm (gut region), ectoderm (epidermis, nerve tissue, etc.) and mesoderm (muscles, blood cells, etc.).

Prior to this study, the prevailing view was that low-quality iPSC lines were not pluripotent and could not form proper teratomas. This hypothesis had not been tested because of the expense of implanting all these iPSC lines into nude mice. To test this hypothesis, Lutzko and her colleagues tested if all iPSC lines, both high and low quality lines, could generate teratomas. Their tests showed that both genetically stable and unstable iPSC lines formed teratomas with cells from all three germ layers. Although genetically unstable iPSC lines demonstrated pluripotency, the concern in a clinical context would be that they also could result in cancer – again emphasizing the need for safe reprogramming methods, according to study authors.

The enormous amount of data generated by these experiments required sophisticated computing for high-level computational analyses. First author, Nathan Salomonis, PhD, a researcher in the Division of Biomedical Informatics at Cincinnati Children’s. Salomonis used computational approaches to collate, examine, and analyze the data and produce large data sets that can compare the different methods of cell programming, the differences in gene regulation between lines, and the functional quality of each iPSC line.

According to Salomonis, his robust data sets uncovered those iPSC lines that had lost their ability to differentiate into particular adult cell types. This massive collection of raw processed data is available through the online web database.

Salomonis said that, in the future, members of this research consortium will test the ability of each iPSCs line to differentiate into specific cell types – such as brain, heart, lung and other cells in the human body. After these data are verified and published, this information will be added to the online database as a public resource.

Engineered Brown Fat For Metabolic Disorders


BioRestorative Therapies, Inc, (BRTX) has announced what they termed as “promising data” on the transplantation of human stem cell-derived tissue engineering from brown fat.

This study is in a meeting abstract at the moment, so there is no paper to reference at the time. The presentation entitled, “In Vitro Evaluation of an Encapsulation System for the Transplantation of Human Stem Cell-Derived Tissue Engineered Brown Fat,” was given at the International Society for Cellular Therapy meeting in Singapore.

In a nutshell, BRTX scientists examined a technique in which they isolated brown fat-derived stem cell populations, and then differentiated those cells in a step-wide fashion into three-dimensional brown fat assemblages. These brown fat constructs were loaded into tiny microcapsules that can potentially deliver these cells into the tissues of a living organism.

Brown fat cells possess a protein called UCP-1 (Uncoupling Protein-1). This protein short circuits the energy producing machinery of cells and converts that energy into heat instead of the energy-storing molecule ATP. Thus, brown fat cells burn more calories than show regular fat cells and generate a good deal more heat. Increasing brown fat levels in a patient with a weight problem could, in theory, at least, cause that patient to inherently burn more calories.

The laboratory-derived, encapsulated brown fat cells seemed to excellent survival and also expressed respectable amounts of UCP-1.

BRTX would like to, someday, transplant these encapsulated cells into human patients someday, but before that day comes, a good deal of animal experiments are required to demonstrate the safety and efficacy of this product. Then and only then will human experiments be warranted.

Autologous Stem Cell Transplantation With Complete Ablation of Bone Marrow Delays Progression of Multiple Sclerosis in Small Phase 2 Trial


Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system. Around 2 million people, worldwide, suffer from MS. MS results from the patient’s immune system attacking the myelin sheath that surrounds nerve axons. These constant and relentless attacks upon the myelin sheath causes “demyelination,” resulting in loss of the sensory and motor function.

Treatment usually required the use of drugs that suppress the immune response. Some of these drugs work better than others, while other patients have forms of MS that do not respond to common MS treatment.

A new report published in the Lancet, has shown that chemotherapy followed by autologous hematopoietic stem cell transplantation (aHSCT) can completely halt clinical relapses of MS and prevent the development of new brain lesions in 23 of 24 MS patients. Patients who participated in this study experienced a prolonged period without the need for ongoing medication. Eight of the 23 patients had a sustained improvement in their disability 7.5 years after treatment. This is the first treatment to produce this level of disease control or neurological recovery from MS, but, unfortunately, treatment related risks limit its widespread use.

There are a few specialist centers that offer MS patients aHSCT. This treatment involves harvesting bone marrow stem cells from the patient, and then employing chemotherapy to suppress the patient’s immune system and essentially partially wipe it out. The isolated bone marrow is then reintroduced into the blood stream to “reset” the immune system and stop it attacking the body. However, a respectable percentage of MS patients relapse after these treatments. Therefore, these treatments must be refined and tweaked to improve their efficacy.

Drs Harold L Atkins and Mark S Freedman from The Ottawa Hospital and the University of Ottawa, Ottawa, Canada, respectively, and their colleagues, tested if complete destruction, rather than suppression, of the immune system during aHSCT could reduce the relapse rate in patients and increase the long-term rates of disease remission. They enrolled 24 patients aged 18-50 from three Canadian hospitals. All of these subjects had previously undergone standard immunosuppressive therapy, but these treatments had failed to control their MS. These patients all had poor prognosis and their disability ranged from moderate to requiring a walking aid to walk 100 meters (according to their Expanded Disability Status Scale or EDSS score).

Adkins and Freeman and their coworkers used a chemotherapy regimen of busulfan, cyclophosphamide and rabbit anti-thymocyte globulin to wipe out the patient’s bone marrow. Atkins explained that this treatment is “similar to that used in other trials, except our protocol uses stronger chemotherapy and removes immune cells from the stem cell graft product. The chemotherapy we use is very effective at crossing the blood-brain barrier and this could help eliminate the damaging immune cells from the central nervous system.” After being treated with chemotherapy regimen, the patients’ bone marrow was reconstituted with their previously isolated bone marrow.

This study’s primary outcome was activity-free survival at 3 years, using EDSS scores as the means of measuring MS progression, in addition to scanning for brain lesions, and assessing MS symptoms.

Of the 24 patients enrolled, one (4%) died from liver failure and sepsis caused by the chemotherapy. In the 23 surviving patients, prior to treatment, patients experienced 1.2 relapses per year on average, but after aHSCT, no relapses occurred during the follow-up period (between 4 and 13 years). These clinical outcomes were nicely complemented by an absence of newly detected brain lesions (as assessed by MRI images taken after the treatment). Initially, 24 MRI scans of the brains of all 24 subjects revealed 93 brain lesions, and after the treatment only one of the 327 scans showed a new lesion.

Despite the exciting success of this clinical trial, Freedman emphasized the need to interpret these results with caution: “The sample size of 24 patients is very small, and no control group was used for comparison with the treatment group. Larger clinical trials will be important to confirm these results. Since this is an aggressive treatment, the potential benefits should be weighed against the risks of serious complications associated with aHSCT, and this treatment should only be offered in specialist centers experienced both in multiple sclerosis treatment and stem cell therapy, or as part of a clinical trial. Future research will be directed at reducing the risks of this treatment as well as understanding which patients would best benefit from the treatment.”

Dr Jan Dörr, from the NeuroCure Clinical Research Center, Charité-Universitätsmedizin, Berlin, Germany, made this comment about this clinical trial: “These results are impressive and seem to outbalance any other available treatment for multiple sclerosis. This trial is the first to show complete suppression of any inflammatory disease activity in every patient for a long period…However, aHSCT has a poor safety profile, especially with regards to treatment-related mortality.”

He added: “So, will this study change our approach to treatment of multiple sclerosis? Probably not in the short-term, mainly because the mortality rate will still be considered unacceptably high. Over the longer term (and) in view of the increasing popularity of using early aggressive treatment, there may be support for considering aHSCT less as a rescue therapy and more as a general treatment option, provided the different protocols are harmonized and optimized, the tolerability and safety profile can be further improved, and prognostic markers become available to identify patients at risk of poor prognosis in whom a potentially more hazardous treatment might be justified.”

Controlling Mesenchymal Stem Cell Activity With Microparticles Loaded With Small Molecules


Mesenchymal stem cells are the subject of many clinical trials and show a potent ability to down-regulate unwanted immune responses and quell inflammation. A genuine challenge with mesenchymal stem cells (MSCs) is controlling the genes they express and the proteins they secrete.

A new publication details the strategy of one enterprising laboratory to control MSC function. Work by Jeffery Karp from the Harvard Stem Cell Institute and Maneesha Inamdar from the Institute for Stem Cell Biology and Regenerative Medicine in Bangalore, India and their colleagues had use microparticles that are loaded with small molecules and are readily taken up by cultures MSCs.

In this paper, which appeared in Stem Cell Reports (DOI: http://dx.doi.org/10.1016/j.stemcr.2016.05.003), human MSCs were stimulated with a small signaling protein called Tumor Necrosis Factor-alpha (TNF-alpha). TNF-alpha makes MSCs “angry” and they pour out pro-inflammatory molecules upon stimulation with TNF-alpha. However, to these TNF-alpha-stimulated, MSC, Karp and others added tiny microparticles loaded with a small molecule called TPCA-1. TPCA-1 inhibits the NF-κB signaling pathway, which is one of the major signal transduction pathways involved in inflammation.

fx1 (7)

Delivery of these TPCA-1-containing microparticles thinned-out the production of pro-inflammatory molecules by these TNF-alpha-treated MSCs for at least 6 days. When the culture medium from TPCA-1-loaded MSCs was given to different cell types, the molecules secreted by these cells reduced the recruitment of white blood cells called monocytes. This is indicative of the anti-inflammatory nature of TPCA-1-treated MSCs. The culture medium from these cells also prevented the differentiation of human cardiac fibroblasts into collagen-making cells called “myofibroblasts.” Myofibroblasts lay down the collagen that produces the heart scar after a heart attack. This is a further indication of the anti-inflammatory nature of the molecules made by these TPCA-1-treated MSCs.

These results are important because it shows that MSC activities can be manipulated without gene therapy. It is possible that such non-gene therapy-based approached can be used to fine-tune MSC activity and the types of molecules secreted by implanted MSCs. Furthermore, given the effect of these cells on monocytes and cardiac fibroblasts, perhaps microparticle-treated MSCs can prevent the adverse remodeling that occurs in the heart after a heart attack.

Antiaging Glycoprotein Quadruples Viability of Stem Cells in Retina


When pluripotent stem cells are differentiated into photoreceptor cells, and then implanted into the retina at the back of the eye of a laboratory animal, they do not always survive.  However, pre-treatment of those cells with an antiaging glycoprotein (AAGP), made by ProtoKinetix, causes those transplanted cells to be 300 times more viable than cells not treated with this protein according to a study recently accepted for publication.

AAGP was invented by Dr. Geraldine-Castelot-Deliencourt and developed in partnership with the Institute for Scientific Application (INSA) of France. For her work in this area Dr. Castelot-Deliencourt was honored with France’s highest award for scientific accomplishment, the Francinov Award, in 2006.

ProtoKinetix, Incorporated said that a paper submitted by Kevin Gregory-Evans on the company’s AAGP was accepted for publication by the Journal of Tissue Engineering and Regenerative Medicine for publication.

AAGP significantly improves the viable yield of stem cells transplanted in retinal tissue, according to experiments conducted at the University of British Columbia in the laboratory of Dr. Kevin Gregory-Evans.

AAGP seems to protect cells from inflammation-induced cell death. This is based on experiments in which cultured cells that were treated with AAGP were significantly more resistant to hydrogen peroxide, ultraviolet A (wavelengths of 320-400 nanometers), and ultraviolet C (shorter than 290 nm). In addition, when exposed to an inflammatory mediator, interleukin β (ILβ), AAGP exposure reduced COX-2 expression three-fold. COX-2 is an enzyme that is induced by the various stimuli that stimulate Inflammation. It is, therefore, an excellent read-out of the degree to which inflammation has been induced. The fact that AAGP prevented the induction of COX-2 shows that this protein can inhibit the induction of inflammation. These data suggest that AAGP™ may not just be usable in cell and organ storage but also in pharmacological treatments.

First Patient Enrolled in Phase 2 Trial That Tests NSI-189 for Major Depressive Disorder


Neuralstem, Inc. has announced the enrollment of its first patient in its double-blind, placebo-controlled multi-center Phase 2 study of a compound called NSI-189 for the treatment of MDD (major depressive disorder).

MDD usually consists of a persistent feeling of sadness or loss of interest. MDD can also include an inability to sleep or concentrate on tasks, changes in appetite, decreased energy level, and even thoughts of suicide.

MDD is treated with a variety of psychological therapies, such as
cognitive behavioral therapy, Behavior therapy, and Psychotherapy. Cognitive behavioral therapy is a type of talk therapy that focuses on changing a person’s thoughts in order to change their behavior and feelings. Behavior therapy focuses on changing behavior to help people break unhealthy habits. Psychotherapy treats mental or behavioral disorders through talk therapy. A medical procedure called electroconvulsive therapy is also used for some patients. Medications include antipsychotic medicines such as Aripiprazole (Abilify), anxiolytics like buspirone (Buspar), and antidepressants such as Trazodone (Oleptro), Bupropion (Wellbutrin), Duloxetine (Cymbalta) and a host of others.

The medications used to treat MDD regulate the levels of particular neurotransmitters (small molecules used neurons use to communicate with each other) in the brain.

NSI-189 works rather different from these other medications. NSI-189 activates neurogenesis, or the production of new neurons. The drug also activates the formation of new synapses and increases the volume of the hippocampus. All of these processes are thought to play a role in reversing depression. Such neurological outcomes can also enhance cognition and promote neuroregeneration.

NSI-189
NSI-189

This phase 2 trial will randomize 220 patients, in three cohorts, two of whom will receive the drug (40 mg twice a day or 40 mg once a day) and another of which will receive the placebo. Twelve different sites will participate in this MDD trial, all under the direction of Maurizio Fava.

The primary efficacy endpoint is a reduction in depression symptoms. The Montgomery-Asberg Depression Rating Scale (MADRS) will be used to assess thee severity of depression symptoms. Other endpoints will examine cognitive improvement measures.

The trial will last for 12 weeks, with an additional observational follow-up period of six months in order to assess NSI-189 long-lasting durability of benefits.

Neuralstem expects to report the results of this trial in the second half of 2017.

“A new class of treatment is needed in major depression, where existing compounds are not effective for all patients and have high side effect profile, so patients discontinue treatment,” said Fava. “We were encouraged by the signs of improvement in the depression and cognitive symptoms of MDD patients, as witnessed in Phase I with NSI-189, and look forward to validating in Phase 2.”

As mentioned in this statement to the press by Fava, NSI-189 successfully completed a phase I clinical trial for MDD in 2011. In this trial, NSI-189 was administered to 41 healthy volunteers. A phase Ib clinical trial for treating MDD in 24 patients was started in 2012 and completed in July 2014, and the results of this trial were published in December 2015.

NSI-189 works via a new pathway that is different from current antidepressants in that it appears to create long-lasting, positive structural changes in the brain.

In animal experiments, rodents treated with NSI-189 showed significant increases in synaptogenesis, neurogenesis, and hippocampal volume.

In the Phase 1b trial, therapeutic effects were observed in patients after completion of the 28-day dosing, and these improvements persisted for an additional 56 days without the drug. This seems to support the hypothesis of a new mechanism of action that induces long-lasting structural changes in key areas of the brain. In this trial, NSU-189 was shown to be safe and demonstrated large treatment effects in two key depression outcome measures.

The Phase 1b study also showed significant improvement in cognitive symptoms (as measured by the Cognitive and Physical Functioning Questionnaire), compared to placebo.

Brain imaging with quantitative EEGs showed an increase in alpha brain waves in two parts of the brain (left posterior temporal and left parietal region), both of which are involved in depression and cognition, compared to placebo.

No significant adverse effects were observed.

This new clinical trial will test the efficacy of this new drug to treat moderate to severe clinical depression.

A New Way to Prepare Labeled Stem Cells


Researchers from Carnegie Mellon University in the laboratory of Chien Ho have designed a new method for preparing stem cells that can be easily detected on an magnetic resonance imaging (MRI) scan.

This new procedure not only can produce more native stem cells, but labels them with a FDA approval approved iron-oxide nanoparticle that is marketed under the name Feraheme (Ferumoxytol).

Mesenchymal stem cells extracted and isolated from bone marrow or other tissues can readily generate bone, cartilage, and fat in the laboratory. Mesenchymal stem cells (MSCs) are being tested in 360 registered clinical trials. The results of these trials have been mixed, and for these and other reasons, it is important to track implanted stem cells.

Tracking MSCs requires labeling the cells in some manner, and Dr Ho and his group discovered that superparamagnetic iron-oxide (SPIO) contrast agent, which are easily seen on MRI scans. Fortunately, MSCs have an intrinsic capacity to internalize SPIO under the right conditions. Ho and his coworkers devised a way to create the right conditions in culture so that MSCs in culture can readily take up ferumoxytol nanoparticles quite effectively.

Labeling MSCs with Feraheme

This new culture method takes MSCs extracted from bone marrow, isolates them, and then expands them in culture. Then the Carnegie group placed the MSCs into a culture system that mimics the environment cells normally experience inside someone’s body. This “in-vivo” culture method not only drives the MSCs to optimize their size, but it aggrandizes their SPIO internalization abilities.

Size optimization is very important, since smaller MSCs tend to be more effective for regenerative therapies than larger MSCs. This optimization produces high-quality cells that are also labeled and can be tracked after implantation into a patient’s body.

This impressive work was published in the journal Scientific Reports, 2016; 6: 26271 DOI: 10.1038/srep26271.

Making Cartilage to Heal Broken Bones


Gage Crump and his colleagues at the University of Southern California have used the regeneration of zebrafish jawbone to demonstrate that the regeneration of damaged bones does not necessarily require a recapitulation of the same processes that occur during embryonic development. Even though this work used zebrafish as a model system, it may provide some of the underlying principles for treating difficult fractures.

Cartilage production is critical for healing full-thickness bone injuries. In order to understand how this bone-producing cartilage is generated, Crump and his coworkers turned to the genetically malleable and relatively more simple zebrafish system. Zebrafish are vertebrates, like humans, but these animals retain a remarkable capacity to regenerate many of their organs.

When human bones fracture, a small cartilage callus forms that is replaced by bone that bridges small, but not large, gaps in the bone.

In zebrafish, however, the cartilage callus continues to expand and fills even very large gaps in broken bones. This cartilage is replaced throughout the bone by bone. This allows zebrafish to heal even very large fractures.

These days, patients with severe bone fractures may have a surgeon insert metal pins and even plates to help set bone. In more severe cases, bone grafts are used to span gaps, and stem cell-based treatments have been tested in a few clinical trials as well.

About six million people in the U.S. suffer bone breaks each year, and even though most of these patients recover fully, about 300,000 are slow to heal and some may not heal at all. Complications include post-traumatic arthritis, growth abnormalities, delayed union and misaligned union.

Hundreds of professional football players have invested in stem cell treatments to treat injuries, even though the evidence for the efficacy of such treatments is, sometimes, sparse. One report even tells of an NFL linebacker who paid $6,000 for a 1-milliliter vial of donated placenta tissue containing stem cells to be injected into his injured knee.

The bone surface contains thin lining called the “periosteum” that contains a stem cell population that helps maintain bone mass throughout one’s life. In Gage’s laboratory, his team identified a gene called Indian Hedgehog a (IHHa), which is responsible for inducing these periosteal stem cells to switch from bone production to cartilage production. Mutant zebrafish strains that lack the IHHa gene are unable to make cartilage in response to bone injury and heal poorly from bone fractures.

Periosteum

Crump said that an “exciting finding from our work is that, somewhat counterintuitively, cartilage is critical for healing full thickness bone injuries. By understanding how this bone-producing cartilage is generated in the simpler zebrafish model, we hope to find ways to create more of this unique cartilage tissue in patients to better heal their bones.”

According to this paper, which was published in the journal Development, 2016; dev.131292 DOI: 10.1242/dev.121292; instead of the more traditional approach of using bone cells or bone-like materials to heal broken bones, stimulating endogenous bone-based stem cells that make this special kind of fracture-healing cartilage might be a more effective strategy.

Induced Pluripotent Stem Cells – Addressing Safety Concerns


In 2012, John B. Gurdon and Shinya Yamanaka won the Nobel Prize in Physiology or Medicine “for the discovery that mature cells can be reprogrammed to become pluripotent.” Since that time, induced pluripotent stem cells (iPSCs) have largely taken the stem cell scene by storm. Because of the ease with which iPSCs can be made from just about any mature cell type, and because they can be made so more cheaply and faster than embryonic stem cells, they are the perfect pluripotent stem cell for laboratory use. The additional advantage to iPSCs is that can instantly reflect the genetic defect of the patient from whom they are made. Therefore, they are provide excellent model systems for a variety of genetic diseases and provide a kind of “disease in a dish” system by which the cellular and molecular characteristics of a disease can be modeled in cell culture.

In addition to their experimental utility, many scientists have sought to promote iPSCs for clinical purposes. However, before iPSCs can be used in the clinic, their safety must be established beyond question. Despite their success in many animal models (most in rodents), the long-term safety of iPSC derivatives has yet to be firmly demonstrated.

To that end, three different experiments have added to our concerns about the safety of iPSCs. For these and other reasons, several scientists have hypothesized that if iPSCs derivatives are going to be used in a clinical setting, they will need to come from young, healthy donors. In particular, blood cells from umbilical cord blood can be matched to just about any tissue and can be easily converted into iPSCs. Therefore, allogeneic iPSC derivatives seem to be the best way to go about treating particular diseases.

That being said, there are three studies about the safety of iPSC derivatives that make important contributions to the debate.

The first study comes from the laboratory of Shoukhrat Mitalipov at the Oregon Health and Science University. Mitalipov and his team have examined the mitochondrial genomes of iPSCs made from older patients.

Mitochondria are small, vesicles surrounded by two membranes, within cells that are the energy-production structures of most cells (not bacteria). Mitochondria also contain their own DNA molecules that express a variety of mitochondrial-specific genes and their own bacterial-like ribosomes that synthesized the mRNAs made from those genes into proteins. However, the vast majority of mitochondrial proteins are encoded on genes housed in the nucleus.

Mutations in genes encoded by the mitochondrial genome are rather devastating and are responsible for several really nasty (albeit rare) genetic diseases. These mitochondrial genetic diseases include: Mitochondrial myopathy, diabetes and deafness, Leber’s hereditary optic neuropathy (includes visual loss beginning in young adulthood, progressive loss of central vision due to degeneration of the optic nerves and retina), Leigh syndrome subacute sclerosing encephalopathy (disease usually begins late in the first year of life, although onset may occur in adulthood; a rapid decline in function occurs and is marked by seizures, altered states of consciousness, dementia, ventilatory failure), neuropathy, ataxia, retinitis pigmentosa, and ptosis (progressive symptoms as described and dementia), Myoneurogenic gastrointestinal encephalopathy (gastrointestinal pseudo-obstruction and neuropathy), Myoclonic epilepsy with ragged red fibers (progressive myoclonic epilepsy, “Ragged Red Fibers” or clumps of diseased mitochondria accumulate in the muscle fiber, short stature, hearing loss, lactic acidosis, exercise intolerance), mitochondrial myopathy, encephalomyopathy, lactic acidosis, stroke-like symptoms (MELAS).

Mitochondrial DNA mutations have long been thought to be a driving force in aging and age-onset diseases. Therefore, if iPSCs are made from older patients, will their starting cells have these mitochondrial mutations?

Taoseng Huang from Cincinnati Children’s Hospital Medical Center said: “If you want to use iPS cells in a human, you must check for mutations in the mitochondrial genome. Every single cell can be different. Two cells next to each other could have different mutations or different percentages of mutations.”

In this study from Mitalipov’s laboratory, his team derived and sequenced 10 iPS clones from each patient tissue sample to get a better understanding of mitochondrial DNA mutations rates. They took samples of blood and skin samples from healthy subjects and patients with degenerative diseases, who ranged in age from 24-72 years old. In these pools of these sampled cells, the rate of mitochondrial DNA mutations was low.

20 iPS cell lines per patient were profiled. Ten of these lines were derived from skin cells and the other 10 were derived from blood cells. Sequencing of the mitochondrial genomes of the iPSC lines revealed higher numbers of mitochondrial DNA mutations, particularly in cells from patients older than 60 years old. Of the 130 iPSC lines analyzed, 80 percent of them showed mitochondrial mutations and higher percentages of the mitochondria per cell contained mutations.

Such mitochondrial mutations can seriously compromise the ability of derivatives of these iPSC lines to carry out their desired function. Mitalipov in his paper, which was published in Cell Stem Cell, 2016; DOI: 10.1016/j.stem.2016.2016.02.005, that all iPSC lines for use in human patients should be screened for mitochondrial mutations.

Graphical abstract-2

One feature not addressed by Mitalipov and his colleagues is whether or not cells that may not show the signs of aging should be used to derived iPSCs, such as particular bone dormant marrow stem cells.

If mitochondrial mutations aren’t bad enough Jennifer E. Phillips-Cremins and her coworkers at the University of Pennsylvania School of Engineering and Applied Science have found that the chromatin structures of iPSCs might prevent them from properly differentiating into particular derivatives.

As previously mentioned in other blog posts, the DNA in the nuclei of our cells is packaged into a compact structure known as chromatin. Chromatin helps cells express those genes it needs to express and shut down other genes whose expression is not needed.

Occasionally, iPSC lines show an inability to differentiate into particular cell types while others have the ability to differentiate into many cell types. According to this study by Phillips-Cremins and her team, defects in DNA packaging might explain these disparities in iPSC lines.

By using experimental and computational techniques, Phillips-Cremins and her graduate student Jonathan Beagan identified chromatin conformations in a variety of iPSC lines. The DNA topology of embryonic stem cells and neural stem cells were also analyzed as comparisons.

“We know there is a link between the topology of the genome and gene expression,” Jennifer Phillips-Cremins, said in a press release. “So this motivated us to explore how the genetic material is reconfigured in three dimensions inside the nucleus during the reprogramming of mature brain cells to pluripotency. We found evidence for sophisticated configurations that differ in important ways between iPS cells and embryonic stem cells.”

The three-dimensional DNA conformations of pluripotent stem cells are reorganized during differentiation. Phillips-Cremin and others discovered that when mature cells are reprogrammed to pluripotent cells, most pluripotency genes reconnect to their enhancers (which are crucial for their expression). However, when these same iPSCs are differentiated into neural progenitor cells, the interactions between pluripotency gene and their enhancers remain in some lines, which should not occur.

“We found marked differences among the heatmaps we generated for each cell type,” said Jonathan Beagan, a graduate student in Phillips-Cremin’s laboratory at the University of Pennsylvania. “Our observations are important because they suggest that, if we can push the 3D genome conformation of cells that we are turning into IPSCs to be closer to that of embryonic stem cells, then we can possibly generate IPSCs that match gold-standard pluripotent stem cells more rapidly and efficiently.”

This paper was published in Cell Stem Cell (2016), 18(5): 611–624. Therefore, the chromatin structure of iPSCs is also important.

Finally, another paper reports some good news for iPSCs. Research from the Wellcome Trust Sanger Institute tracked the genetic mutations acquired by iPSCs when they are made in the laboratory. These cells came from the blood of a 57-year-old male subject.

This research, led by Allan Bradley, showed that mutations arise 10 times less often in iPSCs than they do in cultured laboratory-grown blood cells. Furthermore, non of the iPSC-acquired mutations were in genes known to cause cancer.

Bradley and his colleagues were able to trace the history of every mutation that each cell acquired from its extraction from the body to its reprogramming in the laboratory and propagation in culture.

The techniques utilized in the Bradley laboratory can surely help scientists evaluate the genetic integrity of laboratory-derived iPSCs.

This work was published in PLOS Genetics, 2016; 12(4): e1005932 DOI: 10.1371/journal.pgen.1005932.

All in all, it seems that it is possible to make sound iPSC lines, but those lines must be properly screened before they can be used in a clinical setting to treat live patients. These three papers provide new ways to screen iPSC lines for ensure high levels of safety and efficacy.

Pluripotent Stem Cells Actively Regulate The Openness of their Heterochromatin


Packaging DNA into a small area like the nucleus of the cell does not occur unless that DNA is tightly wound into compact structures collectively known as chromatin. However, not all regions of the genome show the same degree of compaction. Highly-expressed regions of the genome tend to be less highly compacted and regions of the genes that are not expressed to any degree tend to be squirreled away into tight chromatin.

Pluripotent stem cells tend to have an open and decondensed chromatin organization. In fact, this open and decondensed chromatin configuration is a defining property of pluripotent cells in general. The connection between pluripotency and the is open chromatin organization and the mediators of this chromatin configuration remain shrouded in uncertainty.

A new study from the laboratory of Peter J Rugg-Gunn at the Babraham Institute, in collaboration with scientists from Canada, the United Kingdom, and Japan, has identified two proteins, Nanog and Sall1 that participate in the chromatin structure of pluripotent stem cells. Such an understanding can contribute to making better pluripotent stem cells.

Cells tend to possess regions of the genome that are tightly wrapped into tight heterochomatin. These genomic regions are usually structural in nature and are, typically, not expressed. These include centromeric DNA and pericentromeric DNA, which plays a role in spindle attachment during cell division. These regions are collectively known as “constitutive heterochromatin.” However, previous research has demonstrated that this constitutive heterchromatin is maintained in an open and uncompacted conformation.

Clara Lopes Novo, in Rugg-Gunn’s laboratory and her colleagues discovered that transcription factor NANOG acts as an integral regulator of the conformation of constitutive heterochromatin in mouse embryonic stem cells. When Lopes Novo and others deleted the Nanog genes in mouse embryonic stem cells, the constitutive heterochromatin was remodeled in a manner that led to more intensive chromatin compaction. However, when Lopes Novo and her coworkers forced the expression of the Nanog gene in mouse embryonic stem cells, leading to spikes in the levels of NANOG proteim, the heterochromatin domains showed distinct decompaction.

When Lopes Novo and others determined where NANOG spent its time, they discovered that it was bound to heterochromatin. In particular, NANOG associated with satellite repeats within heterochromatin domains. Heterochromatin that was associated with NANOG had highly dispersed chromatin fibers, low levels of modified histone proteins that are usually associated with chromatin compaction (i.e. H3K9me3), and high levels of transcription.

The second heterochromatin-associated protein, SALL1, seems to work in cahoots with NANOG. In fact, when Lopes Novo and others deleted the Sall1 gene from mouse embryonic stem cells, the Sall1-/- cells recapitulate the Nanog -/- phenotype. However, further work showed that the loss of Sall1 can be rescued by forcing the recruitment of the NANOG to major portions of the heterochromatin (by over-expressing the NANOG protein).

These results demonstrate the connection between pluripotency and chromatin organization. This work seems to say, “embryonic stem cells actively maintain an open heterochromatin architecture.” They do this to stabilize their pluritotency.

Loss of heterochromatin regulation has potential consequences for the long-term genetic stability of stem cells, and the ability of stem cells, and the ability of stem cells to differentiate and mature into specialized cell types.

This work was published in the journal Genes and Development (http://www.genesdev.org/cgi/doi/10.1101/gad.275685.115)