Stem Cells Derived From Amniotic Tissues Have Immunosuppressive Properties

Ever since they were first isolated, amnion-based stem cells have been considered promising candidates for cell therapies because of their ease of access, plasticity, and absence of ethical issues in their derivation and use. However, a Japanese research team has discovered that stem cells derived from human female amnion also have the ability to suppress the inappropriate activation of the immune system and that there are straight-forward ways to enhance their immunosuppressive potential.

The amniotic membrane is a three-layered structure that surrounds the baby and suspends it in amniotic fluid. Amniotic fluid acts as a protective shock-absorber, a lubricant and an important physiological player in the life of the embryo and fetus. Because the fetus is a privileged entity that escapes attack from the mother’s immune system, researchers have been very interested in determining the immunological properties of the amnion cells.

“The human amniotic membrane contains both epithelial cells and mesenchymal cells,” said study co-author Dr. Toshio Nikaido, Department of Regenerative Medicine, Graduate School of Medicine and Pharmaceutical Sciences at the University of Toyama. “Both kinds of cells have proliferation and differentiation characteristics, making the amniotic membrane a promising and attractive source for amnion-derived cells for transplantation in regenerative medicine. It is clear that these cells have promise, although the mechanism of their immune modulation remains to be elucidated.”

In this study by Nikaido and his coworkers, amnion-derived cells inhibited natural killer cell activity and induced white blood cell activation. Nikaido reported that he and his colleagues saw the amnion-derived cells increase production of a molecule called interleukin-10 (IL-10).

“We consider that IL-10 was involved in the function of amnion-derived cells toward NK cells,” explained Dr. Nikaido. “The immunomodulation of amnion-derived cells is a complicated procedure involving many factors, among which IL-10 and prostaglandin E2 (PGE2) play important roles.”

Molecules called “prostaglandins,” such as PGE2, mediate inflammation, smooth muscle activity, blood flow, and many other physiological process. In particular, PGE2 exerts important effects during labor and stimulates osteoblasts (bone-making cells) to release factors that stimulate bone resorption by osteoclasts. PGE2 also suppresses T cell receptor signaling and may play a role in the resolution of inflammation.

When Nikaido and others used antibodies against PGE2 and IL-10, they removed the immunosuppressive effects of the amnion-derived cells on natural killer cells. These data imply that these two factors contribute to the immunosuppressive abilities of amnion-derived cells.

“Soluble factors IL-10 and PGE2 produced by amnion-derived cells may suppress allogenic, or ‘other’ related immune responses,” concluded Dr. Nikaido. “Our findings support the hypothesis that these cells have potential therapeutic use. However, further study is needed to identify the detailed mechanisms responsible for their immodulatory effects. Amnion-derived cells must be transplanted into mouse models for further in vivo analysis of their immunosuppressive activity or anti-inflammatory effects.”

Given the levels of autoimmune diseases on the developed world, these results could be good news for patients who suffer from diseases like Crohn’s disease, systemic lupus erythematosus, or rheumatoid arthritis. While more work is needed, amnion-based cells certainly show promise as immunosuppressive agents.

The study will be published in a future issue of Cell Transplantation.

Induced Pluripotent Stem Cells Differentiated into Intestinal Cells

Even the liver is the main organize when it comes to the metabolization of drugs, the small intestine also plays an important role in all aspects of drug metabolism. Unfortunately, no laboratory system exists at present that serves as a standardized system for evaluating the way drugs interact with the small intestine.

A new study by Tamihide Matsubara and his colleagues from Nagoya City University in Japan has sought to alleviate this problem. Matsubara and his coworkers used human induced pluripotent stem (iPS) cells to produce functional human intestinal enterocytes and showed that they faithfully recapitulated the drug metabolism of normal, human intestinal enterocytes.

To make intestinal enterocytes from iPS cells, Matsubara and others treated these cells with chemicals called activin A and fibroblast growth factor 2 to drive the cells to become intestinal-like stem cells. These cultured intestinal-like stem cells them differentiated into enterocytes when grown in a culture medium that contained epidermal growth factor and other small-molecule compounds.

The differentiated cells expressed intestinal marker genes and drug transporters. For example, they expressed sucrase-isomaltase, an intestine-specific marker, and enterocyte drug-metabolizing enzymes such as CYP1/2, CYP2C9, CYP2C19, CYP2D6, CYP3A4/5, UGT, and SULT. Inhibitor studies showed that the intestinal oligopeptide transporter SLC15A1/PEPT1 was inhibited by the pain reliever ibuprofen, just like in naturally-occurring enterocytes. Also, active forms of vitamin D increased the expression of the enzymes CYP3A4 and CYP3A4/5, which is also observed in naturally-occurring human enterocytes.

These results show that Matsubara and his colleagues have successfully generated enterocyte-like cells that have the same drug metabolizing capacities as naturally-occurring enterocytes. These cells would be very useful for developing novel evaluation systems to predict individual human intestinal drug metabolism.

Compact Spinal Implants to Help Spinal Cord Injured Patients Walk

An interdisciplinary research team at the Ecole Polytechnique Fédérale de Lausanne (EPFL) in Switzerland led by Dr. Grégoire Courtine and Dr. Stéphanie P. Lacour has recently lifted the curtain on their flexible spinal implant called the electronic dura (or e-Dura). According to Courtine and Lacour, this implant greatly improves spinal injury rehabilitation in spinal cord injured rats.

In a paper published by this team earlier in the journal Science this month, the EPFL team showed that, because of its flexibility, this next-generation e-Dura implant lasts longer (up to two months) and causes much less damage than traditional implants.

These latest results are an extension of earlier research in 2012 in which Courtine and Lacour published breath-taking results that showed spinal cord-injured rats that should have been paralyzed had regained the ability to walk, run, and even climb stairs.

Spinal cord injury results in loss of control over the part of the body below the point of injury. Courtine and his coworkers were able to reactivate the spinal cord in rats with a specific combination of drugs plus electrical stimulation to simulate the excitatory input from the brain. The drugs they used, monoamine agonists, bind to receptors and activate them in the same way that such neurotransmitters would in healthy subjects. When the spinal cord was exposed to these drugs plus mild electrical stimulation, the activated nerve cells in the spinal column produced movement in the paralyzed animals.

Spinal Cord Implant to help the Cripples walk

This movement, however, was largely involuntary, since the brain was not able to communicate with the area below the injured spinal cord. However, over time, as the animals trained and repeatedly walked in their harnesses (which kept them safe from falling); they became more confident in their ability to walk again. In fact, the EPFL team noticed a fourfold growth of new nerves in the spinal cord. This new nerve growth eventually restored communication between the brain and the injured area of the spine.

Courtine, whose eyes sparkle as he passionately talks about his research, is eager to take these findings to the clinic to see if they can help human beings who have suffered a catastrophic spinal cord injury. In preparation for clinical trials, however, they came up against another problem and that was the need for a long-term spinal implant that could deliver the chemical and electrical stimulation needed to initiate spinal cord healing. Courtine hopes that his e-Dura can satisfy this need.


The e-Dura meets two important criteria for spinal implants: durability and biocompatibility. In order to reduce the number of surgical procedures an injured patient must undergo, an implantable device needs to last a long time. It also has to be biocompatible and flexible. Early generation implants caused inflammation and the formation of scar tissue, which usually offset any positive results the implant provided.

When tested on laboratory animals, Courtine’s laboratory applied the e-Dura implant beneath the protective dura mater, directly on the spinal cord. Thankfully, the implant did not cause any adverse effects and lasted long enough for the paralyzed animals to complete their rehabilitation. Functionally, the implant also performed very well.

The e-Dura unit contains very small microfluidic channels that are embedded on a flexible silicon substrate. The device delivers precise amounts of drugs directly to the nerve cells in the spinal cord. Cracked gold conducting wires and electrodes that are made of a composite material that consists of silicon and platinum send electrical signals to the injured spinal cord. Electronic circuitry in the implant also provides the opportunity for the EPFL team to monitor the electrical messages sent back and forth to the brain as the new nerves are activated.

Indeed, this research is wonderfully exciting, but it is unclear how well it will work in humans. First of all, humans will probably need a different cocktail of drugs or a distinct electrical stimulation pattern to stimulate the spinal cord to heal itself. As with much clinical research at the beginning stages, there many unanswered questions to date. Advanced clinical trials will hopefully uncover some of these idiosyncrasies that characterize the injured human spinal cord and such answers are an integral part of providing a protocol that applies uses technology to human patients.

While exoskeleton technology also continues to approach consumer markets, it would be better to return to people their natural ability to walk. However you slice it, spinal cord injury patients may have more options in the coming years.

New Technology Reprograms Skin Fibroblasts

Fibroblasts are one of the main components of connective tissue, which is the main reason scientists typically exploit them for experiments. A collaborative team of scientists from the University of Pennsylvania, Boston University, and the New Jersey Institute of Technology have invented a way to reprogram fibroblasts without going through a pluripotent stage.

The senior author of this study, Xiaowei Xu, associate professor of pathology and laboratory medicine at the University of Pennsylvania School of Medicine, said, “Through direct reprogramming, we do not have to go through the pluripotent stem cell stage, but directly convert fibroblasts to melanocytes . So these cells do not have tumorigenicity” (the ability to cause tumors).

Melanocytes are found in the skin and they are responsible for the pigment in our skin. They are in the uppermost layer of the skin, known as the epidermis, and produce melanin, a brown pigment that helps screen against the harmful effects of UV light.

Turning a fibroblast into a melanocytes might seem trivial for a stem cell scientist; just reprogram the fibroblast into an induced pluripotent stem cells and then differentiate it into a melanocytes. However, this procedure utilized direct reprogramming, in which the fibroblast was converted into a melanocytes without traversing through the pluripotent stage. The difficultly with direct reprogramming is finding the right cocktail of genes and/or growth factors that will accomplish the deed.

Xu and his colleagues began their search by examining the genes that are specific to melanocytes. They found 10 different transcription factors that are important for melanocytes development. Next they screened these ten genes for their ability to convert a fibroblast into a melanocytes. They found that of the ten melanocytes-specific genes, three of them, Sox10, MITF, and PAX3 could do the job effectively. They called this gene combination “SMP3.”

When Xu and others tested SMP3 on mouse embryonic fibroblasts, they quickly expressed melanocytes-specific genes. When Xu’s group used SMP3 on human fetal dermal cells, once again, the cells rapidly differentiated into melanocytes. Xu and his team referred to these cells as hi-Mel, which is short for human, induced melanocytes.

When hi-Mel were grown in culture they produced melanin a plenty. When they were implanted into the skin of pigmentless mice, once again they rose to the challenge and made a great deal of pigment. Thus hi-Mel express the same genes as melanocytes and they behave for all intents and purposes as melanocytes.

Xu and his colleagues think that their procedure might be able to treat human patients with a condition called vitiligo in which the skin has patches that are devoid of pigment.

Another potential use of this technology is a way to effectively study melanoma, one of the most dangerous skin cancers known to human medicine. My good friend and SAU colleague died over a year ago from melanoma and having better ways to treat this monster would have been marvelous for Charlie, and his family, who miss him dearly. By generating melanocytes from the fibroblasts of melanoma patients, they can “screen not only to find why these patients easily develop melanoma, but possibly use their cells to screen for small compounds that can prevent melanoma from happening.”.

Also, because so the body contains so many fibroblasts in the first place, this reprogramming technique is well-suited for other cell-based treatments.

Teaching Old Cells New Tricks

The laboratory of Helen Blau at Stanford University has devised a technique to lengthen the sequences that cap the ends of chromosomes in skin cells. This treatment enlivens the cells and makes them behave as though they were younger.

In order to properly protect linear chromosomes from loosing DNA at their ends, chromosomes have a special set of sequences called “telomeres” at their ends. Telomeres consists of short sequences that are repeated many times. A special enzyme called the telomerase replicates the telomeres and maintains them. As we age, telomerase activity wanes and the telomeres shorten. This threatens the genetic integrity of the chromosomes, since a loss of genes from the ends of chromosomes can be deleterious for cells. In young humans, the telomeres may be 8,000 to 10,000 bases long. When the telomeres shorten to a particular length, growth stops and the cells become quiescent.

Human telomeres

Embryonic stem cells have long telomeres at the ends of their chromosomes and they also have robust telomerase activity. Adult stem cells have varied telomerase activity and telomere length, but it seems that the length of the telomeres and the activity of the telomerase correlates with the vitality of the stem cell population and its capacity to heal (see H. Saeed and M. Iqtedar (2013). J. Biosci. 38, 641–649). As we age our stem cell quality decreases as their telomeres shorten.

Blau and her colleagues used a modified type of RNA to lengthen the telomeres of large numbers of cells. According to Blau: “Now we have found a way to lengthen human telomeres by as much as 1,000 nucleotides, turning back the internal clock in these cells by the equivalent of many years of human life. This greatly increases the number of cells available for studies such as drug testing or disease modeling.”

In these experiments, Blau and her coworkers used chemically modified messenger RNA molecules that code for TERT, which is the protein component of the telomerase. The expression of these messenger RNAs in human cells greatly increased the levels of telomerase activity.

This technique devised by Blau and her team have distinct advantages of previously described protocols. First, this technique boosts telomerase activity temporarily. The modified messenger RNA sticks around for several hours and is translated into TERT protein, but this protein only lasts for about 48 hours, after which its activity dissipates. After the telomerase have lengthened the telomeres, they will shorten again after each cell division as before.

“This new approach paves the way toward preventing or treating diseases of aging,” said Blau. “There are also highly debilitating genetic diseases associated with telomere shortening that could benefit from such a potential treatment.”

Blau and her team are testing their technique in other cell types besides skin cells.

Cardiosphere-Derived Injections Improve Heart Function in Children with Hypoplastic Left Heart Syndrome

Hypoplastic Left Heart Syndrome or HLHS accounts for 2 to 3 percent of all congenital heart disease. It shows a prevalence rate of two to three cases per 10,000 live births in the United States. HLHS is the most common form of functional single ventricle heart disease. The National Inpatient Sample database has estimated that there were an estimated 16,781 cases of HLHS among neonates born between 1988 and 2005 in the United States. More males have HLHS than females with the male to female ratio being about 1.5:1. Despite its low incidence relative to other congenital cardiac disorders, HLHS, if left untreated, is responsible for 25 to 40 percent of all neonatal cardiac deaths.

In HLHS patients, the left ventricle (the main pumping chamber of the heart), aorta, and related components are underdeveloped.

Children born with HLHS typically require surgery within a few days of birth and additional long-term treatment is required to address issues associated with right ventricular-dependent circulation.

Results from a clinical trial conducted by researchers at Okayama University and Okayama University Hospital show that children who suffer from HLHS seem to benefit from injections of cardiosphere-derived cells (CDCs).

Apparently in children, cardiac progenitor cells that can differentiate into several different heart-specific cell types are more abundant and self-renewing in children than adults.

The research group, led by Hidemasa Oh, monitored the heart function of seven patients who had received injections of cells and a control group of seven patient who had not received any such injections. They concluded that, “Our prospective controlled study, the first pediatric phase I clinical trial of stem cell therapy for heart disease to our knowledge, suggests that intracoronary infusion of autologous cardiac progenitor cells is a feasible and safe approach to treat children with HLHS.”

The cardiac progenitor cells used in this study came directly from the hearts of the patients. When these heart-specific progenitor cells are isolated and grown in cell culture, they form tiny balls of cells called “cardiospheres.” These patient-derived cardiosphere-derived cells (CDCs) were administered to the experimental subjects in this study after they were confirmed to contain a normal number of chromosomes and express a host of heart-specific genes. The transcoronary administration of the CDCs did not produce any adverse effects.

The heart functions monitored by the research group included the right ventricular ejection fraction or RVEF, end-systolic volume (ESV), which is the volume of blood within the ventricle at the maximum point of contraction, and the end-diastolic volume or EDV, which is the volume of blood at the maximum filling point, stroke volume, and cardiac output. Additionally, the levels of brain natriuretic peptide or BNP (a direct measure of heart failure) were also monitored. BNP is made by the ventricles of the heart in response to excessive stretching of the heart muscle.

Because of the rarity of this disease, this study was necessarily small. This study was also a non-randomized study. Therefore, this study is more of an evaluation of the safety of this procedure rather than its efficacy. However, the improvement in the RVEP in the stem cell-treated patients compared to the non-treated group 18 months after CDC administration provides possible evidence of the efficacy of this treatment.

Clearly more work is needed, but we will know more as the data rolls in.

Human Placenta-Derived Multipotent Cells Modulate Cardiac Injury in Large and Small Animal Models

Placental-derived multipotent cells or PDMCs have been isolated from human term placental tissues. PDMCs have the ability to differentiate into neurons, bone, fat, and liver. Can cells like these help heal a damaged heart?

Men-Luh Yen and his colleagues from the National Taiwan University Hospital, Taipei, Taiwan, have recently published a large study of PDMCs that have examined the characteristics of these cells in culture and in small and large animals.

In culture, when PDMCs are grown with mouse heart muscle cells for eight days that differentiate into cells that look a lot like heart muscle cells.  These cells express the heart-specific gene alpha-sarcomeric actinin.  This is not evidence that PDMCs can differentiate into heart muscle cells, but it is evidence that they differentiate into heart muscle-like cells.  It is possible that these cells might be able to completely differentiate into heart muscle cells with the right signals.

When the culture medium from PDMCs are used to grow human umbilical vein endothelial cells, the human umbilical vein endothelial cells formed blood vessel-like tubes.  This indicates that PDMCs secrete a host of growth factors that induce the formation of blood vessels.  When Yen and his group examined the genes expressed by cultured PDMCs, they discovered that they expressed several growth factors known to induce blood vessel formation, such as hepatocyte growth factor (HGF), interleukin-8 (IL-8), and growth-regulated oncogene (GRO).  When these growth factors were given to cultured umbilical vein endothelial cells, they formed blood vessel-like tubes.  Thus HGF, GRO and IL-6 promote the formation of blood vessels.

When PDMCs were used to treat the heart of mice that had suffered a heart attack.  This part of the paper is less satisfying because many of their mice died as a result of this procedure (5 or 18).  However, the PDMS-treated mice did show a steady improvement in their ejection fractions (percentage of blood volume ejected from the heart) compared to mice that were only injected with culture medium.  These PDMC-injected mice also had extensive capillary beds in their heart tissue, suggesting that the increased heart function was due to the induction of new blood vessels.  In all honesty, this section of the paper should have had better controls and more animals should have been tested.  A sham group should have been included with an untreated group as well.

To extend their experiments in living animals, Yen’s group used a similar experimental strategy in Lanyu minipigs.  Here again, a lack of proper controls and large numbers of dead animals (5 of 17) diminish the clarity of the data.  The PDMC-treated minipigs showed a significant increase in ejection fraction (53.8 plus or minus 4.4 percent in the PDMC-treated minipigs vs. 39.2 plus or minus 2.3 percent in the culture medium-treated minipigs).  Also the blood vessel density in the hearts of the PDMC-treated pigs was over three times that of the other group.  Cell death studies showed that the hearts of the PDMC-treated minipigs that half that of the non-stem cell-treated minipigs.  This shows that PDMCs secrete molecules that promote cell survival.

Finally, Yen and others present what they think is evidence that the injected PDMCs in the hearts of the minipigs differentiated into heart muscle cells.  First of all, implanted PDMCs were observed eight weeks after they were injected.  There is little reason to suppose that these cells would have survived if they were not tightly associated with resident heart cells.  Secondly, these PDMCs expressed two heart-specific genes:  cardiac troponin T (cTNT), which is important for heart muscle contraction, and connexin 43, which is integral for forming gap junctions between heart muscle cells.  Gap junctions allow heart muscle cells to stay electrically connected with one another and allow them to contract as a single unit and these cells were expressing connexin 43 and were apparently integrated into the heart muscle.

I must say that I do not find this convincing, since the fusion of heart muscle cells and injected stem cells can account for such data.  Before I would believe that PDMCs can transdifferentiate into heart muscle cells, I would need to see compelling evidence that the connexin 43, cTNT, and human HLA-expressing cells also do not express minipig-specific genes.  Secondly, I would need to see PDMCs express the genes for the calcium-handling system that is unique to heart muscle cells.  The lack of express of these proteins is the single best reason to doubt that mesenchymal stem cells can transdifferentiate into heart muscle cells.  There is evidence that mesenchymal stem cells that stimulate endogenous heart stem cells to make new heart muscle, but little good evidence that mesenchymal stem cells can form mature, functional heart muscle cells.

All in all, the Yen paper shows some interesting data, even if some of it is not top quality.  Clear PDMCs are interesting cells that have a potential future in regenerative medicine.