Forcing Sugars on the Surfaces of Cord Blood Cell Increases Their Engraftment


When a child or adult needs new bone marrow, a bone marrow transplant from a donor is usually the only way to save their life. Without properly functioning bone marrow, the patient’s blood cells will die off, and there will be too few red blood cells to ferry oxygen to tissues or white blood cells to fight off infections.

An alternative to bone marrow from a bone marrow donor if umbilical cord blood. Umbilical cord blood does not require the rigorous tissue matching that bone marrow requires because the blood making stem cells from cord blood are immature and not as likely to cause tissue rejection reactions.. However, umbilical cord blood cells suffer from two drawbacks: low numbers of stem cells in cord blood and poor engraftment efficiencies.

Fortunately, some progress has been made at expanding blood-making stem cells from umbilical cord blood, and it is likely that such technologies might be ready for common use in the future. As to the poor engraftment efficiencies, a new paper in the journal Blood from the laboratory of Elizabeth J. Shpall at the University of Texas MD Anderson Cancer Center, in Houston, Texas reports a new way to increase cord blood stem cells engraftment efficiencies.

As previously discusses, delayed engraftment is one of the major limitations of cord blood transplantation (CBT). Delayed engraftment seems to be due to the diminished ability of the cord blood stem cells to home to the bone marrow. How are cells channeled to the bone marrow? A protein receptor called P- and E-selectins is expressed on the surfaces of bone marrow blood vessels. Cells that can bind these selectin receptors will pass from the circulation to the bone marrow. Thus binding selectin receptors is kind of like having the “password” for the bone marrow.

What does it take to bind the selectin proteins? Selectins bind to specific sugars that have been attached to proteins. These sugars are called “fucose” sugars. As it turns out, cord blood stem cells do not express robust levels of these fucosylated proteins. Could increasing the levels of fucosylated proteins on the surfaces of cord blood stem cells increase their engraftment? Shpall and her colleagues tested this hypothesis in patients with blood-based cancers.

Patients with blood cancers had their cancer-producing bone marrow stem cells destroyed with drugs and radiation. Then these same patients had their bone marrows refurbished with two units of umbilical cord blood. However, these cells in these cord blood units were treated with the enzyme fucosyltransferase-VI and guanosine diphosphate fucose for 30 minutes before transplantation. This treatment should have increased the content of fucosylated proteins on the surfaces of cells in the hope of enhancing their interaction with Selectin receptors on the surfaces of bone marrow capillaries.

The results of 22 patients enrolled in the trial were then compared with those for 31 historical controls who had undergone double unmanipulated CBT. There was a clear decrease in the length of time it took for cells to engraft into the bone marrow.  For example, the median time to neutrophil (a type of white blood cell) engraftment was 17 days (range 12-34) compared to 26 days (range, 11-48) for controls (P=0.0023). Platelet (a cell used in blood clotting) engraftment was also improved: median 35 days (range, 18-100) compared to 45 days (range, 27-120) for controls (P=0.0520).  These are significant differences.

These findings support show that treating cord blood cells with a rather inexpensive cocktail of enzymes for a short period of time before transplantation is a clinically feasible means to improve engraftment efficiency of CBT.  This is a small study.  Therefore, these data, though very hopeful, must be confirmed with larger studies.

Stem Cell Structure and Obesity


New research conducted at Queen Mary University of London (QMUL) has discovered that the regulation of the length of primary cilia, which are small hair-like projections on the surfaces of most cells, can prevent the production of fat cells taken from adult human bone marrow. Such a discovery might be used to develop a way of preventing obesity.

What are primary cilia?  For many years, almost all attention was focused on cilia that moved because their function was readily observable.  However, Alexander Kowalevsky first reported in 1867 the presence of single (nonmotile) cilia in a variety of vertebrate cells.  These solitary and nonmotile cilia are far more widespread than the motile type.  In humans, only a few cell types have motile cilia, namely epithelial cells in the bronchi and oviducts, and ependymal cells that line brain vesicles.  However, virtually all other cells have a primary cilium.

What makes primary cilia different from the motile form? First, they lack the central pair of microtubules, which would explain the lack of motility.  Primary cilia also seem to lack dynein, one of the molecular motors needed for motility.  In addition, some primary cilia do not project beyond the cell surface, and most, but not all, are very short.  What do these organelles do if they are not sticking out of the cell, or motile?

Further work has shown that primary cilia are important in intracellular transport and also in sensory function for cells.  Now it seems that primary cilia are also important in the process of adipogenesis.

Primary cilia

Adipogenesis refers to the differentiation of stem cells into fat cells. The QMUL research team showed that during adipogenesis, the length of primary cilia increases, which increases the movement of specific proteins associated with the cilia. When the QMUL team genetically restricted primary cilia elongation by genetic means, they were able to stop the formation of new fat cells.

One of the lead authors or this study, Melis Dalbay, said that it was the first time that subtle changes in primary cilia structure can influence the differentiation of stem cells into fat.

Since the length of primary cilia can be influenced by various factors including pharmaceuticals, inflammation and even mechanical forces, this study provides new insight into the regulation of fat cell formation and obesity.

This research points toward a new type of treatment known as “cilia-therapy” where manipulation of primary cilia may be used in the future to treat a growing range of conditions including obesity, cancer, inflammation and arthritis.

Safer Culture Conditions for Stem Cells


Jeanne Loring from the Scripps Institute is the senior author of a very important study that examined the culture conditions for pluripotent stem cells.

Several scientists have discovered that induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) can accumulate cancer-causing mutations when grown in culture for extended periods of time (for example, see Uri Weissbein, Nissim Benvenisty, and Uri Ben-David, J Cell Biol. 2014 Jan 20; 204(2): 153–163). However, some laboratories have managed to keep ESCs in culture for extended periods without observing instabilities.

To try to tease apart why this might be the case, Loring and her group examined various culture methods and determined that some stem cell culture methods are associated with increased incidence of mutations in the DNA of stem cells.

“This is about quality control; we’re making sure these cells are safe and effective,” said Loring, who is a professor of developmental neurobiology at Scripps Research Institute (SRI) in San Diego, CA.

All cells run the risk of accumulating mutations when they divide, but previous research from Loring and her colleagues showed that particular culture conditions could potentially select for faster growth and mutations that accelerate growth. Such growth-enhancing mutations are sometimes associated with tumors.

“Most changes will not compromise the safety of the cells for therapy, but we need to monitor the cultures so that we know what sorts of changes take place,” said Ibon Garitaonandia, who is a postdoctoral research fellow in Loring’s laboratory at SRI.

New research from Loring’s group has shown how particular culture conditions can reduce the likelihood of mutations. Loring and her colleagues tested several different types of surfaces upon which the cells were grown. They also used different ways of propagating or “passaging” the cultures. When cells are grown in culture, the culture dishes must be scraped to get the cells off them and then the cells must be transferred to a fresh culture dish. How you do this matters: do you use enzymes to detach the cells, or do you mechanically scrape them off? Other culture techniques use layers of “feeder cells” that do not divide, but are still able to secrete growth factors that improve the health of the growing stem cells.

Loring and her crew tested various combinations of surfaces, passaging methods and feeder cell populations and grew the cells for three years with over 100 passages. Over the course of this experiment, the cells were sampled and analyzed for the presence of new mutations in their genomes.

It turns out that stem cells grown on feeder cells that are passaged by hand (manually) show the fewest growth-enhancing mutations after being cultured for three years.

Loring’s study also demonstrated the importance of monitoring cell lines over time. In particular, deletion of the TP53 gene, a tumor suppressor gene, in whose absence cancer develops, should be closely watched.

“If you want to preserve the integrity of the genome, then grow your cells under those conditions with feeder cells and manual passaging,” said Loring. “Also, analyze your cells. It’s really easy, she added.

When Thomson made the first human ESC lines, he used feeder cells derived from mouse skin cells.  However, the use of animal materials to make ESCs might pollute them with animal viruses and specific sugars from the surfaces of the animal cells might also contaminate the surfaces of the ESCs, making them unsuitable for regenerative medicine (see Stem Cells 2006; 24:221-229).  To address this problem, several laboratories have made “Xeno-free” ESC lines that were made without touching any animal products.  Some of these Xeno-free lines were made without feeder cells (see C. Ellerström, et al., Stem Cells. 2006 Oct;24(10):2170-6)., but others were made with human feeder cell lines (see K Rajala, et al., Hum Reprod. 2007 May;22(5):1231-8). Therefore, it appears, that the use of human feeder cell lines are preferable to feeder-free systems, given Loring’s findings.  However, it is also possible that such culture systems are also preferable for iPSCs, which do not have the problem of immunological rejection for patients, and do not require the killing of the youngest members of humanity.  Therefore, Loring’s work could very well benefit iPSC cultures as well.

Long-term Tumorgenicity of Induced Pluripotent Stem Cells


A paper from the Okano laboratory has shown that implantation of neural stem cells made from induced pluripotent stem cells can still form tumors ever after a long period of time.

This paper is an important contribution to the safety issues surrounding induced pluripotent stem cells (iPSCs). As noted in previous posts, iPSCs are made from adult cells by means of genetic engineering and cell culture techniques. In short, by introducing four different genes into adult cells and then culturing them in a special culture medium, a fraction of these cells will de-differentiate into cells that resemble embryonic stem cells in many ways, but are not exactly like them.

The Okano laboratory made iPSCs using viruses that integrate into the genome of the host cell, which is not the safest option. However, because in the four-gene cocktail that is normally used to reprogram these cells (Oct-4, Klf-4, Sox2, and c-Myc), the c-Myc gene is often thought to be the main cause of tumor formation. Okano and his collaborators made their iPSCs without the c-Myc gene, but only used the three-gene cocktail of Oct-4, Klf-4, and Sox2. Such a cocktail is much less efficient that the four-gene cocktail, but it supposed to make iPSCs that are altogether safer.

These iPSCs were differentiated into neural stem cells that grew as tiny spheres of cells, and these “neurospheres” were transplanted into the spinal cords of mice that had suffered a spinal cord injury. The implanted cells differentiated into neurons and glial cells and restored some neural function to these mice. However, the mice were observed for a long period of time after the implantations to assess the long-term safety of these implanted cells.

After 105 days, the implanted mice began to show deterioration of their neural function and their spinal cords showed tumors. It is clear that the Oct-4 gene that was used in the reprogramming procedure was the reason for the tumor transformation.

Graphical Abstract 20141213

This experiment, once again, calls into question the safety of any method for iPSC generation that leaves the transfected genes in the reprogrammed cells. I reported in a previous post that skin cells made from iPSCs that had their transgenes left in them were good at causing tumors and not as good as forming skin cells, but iPSCs without their reprogramming transgenes were safer and more effective tools for regenerative medicine.  This experiment also shows that c-Myc is not the only concern with iPSCs.  Any of the transgenes used for reprogramming can cause problems, and they must be removed if iPSCs are going to produce safe, differentiated cells.  Finally, this experiment pretty much shows that the use of retrovirus tools to introduce genes into cells for the sake of reprogramming is a bad idea if those cells are going to be used for regenerative medicine.  Non-integrating tools are much safer and preferable in these cases.

The Okano paper appeared in Stem Cell Reports.

Infertility Treatment with Stem Cells is Unlikely


Because several laboratories have managed to differentiate embryonic stem cells into cells that look very much like human eggs and sperm, many have predicted that infertility will be treated with stem cell treatments (see Volarevic V, et al., Biomed Res Int. 2014;2014:507234). However, new work from the University of Gothenburg and Karolinska Institute has cast doubt on this hope.

At about 24 days of life, large, spherical sex cells are recognizable among the endodermal cells of the umbilical vesicle close to the allantois. These cells are the primordial germ cells and they are the progenitor cells of the sperm in men and eggs in women. As the embryo folds during about the late 4th week, the dorsal portion of the umbilical vesicle is incorporated into the embryo. This incorporation of the umbilical vesicle occur concurrently with the migration of these primordial germ cells along the dorsal mesentery of the hindgut to the gonadal ridges. During the 6th week of life, the primordial germ cells enter the underlying mesenchyme and are incorporated into the gonadal cords. Primordial germ cell migration is mainly regulated by three genes: Stella, Fragilis, and BMP-4.

PGC migration

These primordial germ cells divide as they migrate, and by five months of gestation, embryonic ovaries contain about six to seven million oogonia. Most of these oogonia experience cell death before birth, but the remaining oogonia begin meiosis toward the end of gestation. At this time, the oogonia are called primary oocytes. Meiosis is arrested in prophase of the first meiotic division, and this is the same stage at which spermatogenesis in the male is blocked. Primary oocytes decrease in number throughout a woman’s life. The ovaries of a newborn girl contain about two million primary oocytes and these are all the gametes she will ever have. Each primary oocyte is contained within a hollow ball of cells called the ovarian follicle. By the time a woman reaches puberty, that number of primary oocytes has been reduced to 400,000. Only about 400 of these cells will ovulate during a woman’s reproductive years. The rest will die by means of programmed cell death. Once all the primary oocytes are gone, ovulation stops and the woman undergoes menopause.

Kui Liu from the University of Gothenburg said: “Ever since 2004, the studies on stem cell research and infertility have been surrounded by hype. There has been a great amount of media interest in this, and the message has been that the treatment of infertility with stem cells is about to happen. However, many researchers, including my research group, have tried to replicate these studies and not succeeded. This creates uncertainty about whether it is all possible to create new eggs with the help of stem cells.”

In collaboration with Outi-Hovatta’s laboratory at the Karolinska Institute and Jan-Åke Gustafsson’ research team at the university of Houston in the US, Lui’s research team carried out experiments on mice that failed to demonstrate that functional gametes could be formed from pluripotent stem cells. Essentially, the only gametes that could that the female mice had were the ones they were born with.

In Liu’s opinion, fertility clinics should place their attention on using the eggs that women still have in order to treat infertility.

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.

A Way to Get Stem Cells to Make Living Heart Valve Tissue?


What a benefit it would be to be able to replace diseased and defective heart valves with new heart valves. Thus, living tissue engineered heart valves (TEHV) would be a boon to children who require replacement heart valves that have the capacity to grow with the child and completely integrate into the child’s heart tissue. A persistent challenge for TEHV is accessible human cell source(s) that have the ability to mimic native valve cell phenotypes and possess matrix remodeling characteristics that are essential for long-term function.

Mesenchymal stem cells derived from bone marrow (BMMSC) or adipose tissue (ADMSC) are intriguing cell sources for TEHV. Unfortunately, they have not been compared to pediatric human aortic valve interstitial cells (pHAVIC) in relevant 3-dimensional culture environments.

In a recent study, Bin Duan from the Biomedical Engineering department at Cornell University compared the spontaneous and induced multipotency of ADMSC and BMMSC to that of pHAVIC using different induction culture systems within three-dimensional (3D) bioactive hybrid hydrogels that have similar material properties to those of aortic heart valve leaflets. pHAVICs possessed some multi-lineage differentiation capacity in response to induction media, but these cells were limited to the earliest stages and their differentiation capacity were less potent than either ADMSCs or BMMSCs. ADMSCs expressed cell phenotype markers that were similar to pHAVICs when they were grown in HAVIC growth media spiked with a growth factor called basic fibroblast growth factor (bFGF). BMMSCs generally expressed extra cellular matrix remodeling characteristics similar to pHAVICs.

Duan and his colleagues then chemically attached bFGF to components of the 3D hybrid hydrogels in order to further immobilize them. The immobilized bFGF upregulated vimentin expression and promoted the fibroblastic differentiation of pHAVIC, ADMSC and BMMSC. Since fibroblasts help make heart valves, these changes in gene expression might presage the ability of these cells to form new heart living heart valve tissue.

Thus, these findings show that even though mesenchymal stem cells retain a heightened capacity to form bone in 3D culture, this tendency can be shifted fibroblast cell fates by tethering bFGF to the 3-D matrix. Such a strategy is probably rather important for utilizing stem cell sources in heart valve tissue engineering applications.

This is an important finding.  Even though the production of TEHVs are some ways off, Duan’s findings might provide a strategy to begin cells on the path to making TEHVs.