Making Pancreatic Beta Cells from Embryonic Stem Cells

Type 1 diabetes results from an inability to produce sufficient quantities of the hormone insulin. Without insulin, the body does not receive the signal to take up sugar from the blood, and the result is high blood sugar levels, which are damaging to tissues, and a general wasting of tissues because they cannot take up enough sugar to feed them.

Pancreatic beta cells

The cells in the pancreas that produce insulin are the beta cells, and animal studies have shown that transplantation of new beta cells into diabetic animals can reverse and even in some cases cure the diabetic animals. Therefore researchers have tried to make beta cells from pluripotent stem cells in order to make a source of beta cells for transplantation.

Unfortunately, beta cell production in the laboratory has been fraught with problems. The cells produced by differentiation of embryonic stem cells do not have the characteristics of mature beta cells and they produce little insulin and are not glucose responsive (D’Amour, et al., (2006) Nat Biotechnol 24, 1392-1401).

A different strategy, however, works much better. Instead of differentiating stem cells into beta cells, differentiate them into those cells that will form beta cells and other types of pancreatic cells in the embryo – immature endocrine cell precursors – and then transplant those into the pancreas of diabetic mice. In this case, the endocrine cell precursors differentiate in the bodies of the mice into pancreatic beta cells that greatly resemble normal beta cells.

Why don’t embryonic stem cells for beta cells in culture? This question was pursued by a collaboration between research team led by Maike Sander at UC San Diego and a company called ViaCyte, Inc.

When it comes to endocrine precursors transplanted into mice, Dr. Sander noted that, “We found that the endocrine cells retrieved from transplanted mice are remarkably similar to primary human endocrine cells.” He continued, “This shows that hESCs (human embryonic stem cells) can differentiate into endocrine cells that are almost indistinguishable from their primary human counterparts.”

Well, ESCs can make perfectly fine beta cells in the mouse body, but not in culture. What’s up with that?

Sander and her colleagues examined the gene expression patterns of embryonic stem cells as they were differentiated and compared them with the gene expression patterns in those cells that were transplanted into mice and allowed to differentiate inside the body of the mouse.

What Sander and her team found was astounding. As cells progress through their developmental program, particular genes are brought on-line and expressed, and then turned off as the cells passed through each stage of endocrine cell differentiation. The cellular machinery that shuts off genes after they have been activated consists of a family of proteins that remodel chromatin known as the Polycomb group (PcG). PcG-mediated repression of genes silenced those genes that were only turned on temporarily once they were no longer required.

Two major Polycomb repressive complexes (PRCs) have been described. The PRC2 complex contains the histone methyltransferase enhancer of zeste homologue 2 (EZH2), which together with embryonic ectoderm development (EED) and suppressor of zeste 12 homolog (SUZ12) catalyses the trimethylation of histone H3 at lysine K27 (H3K27me3). The EZH2 SET domain confers this activity. Multiple forms of the PRC1 complex exist and these contain combinations of at least four PC proteins (CBX2, CBX4, CBX7 and CBX8), six PSC proteins (BMI1, MEL18, MBLR, NSPC1, RNF159 and RNF3), two RING proteins (RNF1 and RNF2), three PH proteins (HPH1, HPH2 and HPH3) and two SCML proteins (SCML1 1 and SCML2). Some results have suggested that PRC1 complexes are recruited by the affinity of chromodomains in chromobox (Cbx) proteins to the H3K27me3 mark. PRC1 recruitment results in the RNF1 and RNF2-mediated ubiquitylation of histone H2A on lysine 119, which is thought to be important for transcriptional repression. PC, Polycomb; PSC, Posterior sex combs ; SCML, Sex combs on midleg .
Two major Polycomb repressive complexes (PRCs) have been described. The PRC2 complex contains the histone methyltransferase enhancer of zeste homologue 2 (EZH2), which together with embryonic ectoderm development (EED) and suppressor of zeste 12 homolog (SUZ12) catalyses the trimethylation of histone H3 at lysine K27 (H3K27me3). The EZH2 SET domain confers this activity. Multiple forms of the PRC1 complex exist and these contain combinations of at least four PC proteins (CBX2, CBX4, CBX7 and CBX8), six PSC proteins (BMI1, MEL18, MBLR, NSPC1, RNF159 and RNF3), two RING proteins (RNF1 and RNF2), three PH proteins (HPH1, HPH2 and HPH3) and two SCML proteins (SCML1 1 and SCML2). Some results have suggested that PRC1 complexes are recruited by the affinity of chromodomains in chromobox (Cbx) proteins to the H3K27me3 mark. PRC1 recruitment results in the RNF1 and RNF2-mediated ubiquitylation of histone H2A on lysine 119, which is thought to be important for transcriptional repression. PC, Polycomb; PSC, Posterior sex combs ; SCML, Sex combs on midleg .

In the transplanted endocrine precursors, Sanders and his team noted an orderly progression of genes that were turned on and then turned off once as needed. However, in the embryonic stem cells that were differentiated into beta cells in culture, they discovered that these cells failed to express the majority of genes critical for endocrine cell function. The main reason for this appeared to be that the PcG-mediated repression of genes was not fully eliminated when particular genes had to be expressed at specific developmental stages. Thus these cultured cells failed to fully eliminate PcG-mediated repression on endocrine-specific genes, which contributes to the abnormality of the culture-derived beta cells.

Sander commented: “This information will help devise protocols to generate functional insulin-producing beta cells in vitro. This will be important not only for cell therapies, but also for identifying disease mechanisms that underlie the pathogenesis of diabetes.”

When To Use Umbilical Cord Blood Stem Cells

Umbilical cord blood stem cells (UCB-SCs) have been used in a variety of clinical trials and treatments. Their use in treatment bone marrow-based conditions is very well-known, but they have also been used in other experimental treatments as well.

Treatments with UCB-SCs suffer from inconsistent results that stem from a variable number of viable cells in UCB-SC samples. Establishing high numbers of viable cells in UCB-SC samples is not easy, and there is a great interest in being able to grow UCB-SCs in culture and expand them. However, even though UCB-SCs can be grown in culture, the effects of culturing UCB-SCs is presently unclear.

To address this question in a rigorous fashion, Miguel Alaminos at the University of Granada and his colleagues grew UCB-SCs in culture and analyzed cell viability and gene expression at every passage.

What they discovered was astounding. When UCB-SCs were passaged two or three times, the cells showed signs of cells death, and gene expression studies revealed that many of the cells expressed genes associated with programmed cell death. Cells passaged eight, nine, or ten times also showed extensive cell death. However, cells passaged five or six times showed the highest viability.

This suggests that different studied have used cells that were grown for different periods of time and probably had different viabilities. This explains why UCB-SCs have performed so variably in experiments and clinical trials. This suggests that therapies that utilize UCB-SCs should use them after they are passaged for the fifth or sixth time in order to ensue the highest levels of viability.

Specificity Protein 2 Required for Neuron Formation

In mammals, cells contain a group of proteins known as “specificity factors” that acting during gene expression. Specificity proteins, or Sps, bind to DNA at specific sequences and activate gene expression at those genes that possess the binding target for the Sps. Sps control the expression of a many genes, including house keeping, tissue-specific, development-specific and cell-cycle-regulated genes. There are nine different Sps that have been discovered in mammals, and they are numbered Sp1 to Sp9. For a good article on Sps, see Suske G, Bruford E, Philipsen S (2005) Mammalian SP/KLF transcription factors: bring in the family. Genomics 85: 551–556.

Several of the Sps have been shown to play rather central roles during development. For example, mouse embryos that lack Sp1, die very early (around embryonic day 10.5). Mice that do not have functional Sp3, develop until the end of pregnancy but die immediately after birth due to respiratory failure. Such embryos also show impaired skeletal bone ossification, tooth development, heart development, and abnormal organization of the placenta. Newborn Sp4-deficient mice either die within the first month of life or are severely growth-retarded, and males do not breed and females show delayed in sexual maturation. Sp4 is also required for the development of the conduction system in the heart.

Of the Sps, Sp2 has been very poorly characterized. For this reason, Troy Ghashghaei at North Carolina State University has investigated the role of Sp2 in neural stem cells.

In collaboration with Jon Horowitz, a colleague at the Center for Comparative Medicine and Translational Research, they made mouse neural stem cells that lacked functional Sp2. The neural stem cells without Sp2 were able to divide, but the progeny were unable to differentiate into neurons. Instead the neural stem cells simply divided over and over without ever forming neurons.

This result was unexpected, since Horowitz had shown in an earlier publication that overproducing Sp2 did something similar in skin stem cells. Instead of dividing and forming new skin cells, skin stem cells that expressed excessive amounts of Sp2 continued to divide and without forming new skin cells. Instead they formed tumors (Kim TH, et al., Cancer Res. 2010 Nov 1;70(21):8507-16. doi: 10.1158/0008-5472.CAN-10-1213).

In Horowitz’s view: “We believe that Sp2 must play a fundamental role in the lives of normal stem cells. Trouble ensues when the mechanisms that regulate its activity are overwhelmed due to its excess abundance.”

However, this recent work shows that in a different system, the absence of Sp2 has much the same effect – prevent of stem cells from producing progeny that differentiates into mature cell types and continued, uncontrolled proliferation.

Of this Ghashghaei said: “It’s interesting that both an overabundance of this protein and a total lack of it result in similar disruptions in how stem cells divide. So while this work confirms that Sp2 is absolutely necessary for stem cell function, a lot of questions still remain about what exactly it is regulating, and whether it is present in all stem cells or just a few. We also need to find out if Sp2 deletion or overabundance can produce brain tumors in our mice as in the skin.”

Ghashghaei continued: “Lastly, we are very interested in understanding how Sp2 regulates a very important decision a stem cells has to make: whether to produce more of itself or to produce offspring that can become neurons or skin cells. We hope to address these questions in our future research, because these cellular mechanisms have implications for cancer research, neurodevelopmental diseases and regenerative medicine.”

See Liang H, Xiao G, Yin H, Hippenmeyer S, Horowitz JM, Ghashghaei HT. Neural development is dependent on the function of specificity protein 2 in cell cycle progression.  Development. 2013 Feb;140(3):552-61. doi: 10.1242/dev.085621.

Local Anesthesia Inhibits Mesenchymal Stem Cells

Anyone who has had dental work or particular out-patient procedures has had local anesthesia. Local anesthesia inhibits local sensory nerve function and induces numbness. Several studies have shown that when used at high concentrations, local anesthesia can cause particular cells to die. Therefore, some physicians worry that local anesthesia might affect stem cells, but the effects of local anesthesia on mesenchymal stem cells is largely unknown.

To this end, Michael Zaugg from the University of Alberta and his talented co-workers examined the effects of local anesthesia on mesenchymal stem cells from bone marrow. Their results were from experiments on cultured mesenchymal stem cells.

When mouse bone marrow mesenchymal stem cells were isolated and grown in culture and exposed to 100 micromolar concentrations of three different local anesthetics, lidcocaine, ropivacaine, and bupivacaine, they discovered that the mesenchymal stem cells grew much more slowly. In fact, the stem cells seemed to divide and then give up the ghost. Therefore, local anesthetics seemed to inhibit mesenchymal stem cell proliferation.

Upon further investigation, the stem cells stopped dividing at the point when they were supposed to start making new DNA. This phase of the life of the cell is called the S phase for synthesis phase, and the molecule made by the cell at this time is DNA. However, the mesenchymal stem cells exposed to local anesthetics failed to initiate DNA synthesis.

The next question Zaugg and his team asked was whether or not the stem cells had trouble making energy, which is a common feature of cell exposed to too much local anesthetic. Indeed, the mesenchymal stem cells exposed to local anesthetics showed reduced energy production.

A more sophisticated analysis called “microarray analysis,” which examines the gene expression patterns in a cell by a gene-by-gene basis, showed that those genes necessary for cell membrane synthesis were greatly decreased when the cells were exposed to local anesthetics. Furthermore, the mesenchymal stem cells exposed to local anesthetics differentiated quite poorly, and the microarray analysis confirmed this observation, since those genes necessary for differentiation in mesenchymal stem cells were down regulated in the presence of local anesthetics.

Before conclusions can be drawn about what local anesthetics do to a living creature during wound healing, more work must be done, First of all, these results from cultured cells may not hold true in a living organism. Also, the concentration of anesthetic used in this study is well above what are acknowledged to be toxic levels for these drugs. Therefore, while these results are informative and interesting, the must be interpreted with some caution.

Leprosy Bacterium Reprograms Adult Cells into Stem Cells

Hansen’s disease is another name for the modern known as leprosy. Leprosy is known from old documents, for example, the Bible, but what is described in the Old Testament as leprosy seems to be a combination of various conditions. Plague psoriasis, for example, could fit the biblical description of leprosy. Also, in the Old Testament, a house or fabrics could get leprosy (Leviticus 13-14, which suggests that mildew, or something like it, was regarded as leprosy. Thus leprosy in the Old Testament seems to refer to a broad category of diseases. However, in the New Testament, when leprosy is described, it might be a variant of the modern Hansen’s disease.

Hansen's disease

Hansen’s disease is caused by a microorganism called Mycobacterium leprae. It causes skin lesions, loss of sensation, muscle weakness, and numbness in the hands, arms, feet and legs. The skin lesions are lighter than normal skin color, which have decreased sensation to touch, heat, or pain. These lesions do not heal after several weeks to months.

Leprosy is not very contagious and it has a long incubation period (time before symptoms appear). This makes it rather difficult to know where or when someone caught the disease. Children are more likely than adults to get leprosy.

There are two common forms of leprosy, tuberculoid and lepromatous leprosy. Both forms produce sores on the skin, but the lepromatous form is most severe. It causes large lumps and bumps. Leprosy is common in many countries worldwide, but it is also found in temperate, tropical, and subtropical climates. There are about 100 cases diagnosed per year in the United States, and most are in the South, California, and Hawaii.

Mycobacterium leprae (M. leprae) attacks, among other things, the peripheral nerves. The organism causes the insulating myelin sheath that surrounds the nerve to unravel, thus leaving the nerves without their insulating layer, which causes nerve malfunction. However, recent work has shown that M. leprae unravels the myelin sheath by reprogramming the cells that make the myelin sheath. These myelin-making cells are known as Schwann cells, and M. leprae, reprograms Schwann cells to revert to a stem-cell-like state, which causes them to leave the nerves, leaving the nerves in the lurch.

schwann cells

These finding were published in the prestigious international journal Cell.

Scientists from the laboratory of Anura Rambukkana, who holds a dual appointment at the Rockefeller University in New York and The MRC Centre for Regenerative Medicine in Edinburgh, Scotland, discovered this remarkable finding while examining how leprosy spreads around the body.

The initial target of M. leprae is Schwann cells. To understand how the organism affects Schwann cells, Rambukkana and co-workers isolated Schwann cells from mice and infected them with M. leprae. Once infected with M. leprae, the infecting bacteria reprogrammed the cells into a stem-like state. It turned off genes associated with mature Schwann cells and turned on genes associated with embryonic stages or other developmental stages.

M. leprae seemed to trigger Schwann cells’ plasticity. Plasticity refers to the ability of cells to revert to an immature state and differentiate into new types of cells. In fact, healthy Schwann cells do exactly that in order to help nerves recover and regenerate after an injury.

Rambukkana notes that however the bacteria are reprogramming the Schwann cells, they seem to be employing a “very sophisticated mechanism — it seems that the bacterium knows the mechanistic interaction of the Schwann cell better than we do.”

Upon being reprogrammed, the stem cells can migrate to different locations in the body with the bacterium housed inside then. Once the infected cells reach another tissue, such as skeletal muscle, the stem cells integrate with that tissue’s cells, thus spreading the bacteria. The infected stem cells also attract immune cells by secreting summoning proteins called chemokines. This brings more potential carriers to the bacteria’s doorstep.

What do the bacteria do to trigger a reprogramming event? At this point these researchers do not know, but they suspect that the mechanism could exist in other infectious diseases.

According to Sheng Ding, a stem cell biologist at the Gladstone Institute of Cardiovascular Disease in San Francisco, CA, “Cellular plasticity may represent an underlying mechanism of disease, as other cellular reprogramming events have been shown in cancers and metabolic diseases.”

By understanding these precise mechanisms, scientists could devise precise ways to improve treatment and earlier diagnosis of leprosy itself. These latest findings also provide vital clues about how leprosy spreads throughout the body, and how to catch the disease before it spreads rapidly.

In the future, bacteria or products made by the bacteria could be used to change adult tissue cells into stem cells in the laboratory, thus potentially leading to new regenerative treatments for diseases such as diabetes and Alzheimer’s.

See Masaki, T. et al. Cell 152, 51–67 (2013).

Stem Cells Heal Damaged Baboon Arteries in the Lab

A research group at the Texas Biomedical Research Institute in San Antonio, Texas has reprogrammed embryonic stem cells derived from baboon embryos to completely restore a severely damaged artery. Such results lay the ground work for what might be a new way to completely heal large blood vessels that have been damaged by congenital diseases, the ravages of disease, or simply old age.

John L. VandeBerg, chief scientific officer at the Texas Biomedical Research Institute, said: “We first cultured the stem cells in Petri dishes (culture dishes) under special conditions to make them differentiate into cells that are the precursors of blood vessels, and we saw that we could get them to form tubular and branching structures, similar to blood vessels.”

Since VandeBerg and his colleagues were able to differentiate baboon embryonic stem cells into blood vessel precursors, they wanted to try a much more difficult experiment and try to use these blood vessels precursor cells to repair a damaged blood vessel in a simulated environment.

By removing the cells that line the inside of a baboon artery, VandeBerg and co-workers replaced the lining with the blood vessel precursors derived from baboon embryonic stem cells. Then they connected this artery segment to a plastic tubing inside a device known as a “bioreactor.” Bioreactors are designed to grow cells and tissues under conditions that closely mimic those inside the human body. In this case, the bioreactor also pumped fluid through it as though it were inside a real, living baboon.

The artery was bathed in culture medium, and by three days, the complex inner layer of the artery showed signs of regenerating, and by 14 days, it was perfectly restored to its complex, natural state. In two weeks, the artery had gone from stripped to a fully functioning artery.

VandeBerg said of these experiments: “Just think of what this kind of treatment would mean to a patient who had just suffered a heart attack as a consequence of a damaged coronary artery. And this is the real potential of stem cells regenerative medicine – that is, a treatment with stem cells that regenerates a damaged or destroyed tissue or organ.”

A control experiment also showed that the arteries could not regenerate without the added cell stems, they used an artery that can been internally stripped and hooked it up to the bioreactor without seeding it with stem cells. Under these conditions, no healing occurred.

When the arteries were stained for those proteins normally found in a properly functioning artery, the healed artery showed all the same staining characteristics as arteries that had not been internally stripped. Of this result, VandeBerg noted: “This is evidence that we can harness stem cells to treat the gravest of arterial injuries.”

Researchers such as Vandeberg hope to take a skin cell or a white blood cell, or a cell from just about anywhere else in the body and induce it to differentiate into induced pluripotent stem cells that can be used to differentiate into blood vessel precursors that can be used to repair damaged blood vessels.

Three-Dimensional Scaffolds that Support that Regeneration of Tissues by Stem Cells

When cultured in the laboratory, stem cells can form tissues that are commonly found in our own body. However, the size, shape and organization that what stem cells make in culture tends to not resemble what is observed in our bodies.

There are ways to coax stem cells to make tissues that more closely resemble those in our bodies. This includes growing stem cells on “biomimetic” scaffolds that have the same shape and organization as our own tissues. Such scaffolds direct the growth and organization of the stem cells and the tissues that form so that they more closely resemble our own.

Researchers from Singapore at the Nanyang Technological University of Singapore and collaborators, led by Zu-yong Wang, have invented a clever and innovative method that creates a stretched polymer scaffold that supports complex tissue architecture. By stretching this polymer (poly ε-caprolactone for the interested) thin-film, it can actually produce scaffolds that can support the growth of mesenchymal stem cells.

This stretching process generates a nice three-dimensional with micro-grooves that are oriented on the surface of the film. These grooves direct stem cells to grow in a neatly aligned fashion that can develop into tissues as the stem cells grow on and eventually degrade and absorb the scaffold.  Such a finding advances tissue engineering research, the goal of which is to use stem cells to remake new organs to replace damaged or disease ones.