Blocking Differentiation is Enough to Turn Mature Cells into Stem Cells

Hiroshi Kawamoto led a collaboration between the RIKEN Center for Integrative Medical Science and other institutions in Japan and Europe that examined the possibility that adult cells can be maintained in a stem cell-like state where they can proliferate without undergoing differentiation. They discovered that in immune cells, blocking the activity of one transcription factor can maintain the cells in a stem cell-like state where they continue to proliferate and still have the capacity to differentiate into different mature cell types.

Kawamoto and his team genetically engineered hematopoietic progenitor cells from mice to overexpress the Id3 protein. Id3, or inhibitor of DNA binding 3, is an inhibitory protein that forms nonfunctional complexes with other transcription factors. In particular, Id3 inhibits so-called “E-proteins,” (such as TCF3) which drive the progenitor cells to differentiate into immune cells.

Overexpression of Id3, in addition to soaking the cells in a cocktail of cytokines, cause the cells to continue to divide as stem cells. However, when the cytokines were withdrawn, the cells differentiated into various types of immune cells.

Next, Kawamoto and his collaborators infused these engineered hematopoietic progenitors into mice that had been depleted of white blood cells. They discovered that their Id3-overexpressing cells could expand and replenish the white blood cell population of these.

In a follow-up experiment, Kawamoto and his crew recapitulated this experiment using human umbilical cord blood hematopoietic progenitors. Just like their mouse counterparts, these umbilical cord cells could be maintained in culture, and then, upon change of culture conditions, could differentiate into blood cells.

Because these cells can be kept in an undifferentiated state and can extensively proliferate, this culture system provides a model for studying the genetic and epigenetic basis of stem cell self-renewal. And it might also allow scientists to inexpensively grow large quantities of immune cells for regenerative medicine or immune therapies.

This work was published in Stem Cell Reports, October 2015 DOI: 10.1016/j.stemcr.2015.09.012.

Anti-Aging Protein GDF11: Does it Work?

The protein is called GDF11 and some scientists claim that is can rejuvenate older laboratory animals and make them healthier. Sounds like science fiction, but could it be true?

Several decades ago, in the 1950s, some creative and enterprising scientists connected the circulatory systems of two inbred mice, one of which was old and the second of which was young. The blood from the young mouse seemed to rejuvenate the older mouse. That led to a question: “If blood from younger mouse rejuvenated the older mouse, what was it in the blood that did it?” Further work has landed on GDF11 as the rejuvenating protein, but the experimental path to this protein has been fraught with false starts, bumps, and wrong turns. New work by a team of Harvard University scientists hopes to set the record straight on GDF11.

Work by Harvard stem cell biologist Amy Wagers, cardiologist Richard Lee and the members of their laboratories and their collaborators have discovered that the blood concentrations of GDF11 drop in mice as they age. Such a finding is a correlation, which might be suggestive, but it fall short of proving that GDF11 is an anti-aging protein. However, Wagers and Lee and their colleagues also showed that when older mice are injected with GDF11, the protein partially reverses the thickening of the heart that comes with age. Wagers and her team also showed in two papers that were published in the journal Science that administration of GDF11 can rejuvenate the muscles and brains of older mice.

Wagers’ findings, however, received some push-back in May, 2015. According to Jocelyn Kaiser, writing at the Science web site, David Glass, who works at the Novartis Institutes for Biomedical Research in Cambridge, Massachusetts, and his colleagues have made use of an antibody that specifically binds to GDF11 to detect the protein and measure its concentration in the blood and tissues. Experiments with the anti-GDF11 antibody revealed that blood levels of GDF11 increase as rats and people get older. Also, in the hands of Glass and his team, injected GDF11 protein inhibited muscle regeneration in young mice. Furthermore, work from Steven Houser’s group at Temple University in Philadelphia, Pennsylvania, has shown that injections of GDF11 do not decrease the age-related thickening of the hearts of older mice. Now we have a genuine scientific controversy: so who’s right?

Wagers and Lee have concluded that the specific assay Novartis used to detect GDF11 and a related protein (GDF8 or myostatin) did not work properly. In their own experiments, the combined efforts of the Wagers and Lee teams showed that the main protein detected by the antibody test designed and used by the Glass group is immunoglobulin (antibodies). The levels of antibody proteins in the blood are known to rise in the blood as people get older. As a control, when the Wagers and Lee group used the Novartis-designed test to measure the proteins levels of laboratory mice that do not possess the gene that encodes antibodies, the blood of those mice tested negative. According to Jocelyn Kaiser, these data were published in a paper that appeared in the journal Circulation Research.

Wagers summarized the results of her and Lee’s laboratories, “They actually had very consistent findings to ours with respect to the blood levels of GDF11/8 with the antibody we all used.” However, according to Wagers, “their interpretation was confused by this case of mistaken identity.” To corroborate your point, Wagers cited a recently published study by scientists from the University of California, San Francisco, who found that GDF11/8 blood levels decline as people age, and are low in heart disease patients. These results support the hypothesis that GDF11 has antiaging activity.

The Harvard team’s paper also examined the results from the Houser laboratory. According to Wagers, Houser and his colleagues utilized commercially purchased GDF11, and this source of protein can vary in activity and levels. Wagers noted that it “wasn’t something that affected us early on, but we figured out it was an issue. The variability of commercially purchased GDF11 might explain why Houser and his colleagues were unable to see any results from injected GDF11. Houser and his team were quite careful to make sure that they injected the same dose of GDF11 as the Wagers and Lee. However, Wagers pointed out that if only a fraction of the protein was as active as the protein used by Wagers and Lee, then it is likely that Houser and his group actually used a lower effective dose than the Harvard group. Lee has also noted that he and his group have data that suggests that the GDF11 dose they used was actually higher than they initially thought.

Wagers and others also showed that daily injections of GDF11 can shrink heart muscle in both old and new mice, and, incredibly, the mice also lost weight. “We don’t have much insight into that right now, but we’re looking into it,” Wagers says. Wagers suspects that GDF11 only works within a particular therapeutic concentration, outside of which is will not work and above which it might cause side effects that are harmful.

What does the competition think? Houser thinks that Wager and Lee are probably correct that at least one of the assays used by the Novartis team to measure GDF11 detected immunoglobulin. However, both Houser David Glass have pointed out that the Novartis team used a different GDF11 detection assay whose accuracy was not challenged by the work in this new paper.

Houser remains sanguine about finding molecules that can delay aging.  “I’m going to be 65 in a couple months. I’d love to have something that improves my heart, brain, and muscle function,” said Houser. “I think the field is going to figure this out and this is another piece of the puzzle.”

The jury is still out when it comes to GDF11, but Wagers and Lee have made a positive contribution to a robust and thrillingly interesting scientific discussion.

Repairing Nerves Using Exosomes to Hijack Cell-Cell Communication

Biomedical engineers from Tufts University have discovered a new protocol that can induce mesenchymal stem cells (MSCs) derived from bone marrow to differentiate into neuron-like cells by treating them with exosomes from cultured cells.

PC12 cells are neuron-like progenitor cells derived from rats that can be successfully grown in culture. The Tufts team, led by Qiaobing Xu, found that exosomes extracted from cultured PC12 cells at various stages of differentiation could drive MSCs to differentiate into neuron-like cells.

Exosomes are very small, hollow particles that a wide range of cells types secrete. These tiny vehicles contain proteins, RNA, and other small molecules, and serve as a vehicle for communication between cells. In the nervous system, exosomes guide the direction of nerve growth, and they control nerve connection and direct peripheral nerve regeneration.

Xu and his team showed that these exosomes contain microRNAs (miRNAs), which a small RNA molecules that regulate gene expression and are known to play a role in neuronal differentiation. They hypothesized that these miRNAs activate neuron-specific genes in the MSCs that receive them and this is the reason these cells begin their journey towards differentiating into neurons.

“In combination with synthetic nanoparticles, we may ultimately be able to use these identified miRNAs or proteins to make synthetic exosomes, thereby avoiding the need to use any kind of neural progenitor cell line to induce neuron growth,” said Xu.

This work was published in PLoS ONE 2015; 10(8): e135111 DOI: 10.1371/journal.pone.0135111.

Closing the Door on the STAP Episode

Last year, a group of Japanese researchers, led by scientists from the high-regarded RIKEN Center for Developmental Biology, reported a break-through in stem cell technology. Their so-called STAP or stimulus-triggered acquisition of pluripotency cells could be derived from mature, adult cells by exposing those cells to stressful conditions. Even though the papers that reported these advances were published in the prestigious journal Nature, immediately, people found problems in the papers that could not be easily resolved. Several laboratories tried to replicate the STAP results, with no success. The papers were eventually retracted and an internal investigation by the RIKEN Center also suggested that foul play might have been at work. Amidst all this, a question that hung in the air was this, “Was there something to the original discoveries but it was overstated?”

That question has now been definitely answered in the negative, thus closing the door for good on this whole sordid affair. Two papers were published on 23 September in the journal Nature, which was the same journal that published the original, ill-fated papers early last year that showed that STAP cells should be called NE (never existed) cells.

The original STAP papers were published in January 2014 by a team led by researchers at the RIKEN Center for Developmental Biology (CDB) in Kobe, Japan, in collaboration with scientists from Harvard Medical School in Boston, Massachusetts. These two papers claimed that embryonic-like stem cells could be produced by exposing adult body cells to stress, such as acidic conditions or physical pressure. These papers dubbed their technology “stimulus-triggered acquisition of pluripotency,” or STAP. Unfortunately, other scientists quickly discovered problems with data in the research. These problems then generated an investigation, and these papers were eventually retracted.

The paper retraction, however, did not answer the nagging questions as to whether or not the STAP procedure might have worked, and where the pluripotent stem cells labelled STAP in the RIKEN laboratory came from.

Such questions were addressed by seven teams in four countries who tried to replicate the procedure under various conditions (De Los Angeles, A. et al. Nature (2015). These teams collaborated to generate 133 attempts to produce STAP cells, and all of these attempts failed. One of these teams was led by researchers at Harvard Medical School who had worked with one of the original STAP co-authors. In this laboratory, cells were engineered to express a fluorescent protein when a gene related to pluripotency was expressed. When cells were exposed to stressful conditions, they did find some fluorescence, which suggested that pluripotency genes were expressed when cells were subjected to such conditions. However further testing showed this result to be an artifact since cells can naturally emit light; a phenomenon known as autofluorescence. Six other groups also observed autofluorescence in stressed cells, but no convincing evidence of STAP conversion.

A group of RIKEN researchers that did not include any authors of the original STAP papers analyzed the genomes of purported STAP cell lines that had be derived at the CDB. These scientists discovered multiple instances of contradictory data that probably resulted from contamination of purported STAP cells by other known cell types. The RIKEN group’s analyses showed that all remaining purported STAP stem cell lines, for example, were genetically identical to embryonic stem cell lines that already existed in the laboratory.

Additionally the “chimeric” mice that were reportedly produced by injecting STAP cells into the embryo of a developing mouse were found to have been produced by injecting pre-existing embryonic cell lines, rather than STAP cells, into the embryo. The production of chimeric mouse embryos is an experiment that definitively shows that particular cells are truly pluripotent.

Cell contamination also explains one of the most puzzling features of the original work, and that has to do with why the alleged STAP cells were reported to be capable of forming placental tissue, which is something that embryonic stem cells are not able to do (De Los Angeles, A. et al. Nature 525, 469–478 (2015)). These most recent analyses show that mixtures of trophoblast stem cells (which form the placenta in a developing embryo) were mixed with embryonic stem cells and that this mixture was used in the mouse chimeric experiments, leading to the production of mouse placental and embryonic tissue.

Stem-cell scientist Rudolf Jaenisch of the Massachusetts of Technology in Cambridge, who was part of the replication efforts, originally suggested in April 2014 to Nature’s news team that contamination was the reason for the results in the STAP papers. Unfortunately, he did not have evidence at the time for his hypothesis, but this most recent work has vindicated Jaenisch’s hypothesis.

A lingering question is how these embryonic stem cells and trophoblast stem cells came to replace purported STAP cells when the chimeric mouse experiments were performed. So-called cross-examination, which is the accidental contamination of one cell culture by another type of cell, is a well-known problem in cell culture experiments and biological research that depends on cultured cells. However, to properly explain the results in the original STAP papers, multiple independent contamination events must be invoked. “It is very difficult to reconcile the data with simple contamination or careless mislabeling,” says stem-cell scientist George Daley at Harvard Medical School. Unfortunately, requests for clarifying comments from corresponding authors of the original papers went unanswered.

In a review article published in Nature, Daley, Jaenisch argue that all new reports of new types of pluripotency should be subjected to rigorous “forensic” analysis that examines the genomes of the cells under consideration before publication. According to the authors, besides the failed STAP papers, “numerous groups are reporting ever more nuanced states of pluripotency.” In particular, the article focuses on genomic analyses, which are enabled by advances in sequencing technology, that will help evaluate such cell types.

Daley says that these experiments bring some well-desired closure to the STAP. He ended, however, with a warning to scientists who are looking for ways to reprogram cells to an embryonic-like state: “We will all be a tad more cautious in evaluating such claims.”

Elabela, A New Human Embryonic Stem Cell Growth Factor

When embryonic stem cell lines are made, they are traditionally grown on a layer of “feeder cells” that secrete growth factors that keep the embryonic stem cells (ESCs) from differentiating and drive them to grow. These feeder cells are usually irradiated mouse fibroblasts that coat the culture dish, but do not divide. Mouse ESCs can be grown without feeder cells if the growth factor LIF is provided in the medium. LIF, however, is not the growth factor required by human ESCs, and therefore, designing culture media for human ESCs to help them grow without feeder cells has proven more difficult.

Having said that, several laboratories have designed media that can be used to derive human embryonic stem cells without feeder cells. Such a procedure is very important if such cells are to be used for therapeutic purposes, since animal cells can harbor difficult to detect viruses and unusual sugars on their cell surfaces that can also be transferred to human ESCs in culture. These unusual sugars can elicit a strong immune response against them, and for this reason, ESCs must be cultivated or derived under cell-free conditions. However, to design good cell-free culture media, we must know more about the growth factors required by ESCs.

To that end, Bruno Reversade from The Institute of Molecular and Cell Biology in Singapore and others have identified a new growth factor that human ESCs secrete themselves. This protein, ELABELA (ELA), was first identified as a signal for heart development. However, Reversade’s laboratory has discovered that ELA is also abundantly secreted by human ESCs and is required for human ESCs to maintain their ability to self-renew.

Reversade and others deleted the ELA gene with the CRISPR/Cas9 system, and they also knocked the expression of this gene down in other cells with small interfering RNAs. Alternatively, they also incubated human ESCs with antibodies against ELA, which neutralized ELA and prevented it from binding to the cell surface. However Ela was inhibited, the results were the same; reduced ESC growth, increased amounts of cell death, and loss of pluripotency.

How does ELA signal to cells to grow? Global signaling studies of growing human ESCs showed that ELA activates the PI3K/AKT/mTORC1 signaling pathway, which has been show in other work to be required for cell survival. By activating this pathway, ELA drives human ESCs through the cell-cycle progression, activates protein synthesis, and inhibits stress-induced apoptosis.

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Interestingly, INSULIN and ELA have partially overlapping functions in human ESC culture medium, but only ELA seems to prime human ESCs toward the endoderm lineage. In the heart, ELA binds to the Apelin receptor APLNR. This receptor, however, is not expressed in human ESCs, which suggests that another receptor, whose identity remains unknown at the moment, binds ELA in human ESCs.

Thus ELA seems to act through an alternate cell-surface receptor, is an endogenous secreted growth factor in human

This paper was published in the journal Cell Stem Cell.

Umbilical Cord Blood Contains c-kit+ Cells that Can Differentiate into Heart-like Cells

Bone contains a wide variety of stem cells whose potential are only beginning to be tapped. One cell population possesses a cell surface protein called c-kit, and these c-kit+ progenitor cells seem to support myocardial regeneration. Do c-kit+ cells from umbilical cord blood have the same capacity?

Luciana Teofili from the Catholic University of the Sacred Heart in Rome, Italy and her colleagues purified c-kit+ cells from umbilical cord blood by means of magnetic beads that were coated with c-kit-binding antibodies. Teofili and others induced heart muscle differentiation in these cells with several different protocols. Then the expression of cardiac markers (GATA4, GATA6, β-myosin heavy chain, α-sarcomeric actin and cardiac Troponin T) was investigated, and whole-cell current and voltage-clamp recordings were performed.

The c-kit+ cells from umbilical cord blood showed a rather immature gene profile, and by themselves, they did not differentiate into heart muscle-like cells in culture. In contrast, if whole mononuclear cells from umbilical cord blood were subjected to the same treatment, several if the employed protocols produced large, adherent cells that expressed several heart muscle-specific genes and exhibited an excitability much like that of heart muscle cells.

Formation of these heart muscle-like cells was prevented if the c-kit+ cells were removed from the other cells. Tracking studies showed that the c-kit+ cells were the ones that differentiated into heart muscle-like cells, but they only did so when they were together with c-kit– cells.

Thus umbilical cord blood contains progenitors endowed with the ability to differentiate into heart muscle-like cells. The cells with this potential reside in the c-kit+ fraction but they require the presence of abundant accessory cells to differentiate properly.

These preliminary observations suggest that it is a good idea to consider the storage of the umbilical cord blood of patients with prenatal diagnosis of congenital heart diseases. Such conditions might be potentially amenable to myocardial regenerative therapies with umbilical blood-based stem cells.

This paper was published in the journal Cytotherapy, but it must be said that the evidence that these cells differentiated into heart muscle cells was not completely convincing.

Rejuvenation Factor Discovered in Human Eggs

When the egg is fertilized by a sperm, it is transformed into a single-celled embryo or zygote that is metabolically active and driven to divide and develop. The egg, on the other hand, is a rather inert cell from a metabolic perspective. What is it in the egg that allows it to transform into something so remarkably different?

A new study by Swea-Ling Khaw and others in the laboratory of Ng Shyh-Chang at the Genome Institute of Singapore (GIS) has elucidated two main factors that help rejuvenate the egg and might also help reprogram adult cells into induced pluripotent stem cells (iPSCs).

Eggs express large amounts of a protein called Tcl1. Tcl1 suppresses the function of old, potentially malfunctioning mitochondria (the structure in cells that makes the energy for the cell). This suppression prevents damaged mitochondria from adversely affecting the egg’s transformation from into an embryo.

Remember also that if an adult cell is fused to an egg, it can cause the egg to divide and form an early embryo. Therefore, the egg cytoplasm is able to reprogram adult cells as well, and Tcl1 seems to play a role in this reprogramming capability as well.

In a screen for genes that are important to the reprogramming process, Shyh-Chang’s laboratory isolated two genes, Tcl1 and Tcl1b1. Further investigation of these two proteins showed that Tcl1 affects mitochondria by inhibiting a mitochondrial protein called polynucleotide phosphorylase (PNP). By locking PNP in the cytoplasm rather than the mitochondria, the growth and function of the mitochondria are inhibited. Tcl1b1 activates the Akt kinase, which stimulate cell growth, survival, and metabolism.

In a review article in the journal Stem Cells and Development, Anaïs Wanet and others explain that energy production in pluripotent stem cells is largely by means of glycolysis, which occurs in the cytoplasm. Mitochondria in pluripotent stem cells are immature subfunctional. When adult cells are reprogrammed into iPSCs, mitochondria function is shut down and energy production is largely derived from glycolysis. When the cells differentiate, the mitochondria are remodeled and become functional once again. Tcl1 is the protein that help shut down the mitochondria so that the pluripotent state can ensue and Tcf1b1 gears up the pluripotent stem cells to grow and divide at will.

Given this remarkable finding, can Tcf1 help make better iPSCs? Almost certainly, but how does one use this important factor to make better iPSCs?  That awaits further experimentation.  Additionally, this finding might also help aging and infertility issues as well. Hopefully this work by Shyh-Chang and her colleagues will lead to many more fruitful and exciting experiments.