Making Cartilage to Heal Broken Bones

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

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

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

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

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

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

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

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


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

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

Induced Pluripotent Stem Cells – Addressing Safety Concerns

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Pluripotent Stem Cells Actively Regulate The Openness of their Heterochromatin

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

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

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

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

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

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

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

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

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

This work was published in the journal Genes and Development (