Dying Muscles Leave “Ghost Fibers” that Direct Muscle Regeneration


When muscles are injured, they die off in order to make room for the growth of replacement muscles. However, it turns out that these moribund muscle leave behind small evanescent fibers that have been called “ghost fibers.” Ghost fibers seem to be remnants of the gooey stuff that provides the substratum upon which muscle cells sit. This gooey foundation is called “extracellular matrix” or ECM. The ECM consists of acid sugars called “glycosaminoglycans,” which are given the unfortunate abbreviation of GAGs, proteins to which GAGs are attached called “proteoglycans,” and proteins that glue cells to the ECM, such as fibronectin, laminin, and collagen IV. Cells adhere to the ECM by means of receptors embedded in their cell membranes called integrins.

Extracellular matrix

Dying muscle cells leave collagen fibers in their wake and these collagen fibers constitute these so-called ghost fibers. However, these ghost fibers provide the structure into which new muscle cells are inserted. A new study by research teams at the Carnegie Institution for Science and the National Institute of Child Health and Human Development that was published in the journal Cell Stem Cell has established that ghost fibers guide new muscle cells to grow in place and ultimately heal muscle injury in laboratory mice.

Ghost Fibers
Ghost Fibers

Chen-Ming Fan at the Carnegie Institute of Washington in Baltimore, Maryland and his colleagues, in collaboration with and Jennifer Lippincott-Schwartz and her colleagues from the NIH disabled the hind limb muscles of laboratory mice by means of physical injury (laceration), or the administration of toxins. These insults to the skeletal muscles caused the injured muscle fibers to die and disintegrate. They also confirmed that as the skeletal muscle disappeared, they left networks of collagen ghost fibers in their wake.

Then, this team utilized three-dimensional, time-lapse intravital imaging to directly visualize the process of muscle regeneration in live mice. What they saw was stunning. The extracellular matrix remnants or ghost fibers left by the injured skeletal directed muscle stem/progenitor cell behavior during muscle regeneration. The two-photon imaging and second-harmonic generation microscopy employed by this team enabled them to precisely observe the muscle stem and precursor cells in individual mice orient themselves along the ghost fibers and grow new muscle tissue.

The muscle stem cells were quiescent and did not move in uninjured muscle tissue. Only when muscle cells were injured did the muscle stem cells come to life, move to the site of injury and begin the healing process. Both the cell division of these muscle stem cells and their migration were oriented along the longitudinal axes of the ghost fibers.

ImageJ=1.49m unit=inch
ImageJ=1.49m
unit=inch

If the ghost fibers were artificially reoriented, then the muscle progenitors migrated and divided in different planes and gave rise to disorganized regenerated muscle fibers.

From these results, Fan and his team concluded that “the ghost fiber (1) is a key determinant for patterning muscle stem cell behavior and (2) provides the foundation for proportional regeneration. They concluded that “ghost fibers are autonomous, architectural units necessary for proportional regeneration after tissue injury.” They continued, “This finding reinforces the need to fabricate bioengineered matrices that mimic living tissue matrices for tissue regeneration therapy.”

Regenerating Heart Muscle with Powdered ECM


Healing the heart after a heart attack is a tough venture. Stem cell treatments have shown definite glimmers to success, but a lack of consistency is a persistent problem. Kick-starting the resident stem cell population in the heart is also a possibility but no single strategy has emerged as a tried and true method to treat a sick heart. Tissue engineering remains an engaging possibility and in the laboratory of Amit Patel at the University of Utah, the possibilities push the boundaries on your imagination.

Patel and his colleagues have been hammering at this problem for decades. The problem is how you replace dead tissue in a beating heart with live tissue that can beat in sync with the rest of the tissue. Unfortunately, you cannot ask the heart to take a vacation to help heal itself. Presently, Patel said that “The doctors say, ‘We’ll give you the beta blocker and the aspirin and the Lipitor and we can just hope to maintain you. But short of them getting worse or getting a heart transplant, there’s [sic] not too many options.”

Patel’s work, however, might change all that. He is presently leading trials on an experimental technology that might repair scarred heart tissue and even arrest or, perhaps, reverse heart failure.

His procedure is in a Phase 1 FDA clinical trial. The trial is designed to mix a powder that consists of a mixture of proteins and molecules isolated from heart muscle with saline or water, inject this mixture into the dead portions of the patient’s heart by means of a catheter, and then wait three to six months to determine if the patient’s heart muscle regenerates.

Dr. Amit Patel puts liquid matrix into a syringe in his lab at University Hospital in Salt Lake City on Wednesday, Sept. 16, 2015. (Photo: Laura Seitz, Deseret News)
Dr. Amit Patel puts liquid matrix into a syringe in his lab at University Hospital in Salt Lake City on Wednesday, Sept. 16, 2015. (Photo: Laura Seitz, Deseret News)

“Heart disease is the most common cause of death in the world, and the most prominent problem is heart failure,” said Tim Henry, the director of cardiology at the Cedars-Sinai Heart Institute. “Effectively, it’s basically one of the biggest problems in the U.S.” Curing the heart with stem cells is, according to Henry, “within our reach,” and Patel, is, to Henry’s thinking, “is clearly one of the most experienced stem cell people in the country”

After a heart attack, the dead regions of the heart form a scar that does not contract, does not conduct electrical impulses, and the rest of the heart has to work around. Reviving the heart scar, shrinking it or reprogramming it to live again has been the dream of stem cell therapy and gene therapy research. However, according to Patel, these venues have not proven to be very good at regenerating dead scar tissue.

Patel, however, noted that “endocardial matrix therapy” would probably be cheaper than stem cell or gene therapy, since it requires an off-the-shelf product that has the advantage of being mass-produced, is easily delivered clinically speaking, and can be easily commercialized and marketed.

This leads to a new question: “What is “extracellular matrix therapy?”

The extracellular matrix is a foundational material upon which cells sit. Extracellular matrix or ECM also provides the glue that attaches cells to each other, layers of cells to each other, and binds tissues together. In Patel’s rendering, ECM consists of everything in our tissues and organs except the cells. If you were to break down the ECM to its parts, you would end up with a concoction of proteins, minerals and a whole cadre of small molecules that can provide a scaffold for cells, nerves and vessels to attach.

To emphasize the importance of the ECM for the heart, Patel said: “A heart without scaffolding is just a bag of cells.” That pretty well nails it.

The ECM also plays a very important signaling role, since it acts as a repository for important signaling molecules that tell cells to grow and develop or divide and heal. The ECM is the milieu in which cells live and grow.

The foundational importance of the ECM gave Patel a revolutionary thought: to heal the heart the matrix has to come first before the cells can follow.

The powder form of heart-specific ECM was developed by scientists at the University of California, San Diego. This group removed the heart muscle from pig hearts, washed away all the cells, and then freeze-dried the remaining ECM into a powder. Using this work as their template, Patel and his team have also devised a protocol to make ECM power from human heart muscle.

When you add water or saline to this ECM powder, it forms a gooey substance called a “hydrogel.” This hydrogel has been called “VentriGel” and it is as flexible as native tissue. Hydrogels are the mainstay of tissue engineering experiments. VentriGel and hydrogels like it can mimic the molecular environment in which cells normally grow and develop. Fortunately, VentriGel has already been shown to successfully reduce scar tissue in the hearts of rats and pigs. To test VentriGel in human patients, Patel and his co-workers can come to the forefront.

Patel recruited a Utah woman who had suffered a heart attack six months ago. This episode reduced her overall heart blood pumping ability from 60 percent (normal) to less than 45 percent (well below normal). Patel and his colleagues made a virtual model of the inside of the patient’s heart to determine where her dead heart muscle resided. Then they marked out 18 different injection sites, and used a catheter to inject the matrix into her heart. The matrix injection procedure took less than two hours.

“This first patient was able to be done awake and safe and she’s already back to work,” Patel said. “She went home the next day.”

Patel plans to treat up to eighteen patients with his experimental procedure. Additionally, cardiologists at the Minneapolis Heart Institute in Minnesota, the only other site approved to test the new technology, performed the procedure on a second patient on Tuesday.

The risks of this procedure are well-known: When hydrogels are directly injected into the heart muscle, they can unintentionally interrupt the electrical conduction of the heart and cause irregular heartbeats. Also, the injected matrix can travel to other parts of the body where it can form a clot that could lead to a stroke. Clots in other parts of the body can also cause the patient’s blood vessels could collapse.

“If you go through all the bad things that could happen, you’d be so depressed, you’d be like, ‘Really? You found somebody to go through this?'” Patel said. “The key is that the team that we have here, and many of my collaborators, we’re all at that same level of healthy enthusiasm mixed with extreme paranoia.”

All patients will be examined three and six months after the procedure out for evidence of muscle regrowth and revived heart function.

“We want to treat this before it ends up leading to permanent damage,” Patel said.

If the trial returns positive results, it will represent another step forward in a long journey to eradicate heart disease. Patel estimates, that if everything goes smoothly, the technology could become approved for clinical use within five to seven years.

Human Embryonic Stem Cell Communication Network Discovered


Cells use a variety of mechanisms to talk to each other. These signaling pathways are called “signal transduction” pathways, and they vary extensively from one cell type to another.

Therefore, it should be no surprise that human embryonic stem cells signal to each other. The precise signal transduction pathway that human embryonic stem cells use to communicate with each other is the subject of a research project from a laboratory in Singapore.

Human embryonic stem cells or hESCs can differentiate into any adult cell type. The factors that keep hESCs in their pluripotent state are of interest to stem cell scientists because they might allow them to better direct the differentiation of hESCs or even grow them in culture better.

Cell-to-cell communication is vitally important to multicellular organisms. The coordinated development of tissues in the embryo that culminate in the formation of specific organs requires that cells receive signals and respond accordingly. If there are errors in these signals, the cells will respond differently and the embryo will either be grossly abnormal, or the cell might divide uncontrollably to make a tumor.

Human ESCs communicate by means of a signal transduction pathway known as the extracellular regulated kinase or ERK pathway.  The ERK signal transduction pathway begins with the binding of a growth factor receptor by a growth factor.  These growth factors are almost always bound to the extracellular matrix, which is the goo that surrounds cells and provides a structure in which the cells live.  The binding of the receptor causes the receptor to pair with another copy of itself, and that activates the bits of the receptor found inside the cell (tyrosine kinase domain for the interested).  The activated receptor attaches phosphates to itself, which causes particular proteins to find and bind the receptor, which recruits particular proteins to the cell membrane.  One of the recruited proteins is a protein kinase called RAF.  RAF attaches phosphate groups to the protein kinase MEK, and MEK attaches phosphate groups to the protein kinase ERK.  Once ERK has a phosphate attached to it, it can move into the nucleus and regulate transcription factors involved in the control of gene expression.  Thus a phenomenon that began at the cell membrane culminates in a change in gene expression.

ERK_pathway

Stem cell scientists a A*STAR’s Genomic Institute of Singapore and the Max Planck Institute of Molecular Genetics (MPIMG) in Berlin, Germany studied how genetic information is accessed in hESCs. To do this they mapped the kinase interactions across the entire human genome (kinases are enzymes that attach phosphate groups to other molecules) and discovered that ERK2, a protein that belongs to the ERK signal transduction pathway targets important sites such as non-coding genes, and histones, cell cycle, metabolism, and stem cell-specific genes.

The ERK signaling pathway involves an additional protein called ELK1 that interacts with ERK2. However, this research team discovered that ELK1 has a second, totally opposite function. At genomic sites not targeted by ERK signaling, ELK1 silences genetic information, which keeps the cell in its undifferentiated state.

ELK1 Interaction with ERK2

The authors propose a model that integrates this bi-directional control to keep the cell in the stem cell state, in which genes necessary for differentiation are repressed by ELK1 that is not associated with ERK2, and cell-cycle, translation and other pluripotency genes are activated by ELK1 in association with ERK2 or ERK2 plus other transcription factors.

Model of the Transcriptional Regulatory Network of ERK2 Signaling in hESCsTranscription factors such as ELK1 link ERK2 to sequence-specific regulation of gene expression. ERK2 and ELK1 colocalization defines three distinct modules that target different sets of genes. In this model, combinatorial binding of ERK2 and ELK1 with transcription factors, chromatin regulators, and the basal transcriptional machinery integrates external signaling into the cell-type-specific regulatory network. In hESCs, ERK2 and ELK1 participate in the regulation of pluripotency and self-renewal pathways, whereas differentiation genes are repressed.
Model of the Transcriptional Regulatory Network of ERK2 Signaling in hESCsTranscription factors such as ELK1 link ERK2 to sequence-specific regulation of gene expression. ERK2 and ELK1 colocalization defines three distinct modules that target different sets of genes. In this model, combinatorial binding of ERK2 and ELK1 with transcription factors, chromatin regulators, and the basal transcriptional machinery integrates external signaling into the cell-type-specific regulatory network. In hESCs, ERK2 and ELK1 participate in the regulation of pluripotency and self-renewal pathways, whereas differentiation genes are repressed.

First author Jonathan Göke from Stem Cell and Developmental Biology at the GIS said, “The ERK signaling pathway has been known for many years, but this is the first time we are able to see the full spectrum of the response in the genome of stem cells. We have found many biological processes that are associated with this signaling pathway, but we also found new and unexpected patterns such as this dual-mode of ELK1. It will be interesting to see how this communication network changes in other cells, tissues, or in disease.”

A co-author of this study, Martin Vingron said, “A remarkable feature of this study is, how information was extracted by computational means from the data.”

Professor Ng Huck Hui, managing author of this paper, added, “This is an important study because it describes the cell’s signaling network and its integration into the general regulatory network. Understanding the biology of embryonic stem cells is a first step to understanding the capabilities and caveats of stem cells in future medical applications.”

Silk and Cellulose as Scaffolds for Stem Cell-Mediated Cartilage Repair


When two bones come together, they grind each other into oblivion. This results in inflammation, joint swelling and pain, and scar tissue accumulation, which eventually results in the immobilization of the joint. To prevent this, bone are capped at their ends with a layer of hyaline cartilage that acts as a shock absorber. However, cartilage regenerates poorly and the wear and tear on cartilage, particularly at the knee, causes it to degenerate. The loss of the cartilage cap at the end of long bones causes osteoarthritis . The only way to mitigate the damage of osteoarthritis is to replace the knee with a prosthetic knee-joint.

Stem cells can make a significant contribution to the regeneration of lost cartilage. The Centeno/Schultz group near Denver, Colorado has been using bone marrow-derived mesenchymal stem cells to treat patients for over a decade with positive results. However, finding a way to grow large amounts of cartilage in culture that is the right shape for transplantation has proven difficult.

One way to mitigate this issue is the use of scaffolds for the cartilage-making cells that pushes them into a three-dimensional arrangement that forces them to make cartilage that mimics the cartilage found in a living organism. However a problem with scaffolds is finding the right material for the scaffold.

A recent publication has formed scaffolds from naturally occurring fibers such as cellulose and silk. By blending silk and cellulose fibers together, researchers at the University of Bristol have made a very inexpensive and easily manufactured scaffold for cartilage production.

Silk scaffold
Silk scaffold

When mixed with stem cells, cartilage and silk coax connective tissue-derived stem cells to differentiate into chondrocytes or cartilage-making cells. In the silk/cellulose scaffold, the chondrocytes secrete the extracellular matrix molecules characteristic of joint-specific cartilage.

Wael Kafienah, lead author of this work from the University of Bristol’s School of Cellular and Molecular Medicine, said, “The blend seems to provide complex chemical and mechanical cues that induce stem cell differentiation into preliminary form of chondrocytes without need for biochemical induction using expensive soluble differentiation factors. Kafienah continued: “This new blend can cut the cost for health providers and makes progress towards effective cell-based therapy for cartilage repair a step closer.”

To make the blended silk/cellulose scaffolds, Kafienah and his colleagues used ionic fluids, which effectively dissolve polymers like cellulose and silk, but are also much more environmentally benign in comparison to the organic solvents normally used to process silk and cellulose.

Presently, the U of Bristol team to trying to fabricate three-dimensional scaffolds that can be safely and easily implanted into patients for future clinical studies. Before human clinical studies are commenced, however, they must first be extensively tested in animals and also, the nature of the interactions between the scaffold and the stem cells that drive the cells to form cartilage must be better understood.