Skeletal muscle is the most abundant tissue in the human body, but, strangely, growing large quantities of it in the laboratory have proven rather challenging. While it is possible to reprogram other mature cells into heart muscle cells, or neurons, differentiating cells into skeletal muscle cells has simply not worked. So where do we go from here?
A new study from Brigham and Women’s Hospital (BWH) published in Nature Biotechnology has identified and even mimicked integral cues in the development of skeletal muscle. They used these cues to grow millimeter-long muscle fibers that are capable of contracting in the laboratory. This new method for growing functional muscle fibers in the laboratory potentially offer a better model for studying muscle diseases such as muscular dystrophy and for testing new treatments for these diseases.
Previous studies have used genetic modification techniques to grow small numbers of skeletal muscle cells in the laboratory. However, this new technique, which is the result of a collaboration between BWH and Harvard Stem Cell Institute, has produced a way to grow large numbers of skeletal muscle cells for use in clinical applications.
Olivier Pourquié of Harvard Medical School said, “We took the hard route: we wanted to recapitulate all of the early stages of muscle cell development that happen in the body and recreate that in a dish in the lab. We analyzed each stage of early development, and generated cell lines that glowed green when they reached a each stage. Going step by step, we managed to mimic each stage of development and coax cells toward muscle cell fate.”
The team found that a combination of secreted factors are important at the very early stages of embryonic development to stimulate muscle differentiation. By recapitulation this cocktail in the laboratory, Pourquié and his colleagues were able to mature muscle fibers in the laboratory from mouse or human pluripotent stem cells. Additionally, they produced muscle fibers in mice afflicted with muscular dystrophy by using muscle satellite cells. It is unknown if this method could help humans who suffer from muscular dystrophy, as more research is needed.
“This has been the missing piece: the ability to produce muscle cells in the lab could give us the ability to test out new treatments and tackle a spectrum of muscle diseases,” Pourquié said.
This new method also has the potential to help researchers study other muscle diseases, such as sarcopenia, or degenerative muscle loss and cachexia, the wasting away of muscle that typically occurs during severe illness.
A research laboratory lead by Jean-François Côté at the Institut de Recherches Cliniques de Montréal, Montreal, Canada has identified an elusive protein that mediates the fusion of muscle precursor cells into mature muscle.
The development of skeletal muscles depends on the migration of muscle precursor cells called “myoblasts” to migrate to the right location and then fuse with each other to form the multi-nucleate skeletal muscle cells. This finding has the potential to improve the treatment of muscular diseases such as myopathies and muscular dystrophies.
“For several years, we have been studying myogenesis, a process by which muscles are formed during development,” said Côté.
In the fruit fly Drosophila melanogaster, muscle fusion is rather well understood. A protein called “Myoblast City” and a scaffolding protein called “ELMO” activate the Rac protein in response the muscle precursor cells adhering to surfaces. Rac initiates the intracellular mechanisms that culminate in muscle fusion. In vertebrates, the ELMO protein exists in muscle precursor cells and a vertebrate version of the myoblast city protein called DOCK1. However, identifying the receptor that kicks this process off had proven difficult.
Myoblast fusion plays a central role in muscle development because it determines muscle size. Also, the fusion of existing muscle fibers with muscle stem cells helps regenerate and maintain adult muscles. This fusion process has always been a poorly understood process.
However, Côté and his co-workers have identified a receptor called BAI3 as one of the crucial links in myoblast fusion. BAI3 activates a signaling process that initiates the fusion of nearby myoblasts.
In 2008, Côté and his colleagues elucidated the role of two proteins – DOCK1 and DOCK5 – in the development of muscles. DOCK1 and DOCK5 regulate myoblast fusion. When the interaction between BAI3 and the DOCK signaling proteins is inhibited, myoblast fusion is also inhibited.
Côté pointed out that this work could have far-reaching implications, since the delivery of functional proteins to diseased muscle is typically carried out by introducing genetically engineered stem cells into the muscle that fuse with the disease muscle. By increasing the efficiency of the muscle fusion process, the delivery of genes to diseased muscles could become routine rather than painstakingly inefficient.
Stem cell treatments for muscular dystrophy and other degenerative diseases of muscle might be a realistic possibility, since scientists have discovered protocols to make muscle cells from human pluripotent stem cells.
Tiziano Barberi, Ph.D., chief investigator in the Australian Regenerative Medicine Institute (ARMI) at Monash University in Clayton, Victoria, and Bianca Borchin, a graduate student in the Barberi laboratory, have developed techniques to generate skeletal muscle cells. Barberi and Borchin isolated muscle precursor cells from human pluripotent stem cells (hPSCs), after which they applied a purification technique that allows these cells to differentiate further into muscle cells.
Pluripotent stem cells, such as embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs), have the ability to become any cell in the human body, including skeletal muscles, which control movement. Once the stem cells begin to differentiate, controlling that process is very challenging, but essential in order to produce only the desired cells. Barberi and Borchin used a technique known as fluorescence activated cell sorting (FACS) to identify those cells that contained the precise combination of protein markers that are expressed in muscle precursor cells. FACS also enabled them to successfully isolate those muscle precursor cells.
“There is an urgent need to find a source of muscle cells that could be used to replace the defective muscle fibers in degenerative disease. Pluripotent stem cells could be the source of these muscle cells,” Dr. Barberi said. “Beyond obtaining muscle from hPSCs, we also found a way to isolate the muscle precursor cells we generated, which is a prerequisite for their use in regenerative medicine.”
Borchin said there were existing clinical trials based on the use of specialized cells derived from hPSCs in the treatment of some degenerative diseases, but deriving muscle cells from pluripotent stem cells proved to be challenging. “These results are extremely promising because they mark a significant step towards the use of hPSCs for muscle repair,” she said.
“The production of a large number of pure muscle precursor cells does not only have potential therapeutic applications, but also provides a platform for large-scale screening of new drugs against muscle disease,” Dr. Barberi added.
This study was published early online Nov. 27 in Stem Cell Reports. This study does not address the immune response against dystrophin that has plagued gene therapy and stem cell-based muscular dystrophy clinical trials that has been noted in previous posts. The use of embryonic stem cells, in particular, would create muscles that are not tissue matched to the patient and would generate robust inflammation against the implanted muscles. Thus embryonic stem cells would generate a “cure” that would be much worse than the disease itself. Nevertheless, adapting the Barberi-Borchin protocol to induced pluripotent stem cells would produce skeletal muscle cells that are tissue matched to the patient.
Skeletal muscle – that type of voluntary muscle that allows movement – has proven difficult to grow in the laboratory. While particular cells can be differentiated into skeletal muscle cells, forming a coherent, structurally sound skeletal muscle is a tough nut to crack from a research perspective.
Another problem dogging muscle research is the difficulty growing new muscle in patients with muscle diseases such as muscular dystrophy or other types of disorders that weaken and degrade skeletal muscle.
Now research groups at the Boston Children’s Hospital Stem Cell Program have reported that they can boost the muscle mass and even reverse the disease of mice that suffer from a type of murine muscular dystrophy. To do this, this group use a combination of three different compounds that were identified in a rapid culture system.
This ingenious rapid culture system uses the cells of zebrafish (Danio rerio) embryos to screen for these muscle-inducing compounds. These single cells are placed into the well of a 96-well plate, and then treated with various compounds to determine if those chemical induce the muscle formation. To facilitate this process, the zebrafish embryo cells express a very special marker that consists of the myosin light polypeptide 2 gene fused to a red-colored protein called “cherry.” When cells become muscle, they express the myosin light polypeptide 2 gene at high levels. Therefore, any embryo cell that differentiates into muscle should glow a red color.
Once a cocktail of muscle-inducing chemicals were identified in this assay, those same chemicals were used to treat induced pluripotent stem cells made from cells taken from patients with muscular dystrophy. Those iPSCs were treated with the combination of chemicals identified in the zebrafish embryo screen as muscle inducing agents.
The results were outstanding. Leonard Zon from the Division of Hematology/Oncology, Children’s Hospital Boston and Dana-Farber Cancer Institute and his colleagues showed that a combination of basic Fibroblast Growth Factor, an adenylyl cyclase activator called forskolin, and the GSK3β inhibitor BIO induced skeletal muscle differentiation in human induced pluripotent stem cells (iPSCs). Furthermore, these muscle cells produced engraftable myogenic progenitors that contributed to muscle repair when implanted into mice with a rodent form of muscular dystrophy.
Zon hopes that clinical trials can being soon in order to translate these remarkable results into patients with muscle loss within the next several years. Zon and his co-workers are also screening compounds to address other types of disorders beyond muscular dystrophy.
This paper represents the application of shear and utter genius. However, there is one caveat. The mice into which the muscles were injected were immunodeficient mice whose immune systems are unable to reject transplanted tissues. In human patients with muscular dystrophy, an immune response against dystrophin, the defective protein, has been an enduring problem (for a review of this, see T. Okada and S. Takeda, Pharmaceuticals (Basel). 2013 Jun 27;6(7):813-836). While there have been some technological developments that might circumvent this problem, transplanting large quantities of muscle cells might be beyond the pale. Muscular dystrophy results from disruption of an important junction between the muscle and substratum to which the muscle is secured. This connection is mediated by the “dystrophin-glycoprotein complex.” Structural disruptions of this complex (shown below) lead to unanchored muscle that cannot contract properly, and eventually atrophies and degrades.
This is a remarkable advance, but until the host immune response issue is satisfactorily addressed, it will remain a problem.