New Method Derived Skeletal Muscle Cells from Pluripotent Stem Cells

A University of Wisconsin research team led by Masatoshi Suzuki has devised a new protocol for the production of large quantities of skeletal muscle cells from pluripotent stem cells.

Suzuki and his team used embryonic stem cells lines and induced pluripotent stem cells to generate large quantities of muscles and muscle progenitor.

Suzuki adapted a technique used to make brain cells to derive his muscle cells in culture. He grew the stem cells as floating spheres in high concentrations of two growth factors: fibroblast growth factor-2 (FGF2) and epidermal growth factor (EGF). This combination of growth factors directed the stem cells to differentiate into skeletal muscle cells and muscle progenitors.

To replace damaged or diseased muscles in the clinic, physicians will require large quantities of muscle cells. Therefore, there was an ardent search to design a technique that was efficient, but also fast and relatively simple. Even though several protocols have been devised to differentiate pluripotent stem cells into muscle cells, not all of these protocols are practical for clinical use. For example, some protocols are simply too cumbersome for clinical use. Still others make use of genetically engineered cells that have not been approved for clinical use.

Earlier, Suzuki transplanted lab-engineered skeletal muscle into mice that had a form of amyotrophic lateral sclerosis. These animals had better muscle function and survived better than the control animals.

The muscle progenitors generated in Suzuki’s laboratory could potentially play a similar role in human patients with Lou Gehring’s disease. Suzuki’s method can grow muscle progenitor cells, which can grow in culture, from induced pluripotent stem cells, which are derived from the patient’s own cells. Such cells could be used as a model system to study the efficacy of particular treatments on the patient’s muscles, or they could be used to treat patients who have muscle defects.

“Our protocol can work in multiple ways and so we hope to provide a resource for people who are exploring specific neuromuscular diseases in the laboratory,” said Suzuki.

The advantages of Suzuki’s protocol are manifold. First, the cells are grown in a defined medium devoid of animal products. Secondly, the stem cells are grown as spheres, and these grow faster when grown as spheres than they do with other techniques. Third, 40-60 percent of the cells grown in this culture system differentiate into skeletal muscle cells or muscle progenitor cells. This is a very high proportion of muscle cells when compared to other protocols.

Suzuki hopes that by toying with the culture system, he and his colleagues can increase this proportion of muscle cells that form from the initial stem cell culture. This would enhance the potential of using these cells for clinical purposes.

Induced Pluripotent Stem Cells Recapitulate ALS in Culture and Suggest New Treatment

Induced pluripotent stem cells are made from the adult cells of an individual by means of genetic engineering techniques. After introducing four different genes into adult cells, some of the cells de-differentiate to form cells that grow indefinitely in culture and have most of the characteristics of embryonic stem cells. However, if iPSCs are made from a patient who suffers from a genetic disease, then those stem cells will have the same mutation as the patient, and any derivatives of those iPSCs will show the same behaviors and pathologies of the tissues from the patient. This strategy is called the “disease in a dish” model and it is being increasingly used to make seminal discoveries about diseases and treatment strategies.

Scientists from Cedars-Sinai Regenerative Medicine Institute have used iPSC technology to study Lou Gehrig’s disease, and their research has provided a new approach to treat this horrific, debilitating disease.

Because I have previously written about Lou Gehrig’s disease or Amyotrophic Lateral Sclerosis (ALS), I will not describe it further.

Cedar Sinai scientists isolated skin scrapings from each patient and used the skin fibroblasts from each sample to make iPSCs. According to Dhruv Sareen, the director of the iPSC facility and faculty research scientist with the Department of Biomedical Sciences and the first author on this article, skins cells of patients who have ALS were converted into motor neurons that retained the genetic defects of the disease, thanks to iPSC technology. Then they focused on gene called C9ORF72, which was found to be the most common cause of familial ALS and frontotemporal lobar disease, and is even responsible for some cases of Alzheimer’s and Parkinson’s disease.

Mutations in a gene that has the very non-descriptive name “chromosome 9 open reading frame 72” or C9ORF72 for short seems to play a central role in the onset of Lou Gehrig’s disease. Mutations in C9orf72 have been linked with familial frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). FTD is a brain disorder that typically leads to dementia and sometimes occurs in tandem with ALS.

Mutations in C9ORF72 result from the expansion of a hexanucleotide repeat GGGGCC. When the C9ORF72 gene is replicated, the enzyme that replicates DNA (DNA polymerase) has a tendency to slip when comes to this stretch of nucleotides and this polymerase slip causes the hexanucleotide GGGGCC sequence to wax and wane (expand and shrink). Normally, there are up to 30 repeats of this GGGCC sequence, but in people with mutations in C9ORF72, this GGGGCC repeat can occur many hundreds of times. Massive expansions of the GGGGCC repeat interferes with normal expression of the protein made by C9ORF72. The presence of messenger RNAs (mRNAs) with multiple copies of GGGGCC in the nucleus and cytoplasm is toxic to the cell, since it gums up protein synthesis, RNA processing and other RNA-dependent functions. Also the lack of half of the C9ORF72 protein contributes to the symptoms of this conditions.

Robert Baloh, director of Cedars-Sinai’s Neuromuscular Division and the lead researcher of this research project, said, “We think this buildup of thousands of copies of the repeated sequence GGGGCC in the nucleus of patient’s cells may become toxic by altering the normal behavior of other genes in the motor neurons. Because our studies supported the toxic RNA mechanism theory, we used to small segments of genetic material called antisense oligonucleotides – ASOs – to block the buildup and degrade the toxic RNA. One ASO knocked down overall C9ORF72 levels. The other knocked down the toxic RNA coming from the gene without suppressing overall gene expression levels. The absence of potentially toxic RNA, and no evidence of detrimental effect on the motor neurons, provides a strong basis for using this strategy to treat patients suffering from these diseases.”

Baloh continued: “In a sense, this represents the full spectrum of what we are trying to accomplish with patient-based stem cell modeling. It gives researchers the opportunity to conduct extensive studies of a disease’s genetic and molecular makeup and develop potential treatments in the laboratory before translating them into patient trials.”

Researchers from another institution recently began a phase one clinical trial that used a similar ASO strategy to treat ALS caused by a different mutation. No safety issues were reported in this clinical trial.

Clive Svendsen, director of the Regenerative Medicine Institute and one of the authors, has investigated ALS for more than a decade, said, “ALS may be the cruelest, most severe neurological disease, but I believe the stem cell approach used in this collaborative effort holds the key to unlocking the mysteries of the and other devastating disorders. Within the Regenerative Medicine Institute, we are exploring several other stem cell-based strategies in search of treatments and cures.”

ALS affects 30,000-50,000 people in the US alone, but unlike other neurodegenerative diseases, it is almost always fatal within three to five years.

Stephen Hawking Visits UCLA Stem Cell Laboratory

Stephen Hawking
Stephen Hawking

On Tuesday, Stephen Hawking toured a stem cell laboratory where scientists are studying ways to slow the progression of Lou Gehrig’s disease, a neurological disorder that has left the British cosmologist almost completely paralyzed.

After the visit, the 71-year-old Hawking urged doctors, nurses and staff at Cedars-Sinai Medical Center to support the research.

Hawking recalled how he became depressed when he was diagnosed with the disease 50 years ago and initially didn’t see a point in finishing his doctorate. But his attitude changed when his condition didn’t progress as fast and he was able to concentrate on his studies.

“Every new day became a bonus,” he said.

The hospital last year received nearly $18 million from California’s taxpayer-funded stem cell institute to study the debilitating disease also known as amyotrophic lateral sclerosis. ALS attacks nerve cells in the brain and spinal cord that control the muscles. People gradually have more and more trouble breathing and moving as muscles weaken and waste away.

There’s no cure and no way to reverse the disease’s progression. Few people with ALS live longer than a decade.

Diagnosed at age 21 while a student at Cambridge University, Hawking has survived longer than most. He receives around-the-clock care, can only communicate by twitching his cheek, and relies on a computer mounted to his wheelchair to convey his thoughts in a distinctive robotic monotone.

A Cedars-Sinai patient who was Hawking’s former student spurred doctors to invite the physicist to glimpse their stem cell work.

“We decided it was a great opportunity for him to see the labs and for us to speak to one of the preeminent scientists in the world,” said Dr. Robert Baloh, who heads the hospital’s ALS program.

Cedar-Sinai scientists have focused on engineering stem cells to make a protein in hopes of preventing nerve cells from dying. The experiment so far has been done in rats. Baloh said he hopes to get governmental approval to test it in humans, which would be needed before any therapy can be approved.

Hawking is famous for his work on black holes and the origins of the universe. His is also famous for bringing esoteric physics concepts to the masses through his best-selling books including “A Brief History of Time,” which sold more than 10 million copies worldwide. Hawking titled his speech to Cedars-Sinai employees “A Brief History of Mine.”

Despite his diagnosis, Hawking has remained active. In 2007, he floated like an astronaut on an aircraft that creates weightlessness by making parabolic dives.

Doctors don’t know why some people with Lou Gehrig’s disease fare better than others. Dr. Baloh said he has treated patients who lived for 10 years or more.

“But 50 years is unusual, to say the least,” he said.

FDA green lights stem cell clinical trial for Lou Gehrig’s disease

Lou Gehrig’s Disease is a really nasty disease. The patient experiences a progressive degeneration of the nervous system that affects the brain and the spinal cord. The progressive degeneration destroys the nerves that help move muscles and causes a gradual loss of the ability to move. It eventually leads to the death of those neurons. Paralysis slowly sets in right before the patient’s eyes. Then other basic abilities slowly leave, like the ability to talk, void the bladder, control bowel movements and so on. It kills its victims slowly and horribly.

Neuralstem, a company in Rockville, Maryland, has received US Food and Drug Administration (FDA) permission to test spinal cord stem cells in twelve patients with Lou Gehrig’s disease (amyotrophic lateral sclerosis).  This approval comes approximately one month after the FDA placed Geron’s planned clinical trial on hold for a second time. Neuralstem’s trial had also previously been placed on hold by the FDA in February before it received the go-ahead in September.

Though both trials involve placing cells into the spinal cord, Neuralstem’s product is made of cultured neural stem cells derived from a single eight-week-old fetus, whereas Geron’s product, intended to treat spinal cord injury, is derived from embryonic stem cells that have been differentiated into precursors of neuron-support cells.

Lucie Bruijn, a scientist at the ALS Association stated that this is the first stem cell approach for Lou Gehrig Disease.  The chief science office at Neuralstem, Karl Johe, says tests of large animal models show that the transplanted neural stem cells are able to protect motor neurons, although, it’s not entirely clear how.  Neuralstem and their collaborators showed in a rat model of Lou Gehrig’s Disease that transplanted cells could develop into interneurons that formed connections with the rats” motor neurons.

Nevertheless, this approved trial will assess safety rather than efficacy. The first few patients selected for the procedure will be those who are no longer able to walk.  Since the injected cells protect rather than replace motor neurons, these sicker patients are less likely to benefit from treatment, but they are also less able to lose function if something goes wrong. Cells will be injected only on one side of the spinal cord in order to minimize the number of injection sites. Only one patient will be treated each month so that researchers can monitor effects over a longer period of time. According to Johe, the goal is to be able to inject cells in both lower and upper regions of the spinal cord in healthier patients and see if the injections can help motor neurons survive.

Other companies using neural cells include ReNeuron, which received permission from UK authorities this January to start clinical trials for stroke. Its cell product is made from genetically modified cultures of neural stem cells, also of fetal origin.

StemCells Inc. is conducting trials in Batten disease, a neurodegenerative disorder that strikes children, and recently received approval for a clinical trial in a similar disease. It also uses neural stem cells from material originally derived from fetuses and has recently published research showing that its cell product delayed some symptoms of the disease by about three weeks.

It is really a shame that fetuses had to die to give us these cells.  I know that people will argue that the mother’s decision to “terminate her pregnancy” had nothing to do with the use of this person’s cells for research, but the fact remains that a doctor probably killed this very young baby and now his or her neural stem cells are being used in clinics.  Is this the way to value the youngest and most valuable members of our society?  Forgive me, but I find this shameful.

There is another experimental treatment that does not need to use dead babies.  It uses mesenchymal stem cells derived from the patients who receive them.  Read more about it here.