ARTICLE IN BRIEF
Scientists generated induced pluripotent stem cell (iPSC) lines from skin samples of patients with primary progressive multiple sclerosis and then developed an accelerated protocol to induce these stem cells into becoming oligodendrocytes, the myelin-forming cells of the central nervous system implicated in multiple sclerosis and many other diseases.
A team of New York scientists has created a reproducible, functional and efficient population of oligodendrocyte progenitor cells (OPCs) in half the time of previous efforts, providing a more feasible tool to study both how myelin is damaged in multiple sclerosis (MS) and how to use the cells in autologous cell therapies.
The scientists also made several populations of induced pluripotent stem cells (iPSCs) from patients with primary progressive MS and developed oligodendrocytes that were then injected into a mouse that normally does not produce the myelin needed to insulate nerve cells. These human cells were able to sheath the nerve cells, according to the Aug. 12 study published in Stem Cell Reports.
The key to the researchers' success — shortening the production process to 75 days, compared with past efforts to produce myelinogenic OPCs in 120 to 130 days — was due in large part to studying the available protocols from oligodendrocyte differentiation from neural stem cells to mature and functional oligodendrocyte progenitors.
The investigators determined that adding retinoids to the recipe at the beginning of differentiation, then sonic hedgehog, and finally, the requisite list of growth factors from day 20 onward could speed up the process. They also used a sorting method to purify the mature oligoprogenitors from the cultures.
They followed the same process with iPSC lines derived from skin biopsies from four patients with primary progressive multiple sclerosis, and successfully accelerated the production of mature OPCs.
Valentina Fossati, PhD, an endowed New York Stem Cell Foundation-Helmsley investigator, who led these studies, noted that scientists have found it challenging to create healthy populations of oligodendrocytes from stem cells. “There is a very protracted phase to move from oligodendrocyte transcription factor 2 (OLIG2) progenitors to immature oligodendrocytes. We have always wondered why it is so difficult and slow. We needed to understand and mimic what goes on in the human brain,” she explained.
By the end of the differentiation process at day 75, 40 to 70 percent of the cells were OPCs expressing the characteristics of mature cells. Around 34 percent of those cells can further differentiate into mature myelin basic protein oligodendrocytes. Other scientists have had efficiency rates of 9 to 11 percent at 120 to 150 days.
To observe the response to the OPCs, the researchers injected the cells into the shiverer mouse, which has a poorly myelinated nervous system. They observed human myelin forming around the animal's nerve cells. By six weeks, the cells were engrafted and myelinating.
So far, in all of their preliminary studies the progenitor cells did not give rise to tumor cells, which is a worry when promoting stem cell therapies in humans.
With success in the animal models, Dr. Fossati teamed up with neurologist Saud Sadiq, MD, FAAN, director of the Tisch Multiple Sclerosis Research Center in New York City, to see whether they could generate iPSCs from patients with primary progressive MS and use those cells to make mature oligodendrocytes.
Dr. Fossati and her colleagues used the skin cells from 12 patients to make iPSCs. In this first report, they made OPCs from four of the patients with primary progressive MS. Four others have relapsing and remitting MS and four have secondary progressive MS.
“This is a first step to a potential therapy for patients with primary progressive MS,” said Dr. Sadiq, whose center spent six years obtaining federal approval to begin a phase 1 clinical trial using adult bone marrow that is replete with mesenchymal stem cells for the treatment of MS. As of August, they have infused the cells into the spinal cord in three patients.
Cell therapy using populations of a patient's own OPCs would be a more targeted approach to remyelinating the damaged nerve cells, he said.
So far, the oligodendrocytes made from the iPSCs from patients with primary progressive MS do not appear to be any different than iPSCs generated from skin cells of healthy people, said Dr. Fossati. But they are just beginning to design studies to compare these patient-derived cell lines under different conditions. They will also conduct gene expression studies from the cells made from patients and healthy volunteers to identify any differences. They are also forming collaborations with other scientists to study the OPCs in a lab dish and screen potential MS drugs.
“I know that myelination in a dish is not real myelination, but it will be very useful in testing drugs and for understanding basic mechanisms of MS,” Dr. Fossati added. “There are a lot of questions that need answers before we attempt to use oligoprogenitors in patients. Even if they appear normal, they may not be able to accomplish their function due to the inflamed environment when infused into the brain.”
Dr. Fossati is also collaborating with James Goldman, PhD, a glial cell biologist and pathologist at Columbia University. Dr. Goldman has helped Dr. Fossati determine whether the cells she sees under the microscope are mature oligodendrocytes.
Steven A. Goldman, MD, PhD, a University of Rochester Medical Center distinguished professor of neurology, reported in a 2013 paper published in Cell Stem Cell on successful efforts to generate new nerve-insulating myelin in the shiverer mouse using human induced pluripotent stem cells from which they produced OPCs.
Dr. Goldman said that the technique used in the current study is definitely useful. “This is a good and careful study,” he said. “The technical improvements mean a faster production of oligodendrocyte progenitors than pre-existing protocols and they sorted out the cells to maximize the population of oligodendrocytes for transplant.”
As for the creation of iPS cells from MS patients, a technique that many scientists have used for other diseases, “it may be useful in identifying risk factors, but without having the immune system reconstituted it is hard to know if much information can be gleaned from those types of studies. MS is not a disease of oligodendrocytes, but an autoimmune disease that targets oligodendrocytes.”
What's more, it is not clear whether primary progressive MS, a condition that represents 10 percent of MS patients, is caused by a neurodegenerative process or an autoimmune one. Further testing with the OPCs from patients with primary progressive MS may help scientists address this question.
Like Dr. Goldman, Paul J. Tesar, PhD, the Dr. Donald and Ruth Weber Goodman professor in innovative cancer therapeutics at Case Western Reserve University School of Medicine, is one of the pioneers in transforming stem cells into oligodendrocytes. “This is an elusive cell type,” he said. “It's been a tremendous challenge in a laboratory setting to study and utilize these cells. We were impatient for faster protocols.”
“Valentina has taken it one step farther and it will change our ability to work with these cells in the lab,” he added. “Still, there is a lot we don't know about the true lineage of these cells. In neurons, we have well-defined types of cells. It is now emerging that there are distinct populations of oligodendrocytes, and while that would not be surprising, we need to understand the properties of these different cell types before you start using these cells in patients.”
But Jonah Chan, PhD, the Debbie and Andy Rachleff distinguished professor of neurology at the University of California, San Francisco, calls this latest paper by Dr. Fossati “a breakthrough” because of the researchers' ability to produce these cells more quickly and with high efficiency. “The data are more convincing than I have seen,” he said.
Dr. Fossati plans to collaborate with Dr. Chan, who has created axonal-like fibers that can be laid onto scaffolds and used in the laboratory to test myelin-making cells. The model was developed for high-throughput screening.
Mahendra Surendra Rao, MD, PhD, former director of the NIH Center for Regenerative Medicine and current vice president of strategic affairs at a stem cell company called Q Therapeutics, said, “A lot of people have been trying to get human OPC lines done faster. These scientists have figured out the timing and the sorting allows for a purer population of cells.”
A PERSONAL QUEST TO SOLVE THE STEM CELL PROBLEM
Valentina Fossati, PhD, an endowed NYSCF-Helmsley investigator at the New York Stem Cell Foundation, began her career studying B cell development from hematopoietic stem cells. She was working on her doctorate at the University of Bologna and in 2006 moved to Mount Sinai School of Medicine to finish her studies. She was interested in leukemia. It had been eight years since James Thompon, PhD, and his colleagues at the University of Wisconsin Medical Center created the first human embryonic stem cells. Scientists worldwide were focused on creating human stem cell lines. She joined her mentor, Hans-Willem Snoeck, MD, PhD, at that time professor of medicine in microbiology and immunology at Sinai, in studying thymic epithelial cells from embryonic stem cells. Dr. Snoeck is now at Columbia University.
Then, on her 30th birthday, Dr. Fossati woke up with a strange sensation in her leg. She was cold. Her leg was hot, numb, painful and tingling. “I was awake but my leg did not wake up with me,” she said. A brain scan showed small lesions characteristic of MS. She went home and searched the Internet for the latest scientific advances in the field. She stopped her search when she read about on going work that had succeeded in producing oligodendrocytes to treat MS.
She was already immersed in stem cells. Now, it wasn't just science. It was personal. Dr. Fossati would change the direction of her research. A year after her diagnosis, in 2010, she accepted a five-year fellowship at the New York Stem Cell Foundation and set out a plan of attack on multiple sclerosis.
Outside of the lab, Dr. Fossati doesn't let the diagnosis slow her down. The initial episode led to a number of drug treatments and she has not had another flare up. She recently gave birth to a daughter. She is not shy in sharing her diagnosis with colleagues. “It is an important part of who I am as a scientist,” she said. “And what I am doing to tell this disease that I am here to fight it.”
EXPERTS: ON PRODUCING NEW MYELINATED CELLS
LINK UP FOR MORE INFORMATION:
•. Douvaras P, Wang J, Zimmer M, et al. Efficient generation of myelinating oligodendrocytes from primary progressive multiple sclerosis patients by induced pluripotent stem cells. Stem Cell Rep 2014; 3:1–10.
•. Wang S, Bates J, Li X, Schanz S, et al. Human iPSC-derived oligodendrocyte progenitor cells can myelinate and rescue a mouse model of congenital hypomyelination. Cell Stem Cell
2013; 12: 252–264.
•. Najm FJ, Lager AM, Zaremba, et al. Transcription factor-mediated reprogramming of fibroblasts to expandable, myelinogenic oligodendrocyte progenitor cells. Nat Biotechnol
•. Lee S1, Leach MK, Redmond SA, et al. A culture system to study oligodendrocyte myelination processes using engineered nanofibers. Nature Meth 2012; 9(9):917–922.© 2014 American Academy of Neurology
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