One Step Closer To Stem Cell Treatment for Multiple Sclerosis


Valentina Fossati and her colleague Panagiotis Douvaras from the New York Stem Cell Foundation (NYSCF) Research Institute have brought us one step closer to creating a viable stem cell-based therapy for multiple sclerosis from a patient’s own cells.

Valentina Fossati, Ph.D.
Valentina Fossati, Ph.D.

NYSCF scientists have, for the first time, produced induced pluripotent stem cell (iPSCs) lines from skin samples of patients who suffer from primary progressive multiple sclerosis. Fossati, Douvaras and colleagues also developed an accelerated protocol to differentiate iPSCs into oligodendrocytes, which are the myelin-making cells that insulate axons of central nervous system neurons. Destruction of the insulating myelin sheath is one of the hallmarks of multiple sclerosis, and oligodendrocyte progenitor cells or OPCs can replace damaged myelin sheath material.

Previously, producing oligodendrocytes from pluripotent stem cells required almost half a year to produce, which limited research on these cells and the development of treatments. This present study, however, has reduced the time required to make oligodendrocytes by half. This increases the feasibility of making these cells and using them in research and, potentially, for treatments.

Oligodendrocytes

By making oligodendrocytes from multiple sclerosis patients, researchers can use these cells to observe, in a culture dish, how multiple sclerosis develops and progresses. The improved protocol for deriving oligodendrocytes from iPSCs will also provide a platform for disease modeling, drug screening, and for replacing the damaged cells in the brain with healthy cells generated using this method.

“We are so close to finding new treatments and even cures for MS. The enhanced ability to derive the cells implicated in the disease will undoubtedly accelerate research for MS and many other diseases” said Susan L. Solomon, NYSCF Chief Executive Officer.

Valentina Fossati, NYSCF – Helmsley Investigator and senior author on the paper, said, “We believe that this protocol will help the MS field and the larger scientific community to better understand human oligodendrocyte biology and the process of myelination. This is the first step towards very exciting studies: the ability to generate human oligodendrocytes in large amounts will serve as an unprecedented tool for developing remyelinating strategies and the study of patient-specific cells may shed light on intrinsic pathogenic mechanisms that lead to progressive MS.”

NYSCF scientists established in this study that their improved the protocol for making myelin-forming cells worked and that the oligodendrocytes derived from the skin of these patients are functional, and able to form their own myelin when put into a mouse model. This is a definite step towards developing future autologous cell transplantation therapies in multiple sclerosis patients. These results also present new research venues to study multiple sclerosis and other diseases, since oligodendrocytes are implicated in many disorders. Therefore, Fossati and others have not only moved multiple sclerosis research forward, but also research on all demyelinating and central nervous system disorders.

“Oligodendrocytes are increasingly recognized as having an absolutely essential role in the function of the normal nervous system, as well as in the setting of neurodegenerative diseases, such as multiple sclerosis. The new work from the NYSCF Research Institute will help to improve our understanding of these important cells. In addition, being able to generate large numbers of patient-specific oligodendrocytes will support both cell transplantation therapeutics for demyelinating diseases and the identification of new classes of drugs to treat such disorders,” said Dr. Lee Rubin, NYSCF Scientific Advisor and Director of Translational Medicine at the Harvard Stem Cell Institute.

Multiple sclerosis is a chronic, inflammatory, demyelinating disease of the central nervous system, distinguished by recurrent episodes of demyelination and the consequent neurological symptoms. Primary progressive multiple sclerosis is the most severe form of multiple sclerosis, characterized by a steady neurological decline from the onset of the disease. Currently, there are no effective treatments or cures for primary progressive multiple sclerosis and treatments rely merely on symptom management.

Skin Tissue as a Treatment for Multiple Sclerosis


Italian researchers have derived stem cells from skin cells that can reduce the damage to the nervous system cause by a mouse version of multiple sclerosis. This experiment provides further evidence that stem cells from patients might be a feasible source of material to treat their own maladies.

The principal investigators in this work, Cecilia Laterza and Gianvito Martino, are from the San Raffaele Scientific Institute, Milan and the University of Milan, respectively.

Because multiple sclerosis results from the immune system attacking the myelin sheath that surrounds nerves, most treatments for this disease consist of agents that suppress the immune response against the patient’s own nerves. Unfortunately, these treatments have pronounced side effects, and are not effective in the progressive phases of the disease when damage to the myelin sheath might be widespread.

The symptoms of loss of the myelin sheath might one or more of the following: problems with touch or other such things, muscle cramping and muscle spasms, bladder, bowel, and sexual dysfunction, difficulty saying words because of problems with the muscles that help you talk (dysarthria), lack of voluntary coordination of muscle movements (ataxia), and shaking (tremors), facial weakness or irregular twitching of the facial muscles, double vision, heat intolerance, fatigue and dizziness; exertional exhaustion due to disability, pain, or poor attention span, concentration, memory, and judgment.

Clinically, multiple sclerosis is divided into the following categories on the basis of the frequency of clinical relapses, time to disease progression, and size of the lesions observed on MRI.  These classifications are:

A)         Relapsing-remitting MS (RRMS): Approximately 85% of cases and there are two types – Clinically isolated syndrome (CIS): A single episode of neurologic symptoms, and Benign MS or MS with almost complete remission between relapses and little if any accumulation of physical disability over time.

B)         Secondary progressive MS (SPMS)

C)         Primary progressive MS (PPMS)

D)        Progressive-relapsing MS (PRMS)

The treatment of MS has 2 aspects: immunomodulatory therapy (IMT) for the underlying immune disorder and therapies to relieve or modify symptoms.

To treat acute relapses:

A)    Methylprednisolone (Solu-Medrol) can hasten recovery from an acute exacerbation of MS.

B)    Plasma exchange (plasmapheresis) for severe attacks if steroids are contraindicated or ineffective (short-term only).

C)    Dexamethasone is commonly used for acute transverse myelitis and acute disseminated encephalitis.

For relapsing forms of MS, the US Food and Drug Administration (FDA) include the following:

A)    Interferon beta-1a (Avonex, Rebif)

B)    Interferon beta-1b (Betaseron, Extavia)

C)    Glatiramer acetate (Copaxone)

D)    Natalizumab (Tysabri)

E)    Mitoxantrone

F)    Fingolimod (Gilenya)

G)    Teriflunomide (Aubagio)

H)    Dimethyl fumarate (Tecfidera)

For aggressive MS:

A)    High-dose cyclophosphamide (Cytoxan).

B)    Mitoxantrone

In order to treat multiple sclerosis, restoring the damaged myelin sheath is essential for returning patients to their former wholeness.

In this study, this research team reprogrammed mouse skin cells into induced pluripotent skin cells (iPSCs), and then differentiated them into neural stem cells. Neural stem cells can differentiate into any cell type in the central nervous system.

(a) Bright field image of miPSC-NPCs obtained from miPSC Sox2βgeo. Scale bar, 50 μm. (b) GFP expression on miPSC-NPCs upon LV infection. Scale bar, 50 μm. (c–h) Immunostaining for Nestin (c), Vimentin (d), Olig2 (e), Mash1 (f), Sox2 (g) and GLAST (h). Scale bar, 50 μm. (i) Growth curve of miPSC-NPC. (j–l) Differentiation of miPSC-NPCs in three neural-derived cell populations, neurons (j), astrocytes (k) and oligodendrocytes (l). Scale bar, 50 μm. (m–o) Expression of the adhesion molecule CD44 (m), the chemokine receptor CXCR4 (n) and the integrin VLA-4 (o) on in vitro cultured miPSC-NPCs analysed by flow-activated cell sorting (FACS). Grey line represents the isotype control, whereas red line indicates the stained cells.
(a) Bright field image of miPSC-NPCs obtained from miPSC Sox2βgeo. Scale bar, 50 μm. (b) GFP expression on miPSC-NPCs upon LV infection. Scale bar, 50 μm. (c–h) Immunostaining for Nestin (c), Vimentin (d), Olig2 (e), Mash1 (f), Sox2 (g) and GLAST (h). Scale bar, 50 μm. (i) Growth curve of miPSC-NPC. (j–l) Differentiation of miPSC-NPCs in three neural-derived cell populations, neurons (j), astrocytes (k) and oligodendrocytes (l). Scale bar, 50 μm. (m–o) Expression of the adhesion molecule CD44 (m), the chemokine receptor CXCR4 (n) and the integrin VLA-4 (o) on in vitro cultured miPSC-NPCs analysed by flow-activated cell sorting (FACS). Grey line represents the isotype control, whereas red line indicates the stained cells.

Next, Laterza and her colleagues administered these neural stem/progenitor cells “intrathecally,” which simply means that they were injected into the spinal cord underneath the meninges that cover the brain and spinal cord to mice that had a rodent version of multiples sclerosis called EAE or experimental autoimmune encephalomyelitis.

EAE mice are made by injecting them with an extract of myelin sheath. The mouse immune system mounts and immune response against this injected material and attacks the myelin sheath that surrounds the nerves. EAE does not exactly mirror multiple sclerosis in humans, but it comes pretty close. While multiple sclerosis does not usually kill its patients, EAE either kills animals or leaves them with permanent disabilities. Animals with EAE also suffer severe nerve inflammation, which is distinct from multiple sclerosis in humans in which some nerves suffer inflammation and others do not. Finally, the time course of EAE is entirely different from multiple sclerosis. However, both conditions are caused by an immune response against the myelin sheath that strips the myelin sheath from the nerves.

The transplanted neural stem cells reduced the inflammation within the central nervous system. Also, they promoted healing and the production of new myelin. However, most of the new myelin was not made by the injected stem cells. Instead the injected stem cells secreted a compound called “leukemia inhibitory factor” that promotes the survival, differentiation and the remyelination capacity of both internal oligodendrocyte precursors and mature oligodendrocytes (these are the cells that make the myelin sheath). The early preservation of tissue integrity in the spinal cord limited the damage to the blood–brain barrier damage. Damage to the blood-brain barrier allows immune cells to infiltrate the central nervous system and destroy nerves. By preserving the integrity of the blood-brain barrier, the injected neural stem cells prevented infiltration of blood-borne of the white blood cells that are ultimately responsible for demyelination and axonal damage.

(a) EAE clinical score of miPSC-NPC- (black dots) and sham-treated mice (white dots). Each point represents the mean disease score of at least 10 mice per group (±s.e.m.); two-way ANOVA; *P<0.05; **P<0.01. (b) Quantification of spinal cord demyelination and axonal damage at 80 dpi in miPSC-NPC- (black bars) versus sham-treated (white bars) EAE mice (N=6 per group). Data (mean values ±s.e.m.) represent the percentage of damage. Student’s t-test; *P<0.05; **P<0.01. (c,d) Representative images of spinal cord sections stained with Luxol Fast Blue (c) and Bielschowsky (d). Red dotted lines represent areas of damage. Scale bar, 200 μm. (e) Quantitative analysis of the localization of miPSC-NPCs upon transplantation into EAE mice (N=3). (f–k) Immunostaining for CD45, MBP, Nestin, Ki67, Olig2, GFAP, βIII tubulin and GFP shows accumulation of transplanted cells (arrowheads) within perivascular spinal cord damaged areas. Dotted lines represent vessels. Nuclei are visualized with DAPI. Scale bars, 50 μm. (l) Percentage (±s.e.m.) of the miPSC-NPCs expressing the different neural differentiation markers upon transplantation into EAE mice; the majority of transplanted miPSC-NPCs remained undifferentiated. (N=3 mice per group).
(a) EAE clinical score of miPSC-NPC- (black dots) and sham-treated mice (white dots). Each point represents the mean disease score of at least 10 mice per group (±s.e.m.); two-way ANOVA; *P<0.05; **P

“Our discovery opens new therapeutic possibilities for multiple sclerosis patients because it might target the damage to myelin and nerves itself,” said Martino.

Timothy Coetzee, chief research officer of the National Multiple Sclerosis Society, said of this work: “This is an important step for stem cell therapeutics. The hope is that skin or other cells from individuals with MS could one day be used as a source for reparative stem cells, which could then be transplanted back into the patient without the complications of graft rejection.”

Obviously, more work is needed, but this type of research demonstrates the safety and feasibility of regenerative treatments that might help restore lost function.

Martino added, “There is still a long way to go before reaching clinical applications but we are getting there. We hope that our work will contribute to widen the therapeutic opportunities stem cells can offer to patients with multiple sclerosis.”

See Cecilia Laterza, et al. iPSC-derived neural precursors exert a neuroprotective role in immune-mediated demyelination via the secretion of LIF. NATURE COMMUNICATIONS 4, 2597: doi:10.1038/ncomms3597.

The Transformation of Ordinary Skin Cells into Functional Brain Cells


A paper in Nature Biotechnology from research groups at Case Western Reserve School of Medicine describes a technique that directly converts skin cells to the specific type of brain cells that suffer destruction in patients with multiple sclerosis, cerebral palsy, and other so-called myelin disorders. This particular breakthrough now enables “on demand” production of those cells that wrap or “myelinate” the axons of neurons.

Myelin is a sheath that wraps the extension of neurons called the axons. Neurons are the conductive cells that initiate and propagate nerve impulses. Neurons contain cell extensions known as axons that connect with other neurons. The nerve impulse runs from the base of the cell body of the neurons, down the axon, to the neuron to which it is connected. An insulating myelin sheath that surrounds the axon increases the speed at which nerve impulses move down the axon. When this myelin sheath is damaged, nerve impulse conduction goes awry as does nerve function. For example, patients with multiple sclerosis (MS), cerebral palsy (CP), and rare genetic disorders called leukodystrophies, myelinating cells are destroyed are not replaced.

neuron

The new technique discussed in this Nature Biotechnology paper, directly converts skin cells called fibroblasts, which are rather abundant in the skin and most organs, into oligodendrocytes, the type of cell that constructs the myelin sheath in the central nervous system.

Oligodendrocyte

“Its ‘cellular alchemy,'” explains Paul Tesar, PhD, assistant professor of genetics and genome sciences at Case Western Reserve School of Medicine and senior author of the study. “We are taking a readily accessible and abundant cell and completely switching its identity to become a highly valuable cell for therapy.”

Tesar and his group used a technique called “cellular reprogramming,” to manipulate the levels of three naturally occurring proteins to induce the fibroblasts to differentiate into the cellular precursors to oligodendrocytes (called oligodendrocyte progenitor cells, or OPCs).

OPCs

Led by Case Western Reserve researchers and co-first authors Fadi Najm and Angela Lager, Tesar’s research team rapidly generated billions of these induced OPCs (called iOPCs). They demonstrated that iOPCs could regenerate new myelin coatings around nerves after being transplanted to mice—a result that offers hope the technique might be used to treat human myelin disorders.

Demyelinating diseases damage the oligodendrocytes and cause loss of the insulating myelin coating. A cure for these diseases requires replacement of the myelin coating by replacement oligodendrocytes.

Until now, OPCs and oligodendrocytes could only be obtained from fetal tissue or pluripotent stem cells. These techniques have been valuable, but have distinct limitations.

“The myelin repair field has been hampered by an inability to rapidly generate safe and effective sources of functional oligodendrocytes,” explains co-author and myelin expert Robert Miller, PhD, professor of neurosciences at the Case Western Reserve School of Medicine and the university’s vice president for research. “The new technique may overcome all of these issues by providing a rapid and streamlined way to directly generate functional myelin producing cells.”

Even though this initial study used mouse cells, the next critical next step is to demonstrate feasibility and safety of human cells in a laboratory setting. If successful, the technique could have widespread therapeutic application to human myelin disorders.

“The progression of stem cell biology is providing opportunities for clinical translation that a decade ago would not have been possible,” says Stanton Gerson, MD, professor of Medicine-Hematology/Oncology at the School of Medicine and director of the National Center for Regenerative Medicine and the UH Case Medical Center Seidman Cancer Center. “It is a real breakthrough.”

Induced Pluripotent Stem Cells Make Stem Cells to Treat MS


Many nerves inside and outside the central nervous system are insulated by a sheath rich in a protein called “myelin.” This myelin-enriched sheath greatly increases the speed at which nerve impulses travel through these nerves. You have probably experienced such fast nerve impulse conduction. Remember the last time you had your hands in water that was overly hot. First there was a very sharp pain that caused you to withdraw your hand as fast as you could, but it was followed by a dull ache that became more and more painful until it abated. This is an example of the fast-moving pain impulses that help protect our limbs from further damage and the slower moving pain impulses that convey the dull ache associated with soft tissue damage.

In some cases the myelin sheath is damaged, or, in some cases, people are born with damaged myelin sheaths. Either way, such a condition is catastrophic, and multiple sclerosis is an example of a disease that results from progressive damage to and loss of the myelin sheath. Spinal cord injuries also strip the myelin sheath from many neurons, thus decreasing the effectiveness with which nerve impulses are conducted. The loss of the myelin sheath can also, in some cases, causes the death of the nerve.

Can the myelin sheath be replaced? Almost certainly. Cells make the myelin sheath and this is a cue for regenerative medicine. Many different types of stem cells can differentiate into myelin sheath-making cells. Embryonic stem cells, for example, can be differentiated into myelin sheath-making cells. This was the basis for Geron Corporation’s clinical trial with embryonic stem cell (ESC)-derived cells that could make myelin sheaths. Myelin sheath-making cells in the central nervous system are known as “oligodendrocytes,” and “oligodendrocyte progenitor cells,” which are mercifully abbreviated OPCs, give rise to oligodendrocytes. Differentiation of ESCs into OPCs led to the Geron clinical trial. However, Geron prematurely terminated this trial, and it is unclear if these embryonic stem cell-derived OPCs can restore sensation and nerve function to spinal cord injury patients.

Oligodendrocyte-Progenitor-Cell-OPC

Other cells, however, can form OPCs, and one of these is induced pluripotent stem cells (iPSCs). Since these cells are derived from the patient’s own cells, they should be recognized by the immune system as part of the patient’s own tissue and not a foreign group of cells.

Su Wang and colleagues from Steven Goldman’s lab at the Center for Translational Neuromedicine at the University of Rochester in Rochester, NY, have made patient-specific iPSCs from which they made patient-specific OPCs. Wang and his colleagues devised a protocol to differentiate human induced pluripotent stem cells (hiPSCs) into OPCs.

In this publication, Wang and others made three hiPSC lines, from which they made human OPCs. They used a very convenient methods to isolate the OPCs – fluorescence-activated cell sorting. hiPSC OPCs differentiated very efficiently into oligodendrocytes and other cell types found in the nervous system.

Next, Wang and others used their iPSC-derived OPCs to recoat nerves of mutant mice that lack myelin sheaths. Mice that have the “shiverer” mutation lack meylin sheaths, and they shake and shiver as a result of it. When implanted with Wang and companies’ iPSC-derived OPCs, these cells recoated with nerves very efficiently. When they compared the efficiency of the iPSC-derived OPCs with that of fetal OPCs, the iPSC-derived OPCs were clearly superior. The recoating of the nerves definitely increased the survival of the siverer mice. No tumors were observed in any of the mice implanted with iPSC-derived OPCs. implanted mice.

Goldman said of this study, “This study strongly supports the utility of hiPSCs as a feasible and effective source of cells to treat myelin disorders.” Goldman continued: “The new population of OPCs and oiligodendrocytes was dense, abundant, and complete. In fact, the re-myelination process appeared more rapid and efficient than with other cell sources.” This is significant because Goldman’s team also made OPCs from ESCs and their iPSC-derived OPCs outperformed the ESC-derived OPCs as well.

Goldman is part of a collaborative research consortium with scientists from Rochester, Syracuse, and Buffalo that wants to conduct a clinical trial that uses OPCs to treat patients with multiple sclerosis. This research group is called the Upstate MS Consortium and the early stages of this study are scheduled to begin in 2015, and it will focus on cells derived from various tissue sources. Goldman anticipates that his HiPSCs-derived OPCs will be included in this project.