Isolation of skin fibroblasts from MS patients for reprogramming into induced pluripotent stem cells

Multiple sclerosis (MS) is an inflammatory, neurodegenerative disease initially
characterized by relapsing remitting (RR) clinical episodes. The relapses
reflect the progression of lesions within the CNS featuring inflammation,
myelin destruction and axonal loss. After each relapse, axonal damage is
restricted by the remyelination activity of local OPCs. The transition of RRMS
to the secondary progressive form of MS marks the limit of brain plasticity
where OPCs are no longer able to remyelinate axons and axonal loss can no
longer be compensated by parallel connections. In primary progressive MS
(PPMS), a steady neurological deterioration occurs already from the moment of
diagnosis suggesting that OPC recruitment and proper remyelination fails from
the beginning. Although cell-mediated immune mechanisms play a prominent role
in disease progress, it is suggested that intrinsic aberrations in
oligodendrocyte physiology in MS patients may be a primary factor. The
groundbreaking discovery that somatic cells (e.g. skin fibroblasts) can be
reprogrammed into pluripotent stem cells (IPS cells) that can be differentiated
in any cell type has offered an unprecedented source for patient-derived cells.
Such patient-derived IPS cells appear not only a promising source for cell
replacement therapies, but particularly for disease-modeling and, so far, IPS
cells have been generated from patients with neurological disorders (e.g.
Huntington, ALS, SCA). MS is not a genetic disorder with known single or
multiple gene mutations. However, a specific (epi) genetic profile present in
MS patients is suggested to underlie an aberrant physiology and behavior of
oligodendrocytes resulting in abnormal responses to local stress and/or
abnormal myelin production. IPS technology now offers a unique opportunity to
examine this with MS patient derived OPCs in vitro.

In this study we aim to address the hypothesis that oligodendrocytes and
oligodendrocyte precursor cells from MS patients exhibit epigenetically
determined differences in protein expression profiles and functional behavior
in comparison to those from healthy controls. To investigate this we make use
of MS patient-derived IPS cells.
We have the following specific objectives:
Aim 1: To isolate skin fibroblasts from MS patients by punch biopsy under local
anesthesia and store them after in-vitro multiplication.
Aim 2: To reprogram the patient-derived skin fibroblasts into induced
pluripotent stem (IPS) cells and, after full IPS characterization/verification,
differentiate them in-vitro into oligodendrocyte precursor cells (OPCs) and
oligodendrocytes with a protocol developed in our lab.
Aim 3: To compare the expression profiles of MS patient-derived OPCs and
oligodendrocytes with control ones using RNA sequencing, proteomics and
epigenetic characterization.
Aim 4: To expose MS patient and control-derived OPCs and oligodendrocytes to
different types of stress (excitotoxic, oxidative and inflammatory) in vitro
and compare their responses by analyzing their behavior and expression
profiles.
Aim 5: To co-culture MS patient and control-derived OPCs with rat DRG neurons,
a well-known model to examine myelin formation in detail. In this set-up,
myelin production by MS patient-derived oligodendrocytes will be analyzed in
detail with techniques mentioned above.

For the whole study, fibroblasts from two groups of MS patients will be used: 5
RRMS patients, 5 PPMS patients (as controls IPS cells from 5 healthy
age-matched persons will be directly obtained from the IPS facilities, LUMC,
Leiden and from the department Neuroscience, UMCG).
After patient selection, a skin punch biopsy will be performed according to
standard procedures under sterile conditions and after local anesthesia at the
department of Dermatology, UMCG. The skin biopsies will be collected in cold
sterile basic culture medium and transported to the lab of the dept.
Neurosciences, UMCG. After tissue dissociation, fibroblasts will be cultured
and multiplied under fibroblast specific/selective culture conditions. Part of
these fibroblasts will be stored frozen, another part will be used for
reprogramming into IPS cells and subsequently differentiated into OPCs
according to procedures developed in the dept. Neurosciences, UMCG.
Subsequently, the IPS derived OPCs will be subjected to extensive analyses,
comprising mRNA sequencing, proteomics and epigenetic profiling (e.g. DNA
methylation patterns). Myelination capacity of the various IPS derived
oligodendrocytes will be examined and compared in vitro.

After patient selection, a skin punch biopsy ( diameter 4mm) will be performed
according to standard procedures under sterile conditions and after local
anesthesia at the department of Dermatology, UMCG.

From the MS patients a small (6mm) skin biopsy will be taken from the innerside
of the upper arm using a standard punch technique under local anesthesia. The
only minimal risk involved in particpation may be the occurrence of infection
(despite intense desinfection of the site of biopsy) (<1% of cases) ; effective
treatment with antibiotic ointment.