Mesenchymal Stem Cells for the Treatment of MS

This study, is designed as a phase 1/2 open-safety clinical trial. At its first phase, 15 consenting patients with MS are included.

Inclusion Criteria:

Consenting patients fulfilled the following 4 inclusion criteria for this study:

1. the clinical criteria of Poser et alfor definite MS;
2. men and nonpregnant women aged 25 to 65 years;
3. duration of disease longer than 5 years; and
4. failure to respond to the currently available and registered agents for MS (ie, interferons, glatiramer acetate [Copaxone], and immuno-suppressors), as manifested by an increase of at least 1 degree in the EDSS score during the past year or the appearance of at least 2 major MS relapses during the same period.

Exclusion criteria:

1. patients who were treated with cytotoxic medications (ie, cyclophosphamide, mitoxantrone, and azathioprine) during the 3 months before the trial;
2. patients with significant cardiac, renal, or hepatic failure or any other disease that may interfere with the ability to interpret the results of the study;
3. patients with an active infection; and
4. patients who showed severe cognitive decline or were unable to understand and sign the informed consent.

Bone marrow aspiration is performed under short general anesthesia with puncture from the posterior superior iliac crest while the patient was lying in a left or a right lateral position. Approximately 200 mL of bone marrow inocula are obtained from each patient.

MSC Preparation and Culture A culture of purified MSCs is prepared under aseptic conditions (positively pressurized clean rooms) using filtered sterilized Dulbecco modified Eagle medium with low glucose lev- els (Qiagen, Valencia, California) supplemented with 10% fetal bovine serum, 1% L-glutamine, and 1% penicillin-streptomycin- nystatin solution (all from Biological Industries, Kibbutz Beit- Haemek, Israel).

Mesenchymal cells are cultured for 40 to 60 days, until they reach confluency, and then harvested and cryopreserved in 10% dimethyl sulfoxide-containing medium in liquid nitrogen (-196°C). At 2 weeks, a sample is taken for sterility testing and quality control. After sterility confirmed, the MSCs are transferred to the laboratory on dry ice, thawed in a 37°C water bath, and washed twice with normal saline solution to remove any residual dimethyl sulfoxide. The cells are then resuspended in normal saline at a concentration of 10x106/mL to 15x106/mL. Two-thirds of the total number of cells (usually 60x106 to 100x106) are injected intrathecally, and one-third, intravenously. A sample of the cells to be injected is tested by fluorescence-activated cell sorter (FACS) analysis; cells (98%) should express the surface markers characteristic of MSCs (CD29, CD73, CD90, CD105, and CD166) and be negative for CD34, CD45, and CD14.

Treatment Protocol All patients receive an intrathecal injection via a standard lumbar puncture (mean of 1 million cells per Kg of body weight) and an intravenous injection of 0.3-1 million cells per kg, intravenously..

An extension phase is scheduled for patients completing the first phase of this trial as an open prospective study with repeated intrathecal or intravenous injections of autologous MSC in patients from the initial trial and 10 additional ones (total up to 24 patients) with progressive forms of MS (secondary progressive, primary progressive or relapsing-progressive). Patients should be defined as failures to first and second lines of immunomodulatory treatments experiencing deterioration (at least 0.5 degree in the EDSS scale) during the year preceding their inclusion to our study or had at least one major relapse without sufficient recovery. Patients will be treated with 1x10 million MSC per kg of body weight, intrathecally and intravenously and subsequently with up to 8 courses of IT- or IV-injections of MSC (at the same dose), at intervals of 6-12 months. The duration of the trial is 4 years.

Patients will be followed up every 3 months for the whole duration of the trial, for safety assessment and changes in the disability scores (EDSS).

Immunological analysis will be performed at 4 time points (day 1, month 1, month 3 and month 6) following the first MSC-treatment and will include a fluorescent cell sorter (FACS) analysis to evaluate the proportions of the lymphocytes expressing markers of immune activation or of regulatory cell phenotype.