Aspirated bone marrow inoculum from multiple sclerosis patients, participating in our phase I/II clinical trial (NIH registration number: NCT00781872), was proceeded according to classical methods for mesenchymal stem cells isolation (17). Briefly, a purified mesenchymal stem cells culture was prepared using plastic adherence method in filtered sterilized low glucose-DMEM medium supplemented with 10% of Foetal Bovine Serum (FBS), 1% L-Glutamine and 1% L-Pen-Strep-Nystatin (PSN) solution (all from Biological Industries, Israel).

hMSC cultures were incubated for 48 hours prior to use with 0.25 l/ml Feridex^© (11.3 mg/ml, Berlex, USA), and 375 ng/ml of the cationic polymer poly-L-lysine (PLL, Sigma, Israel) in low-glucose DMEM containing 10% FBS, 1% L-Glutamine and 1% PSN.

Flow cytometry: hMSC with and without Feridex were harvested with Trypsin/EDTA Solution (Biological Industries, Israel), divided into polystyrene fluorescence activated cell sorter (FACS) tubes, and labeled with anti human CD45-FITC, anti human CD90-PE and anti human CD105-PE (Beckman Coulter, Israel) for 45 minutes in the dark. Fluorescence data were collected from 30,000 cells and measurements and analysis were performed using a Beckman Coulter FACS machine.

Immunoflurecense: hMSC with and without PLL-coated Feridex were cultured on non-treated coverslips (NUNC Inc., USA) for 48 hours, washed (×3) with PBS and stained with labeled anti human CD45-FITC (Beckman Coulter, Israel), anti human CD90-Texas Red and anti human CD105-Texax Red (Santa Cruz Biotechnology, USA). Fluorescence was evaluated under florescence microscope.

To induce hMSC to differentiate into various cell phenotypes, 2×10⁵ cells were plated in culture dishes and allowed to reach confluence. Osteogenic differentiation medium, consisting of 10% Eagle minimum essential medium/alpha medium and fetal bovine serum, supplemented with 50-mg/mL ascorbic acid, 10 mM β-glycerol-phosphate, and 10⁸Mdexamethasone (all from Sigma, Israel), was exchanged twice a week for 3 weeks. For adipocytic differentiation, commercially adipogenic differentiation kit was used according to the manufacture instructions (STEMCELL Technologoies, Canada). Chondrogenic differentiation was induced by culturing the cells with media containing MEM-alpha, 5% FBS, 1% L-Glutamine, 1% non-essential amino acids, 1% non-essential vitamins, 1% PSN (all from Biological Industries, Israel) and 10 ng/ml TGF-β3 (Sigma, Israel) for 14 days with medium exchange twice a week.

For neural-glial differentiation, hMSC were seeded on fibronectin coated (1 μg/ml, Sigma, Israel) 24-well plates and cultured in Eagle minimum essential medium/alpha medium enriched with 7% FBS, 1% glutamine, 1% vitamins, 1% PSN solution, 20 ng/mL brain-derived neurotrophic factor (Peprotech, Israel), 10 ng/mL fibroblast growth factor-2 (Peprotech, Israel), 20 ng/mL fibroblast growth factor 8 (Peprotech, Israel), 10 ng/ml nerve growth factor (Peprotech, Israel) and 10 ng/ml nurotrophin-4 (Peprotech, Israel). The cells were cultured under these conditions for 18 day. Then the culture media were changed while being depleted of FBS and cultured for more 5∼7 days.

Freshly prepared Perls’ reagent by dissolving 1 gram potassium cyanide in 42ml deionized water+8 ml of 37.5% HCl. For nuclear fast red (NFR) counterstain, 0.1 gram of NFR was dissolved in 100 ml deionized water+5 gram of aluminium sulfate (all from Sigma, Israel). Cells for staining were washed (×3) with PBS and incubated 30 minutes in the dark, washed again (×3) with PBS and incubated with NFR counterstain for 5 minutes, They were then rinsed (×3) in deionized water, dried overnight in dark and embedded in Permount with coverslip.

To detect osteoblastic differentiation, NBT/BCIP staining which shows the alkaline phosphatase activity was used. NBT/BCIP solution (Sigma, Israel) was added to the cells for 30 min at 37°C in a 5% CO₂ incubator. After incubation, cells were washed with PBS (×3) and fixed with 4% PFA. To detect adipogenic differentiation, 10 mg/mL oil red-O (Sigma, Israel) was added and left for 20 minutes at room temperature. The cells were washed 3 times with PBS and fixed with PFA 4%, for 20 minutes. For the detection of chondrogenic differentiation, alcian blue staining was used. The cells were then washed with PBS and fixated with 4%. Then, the cells were rinsed with alcian blue solution, pH 2.5 (Sigma, Israel) for 30 min at RT and washed in running tap water for 2 min and in distilled water for another 2 min. Extracellular matrix components secreted by chondrocytes were then stained blue.

The medium was aspirated and the cells were washed gently with 0.05% Tween 20 diluted in PBS and then fixed with 4% fresh PFA (Sigma, Israel) for 20 minutes at room temperature. To stain the intracellular components, the cells were permeabilized with 0.1% Triton X-100 (Sigma, Israel) for 10 minutes. For blocking nonspecific binding, the cells were rinsed with 5% bovine serum albumin in PBS for 60 minutes at room temperature on a slowly rotated plate. Then, they were washed 3 times with 0.05% Tween 20 (Sigma, Israel) diluted in PBS, and incubated with the following primary antibodies: anti human myelin associated protein 2 (MAP-2), anti human β-tubulin type III, anti human glial fibrillary acidic protein (GFAP), and anti human Galactocerebrosidase (GalC), (all from Chemicon, USA) diluted to the required concentrations with buffer containing 1% bovine serum albumin (Sigma, Israel). After washing, the cells were incubated with goat anti mouse fluorescein isothiocyanate-conjugated and goat anti rabbit tetramethylrhodamine isothiocyanate-conjugated secondary antibodies (Jackson Immunoresearch, USA) diluted in bovine serum albumin buffer, 1%, on a slowly rotated plate for 45 minutes in the dark at room temperature. The cells were mounted on slides with mounting solution and examined under fluorescence microscope. Some of the slides stained for GalC were mounted with mounting media containing DAPI that stains nuclear DNA in blue.

Lymphocytes were isolated from healthy blood donors using standard Ficol separation gradient. All cultures were carried out in triplicates in 96-well, flat-bottom, microtiter plates. The assay was carried out by seeding 2×10⁵ cells/well in 0.2 ml of RPMI medium supplemented with 5% fetal calf serum, 1% L-glutamine, and 1% PSN (all from biological industries, Israel). To the lymphocytes, 10×10³ and 30×10³ hMSC (with and without Feridex) were added. ³H-thymidine (Amersham, UK) incorporation was determined in response to PHA (4 μg/ml, Sigma, Israel). The cultures were incubated for 48 hours in a humidified atmosphere of 5.0% carbon dioxide at 37°C and then pulsed for 16 hours with ³H-thymidine (1 μCi/well). Cells were harvested on fiberglass filters (Whatman, USA) using a multiharvester and the radioactivity was counted.

hMSCs (Fig. 1A), could be effectively labeled by Feridex and showed in vitro positive staining with Prussian blue (Fig. 1B). Naïve hMSC and Feridex-labeled hMSCs (Fig. 2A) were similarly stained for the markers CD45, CD90 and CD105. Both were shown to express the typical hMSC markers profile including negativity for CD45 and positive staining for CD90 and CD105 (Fig. 2A). Immuno-histochemical analysis showed negative staining for CD45 and positive staining for CD90 and CD105 with preservation of the typical for hMSC, fibroblast-like shape (Fig. 3).

To prove their mesodermal nature, we cultured hMSC in adipogenic and osteogenic media. When induced to differentiate into osteoblasts (using medium containing osetegenic differentiation factors: see methods), both unlabeled and labeled cells exhibited alkaline phosphatase activity as visualized by NBT-BCIP staining (Fig. 4A, B). Adipogenesis was evident morphologically by their transformation into rounded cells containing lipid vesicles, visualized by oil red O. Both naïve and labeled cells showed adipogenic differentiation and formation of lipid vacuoles (Fig. 4C, D). Chondrogenic differentiation was tested by using alcian blue staining. After classical culture with chondrogenic media (including TGF-beta-3), only the naïve and not the Feridex-labeled hMSC were stained blue, indicative of chondrogenic differentiation (Fig. 4E, F).

Naïve and Feridex-labeled hMSC were cultured on fibronectin-coated slides with neural differentiation media for 3 weeks. The differentiation media was changed twice a week while changing the differentiation factors’ coktail weekly under a controlled and specified protocol (as detailed in Materials and Methods). After 3 weeks the cells were stained for the neural and glial markers, tubulin-beta-III, MAP2, GFAP and GalC (Fig. 5). Both unlabeled and labeled hMSCs were able to differentiate into neural-like cells as demonstrated by positive staining with Tubulin-beta-III (20∼30% of cells) and MAP2 (30∼50% of the cells) (Fig. 5). Astrocytic-like cells was identified by the positive GFAP staining (30∼50% cells) (Fig. 5). Similar staining, at low percentages, was found in both unlabeled and labeled hMSCs, for the oligodendrocytic cells marker GalC (1∼5% of the cells), DAPI staining for the DNA was used in (Fig. 5H) in order to clarify the differentiated cell morphology.

The well known ability of hMSCs to suppress lymphocytes proliferation was tested in a co-culture system. Both naïve and Feridex-labeled hMSC were cultured with peripheral blood lymphocytes obtained from a healthy donor. Three experimental controls were used: (1) lymphocytes were cultured with medium without PHA (presented in Fig. 6 as LYM+MED) (2) lymphocytes that were cultured with medium containing PHA (presented in Figure 6 as LYM+PHA) and (3) Lymphocytes cultured in the presence of mesenchymal stem cells in the absence of stimulation mitogen. Lymphocytes cultured without mitogen did not show any proliferation. In the positive control, lymphocytes cultured with media containing mitogen show high proliferative ability. Lymphocytes cultured with hMSC in the absence of mitogen did not show any proliferation. Two doses of hMSC were used: 10×10³ cells and 30×10³ cells, mixed with 20×10⁴ lymphocytes under mitogen (PHA) induction. It was found that both types of cells were able to suppress the proliferation of lymphocytes in a similar dose dependent manner (Fig. 6). There was a minor and not statistically significant difference, in favor of the naïve hMSC, in terms of their proliferation blocking effect.