Amelioration of experimental autoimmune encephalomyelitis through transplantation of placental derived mesenchymal stem cells
Placental derived mesenchymal stem cells (PMSCs) have been suggested as a possible source of cells to treat multiple sclerosis (MS) due to their immunomodulatory functions, lack of ethical concerns, and potential to differentiate into neurons and oligodendrocytes. To investigate whether PMSCs share similar characteristics with embryonic mesenchymal stem cells (EMSCs), and if transplanted PMSCs have the ability to integrate and replace degenerated neural cells, we transplanted rat PMSCs and EMSCs into the central nervous system (CNS) of Lewis rats with experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Our findings demonstrated that transplanted PMSCs, similar to EMSCs, were effective in decreasing infiltrating inflammatory cells, preserving axons, and ameliorating demyelination, thereby improving the neurological functions of animals. Moreover, both PMSCs and EMSCs had the ability to migrate into inflamed tissues and express neural–glial lineage markers. These findings suggest that PMSCs may replace EMSCs as a source of cells in MS stem cell therapy.PMSCs have the potential to differentiate into all cell types, depending on the local microenvironment15. To test the ability of our PMSCs to differentiate into neural cells before the transplantation, we cultured cells in the neural differentiation medium, and stained the cells with specific neural markers. As expected, our cultured PMSCs extensively co-expressed the mesenchymal stem cell marker CD44 (red) along with the astrocyte specific marker GFAP (green, Figure S1A–D), oligodendrocyte specific marker Olig1 (green, Figure S1E–H), or neuron specific marker NF-200 (green, Figure S1M–Q). Partial expression of the microglia/macrophage specific marker CD68 (green, Figure S1I–L) was also present. The results suggest that our PMSCs have the potential to differentiate into both neuronal and glia cells in vitro.Placental MSCs were extracted from the placenta as previously described15. The placentas of healthy 18-day pregnant naïve/GFP-rats were collected after Caesarean section. The minced tissue was transferred to a 50 mL centrifuge tube containing DMEM (Invitrogen, Carlsbad, CA, USA) plus 0.1% bovine serum albumin (BSA) (Sigma-Aldrich, St. Louis, MO, USA) and digested for 45 min at 37 °C with a combination of 0.1% collagenase type IV (Sigma-Aldrich, St. Louis, MO, USA), 200 μg/mL DNAse I (Worthington, Lakewood, NJ, USA), and 0.1% dispase (Invitrogen Carlsbad, CA, USA). The tissue was triturated every 10 min using 10 mL plastic serological pipettes. The resulting homogenate was successively filtered through a 100 μm Nytex mesh (BD Falcon, MD, USA), placed in a standard laboratory funnel, and then centrifuged at 800 g for 20 min. The cell pellet was resuspended twice and plated on dishes coated with human placental fibronectin (100 ng/mL; Sigma-Aldrich, St. Louis, MO, USA) at a density of 1 × 106 cells/80 cm2 (i.e., 1 × 10 cm dish) in DMEM (Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS) (Biological Industries, Beit Haemek, Israel), 1:100 non-essential amino acids (Biological Industries, Beit Haemek, Israel), 55 μM beta-mercaptoethanol (Sigma-Aldrich, St. Louis, MO, USA), 10 μg/mL ciprofloxacin (Bayer, Leverkusen, Germany), and 10 μg/mL amphotericin B (Invitrogen, Carlsbad, CA, USA).GFP-conjugated Embryonic Stem Cell-Derived Mesenchymal Stem Cells (EMSCs) was purchased from Cyagen Biosciences and cultured in Murine Mesencult medium (Stem Cell Technologies, Vancouver, BC). Cells were kept in a humidified 5% CO2 incubator at 37 °C, and the medium were refreshed every 3 to 4 days, for 4 to 5 weeks.This work was funded by the Zhejiang Provincial Natural Science Foundation of China (project nos LY16H09002 and R2110025), Foundation of Zhejiang Provincial Education Department (Y201431129) and by the National Natural Science Foundation of China (project no. 81271333).The authors declare no competing financial interests.Author Contributions H.J. and S.H. designed the study. H.J. and B.B.W. collected and analysed the data. Y.Y.Z. and K.W.T. carried out the histological staining and analysis. S.H. drafted and wrote the manuscript. All authors gave intellectual input to the study and approved the final version of the manuscript.