Comparative Study on the Therapeutic Potential of Neurally Differentiated Stem Cells in a Mouse Model of Multiple Sclerosis

Background: Transplantation of neural stem cells (NSCs) is a promising novel approach to the treatment of neuroinflammatory diseases such as multiple sclerosis (MS). NSCs can be derived from primary central nervous system (CNS) tissue or obtained by neural differentiation of embryonic stem (ES) cells, the latter having the advantage of readily providing an unlimited number of cells for therapeutic purposes. Using a mouse model of MS, we evaluated the therapeutic potential of NSCs derived from ES cells by two different neural differentiation protocols that utilized adherent culture conditions and compared their effect to primary NSCs derived from the subventricular zone (SVZ). Methodology/Principal Findings: The proliferation and secretion of pro-inflammatory cytokines by antigen-stimulated splenocytes was reduced in the presence of SVZ-NSCs, while ES cell-derived NSCs exerted differential immunosuppressive effects. Surprisingly, intravenously injected NSCs displayed no significant therapeutic impact on clinical and pathological disease outcomes in mice with experimental autoimmune encephalomyelitis (EAE) induced by recombinant myelin oligodendrocyte glycoprotein, independent of the cell source. Studies tracking the biodistribution of transplanted ES cellderived NSCs revealed that these cells were unable to traffic to the CNS or peripheral lymphoid tissues, consistent with the lack of cell surface homing molecules. Attenuation of peripheral immune responses could only be achieved through multiple high doses of NSCs administered intraperitoneally, which led to some neuroprotective effects within the CNS

Effect of intravenous NSC transplantation on the clinical course of EAE.

<p>(A) C57Bl/6 mice were immunized with recombinant myelin oligodendrocyte glycoprotein and treated with intravenous injections of GS-N cells, 46C-NS cells or SVZ-NSCs on days 8, 10 and 12 post immunization. (B) Proliferative response of splenic T-cells from cell-treated mice and controls to rMOG and non-specific mitogens expressed as the mean counts per minute (CPM). Data are expressed as the mean ± SEM (n = 5–7 mice). (C) Decreasing amounts of DNA from GFP⁺ GS-N cells were titrated with DNA extracted from EAE mice and the minimum amount of GFP⁺ DNA detectable was determined by PCR using primers specific for GFP. (D) GFP⁺ GS-N cells or fibroblasts were injected intravenously into EAE mice at 12 days post disease induction and PCR was used to detect GFP⁺ cells. PCR reactions performed without DNA served as a negative control and DNA from C57Bl/6-GFP chimeric mice served as a positive control.</p

Suppression of <i>in vitro</i> MOG-specific T-cell responses.

<p>(A) Fixed numbers of splenocytes from 2D2 mice stimulated with MOG_35–55 were cultured in the presence of varying numers of GS-N cells, 46C-NS cells or SVZ-NSCs (expressed as NSC: splenocyte ratio). Proliferative responses were measured by ³H-thymidine incorporation and expressed as the mean counts per minute (CPM). (B–G) Supernatants were collected from co-cultures after 48 hrs and the level of pro-inflammatory cytokines was quantified by cytometric bead array. Data represent the mean ± SEM (n = 4 mice). *P&lt;0.05, **P&lt;0.01, ∧P&lt;0.005, ^#P&lt;0.001 vs MOG_35–55 stimulated splenocytes unless otherwise indicated.</p

Clinical and pathological outcome of EAE mice.

<p>Data are expressed as mean ± SEM.</p>*<p>p&lt;0.05,</p>**<p>p&lt;0.01 vs PBS.</p

Cell surface expression of homing molecules.

<p>Flow cytometric analysis was used to examine cell surface expression of homing molecules by GS-N cells (A), 46C-NS cells (B) and SVZ-NSCs (C). Representative histograms are shown in grey and respective isotype controls are indicated by the black curve.</p

Characterization of 46C-NS cells.

<p>(A–C) Immunofluorescence staining showed 46C-NS cells expressed nestin, red (A) and 3CB2, red (B) but not GFAP, green (A) or MAP2, green (B). (<b>D–E</b>) Following neuronal or astrocytic differentiation, 46C-NS cells expressed βIII-Tubulin, red and MAP2, green (C) or GFAP (D), respectively.</p