Immunomodulation by transplanted human embryonic stem cell-derived oligodendroglial progenitors in experimental autoimmune encephalomyelitis

Abstract

Transplantation of embryonic stem cells and their neural derivatives can lead to amelioration of the disease symptoms of experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis (MS). Oligodendroglial progenitors (OPs), derived from human embryonic stem cells (hESC, HES-1), were labeled with superparamagnetic iron oxide and transduced with luciferase. At 7 days following induction of EAE in C57/BL6 mice, 1 × 10(6) cells were transplanted in the ventricles of C57/BL6 mice and noninvasively monitored by magnetic resonance and bioluminescence imaging. Cells were found to remain within the cerebroventricular system and did not survive for more than 10 days. However, EAE mice that received hESC-OPs showed a significant improvement in neurological disability scores (0.9 ± 0.2; n = 12) compared to that of control animals (3.3 ± 0.4; n = 12) at day 15 post-transplantation. Histopathologically, transplanted hESC-OPs generated TREM2-positive CD45 cells, increased TIMP-1 expression, confined inflammatory cells within the subarachnoid space, and gave rise to higher numbers of Foxp3-positive regulatory T cells in the spinal cord and spleen. Our results suggest that transplantation of hESC-OPs can alter the pathogenesis of EAE through immunomodulation, potentially providing new avenues for stem cell-based treatment of MS.

Figure 1

(A): In vitro characterization and quantification of hESC-OPs. Cells were stained for nestin (B), βIII-tubulin (C; TUJ1), NG2 (D), and GFAP (E). Red fluorescence was seen in the human embryonic stem cell-derived oligodendroglial progenitors (hESC-OPs) after 1-day incubation with Molday ION Rhodamine B (F). In vitro bioluminescent imaging of genetically engineered hESC-OPs shows increasing luciferase activity with cell density (G). Data were obtained in triplicate, showing a linear correlation between total photon flux and cell number (H) (R² = 0.99). Abbreviation: GFAP, glial fibrillary acidic protein.

Figure 2

In vivo bioluminescent imaging (BLI) of human embryonic stem cell-derived oligodendroglial progenitors (hESC-OPs). The cells, transplanted intracerebroventricular at day 7 post experimental autoimmune encephalomyelitis induction, do not survive for more than 10 days PT. (A): Representative images obtained at days 1, 5, and 10 PT. Color bar indicates the photon flux intensity level of luciferase. (B): Quantification of BLI signal. Each time point represents the mean of five animals ± SEM. Abbreviation: PT, post-transplantation

Figure 3

Clinical course and disease parameters in mice transplanted with hESC-OPs. (A): A significant EAE progression was observed in hESC-derived OPs-transplanted animals compared to EAE mice that received dead cells or no cells (*, p < .05). Although it appears that the dead cell-transplanted group had a small improvement in clinical scoring over the control (no cell) group, this difference was not significant (p > .05%). Clinical parameters [incidence hind limb paralysis (B), maximum clinical score (C), duration of severe paralysis (D), cumulative clinical score (E), and recovery rate (F)] were analyzed in EAE mice receiving live cells and no cells, and monitored until day 25 PT (i.e., at day 32 post EAE induction). *, p < .05. Abbreviations: EAE, experimental autoimmune encephalomyelitis; hESC-OPs, human embryonic stem cell-derived oligodendroglial progenitors.

Figure 4

Effect of transplanted hESC-derived OPs on migration of inflammatory cells in EAE. Transplanted hESC-derived OPs retain CD45-positive cells within the subarachnoid space of the spinal cord. Anti-CD45 immunofluorescent staining was performed at days 5, 15, and 25 PT of hESC-OPs. A heavy infiltration of CD45-positive cells in the white matter of the spinal cord of EAE control animals can be seen. Scale bar = 200 μm. Abbreviations: EAE, experimental autoimmune encephalomyelitis; hESC-OPs, human embryonic stem cell-derived oligodendroglial progenitors; PT, post-transplantation.

Figure 5

Effect of transplanted hESC-derived OPs on macrophage activation and demyelination. In EAE control animals, activation of macrophages (IB4-positive cells) and demyelination (MBP-negative area, arrowheads) were detected in the white matter. In the groups that received cells, there was a limited activation of macrophages and demyelination in the white matter. Scale bars = 80 μm. Abbreviations: EAE, experimental autoimmune encephalomyelitis; hESC-OPs, human embryonic stem cell-derived oligodendroglial progenitors; IB4, Isolectin B4; MBP, myelin basic protein.

Figure 6

Cytokine/chemokine array of brains and spinal cords from EAE control and hESC-derived OPs-transplanted EAE mice. As compared to untreated EAE controls, hESC-OPs increase the expression of certain chemokines in the brain and spinal cord at 5 days PT. Note the increased level of TIMP-1 in the central nervous system (CNS) of animals receiving live hESC-OPs. TIMP-1 is a molecule related to oligodendrocyte differentiation that enhances myelination and reduces blood-brain breakdown by matrix metalloproteases. Abbreviations: EAE, experimental autoimmune encephalomyelitis; hESC-OPs, human embryonic stem cell-derived oligodendroglial progenitors.

Figure 7

Induction of TREM2 expression in EAE mice after hESC-OP transplantation. (A): TREM2 immunostaining in the spinal cord. There was a limited number of TREM2-positive cells in the EAE control animals, while an increased number of TREM-2 positive cells was detected in the subarachnoid space of the spinal cord in the group of animals that received live hESC-OPs. Scale bars = 100 μm. (B): Representative high magnification images of the insets in (A). Scale bar = 50 μm. (C): Semiquantitative analysis of TREM2-positive cells in EAE control (Cont) and animals that received cells (Tx). Each point is the mean of six spinal cords ± SEM. Data were analyzed using one-way ANOVA followed by the Student–Newman–Keuls post hoc test for multiple comparisons. **, p < .01 versus the other points; ***, p < .001 versus the other points. Abbreviations: EAE, experimental autoimmune encephalomyelitis; hESC-OPs, human embryonic stem cell-derived oligodendroglial progenitors; PT, post-transplantation; TREM, triggering receptor expressed on myeloid cells.