Hepatocyte growth factor mediates mesenchymal stem cell–induced recovery in multiple sclerosis models

Journal name:
Nature Neuroscience
Volume:
15,
Pages:
862–870
Year published:
DOI:
doi:10.1038/nn.3109
Received
Accepted
Published online

Abstract

Mesenchymal stem cells (MSCs) have emerged as a potential therapy for a range of neural insults. In animal models of multiple sclerosis, an autoimmune disease that targets oligodendrocytes and myelin, treatment with human MSCs results in functional improvement that reflects both modulation of the immune response and myelin repair. Here we demonstrate that conditioned medium from human MSCs (MSC-CM) reduces functional deficits in mouse MOG35–55-induced experimental autoimmune encephalomyelitis (EAE) and promotes the development of oligodendrocytes and neurons. Functional assays identified hepatocyte growth factor (HGF) and its primary receptor cMet as critical in MSC-stimulated recovery in EAE, neural cell development and remyelination. Active MSC-CM contained HGF, and exogenously supplied HGF promoted recovery in EAE, whereas cMet and antibodies to HGF blocked the functional recovery mediated by HGF and MSC-CM. Systemic treatment with HGF markedly accelerated remyelination in lysolecithin-induced rat dorsal spinal cord lesions and in slice cultures. Together these data strongly implicate HGF in mediating MSC-stimulated functional recovery in animal models of multiple sclerosis.

At a glance

Figures

  1. Conditioned growth medium from human MSCs biases the development of neurosphere derived cells toward oligodendrocytes and neurons, and promotes functional recovery in MOG35-55-induced EAE.
    Figure 1: Conditioned growth medium from human MSCs biases the development of neurosphere derived cells toward oligodendrocytes and neurons, and promotes functional recovery in MOG35–55-induced EAE.

    (a) In the presence of human MSC-CM, the proportion or GFAP+ astrocytes is reduced and the proportion of oligodendrocyte-lineage cells and neurons is increased. (b) Quantitation of cell types in the presence and absence of MSC-CM. *Control versus MSC-CM: A2B5 P < 0.01, O4 P < 0.05, β-tubulin (β-tub) P < 0.05, GFAP P = 0.005. Mean ± s.e.m. of duplicate preparations taken from three independent experiments. Note there are both overlapping expression and unlabeled cells in these preparations. (c) Treatment with MSC-CM (0.5 mg) (arrow), but not PBS, at peak of disease after MOG35–55 immunization results in functional improvement in EAE. Mean ± s.e.m. of all mice, n = 11. (d) The functional improvement is correlated with a reduction in myelin loss and tissue damage seen with luxol fast blue staining of spinal cord sections; lesions are outlined. Scale bars: 50 μm in a, 500 μm in d.

  2. The activity of MSC-CM to enhance functional recovery in EAE is dependent on a 1-100-kDa fraction.
    Figure 2: The activity of MSC-CM to enhance functional recovery in EAE is dependent on a 1–100-kDa fraction.

    (a) Treatment with MSC-CM100kDa (0.5 mg per mouse, n = 11), but not control conditioned medium (CTL-CM), enhances functional recovery in mice with EAE. Error bars, s.e.m. (b) Growth of neurosphere-derived cultures in MSC-CM100kDa biases cell development in favor of oligodendrocytes and neurons compared to growth in CTL-CM. (c) Treatment with MSC-CM100kDa reduces proinflammatory cytokine expression by spinal cord–derived mononuclear cells in EAE mice. Significantly reduced expression of IFN-γ, IL-17, TNF-α, IL-2 and IL-12p70 and increased expression of IL-10 and IL-4 were seen in mice treated with MSC-CM100kDa. *P = 0.05, **P = 0.01. Mean ± s.e.m. from three independent experiments.

  3. Human MSC-CM100kDa contains HGF and HGF promotes functional and histological recovery in EAE.
    Figure 3: Human MSC-CM100kDa contains HGF and HGF promotes functional and histological recovery in EAE.

    (a) Western blot of three samples of active MSC-CM100kDa shows the presence of HGF. Ctl, unconditioned control medium; Mkr, size marker (kDa). (b) Treatment with HGF (one injection every other day for a total of three over a 5-d period, n = 10) results in functional improvement compared to vehicle control treatment. Arrows indicate treatment initiation day. Error bars, s.e.m. (c) HGF improves tissue histology. Sections after 100 ng i.v. at 30 d after immunization with MOG35–55. The improvement in myelination is apparent by luxol fast blue (LFB) staining. The reduction in immune cell infiltrates is evident in hematoxylin and eosin (H&E) and anti-CD3-labeled sections and confirmed in toluidine blue (tol blue)-stained 1-μm sections. EM, representative electron micrographs through spinal cord lesion areas in EAE control and HGF-treated mice 17 d after initiation of treatment. Outlines define lesion area. (d) Top: analysis of myelin thickness versus axon diameter in lesion areas of control and HGF-treated mice demonstrates thicker myelin in HGF-treated animals compared to controls. Bottom: comparison of the relative axon diameters in lesion areas of control EAE and HGF-treated mice demonstrates a reduction in small-diameter fibers and an increase in medium-diameter fibers in HGF-treated animals. Scale bars: 500 μm in c LFB (top); 50 μm in c LFB (second panel), H&E, CD3 and toluidine blue; 2 μm in EM.

  4. Inhibition of HGF signaling with cMet antibodies negates the capacity of both HGF and MSC-CM to induce functional recovery and reverses EAE-induced changes in cytokine expression.
    Figure 4: Inhibition of HGF signaling with cMet antibodies negates the capacity of both HGF and MSC-CM to induce functional recovery and reverses EAE-induced changes in cytokine expression.

    (a) Two injections of function-blocking cMet antibodies 12 h apart (blue arrows), delivered 1 d before initiation of HFG treatment regimen (yellow arrows) inhibits functional recovery. (b) Similarly, cMet inhibition blocks MSC-CM100kD-stimulated functional recovery. Two injections of function-blocking cMet antibody (purple arrows) at peak of disease or anti-HGF (black arrow) inhibits recovery. (c,d) Treatment with cMet antibodies increases proinflammatory cytokine expression and reduces anti-inflammatory cytokine expression as shown by cytokine profile in mice treated with HGF (c) or MSC-CM100kDa (d). (e,f) ELISPOT analysis on spinal cord–derived cells demonstrates that treatment with cMet antibodies increases the frequencies of TH1 and TH17 cells compared to those in mice treated with HGF alone (e) or MSC-CM100kDa alone (f). The delay in disease onset reflects the use of a different preparation of MOG35–55 peptide and not anti-HGF treatment. The data are representative from triplicate studies; ELISPOT counts represent the mean ± s.d. from one of three experiments. *P = 0.05, **P = 0.01; error bars, s.d.

  5. Inhibition of HGF signaling with cMet or anti-HGF blocks the ability of MSC-CM100kDa and HGF to alter the development and migration of neural cells from neurospheres.
    Figure 5: Inhibition of HGF signaling with cMet or anti-HGF blocks the ability of MSC-CM100kDa and HGF to alter the development and migration of neural cells from neurospheres.

    (a) The proportions of A2B5+, O4+, β-tubulin+ and GFAP+ cells are altered in the presence of MSC-CM100kDa and HGF, and reversed by cMet and anti-HGF. (b) Quantification of cell types in the presence and absence of MSC-CM100kDa and HGF, with or without cMet antibodies. Compared to that in controls, the proportion of O4+ and β-tubulin+ cells is increased by MSC-CM and HGF (P < 0.05 for both), and this increase is blocked by cMet antibodies (P < 0.01 for both). Mean ± s.d. of the proportion of individual cell types taken from five random fields from at least two independent experiments. Note that there are both unlabeled cells and overlap of antigen expression on individual cells. (c) Treatment with HGF stimulates migration of neuronal precursors from adult SVZ-derived neurospheres of EAE mice, and this effect is blocked by anti-cMet. (d) Treatment with MSC-CM100kDa stimulates migration of OPCs and neuronal precursors from adult EAE SVZ-derived neurospheres, and this effect is blocked by cMet and anti-HGF. (e) The mobilization of PLP+ OPCs into EAE lesions is enhanced in mice treated with HGF. The number of EGFP-PLP cells that populate lesions increases upon HGF treatment. Outlining defines neurosphere edges. Scale bars: 20 μm in a, 100 μm in c,d. 50 μm in e. Lesion areas are outlined in e.

  6. Systemic HGF treatment stimulates remyelination of rat spinal cord LPC lesions.
    Figure 6: Systemic HGF treatment stimulates remyelination of rat spinal cord LPC lesions.

    (a,b) Treatment with 0.8 mg kg−1 HGF 5, 9 and 11 dpl results in smaller demyelinated lesions at 14 dpl as shown by luxol fast blue. (ch) Immunohistochemical labeling of frozen sections, demonstrating an increase in MBP labeling (c,d), increase in NG2+ cells (e,f) and a decrease in GFAP expression (g,h) in lesion areas from HGF-treated rats 14 dpl. (i,j) Toluidine blue sections 1 μm thick show extensive remyelination at 14 dpl in HGF-treated rats but not in controls, and ultrastructural analyses shows that axons of different caliber have myelin sheaths of different thicknesses, indicative of ongoing repair, in HGF (l,m) but not control lesions (k). (m) G ratios showing thin myelin sheaths in HGF-treated rats. Scale bars: af, 100 μm; g,h, 50 μm; i,j, 5 μm; k, 1 μm; l, 2 μm; m, 0.5 μm.

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Author information

Affiliations

  1. Center for Translational Neuroscience, Department of Neurosciences, Case School of Medicine, Case Western Reserve University, Cleveland, Ohio USA.

    • Lianhua Bai,
    • Anne DeChant,
    • Jordan Hecker,
    • Janet Kranso,
    • Anita Zaremba &
    • Robert H Miller
  2. Skeletal Research Center, Case Western Reserve University, Cleveland, Ohio, USA.

    • Donald P Lennon &
    • Arnold I Caplan

Contributions

L.B., A.I.C. and R.H.M. conceived the study and experimental design. D.P.L. and A.I.C. prepared and processed the mesenchymal stem cells. L.B. performed all EAE experiments, immunohistochemistry and data analysis. A.Z. designed and conducted the slice and culture studies. J.H. and J.K. conducted the LPC lesion studies. L.B., A.I.C., A.D. and R.H.M. wrote the paper and designed the figures. All authors discussed the results and implications and commented on the manuscript at all stages.

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The authors declare no competing financial interests.

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