Intrathecal delivery of human ESC-derived mesenchymal stem cell spheres promotes recovery of a primate multiple sclerosis model

Abstract

Nonhuman primate experimental autoimmune encephalomyelitis (EAE) is a valuable model for multiple sclerosis, an inflammatory demyelinating disease in the central nervous system (CNS). Human embryonic stem cell-derived mesenchymal stem cells (EMSC) are effective in treating murine EAE. Yet, it remains unknown whether the EMSC efficacy is translatable to humans. Here we induced a primate EAE model in cynomolgus monkeys and delivered EMSC in spheres (EMSC_sp) to preserve the cell viability during long-distance transportation. EMSC_sp intrathecally injected into the CNS, remarkably reduced the clinical symptoms, brain lesions, and neuronal demyelination in the EAE monkeys during a 3-month observation. Whereas, symptoms in the vehicle control-injected EAE monkey remained and reduced slowly and MRI lesions in brain expanded. Moreover, EMSC could transdifferentiate into neural cells in vivo in the CNS of the treated animals. Supporting evidence demonstrated that EMSC_sp cells cultured in cerebrospinal fluid from the EAE monkeys largely converted to neural cells with elevated expression of genes for neuronal markers, neurotrophic factors, and neuronal myelination. Thus, this study demonstrates that EMSC_sp injected directly into the CNS, can attenuate the disease progression in the primate EAE model, highly encouraging for clinical translation.

Fig. 1. EAE induction, EMSC treatment, and cerebral lesions in cynomolgus monkeys.

a EAE induction in monkeys and subsequent EMSC treatment. b MRI images of all the EAE-induced monkeys. Displayed are representative images of T2-weighted lesions marked with red dotted lines

Fig. 2. Histological analyses of EAE-induced monkeys.

A LFB/eosin staining was performed to assess demyelination in the brain (a) and spinal cord (b) of EAE-induced monkeys. Immunostaining for MBP and GFAP indicates astrocytes activation surrounding lesions in the brain (c) and spinal cord (d) in the EAE-induced monkey C1 but not the normal control C8. B Immunostaining for αSMA, MBP, and GFAP shows (a) damages of blood–brain barrier in a lesioned area (lower panels) compared with a normal area (upper panels) in the brain of the EAE-induced monkey C5. Immunostaining for CD3 and oligodendrocytes shows (b) lymphocyte infiltration from a blood vessel to the brain of the EAE-induced monkey C1. Scale bars, 50 μm

Fig. 3. Treatment of EAE-induced monkeys with GFP⁺ EMSC_sp via i.t. injection.

a A scheme for ambient transportation of GFP⁺ EMSC_sp and i.t. injection of the EMSC_sp into EAE-induced monkeys with a table for the experimental overview shown below. b Reduction of lesions, marked with red dotted lines, in the brain of the EAE-induced monkey C5 and C7 detected via T2-weighted MRI compared with PBS-treated monkey C4

Fig. 4. Histological evidence for the myelin-protective effect and presence of EMSC in the CNS of treated monkeys.

a LFB & Eosin staining of spinal cord of C4-C7. EMSC_sp treatment reduced the demyelination in the white matter of spinal cord in EAE monkeys. b Tracking of EMSC in the brain of EAE-induced monkey C5, C6, and C7 after the third i.t. injection with EMSC_sp via immunostaining for human neural cell marker Stem121, GFP, myelin marker MBP. Scale bars, 20 μm. c Immunostaining showed Ki67+ cells in lesioned region of C5 brain. Scale bar, 20 μm

Fig. 5. Transdifferentiation of EMSC in CSF in vitro.

a Scheme for the experimental overview. b Morphology of mesenchymal and neural-like cells observed after re-plated EMSC_sp cells were cultured in the MSC medium and CSF, respectively, for 14 days. Scale bar stands for 200, 100, and 20 μm in panels 1, 2, and 3, respectively. c Immunostaining reveals TUJ1⁺ cells among re-plated GFP⁺ EMSC_sp cells after culture in CSF for 14 days. Scale bars, 20 μm. d RT-PCR shows increasing expression of genes for neural cells and oligodendrocytes in re-plated EMSC_sp cells cultured in CSF for 7 and 14 days

Fig. 6. Microarray of re-plated EMSC_sp cells cultured in CSF.

a Overview of the transcriptomic profiles of EMSC, EMSC_sp-AC/d2, and EMSC_{sp-AC/d2-CSF/d7}. The transcript level of each gene is determined via normalization of its value in each sample by the smallest value among the three samples. The color key stands for log2 of the normalized values. b Pairwise gene function/pathway enrichment analyses among the three samples. P-value at –log10 is displayed in a bar chart with red bars to indicate terms enriched for upregulated genes and blue bars to indicate terms enriched for downregulated genes under comparison between EMSC_{sp-AC/d2-CSF/d7} and EMSC (a). Normalized transcript levels for genes under three designated functions/pathways are displayed in b–d. The color key stands for log2 of the normalized values

Fig. 7

Scheme for an overview of the study