Int J Stem Cells.  2018 Jun;11(1):48-60. 10.15283/ijsc17062.

Transcriptional Profiling of Mesenchymal Stem Cells Identifies Distinct Neuroimmune Pathways Altered by CNS Disease

• Sargent A
• Shano G
• Karl M
• Garrison E
• Miller C
• Miller RH

• KMID: 2413500
• DOI: http://doi.org/10.15283/ijsc17062

Abstract

BACKGROUND AND OBJECTIVES
Bone marrow mesenchymal stem cells (BM-MSCs) are an attractive cell based therapy in the treatment of CNS demyelinating diseases such as multiple sclerosis (MS). Preclinical studies demonstrate that BM-MSCs can effectively reduce clinical burden and enhance recovery in experimental autoimmune encephalomyelitis (EAE), a commonly used animal model of MS. However, a number of recent clinical trials have not shown significant functional benefit following BM-MSC infusion into MS patients. One possibility for the discrepancy between animal and human studies is the source of the cells, as recent studies suggest BM-MSCs from MS patients or animals with EAE lack reparative efficacy compared to naïve cells. We sought to define important transcriptional and functional differences between diseased and naïve MSCs.
METHODS AND RESULTS
We utilized RNA Sequencing (RNA-Seq) to assess changes in gene expression between BM-MSCs derived from EAE animals and those derived from healthy controls. We show that EAE alters the expression of a large number of genes in BM-MSCs and changes in gene expression are more pronounced in chronic versus acute disease. Bioinformatic analysis revealed extensive perturbations in BM-MSCs in pathways related to inflammation and the regulation of neural cell development. These changes suggest that signals from EAE derived BM-MSCs inhibit rather than enhance remyelination, and in-vitro studies showed that conditioned medium from EAE MSCs fails to support the development of mature oligodendrocytes, the myelinating cells of the CNS.
CONCLUSIONS
These data provide insight into the failure of autologous BM-MSCs to promote recovery in MS and support the concept of utilizing non-autologous MSCs in future clinical trials.

Keyword

Mesenchymal stem cells (MSCs); Multiple sclerosis (MS); Experimental autoimmune encephalomyelitis (EAE); Oligodendrocyte; Astrocyte

MeSH Terms

Acute Disease
Animals
Astrocytes
Bone Marrow
Central Nervous System Diseases*
Computational Biology
Culture Media, Conditioned
Demyelinating Diseases
Encephalomyelitis, Autoimmune, Experimental
Gene Expression
Humans
Inflammation
Mesenchymal Stromal Cells*
Models, Animal
Multiple Sclerosis
Myelin Sheath
Oligodendroglia
Sequence Analysis, RNA
Culture Media, Conditioned

Figure

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  Fig. 1 RNA-Seq analysis of gene expression changes in BM-MSCs in EAE. (A) Volcano plots depicting differentially expressed genes (DEGs) measured by RNA-Seq analysis of peak EAE-MSCs versus naïve MSCs (left) or chronic EAE-MSCs versus naïve MSCs (right). DEGs were defined by a magnitude fold change of 1.5 or greater, q-value ≤0.05, and a normalized mean expression count ≥10. (B) Unsupervised hierarchical clustering of samples using Pearson correlation with average linkage, along with corresponding dendrogram and heatmap of DEGs. Peak EAE-MSC and chronic EAE-MSC replicates cluster together independently of naïve MSC replicates.

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  Fig. 2 Distribution of DEGs in different cellular locations. Top five specific cellular locations associated with the DEGs found between peak EAE-MSCs and naïve MSCs (left) or chronic EAE-MSCs and naïve MSCs (right). The number of DEGs associated with that location is given in parenthesis.

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  Fig. 3 EAE-MSCs show changes in genes associated with inflammation and immune activation. (A) Top 20 gene ontology terms (ranked by p-value) for DEGs found between peak EAE-MSCs and naïve MSCs (left) or chronic EAE-MSCs and naïve MSCs (right). Most GO terms relate to immune processes, particularly immune activation and immune response. (B) Heatmap showing immune genes differentially expressed between peak EAE-MSCs and naïve MSCs (left) or chronic EAE-MSCs and naïve MSCs (right). Many inflammatory genes, including chemokines, cytokines and elements of the complement system, are up regulated in EAE-MSCs.

• 

  Fig. 4 EAE-MSCs differentially regulate neural cell development and oligodendrocyte formation. (A) Top pathways (ranked by p-value) associated with the DEGs found between peak EAE-MSCs and naïve MSCs (left) or chronic EAE-MSCs and naïve MSCs (right). The number in parenthesis next to the pathway identifies statistical ranking assigned by MetaCore. Notable immune and neural cell pathways have been highlighted in green and red respectively. (B) Heatmap showing expression changes between peak EAE-MSCs and naïve MSCs (left) or chronic EAE-MSCs and naïve MSCs (right) for genes important in regulating neural cell development. (C) Neural cell cultures treated with conditioned medium (CM) from naïve MSCs had significantly higher number of MBP+ oligodendrocytes compared to control cultures treated with unconditioned medium, whereas cultures treated with EAE-MSC CM had a higher number of GFAP+ astrocytes relative to controls and they appear more reactive (insert). Scale bar=30 μm, data shown in graph=mean+SEM, **p<0.01, One-way ANOVA.

• 

  Fig. 5 Changes in gene expression in BM-MSCs throughout EAE. (A) Volcano plot depicting differentially expressed genes (DEGs) measured by RNA-Seq analysis of peak EAE-MSCs versus chronic EAE-MSCs. (B) Top 10 gene ontology (GO) terms ranked by p-value associated with the DEGs found between peak and chronic EAE-MSCs. (C) Heatmap showing immune genes differentially expressed between peak EAE-MSCs and chronic EAE-MSCs. Many inflammatory genes are up regulated in chronic EAE-MSCs compared to peak EAE-MSCs. (D) Venn diagram illustrating similarities in DEGs found between naïve, peak, and chronic EAE-MSCs. (E) Top 10 transcriptional regulators (identified through MetaCore) associated with the DEGs found between peak EAE-MSCs and naïve MSCs (left) or chronic EAE-MSCs and naïve MSCs (right). Transcription factors (TF) are ranked according to their respective z-score (the level of connectivity of the TF to the DEG list), with the number of DEGs associated with that TF indicated above each bar.

Reference

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