Immunomodulation with Human Umbilical Cord Blood Stem Cells Ameliorates Ischemic Brain Injury - A Brain Transcriptome Profiling Analysis

Abstract

Our group previously demonstrated that administration of a CD34-negative fraction of human non- hematopoietic umbilical cord blood stem cells (UCBSC) 48 h after ischemic injury could reduce infarct volume by 50% as well as significantly ameliorate neurological deficits. In the present study, we explored possible mechanisms of action using next generation RNA sequencing to analyze the brain transcriptome profiles in rats with ischemic brain injury following UCBSC therapy. Two days after ischemic injury, rats were treated with UCBSC. Five days after administration, total brain mRNA was then extracted for RNAseq analysis using Illumina Hiseq 2000. We found 275 genes that were significantly differentially expressed after ischemic injury compared with control brains. Following UCBSC treatment, 220 of the 275 differentially expressed genes returned to normal levels. Detailed analysis of these altered transcripts revealed that the vast majority were associated with activation of the immune system following cerebral ischemia which were normalized following UCBSC therapy. Major alterations in gene expression profiles after ischemia include blood-brain-barrier breakdown, cytokine production, and immune cell infiltration. These results suggest that UCBSC protect the brain following ischemic injury by down regulating the aberrant activation of innate and adaptive immune responses.

Keywords: human umbilical cord blood; immunomodulation; macrophage; microglia; neuroinflammation; stem cells; stroke.

Fig 1.

UCBSC therapy ameliorates neurological deficits induced by MCAO. (A) The Neurological Severity Scores (NSS) were similar for the two groups of animals 2 days post MCAO but prior to the UCBSC therapy. Five days after UCBSC cell therapy (day 7 after MCAO), NSS scores improved significantly in the UCBSC treated group (t-test P = 0.046). (B, C, and D) PCA of RNA-seq data show MCAO-treated samples cluster separately from Normal and MCAO+UCBSC groups.

Fig 2.

Gene expression analysis following MCAO and UCBSC treatment. (A) Scatter plot of significantly DE genes that return to normal expression levels after UCBSC treatment. Black dotted line represents expression levels of transcripts from normal brain. Red dots are expression levels of transcripts from MCAO group. Aqua colored circles are expression levels of transcripts after UCBSC treatment. X-axis indicates log10 counts per million (cpm) level from normal brain. Y-axis represents corresponding expression levels for the same transcript from MCAO group or UCBSC group. (B) Gene expression patterns following MCAO and UCBSC treatment.

Fig 3.

Heatmap of (A) BBB, (B) ECM, (C) apoptosis, and (D) cytokine associated genes. The expression pattern of these genes return to normal expression levels after UCBSC treatment. Red indicates relative increase in expression and blue indicates relative decrease in expression.

Fig 4.

CIBERSORT analysis of control, MCAO, and UCBSC treatment. The relative percentage of different immune cell types were deconvoluted from the RNA-seq data using CIBERSORT.

Fig 5.

Box plot showing RNA expression levels change of (A) Neutrophil, (B) Natural Killer cell, (C) Macrophage, (D) B-cell, (E) Mast cell, (F) T-cell, and (G) Regulatory T-cell related gene transcripts after MCAO (Red) and UCBSC treatment (Green). * denotes statistical significance between the three groups P < 0.05 (edgeR, false-discovery-rate adjusted).