miRNA profiling of NurOwn®: mesenchymal stem cells secreting neurotrophic factors

Abstract

Background: MSC-NTF cells are Mesenchymal Stromal Cells (MSC) induced to express high levels of neurotrophic factors (NTFs) using a culture-medium based approach. MSC-NTF cells have been successfully studied in clinical trials for Amyotrophic Lateral Sclerosis (ALS) patients. MicroRNAs (miRNA) are short non-coding RNA molecules that coordinate post-transcriptional regulation of multiple gene targets. The purpose of this study was to determine whether the miRNA profile could provide a tool for MSC-NTF cell characterization and to distinguish them from the matched MSC from which they are derived.

Methods: NTF secretion in the culture supernatant of MSC-NTF cells was evaluated by ELISA assays. The Agilent microarray miRNA platform was used for pairwise comparisons of MSC-NTF cells to MSC. The differentially expressed miRNAs and putative mRNA targets were validated using qPCR analyses.

Results: Principal component analysis revealed two distinct clusters based on cell type (MSC and MSC-NTFs). Nineteen miRNAs were found to be upregulated and 22 miRNAs were downregulated in MSC-NTF cells relative to the MSC cells of origin. Further validation of differentially expressed miRNAs confirmed that miR-3663 and miR-132 were increased 18.5- and 4.06-fold, respectively while hsa-miR-503 was reduced more than 15-fold, suggesting that miRNAs could form the basis of an MSC-NTF cell characterization assay. In an analysis of the miRNA mRNA targets, three mRNA targets of hsa-miR-132-3p (HN-1, RASA1 and KLH-L11) were found to be significantly downregulated.

Conclusions: We have demonstrated that MSC-NTF cells can be distinguished from their MSCs of origin by a unique miRNA expression profile.

Trial registration: Clinicaltrial.gov identifier NCT01777646 . Registered 12 December 2012.

Keywords: Amyotrophic Lateral Sclerosis; Mesenchymal Stromal Cells; MicroRNAs; Neurotrophic Factors.

Fig. 1

NTF secretion by MSC and MSC-NTF cells of the same patient/donor. MSC of six ALS patients and two healthy donors (D13 and D9) were induced to differentiate into MSC-NTF cells and secretion of neurotrophic factors GDNF, VEGF and HGF was measured in the culture supernatant by ELISA before and after differentiation. Average fold change MSC-NTF/MSC 6.56, 6.01, and 7.85 respectively (p < 0.005). GDNF glial-derived neurotrophic factor, HGF hepatocyte growth factor, MSC mesenchymal stromal cells, NTF neurotrophic factors, VEGF vascular endothelial growth factor

Fig. 2

Comparison analysis of the MSC and MSC-NTF cell types based on all 160 detected miRNAs with cell type and donor ID indicated. a Representation of the eight cellular matched miRNA profiles of the four ALS patients in a 3D PCA projection, including donor ID (02, 03, 05, and 07); b Representation of the eight cellular miRNA profiles as a heatmap clustergram plot after hierarchical clustering, including donor ID. MSC mesenchymal stromal cells, NTF neurotrophic factors, PCA principal component analysis

Fig. 3

Differentially expressed miRNAs. Expression profiles of the 19 upregulated miRNAs (left panel) and the 22 downregulated miRNAs in MSC-NTF versus MSC on a log2 scale (right panel). The miRNAs most strongly down- and up-regulated in MSC-NTFs are highlighted with red ovals. When the expression of a miRNA was below the level of detection for the arrays, a nominal intensity value is given to these data points to avoid errors arising from non-computable mathematical operations during subsequent data analyses. MSC mesenchymal stromal cells, NTF neurotrophic factors

Fig. 4

Validated differentially expressed miRNAs. Differential expression of miRNAs identified in the microarray was validated by qPCR analysis of MSC and MSC-NTF cells of eight different donors (six ALS patients and two healthy donors). ***p < 0.001, two-sided t test. FC fold change, MSC mesenchymal stromal cells, NTF neurotrophic factors

Fig. 5

Differential expression of mRNAs targets of hsa-miR-132-3p. Expression of mRNA targets of miR-132-3p were compared in MSC and MSC-NTF cells of MSC and MSC-NTF cells of eight different donors (six ALS patients and two healthy donors) by qPCR analysis. HN-1, RASA1 and KLH-L11 were significantly downregulated (11.86 fold, 3.73 -fold and 1.5-fold respectively, p < 0.05). MSC mesenchymal stromal cells, NTF neurotrophic factors