SMA Human iPSC-Derived Motor Neurons Show Perturbed Differentiation and Reduced miR-335-5p Expression

Abstract

Spinal Muscular Atrophy (SMA) is a neuromuscular disease caused by mutations in the Survival Motor Neuron 1 gene, resulting in very low levels of functional Survival of Motor Neuron (SMN) protein. SMA human induced Pluripotent Stem Cells (hiPSCs) represent a useful and valid model for the study of the disorder, as they provide in vitro the target cells. MicroRNAs (miRNAs) are often reported as playing a key role in regulating neuronal differentiation and fate specification. In this study SMA hiPSCs have been differentiated towards early motor neurons and their molecular and immunocytochemical profile were compared to those of wild type cells. Cell cycle proliferation was also evaluated by fluorescence-activated cell sorting (FACS). SMA hiPSCs showed an increased proliferation rate and also higher levels of stem cell markers. Moreover; when differentiated towards early motor neurons they expressed lower levels of NCAM and MN specific markers. The expression of miR-335-5p; already identified to control self-renewal or differentiation of mouse embryonic stem cells (mESCs); resulted to be reduced during the early steps of differentiation of SMA hiPSCs compared to wild type cells. These results suggest that we should speculate a role of this miRNA both in stemness characteristic and in differentiation efficiency of these cells.

Keywords: SMA; early motor neuron; hiPSCs; miRNA.

Figure 1

Spinal Muscular Atrophy (SMA) human induced Pluripotent Stem Cells (hiPSCs) show an increased proliferation rate and expression levels of stem cell markers compared to wild type (WT) ones. (A) Flow cytometry analysis of bromodeoxyuridine (BrdU)-positive cells of SMA- and WT-derived hiPSCs incubated with BrdU for 15, 60 and 180 min. Representative dot plots of cell cycle reporting the BrdU-positive cells are shown. Three regions have been drawn in each dot plot to identify G0/G1, S and G2/M subpopulations; (B) The column bar graph reports the percent of cells in G0/G1, S and G2/M phases of SMA- and WT-derived hiPSCs after 15, 60 and 180 min of BrdU incubation. The results (mean ± standard deviation) are representative of three independent experiments (** p < 0.01, *** p < 0.001); (C) Real time-qPCR analysis of OCT4, NANOG and SOX2 in SMA and WT-hiPSCs using the expression of WT sample as the reference. The data were normalized to 5S ribosomal RNA expression. Data are representative of three independent replicates; values represent mean ± SD; *** p < 0.001, ** p < 0.01, * p < 0.05.

Figure 2

Differential expression of Neural Cell Adhesion Molecule (NCAM) and MN-specific transcription factors along differentiation of SMA and WT hiPSCs. (A) RT-qPCR analysis of NCAM expression in WT- and SMA-derived α-motor neurons (MNs) after 14 and 22 days using hiPSCs as a reference. The data are normalized to 5S ribosomal RNA and the expression in hiPSCs was set as =1 in each genotype. Data are representative of three independent replicates; values represent mean ± SD; when comparing WT versus SMA at each time point, *** p < 0.001, ** p < 0.01; (B) The induction of MN differentiation results in transcriptional boost of Isl1, Lhx3 and HB9 in WT cells, while it is lower in SMA ones. The data are normalized to 5S ribosomal RNA and the expression levels in hiPSCs were used as a reference in each genotype. Data are representative of three independent replicates; values represent mean ± SD; (C,D) Representative immunofluorescence images of in WT- and SMA-derived MNs after 22 days of differentiation, expressing β-III tubulin (TUJ1, green) and LIM3 (red). DAPI nuclear staining is in blue. Scale bars, 50 µm.

Figure 3

Early MN derived from SMA hiPSCs express reduced levels of miR-335-5p. RT-qPCR of miR-335-5p both in hiPSCs and in early MNs (EB day 14 and EB day 22). The data were normalized to the expression of snRNA U6. Data are representative of three independent replicates; values represent mean ± SD; *** p < 0.001, ** p < 0.01.