An Example of Neuro-Glial Commitment and Differentiation of Muse Stem Cells Obtained from Patients with IQSEC2-Related Neural Disorder: A Possible New Cell-Based Disease Model

Abstract

Although adult stem cells may be useful for studying tissue-specific diseases, they cannot be used as a general model for investigating human illnesses given their limited differentiation potential. Multilineage-differentiating stress-enduring (Muse) stem cells, a SSEA3(+) cell population isolated from mesenchymal stromal cells, fat, and skin fibroblasts, may be able to overcome that restriction. The Muse cells present in fibroblast cultures obtained from biopsies of patients' skin may be differentiated into cells of interest for analyzing diseases. We isolated Muse stem cells from patients with an intellectual disability (ID) and mutations in the IQSEC2 gene (i.e., BRAG1 gene) and induced in vitro neuroglial differentiation to study cell commitment and the differentiation of neural lineages. The neuroglial differentiation of Muse cells revealed that IQSEC2 mutations may alter the self-renewal and lineage specification of stem cells. We observed a decrease in the percentage of SOX2 (+) neural stem cells and neural progenitors (i.e., SOX2+ and NESTIN+) in cultures obtained from Muse cells with the mutated IQSEC2 gene. The alteration in the number of stem cells and progenitors produced a bias toward the astrocytes' differentiation. Our research demonstrates that Muse stem cells may represent a new cell-based disease model.

Keywords: adult stem cells; disease model; intellectual disability.

Figure 1

Markers of Muse stem cells isolated from patients’ fibroblasts. Panel (A): Micrographs showing the typical morphology of Muse stem cells grown in suspension. The black bar corresponds to 100 µm. Panel (B): Representative flow cytometry plots of CD73, CD90, CD105, CD45, and CD44 on Muse cells isolated from healthy donors and patients. The histograms indicate the percentage of positive cells for every analyzed marker. Panel (C): Representative images of immunocytochemistry analysis to detect SSEA3 (i.e., green), SOX2 (i.e., red), NANOG (i.e., green), and OCT3/4 (i.e., red) in Muse stem cells (200× magnification). Cell nuclei were identified with DAPI (i.e., blue). The graphs on the right indicate the percentages of positive cells in the different samples. For all experiments, the symbols *** (i.e., p < 0.001), ** (i.e., p < 0.01), and * (i.e., p < 0.05) indicate statistical significance between the healthy control and the patients’ samples. For every sample, three biological replicates were performed, and data are reported with SDs.

Figure 2

Biological parameters of patients’ Muse cells. Panel (A): Representative plots of cell cycle analysis on Muse stem cells. Panel (B): Example of annexin assay performed on Muse stem cells. The percentage of apoptotic cells in the different samples is shown in the graph. Panels (C,D): Representative images of beta-galactosidase assay (C) and Ki67 immunostaining (D) performed on Muse stem cells (200× magnification). The immunostaining identified the Ki67 positive cells, shown in red, while the nuclei were stained with DAPI in blue. In Panel (C), some senescent cells in blue are indicated with an arrow. The graphs indicate the percentages of (beta-gal+) senescent cells and (Ki67+) cycling cells, respectively. For all of the experiments, the symbols *** (i.e., p < 0.001), ** (i.e., p < 0.01), and * (i.e., p < 0.05) indicate statistical significance between the healthy control and the patients’ samples. For every sample, three biological replicates were performed, and data are reported with SDs.

Figure 3

Neuroglial differentiation of Muse stem cells. Panel (A): Graph showing the percentages of multipotent Muse stem cells (SOX2+), neural stem cells (i.e., SOX2+ and NESTIN+), neuronal (MAP2+), astrocytes (GFAP+), and oligodendrocyte-committed progenitors (O1+) following neuroglial differentiation of Muse stem cells obtained from a healthy control and Patient 1. The micrographs show representative images of immunostaining on differentiated samples (200× magnification). Green and red dots in the background are due to poly-L-lysine coating present in the samples. In Muse-CT sample, the white arrow indicates SOX2/NESTIN-positive cells. Panel (B): Histogram showing the mRNA expression level of the indicated genes. The mRNA levels were normalized to GAPDH mRNA expression, which was selected as an internal control. NES = NESTIN; VIM = VIMENTIN. Panel (C): mRNA levels of reactive astrocyte markers identified by qRT-PCR. The expression level of markers was set as 1 in the sample obtained from a healthy control (i.e., Muse-CT). For all of the experiments, the symbols *** (i.e., p < 0.001), ** (i.e., p < 0.01), and * (i.e., p < 0.05) indicate statistical significance between the control and Patient 1 (n = 3 biological replicates), and data with SD are reported.

Figure 4

Astrocyte differentiation of Muse stem cells. Panel (A): Examples of immunostaining on Muse stem cells following astrocyte differentiation (200× magnification). The staining for several astrocyte markers was evaluated in non-differentiated and differentiated Muse stem cells (ND-Muse; Diff-Muse). Some green background dots are due to Matrigel present on the samples. The cells’ appearance under light microscopy is also shown. The histogram reports the percentages of cells expressing the analyzed markers. For all experiments, the symbol *** (p < 0.001) indicates statistical significance between the differentiated and non-differentiated cells (n = 3 biological replicates), and data with SD are reported. Panel (B): Phase contrast microscope images of fibroblasts (FIB) isolated from a healthy control and Patient 1 (200× magnification). The figure also shows the undifferentiated Muse cells (Muse) isolated from the corresponding fibroblast cultures and the Muse cells induced to become astrocytes through a four-step procedure: the 1st and 2nd steps of neural induction, neurosphere induction, and mature astrocytes. The patient’s samples failed to become mature astrocytes, and the impairment of differentiation process was already evident at neural induction 2nd step.