Integrin α5β1 Mediated Cellular Reorganization in Human Mesenchymal Stem Cells During Neuronal Differentiation

Abstract

Background/aim: Mesenchymal stem cells (MSCs) have been widely used for yielding neurons in culture to study nervous system pathologies and develop regenerative approaches. In this study, cellular rearrangements of human MSCs related to the expression of the fibronectin common receptor integrin α5β1 and its cell surface localization during neuronal differentiation, were examined.

Materials and methods: Proliferation kinetics of neuronal induced hMSCs (hMd-Neurons) were quantified by BrdU assay, and hMd-Neurons were immunostained for neuronal marker expression. Additionally, cDNA and protein samples were collected at different time points for integrin α5β1 expression analysis.

Results: Endogenous integrin α5β1 expression was significantly upregulated by day 6 and maintained until day 12. Cell surface localization of α5β1 integrin was increased by day 6; the integrin was internalized into the cytosol by day 12.

Conclusion: Integrin dynamics around day 6 of differentiation might be involved in neuronal differentiation and maturation or specification of hMd-Neurons.

Keywords: Mesenchymal stem cells; integrin; integrin α5β1; neuronal differentiation.

Figure 1. Derivation of neurons from hMSCs (hMd-Neurons). A) hMSC immunophenotyping by flow cytometry shows that cells are positive for MSC markers CD44, CD73, CD90, and CD105 while lacking hematopoietic lineage markers CD34 and CD45. B) Bright field images of hMd-Neurons and uninduced hMSCs (40×). C) Immunoflourescence images of NeuN and Neurofilament (NF) in hMd-Neurons at day 12 (40×). D) Caspase 3 assay for induced, uninduced hMSCs as well as CAM treated (apoptosis inducer; positive control) and Z-DEVD-FMK treated (apoptosis inhibitor; negative control) hMSCs at days 2-12. CAM treated hMSCs show significant caspase 3 activity. Data represent mean±SE; p<0.0001.

Figure 2. Cellular rearrangements during hMSC differentiation into neurons. A) BrdU assay showing that hMSC proliferation decreases by neuronal differentiation within 48 h after neuronal induction. B) Florescent images of DAPI staining in uninduced hMSCs and hMd-Neurons (40×). Data represents mean±SE. C) Violin plot indicating changes in nucleus diameter in hMSCs when reaching day 12 of neuronal differentiation. Each dot represents a single cell. Plot width is proportional to cell frequency at a given diameter. p<0.001 and p<0.0001 using Welch’s t-test.

Figure 3. Integrin α5β1 expression dynamics of hMd-Neurons. A) Transcripts of α5 and β1 in hMSCs and hMd-Neurons at day 12. B) Endogenous protein expression levels of integrin α5β1 and C-E) fluorescent images of integrin α5β1 localization during neuronal differentiation of hMSCs. F) Plots indicate percentage of integrin α5β1 cell surface localization among stained cells at days 2, 6, and 12 of differentiation. One hundred cells from three different regions were counted and data represent mean±SE, p<0.001.