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. 2020 Apr;23(4):431-438.
doi: 10.22038/ijbms.2020.39797.9434.

PuraMatrix hydrogel enhances the expression of motor neuron progenitor marker and improves adhesion and proliferation of motor neuron-like cells

Marzieh Darvishi  1   2 , Hatef Ghasemi Hamidabadi  3   4 , Sajad Sahab Negah  5   2 , Ardeshir Moayeri  1 , Taki Tiraihi  6 , Javad Mirnajafi-Zadeh  7 , Ali Jahanbazi Jahan-Abad  2 , Amir Shojaei  7   8
Affiliations

PuraMatrix hydrogel enhances the expression of motor neuron progenitor marker and improves adhesion and proliferation of motor neuron-like cells

Marzieh Darvishi et al. Iran J Basic Med Sci. 2020 Apr.

Abstract

Objectives: Cell therapy has provided clinical applications to the treatment of motor neuron diseases. The current obstacle in stem cell therapy is to direct differentiation of stem cells into neurons in the neurodegenerative disorders. Biomaterial scaffolds can improve cell differentiation and are widely used in translational medicine and tissue engineering. The aim of this study was to compare the efficiency of two-dimensional with a three-dimensional culture system in their ability to generate functional motor neuron-like cells from adipose-derived stem cells.

Materials and methods: We compared motor neuron-like cells derived from rat adipose tissue in differentiation, adhesion, proliferation, and functional properties on two-dimensional with three-dimensional culture systems. Neural differentiation was analyzed by immunocytochemistry for immature (Islet1) and mature (HB9, ChAT, and synaptophysin) motor neuron markers.

Results: Our results indicated that the three-dimensional environment exhibited an increase in the number of Islet1. In contrast, two-dimensional culture system resulted in more homeobox gene (HB9), Choline Acetyltransferase (ChAT), and synaptophysin positive cells. The results of this investigation showed that proliferation and adhesion of motor neuron-like cells significantly increased in three-dimensional compared with two-dimensional environments.

Conclusion: The findings of this study suggested that three-dimension may create a proliferative niche for motor neuron-like cells. Overall, this study strengthens the idea that three-dimensional culture may mimic neural stem cell environment for neural tissue regeneration.

Keywords: Motor neuron-like cells; Nanoscaffolds; Proliferation; Stem cell therapy; Three-dimension culture; Tissue engineering.

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Figures

Figure 1
Figure 1
Differentiation capacity of ADSCs was assessed by specific markers. A-1) Phase contrast images of the third passage of the ADSCs culture. A-2, 3) Lipogenic and osteogenic differentiation of ADSCs stained with Oil red and Alizarin red stain, respectively. Images of differentiated adipogenic and osteogenic cultures showing phenotypic changes as well as lipid droplet accumulation and mineralization of the cultures. B) ADSCs were labeled with primary antibodies; CD105, CD90, CD49d, CD31, CD106, and CD45. Immunocytochemistry showing positive staining for CD105, CD90, and CD49d markers used to confirm ADSCs stemness. C) Immunocytochemistry of neural stem cell markers (SOX2, NT68, and Nestin) was performed. Markers are shown in green, while the cell nuclei, counterstained with propidium iodide (PI), are shown in red
Figure 2
Figure 2
Electrophysiological properties of MNLCs at the end of induction assessed by whole-cell patch clamp recording. A) Phase contrast image of a patch pipette attached to the membrane of a cultured MNLCs. Representative traces of membrane potential in response to 200, 400, and 600 as the pale traces and 800 pA as the dark trace in depolarizing current (660 ms) before (B) and after (C) 1 μM of tetrodotoxin (TTX) treatment. MNLCs at the end of induction (n=15) showed single action potential like spikes. Treatment with 1 μM of TTX as a blocker of voltage-gated Na+ channels inhibited spike firing confirming sodium action potential-like event in these cells
Figure 3.
Figure 3.
Immunofluorescence studies were performed to investigate the expression of the motor neuron markers in two different groups. A) MNLCs were immunostained with primary antibodies; islet-1, HB9, ChAT, and synaptophysin. Markers are shown in green and the cell nuclei (counterstained with PI) are shown in red. B) Quantitative data of positive cells in 2D and 3D groups. Our results showed that islet-1 significantly increased in 3D compared with the 2D group. The higher expression of HB9, ChAT, and synaptophysin was observed in 2D more than in 3D. Data are presented as mean±SD. *P<0.05
Figure 4
Figure 4
Characterization of the MNLCs was assessed by co-culturing with myotubes (C2C12) on 2D (A and B) and 3D (C and D) culture plates. A and C) Myotubes co-cultured with MNLCs was stained by Cresyl violet on 2D (A) and 3D (C) culture plates (black star is Myotubes and white star is MNLCs). B and D) Myotubes were stained with PKh67 (green) and co-cultured with the MNLCs were stained with PKh26 (red) on 2D (B) and 3D (D) culture plates
Figure 5
Figure 5
Assessment of the synaptic vesicles of the MNLCs seeded in 3D and 2D culture were performed by FM1-43 staining. A) The phase contrast image of the MNLCs cultured on 2D and 3D culture plate. A fluorescence image of the same field in A1-5, photographed after 1 min and 10 min following the de-staining on 2D plates. A4-6 were also photographed after 1 min and 10 min following the de-staining of MNLCs on 3D culture plate. B and C represent the curve of the monoexponential decay model for the time points following de-staining of MNLCs culture on 2D (y= 80.7e-0.265x ) and 3D (y=70.4e-0.231x ) systems, respectively
Figure 6
Figure 6
The Ca2+ concentration of MNLCs was detected by Fluo-4 NW assay. A represents the MNLCs stained with Fluo-4 NW followed by their stimulation on 2D culture plate and B on 3D culture. Both groups of serial image show changes in the color of the cells following a shift in the intracellular Ca2+
Figure 7
Figure 7
The staining of the MNLCs with voltage-sensitive dye (RH795) followed by their stimulation on 2D and 3D culture systems. A shows the membrane depolarization and repolarization of the MNLCs with change of color in 2D, and B represents the same field photographed serially demonstrated membrane action potential with same mechanism on 3D culture plates
Figure 8
Figure 8
Initial cell attachment was evaluated by DAPI stain at 2 hr post-plating. A and B represent immunofluorescent staining of two groups. C shows the quantitative data of adherent cells. The average percentages were calculated using five different views. Data are shown as mean±SD. *P<0.05
Figure 9
Figure 9
Proliferation of MNLCs was detected by MTS assay on days 1, 7, and 14. The proliferation assay showed significant difference between groups in 2D and 3D cultures. Data are shown as mean±SD. *P<0.05
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