Neurogenic differentiation of human dental pulp stem cells by optogenetics stimulation

Introduction: Human dental pulp stem cells (hDPSCs), a promising source for autologous transplantation in regenerative medicine, have been shown to be able to differentiate into neural precursors. Optogenetics is considered as an advanced biological technique in neuroscience which is able to control the activity of genetically modified stem cells by light. The purpose of this study is to investigate the neurogenic differentiation of hDPSCs following optogenetic stimulation. Methods: The hDPSCs were isolated by mechanical enzymatic digestion from an impacted third molar and cultured in DMEM/F12. The cells were infected with lentiviruses carrying CaMKIIa-hChR2 (H134R). Opsin-expressing hDPSCs were plated at the density of 5 � 104 cells/well in 6-well plates and optical stimulation was conducted with blue light (470 nm) pulsing at 15 Hz, 90 Duty Cycle and 10 mW power for 10 s every 90 minutes, 6 times a day for 5 days. Two control groups including non-opsin-expressing hDPSCs and opsin-expressing hDPSCs with no optical stimulation were also included in the study. A day after last light stimulation, the viability of cells was analyzed by the MTT assay and the morphological changes were examined by phase contrast microscopy. The expression of Nestin, Microtubule-Associated protein 2 (MAP2) and Doublecortin (DCX) were examined by immunocytochemistry. Results: Human DPSCs expressed the reporter gene, mCherry, 72 hours after lentiviral infection. The result of MTT assay revealed a significant more viability in optical stimulated opsin-expressing hDPSCs as compared with two control groups. Moreover, optical stimulation increased the expression of Nestin, Doublecortin and MAP2 along with morphological changes from spindle shape to neuron-like shape. Conclusion: Optogenetics stimulation through depolarizing the hDPSCs can increase the cells viability and/or proliferation and also promote the differentiation toward neuron-like cells. © 202

Three-Dimensional Imaging in Stem Cell-Based Researches

Stem cells have an important role in regenerative therapies, developmental biology studies and drug screening. Basic and translational research in stem cell technology needs more detailed imaging techniques. The possibility of cell-based therapeutic strategies has been validated in the stem cell field over recent years, a more detailed characterization of the properties of stem cells is needed for connectomics of large assemblies and structural analyses of these cells. The aim of stem cell imaging is the characterization of differentiation state, cellular function, purity and cell location. Recent progress in stem cell imaging field has included ultrasound-based technique to study living stem cells and florescence microscopy-based technique to investigate stem cell three-dimensional (3D) structures. Here, we summarized the fundamental characteristics of stem cells via 3D imaging methods and also discussed the emerging literatures on 3D imaging in stem cell research and the applications of both classical 2D imaging techniques and 3D methods on stem cells biology