ABSTRACT
In vertebrates, most somatosensory pathways begin with the activation of dorsal root ganglion (DRG) neurons. The development of an appropriate DRG culture method is a prerequisite for establishing in vitro peripheral nerve disease models and for screening therapeutic drugs. In this study, we compared the changes in morphology, molecular biology, and transcriptomics of chicken embryo DRG cultured on tissue culture plates (T-DRG) versus three-dimensional collagen hydrogels (C-DRG). Our results showed that after 7 days of culture, the transcriptomics of T-DRG and C-DRG were quite different. The upregulated genes in C-DRG were mainly related to neurogenesis, axon guidance, and synaptic plasticity, whereas the downregulated genes in C-DRG were mainly related to cell proliferation and cell division. In addition, the genes related to cycles/pathways such as the synaptic vesicle cycle, cyclic adenosine monophosphate signaling pathway, and calcium signaling pathway were activated, while those related to cell-cycle pathways were downregulated. Furthermore, neurogenesis- and myelination-related genes were highly expressed in C-DRG, while epithelial-mesenchymal transition-, apoptosis-, and cell division-related genes were suppressed. Morphological results indicated that the numbers of branches, junctions, and end-point voxels per C-DRG were significantly greater than those per T-DRG. Furthermore, cells were scattered in T-DRG and more concentrated in C-DRG, with a higher ratio of 5-ethynyl-2'-deoxyuridine (EdU)-positive cells in T-DRG compared with C-DRG. C-DRG also had higher S100 calcium-binding protein B (S100B) and lower α-smooth muscle actin (α-SMA) expression than T-DRG, and contained fewer terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells after 48 hours of serum starvation. After cryopreservation, C-DRG maintained more intact morphological characteristics, and had higher viability and less TUNEL-positive cells than T-DRG. Furthermore, newly formed nerve bundles were able to grow along the existing Schwann cells in C-DRG. These results suggest that C-DRG may be a promising in vitro culture model, with better nerve growth and anti-apoptotic ability, quiescent Schwann cells, and higher viability. Results from this study provide a reference for the construction, storage, and transportation of tissue-engineered nerves. The study was approved by the Ethics Committee of Aier School of Ophthalmology, Central South University, China (approval No. 2020-IRB16), on March 15, 2020.
ABSTRACT
Human adipose-derived stem cells (hASCs) become an appealing source for regenerative medicine. However, with the multi-passage or cryopreservation for large-scale growth procedures in terms of preclinical and clinical purposes, hASCs often reveal defective cell viability, which is a major obstacle for cell therapy. In our study, the effects of induced pluripotent stem cells-derived conditioned medium (iPS-CM) on the proliferation and anti-apoptosis in hASCs were investigated. hASCs at passage 1 were identified by the analysis of typical surface antigens with flow cytometry assay and adipogenic and osteogenic differentiation. The effect of iPS-CM on the proliferation in hASCs was analyzed by cell cycle assay and Ki67/P27 quantitative polymerase chain reaction analysis. The effect of iPS-CM on the anti-apoptosis of hASCs irradiated by 468 J/m(2) of ultraviolet C was investigated by annexin v/propidium iodide analysis, mitochondrial membrane potential assay, intracellular reactive oxygen species assay, Western blotting and caspase activity assays. The effect of iPS-CM on the surface antigen expressions of hASCs was analyzed using flow cytometry assay. The levels of Activin A and bFGF in culture supernatant of hASCs with different treatments were also detected by enzyme-linked immunosorbent assay. iPS-CM promoted proliferation and inhibited apoptosis of hASCs. This discovery demonstrates that iPS-CM might be used as one of the available means to overcome the propagation obstacle for hASCs and make for large-scale growth procedures in terms of preclinical and clinical purposes.
