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Abstract: We have previously demonstrated that amniotic epithelial cells (AECs) can enhance survival and neural differentiation of neural stem cells (NSCs) when co-cultured in basal media. In addition, the presence of basic fibroblast growth factor (bFGF) enhances this AEC function. The aim of the present study was to extend those findings and investigate whether AECs modified with the bFGF gene will also enhance NSCs survival and neural differentiation in vivo and promote repair of the injured spinal cord. Female Wistar rats were used for a contusive spinal cord injury (SCI) model. Contusive SCIs were induced using a weight-drop device at levels T9–T11. Seven days following contusion, rats received grafts of NSCs only, NSCs with AECs/pLEGFP-hbFGF, or NSCs with AECs/pLEGFP-C1 into the injured region. Significant locomotor improvement was observed in the NSCs/AECs co-graft group beginning at 3 weeks compared with the NSCs or NaCl only groups. These results were confirmed and extended in an electrophysiological analysis. An immunohistological analysis revealed that AECs/pLEGFP-hbFGF promoted the survival (vs NaCl group: 194±9.17 vs 103.6±13.05) and neural differentiation (vs NaCl group: 14.24±1.11 vs 7±0.63) of co-transplanted NSCs. We also confirmed that AECs could promote the survival of host neurons. These results suggest that AECs/pLEGFP-hbFGF improve the NSCs survival and differentiation microenvironment and may be useful as a source of sustained trophic supported to improve NSCs differentiation into neurons in vivo. These findings suggest that a cograft of AECs/pLEGFP-hbFGF and NSCs may have benefits for SCI. [Copyright &y& Elsevier]

Vascularization is a key step to achieve pulp tissue regeneration and in vitro pre-vascularized dental pulp tissue could be applied as a graft substitute for dental pulp tissue repair. In this study, human dental pulp stem cells (DPSCs) and human umbilical vein endothelial cells (hUVECs) were co-cultured in 3D Matrigel and 150 mV/mm electric fields (EFs) were used to promote the construction of pre-vascularized dental pulp tissue. After optimizing co-cultured ratio of two cell types, immunofluorescence staining, and live/dead detection were used to investigate the effect of EFs on cell survival, differentiation and vessel formation in 3D engineered dental pulp tissue. RNA sequencing was used to investigate the potential molecular mechanisms by which EF regulates vessel formation in 3D engineered dental pulp tissue. Here we identified that EF-induced pre-vascularized engineered dental pulp tissue not only had odontoblasts, but also had a rich vascular network, and smooth muscle-like cells appeared around the blood vessels. The GO enrichment analysis showed that these genes were significantly enriched in regulation of angiogenesis, cell migration and motility. The most significant term of the KEGG pathway analysis were NOTCH signaling pathway and Calcium signaling pathway etc. The PPI network revealed that NOTCH1 and IL-6 were central hub genes. Our study indicated that EFs significantly promoted the maturation and stable of blood vessel in 3D engineered pulp tissue and provided an experimental basis for the application of EF in dental pulp angiogenesis and regeneration., Competing Interests: The authors declare that they have no competing interests., (© 2024 The Author(s).)

Objective: To explore a new method to treat brachial plexus root avulsion experimentally by reimplantation combined with transplantation of neural stem cells (NSCs) modified by neurotrophin-3 gene (NT-3)., Methods: The total RNA was extracted from neonatal rat striatum and the NT-3 cDNA was obtained by reverse transcription and amplified by polymerase chain reaction. The NT-3 gene was transferred into NSCs via the pLEGFP-C1, an expression plasmid vectors. The untransfected NSCs, the pLEGFP-C1 treated NSCs, and the pLEGFP-C1-NT-3 treated NSCs were transplanted into corresponding spinal cord segment with brachial plexus root avulsion. The survival, differentiation, and migration of the transplanted cells were determined under confocal laser scanning microscope or by immunohistochemistry method. The nerve regeneration was evaluated by gross observation, electrophysiological examination and reverse horseradish peroxidase tracing., Results: The NT-3 gene was successfully amplified and transferred into neural stem cells via the plasmid vectors. The transplanted cells survived, differentiated, and migrated and NT-3 was expressed within the spinal cord. The animals regained some muscle strength which was less than 3-degree muscular strength according to the British Medical Research Council (BMRC) evaluating system. The results of electrophysiological examination and reverse horseradish peroxidase tracing were superior in the pLEGFP-C1-NT-3 group to the NSCs untransfected group or the pLEGFP-C1 group., Conclusion: Transplantation of NSCs modified by NT-3 gene combined with reimplantation is a relatively effective way to treat brachial plexus root avulsion experimentally. It still need further study to improve the results.

Aim: To construct rat Islet-1 gene recombinant retroviral expression vector and to transduce Islet-1 gene into neural stem cell (NSC) by the vector., Methods: The cDNA encoding the rat Islet-1 gene was isolated by RT-PCR method, the amplified gene fragment was subcloned into the retroviral vector plEGFP-C1. Islet-1 gene was transduced into NSC by PA317 packaging cells, then the expression of Islet-1 in NSC was observed., Results: The recombinant Islet-1 retroviral vector was constructed successfully, as shown by PCR, restriction enzymes digestion and fluorescence detection, and the positive expression of Islet-1 was found in NSC by immunocytochemical staining. The variant of Islet-1 gene was also discovered in this study., Conclusion: The rat Islet-1 recombinant retroviral vector has been successfully prepared, which can be used to study the function of Islet-1 gene in NSC differentiation into cholinergic neurons.

Background: Velvet antler polypeptides (VAPs), which are derived from the antler velvets, have been reported to maintain survival and promote growth and differentiation of neural cells and, especially the development of neural tissues. This study was designed to explore the influence of VAPs on neural stem cells in vitro derived from embryonic rat brain., Methods: Neural stem cells derived from E12-14 rat brain were isolated, cultured, and expanded for 7 days until neural stem cell aggregations and neurospheres were generated. The neurospheres were cultured under the condition of different concentration of VAPs followed by immunocytochemistry to detect the differentiation of neural stem cells., Results: VAPs could remarkably promote differentiation of neural stem cells and most neural stem cells were induced to differentiate towards the direction of neurons under certain concentration of VAPs., Conclusion: Neural stem cells can be successfully induced into neurons by VAPs in vitro, which could provide a basis for regeneration of the nervous system.