Nontoxicity of lentiviral vector infection to viability, migration, apoptosis, and differentiation of postnatal rat enteric neural crest-derived cells

Abstract

Lentiviral vector infection of enhanced green fluorescent protein fluorescence reporter genes in enteric neural crest-derived cells maintained efficient, stable, long-term labeling and the infected enteric neural crest-derived cells could survive, proliferate, and express fluorescent reporter genes. However, the method does not show whether there is some defined or undefined toxicity to the enteric neural crest-derived cells, which may affect enteric neural crest-derived cells' properties. Here, we evaluated the enteric neural crest-derived cells properties under the influence of lentivirus infection of enhanced green fluorescent protein fluorescence reporter genes. This study used the cell count kit-8 for measurement of vitality, transwell for cell migration, immunocytochemistry for cell count and identification, and tested the apoptosis of the enteric neural crest-derived cells with flow cytometry. The enteric neural crest-derived cells with or without lentivirus and their derivative enteric neural crest-derived cells could form characteristic neurospheres, and maintain their level of fluorescent label steady. When cultured under inducing conditions, enteric neural crest-derived cells differentiated into neurons and glia. The results showed that the enteric neural crest-derived cells with or without lentivirus showed no significant difference in viability, migration, apoptosis, neuronal, and glial ratio. The study identified that lentivirus can be used in a nontoxic manner for infection of enhanced green fluorescent protein fluorescence reporter genes into enteric neural crest-derived cells.

Inhibition of ROCK signaling pathway accelerates enteric neural crest cell‐based therapy after transplantation in a rat hypoganglionic model

“…For cell‐based therapy, labeling the cells to be grafted could ensure continuous tracking after transplantation in the animal model. Lentiviral transduction enables efficient, stable, and long‐term labeling of ENS stem cells that is retained after transplantation into the gut in vivo 28 and does not induce toxicity 29 . Moreover, the lentiviral approach could offer the possibility to transfect with a construct containing specific genes of interest in future experiments, such as genes containing mutations or rescue constructs for gene therapy 28 …”

“…Lentiviral transduction enables efficient, stable, and long-term labeling of ENS stem cells that is retained after transplantation into the gut in vivo 28 and does not induce toxicity. 29 Moreover, the lentiviral approach could offer the possibility to transfect with a construct containing specific genes of interest in future experiments, such as genes containing mutations or rescue constructs for gene therapy. 28 For our experiments, we decided to use a GFP-expressing lentiviral construct and started by determining the optimal multiplicity of infection (MOI) among 5×, 25×, 125×, and 250×.…”