Reference - Detail
|Zhou Z, Kohda K, Ibata K, Kohyama J, Akamatsu W, Yuzaki M, Okano HJ, Sasaki E, Okano H.
|Reprogramming non-human primate somatic cells into functional neuronal cells by defined factors.
BACKGROUND:The common marmoset (Callithrix jacchus) is a New World primate sharing many similarities with humans. Recently developed technology for generating transgenic marmosets has opened new avenues for faithful recapitulation of human diseases, which could not be achieved in rodent models. However, the longer lifespan of common marmosets compared with rodents may result in an extended period for in vivo analysis of common marmoset disease models. Therefore, establishing rapid and efficient techniques for obtaining neuronal cells from transgenic individuals that enable in vitro analysis of molecular mechanisms underlying diseases are required. Recently, several groups have reported on methods, termed direct reprogramming, to generate neuronal cells by defined factors from somatic cells of various kinds of species, including mouse and human. The aim of the present study was to determine whether direct reprogramming technology was applicable to common marmosets.
RESULTS:Common marmoset induced neuronal (cjiN) cells with neuronal morphology were generated from common marmoset embryonic skin fibroblasts (cjF) by overexpressing the neuronal transcription factors: ASCL1, BRN2, MYT1L and NEUROD1. Reverse transcription-polymerase chain reaction of cjiN cells showed upregulation of neuronal genes highly related to neuronal differentiation and function. The presence of neuronal marker proteins was also confirmed by immunocytochemistry. Electrical field stimulation to cjiN cells increased the intracellular calcium level, which was reversibly blocked by the voltage-gated sodium channel blocker, tetrodotoxin, indicating that these cells were functional. The neuronal function of these cells was further confirmed by electrophysiological analyses showing that action potentials could be elicited by membrane depolarization in current-clamp mode while both fast-activating and inactivating sodium currents and outward currents were observed in voltage-clamp mode. The 5-bromodeoxyuridine (BrdU) incorporation assay showed that cjiN cells were directly converted from cjFs without passing a proliferative state.
CONCLUSIONS:Functional common marmoset neuronal cells can be obtained directly from embryonic fibroblasts by overexpressing four neuronal transcription factors under in vitro conditions. Overall, direct conversion technology on marmoset somatic cells provides the opportunity to analyze and screen phenotypes of genetically-modified common marmosets.
|Animals Biomarkers / metabolism Calcium / metabolism Callithrix / embryology Callithrix / metabolism* Cell Differentiation / drug effects Cell Proliferation / drug effects Cellular Reprogramming* Doxycycline / pharmacology Electric Stimulation Fibroblasts / cytology* Fibroblasts / drug effects Fibroblasts / metabolism Gene Expression Regulation / drug effects Glutamic Acid / metabolism Humans Immunohistochemistry Lentivirus / drug effects Lentivirus / metabolism Mice Mitosis / drug effects NIH 3T3 Cells Neurons / cytology* Neurons / drug effects Neurons / metabolism Reproducibility of Results Skin / cytology Skin / embryology Transcription Factors / metabolism Transgenes
|pCMV-VSV-G-RSV-Rev (RDB04393) pCAG-HIVgp (RDB04394).