Reprogramming non-human primate somatic cells into functional neuronal cells by defined factors

Zhi Zhou, Kazuhisa Kohda, Keiji Ibata, Jun Kohyama, Wado Akamatsu, Michisuke Yuzaki, Hirotaka James Okano, Erika Sasaki, Hideyuki Okano

Research output: Contribution to journalArticle

15 Citations (Scopus)

Abstract

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.

Original languageEnglish
Article number24
JournalMolecular Brain
Volume7
Issue number1
DOIs
Publication statusPublished - 2014 Apr 3

Fingerprint

Callithrix
Primates
Technology
Voltage-Gated Sodium Channel Blockers
Rodentia
Transcription Factors
Fibroblasts
Tetrodotoxin
Bromodeoxyuridine
Electric Stimulation
Action Potentials
Reverse Transcription
Up-Regulation
Sodium
Immunohistochemistry

Keywords

  • Cell-fate plasticity
  • Common marmoset
  • Direct reprogramming
  • Disease modeling
  • Induced neuronal cells
  • Regenerative medicine
  • Transcription factor
  • Transdifferentiation

ASJC Scopus subject areas

  • Cellular and Molecular Neuroscience
  • Molecular Biology

Cite this

Reprogramming non-human primate somatic cells into functional neuronal cells by defined factors. / Zhou, Zhi; Kohda, Kazuhisa; Ibata, Keiji; Kohyama, Jun; Akamatsu, Wado; Yuzaki, Michisuke; Okano, Hirotaka James; Sasaki, Erika; Okano, Hideyuki.

In: Molecular Brain, Vol. 7, No. 1, 24, 03.04.2014.

Research output: Contribution to journalArticle

Zhou, Zhi ; Kohda, Kazuhisa ; Ibata, Keiji ; Kohyama, Jun ; Akamatsu, Wado ; Yuzaki, Michisuke ; Okano, Hirotaka James ; Sasaki, Erika ; Okano, Hideyuki. / Reprogramming non-human primate somatic cells into functional neuronal cells by defined factors. In: Molecular Brain. 2014 ; Vol. 7, No. 1.
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