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

Abstract

BackgroundThe 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.ResultsCommon 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.ConclusionsFunctional 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.

Functional Neurons Generated from T Cell-Derived Induced Pluripotent Stem Cells for Neurological Disease Modeling

“…We recorded voltage-sensitive currents in 30- to 60-day-old TiPSC- and aHDF-iPSC-derived neurons. iPSC-derived neurospheres were infected with a lentivirus expressing human synapsin promoter-driven GFP (CSIV–hSynI-GFP-IRES2-NeoR) ( Zhou et al., 2014 ) and differentiated into neurons. TTX-sensitive voltage-gated membrane currents were indistinguishable between neurons derived from TiPSCs and those derived from aHDF-iPSCs ( Figure S6 A).…”

Modeling neurological diseases with induced pluripotent cells reprogrammed from immortalized lymphoblastoid cell lines

“…We recorded voltage-sensitive currents in 60-day-old LiPSC-derived neurons to confirm their electrophysiological properties. LiPSC-derived NSs were infected with a lentivirus expressing a human Synapsin I promoter-driven GFP (CSIV–hSynI-GFP-IRES2-NeoR) soon after single dissociated NS cells were plated [ 27 ]. After neuronal maturation, voltage-dependent Na + and K + currents and TTX-sensitive voltage-gated membrane currents were detected (Fig.…”

Standards for Deriving Nonhuman Primate-Induced Pluripotent Stem Cells, Neural Stem Cells and Dopaminergic Lineage

“…In fact, specific culture conditions have been developed to maintain the pluripotent stem cells (PSCs) in an extended PSC stage (EPSC) with widespread chimeric contribution to embryonic and extraembryonic lineages in vivo [ 28 ]. The derivation and tendency of the marmoset pluripotent stem cells to differentiate into neural lineages has been reported [ 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 ] using variable techniques for the neuralization based on the initial protocol developed for human embryonic stem cells [ 38 , 39 , 40 , 41 , 42 , 43 ]. In this study, we demonstrated that using the NN1 medium supplemented with growth factors, we were able to isolate NSCs that are responsive to mitogenic growth factor.…”