Signaling adaptor protein SH2B1 enhances neurite outgrowth and accelerates the maturation of human induced neurons

Abstract

Recent advances in somatic cell reprogramming have highlighted the plasticity of the somatic epigenome, particularly through demonstrations of direct lineage reprogramming of adult mouse and human fibroblasts to induced pluripotent stem cells (iPSCs) and induced neurons (iNs) under defined conditions. However, human cells appear to be less plastic and have a higher epigenetic hurdle for reprogramming to both iPSCs and iNs. Here, we show that SH2B adaptor protein 1β (SH2B1) can enhance neurite outgrowth of iNs reprogrammed from human fibroblasts as early as day 14, when combined with miR124 and transcription factors BRN2 and MYT1L (IBM) under defined conditions. These SH2B1-enhanced iNs (S-IBM) showed canonical neuronal morphology, and expressed multiple neuronal markers, such as TuJ1, NeuN, and synapsin, and functional proteins for neurotransmitter release, such as GABA, vGluT2, and tyrosine hydroxylase. Importantly, SH2B1 accelerated mature process of functional neurons and exhibited action potentials as early as day 14; without SH2B1, the IBM iNs do not exhibit action potentials until day 21. Our data demonstrate that SH2B1 can enhance neurite outgrowth and accelerate the maturation of human iNs under defined conditions. This approach will facilitate the application of iNs in regenerative medicine and in vitro disease modeling.

Keywords: Neural differentiation; Neural induction; Neuron; Reprogramming; SH2B1.

Figure 1.

Generation of SH2B1-positive HFFs whose SH2B1 expression can be induced by doxycycline. (A): Schematic drawing showing the generation of SH2B1-positive HFFs by FACS. So-HFF indicates HFFs that were transfected with doxycycline-inducible SH2B1 plasmid and TetOn plasmid. These two plasmids were selected by puromycin and blasticidin, respectively. SoS-HFFs indicates So-HFFs that were cultured in the presence of doxycycline for 2 days and collected for SH2B1GFP-positive cells using FACS. (B): Schematic drawing showed the protocol for the induction of SH2B1-GFP expression. SoS-HFFs were cultured either with or without doxycycline and collected for FACS analyses on days 2, 4, and 6. (C): A representative figure and summary table showed the percentage of SH2B1-positive cells. The GFP(+) cells in the Dox(−) culture ranged from 4.0%–4.7%, and increased to 81.6%–86.9% in the Dox(+) culture during day 2 to day 6 following the stimulation by doxycycline. Abbreviations: Bsd, blasticidin; Dox, doxycycline; D2–D6, days 2–6; FACS, fluorescence activated cell sorting; HFF, human foreskin fibroblast; Puro, puromycin; SH2B1, SH2B adaptor protein 1β; So-HFF, human foreskin fibroblasts transfected with doxycycline-inducible SH2B1 plasmid and TetOn plasmid.

Figure 2.

SH2B1 enhances the length of neurite outgrowth and the numbers of processes and branches of human iNs induced by IBM. (A): Schematic showing the experimental protocol. (B): A representative figure showing that after 28 days of infection, SH2B1, miR124, BRN2, and MYT1L (S-IBM)-infected human fibroblasts were more efficiently converted to iNs than IBM iNs. Red arrows indicate the successfully reprogrammed iNs. (C): Within 28 days of infection, S-IBM iNs showed twofold more total neurite outgrowth than IBM iNs at days 14, 21, and 28. n = 3; ∗∗∗, p < .001. (D): A representative figure showing that after 28 days of infection, S-IBM iNs showed significantly more neuronal processes and branches than IBM iNs. Red arrows indicate the successfully reprogrammed iNs. (E): Within 28 days of infection, S-IBM iNs showed significantly more neuronal processes and branches than IBM iNs at days 14, 21, and 28. n = 3, ∗∗∗, p < .001. Abbreviations: BDNF, brain-derived neurotrophic factor; Dox, doxycycline; D7–D28, days 7–28; FACS, fluorescence activated cell sorting; FGF, fibroblast growth factor; GDNF, glia-derived growth factor; HFF, human foreskin fibroblast; IBM, transcription factors BRN2 and MYT1L; iNs, induced neurons; SH2B1, SH2B adaptor protein 1β; S-IBM, SH2B1-enhanced iNs; SoS-HFF, SH2B1GFP-expressing cells.

Figure 3.

Human iNs infected with IBM or S-IBM exhibited neuronal markers and markers for neurotransmission. (A): TuJ1-stained human iNs 28 days after infection of human fibroblasts with IBM (left) and S-IBM (right). GFP-positive cells indicate miR124-infected cells. (B): NeuN-stained human iNs 28 days after infection of human fibroblasts with IBM and S-IBM. GFP-positive cells indicate miR124-infected cells. (C): TH-stained human iNs 28 days after infection of human fibroblasts with IBM and S-IBM. GFP-positive cells indicate miR124-infected cells. (D): S-IBM iNs expressing vGluT2. (E): S-IBM iNs expressing GABA. (F): S-IBM iNs expressing synapsin. (G): Within 28 days of infection, S-IBM iNs showed significantly higher expression levels of TuJ1, NeuN, TH, and GABA activities than IBM iNs at days 14, 21, and 28, and the expression levels of vGluT2 and synapsin remained unchanged. The percentages were the ratios of the numbers of marker-positive cells divided by the numbers of cells remaining in the culture plates following the induction protocol. Data were collected from three independent experiments; ∗, p < .05; ∗∗, p < .005. Scale bars = 200 μm. Abbreviations: BDNF, brain-derived neurotrophic factor; D, day; Dox, doxycycline; FACS, fluorescence activated cell sorting; GFP; green fluorescent protein; GDNF, glia-derived growth factor; IBM, transcription factors BRN2 and MYT1L; iNs, induced neurons; SH2B1, SH2B adaptor protein 1β; S-IBM, SH2B1-enhanced iNs; SoS-HFF, SH2B1GFP-expressing cells; TH, tyrosine hydroxylase.

Figure 3.

Human iNs infected with IBM or S-IBM exhibited neuronal markers and markers for neurotransmission. (A): TuJ1-stained human iNs 28 days after infection of human fibroblasts with IBM (left) and S-IBM (right). GFP-positive cells indicate miR124-infected cells. (B): NeuN-stained human iNs 28 days after infection of human fibroblasts with IBM and S-IBM. GFP-positive cells indicate miR124-infected cells. (C): TH-stained human iNs 28 days after infection of human fibroblasts with IBM and S-IBM. GFP-positive cells indicate miR124-infected cells. (D): S-IBM iNs expressing vGluT2. (E): S-IBM iNs expressing GABA. (F): S-IBM iNs expressing synapsin. (G): Within 28 days of infection, S-IBM iNs showed significantly higher expression levels of TuJ1, NeuN, TH, and GABA activities than IBM iNs at days 14, 21, and 28, and the expression levels of vGluT2 and synapsin remained unchanged. The percentages were the ratios of the numbers of marker-positive cells divided by the numbers of cells remaining in the culture plates following the induction protocol. Data were collected from three independent experiments; ∗, p < .05; ∗∗, p < .005. Scale bars = 200 μm. Abbreviations: BDNF, brain-derived neurotrophic factor; D, day; Dox, doxycycline; FACS, fluorescence activated cell sorting; GFP; green fluorescent protein; GDNF, glia-derived growth factor; IBM, transcription factors BRN2 and MYT1L; iNs, induced neurons; SH2B1, SH2B adaptor protein 1β; S-IBM, SH2B1-enhanced iNs; SoS-HFF, SH2B1GFP-expressing cells; TH, tyrosine hydroxylase.

Figure 4.

Electrophysiological characterization of S-IBM and IBM iNs. Human foreskin fibroblasts were infected with three factors (IBM; upper panel) or three factors plus SH2B1 (S-IBM; lower panel) for 14 (A), 21 (B), and 28 (C) days. Traces of spontaneous (left) and evoked (right) action potential recorded in current-clamp configuration. For the measurement of evoked action potential firing in current clamp, membrane potentials were held at −60 mV. Recordings of membrane potential in response to five current steps (in 10-pA increments) from holding membrane potential were applied. The spontaneous and evoked activities of the iNs are illustrated in (D) and (E). For the measurement of evoked firing, cells were recorded at −60 mV, and then a +50-pA current was injected to elicit spikes. For the measurement of spontaneous firing, the cell membrane potential was held at either −50 or −60 mV without current stimulation. Data were collected from three independent experiments. Abbreviations: IBM, transcription factors BRN2 and MYT1L; iNs, induced neurons; SH2B1, SH2B adaptor protein 1β; S-IBM, SH2B1-enhanced iNs; SoS-HFF, SH2B1GFP-expressing cells.

Figure 5.

SH2B adaptor protein 1β-enhanced induced neurons showed calcium influx activity upon glutamate stimulation at day 28. Images before (A) and after (B–T) glutamate injection were taken at consecutive intervals of 3 seconds.