Retrograde monosynaptic tracing through an engineered human embryonic stem cell line reveals synaptic inputs from host neurons to grafted cells

Qi Xing; Aiping Lin; Zhenghui Su; Chunhua Liu; Wenhao Huang; Wenjing Guo; Guangjin Pan; Yiping Guo; Xiaofen Zhong

doi:10.1016/j.cr.2019.01.002

Retrograde monosynaptic tracing through an engineered human embryonic stem cell line reveals synaptic inputs from host neurons to grafted cells

Cell Regen. 2019 Mar 1;8(1):1-8. doi: 10.1016/j.cr.2019.01.002. eCollection 2019 Jun.

Authors

Qi Xing^{1

2

3

4}, Aiping Lin^{1

2

3}, Zhenghui Su^{1

2

3}, Chunhua Liu^{1

2

3}, Wenhao Huang^{1

2

3}, Wenjing Guo^{1

2

3}, Guangjin Pan^{1

2

3}, Yiping Guo^{1

2

3}, Xiaofen Zhong^{1

2

3}

Affiliations

¹ CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China.
² Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences; Guangzhou Medical University, Guangzhou 510530, China.
³ Guangzhou Regenerative Medicine and Health Laboratory, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China.
⁴ Institute of Physical Science and Information Technology, Anhui University, Hefei, 230601, China.

Abstract

Retrograde monosynaptic tracing with EnvA-pseudotyped rabies virus has been employed to identify the afferent and efferent connectivity of transplanted human embryonic stem (hES) cell-derived neurons in animal models. Due to the protracted development of transplanted human neurons in host animals, it is important that those transplanted cells express avian leukosis and sarcoma virus subgroup A receptor (TVA) and rabies glycoprotein G (Rgp) for a period of up to several months to enable identification of the synaptic inputs from host neurons to grafted neurons through this rabies virus-based method. Here, we report the generation of an engineered hES cell line through CRISPR/Cas9-mediated targeting to the AAVS1 locus of an EnvA-pseudotyped rabies virus-based tool for retrograde monosynaptic tracing. This engineered hES cell line, named H1-CAG-GTRgp, expresses GFP, TVA and Rgp. Upon transplantation of H1-CAG-GTRgp-derived neural progenitor cells (NPCs) into the rat brain after traumatic injury, the grafted neurons derived from H1-CAG-GTRgp cells expressed GFP, TVA, and Rgp stably for up to 6 months post-transplantation and received robust synaptic inputs from host neurons in the target regions of the orthotopic neural circuitry. The retrograde monosynaptic tracing hES cell line provides an efficient approach to analyze transplant connectivity for the comprehensive assessment of host-donor cell innervation.

Keywords: AAVS1 locus; EnvA-pseudotyped rabies virus; Neural progenitors; Retrograde monosynaptic tracing; Transplantation.