Direct conversion of human fibroblasts to functional excitatory cortical neurons integrating into human neural networks

Stem Cell Research & Therapy

Niels Bjarne Woods¹  Johan Bengzon²  Giedre Miskinyte³  Karthikeyan Devaraju³  Daniel Tornero³  Zaal Kokaia³  Emanuela Monni³  Olle Lindvall³  Marita Grønning Hansen⁴  Henrik Ahlenius⁵ 

[1]Division of Molecular Medicine and Gene Therapy, Lund University

[2]Division of Neurosurgery, Department of Clinical Sciences Lund, University Hospital

[3]Laboratory of Stem Cells and Restorative Neurology, University Hospital

[4]Lund Stem Cell Center, University Hospital BMC B10, Lund University

[5]Stem Cells, Aging and Neurodegeneration Group, University Hospital

关键词: Cortical projection neurons;    Direct conversion;    Human adult cortical slices;    Human fibroblasts;   

DOI  :  10.1186/s13287-017-0658-3

来源: DOAJ

【 摘 要 】

Abstract Background Human fibroblasts can be directly converted to several subtypes of neurons, but cortical projection neurons have not been generated. Methods Here we screened for transcription factor combinations that could potentially convert human fibroblasts to functional excitatory cortical neurons. The induced cortical (iCtx) cells were analyzed for cortical neuronal identity using immunocytochemistry, single-cell quantitative polymerase chain reaction (qPCR), electrophysiology, and their ability to integrate into human neural networks in vitro and ex vivo using electrophysiology and rabies virus tracing. Results We show that a combination of three transcription factors, BRN2, MYT1L, and FEZF2, have the ability to directly convert human fibroblasts to functional excitatory cortical neurons. The conversion efficiency was increased to about 16% by treatment with small molecules and microRNAs. The iCtx cells exhibited electrophysiological properties of functional neurons, had pyramidal-like cell morphology, and expressed key cortical projection neuronal markers. Single-cell analysis of iCtx cells revealed a complex gene expression profile, a subpopulation of them displaying a molecular signature closely resembling that of human fetal primary cortical neurons. The iCtx cells received synaptic inputs from co-cultured human fetal primary cortical neurons, contained spines, and expressed the postsynaptic excitatory scaffold protein PSD95. When transplanted ex vivo to organotypic cultures of adult human cerebral cortex, the iCtx cells exhibited morphological and electrophysiological properties of mature neurons, integrated structurally into the cortical tissue, and received synaptic inputs from adult human neurons. Conclusions Our findings indicate that functional excitatory cortical neurons, generated here for the first time by direct conversion of human somatic cells, have the capacity for synaptic integration into adult human cortex.

【 授权许可】

Unknown