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

Miskinyte, Giedre LU ; Devaraju, Karthikeyan LU ; Grønning Hansen, Marita LU ; Monni, Emanuela LU ; Tornero, Daniel LU ; Woods, Niels Bjarne LU ; Bengzon, Johan LU ; Ahlenius, Henrik LU ; Lindvall, Olle LU and Kokaia, Zaal LU (2017) In Stem Cell Research and Therapy 8(1).
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... (More)

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.

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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Cortical projection neurons, Direct conversion, Human adult cortical slices, Human fibroblasts
in
Stem Cell Research and Therapy
volume
8
issue
1
publisher
BioMed Central
external identifiers
  • scopus:85030176641
  • wos:000412195400011
ISSN
1757-6512
DOI
10.1186/s13287-017-0658-3
language
English
LU publication?
yes
id
a4db4b27-cebb-4efe-943d-b5697734bc76
date added to LUP
2017-10-25 09:20:22
date last changed
2018-07-08 04:27:52
@article{a4db4b27-cebb-4efe-943d-b5697734bc76,
  abstract     = {<p>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.</p>},
  articleno    = {658},
  author       = {Miskinyte, Giedre and Devaraju, Karthikeyan and Grønning Hansen, Marita and Monni, Emanuela and Tornero, Daniel and Woods, Niels Bjarne and Bengzon, Johan and Ahlenius, Henrik and Lindvall, Olle and Kokaia, Zaal},
  issn         = {1757-6512},
  keyword      = {Cortical projection neurons,Direct conversion,Human adult cortical slices,Human fibroblasts},
  language     = {eng},
  month        = {09},
  number       = {1},
  publisher    = {BioMed Central},
  series       = {Stem Cell Research and Therapy},
  title        = {Direct conversion of human fibroblasts to functional excitatory cortical neurons integrating into human neural networks},
  url          = {http://dx.doi.org/10.1186/s13287-017-0658-3},
  volume       = {8},
  year         = {2017},
}