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

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Direct conversion of human fibroblasts to functional excitatory cortical neurons integrating into human neural networks. / Miskinyte, Giedre; Devaraju, Karthikeyan; Grønning Hansen, Marita; Monni, Emanuela; Tornero, Daniel; Woods, Niels Bjarne; Bengzon, Johan; Ahlenius, Henrik; Lindvall, Olle; Kokaia, Zaal.

In: Stem Cell Research and Therapy, Vol. 8, No. 1, 658, 29.09.2017.

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T1 - Direct conversion of human fibroblasts to functional excitatory cortical neurons integrating into human neural networks

AU - Miskinyte, Giedre

AU - Devaraju, Karthikeyan

AU - Grønning Hansen, Marita

AU - Monni, Emanuela

AU - Tornero, Daniel

AU - Woods, Niels Bjarne

AU - Bengzon, Johan

AU - Ahlenius, Henrik

AU - Lindvall, Olle

AU - Kokaia, Zaal

PY - 2017/9/29

Y1 - 2017/9/29

N2 - 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.

AB - 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.

KW - Cortical projection neurons

KW - Direct conversion

KW - Human adult cortical slices

KW - Human fibroblasts

UR - http://www.scopus.com/inward/record.url?scp=85030176641&partnerID=8YFLogxK

U2 - 10.1186/s13287-017-0658-3

DO - 10.1186/s13287-017-0658-3

M3 - Article

C2 - 28962665

AN - SCOPUS:85030176641

VL - 8

JO - Stem Cell Research and Therapy

JF - Stem Cell Research and Therapy

SN - 1757-6512

IS - 1

M1 - 658

ER -