Functional control of transplantable human ESC-derived neurons via optogenetic targeting.
Current methods to examine and regulate the functional integration and plasticity of human ESC (hESC)-derived neurons are cumbersome and technically challenging. Here, we engineered hESCs and their derivatives to express the light-gated channelrhodopsin-2 (ChR2) protein to overcome these deficiencies. Optogenetic targeting of hESC-derived neurons with ChR2 linked to the mCherry fluorophore allowed reliable cell tracking as well as light-induced spiking at physiological frequencies. Optically induced excitatory and inhibitory postsynaptic currents could be elicited in either ChR2(+) or ChR2(-) neurons in vitro and in acute brain slices taken from transplanted severe combined immunodeficient (SCID) mice. Furthermore, we created a clonal hESC line that expresses ChR2-mCherry under the control of the synapsin-1 promoter. On neuronal differentiation, ChR2-mCherry expression was restricted to neurons and was stably expressed for at least 6 months, providing more predictable light-induced currents than transient infections. This pluripotent cell line will allow both in vitro and in vivo analysis of functional development as well as the integration capacity of neuronal populations for cell-replacement strategies.
Weick
Jason P
JP
Waisman Center, University of Wisconsin-Madison, Madison, Wisconsin 53705, USA.
Johnson
M Austin
MA
Skroch
Steven P
SP
Williams
Justin C
JC
Deisseroth
Karl
K
Zhang
Su-Chun
SC
D000818:Animals; D002478:Cells, Cultured; D004594:Electrophysiology; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism; D006801:Humans; D051379:Mice; D016513:Mice, SCID; D009474:Neurons / Q000166:cytology* / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D012243:Rhodopsin / Q000378:metabolism; D016704:Synapsins / Q000235:genetics; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid / Q000378:metabolism; D005680:gamma-Aminobutyric Acid / Q000378:metabolism
D016704:Synapsins; D005680:gamma-Aminobutyric Acid; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; D012243:Rhodopsin
Stem Cells. 2008 Jul;26(7):1705-12
18420830
J Neurosci. 2007 Dec 26;27(52):14231-8
18160630
Nature. 2002 Jul 4;418(6893):50-6
12077607
Brain Res Mol Brain Res. 2005 Mar 24;134(1):34-51
15790528
Science. 2009 Apr 17;324(5925):354-9
19299587
Nat Neurosci. 2005 Sep;8(9):1263-8
16116447
J Vis Exp. 2009 Mar 16;(25):
19289998
Proc Natl Acad Sci U S A. 2009 Mar 3;106(9):3225-30
19218428
Brain Res. 1979 Jul 20;170(3):409-26
466421
Nat Protoc. 2006;1(4):2075-81
17487197
Nat Biotechnol. 2007 Jun;25(6):681-6
17529971
Stem Cells. 2009 Dec;27(12):2952-61
19750538
Nat Neurosci. 2008 Aug;11(8):901-7
18622400
J Neurosci. 2007 Mar 21;27(12):3069-77
17376968
J Neural Eng. 2007 Sep;4(3):S143-56
17873414
Neuron. 2004 May 27;42(4):535-52
15157417
J Neurosci Res. 2006 Nov 1;84(6):1165-76
16941479
Proc Natl Acad Sci U S A. 2003 Nov 25;100(24):13940-5
14615590
Phys Med Biol. 2002 Jun 21;47(12):2059-73
12118601
Nat Methods. 2006 Oct;3(10):785-92
16990810
Biotechnol Genet Eng Rev. 2007;24:297-309
18059639
Nat Biotechnol. 2005 Feb;23(2):215-21
15685164
Cell. 2010 Apr 2;141(1):154-165
20303157
Proc Natl Acad Sci U S A. 2007 May 8;104(19):8143-8
17483470
Nature. 2006 Feb 2;439(7076):589-93
16341203
Nat Biotechnol. 2001 Dec;19(12):1129-33
11731781
Gene Ther. 2003 Feb;10(4):337-47
12595892
J Neurosci. 2003 Aug 6;23(18):7075-83
12904468
J Neurosci Methods. 1994 Jan;51(1):107-16
8189746
Nature. 1982 Nov 18;300(5889):260-2
7144881
Proc Natl Acad Sci U S A. 2002 Jun 25;99(13):8689-94
12060707
Nat Neurosci. 2007 May;10(5):663-8
17435752
Nat Methods. 2007 Feb;4(2):139-41
17195846
Nat Protoc. 2008;3(10):1559-68
18802437
Proc Natl Acad Sci U S A. 2008 Apr 15;105(15):5856-61
18391196
Nature. 2007 Apr 5;446(7136):633-9
17410168
J Biol Chem. 2008 Sep 5;283(36):24300-7
18599484
J Neural Eng. 2009 Oct;6(5):055007
19721185
Stem Cells. 2009 Dec;27(12):2906-16
19725137
Brain Res. 1985 Jun 17;336(2):302-7
4005587
Proc Natl Acad Sci U S A. 2005 Dec 20;102(51):18644-8
16352714
Neuron. 2006 Apr 6;50(1):23-33
16600853
Curr Biol. 2005 Dec 20;15(24):2279-84
16360690