Supplemental Information Generation of Human Striatal

Neuron, Volume 84
Supplemental Information
Generation of Human Striatal
Neurons by MicroRNA-Dependent
Direct Conversion of Fibroblasts
Matheus B. Victor, Michelle Richner, Tracey O. Hermanstyne, Joseph L. Ransdell,
Courtney Sobieski, Pan-Yue Deng, Vitaly A. Klyachko, Jeanne M. Nerbonne,
and Andrew S. Yoo
A
B
80
pTIGHT
Human Postnatal
Dermal Fibroblasts
Bcl-xL
DAPI
+DOX
miR-9/9*-124
PID 7
Percent of remaining cells
PID 7
***
60
40
20
+DOX
L
Bc
l-x
FP
iR
N
As
+
tR
+
iR
m
m
As
miR-9/9*-124
0
N
tRFP
DAPI
Human Postnatal
Dermal Fibroblasts
pTIGHT
Figure S1 (Related to Figure 1). Effect of Bcl-xL on the number of remaining cells during
miR-9/9*-124 -mediated neuronal reprogramming
(A) Starting with approximately 100,000 cells seeded per well of a single 24-well plate, human fibroblasts
were transduced with lentivirus to express miR-9/9*-124 with either Bcl-xL (top) or turbo red flourescent
protein (tRFP). At 7 days post-infection (PID), the percentage of remaining cells were compared between
Bcl-xL and tRFP-expressing cells.
(B) We found a significantly higher number of surviving cells with Bcl-xL expression compared to tRFP.
The graph represents quantifications of DAPI counts from 4 biological replicates. Scale bar = 50 μm.
Error bars = s.e.m. *** = p<0.001 by student t-test.
G
Endogenous miR-9/9* and miR-124 are
Expressed in miR-9/9*-124-CDM
Converted MSNs
H
700
600
Relative mRNA (A.U.)
D
ay
O
2
ff
D
ay
4
n
D
ox
O
D
ox
Non-Transduced Fibroblasts
miR-9/9*-124+CDM (Dox off)
miR-9/9*-124+CDMn (Dox on)
9900
N.S. p = 0.7207
7600
N.S. p = 0.4109
5300
3000
2
-1
24
0
Continuous expression of miR-9/9*-124 for 30 days is necesssary
to achieve high conversion efficiency
miR-9/9*-124 Post-Infection Day 35
**
400
MAP2/DAPI
500
**
300
200
100
2
0
m
miR-9/9*-124+CDM
Dox Off at PID 15
Dox On for 30 days
im
ar
y
Non-Transduced Fibroblasts
Pr
Pr
im
ar
y
m
iR
iR
-9
/
-1
24
9*
Relative Expression
800
N.S. p = 0.9070
iR
**
160000
130000
100000
70000
m
*
Dox withdrawal does not significantly
reduce mature miRNAs levels
*
0.5
Pr Tr
i-m an
iR sge
-9 n
/9 ic
*12
4
Bc
l-x
L
Dox Off at PID 30
0.5
-9
1.0
0.0
Dox On
1.0
4
D
ay
1
O
ff
D
ay
1.5
F
-9
MAP2/DAPI
miR-9/9*-124 + CDM Post-Infection Day 38
Dox On
Dox Off
iR
2.0
m
E
RNA Quantity (A.U.)
Expression of transgenic miR-9/9*-124 and Bcl-xL
is suppressed upon doxycycline withdrawl
Mature miR-9
1.5
0.0
D
ox
0.0
8
2
7
O
ff
6
D
ay
5
O
n
4
D
ox
D
3
D
ox
Human Postnatal
Dermal Fibroblasts
2
Dox Off Day 1
Dox On Day 2
1
-DOX
0
+DOX
Days
Transgenic Pri-miR-9/9*-124
0.5
D
ox
miR-9/9*-124
X
Dox Off
-DOX
Relative mRNA (A.U.)
Bcl-xL
2.0
1.0
Dox Off Day 4
pTIGHT
Mature miR-9 turnover
in response to doxycycline withdrawn
iR
Transgenic
Pri-miR-9/9*-124
O
ff
+DOX
C
Transgenic Primary miRNA transcriptional kinetics
in response to doxycycline
m
B
pTight-miR-9/9*-124 transcriptional kinetics
in response to doxycycline
Relative mRNA (A.U.)
A
Figure S2 (Related to Figure 1). Kinetics of doxyxycline-dependent miR-9/9*-124 expression
(A-C) (A) Diagram illustrating the doxycycline-dependent vector used to express miRNAs and Bcl-xL and
the design of our experiment to test the transcriptional kinetics of this vector in response to doxycycline (Dox)
Using a primer-set specific to the synthetic miRNA hairpin (Illustrated in orange), we tested the kinetics of Dox
withdrawal on vector transcriptional activity in (B). We also determined that levels of mature miR-9 are reduced
via qPCR with probes specific for the mature sequence in (C).
(D-E) Human fibroblasts were transduced with miR-9/9*-124 + CDM and at 30 post-infection days (PID)
withdrawn from Dox (Dox Off). At 38 PID, cells were lysed and RNA harvested to determine expression levels
of transgene in comparison to cells that were maintained in Dox (Dox On). Data quantified in (E), in which we
observe a reduction of over 70% in transgene expression.
(F) qPCR levels of mature miRNAs persist even 8 days after Dox withdrawl.
(G) Levels of endogenous primary miRNA transcripts are expressed at PID 38. Primers for miR-9/9* were
designed for human loci 3, and miR-124 for loci 1.
(H) Continuous expression of miRNAs for at least 30 days is necessary for efficient neuronal conversion.
Error bars = s.e.m. Student’s t-test, N.S. = Not significant, * = p<0.05; ** = p<0.01 Scale bar = 20 μm.
A
+FOXP1
+RARB + RXRG
+POU6F1
+GSH2
+ISL1
+LHX8
+CTIP2
+MYT1L
MAP2/DAPI
miR-9/9*-124 Only
MAP2/DAPI
+ZFP503
+DLX1 + DLX2
MAP2/DAPI
+MEIS2
MAP2
DARPP-32
100
75
50
Fibroblast
Sample 4
Fibroblast
Sample 3
0
Fibroblast
Sample 2
25
Fibroblast
Sample 1
Percent of Cells Positive
+CTIP2+ DLX1/2 + MYT1L C
MAP2/DAPI
B
D Other combinations tested but not shown:
CTIP2, FOXP1, FOXP2, ZFP503, RARB, RXRG (+/- MYT1L)
CTIP2, DLX1, DLX2, FOXP1, FOXP2, ZFP503 (+/- MYT1L)
CTIP2, DLX1, DLX2, ZFP503 (+/- MYT1L)
CTIP2, DLX1, DLX2, RARB, RXRG (+/- MYT1L)
CTIP2, DLX1, DLX2, ZFP503, RARB, RXRG
CTIP2, DLX1, DLX2, FOXP1, FOXP2, ZFP503, RARB, RXRG
CTIP2, RARB, RXRG (+/- MYT1L)
CTIP2, DLX1, DLX2, FOXP1, FOXP2RARB, RXRG (+/- MYT1L)
CTIP2, DLX1, DLX2, FOXP1, FOXP2 (+/- MYT1L)
CTIP2, FOXP1, FOXP2, RARB, RXRG (+/- MYT1L)
CTIP2, FOXP1, FOXP2, ZFP503
CTIP2, ZFP503, RARB, RXRG (+/- MYT1L)
CTIP2, FOXP1, FOXP2, DLX1, DLX2, RARB, RXRG, GSH2, ZFP503 (+/- MYT1L)
CTIP2, FOXP1, FOXP2, RARB, RXRG (+/- MYT1L)
CTIP2, GSH2, ZFP503, DLX1, DLX2 (+/- MYT1L)
CTIP2, GSH2, ZFP503 (+/- MYT1L)
CTIP2, MYT1L, FOXP1
CTIP2, MYT1L, ZFP503
CTIP2, MYT1L, RARB, RXRG
Figure S3 (Related to Figure 1). Representative images from testing striatal factors with miR-9/9*-124
(A) MAP2 and DAPI staining of miR-9/9*-124 only and in conjuction with single or multiple factors listed above
each image. (B) The final cocktail, miR-9/9*-124 in combination with factors CDM is highly neurogenic.
(C) Fibroblasts from multiple donors can be reprogrammed with similar efficiencies. Samples 1 and 2: postnatal
fibroblasts. Data generated from sample 1 is displayed in Figure 1 of the main text. Sample 3 and 4: adult
fibroblasts (from a 42 year old and a 22 year old individuals, respectively). Data generated from sample 3 is
displayed in Figure 4 of the main text while data generated from sample 4 is displayed in Figure 5.
(D) Additional factors tested in conjuction to miR-9/9*-124. Error bars = s.e.m. Scale bar = 20 μm.
N.S.
p= 0.7350
N.S.
p= 0.2400
1.0
0.5
1.0
p= 0.0115
*
N.S.
p= 0.7453 N.S.
p= 0.1436
0.5
0.0
Doxycycline-inducible transcriptional activation of CTIP2
FSP1/CTIP2
- DOX (Control)
Ectopic expression of CDM
does not induce miR-132
m
iR
-9
m
iR
-9
*
m
iR
-1
24
-1
24
iR
-9
*
m
iR
m
m
iR
-9
0.0
Non-Transduced Fibroblasts
Fibroblast+CDM
2.0
1.5
C
Relative RNA Expression
Non-Transduced Fibroblasts
Fibroblasts + CTIP2
N.S.
p= 0.2196
FSP1/CTIP2
D
Mature miR-9/9* or miR-124 Levels
Upon Ectopic Expression of CDM
Relative RNA Expression
1.5
B
+ DOX (5 days since induction)
2.0
N.S.
p= 0.0821
Non-Transduced Fibroblasts
Fibroblast+CDM
1.5
1.0
0.5
0.0
miR-132
Ectopic expression of CTIP2 does
not induce Bcl-xL
Relative RNA Expression
Mature miR-9/9* or miR-124 Levels
Upon Ectopic Expression of CTIP2
Relative RNA Expression
A
1.5
N.S.
p= 0.8584
DOX OFF
DOX ON
1.0
0.5
0.0
Bcl-xL
Figure S4 (Related to Figure 1). Striatal factors without miR-9/9*-124 do not induce neuronal miRs or Bcl-xL
(A) Human dermal fibroblasts were transduced with CTIP2 alone and the levels of mature miRNAs were
analyzed by qPCR with TaqMan Probes 7 days post-transduction. Results indicate levels of miR-9, miR-9*
and miR-124 are not significantly changed in comparison to non-transduced fibroblast controls.
(B) Similarly, CDM factors alone do not induce the expression of miR-9/9* and miR-124 as well as another
neuronal miRNA, miR-132 (C), further supporting our conclusion that CDM factors without concurrent
expression of miR-9/9*-124 are not capable of inducing neuronal conversion.
(D) To investigate the possible link between CTIP2 and Bcl-xL in our system, we constructed a
doxycycline (DOX)-dependent CTIP2 lentiviral vector. Human neonatal fibroblasts were transduced with this
vector and cultured in the absence or presence of DOX as shown in the immunostaining data.
(FSP1 is a marker specific to fibroblasts and used to generate contrast). After 5 days of DOX exposure, cells
were lysed and the relative expression of Bcl-xL was analyzed by qPCR showing no significant change.
Error bars = s.e.m. Student’s t-test, N.S. = Not significant. Scale bar = 20 μm.
A
GABA/DAPI
GABA/DAPI
human fibroblasts
B
GAD67/MAP2/DAPI
GAD67/MAP2/DAPI
human
human fibroblasts
fibroblasts
Figure S5 (Related to Figure 1). A representative overall field of view of human postnatal fibroblasts
converted into GABAergic neurons
(A) Immunostaining with an antibody against GABA.
(B) Immunostaining with antibodies against GAD67 and MAP2.
Blue color marks DAPI signal. Insets in each figure represent complementary immunostaining data from human
postnatal fibroblast controls. Scale bar = 20 μm.
A
converted cells
VGLUT1/MAP2/DAPI
B
human cortical neurons
VGLUT1/MAP2/DAPI
Figure S6 (Related to Figure 1). miR-9/9*-124-CDM-converted cells immunostained for excitatory neurons
(A) Immunostaining with antibodes against VGLUT1, a marker for glutamatergic neurons, and MAP2.
(B) Human cortical neurons stained for VGLUT1. Note the absence of VGLUT1-positive cells the converted
cells in comparison to human cortical neurons. Scale bar = 20 μm.
A
DARPP-32/GABA
DARPP-32/GABA
human fibroblasts
B
DLX5/TUBB3/DAPI
DLX5/TUBB3/DAPI
human fibroblasts
Figure S7 (Related to Figure 1). Representative overall field of view of medium spiny neurons induced from
human postnatal fibroblasts
(A) Immunostaining with antibodes against DARPP-32 and GABA.
(B) Immunostaining with an antibodies against DLX5 and TUBB3.
Blue color marks DAPI signals. Insets in each figure represent complementary immunostaining data from starting
human postnatal fibroblasts. Scale bar = 20 μm.
A
Relative RNA Expression
60
Non-Transduced Fibroblasts
**
50
miR-9/9*-124+CDM
40
30
20
N.S.
p = 0.1715
10
3'
2
LX
D
D
LX
1
3'
U
U
TR
TR
0
Figure S8 (Related to Figure 2). Endogenous DLX1 but not DLX2 is activated during
miR-9/9*-124+CDM-mediated reprogrammingof human fibroblasts to medium spiny neurons
(A) In order to determine if the endogenous transcripts of DLX1 and DLX2 are induced with ectopic
expression of miR-9/9*-124 + CDM, we designed primers to target the 3’ UTRs of DLX1 and DLX2
since the transgenic cDNAs do not contain 3‘ UTRs of these genes. At 38 days post-infection, we
extracted RNA and determined the levels of endogenous transcripts. Interestingly, we detect a large
level of DLX1, but no increase in the expression of DLX2 in comparison to non-transduced fibroblasts.
This data suggests that the endogenous DLX1 is activated during our reprogramming.
Error bars = s.e.m. Student’s t-test, N.S. = Not significant, ** = p<0.01
A
miR-9/9*-124 + CDM
DARPP-32/GABA
B
miR-9/9*-124 + DM
DARPP-32/GABA
Figure S9 (Related to Figure 1). Demonstration of CTIP2 in promoting the fate of DARPP-32-positive cells
(A) Human postnatal fibroblasts were converted using miR-9/9*-124 and CDM (CTIP2, DLX1/2 and MYT1L)
and immunostained with antibodies against DARPP-32 and GABA.
(B) Human postnatal fibroblasts were converted using miR-9/9*-124 and DM (DLX1/2 and MYT1L), and
immunostained with antibodies againstDARPP-32 and GABA. Note that in DM condition, converted cells are
GABAergic, but deficient in DARPP-32-positive cells. Scale bar = 20 μm.
Laser Microdissected Human MSNs
R2 = 0.769
Reprogrammed MSNs
Figure S10 (Related to Figure 2). Pairwise comparison of sample groups from single-cell gene
expression profile
The degree of similarity in gene expression between single-cells microdisseced from postmortem
human striatum and single-cells collected from reprogrammed MSNs was quantified in a pairwise
comparison with a coefficient of determination (R2) of 0.769. Gene expression is represented
as limit of detection (LOD) Ct value of 26 subtracted by observed Ct values of each
gene tested, in reprogrammed cells (X axis) and laser-microdissected human MSNs (Y axis).
220000
**
180000
****
140000
FB
LCM
100000
2500
****
1500
m
iR
-1
*
-9
iR
m
iR
m
24
2
1
0
-9
Relative RNA Expression
260000
Figure S11 (Related to Figure 2). Mature miR-9/9* and miR-124 are expressed in human striatum
To determine the levels of mature miR-9, miR-9* and miR-124 in native human medium spiny neurons,
we conducted qPCR analysis using TaqMan probes in human postmortem striatum tissue from 89 yearold heathly individual. Results indicate that the miR-9/9*-124 are highly expressed in the human striatum.
Error bars = s.e.m. Student’s t-test, ** = p<0.01; *** = p<0.001
MSNs derived from human neonatal fibroblasts – 12 weeks (monoculture)
B
20 mV
A
2 nA
300 ms
-56 mV
5 ms
MSNs derived from human neonatal fibroblasts – 6 weeks (monoculture)
D
200 pA
20 mV
C
500 pA
20 ms
-58 mV
5 ms
Peak Na Amplitude (pA)
2500
***
2000
F
G
+GABA
1500
200 pA
E
200 pA
Functional maturation with time in vitro and in co-culture with primary neurons
1000
500
0
1s
6 weeks 12 Weeks
Figure S12 (Related to Figure 3). Additional representative traces from patch-clamp recordings
in tissue culture
(A-D) MSNs derived from postnatal fibroblast at 12 weeks (A-B) and at 6 weeks (C-D) post-transduction.
Left and right panels of each figure correspond to current-clamp and voltage-clamp traces, repectively.
(E) Peak Na+ amplitude increases significantly with time in vitro.
(F) EGFP-tagged converted cells increase in morphological complexity upon co-culturing.
(G) Application of 5 μM GABA induces hyperpolarizing currents indicative of functional
GABA receptors. Error bars = s.e.m. Student’s t-test, *** = p<0.001 Scale bar = 10 μm.
A
Neonatal
Fibroblasts
6 weeks
12 weeks
6 weeks - coculture
N
AP
RF
Vrest (mV)
Cm (pF)
Rm (GΩ)
13
13
12
11
11
12
0
6
12
-45.45 ± 1.53
-45.52 ± 2.19
-58.35 ± 3.08
30.95 ± 6.06
16.29 ± 1.96
16.99 ± 2.34
1.7 ± 0.31
1.0 ± 0.14
1.7 ± 0.16
B
Spontaneous Activity (Culture)
Neonatal Fibroblasts -12 Weeks
N
11
Response
8
C
Acute Slice
N
EGFP
7
Non-EGFP
11
Input
Resistance
(Mohm)
Vrest
(mV)
Delay to
first spike
(ms)
Current step
to first spike
(pA)
184.97 ±
40.67
114.65 ±
12.48
-83.29 ±
2.40
-85.205 ±
1.67
271.50 ±
57.35
235.75 ±
27.97
125.71 ±
34.00
177.5 ±
15.78
AP
Threshold
(mV)
Ramp
-42.70 ±
3.36
-40.57 ±
4.42
AP
Threshold
(mV) step
-44.85 ±
2.23
-45.62 ±
2.97
D
Spontaneous Activity (Slice)
16 weeks post-transplantation
N
EGFP: 7
Non-EGFP: 11
Response
7
11
Table S1 (Related to Figures 3, 4 and 6). Electrophysiology recordings summary
table. (A) Induced medium spiny neurons derived from nenonatal human fibroblasts at 6
and 12 weeks post-transduction as well as medium spiny neurons recorded at 6 weeks
post-transduction in the presence of primary rat cortical neurons (Coculture).
Abbreviations: N- Total number of cells recorded; AP- Total number of cells that fired
action potentials (APs); RF- Total number of cells that showed repetitive firing; VrestResting membrane potential; Cm- Membrane capacitance; Rm- Membrane resistance.
Data reported as mean ± S.E.M. (B) Spontaneous activity in reprogrammed cells cocultured with primary rat cortical neurons. (C) Intrinsic membrane properties of neurons
recorded in slice. AP threshold and amplitude (measured from threshold to the peak)
from ramp injection. (D) Spontaneous activity recorded from slice preparation. All data
acquired from slice recordings has been quantified and presented in Figure 3.
Gene Symbol
ANK2
ASCL1
BCL11B
BDNF
CALB2 (Calretinin)
CHAT
CHRM4
DBH
DCX
DDC
DLG4 (PSD95)
DLX1
DLX2
DRD1
DRD2
EOMES (TRB2)
GAD1
GAD2
GAPDH
GPR6
GRP
HPRT1
HTR2C
HTR3A
LHX6
MAP2
MAPT
MYT1L
NCAM1
NES
NEUROD1
NGFR
NKX2-1
OPRM1
OTX1
OTX2
PAX6
PCP2
PDYN
PENK
POU3F1
PPP1R1B (DARPP-32)
PRPH
PVALB
RARB
RPS18
SCN2A
SCN3A
SHANK3
SLC17A6 (VGLUT2)
Forward
TCACAACTGAGTCATCCATCAC
TGGTGCGAATGGACTTTGGAA
CAACCCGCAGCACTTGTC
TGGCTGACACTTTCGAACAC
TCACTCTTTGACATCATGCCA
CCGGTTTGTCCTCTCCACTA
TCTTTGCCATTCTGCTAGCC
GAGTGGGAGATCGTGAACCA
ATCTCTACGCCCACCAGTCC
CACCCTGGGGACCACAAC
AGCTGGAGCAGGAGTTCAC
GGAAGGGCTCAGGAGGAAAC
TTCGTCCCCAGCCAACAA
GCCCTTTGGGTCCTTCTGTA
CCTGAACTTGTGTGCCATCA
CTGTGGCAAAGCCGACAATA
ATCCTGGTTGACTGCAGAGAC
CAAACATTTATCAACATGCGCTTC
ACACCATGGGGAAGGTGAAG
CCAAAGTCAGACTCACACCAT
ACCGTGCTGACCAAGATGTA
GCTTTCCTTGGTCAGGCAGTA
CATGCACCTCTGCGCTATA
ATGTGGCTGCAGTGGTTT
ACGCTCAGACGCTGCAGAA
CAACGGAGAGCTGACCTCA
GAAGATTGGGTCCCTGGACAATA
GCTCTGTGCTATCCTGATACC
CCGTCATCCTGCTTGATCAG
GCTGCGGGCTACTGAAAA
GCCCCAGGGTTATGAGACTA
CGACAACCTCATCCCTGTCTA
GATGGTACGGCGCCAAC
CAGCCATTGGTCTTCCTGTA
CTGCTGGCGGCATTTGG
CATTCTGCTGTTGTTGCTGTT
TTGCCCGAGAAAGACTAGCA
CGGGCCAGACCACCAA
CATCTCTCCCATTCCTCAGA
TTCCTCATTATCACTGCCATCC
CCGCCAGTGTGTACATATCTC
TCTCAAGTCGAAGAGACCCAAC
CACAACCTCGTGCTCTTCC
GCTGAACGCTGAGGACATCAA
ACCATCGCAGACCAAATTACC
CGGAAAATAGCCTTTGCCATCA
TGTGGTGGTCATTCTCTCCA
CTTTGTGGTGGTGATTCTCTCC
CATAATCGCTGTGTGAGGTGA
TGGGGCTACATCATCACTCA
Reverse
ATTACCTGCGATACAGCTTGG
CTCCCAACGCCACTGACAA
CCTCGTCTTCTTCGAGGATGG
ATCACCCTGGACGTGTACAA
GGAAATATGGAAGCACTTTGACG
ATTTGGGACCACAGGACCATA
CTCTGGCAGAAGGTGTTCAC
ACCGACACGACCTTCTTCAA
AGCGAGTCCGAGTCATCCAA
TGCATCAACGTGCAGCCATA
ACACGCTTCACCTTGTGGTA
TAGCTTCTTGGTGCGCTGAA
TGGCTTCCCGTTCACTATCC
CAGAGGTTGAGGATGGATGCA
GAGCTGTAGCGCGTATTGTA
CTCATCCAGTGGGAACCAGTA
CCAGTGGAGAGCTGGTTGAA
CTATGACACTGGAGACAAGGC
GTGACCAGGCGCCCAATA
TCCTCTCACCAACACCACA
GCTCCCTCTCTCAGAAACAGAA
ACTTCGTGGGGTCCTTTTCAC
ATCACAGGGATAGGAACTGATAC
CTGGTTCTGGAGAGAATCGC
GCCCGGCAGTTTTGAAACCA
CTACAGCCTCAGCAGTGACTA
AGGTCAGCTTGTGGGTTTCA
GCTGTGATGGTTCTGGACAT
GAGTTCAAGACGCAGCCA
CTGAGCGATCTGGCTCTGTA
TCTGTCCAGCTTGGAGGAC
TTCTGCTTGCAGCTGTTCC
CCATGCCGCTCATGTTCA
TCAGCAGGTTTTCCCAGTAC
ACTCGCTACCCTGACATCT
CTTCGGGTATGGACTTGCTG
TCTCCATTTGGCCCTTCGATTA
TGTCACACGTTGGTCATCCA
GGTGCTCCTTGTGTGCT
CATGAAGAAGGATGCAGAGGAG
CTTCCCTCTGCTCCCTTTC
TGCAGGTGAGACTCAGCAA
CTCAATCTTGCGCTCTAGTTCC
TTCAGGCCGACCATTTGGAA
TGGGGTATACCTGGTGCAAA
TCCTCAACACCACATGAGCATA
AACAGGGTAGGGGACACAAA
GGAACAAGGTAGGGGACACA
GTGGAGGAAGTGCAGATGAG
GAAGTATGGCAGCTCCGAAA
SLC17A8 (VGLUT3)
SLC6A3 (DAT)
SLC6A4 (SERT)
SST
TAC1
TBR1
TH
TPH2
TUBB3
AACCACAACTGCTGTCAGAAA
TTCCCCTACCTGTGCTACAAAA
TGCTGGCTTTTGCTAGCTAC
GTTCCAGGGCATCATTCTCC
TTGGCACAATATGAAAAATAAACACTT
ACGAACAACAAAGGAGCTTCA
TCACCAAGTTCGACCCTGAC
ATGGCTCAGATCCCCTCTACA
CCTCCGTGTAGTGACCCTT
AAAGCCAACCACCAGGAGTA
GAAAAGTGGCATCCCAGCAA
GAAGCTCGTCATGCAGTTCA
AGACTCCGTCAGTTTCTGC
TTCATTTGTGTCAATGGGCAAT
TGGTACTTGTGCAAGGACTGTA
CGATCTCAGCAATCAGCTTCC
GGATCCGCAAGTAGTGGAACA
GGCCTTTGGACATCTCTTCAG
Bcl-xL
GACATCCCAGCTCCACATC
GTTCCCATAGAGTTCCACAAAAG
DLX1 3'UTR
GGCTGTTTGCCAATTCAGGG
CTCCCCGTGCGCTTAAAGTA
DLX2 3'UTR
CCTTATCTTACCCCCACCGC
TCCGCAAAGGCACCTAAACT
Transgenic Pri-miR9124
Pri-miR-9 Loci 3
TGTCGGTCCCCTCTGGCTCT
AGAAACGGGCCTCCCATTCG
AAGAGCCAGAGGAGCCGA
GAGAGAGCCGAGGTCAGGTA
Pri-miR-124 Loci 1
CAAGGAAGGAGCGACCGAC
TGCATCTCTAAGCCCCTGTC
Table S2 (Related to Figure 2). Primer sequences from single-cell multiplex qPCR
analysis and RT-qPCR. The bottom table provides primer sequences used for RT-qPCR
(Figures S2, S4, S8, S11).
Supplemental Experimental Procedures
Plasmid Construction and Virus Preparation
We modified the previously validated lentiviral construct to drive the expression of both
miR-9/9* (NCBI and miRBASE accession numbers MIMAT0000441 and
MIMAT0000442) and miR-124 (accession number MIMAT0000422) by replacing
TurboRFP with Bcl-xL (NCBI Reference Sequence: NM_138578.1) to promote neuronal
survival (Alavian et al., 2011) during neuronal conversion. MiR-9/9*-124 and Bcl-xL
were co-driven by a doxycycline- responsive promoter, pTight (Open Biosystems) where
doxcycycline was usually removed approximately 4 weeks post-infection. cDNA of
CTIP2 (NCBI Reference Sequence: NM_001079883.1), DLX1 (NCBI Reference
Sequence: NM_178120.4), DLX2 (NCBI Reference Sequence: NM_004405.3), MYT1L
(NCBI Reference Sequence: NM_015025.2) were cloned downstream of the EF1α
promoter in a separate lentiviral construct with neomycin selection. For doxycycline
experiments, human fibroblasts were first infected with a lentiviral construct expressing
rtTA under the EF1α promoter and stably selected with hygromycin, followed by
transduction with pTight-miR-9/9*-124-Bcl-xL and neural factor-expressing lentiviruses.
Infectious lentiviruses were collected 60-70 h after transfection of Lenti-X 293LE cells
(Clontech) with appropriate amounts of lentiviral vectors, psPAX2 and pMD2.G
(Addgene) using polyethyleneimine (Polysciences). Collected lentiviruses were pushed
through a 0.45 μm PES filter and concentrated either by high-speed centrifugation at
70,000 x g for 2 hours or Lenti-X Concentrator (Clontech) following the manufacturer’s
protocol.
Cell Culture
All fibroblast cultures (human postnatal foreskin fibroblasts (ATCC, PCS-201-010 and
ScienCell, 2310) and adult dermal fibroblast (ATCC, PCS-201-012 and Coriell Cell
Repositories, GM02171)) were maintained in fibroblast media (Dulbecco’s Modified
Eagle Medium; Invitrogen) containing 10% fetal bovine serum (FBS; Hyclone), 0.01% βmercaptoethanol, 1% non-essential amino acids, 1% sodium pyruvate, 1% GlutaMAX,
1% 1M HEPES buffer solution and 1% penicillin/streptomycin solution (all from
Invitrogen). The day before lentiviral infection, human fibroblasts were seeded at 2x105
cells/well onto gelatin-coated 12-well tissue culture dishes (Corning). Next day, cells
were infected with the lentiviral supernatant in the presence of polybrene (Sigma-Aldrich,
8 μg /ml), spun at 1000 x g for 30 minutes, and left in the incubator overnight. Fresh
fibroblast media with doxycycline (Sigma Aldrich, 1 μg/mL) (to induce miR-9/9*-124
expression) were added the next morning and left on for two days. Three days after
infection, cells were trypsinized (0.25% Trypsin, Invitrogen) and re-plated onto polyornithine (Sigma-Aldrich, 0.01% solution)/laminin (Roche, 5 μg/mL)/fibronectin (SigmaAldrich, 2 μg/mL)-coated glass coverslips in fibroblast media with doxycycline. The day
after re-plating, the medium was changed to Neuronal Media (ScienCell) supplemented
with valproic acid (Calbiochem, 1 mM), dibutyryl cAMP (Sigma-Aldrich, 400 μM), human
BDNF (Peprotech, 10 ng/ml), human NT-3 (Peprotech, 10 ng/ml), and retinoic acid
(Sigma-Aldrich, 1 μM) with appropriate antibiotics. Doxycycline was replenished every
two days for approximately 4 weeks. Media was replenished every four days by pipetting
out half of the media and replacing an equal volume of fresh media. Addition of
antibiotics was terminated after two weeks.
Immunocytochemistry
The following antibodies were used for the immunofluorescence studies: chicken antiMAP2 (Abcam, 1:10,000), mouse anti-β-III tubulin (Covance, 1:1,000), chicken anti-
NeuN (Aves, 1:500), rabbit anti-SCN1a (Abcam, 1:500), rabbit anti-synapsin1 (Millipore,
1:800), mouse anti-Ankyrin G (Santa Cruz Biotechnology, 1:200), rabbit anti-GABA
(Sigma, 1:2,000), mouse anti-GABA (Sigma, 1:500), mouse anti-GAD67 (Millipore,
1:1,000), rabbit anti-FOXP1 (Abcam, 1:500), rabbit anti-DLX5 (Abcam, 1:2,000), rabbit
anti-DARPP32 (Santa Cruz Biotechnology, 1:400), and rabbit anti-VGLUT1 (Synaptic
Systems, 1:1,000). The secondary antibodies were goat anti-rabbit, mouse, or chicken
IgG conjugated with Alexa-488, -594, or -647 (Invitrogen). For SCN1a, biotinylated
secondary antibodies were detected using TSA amplification kit (Invitrogen). Images
were captured using a Leica SP5X white light laser confocal system with Leica
Application Suite (LAS) Advanced Fluorescence 2.7.3.9723.
Immunohistochemistry
Mice were anesthetized with 200mg/kg Avertin (Sigma #T48402) and transcardially
perfused with ice-cold 4% paraformaldehyde (vol/vol) in 0.1M phospate buffer saline
(PBS). Brains were dissected, post-fixed for an additional 24 hours and cryoprotected in
30% sucrose solution for 48-72 hours. Brains were embedded in Tissue-Tek O.C.T. and
once frozen, cut into 35μm slices. Free-floating slices were washed three times in PBS,
permeabilized in 0.2% Triton-X for 10 minutes, and blocked in 5% BSA and 1% goat
serum for 2 hours at room temperature. Sections were then incubated for 48 hours in
chicken anti-MAP2 (Abcam, 1:500), rabbit anti-DARPP-32 (Cell Signaling, 1:200), rabbit
anti-FOXP1 (Abcam, 1:400), mouse anti-GABA (Sigma, 1:500), rat anti-CTIP2 (Abcam,
1:500) and mouse anti-tyrosine hydroxylase (Millipore, 1:200). Following incubation,
sections were washed three times in PBS and then incubated for 2 hours at room
temperature with goat anti-rabbit, mouse, rat, or chicken IgG conjugated with Alexa-488,
-594, or -647 (Invitrogen, 1:200). Sections were once again washed with PBS and
incubated with DAPI (Sigma, 1:10,000) for 5 minutes followed by one last PBS wash.
Sections were mounted on glass slides with Vectashield (Vector Labs) and images were
captured using a Leica SP5X white light laser confocal system with Leica Application
Suite (LAS) Advanced Fluorescence 2.7.3.9723.
Single Cell Quantitative PCR
Converted cells at 5 weeks were harvested by Trypsin 0.05% (Gibco) and single cells
were collected by FACS sorting using BD Aria II (BD Biosciences) in 5 μl of reverse
transcription reaction mix containing VILO Reaction Mix (Invitrogen), SUPERase-In
(Ambion), NP-40 (Thermo Scientific) and nuclease-free water. Sorted samples were
denatured for 90 seconds at 65°C and reverse-transcribed by adding SuperScript
Enzyme Mix (Invitrogen) and T2 Gene 32 Protein (New England Biolabs). First strand
cDNAs were then pre-amplified for 20 cycles in Preamplification mix containing 50 nM of
all primers for genes of interest (see Table S2) and TaqMan Preamp Master Mix
(Applied Biosystems). Pre-amplified samples were treated with Exonuclease I (New
England Biolabs), diluted (5x) with DNA Suspension Buffer (TEKnova) and stored at 20°C. Sample and assay (primer pairs) preparation for 96.96 Fluidigm Dynamic arrays
was done according to the manufacturer’s recommendation (Genome Technology
Access Center, Washington University School of Medicine in St. Louis). Briefly, sample
was mixed with 20x DNA Binding Dye Sample Loading Reagent (Fluidigm), 2x Sso Fast
EvaGreen supermix with Low ROX (Bio-Rad Laboratories). Assays were mixed with 2x
Assay loading reagent (Fluidigm) and DNA Suspension Buffer (Teknova) to a
concentration of 500 nM in the final reaction. The 96.96 Fluidigm Dynamic Arrays
(Fluidigm) were primed and loaded on an IFC Controller MX (Fluidigm Corp.) and qPCR
experiments were run on the Biomark HD System (Fluidigm). Data was collected and
analyzed using the Fluidigm Real-Time PCR Analysis software (v3.1.3). Melting curves
were used to determine specificity of each reaction and assay primers were also verified
by using four standard dilutions of human cDNA library prepared from human neurons
(ScienCell). The collected cells were confirmed based on RSG18 (18S small ribosomal
subunit), GAPDH and HPRT expression.
Quantitative PCR
Total RNA was extracted using TRIzol (Invitrogen, USA) according to the manufacturer's
instruction. The RNA quality was determined by the ratio of absorbance at 260 nm and
280 nm to be approximately 2.0. Reverse-transcribed complementary DNA (cDNA) was
synthesized from 1μg of RNA with SuperScript III First-Strand Synthesis SuperMix
(Invitrogen, USA) or from 10 ng of microRNAs using specific stem-loop primer probes
from TaqMan MicroRNA Assays (Invitrogen, USA). Subsequently, the cDNA was
analyzed on a StepOnePlus Real-Time PCR System (AB Applied Biosystems,
Germany). Expression data were normalized to housekeeping genes HPRT1 and
RNU44 for coding genes and microRNAs, respectively, and analyzed using the 2-ΔΔCT
relative quantification method. Primer sequences are provided in Supplemental Table
S2.
Laser Microdissection
Striatal putamen sections were obtained from an autopsy sample of an 89 year-old
individual. The frozen samples were sectioned onto Polyethylene naphthalate (PEN)
membrane coated glass slides (Leica) at a thickness of 5 μm and stored at -80°C.
Tissue sections were fixed in cold 75% ethanol for 2 minutes and stained with Cresyl
Violet (Sigma Aldrich), gradually incubated in 75%, 90% and 100% ethanol. After
samples were completely dried, fixed samples were loaded onto Leica Laser
Microdissection system (LMD6500). Cresyl Violet-stained single cells were laserdissected and collected into an individual tube to be used for reverse transcription and
gene expression analysis as described in Single Cell Quantitative PCR.
Electrophysiology in Tissue Culture
Whole-cell patch-clamp recordings were performed at 6 or 12 weeks after transduction
with miR-9/9*-124-CDM, in cells of postnatal origin. Cells converted from human adult
fibroblasts were analyzed at 6 weeks in the presence or absence of primary human
neurons (ScienCell) as described in the main text. Data was acquired using pCLAMP 10
software with multiclamp 700B amplifier and Digidata 1550 digitizer (Molecular Devices).
Electrode pipettes were pulled from borosilicate glass (World Precision Instruments) and
typically ranged between 3–6 MΩ resistance. Intrinsic neuronal properties were studied
using the following solutions (in mM): Extracellular: 140 NaCl, 3 KCl, 10 Glucose, 10
HEPES, 2 CaCl2 and 1 MgCl2 (pH adjusted to 7.25 with NaOH). Intracellular: 130 KGluconate, 4 NaCl, 2 MgCl2, 1 EGTA, 10 HEPES, 2 Na2-ATP, 0.3 Na3-GTP, 5 Creatine
phosphate (pH adjusted to 7.5 with KOH). Membrane potentials were typically kept at 60 mV to -70 mV. In voltage-clamp mode, currents were recorded with voltage steps
ranging from -20 mV to +90 mV. In current-clamp mode, action potentials were elicited
by injection of step currents that modulated membrane potential from +10 mV to +80
mV. Local application of 5 μM GABA (Sigma #A5835) was achieved using a multibarrel
perfusion system with a port placed within 0.5 mm of the patched cell. GABA-evoked
currents were recorded using the previously listed external solution and the following
intracellular solution (in mM): 130 CsCl, 2 NaCl, 10 HEPES, 0.1 EGTA (pH adjusted to
7.25 with NaOH). Postsynaptic potentials were detected spontaneously or evoked by
perfusion of 200mM sucrose. Data was collected in Clampex and initially analyzed in
Clampfit (Molecular Devices). Further analysis was done in GraphPad Prism 6
(GraphPad Software). Liquid junction potential was calculated to be 15.2 mV and
corrected in calculating resting membrane potential according to previously published
methods (Barry, 1994).
Animals
Immunodeficient NOD scid gamma (NSG) mice used for transplantation studies and
breeding were purchased from The Jackson Laboratory. Once transplanted, mice were
kept on doxycycline chow (LabDiet 2000ppm) to ensure proper expression of miR-9/9*124 which was cloned under the doxycycline-responsive promoter, pTight. Doxycycline
chow was removed 30 days after transplantation and replaced with regular chow.
Cell Harvesting and Transplantation
Reprogrammed cells for transplantation were typically transduced with a Synapsin
promoter-EGFP lentivirus at reprogramming day 2 and harvested for transplantation
studies at reprogramming day 14. Adherent cell-cultures were mechanically dissociated
and concentrated by centrifugation (4 minutes at 1000 RPM). Concentrated cell
suspensions (~104 cells μl-1) were loaded into a 5μl Hamilton syringe (26s/2/2
Gauge/Length/Point) and 2μl were injected into the right striatum of NSG mice (P0-P1).
Injection coordinates were guided by cranial transillumination and first verified in
preliminary dye studies.
Brain Slice Preparation and Recordings
Coronal striatum slices were prepared as described previously (Andre et al., 2011;
Ellender et al., 2011). Briefly, after being deeply anesthetized with with 1.25% Avertin,
animals were perfused transcardially with ice cold cutting solution containing (in mM):
240 sucrose, 2.5 KCl, 1.25 NaH2PO4, 25 NaHCO3, 0.5 CaCl2, and 7 MgCl2 and saturated with
95% O2 and 5% CO2. Mice were decapitated and their brains were rapidly dissected out
into ice cold cutting solution. Brain slices (275μm) were cut in ice cold cutting solution
using a microtome (Leica VT1000S) and placed in a holding chamber where they were
incubated in artificial cerebrospinal fluid (ACSF) containing (in mM): 130 NaCl, 24
NaHCO3, 3.5 KCl, 1.25 NaH2PO4, 0.5 CaCl2, 5.0 MgCl2, and 10 glucose, saturated with
95% O2/5% CO2, at 33°C for 30 minutes and then at 22-23°C for at least 30 min before
transfer to the recording chamber. Electrophysiological recordings were made using an
Axopatch 700B amplifier (Molecular Devices) interfaced with a Digidata 1332 and the
pCLAMP 10.4 software (Axon Instruments). Data signals were acquired at 20kHz and
filtered at 10kHz, prior to digitization. Recordings were made in whole-cell mode from
GFP-positive striatal neurons and native medium spiny striatal neurons, visually
identified with florescence and DIC microscopy as described previously (Deng et al.,
2013). All recordings were conducted at room temperature (22°C). The recording
electrodes were filled with the following (in mM): 144 K-gluconate, 0.2 EGTA, 3 MgCl2, ,
4 MgATP, 0.5 NaGTP, and 10 HEPES, pH was brought to 7.3 using KOH. The
extracellular solution (ACSF described above) was continuously perfused and saturated
with 95% 02 and 5% CO2. A liquid-junction potential correction of -15mV was applied to
voltage- and current-clamp recordings prior to analysis as previously described (Barry,
1994). AP threshold was calculated using the first derivative of the AP waveform (first
evoked AP) and measured as the first point greater than 10 mV/ms. Spontaneous PSPs
were measured in current-clamp at the resting membrane potential. To compare
recorded properties between native mouse cells and human reprogrammed cells, a
student’s t-test was used for p-values reported. In non-EGFP tagged mouse cells, two
cells were excluded from analysis due to poor recording quality.
Supplemental References
Alavian, K.N., Li, H., Collis, L., Bonanni, L., Zeng, L., Sacchetti, S., Lazrove, E., Nabili, P.,
Flaherty, B., Graham, M., et al. (2011). Bcl-xL regulates metabolic efficiency of neurons through
interaction with the mitochondrial F1FO ATP synthase. Nature cell biology 13, 1224-1233.
Andre, V.M., Cepeda, C., Fisher, Y.E., Huynh, M., Bardakjian, N., Singh, S., Yang, X.W., and
Levine, M.S. (2011). Differential electrophysiological changes in striatal output neurons in
Huntington's disease. The Journal of neuroscience : the official journal of the Society for
Neuroscience 31, 1170-1182.
Barry, P.H. (1994). JPCalc, a software package for calculating liquid junction potential corrections
in patch-clamp, intracellular, epithelial and bilayer measurements and for correcting junction
potential measurements. Journal of neuroscience methods 51, 107-116.
Deng, P.Y., Rotman, Z., Blundon, J.A., Cho, Y., Cui, J., Cavalli, V., Zakharenko, S.S., and
Klyachko, V.A. (2013). FMRP regulates neurotransmitter release and synaptic information
transmission by modulating action potential duration via BK channels. Neuron 77, 696-711.
Ellender, T.J., Huerta-Ocampo, I., Deisseroth, K., Capogna, M., and Bolam, J.P. (2011).
Differential modulation of excitatory and inhibitory striatal synaptic transmission by histamine. The
Journal of neuroscience : the official journal of the Society for Neuroscience 31, 15340-15351.

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