doi: 10.1038/nature22330.
Epub 2017 Apr 26.
Assembly of functionally integrated human forebrain spheroids
Affiliations
- PMID: 28445465
- PMCID: PMC5805137
- DOI: 10.1038/nature22330
Item in Clipboard
Assembly of functionally integrated human forebrain spheroids
Nature.
.
Abstract
The development of the nervous system involves a coordinated succession of events including the migration of GABAergic (γ-aminobutyric-acid-releasing) neurons from ventral to dorsal forebrain and their integration into cortical circuits. However, these interregional interactions have not yet been modelled with human cells. Here we generate three-dimensional spheroids from human pluripotent stem cells that resemble either the dorsal or ventral forebrain and contain cortical glutamatergic or GABAergic neurons. These subdomain-specific forebrain spheroids can be assembled in vitro to recapitulate the saltatory migration of interneurons observed in the fetal forebrain. Using this system, we find that in Timothy syndrome-a neurodevelopmental disorder that is caused by mutations in the CaV1.2 calcium channel-interneurons display abnormal migratory saltations. We also show that after migration, interneurons functionally integrate with glutamatergic neurons to form a microphysiological system. We anticipate that this approach will be useful for studying neural development and disease, and for deriving spheroids that resemble other brain regions to assemble circuits in vitro.
Conflict of interest statement
H. C.F. and K.C.M. were employees of BD Genomics during this study.
Figures
Two anti–PV antibodies have been used to validate
specificity; co-localization with the neuronal marker DCX (day 209).
(a) Distribution of expression of the neuronal marker
STMN2, (b) the progenitor marker VIM
and of (c) a set of genes associated with the M cell cycle phase
(AURKB, TPX2, UBE2C,
HMMR, TOP2A, CCNB1,
NUSAP1, NUF2, CDC6,
HIST1H4C, BIRC5,
CKS2). (d) Boxplots for genes enriched in each t-SNE
cluster shown in Fig. 1j.
(e–l) Top 25 genes in each of the 8 clusters shown in Fig. 1j (proportion of molecules per
cells). (m) Scatter plot showing the number of genes detected versus the
number of useful reads.
(a) Schematic illustrating the differentiation conditions for
deriving hCS or hSS: IS, ISA and and ISRA. (b) Representative traces of
intracellular calcium measurements (Fluo-4) demonstrating spontaneous
activity in hSS at ~day 50 of differentiation. (c) Average calcium
spike frequency in 3 distinct hSS differentiation conditions: IS (n=
114 cells), ISA (n= 327 cells), ISRA (n= 136 cells); cells
derived from 3 hiPSC lines; one-way ANOVA, F(3, 719)= 5.86,
***P< 0.001. (d) Gene expression (qPCR,
normalized to GAPDH) showing down-regulation of
OCT4 and the lack of mesoderm (BRACH)
and endoderm (SOX17) markers following differentiation of
hiPSC into hCS and hSS conditions. (e) Gene expression (qPCR, fold change
versus hiPSC and normalized to GAPDH) showing upregulation
of forebrain markers (SIX3, FOXG1) but not
midbrain (LMX1B), hypothalamus (RAX1) or
spinal cord (HOXB4) markers. (f) Expression of ventral
forebrain genes in hSS and hCS (qPCR, normalized to GAPDH)
at day 25. (g) Average percentage of the proportion of NKX2–1 by
immunostaining in dissociated hCS or hSS at day 25. (h) Expression of
ventral forebrain genes in hSS (qPCR, normalized to GAPDH)
at day 60. (i) Expression of glutamatergic, GABAergic, dopaminergic and
cholinergic neurotransmitter identify genes in hSS (qPCR, normalized to
GAPDH) at day 60. (j) Average percentage of the
proportion of MAP2/Hoechst and GAD67/MAP2 by immunostaining in dissociated
hSS at day 60. (k, l) Expression of interneuron subtypes genes and markers
associated with interneuron migration in hSS (qPCR, normalized to
GAPDH) at day 60. Number of lines hiPSC used indicated
on each column. Data are mean ± s.e.m.
(a) Representative EPSC traces of recordings from hCS neurons
(sliced preparation) before (black) and during (green) exposure to the
glutamate receptor blocker kynurenic acid (1 mM) (Mann-Whitney U-test,
**P= 0.007). (b) Overlap of averaged EPSCs (red)
recorded in hCS neurons (n= 6 cells) and averaged IPSCs (black)
recorded in hSS (n= 5 cells). Data are
mean ± s.d.
(a, b) Representative immunostaining in cryosections of hSS showing
co-expression of Dlxi1/2::eGFP and GAD67 and GABA. (c) Quantification by
immunostaining of the proportion of Dlxi1/2::eGFP+ cells
that co-express GAD67 in hSS derived using the ISA or ISRA condition
(n= 3 hiPSC lines; t-test, P= 0.35). (d) Proportion of
Dlxi1/2::eGFP and GAD67 positive cells in hSS derived using the ISA or ISRA
condition that co-express SST (t-test, P= 0.48), CR (t-test,
*P= 0.04) or CB (t-test, P= 0.43); n = 3
hiPSC lines. (e) Representative images of hSS-hCS at day 60 showing
migration of Dlxi1/2b::eGFP+ cells (from fluorescently
labeled hSS) in fused hSS-hCS but not in hSS-hSS over time. (f) The number
of Dlxi1/2b::eGFP+ (hSS-derived) or hSYN1::mCherry cells
(hCS-derived) that moved in hSS-hCS or hSS-hSS was quantified in snapshots
of live, intact spheroids at different time points (from day 3 to 25). The
values shown are absolute number of cells that migrated to the other side
(approximately the same area and thickness was imaged in each session);
one-way ANOVA for cells at 17 days after assembly; F(2, 32)= 8.24,
P= 0.001. (g) Representative images of fused hSS-hCS at day 91
showing migration of Dlxi1/2b::eGFP+ cells (from
fluorescently labeled hSS) into fused hSS-hCS. (h) Representative image of
an hSS that was plated on a glass coverslip and cultured for ~7
days. (i) Percentage of Dlxi1/2::eGFP inside the coverslip-plated hSS, in
the rim (0–200 μm) or beyond this region (> 200
μm). (j) Quantification of the number of saltations of
Dlxi1/2b::eGFP+ cells (n= 32 cells) inside
the one-week coverslip-plated hSS, in the rim and outside this region
(one-way ANOVA, interaction F (2, 30)= 22.12, P< 0.001;
Bonferroni post-hoc ***P< 0.0001). (k)
Diagram showing the angle of movement of migrating
Dlxi1/2b::eGFP+ cells at 8–9 days after
assembly of hSS-hCS. The angle was calculated between the leading process of
Dlxi1/2b::eGFP+ cells that have moved into hCS and
the fusion interface (n= 92 cells from 5 hiPSC lines). (l) Histogram
showing the distribution of the distance of migrated
Dlxi1/2b::eGFP+ cells relative to the edge of hCS in
hSS-hCS at 30–50 days after assembly. The distance was measured in
fixed 18 μm cryosections (n= 73 cells from 2 hiPSC lines).
(m, n, o) Representative examples of Dlxi1/2b::eGFP+
cells migrated in the hCS that moved within a VZ-like region. The VZ-like
region contains GFAP-expressing cells, is surrounded by
TBR1+ cells and the migrated cells show GABA
expression. Supplementary
Video 2 shows movement of Dlxi1/2b::eGFP+
cells that is reminiscent of the ventricular-directed migration described in
rodents.
(a) Scheme showing the isolation by dissociation and
fluorescence-activated cell sorting (FACS) of
Dlxi1/2b::eGFP+ cells from hSS or hCS for single cell
transcriptional analysis. (b) Violin plots showing expression in
Dlxi1/2b::eGFP+ cells of selected genes associated
with cortical, striatal and olfactory interneurons in hSS (light green,
n= 123 cells) or hCS (dark green; n= 106 cells) at 2 weeks
after assembly of hSS-hCS. (c) Violin plots showing expression in
Dlxi1/2b::eGFP+ cells (at 4 weeks after assembly of
hSS-hCS) in clusters A, B, and C (likelihood ratio test;
GAD1, CELF4: P> 0.05;
PBX3: P< e−7 for A versus B
& C; NNAT: P< e−16 for C
versus A & B, P< e−16 for B versus A
& C; MALAT1: P< e−9 for
C versus A & B; SOX11: P<
e−16 for B versus A & C, P<
e−9 for A versus B & C;
GRIP2: P< e−8 for B versus A
& C). (d) Scatter plot showing the number of genes detected
(≥ 10 reads cutoff) versus the number of reads (n= 410 cells
from combined single cell RNA-seq experiments after 2 weeks or 4 weeks of
assembly in hSS-hCS). (e) Graph illustrating biologically variable
transcripts (red circles) and non-variable transcripts (black circles) along
with the technical noise from the ERCC spike in RNAs (blue dots). Green line
shows the technical noise fit.
(a, b, c, d) Representative images of immunostainings for SST,
GAD67, GABA, CR, CB in Dlxi1/2b::eGFP+ cells after
migration in fused hSS-hCS. (e) Scheme illustrating the pharmacological
manipulation of Dlxi1/2b::eGFP+ cells that are migrating
in hSS-hCS. (f, g, h, i) Quantification of
Dlxi1/2b::eGFP+ cell migration before and after
exposure to 100 nM of the CXCR4 antagonist AMD3100 (n= 8 cells from
2 hiPSC lines; paired t-tests, *P= 0.03 for number of
saltations, **P= 0.006 for saltation length,
**P= 0.006 for speed when mobile,
*P= 0.02 for path directness). (j) Plot illustrating the
trajectory of Dlxi1/2b::eGFP+ cells in fused hSS-hCS
before and after exposure to AMD3100. Data are
mean ± s.e.m.
(a, b, c) Representative images of human fetal cortex at GW20
showing immunostaining with antibodies against GFAP, BCL11B (CTIP2) and
GABA. (d) Representative image showing cell labeling with the
Dlx2i1/2b::eGFP reporter in human forebrain at GW18 (6 days after lentivirus
infection) (e, f) Representative immunostainings in cryosections of human
tissue at GW18 showing co-localization of Dlx2i1/2b::eGFP with NKX2-1 and
GABA. (g) Representative images showing cell labeling with the
Dlx2i1/2b::eGFP reporter in hSS-hCS (9 days after assembly), in human
forebrain (GW18) and in mouse slice cultures (E18). (h, i) Comparison of
Dlx2i1/2b::eGFP+ cell size and quantification of the
ratio of soma diameter to the length of the leading process in fused hSS-hCS
(n= 25 cells from 4 hiPSC lines), human forebrain at GW18
(n= 19 cells; black) and GW20 (n= 15 cells; gray),
hSS-derived cells cultured on E14 mouse forebrain slices (n= 14
cells), and E18 mouse forebrain slices (n= 30 cells from 2 litters)
(one-way ANOVA, interaction F(3, 97)= 11.61, P= 0.001,
Bonferroni post-hoc ***P< 0.001,
**P< 0.05). (j, k, l) Comparison of the number of
saltations (n= 56 cells from 2 hiPSC lines; one-way ANOVA,
interaction F(2, 103)= 29.27, P= 0.001, Bonferroni post-hoc
***P< 0.001), saltation length (n=
44 cells from 3 hiPSC lines; one-way ANOVA, interaction F(2, 91)=
3.0, P= 0.50), speed when mobile (n= 38 cells from 3 hiPSC
lines; one-way ANOVA, interaction F(2, 83)= 11.38, P= 0.001,
Bonferroni post-hoc ***P< 0.001) for
Dlx2i1/2b::eGFP+ in fused hSS-hCS, human fetal
forebrain (GW18: n= 19 cells; GW20: n= 15 cells), and E18
mouse forebrain slices (n= 14 cells for saltation length and speed,
n= 16 cells for number of saltations from 2 litters). Data are
mean ± s.e.m.
(a) Sequencing of PCR-amplified DNA showing the p.G406R mutation in
exon 8a of CACNA1C in TS (subject: 8303). (b)
Representative images of hiPSC colonies expressing pluripotency markers
(OCT4, SSEA4) in one TS subject (c) Level of gene expression (RT-qPCR,
normalized to GAPDH) for NKX2-1 showing no
defects in ventral forebrain induction in TS (two-way ANOVA; interaction
F(2,15)= 0.20, P= 0.81; TS versus Ctrl F(1,15)=
0.16, P= 0.68). (d–g) Representative immunostainings in
cryosections of TS hSS (day 60) showing expression of NKX2-1, GABA, MAP2,
GAD67, SST and CR. (h) Calcium imaging (Fura-2) in dissociated hCS derived
from TS subjects and controls (Ctrl: n= 81 cells from 2 subjects;
TS: n= 147 cells from 2 subjects). Quantification of residual
intracellular calcium
([Ca2+]i) following 67 mM KCl
depolarization of Ctrl and TS cells in hCS cells. Residual
[Ca2+]i was calculated by
dividing the plateau calcium (C–A) level by the peak calcium
elevation (B−A); (t-test, ***P<
0.001). (i) Quantification of
[Ca2+]i following
depolarization of Ctrl and TS cells in hSS (t-test,
***P< 0.001). (j) Representative image of
fused TS hSS-hCS showing Dlxi1/2b::eGFP expression and migration. (k, l)
Quantification of the number of saltations and saltation length of
Dlx2i1/2b::eGFP cells in fused hSS-hCS across multiple Ctrl and TS lines
(related to Fig. 3d, e). (m)
Quantification of the speed when mobile of Dlxi1/2b::eGFP cells in fused
hSS-hCS (Ctrl: n= 21 cells from 3 hiPSC lines derived from 3
subjects; TS: n= 29 cells from 3 hiPSC lines derived from 3
subjects; TS-Ctrl hybrid: n= 12 cells from 3 hiPSC line shown
combinations; one-way ANOVA with Dunnett’s multiple comparison test;
***P< 0.001). (n) Electroporation of cDNA
encoding the TS– and WT– CaV1.2 channels into
slices of mouse E14 ganglionic eminences (GE). (o) Representative example of
time-lapse live imaging depicting the saltatory migration of
GFP+ cells in slices electroporated with CAG::GFP and
either the WT–or the TS– CACNA1C. (p, q)
Quantification of the number of saltations (t-test;
**P< 0.01) and saltation length (t-test;
***P< 0.001) of GFP+
cells in electroporated mouse forebrain slices (WT: n= 33 cells; TS:
n= 23 cells; from 3 litters). (r) Scheme illustrating
pharmacological manipulation of LTCC during live imaging of fused hSS-hCS.
(s) Quantification of speed when mobile following exposure to the LTCC
blocker nimodipine (5 μM) (paired t-test; Ctrl: n= 13 cells
from 3 hiPSC lines derived from 3 subjects,
***P< 0.001; TS: n= 12 cells from 2
hiPSC lines derived from 2 subjects, **P< 0.005).
(t) Quantification of saltation length following exposure to roscovitine (15
μM) (paired t-test; Ctrl: n= 7 cells from 2 hiPSC lines
derived from 2 subject, **P< 0.005; TS: n=
12 cells from 2 hiPSC lines derived from 2 subjects;
***P< 0.001). (u) Quantification of speed
when mobile following exposure to roscovitine (15 μM) (paired
t-tests; Ctrl: n= 9 cells from 2 hiPSC lines derived from 2
subjects, ***P< 0.001; TS: n= 12
cells from 2 hiPSC lines derived from 2 subjects; P= 0.05). Data are
mean ± s.e.m.
(a) Representative images of 3D–reconstructed
Dlxi1/2b::eGFP+ cell morphologies before and after
migration from hSS into hCS. (b) Quantification of dendritic branching of
Dlxi1/2b::eGFP+ cells in hSS (n= 58 cells)
and in hCS (n= 55 cells) of fused hSS-hCS (two-way ANOVA;
interaction F(2, 129)= 11.29, P< 0.001; Bonferroni post-hoc
*P< 0.05, ***P< 0.001). (c)
Representative examples of action potentials (slice recordings) in
Dlxi1/2b::eGFP+ cells in unfused hSS, in hSS of fused
hSS-hCS and in hCS after migration in fused hSS-hCS. (d) AT showing
expression of the GABAergic synapse marker GPHN (green) colocalized with
SYN1 (red) in hCS of fused hSS-hCS but not in unfused hCS; the glutamatergic
marker PSD95 (cyan) colocalized with SYN1 is found in both fused and unfused
hCS (equal volumes 1.2 μm deep). (e) AT of a
Dlxi1/2b::eGFP+ synapse illustrating the
colocalization with SYN1 (red), GPHN (cyan), and VGAT (white); 5 consecutive
70 nm sections (3 × 3 μm). (f) Representative examples of
whole-cell voltage clamp recordings of IPSCs and EPSCs from
Dlxi1/2b::eGFP+ cells in unfused hSS, in fused
hCS-hSS, or after migration in hCS (g) Representative examples of whole-cell
voltage clamp recordings of IPSCs and EPSCs in cells recorded from unfused
hCS cells and fused hCS cells. (h) Electrical stimulation and patch clamp
recording in fused hSS-hCS showing evoked EPSCs and IPSCs before (black) and
after exposure to 10 μM gabazine (red). (i) Average peri-stimulus
synaptic events (IPSCs and EPSCs) in Dlxi1/2::eGFP+ cells
recorded in the hCS side of fused hSS-hCS before and after electrical
stimulation (paired t-test, *P< 0.05). Data are
mean ± s.e.m.
(a) Generation of hCS and hSS. (b) Fold changes
(relative to gene expression in hPSC; normalized to GAPDH) of
NKX2–1 (n= 6 hPSC lines; Mann-Whitney test,
P= 0.002), FOXG1 (n= 5 hPSC lines; t-test,
P= 0.35) and EMX1 (n= 4 hPSC lines;
Mann-Whitney test, P= 0.02) in hCS and hSS at day 25. (c,
d) Immunostaining of hSS for NKX2–1, (e,
f) GABA, GAD67 and MAP2, and (g, h)
SST, CR, CB, PV. (i, j) Single cell profiling of hCS and hSS.
(k) AT volume in hSS for MAP2, GFAP, SYN1 and VGAT.
(l) Patch clamping in sliced hSS and a representative trace of
whole-cell current-clamp recording. (m, n) Spontaneous IPSCs before
(black) and during (blue) application of gabazine in an hSS slice (paired
t-test, **P= 0.004).
(a) Assembly of hCS and hSS. (b) Morphology before and
after assembly. (c) Time-lapse of migration from hSS into hCS.
(d) Assembly of hCS (AAV-hSYN1::mCherry) and hSS
(Lenti-Dlxi1/2b::eGFP). (e) iDISCO-cleared hSS-hCS. (f,
g) Saltatory migration of Dlxi1/2b::eGFP+ cells in
fused hSS-hCS and (h) nucleokinesis. (I, j) Saltatory
migration of Dlxi1/2b::eGFP+ cells in human fetal forebrain
and (k) nucleokinesis.
(a) TS mutation in Cav1.2. (b) Calcium
imaging in dissociated hSS (Ctrl: n= 38 cells from 2 subjects; TS:
n= 68 cells from 2 subjects). (c) Migration of
Dlxi1/2b::eGFP+ cells in TS and control hSS-hCS.
(d, e) Number of saltations (Ctrl: n= 48 cells from 3
hiPSC lines derived from 3 subjects; TS: n= 51 cells from 3 hiPSC lines
derived from 3 subjects; TS-Ctrl hybrid: n= 24 cells from 5 hiPSC line
combinations from 2 TS and 2 Ctrl subjects), and saltation length (Ctrl:
n= 21 cells from 3 hiPSC lines derived from 3 subjects; TS: n=
29 cells from 3 hiPSC derived from 3 subjects; TS-Ctrl hybrid: n= 12
cells from 3 hiPSC line combinations from 2 TS and 3 Ctrl subjects); one-way
ANOVA with Dunnett’s multiple comparison test
(***, P< 0.001). (f) Migration of
TS and control Dlxi1/2b::eGFP+ cells in fused hSS-hCS
(two-way ANOVA, interaction F(24, 408)= 17.71, P< 0.0001).
(g) Saltation length following exposure to nimodipine (paired
t-test; Ctrl: n= 13 cells from 3 hiPSC lines derived from 3 subjects,
***P< 0.001; TS: n= 12 cells from 2
hiPSC lines derived from 2 subjects, ***P<
0.001).
(a) Isolation of Dlxi1/2b::eGFP+ cells for
transcriptional analysis. (b) t-SNE visualization of single cell
gene expression at day 121 (4 weeks after hSS-hCS assembly). (c)
Distribution across clusters (χ2-test,
χ2= 43.39, P< 0.0001). (d)
Expression of ERBB4, NXPH1, IGF1,
TCF4, FOS, RAD1. (e)
Morphology of Dlxi1/2b::eGFP+ cells before and after
migration into hCS (f) Action potential generation in
Dlxi1/2b::eGFP+ cells (one-way ANOVA, F(2, 30)=
1.25; ***P< 0.001; Bonferroni post-hoc,
**P< 0.01; ***P< 0.001).
(g) GABAergic synapse (by AT) on the pallial side of hCS-hSS
with SYN1, GPHN and VGAT. (h) EPSCs and IPSCs in
Dlxi1/2b::eGFP+ cells after migration. (i)
Synaptic responses in Dlxi1/2b::eGFP+ cells (two-way ANOVA,
interaction F(2, 61)= 18.46, P< 0.0001; Bonferroni post-hoc for
EPSCs, ***P< 0.0001,
**P< 0.001). (j) Synaptic responses in
excitatory cells (two-way ANOVA, cortical neurons in hCS before and after
assembly F(1, 26)= 5.6, P< 0.05; Bonferroni post-hoc for IPSC,
*P< 0.05).
Comment in
-
Human development: Advances in mini-brain technology.Nature. 2017 May 3;545(7652):39-40. doi: 10.1038/545039a. Nature. 2017. PMID: 28470206 No abstract available.
-
Modeling human brain development.Sci Transl Med. 2017 Jun 7;9(393):eaan4295. doi: 10.1126/scitranslmed.aan4295. Sci Transl Med. 2017. PMID: 28592564
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You Have Brains in Your Head, You Have Organoids in Your Dish, You Can Steer Yourself in any Direction You Wish.Epilepsy Curr. 2017 Sep-Oct;17(5):311-313. doi: 10.5698/1535-7597.17.5.311. Epilepsy Curr. 2017. PMID: 29225549 Free PMC article. No abstract available.
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