Science - USA (2022-01-28)

To test this, we subclustered all progenitors
(fig. S11, G and H) and found that 98% of fetal
cells coclustering with the expanded CGE pro-
genitors originated from gestational week 22
(GW22) (fig. S11, G, H, and I). We calculated
gene modules ( 33 ) in progenitors (fig. S11J)
andcomparedorganoidandfetalprogenitors
of various stages. Quiescent CGE progenitor
cells showed the highest correlation with fetal
progenitors from GW22 (+0.74) (fig. S11, K
and L). These data suggest that the expanded
CGE progenitors emerge around late mid-
gestation, a time when cortical tubers and
subependymal tumors are first detected in
TSC patients ( 20 ). On the basis of their CGE
origin and late emergence, we named the
expanded cell type caudal late interneuron
progenitor (CLIP) cells.

CLIP cells and their progeny are abundant

in tumors

To independently validate our observations in

the scRNA-seq experiments, we investigated

expression of markers for CGE, medial gangli-

onic eminence (MGE), and excitatory cells

through immunostaining of patient tumor

primary tissue and organoids.

We confirmed expression of EGFR in tumors

in organoids and in surgically resected SEGAs

(Fig.4Aandfig.S12).Consistentwiththe

scRNA-seq results, the CGE interneuron progen-

itor markers DLX2, COUP-TFII, and SP8 were

found in tumors in organoids from three TSC

patients(Fig.4,BandD,andfigs.S12andS13).

By contrast, NKX2.1, a marker for MGE progen-

itors or SATB2, specific for excitatory neurons,

wasnotexpressed(Fig.4Dandfig.S14,AandB).

To investigate the origin of TSC tumors in

patients, we stained 35GW SENs. Fetal tumors

such as their organoid counterparts were en-

riched in CGE cells, whereas NKX2.1-expressing

cells were rare (Fig. 4, E and F, and fig. S14, C

and D). By contrast, NKX2.1 has been shown

to be expressed in postnatal SEGAs ( 34 ). We

confirmed expression of NKX2.1 (fig. S14E) but

found that postnatal tumor cells also expressed

CGE markers (fig. S14, E to G). Thus, although

fetal SENs consisted mostly of CGE cells, co-

expression of NKX2.1 and CGE markers in

postnatal SEGAs could suggest aberrant dif-

ferentiation at later stages or the involvement

of other lineages.

To test which lineages can generate tumors

in TSC organoids, we used patterning proto-

cols to generate dorsal and ventral forebrain

Eichmülleret al.,Science 375 , eabf5546 (2022) 28 January 2022 5 of 10

1

2

4

9

8

7

13

14 15

17 16

18

19

20

12

11

10

6

5

3

Upper Layer EN

immature EN

Dividing CGE prog. 1

oRGs

Deep Layer EN

immature CGE IN

quiescent CGE prog.

CGE-IN

mature CGE IN 1

1 2 3 4 5 6 7 8 9

S-Phase CGE prog.

dividing oRGs

IPC

Dividing IPCs

Dividing CGE prog. 2

pre-OPCs

mature CGE IN 2

Cajal-Retzius

EN

10

11

12

13

14

15

16

17

18

unclear

unclear

19

20

19

23

(^1816)
6
(^97)
14
5
14
(^6445)
3
9
3
9
(^32332)
2223
12
1 2
55
0 1 0 1 2 002 0
L-M. d110
0
10
20
0
10
20
0
10
Percentage of Dataset^20
Clusters
L-M. Ctrl
L-M. TSC2
H-M. Ctrl
H-M. TSC2
d220
TSC Tumor
1 2 3 4 5 6 7 8 910111213141516
1 2 3 4 5 6 7 8 910111213141516
ABCluster distribution of datasets
CD E
GW 10
GW 6
GW 22
GW 14
GW 18
Organoid Cells
RNA Velocity Pseudotime CGE Lineage Fetal ages in co-clustering
2
Vim
HOPX
EDNRB
S100B
TNC
PTGDS
GFAP
EGFR
ASCL1
DLX1
DLX5
SCGN
DLX6-AS1
SP8
1
0
-1
-2
03471 2 56 89
d220
Tumor
H-M. d110
Fig. 3. Late CGE progenitors give rise to TSC phenotypes.(A) UMAP
projection of 220-day-old TSC tumor organoids with 110-day-old control and
TSC2+/−-derived organoids in H- and L-medium. All cell types of the dorsal
lineage were present, with radial glia (RG; clusters 3 and 10), intermediate
progenitors (IPCs; clusters 12 and 14), and excitatory neurons (EN; clusters 1,
2, 4, and 18). A separate lineage of CGE-derived cells was identified with quiescent
CGE progenitors (cluster 7), dividing CGE progenitor cells (clusters 5, 11, and 13),
and CGE interneurons (IN; clusters 6, 8, 9, 16, and 17). Pre-OPC-like cells (cluster 15)
cluster close to quiescent CGE progenitors. (B) Contribution of different datasets
to clusters shown in (A). TSC tumors 220 days old only contributed to CGE
progenitors and their progeny (clusters 5, 6, 7, 8, 9, 11, and 13). These clusters
were also increased in 110-day-oldTSC2+/−-derived organoids. H-medium enriched
for progenitors. L-medium organoids had more mature CGE-INs (clusters 9
and 16). (C) RNA velocity projected in two dimensions. The cluster annotation
corresponds to (A). From the increased CGE progenitor population, two
trajectories emerge: a small trajectory toward pre-OPC cells (cluster 15) and a
larger trajectory toward interneurons (clusters 6 and 9). (D) Expression of genes
along pseudotime in CGE lineage. Genes enriched along pseudotime were
calculated, and cells were binned into 10 groups (xaxis) (fig. S9B). All genes
with enriched expression along the trajectory were ordered by using a sliding
average. (Top left) Genes enriched in progenitors. (Bottom right) Genes in mature
interneurons. Selected genes are highlighted. (E)UMAPofintegrationof
organoid and fetal cells color-coded for gestational ages shows that GW22 cells
coclustered with (top right) quiescent CGE progenitor cluster.
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