Development 143: doi:10.1242/dev.132209: Supplementary information
A
E
ASGR1 (Alexa 594)
2nd ab (Alexa 488)
91.1%
FSC-A
0.1%
83.7%
0.1%
39.1%
2nd ab only
Albumin (Alexa 488)
Isotype (Alexa 594)
ASGR1 (Alexa 594)
2nd ab only
Albumin (Alexa 488)
Isotype (Alexa 594)
ASGR1 (Alexa 594)
21.82%
0.12%
2.38%
21.82%
0.12%
Alexa 594
2.38%
Unstained
Alexa
488
2nd ab only
AlbuminAlexa
(Alexa
647)
594
Unstained
2nd ab only
Albumin (Alexa 647)
Isotype (Alexa 594)
0.3%
53.85%
Isotype (Alexa0.3%
594)
ASGR1 (Alexa
594)
53.85%
Isotype (Alexa 594)
ASGR1 (Alexa 594)
ASGR1 (Alexa 594)
0.25%
0.0%
17.2%
99.6%
0.12%
82.8%
2nd ab (Alexa 488)
0.0% 0.1%
Albumin (Alexa 488)
0.0%
0.0%
0.4%
0.05%
98.9%
1.04%
48.6%
51%
70
Percent ALB+ cells
SSC
54.0%
G
Alexa 647
SSC
26.9%
Alexa 488
Alexa 647
2nd ab only
60.7%
F
60
2nd ab only
0.0%
16.6%
Alexa 594
0.25%
0.25%
Alexa 488
0.0%
HNF4A (Alexa 488)
ASGR1 (Alexa 594)
0.1% 2.5%
Alexa 488
SSCSSC
SSC
C
0.1%
FSC-A
SSCSSC
B
HNF4A (Alexa 488)
2nd ab (Alexa 594)
16.1%
Alexa 594
90.5%
FSC-W
SSC
Single cells
Albumin
50
40
30
20
SSC
10
0.27%
0.4%
18.39%
0.4%
18.39%
0
Alexa 594
0.27%
HUES1
(14.1)
HUES9
(37.6)
FHS-1
(50.5)
1016
(55.3)
Alexa 594
H
Mean percentage ALB+
*
50
70
ASGR1 (cell surface)
40
60
30
ALB
20
ASGR1
*
10
0
IMH
MH
Percent ASGR1+ cells
Percent positive cells
D
50
40
30
20
10
0
HUES9
(11.6)
1016
(15.1)
FHS-1
(31.4)
Mean percentage ASGR1+
Figure S1. Flow cytometry analysis of HLC differentiation, related to Figure 1. (A)
Gating used for flow cytometry analyses; single cells were defined by FSC-A/SSC (cell size
and granularity) and FSC-W (cell width) to exclude debris, cell clumps, and doublets. (B)
Albumin and ASGR1 expression after the IMH differentiation stage with corresponding
staining controls. Alexa 488 and Alexa 594 conjugated secondary antibodies were used with
albumin and ASGR1 primary antibodies respectively; gating for albumin and ASGR1 positive
cells was performed using secondary-only and isotype-control staining conditions
Development • Supplementary information
HUES1
(2.7)
Development 143: doi:10.1242/dev.132209: Supplementary information
respectively. (C) Albumin and ASGR1 expression after the MH differentiation stage with
corresponding staining controls. Alexa 647 and Alexa 594 conjugated secondary antibodies
were used with albumin and ASGR1 primary antibodies respectively; gating for albumin and
ASGR1 positive cells was performed using secondary-only and isotype-control staining
conditions respectively. A secondary-only staining control is also shown for ASGR1. (D)
Kinetics of albumin and ASGR1 expression during the final stages of HLC differentiation as
determined by intracellular flow cytometry. Shown are mean percent positive cells at the IMH
and MH stages based on multiple independent differentiations (n = 5 - 15 replicates per
marker, per stage). Error bars represent s.e.m. Asterisks indicate statistically significant
differences in mean percentage of positive cells at the IMH and MH stages for each marker
by Student’s t-test. *, P < 0.05. (E) Flow cytometry analysis of ASGR1 and HNF4A coexpression with staining controls. Alexa 594 and Alexa 488 conjugated secondary antibodies
were used for ASGR1 and HNF4A staining respectively. Gating was performed based on
secondary-only (fluorescence minus one) staining conditions. (F) Flow cytometry analysis of
ASGR1 and albumin co-expression with staining controls. Alexa 594 and Alexa 488
conjugated secondary antibodies were used for ASGR1 and albumin staining respectively.
Gating for ASGR1 and albumin positive cells was performed based on isotype-control and
secondary-only staining conditions respectively. (G-H) Efficiency of HLC differentiation with
four different hPSC lines over multiple independent experiments. Shown are percent
albumin-positive (G) and surface ASGR1-positive (H) cells determined by flow cytometry
Development • Supplementary information
analysis after the MH differentiation stage, illustrating a range of differentiation efficiencies.
Development 143: doi:10.1242/dev.132209: Supplementary information
Figure S2. Detailed results of ASGR1 FACS and hepatocyte marker gene expression
analysis, related to Figure 2. (A) Strategy and staining controls for FACS isolation of
ASGR1+ HLCs. Shown are results of a representative differentiation and FACS experiment.
expressing the hepatocyte marker alpha-1 antitrypsin (AAT) among unsorted HLCs, surface
ASGR1-negative cells, and surface ASGR1-positive cells was quantified by intracellular flow
cytometry. Right: mean percent AAT-positive cells by flow cytometry, among unsorted HLCs,
surface ASGR1-negative cells, and surface ASGR1-positive cells (n = 2 differentiations).
Error bars represent s.e.m. (C) ASGR1 FACS and hepatocyte marker gene expression
analysis by qRT-PCR. Gene expression data is displayed as a heatmap in Figure 2C. Three
different hPSC lines were differentiated to HLCs, with two independent differentiations
performed per cell line (differentiations performed in triplicate or greater, n = 6 – 8 biological
replicates per cell line). Data are arranged according to mean percent ASGR1+ cells obtained
in each differentiation. Green bars: percentage surface ASGR1+ cells after the MH
Development • Supplementary information
(B) Left: two different hPSC lines were differentiated to HLCs. The percentage of cells
Development 143: doi:10.1242/dev.132209: Supplementary information
differentiation stage. All other graphs show qRT-PCR gene expression analysis in unsorted
HLCs (“HLC,” red bars) and ASGR1+ cells (“ASGR1+,” blue bars) isolated by FACS.
Expression levels are relative to RPLP0 expression; gene expression levels in ASGR1+ cells
were normalized to level in unsorted HLCs. Shown are normalized mean expression levels for
each differentiation (n = 3 – 5 paired biological replicates per differentiation). Error bars
Development • Supplementary information
represent s.e.m.
Figure S3. Additional analyses of microarray gene expression profiling data from
ASGR1-positive cells, unsorted HLCs, primary human hepatocytes, and HepG2
hepatoma cells, related to figure 3. (A) Hierarchical clustering of ASGR1-positive, HLC, and
PHH samples based on all genes measured by transcriptional microarray and expressed
above background. (B) Heatmap showing pairwise Pearson correlation values for ASGR1-
Development • Supplementary information
Development 143: doi:10.1242/dev.132209: Supplementary information
Development 143: doi:10.1242/dev.132209: Supplementary information
positive, HLC, and PHH samples based on the same expression data used in part A. Yellow,
orange, and red color denotes lower, intermediate, and higher correlation respectively. (C)
Heatmap of hierarchical clustering performed on all genes differentially expressed between
ASGR1-positive cells and HLCs at a 5% FDR. Blue, below average expression, red, above
average expression. 813 probesets differentially expressed between ASGR1+ and unsorted
HLC at 5% FDR; 318 upregulated and 495 downregulated in ASGR1+ vs. unsorted HLCs
respectively. (D) Functional enrichment analysis of genes differentially expressed in ASGR1positive cells relative to HLCs. (E) Gene set enrichment analysis (GSEA). Microarray gene
expression data was arranged based on greater average expression in ASGR1-positive cells
(red color) or unsorted HLCs (blue color). Vertical black bars represent genes within the liver-
Development • Supplementary information
enriched gene set.
Development 143: doi:10.1242/dev.132209: Supplementary information
AFP
AFP
Normalized gene
expression
1.2
1.2
1
1
0.8
0.8
0.6
0.6
0.4
0.4
0.2
0.2
0
0
24
48
72
Normalized gene
expression
24
ALB
1.5
48
72
96
SIRPINA1
SIRPINA1
1.2
1
1
0.8
0.6
0.5
0.4
0.2
0
24
48
72
TF
0
96
24
Hours after re-plating
TF
1.2
Normalized gene
expression
96
Hours after re-plating
ALB
48
72
96
APOA1
APOA1
1.5
1
0.8
1
0.6
0.4
0.5
0.2
0
24
48
72
0
96
24
Hours after re-plating
Replated HLCs
(iPSC) HLCs
Re-plated
(iPSC)
B
1.4
1.2
1.4
Albumin secretion
Normalized albumin
secretion
FGA
FGA
1
0.8
0.6
0.4
0.2
0
0
48
72
96
Replated HLCs
(hESC) HLCs
Re-plated
(hESC)
1.2
1
0.8
0.6
0.4
0.2
0
24
48
72
96
0
24
48
72
Hours after re-plating Hours after re-plating Hours after re-plating
96
Figure S4. Hepatocyte marker gene expression and albumin secretion declines as
anticipated following re-plating of ASGR1 MACS-enriched HLCs. (A) Changes in
hepatocyte marker gene expression over time were determined by qRT-PCR after re-plating
HLCs differentiated from a representative hESC line. Gene expression levels were calculated
relative to RPLP0 expression and normalized to gene expression level at 24 hours post replating for each gene (n = 3 differentiation wells per time point). Error bars represent s.e.m. (B)
Development • Supplementary information
A
Development 143: doi:10.1242/dev.132209: Supplementary information
Quantification of albumin secretion by ELISA following re-plating of HLCs differentiated from
two hPSC lines. Albumin concentration at the indicated time points were calculated using a
standard curve and normalized to the level at 24 hours post re-plating for each cell line (n = 3
differentiation wells per cell line, per time point). Medium was collected after 24 hours in culture
Development • Supplementary information
for each time point. Error bars represent s.e.m.
Development 143: doi:10.1242/dev.132209: Supplementary information
Table S1. Distribution of liver-enriched genes in gene set enrichment analysis (GSEA)
comparing ASGR1+ cells and unsorted HLCs, related to Figure S3E.
Table S1. Distribution of liver-enriched genes in gene set enrichment analysis (GSEA) comparing ASGR1+ cells and unsorted HLCs
AADAC
ABCG8
ACAA1
ACAA2
ACADSB
ACAT1
ACMSD
ACOX1
ACOX2
ACSL1
ACY1
ADH1A
ADH6
ADK
AFF4
AFM
AGTR1
AGXT
AGXT2
AHSG
AIG1
AKR1A1
AKR1D1
ALAS1
ALB
ALDH6A1
ALDH8A1
ALDOB
AMBP
ANG
ANGPTL3
APOA1
APOA2
APOB
APOC2
APOC3
APOE
APOH
APOM
ARG1
ASGR1
ASGR2
ASS1
ATF5
AZGP1
BAAT
BPHL
BRP44
C1S
C5
C6
CD302
CDO1
CFH
CFI
CIDEB
CLDN1
CPB2
CPN1
CPN2
CPS1
CREB3L3
CRYL1
CYB5A
CYP3A7
CYP4A11
CYP4V2
CYP8B1
DCXR
DDT
DECR1
EPHX1
ETFB
F10
F13B
F2
F5
F7
F9
FAM96A
FBP1
FETUB
FGA
FGB
FGFR4
FGG
FGL1
FMO5
G6PC
GATM
GC
GCHFR
GGH
GSTO1
HABP2
HMGCL
HMGCS2
HNF4A
HP
HPD
HPN
HPX
HRSP12
HSD17B4
HSD3B7
HSPE1
ID2
IGFBP1
IL1RAP
INHBE
INSIG1
ITIH1
ITIH2
KHK
KLB
KLKB1
KNG1
LASS2
LBP
LCAT
LEPR
LIPC
MAT1A
METTL7B
MTHFS
MTTP
MUT
NAT8
NEK6
NIPSNAP1
NIT2
OIT3
ORM1
OTC
PAH
PCBD1
PCCB
PCK2
PEBP1
PECR
PEMT
PGRMC1
PHYH
PIPOX
PKLR
PLA2G12B
PLG
PNPLA3
PRAP1
PRDX4
PROC
PROS1
PROX1
PROZ
PXMP2
RBP4
RCL1
SAA4
SC5DL
SCP2
SEPHS2
SERPINA10
SERPINA4
SERPINA6
SERPINA7
SERPINC1
SERPIND1
SERPINF2
SERPING1
SHMT1
SHMT2
SLC10A1
SLC13A5
SLC22A7
SLC22A9
SLC25A13
SLC27A2
SLC2A2
SLC30A1
SLC35D1
SLC38A3
SLC38A4
SLC39A14
SLC41A2
SLC43A1
SLCO2B1
SORD
SPRYD4
ST6GAL1
SULT2A1
TAT
TDO2
TFR2
TM4SF4
TM4SF5
TMEM176A
TMEM176B
TMEM56
TMPRSS6
TP53INP1
TTR
UGT2B4
UGT3A1
VTN
ZNHIT1
Liver-enriched genes with lower expression
in ASGR1+ cells.
91 of 437 genes had lower expression in
ASGR1+ cells vs. unsorted HLCs
ABCB4
ABCG5
ACAT2
ACOT12
ANXA10
APOC4
AQP9
AS3MT
ATF7IP2
BCO2
C4BPA
C4BPB
CCL16
CD5L
CDC37L1
CES1
CES2
CFP
CLEC4M
COLEC11
CP
CRP
CTH
CYP1A2
CYP26A1
CYP2A6
CYP2B7P1
CYP2C8
CYP2D6
CYP2E1
CYP39A1
CYP4A22
DEFB123
DEPDC7
DHRS1
DMGDH
ERRFI1
ETFDH
FAM167B
FMO3
FMO4
GCGR
GHR
GLS2
GLT1D1
GNE
GNPNAT1
GPR126
GRHPR
GSTZ1
GYS2
HAGH
HAMP
HLF
HPS3
HSD11B1
IBTK
IGFALS
ITIH4
KMO
LARP4
LECT2
LPIN2
MAMDC4
MASP1
MASP2
MBL2
MCL1
MTHFD1
MYO1B
N4BP2L1
PHLDA1
PLGLB2
PON1
PON3
PPAPDC2
PZP
RTP3
SCUBE3
SDS
SEC14L2
SEC14L4
SLCO1B1
SOD1
SPP2
STEAP3
THPO
TNFSF14
TUBB1
UROC1
ZNF281
Development • Supplementary information
Subset of liver-enriched genes with greater expression in ASGR1+ cells.
205 genes contributed most strongly to the enrichement result (core enrichment).
346 of 437 genes had greater expression in ASGR1+ cells vs. unsorted HLCs.