Human pluripotent stem cell-derived hepatocytes with ... · extended culture time provide a robust...
1
0 200 400 600 800 1,000 1,200 d4 d8 d12 d19 d4 d8 d12 d19 d4 d8 d12 d19 24 hr C12 C18 C22 hphep pmol 7-OH-coumarin glucuronide / mg protein / min UGT 0 50 100 150 200 250 d4 d8 d12 d19 d4 d8 d12 d19 d4 d8 d12 d19 24 hr C12 C18 C22 hphep pmol 7-OH-coumarin sulfate / mg protein / min SULT 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 4 6 12 20 Relative mRNA expression Days C12 C18 C22 Human pluripotent stem cell-derived hepatocytes with extended culture time provide a robust and efficient platform for long term toxicology studies Barbara Küppers-Munther 1 , Annika Asplund 1 , Josefina Edsbagge 1 , Liz Quinn 2 , Catharina Ellerström 1 1 Takara Bio Europe AB, Göteborg, Sweden 2 Takara Bio USA, Inc., Mountain View, CA, USA; Corresponding author: [email protected] Human pluripotent stem (hPS) cell-derived hepatocytes have the potential to serve as predictive human in vitro model systems for drug discovery, drug metabolism research, and hepatotoxicity studies, provided they possess relevant hepatocyte functions. Importantly, some hepatocyte applications, like chronic toxicity testing, demand a 2-week usage window, an order of magnitude outside standard culturing practices. Here, we show data for a newly developed maintenance medium allowing culturing of the hPS cell-derived functional hepatocytes for 14 days, and thus enables their use for new applications with longer culture times. We have performed multiple analyses, including RT-qPCR, immunostainings, and functional assays, to investigate if our hepatocyte differentiation and maintenance system will 1) generate mature hepatocytes from multiple hPS cell lines, and then 2) to support their functionality during an extended culture time. Importantly, the hPSC- derived hepatocytes expressed important genes of the drug metabolizing machinery, such as CYPs, phase II enzymes and transporters during the entire culture time. Interestingly, the hepatocytes generated from different hPS cell lines show diverse CYP expression profiles, reflecting the inter-individual variation present in the human population. Next, we exposed these novel hPS cell-derived hepatocytes to known hepatotoxins for up to 14 days and found they respond correctly to these toxic compounds with an increasing sensitivity upon longer exposure, demonstrating their utility for chronic toxicology studies. Taken together, our hepatocyte differentiation system along with the new maintenance medium can reliably generate and maintain hPS cell-derived hepatocytes from different genetic backgrounds. Since we observed that our new maintenance medium substantially extended the life-span of hPS cell-derived hepatocytes, we tested if it also could extend the life- span of human primary hepatocytes. Interestingly, we found that cryopreserved human primary hepatocytes cultured in the new maintenance medium were viable and showed stable activities of several key CYP enzymes for several weeks in conventional 2D cultures, sharply contrasting existing hepatocyte maintenance medias. Thus, it seems that our novel maintenance medium prevents the de-differentiation of primary hepatocytes that usually occurs as an adaptation to 2D culture systems. The novel maintenance medium enables the use of human primary hepatocytes in conventional 2D cultures for applications requiring longer culture times without the need to culture in complicated and less user-friendly 3D systems. Takara Bio USA, Inc. United States/Canada: +1.800.662.2566 • Asia Pacific: +1.650.919.7300 • Europe: +33.(0)1.3904.6880 • Japan: +81.(0)77.565.6999 FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES. © 2017 Takara Bio Inc. All Rights Reserved. All trademarks are the property of Takara Bio Inc. or its affiliate(s) in the U.S. and/or other countries or their respective owners. Certain trademarks may not be registered in all jurisdictions. Additional product, intellectual property, and restricted use information is available at takarabio.com Scan to download your copy of this poster or visit takarabio.com/SoT2017 800.662.2566 Visit us at www.takarabio.com Conclusions Abstract 2 Enhanced hiPS-HEP cells show functional characteristics of mature hepatocytes 4 Up to 20 days in culture, activity of key CYP450 enzymes in enhanced hiPS-HEP cells is stable 1 Enhanced hiPS-HEP cells display hepatic markers over an extended culture time B 3 Up to 20 days in culture, expression of key CYP450 enzymes in enhanced hiPS-HEP cells is comparable to that of human primary hepatocytes 5 Enhanced hiPS-HEP cells show the same response as primary hepatocytes upon chronic toxicity exposure B Figure 1. Differentiation process and staining patterns of enhanced hiPS-HEP cells. Panel A. Enhanced hiPS-HEP cells were derived from the hiPS cell lines ChiPSC12, ChiPSC18, and ChiPSC22 (abbreviated as C12, C18, and C22) using a differentiation process that mimics embryonic development, then cryopreserved. These cryopreserved cells, hereafter referred to as enhanced hiPS-HEP cells, were thawed, cultured in enhanced maintenance medium, and assayed for hepatic markers. This is the second generation of this protocol. Panel B. On Day 12 post-thawing, enhanced hiPS-HEP cells derived from C18 were stained for markers of adult hepatocyte cell fate: hepatocyte nuclear factor 4α (HNF4α), a transcription factor that regulates hepatic genes, as well as the hepatocyte-specific genes α1-antitrypsin (α1AT) and cytokeratin 18 (CK18). Enhanced hiPS-HEP cells derived from the other lines (data not shown) display similar morphological features and staining patterns. Figure 2. Functional characteristics of enhanced hiPS-HEP cells cultured in enhanced maintenance medium. The liver performs over 500 functions, many of which are not present in hepatic cell culture models. Here, we demonstrate that enhanced hiPS-HEP cells have several important functional characteristics of hepatic cells. Panel A. Representative images of enhanced hiPS-HEP cells from C18 12 days after thawing (right) and cryopreserved human primary hepatocytes (hphep) 24 hr after thawing (left). Cells were stained for albumin and DAPI. Panel B. Expression levels of albumin in enhanced hiPS-HEP cells derived from three different hiPSC lines, C12, C18, and C22, between Days 4 and 20 post-thawing. Data is presented as mean values ± SEM (n=2 different batches per hiPSC line). Expression levels were normalized to a calibrator and a reference gene. Panel C. Albumin secretion as measured by ELISA; n=2 for enhanced hiPS-HEP cells, with the exception of C18 at 20 d (n=1), and n=3 donors for hphep. Panel D. Urea secretion as measured by LC/MS; n=2 for enhanced hiPS-HEP cells and n=3 donors for hphep; error bars show SEM. Panel E. Representative images of enhanced hiPS-HEP cells 12 days after thawing and hphep cultured for 24 hr and stained with a periodic acid-Schiff (PAS) stain to detect glycogen. Panel F. mRNA expression of albumin (Alb) and gluconeogenesis genes phosphoenolpyruvate carboxykinase 1 (PCK1) and glucose-6-phosphatase (G6P) in enhanced hiPS-HEP cells after 20 days in culture as compared to hphep (dashed line) after 24 hr in culture. 6 Enhanced hiPS-HEP cells demonstrate effective activity and expression of mature drug-metabolizing machinery over an extended culture time A C D F Metabolize more compounds Metabolize fewer compounds CYP Drug Substrate Metabolite CYP3A4/5 Midazolam α-OH-midazolam CYP2D6 Bufuralol 1′-OH-bufuralol CYP2B6 Bupropion (2S,3R)-OH bupropion CYP2C9 Diclofenac 4′-OH-diclofenac CYP1A Phenacetin Paracetamol CYP2C19 Mephenytoin 4′-OH-mephenytoin Enhanced hiPS-HEP cells from C18 hphep A B A A B C D Albumin DAPI Enhanced hiPS-HEP cells from C18 Donor 1 Donor 2 7 Human primary hepatocytes maintained in enhanced medium retain CYP450 activity for 4 weeks in conventional 2D cultures Albumin DAPI hphep A B C C D A B A 0 5 10 15 4 6 12 20 Relative expression Days CYP3A4 mRNA C12 C18 C22 0 200 400 600 800 4 6 12 20 Relative expression Days CYP3A5 mRNA C12 C18 C22 0 10 20 30 40 4 6 12 20 Relative expression Days CYP3A7 mRNA C12 C18 C22 0 5 10 15 4 6 12 20 Relative expression Days CYP2D6 mRNA C12 C18 C22 0.0 0.5 1.0 1.5 2.0 4 6 12 20 Relative expression Days CYP2B6 mRNA C12 C18 C22 0 2 4 6 8 10 4 6 12 20 Relative expression Days CYP2C9 mRNA C12 C18 C22 0.0 0.1 0.2 0.3 0.4 0.5 0.6 4 6 12 20 Relative expression Days CYP1A2 mRNA C12 C18 C22 0 100 200 300 400 500 4 6 12 20 Relative expression Days CYP2C19 mRNA C12 C18 C22 0.01 0.10 1.00 10.00 100.00 1,000.00 CYP3A4 CYP3A5 CYP3A7 CYP2D6 CYP2B6 CYP2C9 CYP1A2 CYP2C19 mRNA expression at 20 days relative to hpep C12 C18 C22 0.01 0.10 1.00 10.00 100.00 1,000.00 Alb PCK1 G6P Albumin and Gluconeogenesis C12 C18 C22 0.01 0.10 1.00 10.00 100.00 1,000.00 UGT1A1 UGT2B7 mRNA expression at 20 days relative to hpep C12 C18 C22 0.01 0.10 1.00 10.00 100.00 1,000.00 NTCP BSEP MRP2 MDR1 mRNA expression at 20 days relative to hpep C12 C18 C22 Figure 4. CYP450 activity is stable in enhanced hiPS-HEP cells over a 21-day time window. LC/MS was used to analyze CYP450 activity in cultured enhanced hiPS-HEP cells previously derived from the hiPS cell lines ChiPSC12, ChiPSC18, and ChiPSC22 (abbreviated as C12, C18, and C22). CYP3A (Panel A), CYP2C9 (Panel B), CYP1A (Panel C), and CYP2C19 (Panel D) activities in enhanced hiPS-HEP cells are stable over an extended culture time. Cryopreserved human primary hepatocytes (hphep), which are functional in culture for a significantly shorter time than enhanced hiPS-HEP cells, were thawed and cultured for 20 hours, and data are shown as the black bar in each panel. CYP2D6 and CYP2B6 activity was also detected at low levels (data not shown). Figure 6. Enhanced hiPS-HEP cells cultured in enhanced maintenance medium have functional drug-metabolizing machinery. Phase II enzymes and transporters continue the drug-metabolizing process. The modifications made by phase II enzymes like sulfotransferases (SULT) and uridine diphosphate glucuronosyltransferase (UGT) increase the solubility of most compounds, generally leading to their renal excretion. Panel A. mRNA expression of phase II enzymes after 20 days in culture, relative to hphep after 24 hr (dashed line). Panel B. Expression of uptake genes (NTCP) and efflux genes (BSEP, MRP2, and MDR1). Expression of these genes in C12, C18, and C22 was measured after 20 days in culture and compared to hphep after 24 hr in culture (dashed line). Panel C. In sharp contrast to hphep, enhanced hiPS-HEP cells have high SULT activity over time, as evidenced by the accumulation of the SULT metabolic product 7-OH-coumarin sulphate as well as increased accumulation of the UGT metabolite 7-OH-coumarin glucuronide ( Panel D). These samples both represent data pooled from two wells compared to hphep from n=3 donor lines, and levels were measured by LC/MS. Figure 7. Use of new enhanced maintenance medium on hphep preserves CYP450 activity. Panel A. Representative phase contrast images showing morphology of human primary hepatocytes cultured for 28 days post-thawing in the enhanced maintenance medium (n=2 donors). Scale bar = 100 μm. Panel B. CYP1A, 3A, 2C9, 2B6, 2D6, and 2C19 activities in hphep cultured for 28 days post-thawing. Metabolites tested are the metabolites listed in Figure 3, Panel A and were measured by LC/MS; n=2 donors, mean values ± SEM. • Enhanced hiPS-HEP cells grown with modified enhanced medium: o Contain many mature hepatic features, including expression of mature hepatic markers and gluconeogenesis genes. They contain functional features like albumin and urea secretion, and they retain these abilities over an extended culture period. o Show expression and activity of CYP450 genes during an extended culture window o Show expression and activity of additional drug- metabolizing enzymes during an extended culture window • Our new enhanced medium can also maintain key CYP450 enzyme activities in cryopreserved human primary hepatocytes significantly longer than with conventional medium (28 days vs. 3 days). Figure 3. Enhanced hiPS-HEP cells and human primary hepatocytes show similar CYP450 expression. Cytochrome P450 (CYP450) enzymes are the first step in drug metabolism. CYP450 enzymes change compounds to an oxidized state. Later, other enzymes further metabolize the compounds. Taken together, the CYP450 genes represented here can metabolize over 99% of existing drugs. Panel A. Drug substrate and oxidation products of the CYP450 genes, listed in order of the fraction of compounds metabolized by each enzyme. Panel B. For all genes tested, the mRNA expression as quantified by qPCR is either stable or increasing over time. We show the expression levels of eight CYP enzymes in enhanced hiPS-HEP cells derived from three different hiPSC lines (C12, C18, and C22) between Days 4 and 20 post-thawing. Data is presented as mean values ± SEM (n=2 different batches per hiPSC line). Expression levels were normalized to a calibrator and a reference gene. Panel C. mRNA expression of the eight most common drug-metabolizing CYP450 genes in enhanced hiPS-HEP cells after 20 days of culturing was compared to that of cryopreserved human primary hepatocytes cultured for 24 hr. Expression levels of CYP3A4, 3A5, 3A7, 1A2, and 2C19 are similar to that of primary hepatocytes. Expression of hphep is shown as a dashed line, and mRNA levels are presented as values relative to hphep. E B Figure 5. Enhanced hiPS-HEP cells are a high-quality model system for chronic toxicity exposure. Panel A. EC 50 (concentration with 50% loss of viability) of four different compounds metabolized by a variety of CYP enzymes. Similar to hphep, enhanced hiPS- HEP cells show increasing sensitivity to compounds after prolonged exposure. Panel B. Representative plots of the dose-response curves of enhanced hiPS-HEP cells. Enhanced hiPS-HEP cells (from C18 and C22) were dosed with four compounds between Days 4 and 18 post-thawing (eight concentrations per compound) and cell viability was assessed after 2, 7, and 14 days of compound exposure using a CellTiterGlo assay. Supplemental data is available upon request. The 3D spheroid hphep experiments were performed at AstraZeneca and Karolinska Institute (within the Scr & Tox project). HNF4α DAPI CK18 DAPI α1AT DAPI mRNA expression relative to hpep
Transcript of Human pluripotent stem cell-derived hepatocytes with ... · extended culture time provide a robust...
0
200
400
600
800
1,000
1,200
d4 d8 d12 d19 d4 d8 d12 d19 d4 d8 d12 d19 24hr
C12 C18 C22 hphep
pm
ol 7
-OH
-co
um
arin
glu
curo
nid
e/ m
g p
rote
in /
min
UGT
0
50
100
150
200
250
d4 d8 d12 d19 d4 d8 d12 d19 d4 d8 d12 d19 24hr
C12 C18 C22 hphep
pm
ol 7
-OH
-co
um
arin
su
lfat
e / m
g
pro
tein
/ m
in
SULT
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
4 6 12 20
Rela
tive
mRN
A ex
pres
sion
Days
C12 C18 C22
Human pluripotent stem cell-derived hepatocytes with extended culture time provide a robust and efficient platform for long term toxicology studiesBarbara Küppers-Munther1, Annika Asplund1, Josefina Edsbagge1, Liz Quinn2, Catharina Ellerström1
1 Takara Bio Europe AB, Göteborg, Sweden2 Takara Bio USA, Inc., Mountain View, CA, USA; Corresponding author: [email protected]
Human pluripotent stem (hPS) cell-derived hepatocytes have the potential to serveas predictive human in vitro model systems for drug discovery, drug metabolismresearch, and hepatotoxicity studies, provided they possess relevant hepatocytefunctions. Importantly, some hepatocyte applications, like chronic toxicity testing,demand a 2-week usage window, an order of magnitude outside standardculturing practices.
Here, we show data for a newly developed maintenance medium allowingculturing of the hPS cell-derived functional hepatocytes for 14 days, and thusenables their use for new applications with longer culture times. We haveperformed multiple analyses, including RT-qPCR, immunostainings, and functionalassays, to investigate if our hepatocyte differentiation and maintenance systemwill 1) generate mature hepatocytes from multiple hPS cell lines, and then 2) tosupport their functionality during an extended culture time. Importantly, the hPSC-derived hepatocytes expressed important genes of the drug metabolizingmachinery, such as CYPs, phase II enzymes and transporters during the entireculture time. Interestingly, the hepatocytes generated from different hPS cell linesshow diverse CYP expression profiles, reflecting the inter-individual variationpresent in the human population.
Next, we exposed these novel hPS cell-derived hepatocytes to known hepatotoxinsfor up to 14 days and found they respond correctly to these toxic compounds withan increasing sensitivity upon longer exposure, demonstrating their utility forchronic toxicology studies. Taken together, our hepatocyte differentiation systemalong with the new maintenance medium can reliably generate and maintain hPScell-derived hepatocytes from different genetic backgrounds.
Since we observed that our new maintenance medium substantially extended thelife-span of hPS cell-derived hepatocytes, we tested if it also could extend the life-span of human primary hepatocytes. Interestingly, we found that cryopreservedhuman primary hepatocytes cultured in the new maintenance medium were viableand showed stable activities of several key CYP enzymes for several weeks inconventional 2D cultures, sharply contrasting existing hepatocyte maintenancemedias. Thus, it seems that our novel maintenance medium prevents thede-differentiation of primary hepatocytes that usually occurs as an adaptation to2D culture systems. The novel maintenance medium enables the use of humanprimary hepatocytes in conventional 2D cultures for applications requiring longerculture times without the need to culture in complicated and less user-friendly 3Dsystems.
Takara Bio USA, Inc.United States/Canada: +1.800.662.2566 • Asia Pacific: +1.650.919.7300 • Europe: +33.(0)1.3904.6880 • Japan: +81.(0)77.565.6999FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES. © 2017 Takara Bio Inc. All Rights Reserved. All trademarks are the property of Takara Bio Inc. or its affiliate(s) in the U.S. and/or other countries or their respective owners. Certain trademarks may not be registered in all jurisdictions. Additional product, intellectual property, and restricted use information is available at takarabio.com
Scan to download your copy of this poster or visit takarabio.com/SoT2017
800.662.2566Visit us at www.takarabio.com
Conclusions
Abstract 2 Enhanced hiPS-HEP cells show functional characteristics of mature hepatocytes
4 Up to 20 days in culture, activity of key CYP450 enzymes in enhanced hiPS-HEP cells is stable
1 Enhanced hiPS-HEP cells display hepatic markers over an extended culture time
B
3 Up to 20 days in culture, expression of key CYP450 enzymes in enhanced hiPS-HEP cells is comparable to that of human primary hepatocytes
5 Enhanced hiPS-HEP cells show the same response as primary hepatocytes upon chronic toxicity exposure
B
Figure 1. Differentiation process and stainingpatterns of enhanced hiPS-HEP cells. Panel A.Enhanced hiPS-HEP cells were derived from the hiPScell lines ChiPSC12, ChiPSC18, and ChiPSC22(abbreviated as C12, C18, and C22) using adifferentiation process that mimics embryonicdevelopment, then cryopreserved. These cryopreservedcells, hereafter referred to as enhanced hiPS-HEP cells,were thawed, cultured in enhanced maintenancemedium, and assayed for hepatic markers. This is thesecond generation of this protocol. Panel B. On Day 12post-thawing, enhanced hiPS-HEP cells derived fromC18 were stained for markers of adult hepatocyte cellfate: hepatocyte nuclear factor 4α (HNF4α), atranscription factor that regulates hepatic genes, as wellas the hepatocyte-specific genes α1-antitrypsin (α1AT)and cytokeratin 18 (CK18). Enhanced hiPS-HEP cellsderived from the other lines (data not shown) displaysimilar morphological features and staining patterns.
Figure 2. Functional characteristics of enhanced hiPS-HEP cells cultured in enhanced maintenancemedium. The liver performs over 500 functions, many of which are not present in hepatic cell culture models.Here, we demonstrate that enhanced hiPS-HEP cells have several important functional characteristics of hepaticcells. Panel A. Representative images of enhanced hiPS-HEP cells from C18 12 days after thawing (right) andcryopreserved human primary hepatocytes (hphep) 24 hr after thawing (left). Cells were stained for albumin andDAPI. Panel B. Expression levels of albumin in enhanced hiPS-HEP cells derived from three different hiPSC lines,C12, C18, and C22, between Days 4 and 20 post-thawing. Data is presented as mean values ± SEM (n=2 differentbatches per hiPSC line). Expression levels were normalized to a calibrator and a reference gene. Panel C.Albumin secretion as measured by ELISA; n=2 for enhanced hiPS-HEP cells, with the exception of C18 at 20 d(n=1), and n=3 donors for hphep. Panel D. Urea secretion as measured by LC/MS; n=2 for enhanced hiPS-HEPcells and n=3 donors for hphep; error bars show SEM. Panel E. Representative images of enhanced hiPS-HEPcells 12 days after thawing and hphep cultured for 24 hr and stained with a periodic acid-Schiff (PAS) stain todetect glycogen. Panel F. mRNA expression of albumin (Alb) and gluconeogenesis genes phosphoenolpyruvatecarboxykinase 1 (PCK1) and glucose-6-phosphatase (G6P) in enhanced hiPS-HEP cells after 20 days in cultureas compared to hphep (dashed line) after 24 hr in culture.
6 Enhanced hiPS-HEP cells demonstrate effective activity and expression of mature drug-metabolizing machinery over an extended culture time
A
C D
F
Metabolize more compounds
Metabolize fewer compounds
CYP Drug Substrate Metabolite
CYP3A4/5 Midazolam α-OH-midazolam
CYP2D6 Bufuralol 1′-OH-bufuralol
CYP2B6 Bupropion (2S,3R)-OH bupropion
CYP2C9 Diclofenac 4′-OH-diclofenac
CYP1A Phenacetin Paracetamol
CYP2C19 Mephenytoin 4′-OH-mephenytoin
Enhanced hiPS-HEP cells from C18
hphep
A B A
A B
C D
AlbuminDAPI
Enhanced hiPS-HEP cells from C18
Donor 1
Donor 2
7 Human primary hepatocytes maintained in enhanced medium retain CYP450 activity for 4 weeks in conventional 2D cultures
AlbuminDAPI
hphepA B
C
C D
A B
A
0
5
10
15
4 6 12 20Rela
tive
expr
essio
n
Days
CYP3A4 mRNAC12 C18 C22
0
200
400
600
800
4 6 12 20Rela
tive
expr
essio
n
Days
CYP3A5 mRNAC12 C18 C22
0
10
20
30
40
4 6 12 20Rela
tive
expr
essio
n
Days
CYP3A7 mRNAC12 C18 C22
0
5
10
15
4 6 12 20Rela
tive
expr
essio
n
Days
CYP2D6 mRNAC12 C18 C22
0.0
0.5
1.0
1.5
2.0
4 6 12 20Rela
tive
expr
essio
n
Days
CYP2B6 mRNAC12 C18 C22
02468
10
4 6 12 20Rela
tive
expr
essio
n
Days
CYP2C9 mRNAC12 C18 C22
0.00.10.20.30.40.50.6
4 6 12 20Rela
tive
expr
essio
n
Days
CYP1A2 mRNAC12 C18 C22
0
100
200
300
400
500
4 6 12 20Rela
tive
expr
essio
n
Days
CYP2C19 mRNAC12 C18 C22
0.01
0.10
1.00
10.00
100.00
1,000.00
CYP3A4 CYP3A5 CYP3A7 CYP2D6 CYP2B6 CYP2C9 CYP1A2 CYP2C19m
RN
A ex
pres
sion
at 2
0 da
ys r
elat
ive
to h
pep
C12 C18 C22
0.01
0.10
1.00
10.00
100.00
1,000.00
Alb PCK1 G6P
Albumin and Gluconeogenesis
C12 C18 C22
0.01
0.10
1.00
10.00
100.00
1,000.00
UGT1A1 UGT2B7mR
NA
exp
ress
ion
at
20 d
ays
re
lati
ve t
o h
pep
C12 C18 C22
0.01
0.10
1.00
10.00
100.00
1,000.00
NTCP BSEP MRP2 MDR1mR
NA
exp
ress
ion
at
20 d
ays
re
lati
ve t
o h
pep
C12 C18 C22
Figure 4. CYP450 activity is stable in enhanced hiPS-HEP cells over a 21-day time window. LC/MS was used to analyze CYP450 activity in cultured enhanced hiPS-HEP cells previously derived from the hiPS cell lines ChiPSC12, ChiPSC18, and ChiPSC22 (abbreviated as C12, C18, and C22). CYP3A (Panel A), CYP2C9 (Panel B), CYP1A (Panel C), and CYP2C19 (Panel D) activities in enhanced hiPS-HEP cells are stable over an extended culture time. Cryopreserved human primary hepatocytes (hphep), which are functional in culture for a significantly shorter time than enhanced hiPS-HEP cells, were thawed and cultured for 20 hours, and data are shown as the black bar in each panel. CYP2D6 and CYP2B6 activity was also detected at low levels (data not shown).
Figure 6. Enhanced hiPS-HEP cells cultured in enhanced maintenance medium have functional drug-metabolizingmachinery. Phase II enzymes and transporters continue the drug-metabolizing process. The modifications made by phase IIenzymes like sulfotransferases (SULT) and uridine diphosphate glucuronosyltransferase (UGT) increase the solubility of mostcompounds, generally leading to their renal excretion. Panel A. mRNA expression of phase II enzymes after 20 days inculture, relative to hphep after 24 hr (dashed line). Panel B. Expression of uptake genes (NTCP) and efflux genes (BSEP,MRP2, and MDR1). Expression of these genes in C12, C18, and C22 was measured after 20 days in culture and comparedto hphep after 24 hr in culture (dashed line). Panel C. In sharp contrast to hphep, enhanced hiPS-HEP cells have high SULTactivity over time, as evidenced by the accumulation of the SULT metabolic product 7-OH-coumarin sulphate as well asincreased accumulation of the UGT metabolite 7-OH-coumarin glucuronide (Panel D). These samples both represent datapooled from two wells compared to hphep from n=3 donor lines, and levels were measured by LC/MS.
Figure 7. Use of new enhanced maintenance medium on hpheppreserves CYP450 activity. Panel A. Representative phase contrastimages showing morphology of human primary hepatocytes cultured for28 days post-thawing in the enhanced maintenance medium (n=2donors). Scale bar = 100 μm. Panel B. CYP1A, 3A, 2C9, 2B6, 2D6,and 2C19 activities in hphep cultured for 28 days post-thawing.Metabolites tested are the metabolites listed in Figure 3, Panel A andwere measured by LC/MS; n=2 donors, mean values ± SEM.
• Enhanced hiPS-HEP cells grown with modified enhanced medium:
o Contain many mature hepatic features, including expression of mature hepatic markers and gluconeogenesis genes. They contain functional features like albumin and urea secretion, and they retain these abilities over an extended culture period.
o Show expression and activity of CYP450 genes during an extended culture window
o Show expression and activity of additional drug-metabolizing enzymes during an extended culture window
• Our new enhanced medium can also maintain key CYP450 enzyme activities in cryopreserved human primary hepatocytes significantly longer than with conventional medium (28 days vs. 3 days).
Figure 3. Enhanced hiPS-HEP cells and human primary hepatocytes show similar CYP450 expression. Cytochrome P450 (CYP450) enzymes are the first step in drugmetabolism. CYP450 enzymes change compounds to an oxidized state. Later, other enzymes further metabolize the compounds. Taken together, the CYP450 genes representedhere can metabolize over 99% of existing drugs. Panel A. Drug substrate and oxidation products of the CYP450 genes, listed in order of the fraction of compounds metabolized byeach enzyme. Panel B. For all genes tested, the mRNA expression as quantified by qPCR is either stable or increasing over time. We show the expression levels of eight CYPenzymes in enhanced hiPS-HEP cells derived from three different hiPSC lines (C12, C18, and C22) between Days 4 and 20 post-thawing. Data is presented as mean values
± SEM (n=2 different batches per hiPSC line). Expression levels were normalized to a calibrator and a reference gene. Panel C. mRNA expression of the eight most commondrug-metabolizing CYP450 genes in enhanced hiPS-HEP cells after 20 days of culturing was compared to that of cryopreserved human primary hepatocytes cultured for 24 hr.Expression levels of CYP3A4, 3A5, 3A7, 1A2, and 2C19 are similar to that of primary hepatocytes. Expression of hphep is shown as a dashed line, and mRNA levels arepresented as values relative to hphep.
E
B
Figure 5. Enhanced hiPS-HEP cells are a high-quality model system for chronictoxicity exposure. Panel A. EC50 (concentration with 50% loss of viability) of four differentcompounds metabolized by a variety of CYP enzymes. Similar to hphep, enhanced hiPS-HEP cells show increasing sensitivity to compounds after prolonged exposure. Panel B.Representative plots of the dose-response curves of enhanced hiPS-HEP cells. EnhancedhiPS-HEP cells (from C18 and C22) were dosed with four compounds between Days 4 and18 post-thawing (eight concentrations per compound) and cell viability was assessed after 2,7, and 14 days of compound exposure using a CellTiterGlo assay. Supplemental data isavailable upon request. The 3D spheroid hphep experiments were performed atAstraZeneca and Karolinska Institute (within the Scr & Tox project).
HNF4αDAPI
CK18DAPI
α1ATDAPI
mR
NA
expr
essi
on
rela
tive
to h
pep
