introduc)on to hepa)c models€¦ · introduc)on to hepa)c models func%on, cell sources &...
TRANSCRIPT
![Page 1: Introduc)on to Hepa)c Models€¦ · Introduc)on to Hepa)c Models Func%on, Cell Sources & Physiological Models Prof. Yaakov Nahmias Phone: +972 (53) 9227232 Email: ynahmias@cs.huji.ac.il](https://reader031.vdocuments.us/reader031/viewer/2022022018/5b8626877f8b9a195a8c37b1/html5/thumbnails/1.jpg)
Introduc)ontoHepa)cModelsFunc%on,CellSources&PhysiologicalModels
Prof.YaakovNahmiasPhone:+972(53)9227232Email:[email protected]
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TableofContents
1. LiverStructureandFunc%on: Organiza%on,Zona%on,Composi%on
2. Func%onalMaintenance: 3Dcultures,CellInterac%ons,Oxygen
3. CellSource: HepG2/C3A,HepaRG,Upcyte,StemCells
4. OrganonChip: HuREL,TissueDynamics
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TheLiver
v Thelargestinternalorganinthehumanbody,about1.5kginadult
v Canregeneratefrom80%lossofmass
v Responsibleforthesystemicregula%onofglucose,aminoacids,andlipids
v Secretesbileacidsintothegallbladderandintes%ne
v Responsibleorganfordrugmetabolism
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v Blood flows from the portal triad to the central vein, while bile flows between hepatocytes radially out to the bile duct. v Sinusoid is a fenestrated capillary 250 µm long, about 10-11 cell lengths v Average blood flow rate 144 µm/sec, or 1.7 sec residence time
LiverLobuleStructure
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Oxygen and hormone gradients develop from the entrance to the exit of the hepatic sinusoid, leading to the development of metabolic zonation
Organiza)onandZona)onBaharHalpernetal.(2015)
Periportal PerivenusOxygen 60–70mmHg 25-35mmHgHormones High Low
CYP450 Low HighGlucose Gluconeogenesis GlycolysisNitrogen Ureagenesis GlutamineSyn.Lipids LipidOxida)on Lipogenesis
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LiverUltrastructure
v Hepatocytesareabout25μmindiameter,withabout1,500mitochondriaineachcellandnumerousglycogengranules
v Hepatocytesdisplaycomplexaneuploidyandpolyploidyinthenormalliver
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LiverUltrastructure
SpaceofDisse(1.2μm)separateshepatocytesfromendotheliumBilecanaliculi(1μm)aretheapicalsurfaceofhepatocytes
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Cell Type Diameter (µm)
Volume (% Total)
Number (% Total) Type Density
(gr/ml) Hepatocytes 20-25 77.8% 65% Epithelial 1.10-15 Sinusoidal Endothelium 7-11 2.8% 16% Endothelial 1.061-80 Kupffer Cells 10-13 2.1% 12% Macrophages 1.076 Stellate Cells 11-12 1.4% 8% Fibroblasts 1.05 Pit Cells Minor Minor Table 1. Cellular composition of the liver
CellularComposi)onoftheAdultLiver
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TableofContents
1. LiverStructureandFunc%on: Organiza%on,Zona%on,Composi%on
2. Func%onalMaintenance: 3Dcultures,CellInterac%ons,Oxygen
3. CellSource: HepG2/C3A,HepaRG,Upcyte,StemCells
4. OrganonChip: HuREL,TissueDynamics
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CulturingPrimaryHepatocytes
0
10
20
30
40
50
0 2 4 6 8 10 12 14
Alb
umin
(µg/
day)
Days in Culture
Suspension (6 hrs)
Hepatocytes alone Primaryhepatocyteslosemetabolicfunc%onwithinhoursacquirefibroblast-likemorphologyanddieinafewdays
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0
15
30
45
60
0 10 20 30 40 50
Alb
umin
(µg/
day)
Days in Culture
Hepatocyte alone
Collagen 'Sandwich'
Matrigel Spheroids
SpheroidsSandwich
3DLiverCulture
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Endothelial-dependentHepatocyteMigra)on
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Self-AssembledSinusoids
High-resolu%onphaseandfluorescentimagesofself-assembledliversinusoid-likestructures
Rathepatocytes(right)orhumanhepatocytes(bohom)
Albumin / CD31
CK18 / CD31
Nahmiasetal.TissueEngineering2006
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LiverOrganoids
Day2 Day5 Day10 Day28
Nahmias&Odde,NatureProtocols2006
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0
15
30
45
60
0 10 20 30 40 50
Alb
umin
(µg/
day)
Days in Culture
Hepatocyte alone
Collagen 'Sandwich'
Matrigel Spheroids
Organoids
SpheroidsSandwich LiverOrganoids
3DLiverCulture
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HeterotypicInterac)ons
Bioengineering Hebrew University
3T3-J2FibroblastsEndothelialCells
Kidambietal.PNAS.2009Khetanietal.Nat.Biotech2008
0
15
30
45
60
0 2 4 6 8 10 12 14
Alb
umin
(µg/
day)
Days in Culture
Suspension (6 hrs)
Hepatocytes alone
Hep - Endothelial
Hep - Fibroblasts
Micropaherns
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OxygenisaLimi)ngFactor?
0
1
2
3
4
10%Serum Serumfree
CYP4
50Ac)vity
10%Oxygen21%Oxygen60%Oxygen95%Oxygen
10
15
20
25
30
0% 0.5% 1% 5% 10% 20% 30%
Alb
umin
(µg/
day)
Serum Concentration
0
0.5
1
1.5
In Vivo 1 3 5 7
Oxy
gen
Upt
ake
Days in Culture
21%O2
95%O2
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HighOxygenCulture
3T3-J2FibroblastsEndothelialCells
0
30
60
90
120
0 1 2 3 4 5 6 7 8
Alb
umin
(µg/
day)
Days in Culture
Serum Free (95% O2) Serum Free (21% O2) Control
Suspension (6 hrs) Serum Free (95% O2)
Serum Free (21% O2) Control
OR
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DrugClearance
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0 10 20 30 40 50
Dru
g C
once
ntra
tion
(mM
)
Time (Hours)
Buspirone Metoprolol Sildenofil Timolol Antipyrine Carbamazepine
Drug PhaseI
Buspirone 2D6,3A4
Metoprolol 2D6
Timolol 2D6
Sildenafil 3A4,2C9
Carbamazepine 3A4
An%pyrine 1A2,2B62C9,3A4
Kidambietal.PNAS2009
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CYP3A4MetabolismandIVIVC
Buspirone
Metoprolol
Timolol
Sildenafil
Antipyrine
R² = 0.92
0
5
10
15
20
25
30
35
0 2 4 6 8
In V
ivo
Cle
aran
ce
In Vitro Clearance
Kidambietal.PNAS2009
Primaryhepatocytesculturedin95%oxygenunderserumfreecondi%onsshowclearanceratesequivalenttopa%ents
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TableofContents
1. LiverStructureandFunc%on: Organiza%on,Zona%on,Composi%on
2. Func%onalMaintenance: 3Dcultures,CellInterac%ons,Oxygen
3. CellSource: HepG2/C3A,HepaRG,Upcyte,StemCells
4. OrganonChip: HuREL,TissueDynamics
![Page 22: Introduc)on to Hepa)c Models€¦ · Introduc)on to Hepa)c Models Func%on, Cell Sources & Physiological Models Prof. Yaakov Nahmias Phone: +972 (53) 9227232 Email: ynahmias@cs.huji.ac.il](https://reader031.vdocuments.us/reader031/viewer/2022022018/5b8626877f8b9a195a8c37b1/html5/thumbnails/22.jpg)
PrimaryHumanHepatocytes
• Humanhepatocytesarescarceandfailtoproliferateinvitro• Cryopreservedhepatocytesshowmassivebatchtobatchvariability
• Pla%ngefficiencyofcryopreservedcellsokenfallsbelow70%• Cellsaredifficulttopassage
Hepatocytesoncollagengel HepatocytesonMatrigel™
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Aerobic Energetic
Glycolytic Quiescent 0
25
50
75
100
0 10 20 30 40 50
Mito
chon
dria
(OC
R)
Glycolysis (ECAR)
HepG2/C3ACells
• Clonalderiva%veofHepG2selectedforitscontactinhibitedgrowth
• Strongalbuminproduc%onandnitrogenclearance.Highlyglycoly%c
HepG2/C3Acells
ATCC®CRL-1074115yearoldCaucasianMale18-20hrdoubling%me
PHH
HepG2
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HepG2/C3ACells
• Clonalderiva%veofHepG2selectedforitscontactinhibitedgrowth
• Strongalbuminproduc%onandnitrogenclearance.Highlyglycoly%c
HepG2/C3Acells
ATCC®CRL-1074115yearoldCaucasianMale18-20hrdoubling%me
0
2
4
6
8
10
MFC BFC EROD (2E1, 2C9) (3A4, 1A2) (1A)
HepG2
PHH
CYP
450
Act
ivity
(p
mol
e/m
in/m
g pr
otei
n)
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HepaRG™Cells
• IsolatedfromfemaleHCVpa%ent• Possiblehepa%cprogenitorcellorigin• HepaRGcellsdifferen%ateintoamixedpopula%onofhepatocytes
andcholangiocytes
HepaRG(Progenitor)
28days1.7%DMSO
HepaRG(Differen%ated)
Griponetal.PNAS2002
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Aerobic Energetic
Glycolytic Quiescent
HepaRG™Cells
• HepaRGcellsshowpolarizedfunc%on• CYP450ac%vityiscomparableto
cryopreservedhepatocytes• HepaRGcellsshowstrongOCR
HepaRG(Differen%ated)
0
25
50
75
100
0 10 20 30 40 50
Mito
chon
dria
(OC
R)
Glycolysis (ECAR)
PHH
HepaRG
0
2
4
6
8
10
MFC BFC EROD (2E1, 2C9) (3A4, 1A2) (1A)
CYP
450
Act
ivity
(p
mol
e/m
in/m
g pr
otei
n)
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Prolifera)ngHumanHepatocytes
OSM
gp130JAK
STAT3
E6E7
E2F
Rbp53
E6/E7LOW
SphaseentryCondi%onalProlifera%on
MEK
ERK
EMT
U0126STTC
S31
Levyetal.NatureBiotechnology2016
0
2
4
6
8
10
12
14
16
0 6 12 18 24
Popu
la)on
Dou
bling
Times(Days)
E6/E7E6/E7+OSM
HepatocytecoloniesexpressinglowlevelsofE6/E7,showanOSMdependentprolifera%on.Ø Thecellsrapidlydifferen%ate
uponOSMremoval
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Upcyte™Hepatocytes
• Generatedfrombothmaleandfemaledonors,fromdifferentgene%cbackgrounds(Caucasian,Asian,Hispanic)
• Celldifferen%aterapidlyintometabolicallyfunc%onalhepatocytes
UpcyteHepatocytes(Prolifera%ng)
4daysOSMremoval
UpcyteHepatocytes(Differen%ated)
Griponetal.PNAS2002
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0
1
2
3
HNF4α PXR FXR CAR NTCP BSEP UGT1A1 NNMT
Primary Hepatocytes Differentiated Hepatocytes Proliferating Hepatocytes HepG2
0
100
200
300
400
CONTROL INDUCED C
YP
3A4
activ
ity
(pm
ol/m
in/m
g pr
otei
n)
21 26 29 42
Population Doubling
0
10
20
30
40
50
653 740 422 653 740 422 653 740 422
CITCO Omeprazole Rifampicin
Fold
mR
NA
Indu
ctio
n CYP1A2
CYP2B6
CYP3A4
0
2
4
6
8
0 2 4 6 8 10 12 14
Alb
umin
Pro
duct
ion
(µ
g /d
ay/m
g pr
otei
n)
Days Post Differentiation
E-Cadherin / Actin E-Cadherin / Albumin Differentiated (D4) A B
CCDFDA / DiI-LDL Polarized
Non-polarized
D
0
1
2
MRP3 CYP1A2 CYP2C9 CYP2D6 CYP2E1 CYP3A4 OCT1 MDR1
Rel
ativ
e G
ene
Exp
ress
ion
0
50
100
150
200
250
300
CONTROL INDUCED
CY
P2B
6 ac
tivity
(p
mol
/min
/mg
prot
ein)
21 26 29 42
Population Doubling
EF
Upcyte™Hepatocytes Aerobic Energetic
Glycolytic Quiescent 0
25
50
75
100
0 10 20 30 40 50
Mito
chon
dria
(OC
R)
Glycolysis (ECAR)
PHH
Upcyte
0
1
2
3
HNF4α PXR FXR CAR NTCP BSEP UGT1A1 NNMT
Primary Hepatocytes Differentiated Hepatocytes Proliferating Hepatocytes HepG2
0
100
200
300
400
CONTROL INDUCED
CY
P3A
4 ac
tivity
(p
mol
/min
/mg
prot
ein)
21 26 29 42
Population Doubling
0
10
20
30
40
50
653 740 422 653 740 422 653 740 422
CITCO Omeprazole Rifampicin
Fold
mR
NA
Indu
ctio
n CYP1A2
CYP2B6
CYP3A4
0
2
4
6
8
0 2 4 6 8 10 12 14 A
lbum
in P
rodu
ctio
n
(µg
/day
/mg
prot
ein)
Days Post Differentiation
E-Cadherin / Actin E-Cadherin / Albumin Differentiated (D4) A B
CCDFDA / DiI-LDL Polarized
Non-polarized
D
0
1
2
MRP3 CYP1A2 CYP2C9 CYP2D6 CYP2E1 CYP3A4 OCT1 MDR1
Rel
ativ
e G
ene
Exp
ress
ion
0
50
100
150
200
250
300
CONTROL INDUCED C
YP
2B6
activ
ity
(pm
ol/m
in/m
g pr
otei
n) 21
26 29 42
Population Doubling
EF
• Upcytesshowpolarizedfunc%on• CYP450ac%vityiscomparableto
cryopreservedhepatocytes• UpcytecellsshowstrongOCR
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0.1
1
10
100
1000
10000
0.1 1 10 100 1000 10000
Hep653Hep151Hep10Hep422A
PrimaryHepatocytesTC50(μM)
E6/E7HepatocytesTC 5
0(μM)
R2>0.98all
Donor Ethnicity Gender 10 Caucasian Female
151 Caucasian Female 422A Hispanic Male 653 Caucasian Female 740 Asian Female
Upcyte™Hepatocytes
Melatonin
Troglitazone
Chlorpromazine
Cyclosporine
Melatonin
Diclofenac
Acetaminophen
Aflatoxin-B1
TUNEL/DAPI
Melatonin
Amiodarone
Aspirin
Valproate
Nile Red/Hoechst CDFDA/Hoechst
0
10
20
30
Afla
toxi
n B
1
Ace
tam
inop
hen
Dic
lofe
nac
Mel
aton
in
Apo
ptot
ic In
dex
(%)
0
0.1
0.2
0.3
Cyc
losp
orin
A
Chl
orpr
omaz
ine
Trog
litaz
one
Mel
aton
in
Bile
Sec
retio
n
0.7
1
1.3
1.6
Mel
aton
in
Am
ioda
rone
Ace
tyls
alis
ilic
Aci
d
Valp
roic
Aci
d Intr
acel
lula
r Lip
ids
Asp
irin
Valp
roat
e
Apoptosis Cholestasis Steatosis Negative Control
> 5mM > 5mM > 5mM
0 1000 2000 3000 4000 5000
Manntiol Melatonin
Menthol Acetaminophen
Valproic Acid Acetylsalicilic Acid
Diclofenac Cyclosporine A
Troglitazone Chlorpromazine
TC50 Concentration (µM)
0 1 2 3 4 5
Amiodarone Aflatoxin B1
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Reprogramming factors
hPSC-derivedHepatocytes
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hPSC-derivedHepatocytes5% 84.5%
5.5% 5%
HNF4α
Alb
umin
0
4
8
12
16
20
7 9 11 13 15 17
Alb
umin
(µ
g/m
L/m
g)
Differentiation (Days)
PHH secretion
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0.1
1
10
EROD BFC MFC (1A) (3A4, 1A2) (2E1, 2B9)
PHH Control HepG2
CYP
450
Act
ivity
(p
mol
e/m
in/m
g pr
otei
n)
hPSC-HEP
Differen%atedhepatocytesshowfetalphenotype.CellsexpressAFPandCYP3A7butlihletonoCYP3A4.Hepatocytesshowlihleoxida%vefunc%on,andlihlemitochondrialmass
Aerobic Energetic
Glycolytic Quiescent 0
25
50
75
100
0 10 20 30 40 50
Mito
chon
dria
(OC
R)
Glycolysis (ECAR)
PHH
HepG2hPSC-HEP
hPSC-derivedHepatocytes
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MicrobiomeDerivedCues
Lithocholicacid(LCA)
Conjugatedlinoleicacid(CLA)
1
10
100
0 10 20 50
Rel
ativ
e m
RN
A Ex
pres
sion
[Log
]
Lithocholic Acid (µM)
PXR CAR CYP2C9 CYP3A4
*
**
* **
Avioretal.Hepatology2015
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0
2
4
6
8
10
MFC BFC EROD (2E1, 2C9) (3A4, 1A2) (1A)
HepG2 OA + 9CLA PHH
hPSC-HEP
Differen%atedhPSC-derivedhepatocytesmorematurephenotype,becomingoxida%veandeleva%ngCYP450ac%vity.
Aerobic Energetic
Glycolytic Quiescent 0
25
50
75
100
0 10 20 30 40 50
Mito
chon
dria
(OC
R)
Glycolysis (ECAR)
PHH
HepG2
hPSC-HEP
hPSC-derivedHepatocytes
E-Cad / CYP3A4 / DAPI
CYP
450
Act
ivity
(p
mol
e/m
in/m
g pr
otei
n)
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hPSC-derivedHepatocytes
10
100
1000
10000
10 100 1000 10000 Prim
ary
Hep
atoc
ytes
lo
g TC
50 (µ
M)
hESC-derived Hepatocytes log TC50 (µM)
R2=0.94
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TableofContents
1. LiverStructureandFunc%on: Organiza%on,Zona%on,Composi%on
2. Func%onalMaintenance: 3Dcultures,CellInterac%ons,Oxygen
3. CellSource: HepG2/C3A,HepaRG,Upcyte,StemCells
4. OrganonChip: HuREL,TissueDynamics
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Bowel
Liver
Lung
Human-on-a-Chip
1.5cm
Onetechnologythatcanpoten%allygobeyondanimalandcellculturestudiesishuman-on-a-chip.
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MicrodevicesinBiologyandMedicine(2009)
Liver
Cancer
HuRELdevelopedandsellsthefirstmicrochipswithhumanlivercellsusedtopredictdrugmetabolismforpharmaceu%calR&D
HμREL®Technology
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Albumin/CD31 Sensors/DNA
Sensor-embedded microscale tissues (liver, heart, brain) rapidly aggregate in collagen matrix in microwells that mimic the native tissue microenvironment.
TissueDynamics®Mul)OrganonChip
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MetabolicZona)ononChip
0
0.1
0.2
0 0.15 0.3 0.45 0.6
Oxy
gen
(mM
)
Depth (mm)
0.1
0.15
0.2
0 150 300
Oxygen
Sinusoid(μM)
InVivo
0.20
0.15
0.10
0.05
0.00
DAPI/G6Pase/CYP2E1
Microwells protect the Upcyte® cells from shear, while nutrient gradients push cells toward metabolic zonation
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Liver,Cardiac,andBrain
3D vascular organs (liver, heart, brain) created from iPSC-derived hepatocytes, cardiomyocytes and neural stem cells.
//////
iPSC-derivedLiver iPSC-derivedHeart iPSC-derivedBrain
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Rotenone(organicpes)cide)
0
20
40
60
80
100
0 5 10 15 20 25 30
Oxygen(%
Air)
Time(hours)
Control1μM50μM200μM
0
0.2
0.4
0.6
0.8
0.001 0.05 0.2
TimetoOnset
Rotenone(mM)
TTO = 12 min
24 hrs Apoptosis
Rotenoneisapes%cidenaturallyoccurringintheseedsofseveraltypesofplants.Itishighlytoxic,causingdirectdamageinminutes
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Amiodarone(an)arrhythmic)
0
4
8
12
16
0.2 0.5 1
TimetoOnset
Amiodarone(mM)0
20406080
100120
0 4 8 12 16 20 24
Oxygen(%
Air)
Time(hours)
Control 0.2µM200µM 500µM1000µM
24 hrs Fatty Liver Disease
TTO = 4 hrs
Amiodaroneisawidely-usedan%arrhythmicdrug.Itcausessecondarydamagebyslowaccumula%onoflipidsinlivercells
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Troglitazone(an)diabe)c)
Troglitazonewaswithdrawnfromthemarketduetoidiosyncra%clivertoxicity,possiblymediatedbycholestasis.
020406080
100120
0 4 8 12 16 20
Oxygen(%
Air)
Time(hours)
Control 50µM350µM 500µM2000µM
0
0.5
1
1.5
2
0.35 0.5 2
TimetoOnset(H
rs)
Troglitazone(mM)
TTO = 60 min
24 hrs Cholestasis
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TroglitazoneRiskAnalysis
Cellular respiration is disrupted forcing the cells to compensate by glycolysis increasing lactate production. ATP production stays constant but lipogenesis is all but shut down.
Non-Toxic Concentration Troglitazone (15 hours)
0 hrs
15 hrs
GlycolysisRespira%onLipogenesisGlutaminolysis
0
4
8
12
16
20
1
2
3
-5 5 15 25
Oxygen(%
Air)
Lactate/GlucoseRa)
o
Time(hours)
Glycolysis
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TMIHCV(ERCStar)ngGrant)OCLD(ERCConsolidatorGrant)5K01DK080241(NIHNIDDK)HeMiBioConsor%um(FP7&COLIPA)BIRAXRegenera%veMedicine
RobertSchwartz(WeillCornell)MathieuVinken(VUB)MichaelSchwarz(TuebingenU.)MagnusJaeger(Fraunhofer)JanVanfleteren(U.Gent)Mar%nYarmush(Harvard)LeslieBenet(UCSF)
Acknowledgments