introduc)on to hepa)c models€¦ · introduc)on to hepa)c models func%on, cell sources &...

Introduc)ontoHepa)cModelsFunc%on,CellSources&PhysiologicalModels

Prof.YaakovNahmiasPhone:+972(53)9227232Email:[email protected]

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

TheLiver

v  Thelargestinternalorganinthehumanbody,about1.5kginadult

v  Canregeneratefrom80%lossofmass

v  Responsibleforthesystemicregula%onofglucose,aminoacids,andlipids

v  Secretesbileacidsintothegallbladderandintes%ne

v  Responsibleorganfordrugmetabolism

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

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

LiverUltrastructure

v  Hepatocytesareabout25μmindiameter,withabout1,500mitochondriaineachcellandnumerousglycogengranules

v  Hepatocytesdisplaycomplexaneuploidyandpolyploidyinthenormalliver

LiverUltrastructure

SpaceofDisse(1.2μm)separateshepatocytesfromendotheliumBilecanaliculi(1μm)aretheapicalsurfaceofhepatocytes

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

TableofContents





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

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

Endothelial-dependentHepatocyteMigra)on

Self-AssembledSinusoids

High-resolu%onphaseandfluorescentimagesofself-assembledliversinusoid-likestructures

Rathepatocytes(right)orhumanhepatocytes(bohom)

Albumin / CD31

CK18 / CD31

Nahmiasetal.TissueEngineering2006

LiverOrganoids

Day2 Day5 Day10 Day28

Nahmias&Odde,NatureProtocols2006

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

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

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

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

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

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

TableofContents





PrimaryHumanHepatocytes

•  Humanhepatocytesarescarceandfailtoproliferateinvitro•  Cryopreservedhepatocytesshowmassivebatchtobatchvariability

•  Pla%ngefficiencyofcryopreservedcellsokenfallsbelow70%•  Cellsaredifficulttopassage

Hepatocytesoncollagengel HepatocytesonMatrigel™

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

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)

HepaRG™Cells

•  IsolatedfromfemaleHCVpa%ent•  Possiblehepa%cprogenitorcellorigin•  HepaRGcellsdifferen%ateintoamixedpopula%onofhepatocytes

andcholangiocytes

HepaRG(Progenitor)

28days1.7%DMSO

HepaRG(Differen%ated)

Griponetal.PNAS2002

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


CYP

450

Act

ivity

(p

mol

e/m

in/m

g pr

otei

n)

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

Upcyte™Hepatocytes

•  Generatedfrombothmaleandfemaledonors,fromdifferentgene%cbackgrounds(Caucasian,Asian,Hispanic)

•  Celldifferen%aterapidlyintometabolicallyfunc%onalhepatocytes

UpcyteHepatocytes(Prolifera%ng)

4daysOSMremoval

UpcyteHepatocytes(Differen%ated)

Griponetal.PNAS2002

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


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

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

Reprogramming factors

hPSC-derivedHepatocytes

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

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


25

50

75

100

0 10 20 30 40 50

Mito

chon

dria

(OC

R)

Glycolysis (ECAR)

PHH

HepG2hPSC-HEP


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

0

2

4

6

8

10


HepG2 OA + 9CLA PHH

hPSC-HEP

Differen%atedhPSC-derivedhepatocytesmorematurephenotype,becomingoxida%veandeleva%ngCYP450ac%vity.

Aerobic Energetic


25

50

75

100

0 10 20 30 40 50

Mito

chon

dria

(OC

R)

Glycolysis (ECAR)

PHH

HepG2

hPSC-HEP


E-Cad / CYP3A4 / DAPI

CYP

450

Act

ivity

(p

mol

e/m

in/m

g pr

otei

n)


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

TableofContents





Bowel

Liver

Lung

Human-on-a-Chip

1.5cm

Onetechnologythatcanpoten%allygobeyondanimalandcellculturestudiesishuman-on-a-chip.

MicrodevicesinBiologyandMedicine(2009)

Liver

Cancer

HuRELdevelopedandsellsthefirstmicrochipswithhumanlivercellsusedtopredictdrugmetabolismforpharmaceu%calR&D

HμREL®Technology

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

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

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

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

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

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

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

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

introduc)on to hepa)c models€¦ · introduc)on to hepa)c models func%on, cell sources &...

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