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Functional Integrity of the Chimeric (Humanized) Mouse Liver: Enzyme Zonation,

Physiological Spaces, and Hepatic Enzymes and Transporters

Authors: Edwin Chiu Yuen Chow, Jason Zi Yang Wang, Holly P. Quach, Hui Tang, David C. Evans, Albert P. Li, Jose Silva, and K. Sandy Pang

Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto, Canada M5S 3M2 (ECC, JZW, HPQ, HT, KSP)

Pharmacokinetics, Dynamics & Metabolism, Janssen Pharmaceuticals Inc., Spring House, PA, USA 19477 (DCE, JS)

In Vitro ADMET Laboratories, 9221 Rumsey Rd, Suite 8, Columbia, MD 21045 (APL)

Edwin Chiu Yuen Chow present address is Division of Clinical Pharmacology V, Office of Clinical Pharmacology, Office of Translational Sciences, Center for Drug Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD, USA

This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on June 24, 2016 as DOI: 10.1124/dmd.116.070060

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Short Title: Functional Integrity of the Chimeric Mouse Liver

Corresponding author: send inquiries and correspondence to Dr. K. S. Pang, Leslie Dan Faculty of Pharmacy, University of Toronto, 144 College Street, Toronto, ON, Canada M5S 3M2. Email: [email protected]

Text Pages: 36

Abstract: 250 words

Introduction: 714 words

Discussion: 1331 words

References: 38

Figures: 9

Tables: 3

Abbreviations: γdiff , ratio of diffusible/nondiffusible spaces; ACN, acetonitrile; AUC, area under the curve; AUMC, area under the moment curve; E, extraction ratio; ELISA, enzyme-linked immunosorbent assay; Fah, fumarylacetoacetate hydrolase; FRGN, triple knockout of Fah, Rag2, and Il2rg genes on the non-obese diabetic strain background; Fxr/FXR, murine/human farnesoid X receptor; GAPDH, human glyceraldehyde 3-phosphate dehydrogenase; Hct, hematocrit; HG, harmol glucuronide; HPLC, high pressure liquid chromatography; HS, harmol sulfate; k, elimination rate constant; KHB, Krebs-Henseleit Buffer; MID, multiple indicator dilution; Mrp/MRP, murine/human multidrug resistance associated protein; MTT, mean transit time; NTBC, nitisinone or 2-(2-nitro-4-trifluoro-methylbenzoyl)-1,3-cyclohexanedione; Ntcp/NTCP, QB, blood flow rate; Qp, plasma flow rate; Qw, water flow rate; qPCR, quantitative real-time PCR; R, retrograde; RBC, red blood cell; Rosa, mouse hepatocytes derived from 129S7 mouse strain; SMX, sulfamethoxazole; Sult/SULT, murine/human sulfotransferase; t0, vessel transit time; TMX, trimethoprim; Ugt/UGT, murine/human UDP-glucuronosyltransfersases; Vsin, sinusoidal blood volume; Valb, total albumin space; Vsuc, total sucrose space; Vtotal water, total water space; VDisse alb, albumin Disse space; VDisse suc, sucrose Disse space; Vcell water, cellular water space


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Abstract

Chimeric mouse liver models are useful in vivo tools for human drug metabolism studies.

However, liver integrity and the microcirculation remain largely uninvestigated. Hence, we

conducted liver perfusion studies to examine these attributes in FRGN [Fah(-/-), Rag2(-/-), and

Il2rg(-/-), NOD strain] livers (control) and chimeric livers repopulated with mouse (mFRGN) or

human (hFRGN) hepatocytes. In single pass perfusion studies (2.5 ml/min), outflow dilution

profiles of non-eliminated reference indicators (51Cr-RBC, 125I-albumin, 14C-sucrose and 3H-

water) revealed preservation of flow-limited distribution and reduced water and albumin spaces

in hFRGN livers when compared to FRGN livers, a view supported microscopically by tightly

packed sinusoids. With prograde and retrograde perfusion of harmol (50 µM) in FRGN livers, an

anterior sulfation (Sult1a1) over the posterior distribution of glucuronidation (Ugt1a1) activity

was preserved, evidenced by the 42% lower sulfation to glucuronidation ratio (HS/HG) and 14%

higher harmol extraction ratio (E) upon switching from prograde to retrograde flow. By contrast,

zonation was lost in mFRGN and hFRGN livers, with HS/HG and E for both flows remaining

unchanged. Remnant mouse genes persisted in hFRGN livers (10-300% those of FRGN). When

hFRGN livers were compared to human liver tissue, higher UGT1A1 and MRP2, lower MRP3,

and unchanged SULT1A1 and MRP4 mRNA expression were observed. Total

Sult1a1/SULT1A1 protein expression in hFRGN livers was higher than that of FRGN livers,

consistent with higher harmol sulfate formation. The composite data on humanized livers suggest

a loss of zonation, lack of complete liver humanization, and persistence of murine hepatocyte

activities leading to higher sulfation.


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INTRODUCTION

Traditionally, in vitro tools such as cryopreserved hepatocytes are popular means to study

human drug metabolism. However, these preparations are of limited stability and viability,

precluding their long-term use for metabolic or toxicological studies (Brandon et al., 2003). The

transgenic liver model that is often exploited in defining human liver drug metabolism expresses

a limited number of human liver enzymes and lacks human transporters, rendering it unsuitable

for the study of overall human hepatic elimination that requires the interplay of a full

complement of human enzymes and transporters.

Chimeric mouse liver models that are repopulated with human hepatocytes are viewed as

improved models for the study of human drug metabolism since the livers contain the full

complement of human liver genes (Foster et al., 2014; Sanoh and Ohta, 2014). The recently

developed chimeric mouse liver model allows researchers to tailor repopulation of hepatocytes,

humans in particular, to closely resemble the human liver for drug metabolism studies, though

other donor hepatocytes from different species may be used as needed (Strom et al., 2010).

Numerous reports have related that human liver genes do reside in these humanized mouse livers

(Katoh et al., 2004; Katoh et al., 2005a; Katoh et al., 2005b), and that hepatocytes or microsomes

show human metabolic activities (Strom et al., 2010) and metabolites (Liu et al., 2011; Sanoh et

al., 2012; Kitamura and Sugihara, 2014). Other studies show selective induction of enzymes by

human-specific nuclear receptor activators (Katoh et al., 2005a; Sanoh and Ohta, 2014) and

allow for toxicological assessment and prediction of human toxic metabolites (Strom et al., 2010;

Cohen, 2014; Kitamura and Sugihara, 2014). Recently, the model has been suggested to be a

suitable preparation for the examination of human lipoprotein metabolism (Ellis et al., 2013).

Azuma et al. (2007) and others (Katoh et al., 2004; Katoh et al., 2005b) suggest that >90%


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repopulation with human hepatocytes within the mouse liver as a host is achievable, as verified

by the blood/plasma human albumin levels as estimates of the extent of human hepatocyte

repopulation.

The premise of these chimeric liver models is the destruction of host hepatocytes to allow

for implantation of foreign hepatocytes. The FRGN chimeric model is a triple genetic knockout

of the fumarylacetoacetate hydrolase (Fah) gene that disrupts the tyrosine catabolic pathway to

result in accumulation of the toxic metabolite, fumarylacetoacetate, leading to cell death. The

absence of this gene, together with Rag2-/- and Il2rg-/- for disruption of the immune system, in the

triple knockout FRGN mouse allows for repopulation with foreign hepatocytes (Strom et al.,

2010). The irreversible hepatic injury caused by absence of Fah in the FRGN mouse facilitates

the high repopulation of foreign hepatocytes without rejection and reversion to native mouse

hepatocytes. Hepatotoxicity is avoided by administration of 2-(2-nitro-4-trifluoro-

methylbenzoyl)-1,3-cyclohexanedione (NTBC), which blocks tyrosine catabolism upstream of

Fah. This allows for a controllable environment where hepatocyte implantation can be conducted

in series with greater success of liver engraftment (Grompe and Strom, 2013) and allows

researchers to tailor the repopulation of foreign (human, rat or mouse) hepatocytes (Strom et al.,

2010).

The surgical and diet manipulation as well as housing procedure of these chimeric mice

could alter gene expression and therefore drug elimination. In addition, the metabolic zonation of

human genes and the microcirculation in highly repopulated chimeric livers are mostly unknown.

Hence, we examined gene expression in livers repopulated with mouse hepatocytes (mFRGN or

m-chimeric), with the expectation that mouse genes would remain identical to those in FRGN

livers if surgery and diet are not mitigating factors. Because differences in flow patterns existing


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in FRGN/mFRGN/hFRGN livers could result in different drug extraction ratios (Pang and

Rowland, 1977), we investigated liver microcirculation and physiological spaces with the

multiple indicator dilution (MID) technique to trace outflow dilution profiles of non-eliminated

reference indicators (Goresky, 1963). We studied metabolic zonation that also impacts drug

metabolism with harmol, which was used to probe the anterior sulfation (Sult1a1/SULT1A1) vs.

posterior glucuronidation (Ugt1a1/UGT1A1) activities, a pattern that was observed in the rat

liver (Pang et al., 1981; Pang et al., 1983) in prograde (P, from portal to hepatic vein) and

retrograde (R, from hepatic to portal vein) perfusion studies. We further examined mRNA and

protein expression of mouse and human transporters, enzymes, and nuclear receptors in FRGN

and hFRGN livers and human liver tissue.


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MATERIALS AND METHODS

Materials: 125I-Labeled bovine serum albumin (NEX076001MC), 3H2O (NET001B001MC), sodium 51Cr-

chromate (NEZ030001MC), and 14C-labeled sucrose (NEC100X001MC) were purchased from

Perkin Elmer Radiochemicals Inc. (Woodbridge, ON). Harmol hydrochloride

(Cat#AC152270050) was procured from Fisher Scientifics and harmol sulfate, biosynthesized

from rat liver perfusion studies, and purified by preparative HPLC in our laboratory. Antibodies

against mouse and human Sult1a1/SULT1A1 (Cat#ab38411) and human SULT1A1

(Cat#ab124011), mouse and human Ugt1a1/UGT1A1 (Cat#ab62600) and human UGT1A1

(Cat#ab170858), mouse and human Cyp3a/CYP3A (Cat#ab22724) and human CYP3A4

(Cat#ab124921), human MRP2 (Cat#ab3373), and Gapdh/GAPDH (Cat#ab8245) were from

Abcam (Cambridge, MA). The monoclonal antibody against mouse Mrp2 only (NBP1-69023)

was obtained from Novus Biologicals Canada (Oakville, ON). Human liver tissues (Table 1)

were obtained from organs procured but not used for transplantation, provided by In Vitro

ADMET Laboratories from the International Institute for the Advancement of Medicine (Edison,

NJ) and the National Disease Research Interchange (Philadelphia, PA).

FRGN mice, housing and diet conditions: Male FRGN, mFRGN [repopulated with hepatocytes

of the Rosa (129S7) mouse strain, pooled from 8-10 mouse livers] and human repopulated

(hFRGN) mice (4-6 months old from three different human donors; Table 1) were purchased

from Yecuris Corporation (Tualatin, OR). Upon arrival, FRGN, mFRGN and hFRGN mice were

housed in special sterile quarters, and monitored and weighed daily. During the first 3 days after

arrival, all mice received the STATTM high calorie liquid diet and supplement (30% v/v with

sterile water) in a diluted form supplied by the Yecuris Corporation (Tualatin, OR) in the

irradiated LabDiet® 5LJ5 chow. In the first week, all mice received sterile, filtered drinking


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water containing NTBC [8 mg/l final concentration containing 4.8 mg NaHCO3], 3% dextrose,

and an antibiotic mixture [sulfamethoxazole (SMX) and trimethoprim (TMP), 640 and 128

µg/ml final concentration, respectively]. Details of the housing conditions and diets of the

FRGN, mFRGN, and hFRGN mice during the second and subsequent weeks were described in

Figure 1. All studies were performed in accordance to approved animal protocols at University

of Toronto.

Mouse liver perfusion: Single pass mouse liver perfusion was conducted according to St-Pierre

and Pang (1993). CD-1 (30-35 g) mice, obtained from Charles River (Montreal, QC), were

chosen as an additional control because the body and liver weights were similar to those of

FRGN mice. Under anesthetia with ketamine and xylazine i.p. (150 and 10 mg/kg, respectively),

the portal and hepatic veins and bile duct were cannulated with 20-GA, 1.16 inch catheter

placement units (Cat#427401, BD, Mississauga, ON) and PE10 tubing, respectively. The gall

bladder, pyloric vein, hepatic artery and abdominal inferior vena cava above the kidneys were

ligated. After the completion of surgery, flow was immediately adjusted to 2.5 ml/min and the

liver was kept at 37°C with a heating lamp. Oxygenated blood perfusate (95% O2 and 5% CO2 at

1 ml/min) was delivered at 2.5 ml/min once through the mouse liver preparation in situ (St-Pierre

and Pang, 1993). Perfusate consisted of 15-20% freshly washed bovine erythrocytes (RBC) (a

kind gift from Ryding Regency Meat Packers, Toronto, ON), 300 mg dextrose, 1% bovine serum

albumin (Cat#A7030, Sigma Aldrich Canada, ON), and Krebs-Heinseleit-Bicarbonate (KHB)

solution, buffered to pH 7.4.

Multiple Indicator Dilution (MID) technique: The procedure for multiple indicator dilution

studies (Goresky, 1963; St-Pierre et al., 1989) was modified due to the low sampling volume

(41.7 µl/min) stemming from the perfusate flow rate of 2.5 ml/min. After equilibration of the


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murine liver for 10-15 min, two injection doses (~0.16 ml each), one consisting of 14C-sucrose

and 3H-H2O and the second, consisting of 125I-albumin and 51Cr-RBC, were injected into the

portal vein at 8 min apart. For the first dose, 14C-sucrose and 3H-H2O (~1:3 dpm ratio) in blank

blood perfusate (20% RBC in 1% albumin and KHB, same composition as blood perfusate), was

prepared; for the second dose, labeling of the bovine RBC was carried out by incubation of blood

perfusate with sodium 51Cr-chromate at 37°C for 30 min, followed by addition of ascorbic acid

(250 mg/ml) then three times washing with saline and centrifugation of RBCs (St-Pierre et al.,

1989). The labeled RBCs and 125I-labeled albumin were reconstituted into an injectate of

identical composition to that blood perfusate. The hematocrits of the doses and perfusate were

measured to estimate the dose and appropriate flow rates (St-Pierre et al., 1989). Simultaneous

to each injection, a home-built fraction collector was activated to collect outflow fractions at

successive 1 and 2 sec intervals for a total of 200 sec. For 14C- and 3H-analysis, the volume of

the plasma perfusate or plasma dose (10-50 µl) sample was made up to 50 µl volume with blank

plasma perfusate for the same quenching, and directly subjected to beta counting (Beckman

Coulter, Canada, model LSC6500). The 51Cr and 125I cpm in blood perfusate (10-50 µl) or dose

was directly counted by a two channel gamma counter (Perkin Elmer Gamma Counter, Wizard

3; Waltham, MA, USA). The lack of residual dpm from the first dose was confirmed by the

absence of counts in the outflow perfusate sample taken immediately prior to the second

injection. The measured radioactivity (concentration) in the outflow was normalized to the

amount injected (dose in dpm/cpm) to obtain fractional recoveries per ml. After normalization

of the outflow concentration of 51Cr-RBC, 125I-albumin, 14C-sucrose or 3H2O to the amount

injected (dose), the frequency (concentration/dose) was presented against the mid time of

collection. The area under the curve (AUC) and area under the moment curve (AUMC) were


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obtained from trapezoid rule, extrapolated to infinity, as shown in the equations below (where

Clast is the concentration of the last sample and k is the rate constant of log linear decay) to yield

the AUC (area under the curve) and AUMC (area under the moment curve) (St-Pierre et al.,

1989).

0

t lastCAUC AUC

k= + (Eq. 1)

20

1t lastlast

tAUMC AUMC C

k k⎛ ⎞= + +⎜ ⎟⎝ ⎠

(Eq. 2)

The mean transit time (MTT = AUMC/AUC) and the physiological volume (MTT x appropriate

flow rate, blood flow for 51Cr-counts, plasma flow for 125I-albumin and 14C-sucrose; and water

flow for 3H2O) were obtained. A sham experiment was performed without the liver to correct for

the MTT of blood within the catheters. The plasma (Qp) and water (Qw) flow rates were

expressed relative to the blood perfusate flow rate (QB) as QB(1-Hct) (Hct=hematocrit) and

QB[(1-Hct)*0.98+0.7*Hct], respectively, as previously described (St-Pierre et al., 1989). The

physiological liver and Disse volumes were calculated as shown below:

sin = ×B bloodV Q MTT where Vsin is the sinusoidal blood volume (Eq. 3)

alb P albV =Q ×MTT where Valb is the total albumin space (Eq. 4)

suc P sucV =Q ×MTT where Vsuc is the total sucrose space (Eq. 5)

total water w waterV =Q ×MTT where Vtotal water is the total water space (Eq. 6)

Disse alb alb sinV =V -V (1-Hct) where VDisse,alb is the albumin Disse space (Eq. 7)

Disse suc suc sinV =V -V (1-Hct) where VDisse,suc is the sucrose Disse space (Eq. 8)


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cell water total water w sin Disse,waterV = V - Q ×V -V where Vcell water is the cellular water space and VDisse,water is

the Disse water space, or VDisse,suc*0.98, the fraction that is water in perfusate plasma (Eq. 9)

The outflow profiles of diffusible references (albumin, sucrose and water) were then related to

that of RBC that is confined to the sinusoidal blood space by way of a simple, linear

transformation process. Dilution profiles of 125I-albumin, 14C-sucrose and 3H-H2O were

superposed onto the 51Cr-RBC curve, after adjustment of the concentration and time by (1+ γdiff)

and [1/(1+ γdiff)], respectively, as described by Goresky and colleagues (Goresky, 1963; St-Pierre

et al., 1989) as 00

11( )

(1 ) 1diff RBCdiff diff

tC t C t

γ γ⎛ ⎞−= +⎜ ⎟⎜ ⎟+ +⎝ ⎠

. The process was dependent on t0 (large

vessel transit time, which relates to the volume of the large vessels) and γdiff is the ratio of

diffusible/nondiffusible spaces; γalb and γsuc are the ratio of albumin and sucrose Disse spaces to

the plasma space, and γH2O is the ratio of the sum of intracellular water and Disse water spaces to

the sinusoidal water space. Superposition of the transformed data was achieved by splining

(using SOLVER in Microsoft® Excel) and provided γ and t0.

Prograde/Retrograde single-pass harmol liver perfusion: Mouse (FRGN, mFRGN, or hFRGN)

livers were perfused with ~50 µM harmol with single pass prograde (P), followed by retrograde

(R) flow, then P flow, each for 40 min (P-R-P), or in reverse fashion: R-P-R at 2.5 ml/min. The

P-R-P and R-P-R perfusion design was intended for viability check of the liver upon comparison

of the data during the first and third periods.

Harmol perfusion studies and HPLC: Harmol (H), harmol sulfate (HS), and harmol glucuronide

(HG) concentrations were determined by high pressure liquid chromatography (HPLC). An

aliquot of 200 µl blood perfusate was precipitated with 600 µl of acetonitrile, with 4-

methylumbelliferone (4-MU) added as the internal standard. The sample was mixed and spun at


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12,000g for 10 min. The supernatant was removed and dried under nitrogen gas, and

reconstituted with the buffer mix (0.05 M KH2PO4 and MeOH; 70:30). Bile was diluted with

water, then mixed with 4-MU and the buffer mix. The supernatant was centrifuged at 12,000g for

5 min prior to injection. Standards of H and HS were prepared in similar manner. Because HG

standard was unavailable, the concentration of HG was quantified using the standard curve of HS

since a precipitation procedure was used. Standards and samples were injected into the HPLC

machine (Shimadzu 10A HPLC system, Mandel Scientific Company Inc., Guelph, ON) that is

equipped with a 10 µm C18 reverse phase column (4.6 x 250 mm, Altech Associates, Deerfield,

IL) containing a C18 guard column (Waters Bondapak C18/Corasil 37-55 µm). The detection

wavelength was 313 nm. The mobile phase consisted of filtered 0.05 M KH2PO4 (A) and HPLC

grade MeOH (B). A gradient was utilized over 40 min: 0-12 min, 18% solvent B and 1 ml/min

flow; 12-13 min, 18-70% solvent B and 1.1 ml/min flow; 13-23 min, 70% solvent B and 1.1

ml/min flow; 23-24 min, 70-18% solvent B and 1.1 ml/min flow; 24-40 min, 18% solvent B and

1 ml/min flow. H, HS standards and internal standard peaks and areas were used to construct

calibration curves to determine the concentrations.

Calculations: Due to the slight loss of viability of some of the mouse liver preparations at the

end of the third period (between 80 to 120 min perfusion), only data from the first 80 min of

perfusion (periods I and II) were used for comparison. Three reservoir perfusate samples were

removed for the determination of CIn (input concentration); 5 outflow samples, collected within

the last 13 min of each steady-state perfusion period (from 25 to 40 min), were averaged for COut

(output concentration), and bile was collected for 10 min of each perfusion period, all during the

steady state. The steady state extraction ratio (E) of harmol is given by In Out

In

C - CC

. The steady-

state sulfation or glucuronidation rate is given by the summed rate of perfusate outflow and


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biliary excretion rate: conjugate formation rate = QBCOut (HS or HG) + QbileCbile(HS or HG)

where Qbile is the bile flow rate and Cbile(HS or HG) is the concentration of HS or HG in bile.

The ratio of harmol sulfation to glucuronidation rate (HS/HG) and E were expected to decrease

and increase, respectively, with flow switched from P to R flow when an anterior and saturable

sulfation (higher affinity, low capacity) pathway vs. a posterior, glucuronidation (higher

capacity, lower affinity) pathway exist, as observed for the rat liver (Pang et al., 1981; Pang et

al., 1983).

H&E Staining: After flushing of the mouse liver with ice-cold saline, livers were fixed with

10% formalin overnight for H&E staining, as performed by the Toronto Centre for

Phenogenomics (Toronto, ON).

RNA extraction and qPCR: Since there was no difference in mRNA expression data between

perfused and non-perfused livers, all data was pooled from perfused and non-perfused tissues.

Total mRNA in human liver and mouse tissues and mouse hepatocytes (FRGN and Rosa

hepatocytes from the 129S7 mouse strain) was extracted with TRIzol (Chow et al., 2009; Chow

et al., 2014). The mRNA sequences of target gene were obtained from the nucleotide PubMed

database (http://www.ncbi.nlm.nih.gov/pubmed/). Specific mouse and human primers were

designed in Primer-BLAST (http://www.ncbi.nlm.nih.gov/tools/primer-blast/) with exon-exon

junction span and specificity check for appropriate species selected. Primers were checked

against mRNA of mouse liver tissue and human hepatocellular carcinoma cell (HepG2) and

water for specificity (see Supplementary Table 1). cDNA was synthesized from mRNA by a

High Capacity cDNA synthesis kit as described previously (Chow et al., 2014) and quantified by

qPCR (Applied Biosystems® 7500 series) using SYBR Green or Taqman for detection. For

mRNA analyses of mouse liver genes in mouse liver and hepatocytes, the target genes of mouse


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liver and hepatocytes were normalized to that of cyclophilin. For mRNA analyses of mouse liver

genes in FRGN and hFRGN livers, the target gene was normalized to that of mouse β-actin

(specific for mouse only). For mRNA analyses of human liver genes in hFRGN livers and human

liver tissues, the target gene was normalized to that of human GAPDH (detects only human

GAPDH).

Western blotting: Human and mouse livers were homogenized with homogenizing buffer, as

described (Chow et al., 2009). Samples were centrifuged at 9,000g for 20 min at 4 °C, and

protein concentration was quantified, with 25-50 µg used for immunoblotting.

Statistics: Data were expressed as mean ± SEM for mRNA and protein data and for perfusion

and MID data. For comparison between two groups, the 2-tailed paired Student’s t-test was used

for the P-R perfusion data, and the unpaired t-test was used for the MID, mRNA, and protein

data. The value of P<0.05 was set as the level of significance.


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RESULTS

Liver microcirculation. The MID technique was used to appraise the physiological volumes

and flow characteristics. Data from FRGN and hFRGN livers were compared, and CD-1 livers of

similar weights as FRGN livers were further used as alternate liver perfusion controls. The

principle is to simultaneously inject labeled non-eliminating reference indicators (51Cr- RBC,

125I-albumin, 14C-sucrose, and 3H-H2O) into the livers to trace the outflow dilution profiles to

define the physiological volumes (Goresky, 1963; St-Pierre et al., 1989). Dilution profiles of all

of the non-eliminated reference indicators (expressed as concentration/dose vs. mid time of the

collection interval) were asymmetric and unimodal, and profiles of labeled albumin, sucrose and

water were increasingly dispersed compared to that of 51Cr-labeled RBC due to the progressively

larger space of distribution (Figure 2A).

Physiological volumes, estimated by moment analysis (transit time = area under the

moment curve/area under the curve = volume/flow) according to Goresky and colleagues

(Goresky, 1963; St-Pierre et al., 1989), revealed the relatively smaller total (Vtotal water) and

intracellular (Vcell) water spaces, Vsin (sinusoidal blood volume) and total albumin (Valb) and

albumin Disse (VDisse,alb) spaces for hFRGN livers when compared to those for FRGN and CD-1

livers (Table 2). After correction for the dead volume of tubing, we further employed γdiff, the

ratio of the summed Disse+cellular distribution space divided by the sinusoidal plasma volume

or plasma water space for the diffusible reference, and t0, the large vessel transit time, for

correction of the concentration by (1+ γdiff) and the time by 1/(1+ γdiff). Thereafter, the

“corrected” outflow dilution data of 125I-albumin, 14C-sucrose or 3H2O (the diffusible label)

showed that the outflow data for the diffusible label, 00

11( )

(1 ) 1diff RBCdiff diff

tC t C t

γ γ⎛ ⎞−= +⎜ ⎟⎜ ⎟+ +⎝ ⎠


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became superimposable onto that of the 51Cr-RBC curve (Figure 2B) (St-Pierre et al., 1989). The

γalb

, γsuc

, and γH2O

among CD-1, FRGN and hFRGN livers were all similar (Table 2). Despite the

small difference in volume for the hFRGN livers, all non-eliminated reference indicators

displayed delayed-wave forms that are characteristic of flow-limited distribution, showing the

intactness of the microcirculation, and presence of a permeability barrier. Results on reduced

volumes of hFRGN livers were consistent with those from H&E staining. hFRGN livers showed

normal morphology compared to FRGN livers, but the sinusoids were dense and tightly packed

with hepatocytes (Figure 3), showing reduced sinusoidal and Disse spaces (Table 2).

Liver enzymes and zonation: Single pass liver perfusion studies with harmol, delivered by P

and R flows, were performed to assess the metabolic zonation of phase II enzymes (Sult1a1 and

Ugt1a1) towards harmol metabolism (Pang et al., 1983; St-Pierre and Pang, 1993). Steady state

was reached in murine liver for harmol and the conjugates in perfusate and bile after 25 min

perfusion (Figure 4). For FRGN livers, the sulfation rate [4.8 vs. 3.1% rate in; or (perfusate rate

out + biliary excretion rate)/rate in] and the HS/HG ratio (0.078 vs 0.045, P < .05) were higher

for P perfusion, while the extraction ratio (E) of harmol, lower (0.633 vs. 0.723, P < .05),

compared to data during R perfusion (Table 3; Figure 4); there was no change in glucuronidation

rate (63-70%), the major metabolic pathway. Bile flow rate (0.5 to 0.56 μl/min/g liver) was

similar for both directions (P > .05). Although a higher percentage of HS that was formed was

excreted into bile for P vs. R flow (63% vs. 46%), the change was not significantly different; and

the extent of excretion was similar for HG (30% vs.16%). The higher E during R flow was due

to the lessened degree of saturation for sulfation, as found for the rat liver (Pang et al., 1983).

These metabolic data suggest intact zonation for FGRN mouse livers, and an anterior distribution


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of Sult1a1 activity over the posterior distribution of glucuronidation activity (evenly distributed

or perivenous distribution) was observed, as found for the rat liver.

In stark contrast, the HS/HG ratio and E were unchanged for mFRGN livers between P

and R perfusion (Table 3), suggesting loss of zonation for the repopulated mFRGN liver. The

bile flow rates of mFRGN livers were similar to those of FRGN livers. However, HS formation

rate for mFRGN livers was almost tripled that of FRGN livers, though HG rates were lower,

rendering higher HS/HG ratios when compared to those in FRGN livers in both P and R

directions. To explain for these differences, gene profiling was performed on donor Rosa

hepatocytes of mFRGN livers vs. that for FRGN livers, with the intention to correct for any

higher or lower activities associated with these donor (Rosa) hepatocytes. From gene profiling,

we obtained the ratio of Sult1a1 mRNA expression for liver tissue mFRGN/FRGN, and we

found that these ratios correlated well to those found in (Rosa/FRGN) hepatocytes (Figure 5).

The data explained that the higher sulfation activity found in mFRGN liver perfusion studies

(Table 3) was due to the higher sulfation activity in donor (Rosa) hepatocytes used to repopulate

the mFRGN livers. This data verified that housing and the diet (Figure 1) did not alter the

enzymatic activity nor the bile flow.

Similar to mFRGN livers, the HS/HG ratio and E for hFRGN livers remained unchanged

between P and R perfusion. The HS rate of hFRGN livers was similar to that of mFRGN livers

but was double that of FRGN livers; HG rate for hFRGN livers was similar to that of FRGN

livers. Bile flows and excretion rates of HS and HG were, however, significantly lower for

hFRGN livers compared to FRGN and mFRGN livers for both flow directions (Figure 4; Table

3). The bile flow rate for the perfused, humanized liver of hFRGN mice was only slightly lower


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than that found in normal human liver (0.21 vs. 0.28 μl/min/g) (Esteller, 2008), and this could be

explained by the lower blood flow rate chosen for perfusion relative to that in vivo.

Mouse and human gene expression in hFRGN livers. To explain the differences in

sulfation/glucuronidation and excretion activities of harmol conjugates in hFRGN and FRGN

livers, we first examined the presence of remnant mouse genes amidst the human gene

expression in hFRGN livers (Figure 6). We used mouse-specific β-actin primers for

normalization; however levels were lower in hFRGN livers, which may have contributed to the

variability. mRNA levels of murine Hnf-4α, Shp, and albumin in hFRGN livers were observed to

be much higher than those of FRGN livers, though levels of Car, Fxr, Lrh-1, and Hnf-1α were

lower. In addition, mRNA expression of murine enzymes, Cyp1a1 and Sult2a1, and transporters,

Bcrp, Mdr1a, and Mrp4, were higher in hFRGN livers whereas other enzymes (Cyp1a2,

Cyp2b10, Cyp3a11, Cyp2e1, and Ugt1a1) and transporters (Oatp1a1, Oatp1a4, Oatp2b1, Ntcp,

Ostα, Bsep, and Mrp2 were lower (Figure 6). There was no significant difference in the mRNA

expression of murine Lxrα, Pxr, Gsta3-3, Sult1a1, Sult1e1, Mrp3, and Ostβ in FRGN and

hFRGN livers. The persistence of unexpectedly higher levels of mouse gene remaining in the

humanized liver may have indeed constituted as an additional source for the higher conjugation

activity for the hFRGN livers.

We further examined the mRNA expression of the human conjugation enzymes

(SULT1A1 and UGT1A1) and transporters (MRP2, MRP3 and MRP4) that are involved in

harmol conjugation in hFRGN livers vs. those in normal human liver tissue (for information of

human liver tissue, see Table 1) due to the unavailability of original human donor hepatocytes

used for repopulation in hFRGN recipients. When hFRGN livers were compared to human liver


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tissue, the mRNA expressions of MRP2 and UGT1A1 were higher, whereas that for SULT1A1

and MRP4 were similar, and MRP3 was lower (Figure 7).

We then appraised gene variability of human hepatocyte implantation to assess whether

the donor variability could result in the difference in gene expression among hFRGN recipients

(Figure 8). The human mRNA expression of highly repopulated hFRGN livers from the same or

different human donor implanted hepatocytes was compared. Whether variability among hFRGN

donors contributed to the above results was further examined among 11 hFRGN liver

preparations arising from 3 donors (HHM05010, HHF07007, and HH17006). It was observed

that SULT1A1, CAR, SHP, CYP1A1, CYP2D6, GSTA4-4, SULT2A1, and OSTβ expressions

were extremely variable (visually) among hFRGN recipients arising from the same as well as

different donors (Figure 8), independent of the blood human albumin level (Table 1). Much

variability exists for the hFRGN preparations despite that they may originate from the same

donor. This is especially so when the livers serve as donors at various stages to produce different

generations of hFRGN mice, despite that all the hFRGN donor livers have arisen from the same

donor.

Mouse and human protein expression in FRGN, mFRGN, and hFRGN livers. Linearity in

antibody response among samples was first verified in Western blotting that was used for the

determination of relative protein expression. The methodology, however, proved to be

problematic due to cross-reactivity of the antibodies against mouse and human UGT, SULT, and

GAPDH. The same intensity was found for equal amounts of sample containing Gapdh,

GAPDH, or total Gapdh+GAPDH, namely, within the murine or humanized liver samples as

well as human liver sample (FRGN, mFRGN, hFRGN and human liver). Henceforth, the

intensities of lone bands were presented without normalization. Accordingly, the SULT1A1


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relative protein expression of hFRGN and human liver samples were similar, but the rank order

for total (Sult1a1/SULT1A1) protein expression was hFRGN > mFRGN > FRGN; and was

FRGN ≈ mFRGN > hFRGN for total (Ugt1a1/UGT1A1) and mouse Mrp2 protein expression

(Figure 9). These latter observations on the relative protein expression levels were consistent

with the higher HS formation and the slightly lower biliary excretion rates of HS and HG for

hFRGN livers (lower MRP2) compared to FRGN livers (Table 3). The data further suggests that

the higher HS formation in hFRGN was due to the unwanted contamination due to persistence of

remnant, murine Sult1a1.


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DISCUSSION

Humanized (h-chimeric) livers have been hailed as a major, improvement towards

research on anti-hepatitis drugs as well as human drug metabolism and toxicity. Indeed, viral

propagation could be maintained for > 6 months without impacting the health of the animal, and

the model demonstrates unprecedented usefulness towards the understanding the mechanisms of

Hepatitis B and C (Bissig et al., 2010). For human drug metabolism, the model facilitates the

administration of metabolites that are identified as culprits of toxicity/activity, as required in new

drug discovery processes. The preparation serves as useful, pre-clinical tools for drug-drug

interaction (Jaiswal et al., 2014), and for DILI studies on troglitazone (Barnes et al., 2014;

Samuelsson et al., 2014), and bosentin (Xu et al., 2015) and fialuridine (Xu et al., 2014).

However, human drug metabolism and toxicity studies may only be qualitatively

addressed in these chimeric liver mouse models. De Serres et al. (2011) concluded that there is

limited predictability of secondary metabolites with these chimeric mouse models. Similarly,

amide hydrolysis of GDC-0834 exposure levels failed to correlate with human data (Liu et al.,

2011). Sanoh et al. (2012) found correlation only for ibuprofen and (S)-naproxen acryl

glucuronides and other metabolites. A low repopulation in PXB livers and residual mouse

hepatocytes has been found to disrupt allometric scaling (Sanoh et al., 2015). Another drawback

is extrahepatic drug metabolism, e.g. intestinal drug metabolism that is murine in origin,

although this aspect may be partially addressed upon knocking down murine Cyp3a (Kato et al.,

2015). In our present study, we show that the presence of remnant native mouse hepatocytes

(Figure 6) in hFRGN liver could contribute to in vivo liver drug metabolism and confound the

prediction of human drug metabolism and pharmacokinetics. Moreover, we add another factor,

namely, the notable variability in gene expression among hFRGN recipients of the same or


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different donor hepatocytes (Figure 8); there is a cycling process of the hFRGN hepatocytes

when the resultant livers are harvested and implanted into new hFRGN recipients for another

cycle of repopulation. The recycling process not only contribute to the variability of human

genes among hFRGN livers, but may dilute pharmacogenetics aspects pertaining to enzymes

such as CYP2C19 and CYP2C9 towards S-mephenytoin and diclofenac metabolism; it appeared

that pharmacogenetic differences of these enzymes are preserved chimeric TK-NOG mouse

recipients (Hu et al., 2013).

hFRGN livers, comprising of both human SULT1A1 and mouse Sult1a1 (Figure 9),

exhibited a higher sulfation activity towards harmol (Table 3). Since there was little difference

in SULT1A1, UGT1A1, and MRP2 relative protein expression between hFRGN livers and

human liver tissue, differences in sulfation rate in mouse perfusion studies between FRGN and

hFRGN are likely explained by the residual, mouse activity. Despite the higher sulfation rate of

harmol in hFRGN livers, harmol extraction ratio remained to be similar to that of FRGN livers

(Table 3); the lack of difference was due to the fact that harmol is rapidly cleared by the liver,

and its clearance is flow-limited. Because only a low contamination of murine Mrp2 was present

in hFRGN livers (Figure 9A), it may be conjectured that the functionality of murine Mrp2 is

much higher than that of human MRP2, as observed by the higher excretion rates of harmol

conjugates in FRGN livers (Table 3).

Our study suggests that implantation of foreign hepatocytes disrupted and induced loss of

the metabolic zonation for harmol conjugation with Sult1a1 and Ugt1a1 in both mFRGN and

hFRGN livers (4 months old) that had undergone repopulation for >90 days (about 13 weeks)

when compared to the FRGN livers (Table 3). The method of prograde-retrograde mouse liver

perfusion (Pang and Terrell, 1981) provides a sense of metabolic heterogeneity of the hFRGN


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and mFRGN vs. the FRGN liver, but does not discriminate against which isoforms of SULT and

UGT are active towards harmol. Rather, the method provides a sense of the overall metabolic

activity in the anterior vs. posterior regions of the liver (Pang and Terrell, 1981; Pang et al.,

1983). By contrast, Hasegawa et al. (2011) had observed preservation of a perivenous zonation

of human glutamine synthase with immunohistochemistry in TK-NOG mice at 14 weeks after

transplantation. The method is reliant on antibody interaction with the epitope of the protein

target, and reveals little on the reactivity or function of the protein (enzyme or transporter). A

possible drawback of the method is the cross-reactivity among species, and that it is difficult to

find specific antibodies to distinguish between the mouse and human. Nevertheless, our data

from prograde-retrograde perfusion clearly reflects that loss of enzymatic zonation is due to the

repopulation process and not to housing conditions.

Our comprehensive study further revealed that the microcirculation of the repopulated m-

chimeric and h-chimeric mice was intact and showed delayed wave flow patterns. The presence

of tightly packed human hepatocytes (Figure 3), however, has contributed to smaller vascular

and cellular spaces within hFRGN livers (Table 2). The liver sizes for hFRGN livers greatly

exceed those from FRGN livers. Yoshizato et al. (2012) had conjectured the existence of

interspecies difference in human and mouse liver regeneration systems, that a continuous, high

growth rate for the human hepatocytes in h-chimeric livers likely occurs due to absence of the

TGF-β signaling pathway from mouse stellate cells. This renders human hepatocytes to exist in a

highly proliferative state (Yoshizato et al., 2012). Double humanization of both liver and

hematopoietic cells in FRGN mice could overcome this defect, when human macrophages

(Kupffer cells) are found present in modified, hFRGN liver preparations (Wilson et al., 2014). It

was further noted that Fgf15, the hormonal signalling agent produced by mouse intestine and


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controls liver size, may not be recognized by the hFRGN liver, leading to hepatomegaly

(Naugler et al., 2015). Indeed levels of liver Fgf15, detected by ELISA, for FRGN livers were

almost double that of hFRGN (304 ± 49 vs. 173 ± 30 ng/mL), whereas those for FGF19 in

hFRGN livers were undetectable (data not shown).

This comprehensive study of the microcirculation and zonation of enzymes in the

hFRGN and mFRGN vs. the FRGN liver provides additional information on the usefulness of

hFRGN preparation, which has been used to provide qualitative data on human drug metabolism.

Our study suggests that there exist limitations with use of these hFRGN mice when conducting

in vivo pharmacokinetics and drug metabolism studies. First and foremost, there is copious,

residual mouse liver activity (Figure 6) and loss of liver metabolic zonation (Table 3) in hFRGN

livers, although the hepatic flow patterns have remained intact (Table 2). The consequence of

loss of metabolic zonation may lead to altered metabolic pattern of secondary metabolites (Xu et

al., 1989). Persistence of murine liver activity in hFRGN needs to be addressed to determine the

contribution of mouse vs. human liver in assessing human hepatic elimination. Moreover, due to

overpopulation of human hepatocytes in hFRGN livers (Figure 3), an appropriate scaling method

is needed to scale clearance when the number of hepatocyte per g liver is increased, as is the size

of the liver. In addition, recognition of a reduced bile flow rate in hFRGN livers would be

needed for drugs that are eliminated via bile.

We also found a dramatically higher pool size of human bile acids produced by the

humanized chimeric liver (data not shown). The accumulation of highly, human specific bile

acid production in hFRGN livers may further result in changes of the baseline expression of

human transporters and enzymes in hFRGN livers. There is evidence to suggest a

miscommunication between mouse intestine and the humanized liver, and lack of suppression of


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CYP7A1, the rate-limiting enzyme in cholesterol metabolism, in hFRGN liver (Ellis et al., 2013;

Naugler et al., 2015). The lack of communication between the human liver and mouse intestine

may lead to disruption of normal bile acid homeostasis and alteration in the basal expression of

transporters and enzymes in the liver and extrahepatic tissues. Regulation of liver genes by the

farnesoid X receptor (FXR) in the face of bile acid accumulation would also need to be

appraised.


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Acknowledgement We thank Dr. Andreas J. Schwab for discussion on MID superposition.


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Author contributions Participated in research design: Chow, Evans, Silva, Pang. Conducted experiments: Chow, Wang, Quach. Contributed new reagents or analytic tools: Chow, Wang, Quach, Tang, and Li. Performed data analysis: Chow, Wang, Quach, and Pang. Wrote or contributed to the writing of the manuscript: Chow, Wang, Quach, and Pang.

Disclosures: All authors disclose no conflicts.


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Footnote We gratefully acknowledge research funding from Janssen Pharmaceutical Inc., Spring House, Pennsylvania.


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Legends for Figures

Figure 1. Different housing conditions for (A) FRGN and (B) chimeric (mFRGN and hFRGN) mice in second and subsequent weeks. After the first week, FRGN mice were given a weekly cycle of sterile filtered drinking water, either containing NTBC alone or NTBC, 3% dextrose, and SMX/TMP antibiotic mixtures (A), whereas mFRGN and hFRGN mice were given a weekly cycle of sterile filtered drinking water, either containing 3% dextrose and SMX/TMP antibiotic mixtures or water alone (B). In these periods, mFRGN and hFRGN mice were also given a diluted (25% in drinking water) STATTM high calorie liquid diet supplement in 5LJ5 chow, every two or three days. The mFRGN and hFRGN mice were not allowed NTBC in drinking water for a total of 24 days. From days 25 to 27, all mice (including FRGN mice) received only sterile filtered drinking water and 5LJ5 chow prior to the experiment. This house scheduling scheme was recommended by Yecuris Corporation to ensure maximization of liver repopulation in mFRGN and hFRGN livers and minimized NTBC and antibiotic effects in those livers.

Figure 2. Liver microcirculation studies, comprising of (A) MID outflow dilution profiles of 3H-H2O, 14C-sucrose, 51Cr-labeled RBC, and 125I-labeled albumin in representative CD-1 (n=6), FRGN (n=5) and hFRGN (n=6) perfused livers (each graph represents one representation of a mouse), showed that all dilution profiles were unimodal and were increasingly dispersed for albumin, sucrose, and water; (B) superposition of the 3H-H2O, 14C-sucrose, and 125I-labeled albumin curves onto the 51Cr-RBC curve showed delayed wave form flow patterns.

Figure 3. Hematoxylin and eosin (H & E) staining (100x magnification; scaling from 0-250 µm with interval of 50 µm) of CD-1, FRGN, mFRGN, and hFRGN livers (one representation). The H & E staining of 400X magnification (scaling from 0-50 µm with interval of 10 µm) is represented on the top right corner of each picture. Normal morphology was observed in CD-1 and FRGN livers, whereas hFRGN hepatocytes appeared tightly packed. Figure 4. Prograde and retrograde liver perfusion (representative preparation for each flow pattern) of 50 µM harmol in FRGN (n=8), mFRGN (n=6), and hFRGN (n=6) livers for three perfusion periods of 40 min each (either P-R-P or R-P-R). The formation rate of harmol glucuronide (HG), harmol sulfate (HS), summed outflow rates in bile and perfusate at steady-state, expressed as % rate in, and the HS/HG ratio were denoted as orange, purple, and blue solid symbols, respectively. The total HS formation rate and HS/HG ratio were higher in mFRGN and hFRGN livers when compared to those of FRGN livers, and no difference was observed between mFRGN and hFRGN livers.

Figure 5. Mouse liver mRNA expression of nuclear receptors, enzymes, transporters and others in FRGN and mFRGN liver tissues and FRGN and Rosa (pooled 129S7 mouse hepatocytes as donor for mFRGN) hepatocytes (left panel), and the correlation of those genes as a representation of mFRGN/FRGN liver and Rosa/FRGN hepatocyte ratios (right panel). A comparison between the mRNA expression ratios of mFRGN/FRGN liver and Rosa/FRGN hepatocyte was conducted to explain the observed differences in mRNA expression between mFRGN and FRGN livers. For the right panel, each dot denotes the averaged ratio of each gene.


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Data for the right panel show that, after correction for the higher mRNA expression observed for Rosa donor hepatocytes, the mRNA expression ratios of most genes were close to the line of symmetry (dashed line). The data suggests that differences in mRNA expression observed in mFRGN vs. FRGN livers were due to higher gene expression of the implanted (Rosa) hepatocytes. Data was presented as mean±SEM; * P< .05 denotes comparison between FRGN and mFRGN livers using 2-tailed Student’s t-test; # P< .05 denotes comparison between Rosa and FRGN hepatocytes using 2-tailed Student’s t-test.

Figure 6. Remnant mouse mRNA expression in hFRGN (n=11) vs. FRGN livers (n=8). Mouse liver genes, especially Sult2a1, and Mdr1a showed higher mRNA expressions in hFRGN vs. those in FRGN livers; β-actin was used for normalization. Data was presented as mean±SEM; #P<0.05, between FRGN and hFRGN livers, using 2-tailed Student’s t-test. Figure 7. Human mRNA expression for hFRGN livers and human liver tissue. mRNA expression levels of hFRGN (n=11) were compared against those from human liver tissue (n=6); GAPDH was used for normalization. Levels of hFRGN were generally higher than those in human liver tissue, except MRP3. Data was presented as mean±SEM; †P<.05, between human tissue and hFRGN livers, using 2-tailed Student’s t-test. Figure 8. Variability of human liver mRNA expression in hFRGN livers (>80% repopulation) originating from three different, human donors (HHM05010, n=2; HHF07007, n=5; HHF17006, n=3). Note that hepatocytes from hFRGN livers may be harvested for inoculation/implantation of other hFRGN recipient mice to provide the same lineage. Figure 9. Mouse and human protein expression in FRGN (n=4), mFRGN (n=7), hFRGN livers (n=6-8), and human liver tissue (n=6). (A) Both mouse and human Mrp2/MRP2, Sult1a1/SULT1A1, Ugt1a1/UGT1A1 protein expression in FRGN, mFRGN, and hFRGN livers and human liver tissue (n=6) were detected by anti-mouse antibodies, which cross-reacted with human proteins. Note that for total Sult1a1/SULT1A1 protein expression, hFRGN> mFRGN > FRGN; total Ugt1a1/UGT1A1 protein expression was lower for hFRGN compared to that of FRGN. (B) anti-human antibodies were used for the detection of MRP2, SULT1A1, and UGT1A1 for hFRGN livers vs. human liver tissue samples (lone band intensity was presented without normalization to GAPDH; see text for details). Protein expression was generally similar between hFRGN and human liver tissue, except for UGT1A1, which was higher in hFRGN livers. Data was presented as mean±SEM; *P< .05, between FRGN and mFRGN livers; #P<.05, between FRGN and hFRGN livers; †P<.05, between human and hFRGN livers, using a 2-tailed Student’s t-test.


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Table 1. hFRGN human donor and human liver tissue information. All hFRGN livers were >80% human repopulated, as reported by Yecuris Corporation

NA denotes not available

Mouse ID Human

Donors ID Donor Information

Human Albumin (mg/ml)

Experiments

hFRGN livers

hFRGN3 HHF07007 Female, Caucasian, 7 years old 7.2 Harmol perfusion; Liver mRNA & protein



hFRGN6 HHF07007 Female, Caucasian, 7 years old 5.3 Liver mRNA & protein; Histology

hFRGN7 HHF07007 Female, Caucasian, 7 years old 15.5 Liver mRNA & protein

hFRGN10 HHM05010 Male, Caucasian, 5 years old 4.8 Harmol perfusion; Liver mRNA & protein

hFRGN11 HHM05010 Male, Caucasian, 5 years old 4.0 Liver mRNA & protein; Histology

hFRGN12 HHM05010 Male, Caucasian, 5 years old 4.0 Histology





hFRGN21 HHM05010 Male, Caucasian, 5 years old 12.5 MID




hFRGN25 HHF17006 Female, Caucasian, 17 years old 5.1 MID

hFRGN26 HHF17006 Female, Caucasian, 17 years old 4.7 MID

hFRGN6 HHF07007 Female, Caucasian, 7 years old 5.3 mRNA & protein

hFRGN7 HHF07007 Female, Caucasian, 7 years old 15.5 mRNA & protein

hFRGN11 HHM05010 Male, Caucasian, 5 years old 4.0 mRNA & protein

hFRGN13 HHF17006 Female, Caucasian, 17 years old 6.3 mRNA & protein hFRGN15 HHF07007 Female, Caucasian, 7 years old 5.7 mRNA & protein

Human liver tissues

Human Liver 1 H1016 Female, Hispanic, 64 years old NA (not available)a Liver mRNA & protein Human Liver 2 H1028 Male, Hispanic, 43 years old NA Liver mRNA & protein Human Liver 3 H1041 Male, Caucasian, 53 Years old NA Liver mRNA & protein

Human Liver 4 H1047 Male, Hispanic, 44 years old NA Liver mRNA & protein

Human Liver 5 H1057 Female, Caucasian, 33 years old NA Liver mRNA & protein

Human Liver 6 H1072 Female, Caucasian, 40 years old NA Liver mRNA & protein


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Table 2. Physiological liver volumes and stationary/sinusoidal blood or plasma or water space in CD-1, FRGN, and hFRGN livers.

CD-1 FRGN hFRGN

Distribution spaces (ml/g liver)

Total water space = Vtotal water

1.03 ± 0.04 1.02 ± 0.05 0.79 ± 0.04*,#

Total sucrose space = Vsuc

0.28 ± 0.02 0.26 ± 0.03 0.22 ± 0.03

Sinusoidal blood volume Vain

0.19 ± 0.02 0.10 ± 0.03# 0.08 ± 0.02#

Total albumin space = Valb

0.28 ± 0.02 0.26 ± 0.03 0.17 ± 0.02*,#

Sucrose Disse space = VDisse,suc

0.13 ± 0.03 0.17 ± 0.03 0.15 ± 0.02

Albumin Disse space = VDisse, alb

0.13 ± 0.03 0.17 ± 0.01 0.10 ± 0.01*

Cellular water space = Vcell water

0.74 ± 0.04 0.75 ± 0.02 0.56 ± 0.02*,#

Sinusoidal blood or plasma or water space

t0 (H2O), sec 52.0 ± 1.3 49.7 ± 1.2 50.6 ± 1.5

t0 (surcose), sec 56.1 ± 2.1 50.0 ± 2.9 49.8 ± 2.3

t0 (albumin), sec 52.1 ± 0.7 49.0 ± 2.5 51.1 ± 1.1

γH2O

[ratio of (intracellular water + Disse water spaces) to

the sinusoidal water space] 2.94 ± 0.29 3.20 ± 0.41 3.23 ± 0.20

γsuc

(ratio of sucrose Disse space to plasma space) 0.51 ± 0.09 0.36 ± 0.02 0.54 ± 0.09

γalb

(ratio of albumin Disse space to plasma space) 0.19 ± 0.00 0.20 ± 0.04 0.21 ± 0.03

Data was presented as mean±SEM (n=5-6). P < 0.05 using 2-tailed Student’s t-test: #, CD-1 vs. FRGN or hFRGN; *, FRGN vs. hFRGN

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Table 3. Single pass perfused mouse liver data with 50 µM harmol perfused with prograde or retrograde flow at 2.5 ml/min.

FRGN mFRGN hFRGN

Prograde (P) Retrograde (R) Prograde (P) Retrograde (R) Prograde (P) Retrograde (R)

Extraction Ratio (E)a 0.633 ± 0.032 0.723 ± 0.018* 0.610 ± 0.011 0.603 ± 0.023† 0.697 ± 0.023 0.714 ± 0.031

Sulfation Rate (HS)b

(% Rate in) 4.8 ± 0.3% 3.1 ± 0.3%* 13.2 ± 1.5%# 12.1 ± 0.5%† 8.1 ± 0.9% 8.2 ± 2.0%†

Glucronidation Rate (HG)b (% Rate in)

62.9 ± 4.0% 69.6 ± 2.3% 47.4 ± 2.2%# 48.0 ± 2.0%† 62.0 ± 2.7% 63.9 ± 4.5%†

HS/HG Ratio 0.078 ± 0.007 0.045 ± 0.004* 0.303 ± 0.053# 0.263 ± 0.018† 0.133 ± 0.022# 0.142 ± 0.054

Bile flow rate (µl/min/g) 0.56 ± 0.06 0.50 ± 0.03 0.56 ± 0.10 0.52 ± 0.09 0.23 ± 0.04d,# 0.16 ± 0.02d,*,†

% HS excreted into bilec 63.4 ± 3.3% 46.2 ± 1.9%* 65.2 ± 4.8% 55.5 ± 5.9% 8.9 ± 1.1%# 6.0 ± 2.1%†

% HG excreted into bilec 29.7 ± 2.8% 16.4 ± 1.8%* 41.6 ± 6.6%# 33.1 ± 5.5%† 3.5 ± 1.0%# 1.6 ± 0.2%†

Data are mean±SD of 6 perfused livers; HS is harmol sulfate; HG is harmol glucuronide Note: Only data of first and second periods (first 80 min) was reported; data for period three was not used due to a slight loss of viability; the direction of flow: prograde or retrograde was assigned randomly for the first and second periods. P <.05: * P flow vs. R flow FRGN, paired t-test; # FRGN vs. mFRGN or hFRGN, P flow, t-test or ANOVA; † FRGN vs. mFRGN or hFRGN, R flow, t-test or ANOVA a E=(CIn-COut)/CIn. at steady-state, where CIn and COut are the steady-state blood concentrations of harmol b steady-state formation rate, or sum of perfusate rate out (QCOut)+biliary excretion rate, expressed as % rate in (Q x CIn), where Q is the blood perfusate flow rate c steady-state biliary excretion rate divided by formation rate of conjugate




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