Transplantation of endothelial cells corrects the phenotype in hemophilia A mice

ORIGINAL ARTICLE

Transplantation of endothelial cells corrects the phenotypein hemophilia A mice

V. KUMARAN,* D . BENTEN ,* A . FOLLENZ I , * B . JOSEPH ,* R . SARKAR� and S . GUPTA**Marion Bessin Liver Research Center, Departments of Medicine and Pathology, Albert Einstein College of Medicine, Bronx, NY;

and �Department of Genetics, University of Pennsylvania School of Medicine, Philadelphia, PA, USA

To cite this article: Kumaran V, Benten D, Follenzi A, Joseph B, Sarkar R, Gupta S. Transplantation of endothelial cells corrects the phenotype in

hemophilia A mice. J Thromb Haemost 2005; 3: 2022–31.

Summary. Background: The deficiency of factor VIII, a

co-factor in the intrinsic coagulation pathway results in

hemophilia A. Although FVIII is synthesized largely in the

liver, the specific liver cell type(s) responsible for FVIII

production is controversial. Objective: This study aimed to

determine the cellular origin of FVIII synthesis and release in

mouse models. Methods: We transplanted cells into the

peritoneal cavity of hemophilia A knockout mice. Plasma

FVIII activity was measured using a Chromogenix assay

2–7 days after cell transplantation, and phenotypic correction

was determined with tail-clip challenge 7 days following cell

transplantation. Transplanted cells were identified by histologic

and molecular assays. Results: Untreated hemophilia A mice,

as well as mice treated with the hepatocyte-enriched fraction,

showed extensive mortality following tail-clip challenge. In

contrast, recipients of unfractionated liver cells (mixture of

hepatocytes, liver sinusoidal endothelial cells (LSEC), Kupffer

cells, and hepatic stellate cells) or of the cell fraction enriched in

LSECs survived tail-clip challenge (P < 0.001). FVIII was

secreted in the blood stream in recipients of unfractionated liver

cells, LSECs and pancreatic islet-derivedMILESVEN1 (MS1)

endothelial cells. Although transplanted hepatocytes main-

tained functional integrity in theperitoneal cavity, these cells did

not produce detectable plasma FVIII activity. Conclusions:

The assay of cell transplantation in the peritoneal cavity showed

that endothelial cells but not hepatocytes produced phenotypic

correction in hemophilia A mice. Therefore, endothelial cells

should be suitable additional targets for cell and gene therapy in

hemophilia A.

Keywords: endothelial cell, hemophilia A, hepatocyte.

Introduction

The X-linked bleeding disorder, hemophilia A, afflicts one in

5000 males [1]. This disorder results from either absence of or

abnormal forms of factor VIII, a non-enzymatic co-factor in

the coagulation cascade for the serine protease, FIX. Main-

tenance of FVIII levels in people with hemophilia A requires

repeated administration of products derived from pooled

plasma with inactivation of pathogens, although some risk of

disease transmission remains, and use of recombinant FVIII is

expensive. Although the gene therapy approach has shown

promise in a FVIII knockout mouse model [2], progress in this

area has been relatively slow [3–9].

To retarget therapeutic efforts, it will be critical to define

which liver cell type produces FVIII. The liver being the major

source of FVIII synthesis, normal levels of FVIII were restored

following orthotopic liver transplantation in people with

hemophilia A [10,11]. In contrast, transplantation of lymphatic

tissue, lung or spleen was less effective [12–15]. However,

identification of the liver cell type(s) responsible for FVIII

synthesis and storage has been controversial. Previous reports

demonstratedFVIII expression inhepatocytes,whichconstitute

approximately 60%of the livermass, aswell as in less abundant

cells, such as liver sinusoidal endothelial cells (LSEC) and

Kupffer cells [16–19]. The ambiguity concerning the site of

FVIII production seems unresolved, casting doubt over the role

of hepatocytes, as hepatic failure with loss of parenchymal liver

cells resulted in increased, as well as decreased, plasma FVIII

levels [20,21].

Here,weattemptedtodemonstrate the typeofcell responsible

forFVIII synthesis andstorage,with thehope that identification

of appropriate liver cell types will enable cell therapy as a viable

option for hemophilia A. Also, such cells could be targeted for

FVIII gene delivery to pursue the gene therapy approach.

Although FVIII assays have been used to demonstrate FVIII

production in cultured mouse LSEC [18], such assays may not

be informative in all cell types because it is known that gene

expression is rapidly perturbed in culturedprimary hepatocytes.

In view of these limitations, we considered using a simple cell

transplantation assay, where healthy cells are transplanted

into the peritoneal cavity [22]. This permits monitoring of

Correspondence: Sanjeev Gupta, Marion Bessin Liver Research

Center, Albert Einstein College of Medicine, Ullmann 625, 1300

Morris Park Avenue, Bronx, NY 10461, USA.

Tel.: 718-430-3309; fax: 718-430-8975; e-mail: [email protected]

Received 5 August 2004, accepted 28 April 2005

Journal of Thrombosis and Haemostasis, 3: 2022–2031

� 2005 International Society on Thrombosis and Haemostasis

transplanted cell survival, as well as release of secreted proteins

in the blood, which would help identify specific cell type(s) that

may serveas therapeutic targets forhemophiliaA.Weexamined

animal outcomes following transplantation of unfractionated

liver cells, and of cell fractions enriched in either hepatocytes or

LSEC.Moreover,wemeasuredplasmaFVIII activityproduced

by transplanted cells, including cell fractions enriched in

hepatocytes and endothelial cells. We found that transplanted

endothelial cells secreted FVIII and corrected the bleeding

phenotype in hemophilia A mice. In contrast, hepatocytes

engrafted in the peritoneal cavity but did not reconstitute FVIII

deficiency in these mice.

Materials and methods

Animals

TheAnimalCareandUseCommitteeatAlbertEinsteinCollege

of Medicine approved the studies. Hemophilia A mice were

originally producedby inserting aneomycin gene into the 3¢-endof the 16th exon of FVIII gene and were in the 129-C57BL/6J

background [2]. Donor C57BL/6J-TgN(MT-nLacZ)204Bri

mice and 129-Gt(ROSA)26 Sor mice were from Jackson Labs

(Bar Harbor, ME, USA) and wild-type C57BL/6 mice were

from the National Cancer Institute (Bethesda, MD, USA).

In some donor mice, Kupffer cells were labeled with carbon

12 h before cell isolation by intrasplenic injection of carbon

particles enriched from India ink No. 17 (Pelikan, Hannover,

Germany), as described previously [23].

For transplantation, cells were mixed with Cytodex 3

microcarriers (Amersham Pharmacia Biotech, Uppsala,

Sweden) in a ratio of 3 · 106 cells mL)1 rehydrated micro-

carriers and injected intraperitoneally using a 20-gauge needle.

The cells were suspended in RPMI 1640 culture medium

without any serum and transplanted within 1–2 h after

isolation following collagenase perfusion or cell fractionation.

Recipient animals were not treated with FVIII either prior to

or subsequent to cell transplantation. The tail-clip challenge

was designed to analyze hemophilia A mice 1 week after cell

transplantation by cutting 1 cm of the tail and monitoring

animal survival over 24 h [2].

Liver cell fractions

The liver was digested by antegrade perfusion via portal vein at

5 mL min)1 with 0.03% collagenase type-1 (Washington Bio-

chemical Corp., Lakewood, NJ, USA), essentially as described

[22]. To obtain the total liver cell fraction, the cell suspension

obtained from the collagenase-perfused liver was centrifuged

under 350 g for 10 min at 4 �C. To isolate the hepatocyte

fraction, cells were centrifuged under 50 g for 5 min at 16 �Cfollowed by two washes with plain Leffert’s buffer at 4 �C(SigmaChemical Co., St Louis,MO,USA). To obtain the non-

hepatocyte liver cell fraction, the supernatant remaining after

hepatocytes had been pelleted was centrifuged under 350 g for

10 min at 16 �C, washed with plain Leffert’s buffer once and

cells were resuspended in phosphate-buffered saline, pH 7.4

(PBS; Gibco, Grand Island, NY, USA). Finally, to isolate the

LSEC fraction, the non-hepatocyte cell fraction was resuspend-

ed in 2 mL PBS and processed withmodification of procedures

described previously [24,25]. Percoll (Sigma) was mixed with

10X PBS in 1:9 (v/v) ratio and this mixture was used to prepare

75% and 35% Percoll solutions by adding 1X PBS. Percoll

gradientswere preparedby layering 1.5 mLof 35%Percoll over

2 mL of 75% Percoll. Cells suspended in 1 mL PBS were then

layered above Percoll and centrifuged under 900 g for 20 min

without using brakes. Cells separated in two bands, with the

upper band-containing hepatic stellate cells and Kupffer cells,

and the lower band, LSEC. The LSEC layer was carefully

aspirated and resuspended in an equal volume of PBS followed

by centrifugation under 900 g for 10 min.All cell fractionswere

finally resuspended in RPMI 1640 culture medium without

serum.

MS1 cells

This cell line was originally from American Type Culture

Collection (CRL-2279, ATCC, Manassas, VA, USA). MILE

SVEN 1 (MS1) cells were isolated from C57BL/6 mouse

pancreatic islet endothelial cells and transduced with a

temperature-sensitive SV40 large T antigen construct con-

taining the neoR gene [26]. Resistant colonies were isolated

and screened for acetylated low-density lipoprotein (LDL)

uptake. MS1 cells are immortal with properties of endothelial

cells, including uptake of acetylated LDL, expression of

FVIII-related antigen, and low-level expression of vascular

endothelial growth factor (VEGF) and VEGF receptor. The

cells are not tumorigenic in vivo. To mark MS1 cells with a

green fluorescent protein (GFP) transgene, we obtained

vesicular stomatitis virus (VSV)-pseudotyped lentiviral stocks

by co-transfecting pCCL.PPT.hPGK.GFP.Wpre transfer

construct, the third generation packaging constructs

pMDLg/pRRE and pRSV-REV, and the pMD2.G envelope

into 293T cells, as described [27]. Incubation of MS1 cells

with GFP-lentivirus (using MOI of 2, as tittered on HeLa

cells by end-point titration) produced GFP expression in

>90% cells by flow cytometry and these GFP-MS1 cells

were expanded for studies.

Cell culture

Cell viability was analyzed by trypan blue dye exclusion.

Hepatocytes were cultured at 2.5 · 104 cells cm)2 in RPMI

1640 containing 10%fetal bovine serum (FBS), andLSECwere

plated at 5–10 · 104 cells cm)2 on dishes coated with type-1

collagen in 199 medium (Gibco) containing 15% FBS,

100 lg mL)1 of endothelial cell growth supplement (E2759,

Sigma), and antibiotics. MS1 cells were cultured in Dulbecco’s

modified Eagle’s medium with 10% FBS and antibiotics. To

determine FVIII activity, cells were cultured for 24 h and cell

culturemediumwas harvested for analysis with aChromogenix

assay (see below).

Cell therapy for hemophilia A 2023


ICAM-1 and CD31 expression in LSEC

For immunostaining, cells were fixed in cold acetone for

10 min, washed with PBS and incubated in 2% FBS and 1%

bovine serum albumin (BSA). For flow cytometry, cells were

fixed in ethanol for 10 min. For intercellular adhesion

molecule-1 (ICAM-1) staining, cells were blocked in 0.05%

avidin and incubated with biotinylated anti-ICAM-1 (1:500,

BD Pharmingen, San Diego, CA, USA), for 1 h at room

temperature [18]. This was followed by incubation of cells for

1 h in the dark with avidin-fluorescein isothiocyanate (FITC;

1:300, Sigma) at room temperature. Cells were washed thrice

between procedures in PBS (hepatocytes, 50 g; LSEC, 350 g).

For CD31 staining, we incubated fixed cells with rat antimouse

CD31 (BD Biosciences, Mountain View, CA, USA) diluted

1:100 in 5% donkey serum followed by incubation with

Cy2-conjugated donkey antirat IgG (1:100; Jackson Immuno-

Research Labs, Inc., West Grove, PA, USA) for 1 h each at

room temperature. For fluorescence microscopy, cells were

counterstained with 4¢,6-diamidino-2-phenylindole (DAPI-

antifade) (Rainbow Scientific, Windsor, CT, USA).

Cytochemical and GFP stainings

Tissues were collected in either 4% paraformaldehyde in PBS

or frozen in methylbutane at )80 �C. Cryosections were

obtained. Histochemical stainings for glycogen, glucose-

6-phosphatase (G-6-P), and bacterial b-galactosidase (LacZ)

were as previously described [28]. For GFP immunostaining,

tissue samples were frozen in methylbutane at )80 �C and

7 lm cryosections fixed in paraformaldehyde were blocked

with 3% goat serum in PBS containing 0.1% Triton X-100

(PBS-T) for 1 h at room temperature. This was followed by

incubation with rabbit anti-GFP in PBS-T (1:300; Molecular

Probes, Eugene, OR, USA) for 1 h at room temperature.

After washing with PBS, sections were incubated with

FITC-conjugated antirabbit IgG (1:100; Sigma) for 1 h at

room temperature and counterstained with DAPI-antifade.

DNA PCR and RT–PCR analysis

Genomic DNA was extracted with the DNeasy kit (Qiagen,

Valencia, CA, USA) and RNA was extracted with the

Trizol reagent (Invitrogen Corp., Carlsbad, CA, USA).

Reverse transcription (RT) used the Omniscript RT kit

(Qiagen). The assays utilized 0.5–1 lg DNA and Hi-Fi

platinum Taq polymerase (Invitrogen). The primers for

the MT-nLacZ transgene were as described [29] with

DNA denaturing at 94 �C for 3 min, followed by 35 cycles

at 94 �C · 30 s, 55 �C · 1 min, 72 �C · 1 min, and

72 �C · 7 min for extension. RT–polymerase chain reaction

(PCR) for FVIII utilized published sequences with PCR for

40 cycles at 94 �C · 30 s, 51 �C · 30 s, 72 �C · 1 min, and

72 �C · 7 min [18]. RT–PCR for mouse GAPDH used 40

cycles at 94 �C · 30 s, 55 �C · 30 s, 72 �C · 1 min, and

72 �C · 7 min with the following primers: forward,

5¢-GGGTGGAGCCAAACGGGTC-3¢ and reverse,

5¢-GGAGTTGCTGTTGAAGTCGCA-3¢. The expected

product sizes for LacZ, FVIII cDNA and GAPDH were

400, 500, and 532 bp, respectively. PCR for FVIII mutation

in exon 16 knockout mice [2] used 40 cycles at 94 �C · 30 s,

52 �C · 45 s, 72 �C · 1 min, and 72 �C · 7 min with

primers A, 5¢-TGTGTCCCGCCCCTTCCTTT-3¢, B,

5¢-TGCAAGGCCTGGGCTTATTT-3¢, and C, 5¢-GAG-

CAAATTCCTGTACTGAC-3¢, where primer C is a reverse

primer that anneals in both normal and knockout mice,

primer B is a sense primer annealing in normal mouse to

generate a 680 bp product and primer A is a sense primer

that anneals only in the knockout mouse to amplify a

420 bp product.

FVIII assay

Cell culture mediumwas stored at )80 �C until analysis. Blood

samples were rapidly collected from the orbital sinus in tubes

prefilled with 3.8% sodium citrate to the necessary level for

obtaining the final sodium citrate concentration of 0.38% and

plasma was stored at )80 �C.Factor VIII activity was assayed using the Coatest C/4 kit

(Chromogenix, Milan, Italy). The samples were assayed

based on standard curves generated in the low range of

between 0.01 and 0.2 IU mL)1, which corresponded to

between 1% and 20% of normal plasma FVIII activity,

respectively. Standard curves were generated by mixing

pooled plasma from multiple healthy C57BL/6J mice (100%

FVIII activity) with plasma from multiple hemophilia A

mice (0% FVIII activity) to achieve the desired level of

FVIII activity. The assigned percentage values of the

standard dilutions were those obtained from normal plasma

containing 1 IU FVIII activity mL)1 (100%). Normal

mouse plasma was initially prediluted in hemophilia mouse

plasma in the ratios of 1:5 for 20%, 1:10 for 10%, 1:20 for

5%, 1:40 for 2.5%, and 1:100 for 1% content. This was

followed by final dilutions at 1:80 with 25 lL of the

prediluted standard in 2 mL buffer. As a negative control,

undiluted hemophilia mouse plasma was used for 0% of

normal FVIII activity. Coatest assay was carried out by the

acid-stopped method using 100 lL of the mixture (buffer/

plasma). Absorbance was measured at 405 nm. A linear

best-fit graph was generated with these standards to

establish linear relationship between the absorbance at

405 nm and FVIII activity within the range of the curve.

All test samples from mice (25 lL plasma) were diluted in

buffer at 1:80 and assayed with this procedure. Cell culture

medium was not diluted with buffer. The percentage of

FVIII activity for the corresponding absorbance was read

against the standard curve.

Statistics

Data are shown as mean ± SEM when in triplicate and as

mean ± SD otherwise. The significance of differences was

2024 V. Kumaran et al


analyzed by t- and chi-square-tests using SIGMASTAT software

(Jandel Scientific, San Rafael, CA, USA). P-values of <0.05

were considered significant.

Results

Characterization and analysis of cell populations

Typically, we isolated 3–5 · 107 hepatocytes and 1.5–3 · 106

LSEC from mouse livers. Trypan blue dye exclusion showed

that the viability of mouse hepatocytes was lower (65%–85%)

than that of LSEC or hepatic stellate cells (95%–100%,

P < 0.001, v2-test).To define the presence of cross-contaminating cell types in

cell fractions, we analyzed expression of glycogen, G-6-P

(hepatocyte markers), ICAM-1 (non-hepatocyte marker

expressed in endothelial and other cells), CD31 (endothelial

marker), vitamin A autofluorescence (hepatic stellate cell

marker), and carbon particle incorporation (marker of Kupffer

cell activity; Fig. 1). The hepatocyte fraction contained 98%–

99% epithelial cells with only 0.4% ± 0.4%Kupffer cells. No

LSEC with ICAM-1 or CD31 staining and stellate cells with

vitamin A autofluorescence were identified in the hepatocyte

fraction. The LSEC fraction was highly enriched, with only

1.6% ± 1.1% hepatocytes-containing glycogen or G-6-P,

2% ± 0.63% carbon-containing Kupffer cells, and no hepatic

stellate cells. Flow cytometry using anti-ICAM-1 established

that 99% cells in the LSEC fraction were ICAM-1-reactive.

LSEC showed immunoreactivity for CD31. In contrast, the

hepatocyte fraction was negative for ICAM-1-reactive cells,

verifying absence of LSEC or other ICAM-1-expressing cells.

Enriched hepatocytes and LSEC promptly attached and

survived for several days in culture dishes, indicating cell

viability. LSEC showed proliferationwithmore confluent areas

in the presence of endothelial cell growth supplement.

RT–PCR verified that hepatocytes and LSEC expressed FVIII

immediately after cell isolation, as well as after culture for up to

3 days. Cultured MS1 cells expressed FVIII mRNA (see

below). Analysis of FVIII activity in cell culture medium after

24 h in replicate dishes with the Chromogenix assay showed

FVIII production in cultured LSEC (mean, 0.037 IU mL)1/

1 · 106 cells plated), as well asMS1 andGFP-MS1 cells (mean,

0.07 IU mL)1/1 · 106 cells plated), albeit not in cultured

hepatocytes. Taken together, these data indicated that our

cells were suitable for animal studies.

Transplantation of liver cells in hemophilia A mice

We used 42 donor mice and 61 hemophilia A mice grouped as

follows: untreated mice, eight; recipients of the total liver cell

fraction, three; recipients of the hepatocyte fraction, 11;

recipients of non-hepatocyte liver cell fraction, 10; recipients

of LSEC, 23 mice; and recipients of MS1 cells, six mice. As a

proof of principle, our initial goal was to determine whether the

total liver cell fraction would engraft in the peritoneal cavity

and produce FVIII demonstrated by subjecting hemophilic

mice to a tail-clip challenge. Should there be the desired

phenotypic correction, our second objective was to identify the

specific cell fractions capable of FVIII synthesis in animals.

Initial studies in control hemophilia A mice (n ¼ 6) demon-

strated that intraperitoneal injection of either saline or RPMI

1640 culture medium without serum (vehicle) did not produce

overt bleeding or mortality, which permitted us to transplant

cells without perioperative FVIII administration, which is

otherwise needed for surgery in hemophilia A mice. Also,

FVIII activity was undetectable in untreated hemophilia mice

and animals succumbed to bleeding following tail clipping, as

observed previously [2].

To first study the therapeutic potential of cells in

hemophilia A mice, we transplanted the total liver cell

population isolated from individual syngeneic C57BL/6

donors without fractionation (range: 4–8 · 107 cells, viability:

>80%). In recipients of unfractionated total liver cells, tail-

clip challenge led to survival in two of three (66%)

hemophilia A mice, whereas zero of eight (0%) control mice

receiving microcarriers in vehicle alone survived after tail

clipping, P < 0.001, v2-test on percentages. Transplanted

cells were identified histologically in the peritoneal cavity,

where transplanted cell-microcarrier conglomerates were in

proximity to the omentum, pancreas, and liver. Therefore, we

considered that intraperitoneal cell transplantation followed

by tail clip-induced bleeding assay was appropriate for

analyzing the function of cell fractions.

Transplanted cell engraftment and function in hemophilia A

mice

To demonstrate transplanted cell survival, FVIII protein

processing and correction of the hemophilia A phenotype in

the FVIII knockout mouse model, we utilized two strains of

healthy syngeneic donors in the C57BL/6 background. To

identify transplanted cells by PCR, we used MT-nLacZ

transgenic donor mice [29]. In these mice, LacZ is expressed

in some hepatocytes under control of a zinc-inducible promo-

ter, although transgene sequences are present in all cells.

Finally, to identify transplanted cells histologically, we used

Rosa26 donor mice [30], which express LacZ throughout cells

in all tissues in a cell type-independent fashion.

We established several lines of evidence to demonstrate

that transplanted cells survived and engrafted in the perito-

neal cavity of hemophilia A mice. In microcarrier conglom-

erates recovered from hemophilia A mice, 3–7 days after

transplantation of MT-nLacZ cells, LacZ DNA was dem-

onstrated in six of seven samples from hepatocyte recipients

and in four of four samples from LSEC recipients (Fig. 2A).

As microcarrier conglomerates contain transplanted, as well

as stromal and other host cells [22], we co-amplified

sequences from exon 16 of the FVIII gene and neo gene

cassette in exon 16 of the mutated FVIII gene to identify

transplanted cells and native hemophilia mouse cells, respect-

ively (Fig. 2B). These experiments showed both transplanted

and native cells in microcarrier conglomerates. Transplanted



hepatocytes have previously been demonstrated to maintain

hepatic gene expression in the peritoneal cavity, including at

the mRNA and protein levels [22].

Histologic analysis of the recovered microcarriers provided

further evidence for transplanted cell survival (Fig. 3). Trans-

planted hepatocytes were readily identified on the basis of their

distinct morphology, with abundant eosinophilic cytoplasm,

rounded nuclei, and prominent nucleoli (Fig. 3A). Glycogen

staining provided additional morphologic evidence for engraft-

ment of transplanted hepatocytes (Fig. 3B). However, LSEC

are much smaller than hepatocytes (Fig. 1A,D), similar to

stromal cell types, and do not offer characteristic morphologic

features at the light microscopy level. However, use of Rosa26

cells was helpful in identifying transplanted cells in recipients of

both hepatocytes and LSEC (Fig. 3E,F). To verify that MS1

cells survived after transplantation in hemophilia A mice, we

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Fig. 1. Characterization of mouse liver cells. Panels A–C show primary hepatocytes and D–F primary liver sinusoidal endothelial cells (LSEC).

Hepatocytes showed larger size, contained glycogen andwere not contaminated with LSEC (A and B), as further verified by the absence of cells with CD31

staining. Rosa26 hepatocytes-expressing LacZ are shown after 3 days in culture (C). The LSEC fraction contained intercellular adhesion molecule

(ICAM-1-; D) and CD31 (E)-expressing cells, as seen under fluorescence microscopy with DAPI counterstaining of cells in panel (E). Inset in panel (D)

shows magnified view of LSEC with ICAM-1 staining. Panel (F) shows LSEC 3 days after culture with both single cells (arrowheads) and clusters of

proliferating cells (arrows). Flow cytometry for ICAM-1 in LSEC is shown in panel (G), where LSEC not incubated with ICAM-1 antibody (negative

controls) are at the top (G–A and G–C) and samples incubated with anti-ICAM-1 are at the bottom (G–B and G–D). Panels (G–A) and (G–C) show

forward scatter (cell size) and side scatter (cytoplasmic complexity) of the cell preparations, whichwere similar. Panels (G–G) and (G–D) show cell numbers

on the y-axis and fluorescein isothiocyanate (FITC) fluorescence, indicating intensity of ICAM-1 expression on the x-axis. Flow cytometry for the

hepatocyte fraction is not shown because these cells were negative for ICAM-1 staining, similar to panel (G–C). FVIII reverse transcription polymerase

chain reaction (RT–PCR) in cells immediately after isolation and intact liver is shown in panel (H) with a 500-bp size expected product. These data

verified that isolated cells were viable, enriched in hepatocytes and LSEC in respective fractions, and produced FVIII mRNA to proceed with analysis of

cell function in vivo. Original magnification: A–F, ·400; E is at higher magnification.



utilized GFP-transduced cells. The microcarrier conglomerates

recovered from hemophilia mice showed presence of trans-

planted MS1 cells with GFP expression (Fig. 3G–I). These

findings were in agreement with the survival and continued

function in transplanted hepatocytes and endothelial cells in the

peritoneal cavity.

Phenotypic correction in hemophilia A mice

We determined whether cell transplantation altered mortality

in hemophilia Amice following tail-clip challenge. As indicated

earlier, animals treated with onlymicrocarriers and vehicle died

within 6 h after tail clipping. Only one of six mice (17%) that

received hepatocytes from an entire liver, 3–5 · 107 viable cells

each, survived tail clipping (Table 1). In contrast, six of seven

animals (86%) transplanted with the non-hepatocyte cell

fraction, which contained LSEC, Kupffer cells, hepatic stellate

cells, and other cells, survived tail clipping (P ¼ 0.001).

Moreover, recipients of LSEC showed dose-dependent survival

following transplantation of 0.5 · 106 or more endothelial

cells. In some of these animals, plasma FVIII activity was

detected 7 days following cell transplantation, when animals

were subjected to tail-clip challenge, although sufficient plasma

samples were not available from all animals for FVIII assays.

To demonstrate whether transplanted cells secreted FVIII in

the blood stream, we studied additional animals 48 h following

cell transplantation (Fig. 4). In recipients of 3–4 · 107 hepato-

cytes (n ¼ 5), plasma FVIII activity was not detectable in any

of the mice. On the contrary, transplantation of the total non-

hepatocyte cell fraction showed FVIII activity in three of three

mice (1%, 2.4%, and 7.6%). Plasma FVIII activity was also

observed in recipients of 2 · 106 LSEC, where in three of five

mice, showed 1%, 2%, and 5.5% FVIII activity. In mice

receiving five times more MS1 cells (1 · 107 cells each), six of

six animals showed plasma FVIII activity in greater amounts

(6%, 8%, 8%, 10%, 12%, and 12% FVIII activity). In

contrast, FVIII activity was not detected in untreated hemo-

philia A mice serving as negative controls. Overall, these

findings were in agreement with the maintenance of secretory

function in endothelial cells and the ability of these cells to

replenish FVIII in hemophilia mice.

Discussion

In this study, we provide evidence regarding the liver cell type

that can produce and secrete FVIII in vivo. We used liver cell

fractions, including those enriched in either hepatocytes or

LSEC, to determine whether transplanted cells produced FVIII

protein in hemophilia mice. Donor cells with specific reporters

were used to demonstrate engraftment and function of

transplanted cells. Besides identifying LSEC, as well as MS1

cells derived from pancreatic islets, as capable of FVIII

synthesis, we established that primary LSEC corrected the

bleeding phenotype of hemophilia A mice, suggesting that

endothelial cells should be appropriate targets for FVIII cell

and gene therapy.

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Fig. 2. Transplanted cell survival in hemophilia A mice. Polymerase chain reaction (PCR) was used to identify transplanted cells with the LacZ transgene

(MT-nLacZ donors) (A) or wild-type FVIII sequences (B). Data are from animals 7 days after cell transplantation. Panel (A) shows a 400 bp band

expected in MT-nLacZ cells, lane 2; and its absence in hemophilia A mice (lane 1). LacZ sequences were present in DNA from recovered microcarrier-cell

conglomerates after transplantation of MT-nLacZ hepatocytes, lanes 4–7, and MT-nLacZ liver sinusoidal endothelial cells (LSEC), lanes 8–11. (B) PCR

for FVIII gene showed only mutant sequences (420 bp band) in FVIII)/) mouse, lane 1; and both mutant and wild-type bands in a FVIII+/)heterozygous mouse, lane 2. In MT-nLacZ cell recipients, both mutated and wild-type FVIII bands were amplified, indicating transplanted, as well as

native cells, in microcarrier conglomerates.



A

mc

B

D

mc

mc mc

F

mc

H

mc

mc

G

I

C

E

mc

J

1 2 3

MWMarker

-VeCtrl

MS1Cells



A major advantage of transplanting cells into the peritoneal

cavity was the capacity of this space to accommodate

functionally intact cells in large numbers. Previous studies

established that after transplantation of hepatocytes, secreted

proteins were detected within 30–60 min in the blood of

recipient mice [31]. Moreover, transplanted hepatocytes

engrafted in the peritoneal cavity in the presence of microcar-

riers, as shown here, and maintained liver gene expression, as

well as production of secreted proteins for more than 1 week,

as reported previously with rodent, as well as human hepato-

cytes [22,32,33]. These studies included in situ hybridization to

show maintenance of liver gene expression at the mRNA level,

as well as other assays to demonstrate protein expression in

transplanted hepatocytes (e.g. albumin, G-6-P, glycogen) in the

peritoneal cavity. Similarly, non-parenchymal liver cells have

been successfully transplanted into the peritoneal cavity ofmice

[34]. The characteristic morphology of hepatocytes and visu-

alization of liver gene expression permitted us to distinguish

between transplanted cells and native stromal cells in micro-

carrier conglomerates. In addition, our studies here with

LacZ-expressing Rosa26 cells and GFP-expressing MS1 cells

further verified that additional cell types engrafted in the

peritoneal cavity of recipient mice.

Our studies were restricted to the short-term because we first

wished to identify the most effective cell type capable of

producing FVIII. In the adult liver, hepatocytes constitute 60%

and LSEC 20%of the total liver cell mass, which approximates

100 million cells in the mouse [31]. Transplantation of hepato-

cytes in the native liver leads to engraftment of only 20% or

fewer transplanted cells, because of the requirement of

translocation from hepatic sinusoids into the liver plate

structure, with liver reconstitution by, at most, 1%–2%, after

one session of cell transplantation [31]. In contrast, cells

transplanted in the peritoneal cavity are not subject to

clearance because of the restriction imposed by the requirement

for entry into the liver parenchyma.

Transplantation of 20–35 million hepatocytes represented

reconstitution of 30%–60% of the hepatocyte mass in hemo-

philia mice. We observed sheets of transplanted hepatocytes in

the peritoneal cavity. These transplanted hepatocytes are

capable of secreting soluble proteins into the bloodstream, as

shown by previous studies using transgenic hepatitis B virus

140

120

100

80

60

40

20

0n 8 5 3 5 6

MS-1LSECAll livercells

Heps

FVIII activity present

FVIII activity absent

Cell-type transplanted

Frac

tion

of h

emop

hilia

A m

ice

with

FV

III a

ctiv

ity (

% tr

eate

d)

Vehiclealone

Fig. 4. Analysis of plasma FVIII activity in treated hemophilia A mice.

The data show the fraction of animals in various groups with reconstitu-

tion of plasma FVIII activity. Plasma FVIII activity was absent in animals

treated with either vehicle alone or the hepatocyte fraction. In contrast,

100% of the animals treated with all liver cells or MILE SVEN 1 (MS1)

cells showed plasma FVIII activity. In recipients of the liver sinusoidal

endothelial cells (LSEC) fraction, plasma FVIII activity was identified in

three of five animals. The animal number in various groups is indicated.

Asterisks represent P < 0.001, using percentages, v2-test.

Table 1 Phenotypic correction of hemophilia A mice

Intervention Number of mice Survival after tail clip, n (%) P-value (v2-test)�

Vehicle (RPMI 1640 culture medium) 8 0 NA

Total liver cell fraction (3–5 · 107 cells; 60%–65% hepatocytes,

10%–15% LSEC, 2%–4% hepatic stellate cells)

3 2 (67) NA

Hepatocyte fraction (2–3.5 · 107 cells) 6 1 (17) ns

Non-hepatocyte fraction (1–2 · 107 LSEC,

hepatic stellate cells, Kupffer cells, etc.)

7 6 (86) <0.001

LSEC fraction

0.25 · 106 LSEC 6 1 (17) ns

0.5–1 · 106 LSEC 12 8 (67) <0.001

ns, non-significant; NA, not applied; LSEC, liver sinusoidal endothelial cells.�Tested on percentages vs. vehicle alone.

Fig. 3. Identification of transplanted cells in tissues. Seven days after transplantation, transplanted hepatocytes are shown with characteristic morphology

(A), including glycogen (B) in areas adjacent to microcarriers (mc) in the peritoneal cavity of hemophilia mice. (C) LacZ staining of donor Rosa26 liver

demonstrating cytoplasmic blue staining of all cells, including sinusoidal cells (inset of boxed area). LacZ staining was absent in recipients of microcarriers

alone (D). In contrast, 3 days after transplantation of Rosa26 hepatocytes (E) or liver sinusoidal endothelial cells (LSEC; F), LacZ-positive cells (arrows)

were identified in proximity to microcarriers. Panels (G–I) show green fluorescent protein (GFP)-expressing MILE SVEN 1 (MS1) cells 2 days after

transplantationwithDAPI counterstain alone (G), GFP-immunostaining (H), andmerged view (I). Panel (J) shows reverse transcription polymerase chain

reaction (RT–PCR) indicating FVIIImRNAexpression in culturedMS1 cells. Originalmagnification: A, ·200; B–I, ·400; C–F, hematoxylin counterstain.



(HBV) hepatocytes [22,32], where secreted proteins rapidly

appeared in the circulation and persisted for a few weeks.

Nonetheless, despite transplantation of hepatocytes in large

numbers and their known capacity to release secreted proteins

in the bloodstream, we failed to demonstrate phenotypic

correction or plasma FVIII activity in hemophilia A mice.

Previous studies showed that blood loss and survival in

hemophilia A mice after tail-clip challenge correlates with

therapeutic reconstitution of plasma FVIII activity [35].

However, whether hepatocytes could produce FVIII in the

liver microenvironment compared with their inability to do this

in the peritoneal cavity will be best determined by studies of

liver repopulation in hemophilia mice. Such studies will be

facilitated by the development of effective strategies to repop-

ulate the healthy mouse liver, similar to the success in

repopulating the rat liver with transplanted hepatocytes after

genotoxic perturbations of the native liver [36]. Liver repop-

ulation studies will help further exclude whether hepatocytes

could possibly reconstitute FVIII activity. Similarly, long-term

or indefinite correction of FVIII deficiency in hemophilia A

mice will benefit from further strategies, e.g. transplanted

hepatocytes survive lifelong in the rat liver [37], and whether

transplanted endothelial cells could repopulate the liver should

be of interest.

Our studies with endothelial cells, especially MS1 cells,

established that cells transplanted into the peritoneal cavity

secreted FVIII into the bloodstream. Transplantation of

fractions containing all liver cells, mixture of non-hepatocyte

cells, and LSEC ameliorated the hemophilia A phenotype. The

level of plasma FVIII activity in recipients of 0.5–1 million

LSEC was commensurate with the cell number transplanted,

which represented 2.5%–5% of the mouse LSEC mass.

Although plasma FVIII activity in hemophilia mice treated

with primary LSEC was in the low range of detection by the

Chromogenix assay, whether unidentified differences in cell

viabilitymight have interfered with the engraftment or function

of transplanted primary LSEC was not excluded. These

limitations were avoided in our studies with MS1 cells, where

transplantation of 10 million MS1 cells reconstituted up to

12% plasma FVIII activity, which should again be reasonable,

because not all cells would necessarily have survived or

functioned equally in the peritoneal cavity after transplanta-

tion. Also, FVIII production in MS1 should be noteworthy,

considering their origin from pancreatic islets, and was in

agreement with the potential of extrahepatic endothelial cells.

Moreover, in our assays, endothelial cells were more effective

than hepatocytes in producing and secreting FVIII, despite

transplantation of the latter in much larger numbers.

Physiologically, LSEC offer advantages for FVIII expres-

sion, including presence of intracellular mechanisms that

facilitate FVIII synthesis, maturation, and release. For

instance, complexing of FVIII to von Willebrand factor

(VWF), which is produced in endothelial cells and megak-

aryocytes, with storage in Weibel-Palade bodies and

a-granules, respectively, helps concentrate FVIII in sites of

vascular injury and prevents premature proteolysis [38].

Alterations in the intracellular trafficking of VWF may

perturb subcellular FVIII localization [39]. Similarly, mul-

tiple extracellular stimuli, including inflammatory mediators

and vasoactive drugs, such as desmopressin, promote release

of FVIII and VWF from intracellular stores [40]. The

significant role of VWF in FVIII stabilization was further

verified in studies of cultured human hepatocytes, where

FVIII antigen expression was not detected without the

addition of VWF to the culture medium [41]. This is in

agreement with our findings here showing the inability of

transplanted mouse hepatocytes to reconstitute plasma

FVIII activity. Whether the additional provision of VWF

to transplanted hepatocytes could be effective for this

purpose will require suitable in vivo systems, e.g. examina-

tion of liver repopulation with transplanted hepatocytes,

where release of FVIII from transplanted cells and of VWF

released from LSEC within proximity to hepatocytes could

bring FVIII and VWF together.

The liver is clearly not an exclusive source of FVIII, as

endothelial cells derived from additional sources can also

produce FVIII [16,19,42], which will be in agreement with

the efficacy of MS1 cells, and possibly accounted for

relatively lower reconstitution of FVIII activity expected

from the mass of LSEC transplanted in our studies here. In

recent studies, endothelial cells isolated from lungs were

found to engraft in pulmonary tissues [43]. Similarly,

circulating endothelial cells engraft in diseased vessels [44].

Also, hematopoietic stem cells derived from mononuclear

blood cells can produce FVIII [42]. Moreover, hematopoi-

etic stem cells can differentiate into LSEC [45], which

should offer further opportunities for therapeutic targeting

of specific cell subsets in hemophilia A.

Acknowledgements

The authors thank Ms Chaoying Zhang for excellent technical

assistance. Supported in part by NIH grants R01 DK46952

and P30-DK-41296.

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Transplantation of endothelial cells corrects the phenotype in hemophilia A mice

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