Simultaneous characterization of progenitor cell compartments in adult human liver

Simultaneous Characterization of Progenitor Cell

Compartments in Adult Human Liver

Laura Porretti,1 * Alessandra Cattaneo,1 Federico Colombo,1 Raffaella Lopa,1 Giorgio Rossi,2

Vincenzo Mazzaferro,3 Carlo Battiston,3 Gianluca Svegliati-Baroni,4 Francesco Bertolini,5

Paolo Rebulla,1 Daniele Prati1,6

� AbstractThe human liver is a complex tissue consisting of epithelial, endothelial, hematopoietic,and mesenchymal elements that probably derive from multiple lineage-committed pro-genitors, but no comprehensive study aimed at identifying and characterizing intrahe-patic precursors has yet been published. Cell suspensions for this study were obtainedby enzymatic digestion of liver specimens taken from 20 patients with chronic liver dis-ease and 13 multiorgan donors. Stem and progenitor cells were first isolated, amplified,and characterized ex vivo according to previously validated methods, and then opti-mized flow cytometry was used to assess their relative frequencies and characterize theirimmunophenotypes in the clinical specimens. Stem and progenitor cells committed tohematopoietic, endothelial, epithelial, and mesenchymal lineages were clearly identifia-ble in livers from both healthy and diseased subjects. Within the mononuclear liver cellcompartment, epithelial progenitors [epithelial cell adhesion molecule (EpCAM)1/CD49f1/CD291/CD452] accounted for 2.7–3.5% whereas hematopoietic (CD341/CD451), endothelial [vascular endothelial growth factor-2 (KDR)1/CD1461/CD452],and mesenchymal [CD731/CD1051/CD90 (Thy-1)1/CD45 2] stem cells and progeni-tors accounted for smaller fractions (0.02–0.6%). The patients’ livers had higher per-centages of hematopoietic and endothelial precursors than those of the donors. In con-clusion, we identified and characterized precursors committed to four different lineagesin adult human liver. We also optimized a flow cytometry approach that will be usefulin exploring the contribution of these cells to the pathogenesis of liver disease. ' 2009

International Society for Advancement of Cytometry

� Key termsstem and progenitor cells; human liver; flow cytometry

ADULT human liver is a complex tissue consisting of epithelial, endothelial, hemato-

poietic, and mesenchymal elements. This supports the view that the intrahepatic

stem and progenitor cell compartment probably consists of a heterogeneous pool

with various molecular and functional markers. Studies characterizing hepatic stem

cells and progenitors have so far mainly concentrated on the epithelial compartment,

and generally used human fetal or rodent liver (1,2); only a few have identified and

characterized the hematopoietic (3,4), endothelial (5), and mesenchymal lineages

(6), although it is well known that their progenies are deeply involved in the pathoge-

nesis of liver disease (7).

Despite recent growth of interest in liver stem and progenitor cell research and

great expectations concerning the potential of these cells in the field of regenerative

medicine (8), we are unaware of comprehensive studies of the entire stem/progenitor

cell pool. This could be due to the rare occurrence of these cells in liver tissue, and

the possible overlap in the immunological profiles of the cells committed to different

lineages (9–11), thus making their identification difficult.

1Center of Transfusion Medicine, CellularTherapy and Cryobiology, IRCCSFondazione Ospedale Maggiore Policlinico,Mangiagalli e Regina Elena, Milan, Italy

2Liver Transplant Unit, IRCCS FondazioneOspedale Maggiore Policlinico,Mangiagalli e Regina Elena, Milan, Italy

3Gastroenterology Surgery Department,National Cancer Institute, Milan, Italy

4Clinic of Gastroenterology, Universit�aPolitecnica delle Marche, Ancona, Italy

5Hemato-oncology Department, EuropeanOncology Institute, Milan, Italy

6Department of Transfusion Medicineand Hematology, Ospedale A. Manzoni,Lecco, Italy

Received 20 August 2009; RevisionReceived 2 November 2009; Accepted 3November 2009

Additional Supporting Information may befound in the online version of this article.

Grant sponsor: COPEV (Comitato per laPrevenzione dell’Epatite Virale), Milan;Grant sponsor: Associazione ItalianaStudio Fegato (AISF).

*Correspondence to: Laura Porretti, DSc,Center of Transfusion Medicine, CellularTherapy and Cryobiology, IRCCSFondazione Ospedale MaggiorePoliclinico, Mangiagalli e Regina Elena,Via F. Sforza 35, 20122 Milan, Italy

Email: [email protected]

Published online 3 December 2009 in WileyInterScience (www.interscience.wiley.com)

DOI: 10.1002/cyto.a.20834

© 2009 International Society forAdvancement of Cytometry

Original Article

Cytometry Part A � 77A: 31�40, 2010

However, new-generation FCM techniques allowing the

immunological detection of larger numbers of cells have sig-

nificantly improved cell characterization by making it possible

to analyze up to six to eight markers on the same cell. As a

result, FCM has become the gold standard to characterize clin-

ical grade cell preparations in several fields of regenerative

medicine. In our own laboratories, it has allowed the identifi-

cation of rare stem cells and progenitors in peripheral blood

and parenchymal tissues (12–14), thus prompting us to refine

FCM protocols for liver stem cell analysis with a view to future

clinical applications.

The aim of this study was to implement a FCM approach

to investigate the heterogeneous pool of intrahepatic progeni-

tor cells suitable for both healthy and diseased livers. To this

aim, intrahepatic progenitor cells committed to four different

lineages were first isolated, amplified, and characterized ex

vivo using previously validated culture conditions and anti-

body staining techniques (15–18). This enabled us to select an

optimal panel of monoclonal antibodies that was then used to

determine the relative frequencies of the four lineages and fur-

ther characterize them in clinical specimens.

MATERIALS AND METHODS

Tissue Specimens

We analyzed liver samples taken from 13 organ donors

(seven males/six females; median age 56 years, range 33–82) at

the time of organ harvest, and from 20 patients with chronic

liver disease (11 males/9 females; median age 66 years, range

48–82) who had undergone liver resection because of isolated

hepatocellular carcinoma. The liver disease was cryptogenic in

three cases, and related to Hepatitis C virus (HCV) in eleven,

Hepatitis B virus (HBV) in three and alcohol abuse in three.

The study was approved by the local Ethics Committee,

and informed consent was obtained from the patients and the

donors’ relatives.

Cell Isolation

The cells were isolated within 2–8 hours of sample collec-

tion. After tissue digestion (5), the cell suspension was resus-

pended in HBSS (Euroclone, Pero, Italy), and centrifuged at

50 g for 1 min to remove mature hepatocytes. The mononu-

clear liver cells (MNLCs) were pelleted by centrifuging the su-

pernatant at 650g for 5 min. Cell counts and viability were

assessed by means of trypan blue staining.

In Vitro Cell Cultures

The samples from three organ donors were used for spe-

cific culture assays of the four liver stem/progenitor cell com-

partments.

Liver hematopoietic stem cells (L-HSCs). CD341 or

CD1331 cells were immunoselected by means of magnetic cell

separation (Miltenyi Biotech, Florence, Italy), and their purity

was evaluated by FCM. Next, 200 CD341 or CD1331 cells

were added to 2 ml of culture medium (Methocult, StemCells

Technologies, Milan, Italy) in Petri dishes, and then incubated

in humidified air with 5% CO2 at 378C. After 14 days, colonies

were identified using an inverted microscope: Burst Forming

Unit-Erythroid (BFU-E) as closely packed red/orange cells,

Colony Forming Unit-Granulocyte/Monocyte (CFU-GM) as

flat, nonhemoglobinized colonies consisting of translucent

cells, and mixed colonies containing elements of both (4).

Liver endothelial (L-EnPCs) and epithelial (L-EpPCs) pro-

genitor cells. MNLCs were plated at 378C onto 6-well tissue

plates precoated with human collagen (StemCells Technolo-

gies) for L-EnPCs or uncoated for L-EpPCs, and cultured as

described by Yoder et al. (16) and Duret et al. (17), respec-

tively. After 24 hours of culture, nonadherent cells were dis-

carded.

L-EnPCs were cultured with complete endothelial growth

medium 2 (EGM-2, Lonza, Treviglio, Italy); colonies of endo-

thelial cells appeared between 5 and 22 days of culture and

were identified as well-circumscribed monolayer cells.

L-EpPCs were cultured with minimal essential medium

supplemented with 10% FCS (Invitrogen, San Giuliano Mila-

nese, Italy), 20 ng/ml hepatocyte growth factor (Peprotech,

Segrate, Italy), 10 ng/ml epidermal growth factor (Peprotech),

25 mM glucose, 1 lM hydrocortisone, 10 lg/ml insulin, 50

lg/ml albumin-linoleic acid, 0.1 lM selenium acid, 0.75 mg/

ml zinc sulfate, 10 mM nicotinamide (all from Sigma-Aldrich,

Milan, Italy), streptomycin and penicillin (Invitrogen) (L-

EpPC medium). Media were changed three times a week. L-

EnPCs and L-EpPCs were counted at each passage and, at the

first and fifth passages, characterized for the expression of var-

ious markers (1,2,16,17,19–21).

At fifth passage L-EpPCs were cultured in L-EpPC me-

dium without serum (17) for 3 weeks, and then the activity of

cytochrome P450 enzymes was evaluated (15). For this pur-

pose, cells were washed with PBS and then left overnight at

378C at 5% CO2 with 0.02 mM 5-(and 6)-chloromethylfluor-

escein diethyl ether (Invitrogen). This substrate is converted

by cytochrome P450 in a fluorescent product detectable by flu-

orescence microscopy.

Liver mesenchymal stem cells (L-MSCs). Cell suspensions

were plated at a density of 5 3 106/cm2 on plastic flasks. After

24 hours, nonadherent cells were removed and MSC Growth

Medium BulletKit (Lonza) was added and replaced twice a

week (18). After 3 weeks, the confluent cells with a fibroblastic

morphology were split; at the first and fifth passages the cells

were evaluated by FCM and fluorescence microscopy and cul-

tured at the fifth passage under different conditions in order

to evaluate their osteogenic and adipogenic (18), endothelial

(16), and hepatic (17) differentiation capacity. In particular,

cells were then cultured for 3 weeks with the following specific

media: MSC Osteogenic or Adipogenic Differentiation Bullet-

Kit (Lonza), EGM-2 medium and L-EpPC medium, respec-

tively. Differentiation was assessed by Alizarin red (Sigma),

Red-O-oil (Sigma), CD31/CD144/vascular endothelial growth

factor receptor-2 (KDR) expression, and cytochrome P450

activity, respectively.

ORIGINAL ARTICLE

32 Progenitor Cell Characterization in Human Liver

FCM Characterization of Cultured Cells

A total of 1 3 105 liver cells/tube were stained at 48C for

20 min with 10 ll of 7-AAD (Becton Dickinson Biosciences,

BD, San Jose, CA) in order to distinguish dead cells, and with

appropriate mAbs (Supporting Information, Table 1S) conju-

gated with different fluorochromes. The samples were

acquired and analyzed on a FACSCanto I flow cytometer

(standard configuration) with FACSDiva 5.0.3 software (BD).

Quality control included regular check-up with 7-Color Setup

Beads (BD). To avoid channel overlapping, fluorescence com-

pensation was initially assessed creating an automated six

color compensation matrix specific for MNLCs. However, flu-

orescence compensation was further checked and adjusted in

each sample during data analysis.

Immunofluorescence Microscopy of Cultured Cells

Fixed plated cells (4% paraformaldehyde or 70% ethanol)

were permeabilized using HEPES-Triton X-100 buffer and

then immunostained with anti-cytokeratin (CK)-18, anti-a1-

antitrypsin (Sigma), anti-CK19 (Novocastra, Newcastle, UK),

anti-albumin (Dako-Cytomation, Milan, Italy), anti-CD31,

anti-CD146, anti-CD144, and anti-b-catenin (BD) mAbs. The

cell nuclei were stained with DAPI (Sigma) and evaluated with

a fluorescence microscope (DM IRE2, Leica Microsystems,

Wetzlar, Germany).

FCM Characterization of Dissociated Tissues

Samples from liver tissues were used to characterize the

hematopoietic, endothelial, epithelial and mesenchymal com-

partments by means of FCM. Specific analyses were made

with reference to the antigen profiles previously determined

on cultured cells.

Four tubes per sample were set up with at least 106 cells/

tube: Tube 1: Thy-1/ATP Binding Cassette sub-family G mem-

ber 2 (ABCG2)/7-AAD/CD34/CD133-2/CD45 (L-HSCs).

Tube 2: CD146/KDR/7-AAD/CD34/CD133-2/CD45 (L-

EnPCs). Tube 3: CD49f/CD29/7-AAD/CD56/CD133-1/CD45

(L-EpPCs). Tube 4: CD105/CD73/7-AAD/ CD34/ Thy-1/

CD45 (L-MSCs).

Appropriate isotype controls for all mAbs were used to

measure the non-specific binding on MNLCs. The four cell

compartments were further evaluated for the expression of

other antigens (Supporting Information, Table 1S) in order to

characterize their immunophenotype in more detail.

Statistical Analysis

The data are expressed as median and range. The groups

were compared using the Mann-Whitney test; P values of

\0.05 were considered statistically significant.

RESULTS

In Vitro Cell Cultures

L-HSCs. CD341/CD451, but not CD1331/CD451 cells, pro-

duced CFUs, a few myeloid and many erythroid colonies, after

14 days of culture (Fig. 1, panel A).

L-EnPCs. Upon the fifth passage in culture (doubling time,

50–70 hours), the flattened morphology and KDR1/CD1461/

CD311/CD1441/CD452 phenotype of the cells were similar

to those of circulating endothelial colony-forming cells

(ECFCs) (16) (Fig. 1, panels B–D). Moreover, more than 95%

of the cultured L-EnPCs expressed CD34 (Fig. 1, panel D) but

not CD133-2 (data not shown). This feature distinguishes cir-

culating from bone marrow immature endothelial progenitors,

as reported by Mobius-Winkler (22).

L-EpPCs. A confluent monolayer of cells with typical cuboid

morphology was obtained after 3 weeks of culture (Fig. 2,

panel A). The cells were replated five times (doubling time 160

hours), and evaluated for marker expression. Between the first

and fifth passages, they showed uniform CK19 and CK18

staining, membrane-localized b-catenin, and weak albumin

reactivity; they were not a-1-antitrypsin positive after the first

passage and only weakly positive after the fifth passage (Fig. 2,

Table 1. Commitment of intrahepatic stem cells towards different lineages

DONORS (n 510) PATIENTS (n 520) P

Median (range) Median (range)

Hematopoietic stem cells

CD341/CD451 0.05 (0.01–0.2) 0.2 (0.03–1) 0.01

CD133-21/CD451 0.08 (0.01–0.33) 0.08 (0.01–1) NS

Endothelial progenitors

KDR1/CD1461/CD451 0.025 (0–0.22) 0.11 (0–0.17) NS

KDR1/CD1461/CD452 0.02 (0–0.13) 0.13 (0.01–0.77) 0.02

Epithelial stem cells

CD291/CD49f1/CD452 3.5 (0.7–13.6) 2.7 (0.1–18.4) NS

Mesenchymal stem cells

CD731/CD1051/CD452 0.6 (0.4–2.5) 0.6 (0.1–3.7) NS

Median percentages (range) of freshly isolated mononuclear liver cell fraction.

The groups were compared using the Mann-Whitney test.

P values of\0.05 were considered significant.

ORIGINAL ARTICLE

Cytometry Part A � 77A: 31�40, 2010 33

Figure 1. Liver hematopoietic and endothelial progenitor cells. (A) L-HSCs: Erythroid, myeloid, and mixed colonies of CD341/CD451 cells

after 14 days culture in methylcellulose. (B) L-EnPCs: Representative cell morphology of endothelial progenitors at the first (left) and fifth

culture passage (right) (original magnification 320). (C) CD31 (left) and CD144 (right) immunofluorescence staining of cultured L-EnPCs at

the fifth culture passage (original magnification 320). (D) FCM analysis of cultured endothelial progenitors at fifth passage: KDR and

CD146 (upper panel), CD45, CD144, CD31, and CD34 antigen expression (middle and lower panels). In all histograms grey line represents

isotype control. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

ORIGINAL ARTICLE


Figure 2. Liver epithelial progenitors and mesenchymal stem cells. (A) Cell morphology (310) of L-EpPCs at the first (left panel) and fifth

culture passage (middle panel), and L-MSCs morphology at the fifth culture passage (right panel, 320). (B) L-EpPCs immunofluorescence

staining at the first (upper panels) and fifth passage (lower panels): albumin (green)/CK19 (red) (left panels), CK18 (green)/a1-antitrypsin (red)

(middle panels), b catenin (green) (right panels), and DAPI (blue); magnification 320 (for negative control and single staining see Supporting

Information, Fig. 4S). (C) FCM analysis of L-EpPCs: CD29 and CD49f antigen expression after one (upper left panel) and five culture passages

(upper right panel); CD166 overlay expression on first (blue) and fifth (red) passage cells (lower panel). (D) Cytochrome P450 expression after

hepatic differentiation of fifth passage L-EpPCs (left panel), negative control (right panel); original magnification 320. (E) L-MSCs at fifth cul-

ture passage: CD105 and CD73 expression (upper-left panel); Thy-1 (upper-right panel) and CD34 (lower panel) expression on CD1051/CD731

cells. (F) Differentiation capability: osteogenic differentiation (Alizarin red staining, original magnification 320, left panel) and adipogenic dif-

ferentiation (Red-O-oil staining, right panel, original magnification 320) of fifth passage L-MSCs. In all histograms grey line represents isotype

control. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

ORIGINAL ARTICLE


panel B). The CD291/CD49f1/CD452 phenotype was main-

tained by more than 90% of the cells at the fifth passage (Fig.

2, panel C). Moreover, freshly isolated L-EpPCs expressed

epithelial cell adhesion molecule (EpCAM) and CD133-1,

which have recently been identified as markers of hepatic pro-

genitor cells (20,21,23), but this expression progressively

decreased in culture, whereas the expression of neural cell ad-

hesion molecule (NCAM) and CD166 antigens increased dur-

ing the five culture passages (Fig. 2, panel C). The cultured

cells gradually became larger and flatter and a number of

binuclear cells appeared. After hepatic maturation, P450 cyto-

chrome enzymes were expressed in 70% of the cells (Fig. 2,

panel D).

L-MSCs. During culture, the cells acquired their characteristic

spindle shape and showed the CD1051/CD731/Thy-11/

CD452/CD342 antigen profile (Fig. 2, panels A and E). The

cells harvested at the fifth passage had differentiated to osteo-

cytes and adipocytes (Fig. 2, panel F). No mature endothelial

and epithelial markers, including P450 cytochrome activity,

were observed after lineage specific commitments.

Clinical Specimens

The liver specimens weighed between 0.5 g and 388 g

(median 2.64 g). A median of 5.8 3 106 liver cells were iso-

lated per gram of tissue (0.7–55 3 106), and median cell via-

bility was 86% (70–95%). There was no significant difference

in recovery or viability rates between donors and patients. The

analyses of all staining tubes were performed on viable cells

(not stained with 7-AAD) with a scatter profile typical of

stem/progenitor cells: i.e. intermediate/high FSC and low/in-

termediate SSC (10,12,14). Viable MNLCs were then divided

into CD451 and CD452 cells.

Flow cytometry L-HSC analysis. CD341/CD451 cells (Fig. 3,

panel A) were significantly increased in the patients MNLCs

(0.08%) as compared with the donors (0.05%), whereas

CD133-21/CD451 cells accounted for about 0.08%, without

any differences between donors and patients (Table 1). As

shown in Figure 3, panel A, the CD341/CD451 cells were dis-

tinct from the CD133-21/CD451 cells: very few CD451 cells

(1–2%) coexpressed CD34 and CD133-2 antigens. Both sub-

populations expressed the same levels of Thy-1 (17%, 9–53%)

and ABCG2 (4%, 1–35%) (Supporting Information, Fig. 1S).

Flow cytometry L-EnPC analysis. KDR1/CD1461/CD452

cells were the output of cultured endothelial progenitors or

ECFCs (16); another cell population expressing CD45 antigen

(KDR1/CD1461/CD451) and usually indicated as colony

forming unit-endothelial cells (CFU-ECs), was also identified

(16). The analysis therefore included a first gate for KDR1

cells, followed by a quadrant analysis for the expression of

CD146 and CD45 antigens (Fig. 3, panel B). The ECFC subset

was significantly increased in the patients as compared to the

donors (0.13% vs. 0.02%), but there were no between-group

differences in the CFU-EC population (Table 1). CD34 and

CD133-2 antigens were expressed by 30–40% of the ECFCs

and 1–5% of the CFU-ECs (Supporting Information, Fig. 2S).

Flow cytometry L-EpPC analysis. Epithelial progenitor cells,

which were defined by the expression of the two integrins

CD49f and CD29 in the absence of CD45, accounted for 2.7-

3.5% of the MNLCs (Fig. 3, panel C) without differences

between patients and donors (Table 1). About 15–20% of the

CD49f1/CD291/CD452 cells were positive for NCAM anti-

gen, and about 50–70% for CD133-1 (Fig. 3, panel C). All of

the L-EpPCs were EpCAM positive, whereas there was no

CD34 antigen reactivity (Supporting Information, Fig. 3S).

Flow cytometry L-MSC analysis. MSCs, identified as

CD1051/CD731/Thy-11/CD452/CD342 cells (Fig. 3, panel

D), accounted for 1–10% of the CD452 MNLCs.

Table 2 summarizes the immunophenotypical character-

istics of the intrahepatic stem cells and progenitors committed

to different lineages. We found that a combination of five

mAbs (EpCAM/KDR/CD34/Thy-1/CD45) could identify the

four lineages in a single tube reaction on the basis of their dif-

ferent mAb reactivities. Figure 4 represents 3D plots showing

the four lineage-committed stem/progenitor cells.

DISCUSSION

Necroinflammation, fibrogenesis, neoangiogenesis, and

accelerated hepatocellular turnover are common features of

almost all forms of chronic liver disease, and are respectively

sustained by hematopoietic, mesenchymal, endothelial, and

epithelial elements. Although all of these elements derive from

lineage-specific progenitors, there is no comprehensive study

identifying and characterizing the heterogeneous hepatic pro-

genitor cell pool.

This study confirmed that our FCM analysis, based on an

optimized mAb panel, is useful in identifying rare intrahepatic

progenitors, committed to four lineages, both in healthy and

diseased livers. These progenitors are capable of expansion

and differentiation to their lineage commitment.

In order to ensure an adequate number of observations,

at least four million cells were evaluated for each liver speci-

men and characterized on the basis of more than one marker

in order to improve the specificity of the detection (9,24).

We first identified the hematopoietic and endothelial

lineages which, given their common mesodermal origin, share

some antigens. As they differently express CD45, KDR and the

endothelial/mesenchymal marker CD146 (16), these antigens

were used to distinguish them. Our data indicate that the HSC

compartment, firstly evidenced by Crosbie et al. (4), accounts

for 0.05–0.2% of intrahepatic mononuclear cells, 10 times

more than in peripheral blood. Interestingly, we observed that

CD341/CD451 cells were approximately four times more fre-

quent in the patients with chronic liver disease than in the

donors, probably because of increased cell trafficking due to

chronic inflammation.

Unlike bone marrow and peripheral blood, in which

60–70% of CD34bright cells express CD133 antigen (25), less

than 3% of the intrahepatic CD341 cells coexpressed CD133

ORIGINAL ARTICLE


Figure 3. Flow cytometry analysis of mononuclear liver cell suspensions immediately after tissue dissociation. (A) Analysis of L-HSCs:

CD45 expression (upper left panel); CD34 and CD133-2 expression on CD451 cells (upper right panel); isotype controls for APC and PE-Cy7

on CD451 cells (lower panel). (B) Analysis of L-EnPCs: KDR expression (orange dots) on MNLCs (upper left panel); CD45 and CD146 expres-

sion on KDR1 cells (upper right panel); isotype controls for PE on MNLCs and for FITC and APC-Cy7 on KDR1 cells (lower panels). (C) Anal-

ysis of L-EpPCs: CD45 staining for the identification of non-hematopoietic cells (CD452) in MNLCs (upper left panel); CD49f and CD29

expression on CD452 cells (red dots, upper middle panel); expression of CD56 and CD133-1 antigens on CD49f1/CD291 cells (upper right

panel); isotype controls for FITC and PE on CD452 cells and for PE-Cy7 and APC on CD49f1/CD291 cells (lower panels). (D) Analysis of L-

MSCs: CD45 staining (left panel); CD105 and CD73 expression on CD452 cells (right panel). [Color figure can be viewed in the online issue,

which is available at www.interscience.wiley.com.]

ORIGINAL ARTICLE


antigen. However, a cell population bearing a CD1331/

CD451/CD342 phenotype and without hematopoietic poten-

tial in culture was also clearly identifiable. The hematopoietic

colonies were mainly erythroid, which fits well with the evi-

dence that the majority of BFU-E and CFU-mixed are pro-

duced by CD341/CD1332 stem cells (25). A small population

of ‘‘early’’ stem cells (about 4%) coexpressing ABCG2, which

is the molecular determinant of the side population (SP) phe-

notype (26), could be identified in almost all liver specimens,

suggesting the presence of cells with potent blood-repopulat-

ing potential (27) and capable of differentiating into hepato-

cyte-like cells in vitro (28).

Table 2. Patterns of mAb reactivities (mean percentages, n 5 3) on freshly isolated intrahepatic stem cells committed to different

lineages

COMMITMENT OF STEM CELLS

AND PROGENITORS CD34 CD45 CD133 ABCG2 THY-1 KDR CD146 CD31 CD105 CD73 CD29 CD49F EPCAM CD56 CD166

Hematopoietic 33 100 56 4 17 \1 0 0 0 NT NT NT 0 NT NT

Endothelial 35 0 70 NT 8 100 93 100 98 NT NT NT 0 NT NT

Epithelial 0 0 63 \1 \1 0 20 0 0 100 100 99 99 28 45

Mesenchymal 0 0 0 0 100 \1 83 0 87 88 100 0 \1 5 12

NT, not tested.

Figure 4. Three-dimensional plot of liver stem and progenitor cells analyzed in a single tube combination of EpCAM/KDR/7-AAD/CD34/

Thy-1 (CD90)/CD45. FACSDiva files, adequately compensated as reported in the text, were exported as FCS 2.0 in Paint-a-gate software.

The following gating strategy was then applied: L-HSCs (viable CD451/CD341 cells, yellow dots), ECFCs (viable CD452/KDR1 cells, black

dots), CFU-ECs (viable CD451/KDR1 cells, blue dots), L-EpPCs (viable CD452/EpCAM1 cells, green dots), L-MSCs (viable CD452/Thy-11

cells, violet dots). [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

ORIGINAL ARTICLE


Since no studies on liver endothelial progenitors have

been published yet, we took advantage of the recent work by

Yoder et al. (16) and used the same culture conditions to iso-

late and maintain L-EnPCs. The morphology and phenotype

of the CD1461/KDR1/CD311/CD1441/CD452 cell popula-

tion were similar to those of circulating ECFCs, the only sub-

population capable of forming vessels in vivo (16). They were

more frequent in patients than in donors, which is in line with

the current view that hepatic inflammation and fibrogenesis

are frequently associated with angiogenesis (29).

The liver specimens also contained another cell popula-

tion coexpressing endothelial and hematopoietic markers

(KDR1/CD1461/CD451), which represented approximately

50% of the total KDR1 population. This immunophenotype

corresponded to CFU-EC, which are currently considered to

be descendents of hematopoietic stem cells that may facilitate

neoangiogenesis but cannot form perfused vessels in vivo (16).

We first cultivated epithelial progenitor cells under selec-

tive conditions (17) and obtained clusters with a typical mor-

phology that lasted for more than two months. After evaluat-

ing a large panel of markers because no single antigen had

been definitely validated (30), we observed that all of the cul-

tured cells expressed the a6 and b1 integrins, fundamental in

ensuring cell adhesion and clonality of hepatic progenitor cells

(1,2). EpCAM and CD133 antigens were also expressed on

freshly isolated L-EpPCs, but their reactivity progressively

decreased as the cells matured, as also suggested by the gradual

increase in CD166 and a1-antitrypsin positivity during

culture. In line with this, upon the fifth culture passage, the

cells were frequently binucleated and after maturation they

expressed P450 cytochrome, which are characteristic of mature

hepatocytes (31). The typical mesenchymal marker Thy-1, which

was not initially expressed by fresh L-EpPCs, progressively

appeared during culture, thus suggesting epithelial to mesenchy-

mal transition; however, the cells maintained the membrane

expression of b-catenin typical of epithelial cells (32).

In the clinical specimens, L-EpPCs accounted for the lar-

gest cluster of committed lineage within the MNLCs. Almost

all of these cells coexpressed EpCAM and a variable propor-

tion of CD166, thus suggesting the presence of cells at differ-

ent stages of maturation. In line with the results of recent stu-

dies (25,33), CD34 was not expressed at membrane level

whereas NCAM was detected in some cells.

MSCs were originally identified in bone marrow, but it is

currently agreed that they exist in all organs and tissues (34);

however, adult human liver MSCs have so far been poorly

described. Using the classic isolation protocol, we cultured

mesenchymal elements from liver specimens with the typical

hallmarks of MSCs, i.e. the expression of CD73, CD105, and

Thy-1, the absence of hematopoietic markers and osteogenic

and adipogenic differentiation potential (34). Mature liver

mesenchymal cells (mainly stellate cells) provide the basis for

the progression of fibrosis (35). Thus, a better characterization

of L-MSCs seems particularly important to improve our

knowledge of the pathogenesis of chronic liver disease.

In conclusion, healthy and diseased adult human livers

contain not only tissue-specific, but also mesenchymal, endo-

thelial, and hematopoietic progenitors, which can be distin-

guished by immunophenotyping and FCM analysis. A method

of single-tube staining with five-colour monoclonal antibo-

dies, that we defined in this study, can be used for future

investigations aimed at exploring the role and reciprocal inter-

actions of different subfractions of stem/progenitor cells in the

pathogenesis of liver disease.

ACKNOWLEDGMENTS

The authors are grateful to the Nord Italia Transplant

program (NITp) for providing liver tissue from organ donors

unsuitable for transplantation.

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Simultaneous characterization of progenitor cell compartments in adult human liver

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