Fate of Extrahepatic Human Stem and Precursor Cells
After Transplantation into Mouse Livers
Marc Brulport,1,2 Wiebke Schormann,1,2 Alexander Bauer,1,2 Matthias Hermes,1,2 Carolin Elsner,1,2
Friedrich Jakob Hammersen,1,2 Walter Beerheide,3 Dimitry Spitkovsky,4 Wolfgang Härtig,5 Andreas Nussler,6
Lars Christian Horn,7 Jeanett Edelmann,8 Oliver Pelz-Ackermann,9 Jörg Petersen,10 Manja Kamprad,11
Marc von Mach,12 Amelie Lupp,13 Henryk Zulewski,14* and Jan G. Hengstler1,2*
In recent years, a large number of groups studied the fate of human stem cells in livers of
immunodeficient animals. However, the interpretation of the results is quite controversial.
We transplanted 4 different types of human extrahepatic precursor cells (derived from cord
blood, monocytes, bone marrow, and pancreas) into livers of nonobese diabetic/severe
combined immunodeficiency mice. Human hepatocytes were used as positive controls.
Tracking of the transplanted human cells could be achieved by in situ hybridization with alu
probes. Cells with alu-positive nuclei stained positive for human albumin and glycogen.
Both markers were negative before transplantation. However, cells with alu-positive nuclei
did not show a hepatocyte-like morphology and did not express cytochrome P450 3A4, and
this suggests that these cells represent a mixed cell type possibly resulting from partial
transdifferentiation. Using antibodies specific for human albumin, we also observed a second human albumin–positive cell type that could be clearly distinguished from the previously described cells by its hepatocyte-like morphology. Surprisingly, these cells had a
mouse and not a human nucleus which is explained by transdifferentiation of human cells.
Although it has not yet been formally proven, we suggest horizontal gene transfer as a likely
mechanism, especially because we observed small fragments of human nuclei in mouse cells
that originated from deteriorating transplanted cells. Qualitatively similar results were obtained with all 4 human precursor cell types through different routes of administration with
and without the induction of liver damage. Conclusion: We observed evidence not for
transdifferentiation but instead for a complex situation including partial differentiation and
possibly horizontal gene transfer. (HEPATOLOGY 2007;46:861-870.)
Abbreviations: CYP3A4, cytochrome P450 3A4; DAB, 3,3⬘-diaminobenzidine; NOD, nonobese diabetic; PAS, periodic acid Schiff; RAG-2, recombination activation
gene-2; SCID, severe combined immunodeficiency; uPA, urokinase plasminogen activator.
From the 1Leibniz Research Centre for Working Environment and Human Factors, Dortmund, Germany, and 2Center for Toxicology, Institute of Legal Medicine and
Rudolf-Boehm Institute of Pharmacology and Toxicology, University of Leipzig, Leipzig, Germany; 3Siam Life Science, Limited, Suriyawong Bangrak, Bangkok, Thailand;
4Institute for Vegetative Physiology, University of Cologne, Cologne, Germany; 5Department of Neurochemistry, Paul Flechsig Institute for Brain Research, University of
Leipzig, Leipzig, Germany; 6Fresenius Biotech GmbH, Bad Homburg, Germany; 7Institute of Pathology, Division of Gynaecopathology, University of Leipzig, Leipzig,
Germany; 8Institute of Legal Medicine, 9Laboratory of Molecular Medicine, Interdisciplinary Centre for Clinical Research, University of Leipzig, Leipzig, Germany;
10Heinrich Pette Institute for Experimental Virology, University of Hamburg, Eppendorf, Hamburg, Germany; 11Institute of Clinical Immunology, University of Leipzig,
Leipzig, Germany; 12II Medical Department, University of Mainz, Mainz, Germany; 13Institute of Pharmacology and Toxicology, Friedrich Schiller University, Jena,
Germany; 14Division for Endocrinology, Diabetes and Clinical Nutrition, Department of Research, University Hospital Basel, Basel, Switzerland.
Received December 13, 2006; accepted March 26, 2007.
Supported by the Federal Ministry of Education and Research funding priority HepatoSys (31P3131) and by the Interdisciplinary Centre for Clinical Research at the
University of Leipzig (01KS9504, project Z10).
*Jan G. Hengstler and Henryk Zulewski share senior authorship.
Address reprint requests to: Marc Brulport, Leibniz Research Centre for Working Environment and Human Factors, Ardeystraße 67, D-44139 Dortmund, Germany.
E-mail: brulport@ifado.de; or Henryk Zulewski, Division for Endocrinology, Diabetes and Clinical Nutrition, Department of Research, University Hospital Basel,
Hebelstraße 22, CH-4031 Basel, Switzerland. E-mail: zulewskih@uhbs.ch.
Copyright © 2007 by the American Association for the Study of Liver Diseases.
Published online in Wiley InterScience (www.interscience.wiley.com).
DOI 10.1002/hep.21745
Potential conflict of interest: Nothing to report.
Supplementary material for this article can be found on the HEPATOLOGY Web site (http://interscience.wiley.com/jpages/0270-9139/suppmat/index.html).
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n recent years, numerous reports described the generation of hepatocytes or “hepatocyte-like” cells from
various types of extrahepatic stem or precursor
cells.1,2 At first glance, this appears to provide exciting
new opportunities for cell therapy, as some types of stem
cells proliferate efficiently in vitro and therefore may help
to generate a larger supply of human hepatocytes or precursor cells for transplantation. On the other hand, some
studies presenting far-reaching conclusions with respect
to the capacity of stem cell therapy have not yet been
reproduced or may have been interpreted in an overly
optimistic manner.
The first evidence that hematopoietic stem cells might
be capable of differentiating into hepatocytes in rodents
came from Petersen et al.3 Similar results were obtained in
further studies using different animal models and purified
cell types for transplantation (for a review, see Hengstler
et al.1). Later, it was recognized that cell fusion and not
transdifferentiation was responsible in the FAH⫺/⫺
mouse model.4-6 However, other authors using different
mouse models with less severe selection pressure did not
observe evidence for cell fusion.7-9 Therefore, this matter
is still discussed controversially.
The intriguing results obtained in rodents stimulated many groups to study the fate of human stem and
precursor cells in livers of experimental animals (Supplemental Table 1; for a review, see Hengstler et al.1).
Recipients were mostly immunodeficient severe combined immunodeficiency (SCID) or nonobese diabetic
(NOD)/SCID mice. Although different cell types and
routes of injection have been tested, qualitatively similar results have been reported (Supplemental Fig. 1).
All 18 published studies observed either human albumin–positive or HepPar1 (a human hepatocyte antigen) -positive hepatocyte-like cells. Most of these
studies confirmed their results by reverse transcriptase
(RT)-PCR.
Here we analyzed in detail the human albumin–positive cells observed in mouse livers after human precursor
cell transplantation. A new aspect of our study is the combination of cell tracking techniques and marker analysis in
the same cells. Using this technique, we consistently observed 2 different types of human albumin–positive cells
after the transplantation of human cord blood cells. The
first cell type, named type 1, has a human nucleus. However, its morphology is not hepatocyte-like. Some but not
all of the analyzed hepatocyte markers were positive. Type
2 has a perfect hepatocyte-like morphology similar to that
of the cells shown in Supplemental Fig. 1. Surprisingly,
however, type 2 cells had a mouse nucleus and not a
human nucleus. Qualitatively similar results were obtained when the experiments were reproduced with dif-
HEPATOLOGY, September 2007
ferent types of human precursor cells, namely bone
marrow stem cells, monocyte-derived cells, and hepatopancreatic precursor cells. Therefore, no clear transdifferentiation of human precursor cells to hepatocytes was
observed, but instead a complex situation was observed
that may include partial transdifferentiation and horizontal gene transfer.
Materials and Methods
Cells and Culture Conditions. The isolation of human adherently proliferating cord blood cells,10 human
monocyte– derived NeoHep cells,11 nestin-positive hepatopancreatic precursor cells,12,13 mesenchymal bone marrow cells,14 and primary human hepatocytes15 was
performed as described (for a detailed description, see the
supplementary material). Marking with CM-DiI was performed with a commercially available kit (Invitrogen,
Karlsruhe, Germany).
Transplantation into NOD/SCID and Urokinase
Plasminogen Activator (uPA)/Recombination Activation Gene-2 (RAG-2) Mice. Male NOD/SCID or uPA/
RAG-2 mice, 8-12 weeks old,16 were used. Cells
(750,000) suspended in 100 ␮L of a culture medium were
transplanted directly either into the left liver lobe11 or into
the spleen. A detailed description of the transplantation
procedure and preparation of the liver is given in the
supplementary material.
Immunostaining, In Situ Hybridization, and Histological Standard Staining Techniques. For immunohistochemical detection of human albumin antihuman albumin rabbit antiserum (Abcam; diluted
1:1000 in a solution of 0.3% bovine serum albumin in a
tris-buffered saline) and the commercially available ABC
kit (Vector Laboratories, Burlingame, CA) were used. For
in situ hybridization, fluorescein-labeled alu probes (BioGenex, San Ramon, CA) and digoxigenin-labeled major
mouse satellite DNA (a gift from Dr. O. Brüstle) were
used. Prussian blue staining, periodic acid Schiff (PAS)
staining, and Solanum tuberosum lectin staining standard
protocols were used to detect iron deposition, glycogen,
and endothelial cells, respectively. Combined in situ hybridization (using alu probes) and immunostaining and
standard histological staining techniques were performed
on the same slices to analyze hepatocyte marker expression in alu-positive cells. Detailed descriptions of all techniques are given in the supplementary material.
Duplex PCR for the Detection of Human DNA in a
Mouse Background. A recently published protocol for
the quantification of small amounts of human DNA in
mouse tissues was applied.17 The technique is based on
primers specific for the ␣-satellite repeat of human chro-
HEPATOLOGY, Vol. 46, No. 3, 2007
mosome 7 and primers amplifying fragments from the
mouse chromosome 8 gene repeat sequence. Product ratios are determined by the standard electrophoresis of
end-stage PCR reactions.
Microsatellite Analysis for Human Identification.
DNA was extracted from the cord blood cell lines before
transplantation and from formalin-fixed and paraffin-embedded tissue of mouse livers 3 weeks after transplantation as described.18 For PCR amplification, we used the
PowerPlex16 Kit (Promega, Madison, WI), which contained the following short tandem repeat loci: D3S1358,
TH01, D21S11, D18S51, PentaE, D5S818, D13S317,
D7S820, D16S539, CSFPO, PentaD, VWA, D8S1179,
TPOX, FGA, and amelogenin. All resulting PCR products were resolved and detected by capillary electrophoresis (for details, see the supplementary material).
Results
Characterization of Human Stem and Precursor
Cells. Most of our transplantation experiments were performed with adherently proliferating cord blood cells.10
Therefore, we characterized our cells by surface marker
analysis (Supplemental Fig. 2A). The pattern of surface
marker expression was very similar to that obtained in a
previous study using adherently proliferating cord blood
cells.7 For instance, CD10, CD13, CD29, CD90w,
CD105, and CD144 were positive, whereas CD31 and
CD34 did not differ from isotype controls (Supplemental
Fig. 2A). All other cell types were handled according to
published procedures and have already been extensively
characterized in the indicated references: human monocyte– derived NeoHep cells,11,19 human hepatopancreatic
precursor cells,12,13 and mesenchymal bone marrow stem
cells.14 Cord blood, hepatopancreatic, and mesenchymal
stem cells had a fibroblast-like morphology (Supplemental Fig. 2B,C). In contrast, NeoHep cells (Supplemental
Fig. 1D) appeared to be large, round cells interspersed
with a smaller number of spindle-shaped cells, as described by Ruhnke et al.11,19 Cord blood, hepatopancreatic, and mesenchymal cells were negative for albumin
and cytochrome P450 3A4 (CYP3A4) immunostaining;
iron storage was evidenced by Prussian blue staining, glycogen storage by PAS staining. Endothelial cells were detected using S. tuberosum lectin staining (Supplemental
Fig. 3). For all transplantation experiments, freshly isolated primary human hepatocytes were used as positive
controls.
Tracking of the Transplanted Cells in Mouse Livers. The cell types described in the previous paragraph were
transplanted into NOD/SCID mice: 750,000 cells were injected directly into the left liver lobe. A first milestone ana-
BRULPORT ET AL.
863
lyzing the fate of the human cells in the mouse livers was the
tracking of the transplanted cells. As positive controls, primary human hepatocytes were transplanted. Using probes
for alu sequences, we could clearly identify human hepatocytes by in situ hybridization in a mouse liver microenvironment (Fig. 1). Human nuclei were visualized by green
fluorescence and showed a morphology and size characteristic of human hepatocytes. For the staining of mouse nuclei, a
mouse major satellite probe was applied that led to characteristic pink spots within the nuclei of mouse hepatocytes
(Fig. 1). Combined in situ hybridization using alu probes
and mouse major satellite probes also allowed the identification of cell fusion by the presence of green and pink nuclei in
the same cell. As expected, no evidence for cell fusion was
observed after the transplantation of human hepatocytes. In
situ hybridization with alu probes was combined with immunostaining using antibodies specific for human albumin.
This technique identified human cells (by the green fluorescence of the nuclei) that additionally showed a positive albumin staining [visualized by 3,3⬘-diaminobenzidine (DAB)
staining], a scenario that should be expected after the transplantation of human hepatocytes (Supplemental Fig. 4).
Having obtained plausible results for our positive controls, the primary human hepatocytes, we next studied
adherently proliferating human cord blood cells, using
similar techniques. We also observed alu-positive cells 1
day to 3 weeks after the transplantation of cord blood cells
(shown later in Figs. 2 and 4). However, the alu-positive
nuclei were much smaller than those of human hepatocytes (Fig. 1 and Supplemental Fig. 4) and showed an
angular shape. Small clusters of alu-positive cells were
often found in vessels. Because of the morphological differences from human hepatocytes, we performed additional controls to analyze whether the alu-positive cells
were derived from the transplanted cord blood cells:
1. Before transplantation, cord blood cells were
marked with the red fluorescent dye CM-DiI. After transplantation, red fluorescent structures were observed (Fig.
2A) that colocalized with the alu-positive nuclei (Fig. 2B).
2. Using a panel of 16 short tandem repeats for the
forensic identification of individuals, we analyzed 1 of our
cord blood cell lines before transplantation (Supplemental Fig. 5A) and mouse livers 3 weeks after transplantation
with this cell line (Supplemental Fig. 5B and Supplemental Table 3).
For all markers, we typed identical alleles. With the
application of forensic standards, human DNA in the
mouse liver could be shown to derive from the same individual whose cord blood cells had been established. The
probability of compliance was more than 99.9%.
Next, we performed a semiquantitative analysis of alupositive nuclei in the left liver lobe 4 hours, 12 hours, 24
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BRULPORT ET AL.
HEPATOLOGY, September 2007
Fig. 1. Identification of human and mouse
nuclei by in situ hybridization. Slides were incubated with both alu probes (green fluorescence, human nuclei) and mouse major
satellite probes (pink spots, mouse nuclei). (A)
NOD/SCID mouse liver, (B) human liver, and
(C) NOD/SCID mouse liver after the transplantation of 750,000 human hepatocytes into the
left liver lobe. Part C shows the section of the
left liver lobe with the highest density of human
cells. The scale bar is 50 ␮m.
hours, 5 days, and 21 days after transplantation. For this
purpose, we counted alu-positive structures; for instance,
the 2 cell clusters in Fig. 2B were counted as 1 alu-positive
structure (Table 1). We observed an increase between 4
and 12 hours after transplantation, which could be explained by the redistribution of cell pools. Later, a decrease was observed, and only a relatively small fraction of
alu-positive cell clusters remained 3 weeks after transplan-
Fig. 2. Tracking of human cord blood cells 1 day after transplantation
into the left liver lobe of NOD/SCID mice. The cord blood cells were
labeled by the red fluorescent dye CM-DiI before transplantation and
were additionally visualized by in situ hybridization using an alu probe
(green fluorescence). The red fluorescence visualizes CM-DiI (A). Human
nuclei were identified with a human-specific alu probe and visualized
with a Cy2-labeled antibody (green fluorescence). Overlay of red and
green fluorescence together with 4⬘,6-diamidino-2-phenylindole nuclear
staining (blue) to verify the colocalization of nuclei with alu probe–
positive structures (B). The scale bar is 50 ␮m.
tation. To confirm these results by an independent
method, we applied a recently established duplex PCR
technique that allows the quantification of human DNA
in a mouse background.17 These results confirmed the
decrease already seen with in situ hybridization. Nevertheless, human DNA still was detectable 21 days after
transplantation (Supplemental Table 4).
Two Different Types of Human Albumin–Positive
Cells. We applied an immunostaining technique with
antibodies specific for human albumin (Fig. 3A-D).
Much care was taken to establish conditions that minimized false positive results in mock-transplanted mice
(Supplemental Table 5 and Supplemental Fig. 6). Using
this technique, we consistently observed human albumin–positive cells after the transplantation of cord blood
cells (Fig. 3A). However, 2 types of human albumin–
positive cells could be differentiated. One cell type
(named type 1 cell) does not resemble human hepatocytes
(Fig. 3A1,A2). Nuclei of type 1 cells appear smaller than
those of hepatocytes and have an angular shape. Type 1
cells usually occur as cell clusters, are often located in
small vessels, and do not form columnar structures that
are typical for hepatocytes. A second cell type (named type
Table 1. Numbers of alu-Positive Structures 4 Hours, 12
Hours, 24 Hours, 5 Days, and 21 Days After the
Transplantation of 750,000 Human Cord Blood Cells into
the Left Liver Lobe of NOD/SCID Mice
Removal of
Transplanted Liver
After
After
After
After
After
4 hours
12 hours
24 hours
5 days
21 days
Number of
Analyzed
Slices
Mice
per
Group
alu-Positive
Nuclei
(Mean)
Standard
Error
8
8
10
7
8
3
3
3
3
4
78.4
118.1
233.1
37.4
2.6
19.6
22.1
63.3
12.0
1.2
HEPATOLOGY, Vol. 46, No. 3, 2007
BRULPORT ET AL.
865
Fig. 3. Two morphologically different types of human albumin–positive cells were observed 3 weeks
after the transplantation of cord
blood cells, monocyte-derived cells,
hepatopancreatic precursor cells, or
bone marrow– derived stem cells
into the left liver lobe of NOD/SCID
mice. Immunostaining was performed with antibodies specific for
human albumin. Sections from human liver served as positive controls, and sham-transplanted NOD/
SCID mouse livers were used as
negative controls. Twenty-one days
after transplantation, type 2 cells
showed a morphology similar to that
of genuine hepatocytes. In contrast,
type 1 cells did not have a hepatocyte-like morphology. The scale bar
in A1, A2, B1, B3, B4, C1, C4, D1,
and D3 is 20 ␮m; in B2, it is 50
␮m; and in E and F, it is 100 ␮m.
2 cell) has an hepatocyte-like morphology (Fig. 3A3,A4).
Type 2 cells often show 2 nuclei and are well integrated
into the liver tissue. However, they usually occur as single
cells and, therefore, never form liver tissue. Results similar
to those for cord blood cells were obtained after the transplantation of monocyte-derived cells (NeoHep cells),
hepatopancreatic precursor cells, and bone marrow– derived mesenchymal stem cells (Fig. 3B-D).
Combined Cell Tracking and Marker Analysis Reveals Type 1 Cells as Partially Differentiated Stem
Cells. In the next step, we combined the tracking techniques with CM-DiI and alu-probe in situ hybridization
with an analysis of hepatocyte markers in the same cells.
For this purpose, mouse livers 3 weeks after the transplantation of CM-DiI–marked cord blood cells were immunostained with human albumin–specific antibodies
followed by in situ hybridization with alu probes of the
same slices. A representative result of human albumin–
positive type 1 cells is shown in Fig. 4C. The CM-DiI cell
tracker (Fig. 4E,F) and in situ hybridization (Fig 4D,F)
demonstrate that the human albumin–positive cluster of
type 1 cells consists indeed of the transplanted human
cells. Therefore, the hepatocyte marker albumin that is
negative in cord blood cells before transplantation (Supplemental Fig. 3A) becomes expressed after transplantation into the liver microenvironment. A similar procedure
was applied for PAS staining to analyze whether type 1
cells stored glycogen (Supplemental Fig. 7). Indeed, type
1 cells were characterized by a colocalization of PAS-positive staining (Supplemental Fig. 7D) in cells with alu-
positive nuclei (Supplemental Fig. 7E). Therefore,
glycogen is another marker newly expressed after transplantation. However, not all analyzed hepatocyte markers
were positive in type 1 cells. For instance, alu-positive
type 1 cells stained negative for CYP3A4, the major cytochrome P450 in human liver (Supplemental Fig. 8).
In our negative controls for immunostaining, we observed cell clusters with a cauliflower-like, brownish cytoplasmatic stain (Supplemental Fig. 9). These brownish
structures occurred independently of the first and secondary
antibodies and were absent in mock-transplanted mice.
They are relevant from a technical point of view because an
inexperienced operator might misinterpret them as positive
DAB staining. To elucidate the nature of the brownish inclusions, we performed several histological routine staining
techniques and finally identified some type 1 cells as Prussian
blue–positive; this suggested that these cells store iron. Supplemental Fig. 10 demonstrates the colocalization of Prussian blue (Supplemental Fig. 10D) with CM-DiI–positive
and alu-positive nuclei (Supplemental Fig. 10E). However,
only those type 1 cells located between hepatocytes stained
positive with Prussian blue (Supplemental Fig. 10D,E). We
also observed alu-positive CM-DiI cells in the endothelium
of vessels (Supplemental Fig. 10G,H). These endothelial
type 1 cells stained negative for Prussian blue (Supplemental
Fig. 10F), in contrast to some type 1 cells located between
hepatocytes (Supplemental Fig. 10D,E). To further characterize endothelial type 1 cells, we used a costaining technique
with in situ hybridization plus S. tuberosum lectin; the latter
reliably identified endothelial cells of veins (green arrows in
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BRULPORT ET AL.
Supplemental Fig. 11A,B) and sinusoidal lining cells (white
arrows in Supplemental Fig. 11A,B). Supplemental Fig.
11C1,C2 shows an alu-positive endothelial type 1 cell,
which at first glance seems to resemble the host’s endothelial
cells. However, a closer examination by confocal microscopy
reveals a lectin-negative cytoplasm (the yellow arrow in Supplemental Fig. 11C4), in contrast to the lectin-positive genuine endothelial cells of the host (the white arrows in
Supplemental Fig. 11C4). Therefore, endothelial type 1 cells
do not perfectly match the staining pattern of real endothelial cells. In conclusion, our combined tracking/marker analysis technique leads to a preliminary classification of type 1
cells (Table 2) that is in agreement with a partial but not
complete differentiation of cord blood cells into endothelial
cells and hepatocytes. In particular, the difference between
albumin-positive type 1 cells and real hepatocytes is obvious
because of differences in the morphology and marker expression. To analyze whether type 1 cells are apoptotic or proliferate, we performed double stainings of the same slices with
alu-probe in situ hybridization plus either ki67 immunostaining or TUNEL staining. The results show that both
apoptosis (Supplemental Fig. 12) and proliferation (Supplemental Fig. 13) are very rare in type 1 cells.
Human Albumin–Positive Type 2 Cells Have a
Mouse Nucleus but Not a Human Nucleus. At first
glance, type 2 cells (examples are shown in Fig. 3A3,A4)
seem to be promising because of their hepatocyte-like
morphology and clear-cut positive human albumin staining. On the other hand, we feel that these cells should be
interpreted with caution because our in situ hybridization
experiments with alu probes (Fig. 2B) did not show alupositive nuclei that would match the nuclei of type 2 cells.
Therefore, we decided to study human albumin–positive
type 2 cells in detail and performed consecutive in situ
hybridization with alu probes and mouse major satellite
probes followed by albumin immunostaining. A representative result (Fig. 5) shows a typical type 2 cell staining
positive with antibodies specific for human albumin. The
DAB (human albumin) -positive cell in Fig. 5C appears
darker than that in Fig. 5D in comparison with the surrounding DAB negative cells. This is due to the fact that
DAB quenches fluorescence. Nevertheless, the DAB-positive cell could be shown as mouse major satellite–positive
(Fig. 5D). In contrast, in situ hybridization with alu
probes was negative (Fig. 5E). Therefore, the cell shown
in Fig. 5 has a mouse nucleus but not a human nucleus,
despite positive staining for human albumin. A positive
control with human liver that underwent exactly the same
procedure showed clear-cut alu-positive nuclei (Supplemental Fig. 4). We carefully evaluated several type 2 cells
(further examples are shown in Supplemental Fig. 14) but
never could demonstrate intact human nuclei. In a mi-
HEPATOLOGY, September 2007
nority of type 2 cells, small alu-positive fragments (Supplemental Fig. 15) were visible. In addition, dye transfer
from transplanted CM-DiI–marked cord blood cells into
mouse hepatocytes was observed (Supplemental Fig. 16).
A possible relevance of alu-positive fragments and dye
transfer is addressed in the Discussion section.
Selection Pressure and Different Routes of Administration: Qualitatively Similar Results. All experiments shown so far were obtained in NOD/SCID mice
without the induction of a selection advantage for the
transplanted cells. In these experiments, human cells were
injected directly into the left liver lobe. It should be considered that this procedure causes substantial local liver
damage (Supplemental Fig. 17) but no specific selection
advantage. To study a possible influence of selection pressure, we used uPA/RAG-2 mice20-22 that suffered from
permanent deterioration of hepatocytes. Human cord
blood cells (750,000 per mouse) were transplanted into
uPA/RAG-2 mice. Transplantations were performed intrasplenically or by direct injections into the left liver
lobes using three mice per group. Qualitatively, similar
observations were made as already described for NOD/
SCID mice (Supplemental Fig. 18) for both routes of
administration. There seemed to be quantitative differences. However, the latter were not systematically addressed in this study.
Discussion
The limited availability of hepatocytes has been recognized as the major hurdle of liver cell therapy.2 A wider use of
liver cell therapy will not be possible until adequate numbers
of functional cells for transplantation become more readily
available. Although the most relevant cytokines for liver regeneration are known, human hepatocytes cannot yet be
stimulated to proliferate efficiently in vitro.23,24 Also, the
generation of functional hepatocytes from human oval cells
is not yet possible. Therefore, several groups have tried to
differentiate specific types of extrahepatic stem and precursor
cells into hepatocytes (for a review, see Hengstler et al.1). A
strategy for the evaluation of the differentiation capacity of
human stem cells is transplantation into the livers of immunodeficient animals and analysis of whether previously silent
human hepatocyte markers become expressed. This approach offers the advantage of a real liver microenvironment.
A disadvantage may be the human-in-mouse situation with
possible interspecies incompatibilities. However, human
and monkey hepatocytes have been shown to survive and
remain functional in livers of immunodeficient mice.20-22
Therefore, the fate of human stem cells in mouse livers may
give some evidence of their hepatocellular differentiation capacity.
HEPATOLOGY, Vol. 46, No. 3, 2007
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Fig. 4. Combined analysis of human albumin expression, CM-DiI cell tracker, and in situ hybridization with alu probes of the same slice. The left
liver lobe of a NOD/SCID mouse was analyzed 3 weeks after the transplantation of human cord blood cells. Consecutive immunostaining with
antibodies specific for human albumin (C) and in situ hybridization with alu probes (D). Red fluorescence visualizes the position of the cell tracker
CM-DiI because cord blood cells have been CM-DiI–marked before transplantation (E). The merged picture (F) demonstrates that the cluster of human
albumin–positive type 1 cells (C) colocalizes with CM-DiI–positive cells and with alu-positive nuclei. A human liver slice was used as a positive control
for in situ hybridization (A) and in immunohistochemistry (B1), whereas sham-transplanted NOD/SCID mouse liver served as a negative control (B2).
The scale bar is 100 ␮m in A and B and 50 ␮m in C-F.
A large number of groups have transplanted human
extrahepatic stem and precursor cells into immunodeficient animals (for an overview, see Supplemental Table
1). Interestingly, very similar results have been obtained
after transplantation, that is, hepatocyte-like cells expressing human albumin or the human hepatocyte antigen
HepPar1. The impressive consistency between the results
of different groups is illustrated in Supplemental Fig. 1, in
which we collected representative published images of
mouse livers after the transplantation of human stem and
precursor cells. However, the interpretation of these observations is quite controversial (for a review, see Hengstler et al.1). Several authors tend to give an optimistic
Table 2. Classification of Cell Types Observed in Livers of
NOD/SCID Mice After the Transplantation of Extrahepatic
Human Precursor Cells
Type 1 Cells
● Alu-positive (human) nuclei, mouse major satellite staining negative
● No hepatocyte-like morphology (small angular nuclei)
● Expression of human albumin, positive PAS (glycogen) staining
● No expression of CYP3A4
● Accumulation of iron in some type 1 cells
3 Probably result of partial transdifferentiation
Type 2 Cells
● Mouse major satellite–positive (mouse) nuclei, no alu-positive (human)
nuclei
● Perfect hepatocyte-like morphology
● Expression of human albumin
● No accumulation of iron
Fig. 5. Representative example of a human albumin–positive type 2 cell
that has a mouse nucleus but no human nucleus. Consecutive albumin
immunostaining and in situ hybridization with alu probes and mouse major
satellite probes were performed with slices of the left liver lobe of a
NOD/SCID mouse 3 weeks after the transplantation of human cord blood
cells. The immunostaining visualized a human albumin–positive type 2 cell
(C). The same cell proved negative for in situ hybridization with an alu probe
(E) but was positive with a mouse major satellite probe (D), as indicated by
the yellow arrow. Human (A) and sham-transplanted (B) NOD/SCID mouse
liver tissue slices were used as positive controls. The scale bar is 50 ␮m.
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BRULPORT ET AL.
view. For instance, Kogler et al.7 transplanted adherently
proliferating cells isolated from human cord blood into
livers of fetal sheep. The authors observed the expression
of albumin and human hepatocyte–specific antigen after
transplantation and concluded that the human cord
blood cells differentiated into human parenchymal hepatic cells. Newsome et al.25 demonstrated the expression
of the HepPar1 human hepatocyte–specific antigen and
concluded that cells from human cord blood “become
mature hepatocytes” in livers of NOD/SCID mice. Ishikawa et al.26 detected human albumin and the HepPar1
antigen in livers of immunodeficient mice, postulating
that the engrafted cells from human cord blood “functioned as hepatocytes.” However, other scientists10,13,27,28
(for a review, see Hengstler et al.1), including our group,
remained skeptical because we observed the expression of
some but not all hepatocyte markers that should be expressed by a real human hepatocyte. Because of its high
relevance, we decided to study more closely the fate of
human stem cells in mouse livers. For this purpose, we
established cell tracking techniques that reliably allow the
identification of the transplanted cells and additionally
hepatocyte marker analysis in the same cells. In our study,
efficient tracking of transplanted human cells could be
achieved by a combination of in situ hybridization with
alu probes and marking of the cells with CM-DiI before
transplantation. The results obtained with this technique
correlated well with data obtained from a duplex PCR
technique quantifying human DNA in a mouse background. In comparison with the data 4 and 24 hours after
transplantation, a strong decrease in the number of surviving human cells from cord blood was obtained after 21
days (Table 1). Nevertheless, a sufficient number of the
transplanted cord blood cells survived (median: 2.6 alupositive structures per slice) to allow a combined analysis
of alu-probe in situ hybridization and hepatocyte markers. Indeed, alu-positive cells were also positive for human
albumin and for PAS staining, indicating glycogen storage. Importantly, both markers were negative in cord
blood cells before transplantation. Therefore, exposure to
the liver microenvironment caused expression of at least 2
previously silent hepatocyte markers. However, the alupositive cells did not show a hepatocyte-like morphology.
Their nuclei are much smaller than the large, round nuclei
of hepatocytes. Moreover, they stained negative for
CYP3A4, the major cytochrome P450 in human hepatocytes. This scenario suggests that the transplanted cells
have a mixed phenotype that might be explained by partial differentiation. The resulting mixed cell type is not
implausible if we consider the molecular mechanisms
controlling gene expression. The somatic stem and precursor cells, torn out from their physiological surround-
HEPATOLOGY, September 2007
ings and introduced into a new microenvironment, are
likely to be stimulated to express some previously silent
genes. However, because the transplanted cell types represent somatic and not embryonic stem cells, the local
accessibility of genes to the transcriptional machinery
may be limited. For instance, histone modifications determine the accessibility of chromatin and control gene
expression patterns29 (for a review, see Gan et al.30). Our
results suggest that the mere exposure of somatic stem
cells to a liver microenvironment is not sufficient to induce the complex mechanisms (i.e., activation of transcription factors plus establishment of accessible and
responsive chromatin) that finally lead to the expression
of the several hundreds of genes that make up a hepatocyte. Multistep protocols starting with dedifferentiation
steps for the establishment of responsive chromatin by
altering the acetylation and methylation patterns possibly
may be more successful.11,19,31 Also, promising results
have been obtained by multistep protocols for the differentiation of embryonic stem cells into the hepatic lineage.32
Immunostaining for human albumin after the transplantation of human cells into mouse livers showed 2 different
types of albumin-positive cells. Type 1 cells have a human
nucleus and correspond to the previously described mixed
cell types with a non-hepatocyte–like morphology. In contrast, type 2 cells stain positive for human albumin and are
characterized by a hepatocyte-like morphology with its characteristic polygonal shape and large, round nuclei. These 2
human albumin–positive cell types were observed consistently after the transplantation of 4 different extracellular
precursor cell types used in this study, namely, adherently
proliferating cord blood cells, monocyte-derived NeoHep
cells, hepatopancreatic precursors, and mesenchymal stem
cells from bone marrow. Our type 2 cells correspond perfectly to the human albumin–positive or HepPar1-positive
cells in mouse livers observed by several other groups after
human stem cell transplantation (Supplemental Fig. 1). At
first glance, these cells seem to be promising candidates resulting from a transdifferentiation process. However, by
combined in situ hybridization and albumin immunostaining, we have clearly demonstrated that type 2 cells have a
mouse nucleus and not a human nucleus (Fig. 5 and Supplemental Fig. 14). Therefore, type 2 cells cannot result from
transdifferentiation of the transplanted human stem cells.
Cell fusion is improbable because we never observed both a
human nucleus and a mouse nucleus in type 2 cells. Although it has not yet been formally proven, we suggest horizontal gene transfer as the most likely mechanism that could
explain human albumin–positive type 2 cells. We have
shown that the majority of the transplanted human cells
deteriorate within 3 weeks. On the other hand, hepatocytes
HEPATOLOGY, Vol. 46, No. 3, 2007
are able to recognize and internalize apoptotic cells by means
of specific receptors (galactose-specific and mannose-specific
receptor, receptor for phosphatidylserine, and asialoglycoprotein receptor) and by cytoskeletal reorganization that
allows the engulfment of apoptotic bodies.33,34 The identification of apoptotic cells by hepatocytes is achieved by sugar
residues and phosphatidylserine exposed on the surface of
apoptotic bodies. In fact, we have observed small fragments
of human (alu-positive) nuclear fragments in mouse cells and
also have identified dye transfer from the transplanted cells
into hepatocytes of the host. This suggests that chromosomal
fragments from deteriorating transplanted cells may use apoptotic bodies as a Trojan horse to enter hepatocytes, leading
to the expression of some human genes in mouse cells. However, further experiments are needed to prove this hypothesis, such as the transplantation of human stem cells carrying
reporter constructs.
An unexpected result was the detection of alu-positive
cells with an endothelial morphology integrated into the
endothelial layer of vessels. S. tuberosum lectin staining,
a technique that reliably identifies endothelial cells,35
revealed differences in the staining pattern between
alu-positive endothelial-like cells and real endothelial cells
of the host, suggesting that an interpretation of transdifferentiation should still be treated with caution.
Nevertheless, further studies concerning a possible role of
cord blood cells as endothelial cell precursors are indicated.
In conclusion, we did not observe transdifferentiation
of human extrahepatic stem cells to hepatocytes after
transplantation into mouse livers. Instead, we have seen a
complex situation including partial transdifferentiation
and possibly horizontal gene transfer. Presently, we cannot rule out that the reported limitations might be due to
the human-in-mouse situation. Nevertheless, it should be
considered that cells capable of horizontal gene transfer
may be used as vehicles for gene delivery, for instance, in
inherited metabolic liver disease.
Acknowledgment: The authors thank Dr. J. Grosche
for expert technical assistance with the operation of the
confocal microscope.
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