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Prepublished online as a Blood First Edition Paper on January 30, 2003; DOI 10.1182/blood-2002-05-1338.
TRANSPLANTATION
From the Division of Research Immunology/Bone
Marrow Transplantation, Childrens Hospital Los Angeles (CHLA),
Los Angeles, CA; the Department of Pediatrics, University of Southern
California School of Medicine, Los Angeles, CA; and the Childrens
Hospital Los Angeles Research Institute, Congressman Dixon Cellular
Imaging Core, Los Angeles, CA.
Rodent bone marrow cells can contribute to liver. If these
findings are applicable to humans, marrow stem cells could
theoretically be harvested from a patient and used to repair
his/her damaged liver. To explore this potential,
CD34+ or highly purified
CD34+CD38 There is an intriguing literature describing murine
and rat stem cells that display plasticity. In one of the most
definitive studies in the field to date, Lagasse et al reported that
c-kit+ Thy-1.1 (lo) Lin Theise et al also demonstrated that in humans hepatocytes and
cholangiocytes could be derived from bone marrow.5 They
analyzed archival liver specimens from female recipients of bone marrow transplants (BMTs) from male donors, and found Y
chromosome-positive hepatocytes and cholangiocytes in the female BMT
recipients.5 This observation suggests that marrow-derived
stem cells can generate liver in humans. There is little other data
indicating that human counterparts to the rodent stem cells that have
displayed plasticity exist because of a lack, so far, of appropriate in
vivo models for studying human stem cells that may have the capacity to
generate multiple tissues.
In the current studies, we sought to develop a model to extend
the seminal reports on murine stem cell plasticity, to allow examination of the potential for highly purified human
"hematopoietic" stem cells to contribute to liver regeneration in
immunodeficient mice. We used both nonobese diabetic-severe combined
immunodeficiency (NOD/SCID) and NOD/SCID/ After exploring less successful approaches, we determined that human
albumin and cytokeratin 19 (CK19)-expressing cells were best
generated in NOD/SCID/ Isolation of human hematopoietic progenitors
Mice
Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Total cellular RNA was extracted from the human-mouse chimeric hepatocytes, murine tissues, and starting populations of human cells. Samples were quantitated using a spectrophotometer, and equal amounts of RNA from all samples were subjected to cDNA synthesis using Oligo (dT) (Invitrogen, Carlsbad, CA) to prime first-strand synthesis. Human-specific albumin12 and cytokeratin 19 (CK19) primers13 were used to detect human albumin and human CK19 expression in the mouse liver. Primers were selected from 2 different exons with at least one intervening intron to rule out false signals at the expected band size from contaminating DNA strands. The albumin genomic DNA fragment would have been 2019 bp, and the ck19 genomic fragment would have been 1197 bp. PCR conditions for albumin were as follows: 94°C for 1 minute; 55°C, 1.5 minutes; and 72°C, 1.5 minutes; and for CK19: 94°C, 1 minute; 64°C, 1 minute; and 72°C, 2 minutes. PCR products were run on an agarose gel and visualized using an Eagle Eye Gel reader (Stratagene, La Jolla, CA). Product sizes were 422 bp for human albumin and 461 bp for human CK19. In situ hybridization (ISH) and immunohistochemistry staining.
Dissociated liver cells were examined on cytospin slides by in situ
hybridization (ISH) to detect human Alu sequences, with simultaneous
antibody staining for human albumin protein. Human Alu ISH was carried
out using the ISH diaminobenzidine (DAB) kit (Innogenex, San Romon CA),
according to the manufacturer's instructions. Colorfrost/plus glass
slides (Fisher Scientific, Pittsburgh, PA) were used in all studies.
Hepatocyte cytospin slides were fixed in acetone-methanol (1:1) for 30 seconds. To permeabilize, slides were covered by cold ethanol for 10 minutes at Microscopy. Images were viewed with a Leica DMRA microscope (Wetzlar, Germany) using a Plan Apo 40 ×/1.25 NA phase 3 DIC or Plan Apo 63 ×/1.32 oil immersion objective lens. The microscope was equipped with a Sutter LS175W ozone-free xenon arc lamp (Novato, CA). Images were acquired with an Applied Spectral Imaging SkyVision-2/VDS camera from EasyFISH software (Applied Spectral Imaging, Migdal Ha'Emek, Israel) and printed using Microsoft PowerPoint (Redmond, WA). Determination of human serum albumin in the mouse plasma. Plasma was collected for the human serum albumin assays, and ELISA was performed according to the manufacturer's instructions (MD Biosciences, Gewerbesteuer, Zurich, Switzerland). Normal human and nontransplanted murine sera were tested in comparison with the samples in each experiment in addition to the standards included in the kit. Statistical analyses. Analyses were done using Correlation Analysis in the GraphPad Prism software program (San Diego, CA), the Mann-Whitney test, and descriptive statistics analysis in the Minitab Computer Program (State College, PA). Means ± SEM are listed.
Induction of liver damage in immune-deficient mice The initial challenge to studying human hematopoietic stem cell to hepatocyte differentiation was to establish a model of acute liver damage in immunodeficient mice, in which the control mice that did not undergo transplantation would recover from the injury. Hepatic injury in NOD/SCID mice by several different doses of radiation and by intraperitoneal administration of varying doses of allyl alcohol was studied first. There was good survival of the mice in all arms of the allyl alcohol studies, and NOD/SCID and NOD/SCID/ 2M-null mice
tolerated up to 300 RADS without significant mortality. However, no
evidence of human hematopoietic stem cell to hepatocyte (or
albumin-expressing cell) differentiation was observed in those studies
using RT-PCR and the assays detailed in the following sections (data
not shown).
Next, hepatic injury using carbon tetrachloride (CCl4) was
evaluated because it is known to induce periportal lipid peroxidation and protein denaturation after administration, yet will allow endogenous liver regeneration if not given in an excess
dose.14 Mice were screened for engraftment prior to
induction of liver damage. When as few as 0.5% human CD45+
cells were found in the peripheral blood, the mouse was considered engrafted and the CCl4 treatment proceeded. Occasionally, human cells
were not detected in the circulation of the mouse, although the marrow
was later found to be engrafted. These animals were not used in the
current studies. Successfully engrafted NOD/SCID and
NOD/SCID/ Compared with normal age-matched mouse liver (Figure
1A), 0.4 mL/kg CCl4 induced
massive liver damage in the form of numerous foci of periportal
necrosis (Figure 1B), and 75% of the mice died within 4 weeks
(n = 12). Injection of recombinant human HGF enhanced recovery of the
necrotic areas in the liver (Figure 1C). None of the mice died in the
initial CCl4-treated group that was given HGF treatment
(n = 5). In spite of the toxicity to the non-HGF-treated mice, we
chose to use this model for all subsequent studies because the first
evidence of human stem cell to hepatocyte differentiation was
obtained, as will be described in more detail.
Differentiation of human albumin-expressing hepatocyte-like cells in the immune-deficient mouse livers was augmented by injection of recombinant human HGF (rhHGF) Mice were killed 2 months after transplantation with human cells and one month after liver injury using carbon tetrachloride (CCl4), with or without HGF injection. RNA samples were obtained from the livers of the mice in each group; controls that did not receive transplants versus mice that had received stem cell transplants alone, stem cell transplants with CCl4 damage, or stem cell transplants with CCl4 damage plus HGF administration. RT-PCR for human albumin was done in comparison with2M in Oligo (dT) primed cDNA amplified from equivalent amounts of
RNA isolated from each sample. In order to demonstrate the specificity
of the primers to detect human albumin mRNA, we examined the mRNA in
nontransplanted mouse liver and in injured (CCl4 treated)
nontransplanted mouse liver. These samples were negative in every
assay, and an example is shown in Figure
2. Because of the phenomenon known as
"stem cell priming," in which primitive hematopoietic cells have
been shown to express low levels of various mRNA before committing to a
particular blood cell lineage,15,16 we also tested the
human hematopoietic stem/progenitor cell starting populations for
albumin expression. As shown in Figure 2, the starting stem cell
populations did not express albumin when they were freshly isolated
from umbilical cord blood. Equivalent levels of RNA from total cord
blood mononuclear cell preparations, purified CD34+ cells,
and CD34+38 cell samples were all negative
for albumin expression (Figure 2). The same result was observed for
stem cells isolated from human bone marrow (data not shown).
In the mice that received human stem cell transplants that were not
subjected to liver injury by CCl4 injection, there was no
human albumin expression. However, in mice that received human stem
cell transplants that had received CCl4-mediated liver
injury, a portion of the transplanted human stem cells had localized to the liver and had begun to express human albumin when analyzed one
month after the liver damage (Figure 2). The human albumin mRNA levels
in the livers of the mice in this group were positively correlated with
the percentage of human cells in the BM
(r2 = 0.8). Human alpha-fetoprotein
mRNA was not detected in any sample (data not shown). In an attempt to
increase the efficiency of HSC to hepatocyte differentiation,
recombinant human HGF was injected into some of the mice that underwent
stem cell transplantation, following CCl4 treatment. As
shown in an example in Figure 2, there was an increase in human albumin
expression in the HGF-treated mice. Human albumin RNA was detected
specifically in the livers of the mice, but not in other tissues such
as spleen and BM (Figure 2). When densitometry was performed to compare
the levels of human albumin mRNA with the
Morphology of human albumin-expressing cells in the immune-deficient mouse liver Livers from the mice in each experiment were sectioned and used for detection of human DNA by in situ hybridization and human albumin protein by immunohistochemistry. Figure 4A shows detection of human albumin-expressing cells in a paraffinized liver section from one of the CCl4 + HGF-treated mice, as assessed by immunohistochemistry. The human albumin-expressing cells, stained blue, were found to be dispersed throughout some areas of the liver. There was little background staining in the control slides from mice that were injured but that had not undergone transplantation (Figure 4A), and in those samples the blue chromogen did not localize to discrete cells but to the inner linings of vessels or to debris that was on the slide. The photos in Figure 4A (inset) show the human albumin-expressing cells at higher magnification, visualized under a fluorescent scope with the antihuman albumin antibody conjugated to FITC (panels A-B). Panels C and D of Figure 4A inset show the level of background autofluorescence obtained from the liver tissue damaged by CCL4 and HGF, in mice that did not undergo transplantation. It is important to include the injured liver controls shown in Figure 4A because the damaged tissue binds antibodies and probes with much higher background than intact tissue.
The dispersed colony shown in Figure 4A was one of the best areas
located, and the overall detection of albumin-expressing human cells in
the livers of the mice was low. The percentage of human cells in the
livers of the mice was consistently between 1% and 10% when analyzed
by FACS or imunohistochemistry using an antibody that detects all human
major histocompatibility complex class 1 (MHC1) subtypes
(obtained from Sigma). However, the total percentage of human
hepatocyte-like cells in the liver is difficult to quantitate precisely
because of interference from hematopoietic cells that have not been
induced to express albumin. We would estimate, from counting cells on
the double-stained slides, that 1 in 20 of the human DAB+
cells was expressing albumin. This is only an estimate but gives some
indication of the frequency, or lack thereof, of this event. Also, the
level of human MHC+ CD45+ cells was
consistently close to the number of total human MHC+ cells
(data not shown). Therefore, the numbers of cells that had become
CD45 Next, slides were used for both ISH and albumin staining. Cytospin preparations of dissociated liver cells from the treated mice were used to eliminate potential artifacts that might occur from performing double staining on the tissue sections that could be several cells in thickness. Following performance of ISH for human Alu sequences, followed by fluorescein-linked antihuman albumin antibody, slides were evaluated under a fluorescent microscope. Scanning the cytospin slides under bright field, numerous cells with dark DAB-stained nuclei were observed, indicating that they were human cells that contained Alu sequences. No cells with black nuclei were seen in the control mouse slides. When the bright field and fluorescein filters were combined, a portion of the human cells were observed to have human albumin in their cytoplasm, identified by the FITC-conjugated antibody (Figure 4B). The human cells that did not express albumin were likely hematopoietic cells or undifferentiated stem cells. The human albumin-positive cells in the tissues were not round like their human CD45+ hematopoietic counterparts but were often oval or polygonal (Figure 4), and 2 nuclei were sometimes seen in the same cell (Figure 4B), as has been reported for both human and rat binucleate hepatocytes.17-19 To sustain the systemic levels of human HGF in the immune-deficient
mice that received stem cell transplants and were recovering from liver
injury, we implanted human mesenchymal stem cells/stromal cells that
had been engineered to secrete HGF using retroviral vectors into 6 NOD/SCID/
In addition to the mice that had received transplants of human
umbilical cord blood-derived stem cells, 6 NOD/SCID/ Human serum albumin in the plasma of mice that received human stem cell transplants after liver injury The levels of human serum albumin in the plasma of the CCl4-treated mice that underwent transplantation was next evaluated by ELISA. Normal mouse serum was tested and showed no cross-reaction with the ELISA reagents. Human serum albumin was detected in the plasma of mice that received human stem cell transplants, indicating that human hepatocyte-like cells produced in the mouse liver were capable of albumin secretion (Table 1). Mice that had received transplants of stem cells derived from human bone marrow had significantly higher levels of human albumin in their plasma than mice that received transplants of human umbilical cord blood-derived stem cells (Table 1, P = .03), although the engraftment levels with human hematopoietic cells in their livers and bone marrow were comparable between the groups that had or had not been treated with HGF. Although more albumin mRNA had been detected in the mice that had been treated with HGF, there was not a significantly higher level in the serum in those groups. A positive correlation was found between the levels of engraftment of human cells in the bone marrow with the levels of human albumin in the liver, as tested by mRNA and immunohistochemistry, but not in the serum of the mice, as tested by ELISA. There may be additional, rate-limiting factors that control secretion of the albumin produced by the human cells into the bloodstream of the mice. It is possible that there could be different developmental stages of the human hepatocyte-like cells, with the earliest stages able to make albumin but not able to secrete it into the bloodstream, whereas the later stages could do both. A lack of correlation between systemic albumin levels and albumin mRNA detection from the livers could also be due to nonuniform distribution of the human cells in the livers. From the current studies it appears that a certain level of albumin-secreting cells was attained and that this level was not increased by HGF administration. Prevention of the host hepatocytes from outcompeting the human stem cells in the liver may increase the levels of development of the most mature forms of human hepatocytes. Novel methods for suppressing host hepatocyte regeneration have recently been suggested by Wang et al21 and Battaile et al.22
Development of human cytokeratin 19-expressing cells in the mouse liver Finally, expression of the human cholangiocyte marker cytokeratin 19 (CK19) in the livers of the mice that received stem cell transplants was studied. No human CK19 was detected in freshly isolated CD34+ hematopoietic progenitors. No human CK19 was ever detected in NOD/SCID mice that received transplants of human hematopoietic stem/progenitor cells, either with or without liver injury and with or without human HGF supplementation by intravenous injection or MSC-mediated delivery (6 mice per arm tested; data not shown). However, in a different model of immune-deficient mice, the NOD/SCID/2M-null strain, hepatic development of human
CK19+ cells from the transplanted human stem cells was
observed (Figure 6). In 6 NOD/SCID/2M-null mice that underwent transplantation, 2 bands were
observed. The first was murine CK19, which comigrated with the band
from the control mice that did not undergo transplantation (Figure 6).
The second band was a human-specific band of the expected size, which
comigrated with the CK19 band from a human liver control (Figure 6).
The ability of the CK19+ cells to develop in the livers of
the NOD/SCID/2M-null mice, but not the NOD/SCID parental strain,
indicates that different xenograft models may be required to examine
development of different tissues from transplanted stem cells in
"stem cell plasticity" studies. The current studies provide a model
in which the differentiation of human albumin-expressing cells and
CK19-expressing cells from either bone marrow- or umbilical cord
blood-derived hematopoietic stem/progenitor cells can be examined and
provide the first animal model to study differentiation of human HSC
into hepatocyte-like cells.
The concept of stem cell plasticity is a very exciting and intriguing one. We can envision that a simple bone marrow aspirate could be used in the future to repair a patient's damaged liver, heart or skeletal muscle, nervous tissue, or perhaps any somatic tissue. The plasticity studies reported so far in the literature, demonstrating that murine stem cells can differentiate to tissues other than blood, are extremely exciting and provide us with groundbreaking ideas that are changing the way stem cell biologists perceive their field. In the current studies we demonstrate that purified human stem cells from marrow and umbilical cord blood can generate hepatocyte-like cells in the livers of immune-deficient mice. If human cord blood- or bone marrow-derived stem cells, which are relatively easily obtained and tissue matched, can contribute to liver repair in humans, this capability would impact health care in a major way. It has been difficult to achieve sustained engraftment of mature human
hepatocytes into the livers of immune-deficient mice. The mature
hepatocytes initially engraft but die out within weeks after
transplantation. Mature human hepatocytes are also difficult to culture
and sustain in vitro, and cloning can be better accomplished from
regenerating cells in the liver after injury. These data suggest that
the matrix and soluble factors controlling survival and self-renewal of
mature human hepatocytes are not yet fully understood and may be absent
(nonspecies cross-reactive) in mice. The hepatocytes survive better
under the kidney capsule than when injected via the portal vein. Many
groups are working on promoting human hepatocyte survival in vitro for
extended use in bioartificial livers.23-25 Braun et
al recently reported that stimulation of c-Met, the receptor
for hepatocyte growth factor, extended the duration of survival of
mature human hepatocytes in immune-deficient mice to 5 months.26 Part of the challenge in this field may be that
there could be inhibitory factors in adult liver to maintain the
appropriate organ mass, and therefore human prehepatocytes might
engraft there only after chemical- or radiation-induced injury or
partial hepatectomy. The primitive, marrow-derived hepatocyte precursors/stem cells may have a better advantage for survival in the
liver than mature hepatocytes. We have previously demonstrated that
intravenously injected human hematopoietic stem cells home initially to
the liver, as well as lung, spleen, and bone
marrow.9,10,27 The human cells and their progeny persist
for at least 6 months in immune-deficient mice, and human T lymphocytes
are generated from CD34+/CD3 To promote differentiation of the human "hematopoietic" stem cells to hepatocyte-like cells, we developed an immune-deficient mouse liver injury model. We first tried liver injury with radiation, and then allyl alcohol, and were not successful at obtaining significant differentiation of the transplanted human hematopoietic stem cells into hepatocyte-like cells. We then learned that CCl4 is less damaging to small blood vessels in the liver than allyl alcohol. Because it is possible that this may be a critical route for the human hematopoietic cells to influx into the damaged liver, we tried CCl4 to reduce the damage to the liver microenvironment, because it may be key in directing differentiation of stem cells to hepatocytes. Administration of 0.4 mL CCl4 per kilogram body weight was toxic to the immunodeficient mice, resulting in 75% death if left untreated. However, in the groups of mice that were given administration of human hepatocyte growth factor, 100% survival was observed. We used human CD34+ cells or
CD34+/CD38 The NOD/SCID/ The phenomenon of different immune-deficient mouse strains promoting or allowing differential lineage development has been seen in the hematopoietic system. For instance, the proportion of each hematopoietic lineage that develops from the same populations of transplanted human hematopoietic stem and progenitor cells in the NOD/SCID and beige/nude/xid xenograft systems differs dramatically. NOD/SCID mice generate primarily human B lymphocytes from transplanted human CD34+ cells. Human T-lymphocyte development has seldom been observed in NOD/SCID mice in our hands or in other laboratories unless methods are undertaken to reduce residual murine natural killer activity.42-44 In contrast, human cells of all myeloid lineages and T lymphocytes develop in the bone marrow of bnx mice that received transplants of human CD34+ cells. Development of human B cells does occur in bnx mice, but to a much lesser extent than in the NOD/SCID system.20,27,45 It is likely that similar mechanisms may exist for induction of human stem cells toward different tissue-specific lineages in the various immune-deficient mouse strains. Future studies will determine which models are best to study human stem cell plasticity in each tissue. An added advantage to the xenograft experiments described here is that
this system will allow relatively straightforward fluorescence ISH-based murine versus human chromosomal analyses to determine whether cell fusion is contributing to the apparent
"transdifferentiation" phenomena that are being observed in vivo.
Because cells in vitro can fuse when put under strong selective
pressure,46,47 it will be important to determine whether
fusion can also occur in vivo. We have not ruled out cell fusion yet in
the current studies, but have shown only that the damaged, but not
normal, liver microenvironment can promote induction of human albumin
expression from human DNA. In summary, the current studies define a
model to study differentiation of purified human cord blood- and bone
marrow-derived CD34+ and
CD34+/CD38
We very much appreciate the donation of umbilical cord blood samples from Kaiser Permanente, Los Angeles. We thank Sally Worttman, who heads our animal facility; Phillip Herrbrich, who runs our immunodeficient mouse colony; and Dr Janet Baer, for her extremely valuable veterinary assistance and advice. Light microscopy was performed at the CHLA Congressman Dixon Cellular Imaging Core. Giovanni Gaudino (University of Piemonte Orientale, Novara, Italy) kindly supplied the HGF cDNA. We thank Kathy Parker-Ponder (Washington University, St Louis, MO), Bryon Peterson (Gainesville, FL), and Neil Theise (New York University Medical Center, NY), who assisted us greatly with advice on liver damage and regeneration.
Submitted May 8, 2002; accepted December 23, 2002.
Prepublished online as Blood First Edition Paper, January 30, 2003; DOI 10.1182/blood-2002-05-1338.
Supported by National Institutes of Health (NIH), National Heart, Lung, and Blood Institute (NHLBI)/National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) 1 R01DK61848-01 (J.A.N.), NIDDK RO1 DK53041 (J.A.N.), NIDDK RO1DK54567 (G.M.C.), P01CA59318 (G.M.C.), and NIH NHLBI SCOR no. 1-P50-HL54850 (J.A.N., G.M.C.). G.M.C. is a Scholar of the Leukemia, Lymphoma Society and is supported by the Seaver Foundation.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Jan A. Nolta, Washington University School of Medicine, Department of Internal Medicine, Division of Oncology, Section of Stem Cell Biology, 660 S Euclid, Box 8007, St Louis, MO 63110; e-mail: jnolta{at}im.wustl.edu.
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Top
Abstract
Introduction
20°C. Then slides were incubated with probe at 80°C
for 5 minutes, then at 37°C for 90 minutes. Alu probes end-labeled
with multiple fluorescein-linker molecules were obtained from
Innogenex. The Alu probe was diluted to 1:1 in a buffer provided by the
company, and 20 uL of this mixture per slide was used. An
antifluorescein antibody (LINK-1) was used to detect the
labeled probe. To amplify the signal, LINK-2, which carries biotin and
recognizes and binds to the link 1 antibody, was added. To visualize
the antibody/probe complex, horseradish peroxidase-conjugated
streptavidin (label) was added next. Each biotin molecule on LINK-2
binds to a streptavidin moiety of the label. Upon addition of the
chromogen DAB/substrate, an intense dark brown to black color appeared
at the specific site of the hybridized probe. Next, fluorescent
labeling or immunohistochemical detection of human albumin was
performed. Slides were rinsed in Tris
(tris(hydroxymethyl)aminomethane)-buffered saline (TBS, pH 7.5) 2 times for 5 minutes each. Slides were then blocked with Power Block (Innogenex) supplemented with murine
immunoglobulin G (Coulter, Hialeah, FL) for 15 minutes. Rabbit antihuman albumin-fluorescein isothiocyanate
(FITC; DAKO, Carpenteria, CA) was used at a 1:100 dilution to
counterstain the slides for 15 minutes.
