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Bone marrow to liver: the blood of Prometheus
seminars in CELL & DEVELOPMENTAL BIOLOGY, Vol. 13, 2002: pp. 411–417 doi:10.1016/S1084–9521(02)00128-3, available online at http://www.idealibrary.com on
Bone marrow to liver: the blood of Prometheus Neil D. Theise a,∗ and Diane S. Krause b a liver stem cell. The existence of hepatic stem or progenitor cells has thus been hotly debated, though the presumption was that if such cells existed, they would lie within the liver. Consensus that there is an intra-hepatic progenitor cell compartment in animals and humans has only recently been achieved.1 The existence of circulating hepatic progenitors in the blood, that are likely to be, at least in part, bone marrow derived was, to say the least, a surprise.2, 3 Two independent lines of investigation strongly suggest that this is the case. First, it was demonstrated that the intrahepatic progenitor cells that proliferate in response to liver damage, ‘oval cells’ in rodents and ‘ductular reactions’ in humans, sometimes express markers that were previously believed to be hematopoietic stem cell markers, such as Thy1,4 CD34,5 and c-kit.6 These findings led Bryon Petersen to speculate that oval cells may be marrow-derived in adult animals, which he then confirmed in a rat model of acute injury.2 Second, our own structural analysis of the canals of Hering in humans, while showing that they comprised an intraorgan progenitor compartment, could not account for all the experimental findings.7, 8 In order to account for these findings, one needed to hypothesize that there was an extra-organ, circulating population of hepatic progenitor cells. We confirmed this hypothesis in mice in the absence of severe injury.3 Confirmation that circulating cells, at least partly bone marrow-derived, could reconstitute human livers soon followed.9, 10 Thus, liver cell turn over and repair can now be thought of as a three-tier process as diagrammed in Figure 1. The differentiation of marrow-derived cells into hepatic cells has now been demonstrated in a variety of in vivo models2, 3, 9–13b and one ex vivo series of experiments14 (Table 1). While the phenomenon is now rather well accepted, many questions remain unanswered. For example, is there a specific cell in the marrow that is capable of hepatic repopulation or do diverse cells have this capacity? What are the mechanisms whereby cells are mobilized from the marrow, recruited to the liver, and induced to differentiate along hepatocellular (or cholangiocellular) lines?
The existence of hepatic stem or progenitor cells has been controversial for decades, though it was presumed that if such cells existed, they would lie within the liver. There is now consensus, however, that not only do facultative hepatic stem cells exist within the liver, but also that cells from extra-hepatic sites, in particular the bone marrow, can contribute to hepatocyte and cholangiocyte regeneration. Despite confidence that engraftment of marrow cells in the liver occurs, the mechanistic details of this process remain poorly understood. Moreover, the physiological importance and therapeutic utility of this phenomenon remains controversial. Key words: bone marrow / liver / plasticity / progenitor cells / stem cells © 2002 Elsevier Science Ltd. All rights reserved.
Introduction The fact that the liver has extraordinary regenerative capacity was enshrined millenia ago in the Greek myth of Prometheus, whom Zeus punished by chaining him to a rock and having his liver eaten every morning by an eagle. At nightfall the eagle would fly away, sated, and Prometheus’ liver would grow back, so as to be ready for the eagle’s breakfast at the next sunrise. Our understanding of hepatic regeneration took its next leap forward when the microscope enabled investigators to see that mature hepatocytes and cholangiocytes were capable of division, thus elucidating at least part of the mechanism for hepatic regeneration. The observation of mature hepatic cells undergoing division in response to injury seemed to obviate the need for From the a Department of Pathology, New York University School of Medicine, Room 461, 560 First Avenue, New York, NY 10016, USA and b Department of Laboratory Medicine, Yale University School of Medicine, P.O. Box 208035, New Haven, CT 06520-8035, USA. * Corresponding author. E-mails: [email protected], [email protected] © 2002 Elsevier Science Ltd. All rights reserved. 1084–9521 / 02 / $– see front matter
411
Authors
In vivo Petersen et al., 2 Theise et al., 3
Species
Donor cells
412
Theise et al., 9
Rat Mouse Mouse Human
wbm wbm CD34+ lin− wbm
Alison et al., 10 Lagasse et al., 12 Lagasse et al., 12 Lagasse et al., 12 Lagasse et al., 12 Lagasse et al., 12 Lagasse et al., 12 Krause et al., 11
Human Mouse Mouse Mouse Mouse Mouse Mouse Mouse
Korbling et al., 13a
Human
Mallet et al., 13b
Mouse
wbm c-kit+ c-kit− lin+ lin− sca-1+ sca-1− lin− , elutriated, marrow homing Peripheral blood stem cells lin− bm from L-PK-Bcl-2 mice
Mouse
In vitro Schwartz et al., 14
Human, rat, mouse
lin− bm from L-PK-Bcl-2 mice
Marrow stromal cells
Injury type
Severity
Semiquantificative summary of % engraftment data Hepatocytes
Oval cells/ductular reactions
Cholangiocytes
CCl4 , AAF Radiation (12 cGy) Radiation (12 cGy) Cholestasis, biliary strictures post-OLT, or recurrent HCV and PSC NR FAH−/− model FAH−/− model FAH−/− model FAH−/− model FAH−/− model FAH−/− model Radiation (12 cGy)
Severe ? Minimal ? Minimal Various
1+, clustering 1+, no clustering 1+, no clustering 1+ to 3+, clustering
Blood derived Not present Not present Blood derived
0 0 1+ to 3+
NR Severe Severe Severe Severe Severe Severe ? Minimal
1+, clustering Clustering, 3+ (est.) Clustering, 1+ (est.) Clustering, 1+ (est.) Clustering, 3+ to 4+ (est.) Clustering, 3+ to 4+ (est.) Clustering, 1+ (est.) 1+, no clustering
NR NR NR NR NR NR NR Not present
1+ NR NR NR NR NR NR 1+
GVHD, drug toxicity
? Mild
2+, clustering
NR
NR
Radiation (9.5 cGy)
? Minimal
NR
NR
2/3 hepatectomy + Fas agonist antibody Jo2 Sublethal anti-mouse Fas monoclonal antibody, IV, + Fas agonist antibody Jo2
Severe
? 1+, perivascular distribution, no clustering 1+, clustering
NR
NR
? Severe
1+, clustering
NR
NR
NA
NA
Robust differentiation
NA
NA
Notes: wbm: whole bone marrow; CCl4 : carbon tetrachloride; AAF: aminoacetylfluorane; OLT: orthotopic liver transplant; HCV: hepatitis C virus; PSC: primary sclerosing cholangitis; NR: not reported; FAH: fumaryl acetoacetate hydrolase knockout model of tyrosenemia, type I; est.: data not provided in percentile engraftment, semiquantitation is estimated; GVHD: graft versus host disease; NA: not applicable. Semi-quantitative grading of reported engraftment—0: absent; 1+: 0.1–3%; 2+: 3–20%; 3+: 20–50%; 4+: >50%.
N.D. Theise and D.S. Krause
Table 1. Recent reports of liver from bone marrow-derived cells
Bone marrow to liver: the blood of Prometheus
have caused low level injury or modified the ability of hepatocytes to contribute to normal cell turnover. The livers never had histologically demonstrable liver damage following the irradiation, whether examined in the first days after irradiation or months later. In this model, hepatocyte engraftment from the marrow never rose above 2%.3 Human studies in which no more than mild injury was known from clinical or histologic parameters show a greater, though still relatively low level of engraftment: 1 to 5% of hepatocytes, and up to 10% of cholangiocytes.9 With a greater degree of liver injury, there is a broad range of engraftment of marrow-derived cells as mature liver cells, and the degree of engraftment correlates with the degree and type of liver damage. This point is emphasized by Mallet et al. arguing that some selection against native hepatocytes and for circulating cells must be exerted in order to stimulate hepatocyte engraftment from the marrow compartment.13b In addition, there may also be differences in the marrow derived cellular response in different species. In Petersen’s original work, massive hepatocyte injury with carbon tetrachloride in the presence of acetylaminofluorene inhibition of hepatocyte regeneration induced oval cell proliferation and led to no more engraftment than we had observed in mice that had been lethally irradiated prior to bone marrow transplantation.3 However, with the injury induced by Petersen, clusters of marrow-derived hepatocytes were seen.2 In humans with injury severe enough to provoke the human equivalent of oval cell proliferation, the level of engraftment of extrahepatic cells ranged from 5 to 40% for hepatocytes and 5 to 35% for cholangiocytes.9, 10 The most dramatic engraftment of marrow-derived cells as functional liver cells was seen in a murine model of tyrosinemia in which wild type donor cells rescued the mice with reconstitution of >50% of the liver mass.12 Although a systematic comparison of different levels of a single type of injury or of the same injury in different species has not yet been published, it is difficult to refute that marrow-derived cells can engraft in the liver as part of physiologic repair mechanism. In fact, this may be the predominant means of recovery from certain types of experimental liver injury.
Figure 1. Schematic diagrams of normal hepatic tissue maintenance and regeneration in the case of massive hepatic necrosis (possible causes of which include severe acute viral hepatitis or toxic injury). Thick lines represent dominant pathways, thin lines indicate pathways that contribute at no more than low levels. Dotted lines indicate pathways that are hypothesized to exist, but have not yet been firmly demonstrated in clinical or experimental conditions.
There are also areas of controversy regarding the validity of the data that show differentiation of hepatic cells from marrow-derived cells. Could the methods used to detect the phenomenon over or under-estimate its incidence? Could a process of cell fusion, rather than true engraftment and differentiation, account for the experimental findings? This brief review will consider some of these questions and controversies.
Magnitude of hepatic repopulation from the marrow Studies of liver reconstitution from the marrow suggest that the degree and pattern of hepatic reconstitution depend on the level of injury to the liver. In our own original murine study there was no attempt to cause overt liver damage, though the radiation to condition the animals for bone marrow transplant might
Marrow subpopulations with hepatic potential Half of the published studies in this area, and all those involving humans, employed whole bone marrow or large populations of mobilized, circulating 413
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hematopoietic cells as the donor population. However, some studies have specifically addressed the issue of which marrow subpopulations are capable of differentiation into mature liver cells. Our initial murine study included two mice who had been transplanted 8 months previously with 200 CD34+ lin− bone marrow cells. This marrow subpopulation certainly contained hematopoietic stem cells, but may also have harbored CD34+ stromal or endothelial cells. The most detailed data to compare the liver-differentiation ability of different marrow subpopulations are available for the murine tyrosinemia model.12 The liver engrafting capacity was compared for marrow populations with different levels of expression of lineage commitment markers (lin), the Stem Cell Factor receptor c-kit and the stem cell antigen sca-1. Consistent high levels of engraftment that were able to rescue the animals from liver failure were found for the lin− kit+ sca-1+ population, which was able to achieve this with as little as 50 donated cells. They concluded that “only hematopoietic stem cells in adult bone marrow gave rise to hepatocytes.” However, other marrow subpopulations in this study also had some degree of hepatic potential, however small or inconsistent. If one assumes that differentiation processes are stochastic in nature as we have suggested, then we cannot wave these aside as purely a result of ‘contaminants’ because these could represent the rare occurrence of more differentiated cells ‘transdifferentiating’ into hepatic cells.15, 16 The authors state that these data confirm that the marrow-derived hepatic engrafting cells are also hematopoietic. However, confirmation of such plasticity requires experiments demonstrating clonal expansion of a single cell, and even if there is a single cell that is capable of differentiation into both blood and liver cells, this would not be a proof that a cell that was committed to hematopoiesis had changed its gene expression pattern. An alternative explanation would be that the marrow harbors a stem cell population that is both pre-hematopoietic and pre-hepatic. Only two published studies to date demonstrate true plasticity of marrow cells that includes hepatic potential. The first is our murine marrow transplantation study, in collaboration with the laboratory of Saul Sharkis at Johns Hopkins University. In this study, a single bone marrow cell selected by lineage depletion, elutriation and functional marrow homing, whether obtained by limiting dilution or by direct visualization, was capable of producing mesodermal tissues (hematopoiesis), ectodermal tissues (skin and
skin appendages), and endodermal tissue (epithelial cells of liver, lung, and gastrointestinal tract).11 The second study, from the group of Catherine Verfaillie at University of Minnesota, demonstrates that clonally expanded stromal cells from mouse, rat, and human marrow can differentiate in vitro into hepatocytes,14 as well as an array of other endodermal, ectodermal, and mesodermal cell types.17 It seems unlikely that the marrow homing cell in our study and the marrow stromal cell of origin in the Verfaillie studies are the same cell type. Thus, it would seem that marrow-derived cells with at least two different phenotypes at least two types of marrow cells have hepatic potential. Yet, again, these studies are very limited in what they have to teach us about which cell populations physiologically contribute to liver maintenance and repair. In our bone marrow homing study, the single cell is certainly plastic, but, having given rise to the entire bone marrow after engraftment, it tells us little about which marrow subpopulations normally comprise the extrahepatic stem cells for liver maintenance and repair; this is the strength of the study with the tyrosinemia model, its data suggests which populations of the fully developed marrow may be most prepared to act as a reservoir for liver regeneration in severe injury. Whether the marrow stromal cell participates in normal physiologic hepatic maintenance is not addressed at all by the in vitro work.
Mechanisms of engraftment Two patterns of hepatic engraftment from the marrow have been identified. The first is that cells enter the liver from the circulation in response to injury of a severity and type that also activates an oval cell/ductular reaction. Many of these entering cells clearly begin life in the liver with the morphology of these oval cell-like hepatic progenitors and then differentiate into hepatocytes and cholangiocytes as the repair process continues. Because of this mechanism of entry, the hepatocyte engraftment is greater in the periportal region, where these intermediate cell reactions predominantly occur in both animals2 and humans.9 It is tempting to suggest that entry to the liver is site specific, either into the canals of Hering7 or as periductal cells.18 Whether the expression by oval cells/ductular reaction cells of c-kit, thy1, and CD34 are markers of cells that have come from the circulation and whether these molecules are functionally important in this process remains to be determined. 414
Bone marrow to liver: the blood of Prometheus
One mechanism for recruitment to the liver of circulating cells, as oval cell/ductular reactions, has been preliminarily reported by Petersen’s group.19 They suggest that in severe hepatic injury, surviving hepatocytes produce stromal derived factor-1 (SDF-1) and that the circulating cells, entering the liver as oval cell/ductular reactions, express the receptor for this chemokine, CXCR4. This is the same homing mechanism that has been described for hematopoietic stem cells homing to the bone marrow,20 and may be related to the cells that are functionally isolated by marrow-homing in the Sharkis isolation procedure. A second pattern of engraftment has been noted in the absence of oval cell/ductular reactions. Without injury or with mild injury, the engraftment seems to occur without an identifiable oval cell/ductular intermediate and the periportal predominance is not seen: isolated cells or small clusters appear as though randomly scattered in the hepatic lobule.3, 9 Such distribution suggests that these marrow-derived cells engrafted from the circulation into the liver via an alternative pathway. We may speculate that such isolated cells represent an epithelial equivalent to the short term engrafting progenitor cells from the marrow: they differentiate appropriately into mature cell types, but do not have an open-ended ability to replicate. This question is intriguing and certainly amenable to easy investigation. How cells mobilize from the marrow is also an open question. Do they stream steadily from the marrow or are they mobilized in response to injury? Mobilization of cardiac progenitor cells from the marrow and their subsequent engraftment in ischemic myocardium can be induced by SCF and G-CSF administration, thereby increasing marrow contribution to myocyte repopulation after myocardial infarction.21 It is possible that marrow-mobilizing cytokines are secreted in response to severe liver damage as a signaling pathway to stimulate liver reconstitution from marrow.
ever, closer examination revealed that these cells were actually tetraploid fusion products, with XXXY genotype. An accompanying editorial declared “doubt has been shed on adult stem cell research”, suggesting that the studies of marrow engraftment in the liver and in other organs could be an artifact of such fusion events. That stable fusion events are responsible for the liver findings is very unlikely. Diploidy of hepatocytes in acute, let alone in severe injury, has been well documented for many decades.24 Tetraploidy is only seen in aging livers and then predominates in the centrilobular regions, i.e. not where the most significant engraftment of marrow cells has been documented. This does not rule out the possibility of unstable fusion events, in which the tetraploid fusion product immediately redivides into two diploid cells. But if this occurs it would simply be a mechanism for the demonstrated plasticity, since it still requires that genes or chromosomes which were in a circulating cell population now reside in hepatocytes. Although fusion is unlikely, control experiments that directly address this issue should be included in published studies for the near term, at least. The second point of controversy involves the relative capacity of marrow cells to repopulate liver compared with other cells, in particular with transplanted hepatocytes. This issue is raised by cell transplantation rescue with the murine tyrosinemia model for which hepatocytes are clearly a more dependable and efficient population.12 However, it would again be unwise to over interpret this difference: the comparatively greater rescue capacity of hepatocytes probably relates to the specifics of the model. Above, we surmise that large scale hepatic repopulation by marrow cells is probably a receptor dependent, site-specific process. On the other hand, engraftment of transplanted hepatocytes is dependent on where these large cells get trapped within the sinusoids of the liver.25 One may presume that in the tyrosinemia model there is near complete disruption of the microanatomy upon which large-scale marrow engraftment depends and, at the same time, an increase in circulatory sluggishness, favoring hepatocyte trapping and integration. Thus, the model itself may be the selective factor, not the general capacities of the donor cells. It is not clear whether the data for this model will be directly relevant to the treatment of human liver disease. The murine tyrosinemia model is unlike human tyrosinemia in its morphological and temporal course; only rare, very severe instances of massive hepatic necrosis in humans are a pathologic correlate to the
Controversies Three issues have been raised which lend an air of controversy to this field. The most recent arises from publication of two studies in which male adult stem cells (hematopoietic and neural, respectively) were co-cultured with female embryonic stem cells.22, 23 Both experiments resulted in embryonic-type cells containing male chromosomes, suggesting differentiation of adult cells to an embryonic phenotype. How415
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model. Until more data are obtained using different models of liver disease, and studying human tissues from patients with variable pathology, we cannot reach any conclusions regarding which cell populations may be the best to use in the development of cellular therapeutics. A third area of controversy involves differences between engraftment detected by Y-chromosome staining in gender mismatched models and that detected by expression of a transgene specific to the donor population. With the exception of the Lagasse study in which both Y-chromosome and beta-galactosidase expression demonstrate large-scale repopulation, Y-chromosome analysis seems to give higher estimates of engraftment. One explanation for this is that whereas nearly every visualized male cell contains a Y-chromosome, transgene expression may be variable in hepatocytes, either being unexpressed or expressed at low levels within the time course of the experiments. On the other hand, extreme care must be taken to be sure that overlapping or emperipolesis of circulating monocytes does not lead to false positive interpretation of Y-chromosome signals in hepatocytes that are actually female derived.
References 1. Sell S (2001) Heterogeneity and plasticity of hepatocyte lineage cells. Hepatology 33:738–750 2. Petersen BE, Bowen WC, Patrene KD, Mars MW, Sullivan AK, Murase N, Boggs SS et al. (1999) Bone marrow as a potential source of hepatic oval cells. Science 284:1168–1170 3. Theise ND, Badve S, Saxena R, Henegariu O, Sell S, Crawford JM, Krause DS (2000) Derivation of hepatocytes from bone marrow cells in mice after radiation-induced myeloablation. Hepatology 31:235–240 4. Petersen BE, Goff JP, Greenberger JS, Michalopoulos GK (1998) Hepatic oval cells express the hematopoietic stem cell marker Thy-1 in the rat. Hepatology 27:433–445 5. Omori N, Omori M, Evarts RP, Teramoto T, Miller MJ, Hoang TN, Thorgeirsson SS (1997) Partial cloning of rat CD34 cDNA and expression during stem cell-dependent liver regeneration in the adult rat. Hepatology 26:720–727 6. Baumann U, Crosby HA, Ramani P, Kelly DA, Strain AJ (1999) Expression of the stem cell factor receptor c-kit in normal and diseased pediatric liver: identification of a human hepatic progenitor cell? Hepatology 30:112–117 7. Theise ND, Saxena R, Portmann BC, Thung SN, Yee H, Chiriboga L, Kumar A, Crawford JM (1999) The canals of Hering and hepatic stem cells in humans. Hepatology 30:1425–1433 8. Yavorkovsky L, Lai E, Ilic Z, Sell S (1995) Participation of small intraportal stem cells in the restitutive response of the liver to periportal necrosis induced by allyl alcohol. Hepatology 21:1702–1712 9. Theise ND, Nimmakayalu M, Gardner R, Illei P, Morgan G, Teperman L, Henegariu O, Krause DS (2000) Liver from bone marrow in humans. Hepatology 32:11–16 10. Alison MR, Poulsom R, Jeffery R, Dhillon AP, Quaglia A, Jacob J, Novelli M, Prentice G, Williamson J, Wright NA (2000) Hepatocytes from non-hepatic adult stem cells. Nature 406:257 11. Krause DS, Theise ND, Collector MI, Henegariu O, Hwang S, Gardner R, Neutzel S, Sharkis SJ (2001) Multi-organ, multi-lineage engraftment by a single bone marrow-derived stem cell. Cell 105:369–377 12. Lagasse E, Connors H, Al-Dhalimy M, Reitsma M, Dohse M, Osborne L, Wang X, Finegold M, Weissman IL, Grompe M (2000) Purified hematopoietic stem cells can differentiate into hepatocytes in vivo. Nat Med 6:1229–1234 13. (a) Korbling M, Katz RL, Khanna A, Ruifrok AC, Rondon G, Albitar M, Champlin RE, Estrov Z (2002) Hepatocytes and epithelial cells of donor origin in recipients of peripheral-blood stem cells. New Engl J Med 346:738–746; (b) Mallet VO, Mitchell C, Mezey E, Fabre M, Guidotti J-E, Renia L, Couloumbel L, Kahn A, Gilgenkrantz H (2002) Bone marrow transplantation in mice leads to a minor population of hepatocytes tah can be selectively amplified in vivo. Hepatology 35:799–804 14. Schwartz RE, Reyes M, Koodie L, Jiang Y, Blackstad M, Lund T, Lenvik T, Johnson S, Hu W-S, Verfaillie CM (2002) Multipotent adult progenitor cells from bone marrow differentiate into functional hepatocyte-like cells. J Clin Invest 109:1291–1302 15. Theise ND, Krause DS (2001) Suggestions for a new paradigm of cell differentiative potential. Blood Cells Mol Dis 27:625–631 16. Theise ND, Krause DS (2002) Toward a new paradigm of cell differentiation capacity. Leukemia 16:542–548 17. Reyes M, Lund T, Lenvik T, Aguiar D, Koodie L, Verfaillie CM (2001) Purification and ex vivo expansion of postnatal human marrow mesodermal progenitor cells. Blood 98:2615–2625
Conclusion This exciting field of investigation is in its infancy. Conclusions about the uses to which these discoveries will be put or about the relative physiological importance of the events are premature. The list of questions far exceeds the list of answers. And we may add to that list still more intriguing questions. The liver is capable of hematopoiesis during embryogenesis and also occasionally in adulthood:26 is this truly, as has been tacitly presumed, because of an hematopoietic stem cell ‘rest’ in the hepatic vascular compartment, left over from fetal development, which gets reactivated? Or might the liver’s own epithelial, intraorgan stem cell compartment send cells back into the circulation? If so, perhaps some marrow-derived hepatic progenitors are actually originally hepatic derived. Might other organs also participate in such trafficking? While such possibilities may have been unimaginable a few years ago, they must now be considered serious hypotheses that require investigation. The only certainty is that much work lies ahead and that we have barely begun to examine these exciting possibilities, let alone to exploit their implied therapeutic potential. 416
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22. Terada N, Hamazaki T, Oka M, Hoki M, Mastalerz DM, Nakano Y, Meyer EM, Morel L, Petersen BE, Scott EW (2002) Bone marrow cells adopt the phenotype of other cells by spontaneous cell fusion. Nature 416:542–545 23. Ying QL, Nichols J, Evans EP, Smith AG (2002) Changing potency by spontaneous fusion. Nature 416:545–548 24. Gupta S (2000) Hepatic polyploidy and liver growth control. Semin Cancer Biol 10:161–171 25. Gupta S, Rajvanshi P, Sokhi R, Slehria S, Yam A, Kerr A, Novikoff PM (1999) Entry and integration of transplanted hepatocytes in rat liver plates occur by disruption of hepatic sinusoidal endothelium. Hepatology 29:509–519 26. Cardier JE, Barbera-Guillem E (1997) Extramedullary hematopoiesis in the adult mouse liver is associated with specific hepatic sinusoidal endothelial cells. Hepatology 26:165–175
18. Lee JH, Rim HJ, Sell S (1997) Heterogeneity of the “oval-cell” response in the hamster liver during cholangiocarcinogenesis following Clonorchis sinensis infection and dimethylnitrosamine treatment. J Hepatol 26:1313–1323 19. Petersen BE, Hatch HM, Jorgensen ML, Stolz DB (2001) SDF-1 as a potential homing protein for bone marrow derived liver oval cells. FASEB J 15:A1084 20. Voermans C, van Hennik PB, van Der Schoot CE (2001) Homing of human hematopoietic stem and progenitor cells: new insights, new challenges? J Hematother Stem Cell Res 10:725–738 21. Orlic D, Kajstura J, Chimenti S, Limana F, Jakoniuk I, Quaini F, Nadal-Ginard B, Bodine DM, Leri A, Anversa P (2001) Mobilized bone marrow cells repair the infarcted heart, improving function and survival. Proc Natl Acad Sci USA 98:10344– 10349
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Bone marrow to liver: the blood of Prometheus Neil D. Theise a,∗ and Diane S. Krause b a liver stem cell. The existence of hepatic stem or progenitor cells has thus been hotly debated, though the presumption was that if such cells existed, they would lie within the liver. Consensus that there is an intra-hepatic progenitor cell compartment in animals and humans has only recently been achieved.1 The existence of circulating hepatic progenitors in the blood, that are likely to be, at least in part, bone marrow derived was, to say the least, a surprise.2, 3 Two independent lines of investigation strongly suggest that this is the case. First, it was demonstrated that the intrahepatic progenitor cells that proliferate in response to liver damage, ‘oval cells’ in rodents and ‘ductular reactions’ in humans, sometimes express markers that were previously believed to be hematopoietic stem cell markers, such as Thy1,4 CD34,5 and c-kit.6 These findings led Bryon Petersen to speculate that oval cells may be marrow-derived in adult animals, which he then confirmed in a rat model of acute injury.2 Second, our own structural analysis of the canals of Hering in humans, while showing that they comprised an intraorgan progenitor compartment, could not account for all the experimental findings.7, 8 In order to account for these findings, one needed to hypothesize that there was an extra-organ, circulating population of hepatic progenitor cells. We confirmed this hypothesis in mice in the absence of severe injury.3 Confirmation that circulating cells, at least partly bone marrow-derived, could reconstitute human livers soon followed.9, 10 Thus, liver cell turn over and repair can now be thought of as a three-tier process as diagrammed in Figure 1. The differentiation of marrow-derived cells into hepatic cells has now been demonstrated in a variety of in vivo models2, 3, 9–13b and one ex vivo series of experiments14 (Table 1). While the phenomenon is now rather well accepted, many questions remain unanswered. For example, is there a specific cell in the marrow that is capable of hepatic repopulation or do diverse cells have this capacity? What are the mechanisms whereby cells are mobilized from the marrow, recruited to the liver, and induced to differentiate along hepatocellular (or cholangiocellular) lines?
The existence of hepatic stem or progenitor cells has been controversial for decades, though it was presumed that if such cells existed, they would lie within the liver. There is now consensus, however, that not only do facultative hepatic stem cells exist within the liver, but also that cells from extra-hepatic sites, in particular the bone marrow, can contribute to hepatocyte and cholangiocyte regeneration. Despite confidence that engraftment of marrow cells in the liver occurs, the mechanistic details of this process remain poorly understood. Moreover, the physiological importance and therapeutic utility of this phenomenon remains controversial. Key words: bone marrow / liver / plasticity / progenitor cells / stem cells © 2002 Elsevier Science Ltd. All rights reserved.
Introduction The fact that the liver has extraordinary regenerative capacity was enshrined millenia ago in the Greek myth of Prometheus, whom Zeus punished by chaining him to a rock and having his liver eaten every morning by an eagle. At nightfall the eagle would fly away, sated, and Prometheus’ liver would grow back, so as to be ready for the eagle’s breakfast at the next sunrise. Our understanding of hepatic regeneration took its next leap forward when the microscope enabled investigators to see that mature hepatocytes and cholangiocytes were capable of division, thus elucidating at least part of the mechanism for hepatic regeneration. The observation of mature hepatic cells undergoing division in response to injury seemed to obviate the need for From the a Department of Pathology, New York University School of Medicine, Room 461, 560 First Avenue, New York, NY 10016, USA and b Department of Laboratory Medicine, Yale University School of Medicine, P.O. Box 208035, New Haven, CT 06520-8035, USA. * Corresponding author. E-mails: [email protected], [email protected] © 2002 Elsevier Science Ltd. All rights reserved. 1084–9521 / 02 / $– see front matter
411
Authors
In vivo Petersen et al., 2 Theise et al., 3
Species
Donor cells
412
Theise et al., 9
Rat Mouse Mouse Human
wbm wbm CD34+ lin− wbm
Alison et al., 10 Lagasse et al., 12 Lagasse et al., 12 Lagasse et al., 12 Lagasse et al., 12 Lagasse et al., 12 Lagasse et al., 12 Krause et al., 11
Human Mouse Mouse Mouse Mouse Mouse Mouse Mouse
Korbling et al., 13a
Human
Mallet et al., 13b
Mouse
wbm c-kit+ c-kit− lin+ lin− sca-1+ sca-1− lin− , elutriated, marrow homing Peripheral blood stem cells lin− bm from L-PK-Bcl-2 mice
Mouse
In vitro Schwartz et al., 14
Human, rat, mouse
lin− bm from L-PK-Bcl-2 mice
Marrow stromal cells
Injury type
Severity
Semiquantificative summary of % engraftment data Hepatocytes
Oval cells/ductular reactions
Cholangiocytes
CCl4 , AAF Radiation (12 cGy) Radiation (12 cGy) Cholestasis, biliary strictures post-OLT, or recurrent HCV and PSC NR FAH−/− model FAH−/− model FAH−/− model FAH−/− model FAH−/− model FAH−/− model Radiation (12 cGy)
Severe ? Minimal ? Minimal Various
1+, clustering 1+, no clustering 1+, no clustering 1+ to 3+, clustering
Blood derived Not present Not present Blood derived
0 0 1+ to 3+
NR Severe Severe Severe Severe Severe Severe ? Minimal
1+, clustering Clustering, 3+ (est.) Clustering, 1+ (est.) Clustering, 1+ (est.) Clustering, 3+ to 4+ (est.) Clustering, 3+ to 4+ (est.) Clustering, 1+ (est.) 1+, no clustering
NR NR NR NR NR NR NR Not present
1+ NR NR NR NR NR NR 1+
GVHD, drug toxicity
? Mild
2+, clustering
NR
NR
Radiation (9.5 cGy)
? Minimal
NR
NR
2/3 hepatectomy + Fas agonist antibody Jo2 Sublethal anti-mouse Fas monoclonal antibody, IV, + Fas agonist antibody Jo2
Severe
? 1+, perivascular distribution, no clustering 1+, clustering
NR
NR
? Severe
1+, clustering
NR
NR
NA
NA
Robust differentiation
NA
NA
Notes: wbm: whole bone marrow; CCl4 : carbon tetrachloride; AAF: aminoacetylfluorane; OLT: orthotopic liver transplant; HCV: hepatitis C virus; PSC: primary sclerosing cholangitis; NR: not reported; FAH: fumaryl acetoacetate hydrolase knockout model of tyrosenemia, type I; est.: data not provided in percentile engraftment, semiquantitation is estimated; GVHD: graft versus host disease; NA: not applicable. Semi-quantitative grading of reported engraftment—0: absent; 1+: 0.1–3%; 2+: 3–20%; 3+: 20–50%; 4+: >50%.
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Table 1. Recent reports of liver from bone marrow-derived cells
Bone marrow to liver: the blood of Prometheus
have caused low level injury or modified the ability of hepatocytes to contribute to normal cell turnover. The livers never had histologically demonstrable liver damage following the irradiation, whether examined in the first days after irradiation or months later. In this model, hepatocyte engraftment from the marrow never rose above 2%.3 Human studies in which no more than mild injury was known from clinical or histologic parameters show a greater, though still relatively low level of engraftment: 1 to 5% of hepatocytes, and up to 10% of cholangiocytes.9 With a greater degree of liver injury, there is a broad range of engraftment of marrow-derived cells as mature liver cells, and the degree of engraftment correlates with the degree and type of liver damage. This point is emphasized by Mallet et al. arguing that some selection against native hepatocytes and for circulating cells must be exerted in order to stimulate hepatocyte engraftment from the marrow compartment.13b In addition, there may also be differences in the marrow derived cellular response in different species. In Petersen’s original work, massive hepatocyte injury with carbon tetrachloride in the presence of acetylaminofluorene inhibition of hepatocyte regeneration induced oval cell proliferation and led to no more engraftment than we had observed in mice that had been lethally irradiated prior to bone marrow transplantation.3 However, with the injury induced by Petersen, clusters of marrow-derived hepatocytes were seen.2 In humans with injury severe enough to provoke the human equivalent of oval cell proliferation, the level of engraftment of extrahepatic cells ranged from 5 to 40% for hepatocytes and 5 to 35% for cholangiocytes.9, 10 The most dramatic engraftment of marrow-derived cells as functional liver cells was seen in a murine model of tyrosinemia in which wild type donor cells rescued the mice with reconstitution of >50% of the liver mass.12 Although a systematic comparison of different levels of a single type of injury or of the same injury in different species has not yet been published, it is difficult to refute that marrow-derived cells can engraft in the liver as part of physiologic repair mechanism. In fact, this may be the predominant means of recovery from certain types of experimental liver injury.
Figure 1. Schematic diagrams of normal hepatic tissue maintenance and regeneration in the case of massive hepatic necrosis (possible causes of which include severe acute viral hepatitis or toxic injury). Thick lines represent dominant pathways, thin lines indicate pathways that contribute at no more than low levels. Dotted lines indicate pathways that are hypothesized to exist, but have not yet been firmly demonstrated in clinical or experimental conditions.
There are also areas of controversy regarding the validity of the data that show differentiation of hepatic cells from marrow-derived cells. Could the methods used to detect the phenomenon over or under-estimate its incidence? Could a process of cell fusion, rather than true engraftment and differentiation, account for the experimental findings? This brief review will consider some of these questions and controversies.
Magnitude of hepatic repopulation from the marrow Studies of liver reconstitution from the marrow suggest that the degree and pattern of hepatic reconstitution depend on the level of injury to the liver. In our own original murine study there was no attempt to cause overt liver damage, though the radiation to condition the animals for bone marrow transplant might
Marrow subpopulations with hepatic potential Half of the published studies in this area, and all those involving humans, employed whole bone marrow or large populations of mobilized, circulating 413
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hematopoietic cells as the donor population. However, some studies have specifically addressed the issue of which marrow subpopulations are capable of differentiation into mature liver cells. Our initial murine study included two mice who had been transplanted 8 months previously with 200 CD34+ lin− bone marrow cells. This marrow subpopulation certainly contained hematopoietic stem cells, but may also have harbored CD34+ stromal or endothelial cells. The most detailed data to compare the liver-differentiation ability of different marrow subpopulations are available for the murine tyrosinemia model.12 The liver engrafting capacity was compared for marrow populations with different levels of expression of lineage commitment markers (lin), the Stem Cell Factor receptor c-kit and the stem cell antigen sca-1. Consistent high levels of engraftment that were able to rescue the animals from liver failure were found for the lin− kit+ sca-1+ population, which was able to achieve this with as little as 50 donated cells. They concluded that “only hematopoietic stem cells in adult bone marrow gave rise to hepatocytes.” However, other marrow subpopulations in this study also had some degree of hepatic potential, however small or inconsistent. If one assumes that differentiation processes are stochastic in nature as we have suggested, then we cannot wave these aside as purely a result of ‘contaminants’ because these could represent the rare occurrence of more differentiated cells ‘transdifferentiating’ into hepatic cells.15, 16 The authors state that these data confirm that the marrow-derived hepatic engrafting cells are also hematopoietic. However, confirmation of such plasticity requires experiments demonstrating clonal expansion of a single cell, and even if there is a single cell that is capable of differentiation into both blood and liver cells, this would not be a proof that a cell that was committed to hematopoiesis had changed its gene expression pattern. An alternative explanation would be that the marrow harbors a stem cell population that is both pre-hematopoietic and pre-hepatic. Only two published studies to date demonstrate true plasticity of marrow cells that includes hepatic potential. The first is our murine marrow transplantation study, in collaboration with the laboratory of Saul Sharkis at Johns Hopkins University. In this study, a single bone marrow cell selected by lineage depletion, elutriation and functional marrow homing, whether obtained by limiting dilution or by direct visualization, was capable of producing mesodermal tissues (hematopoiesis), ectodermal tissues (skin and
skin appendages), and endodermal tissue (epithelial cells of liver, lung, and gastrointestinal tract).11 The second study, from the group of Catherine Verfaillie at University of Minnesota, demonstrates that clonally expanded stromal cells from mouse, rat, and human marrow can differentiate in vitro into hepatocytes,14 as well as an array of other endodermal, ectodermal, and mesodermal cell types.17 It seems unlikely that the marrow homing cell in our study and the marrow stromal cell of origin in the Verfaillie studies are the same cell type. Thus, it would seem that marrow-derived cells with at least two different phenotypes at least two types of marrow cells have hepatic potential. Yet, again, these studies are very limited in what they have to teach us about which cell populations physiologically contribute to liver maintenance and repair. In our bone marrow homing study, the single cell is certainly plastic, but, having given rise to the entire bone marrow after engraftment, it tells us little about which marrow subpopulations normally comprise the extrahepatic stem cells for liver maintenance and repair; this is the strength of the study with the tyrosinemia model, its data suggests which populations of the fully developed marrow may be most prepared to act as a reservoir for liver regeneration in severe injury. Whether the marrow stromal cell participates in normal physiologic hepatic maintenance is not addressed at all by the in vitro work.
Mechanisms of engraftment Two patterns of hepatic engraftment from the marrow have been identified. The first is that cells enter the liver from the circulation in response to injury of a severity and type that also activates an oval cell/ductular reaction. Many of these entering cells clearly begin life in the liver with the morphology of these oval cell-like hepatic progenitors and then differentiate into hepatocytes and cholangiocytes as the repair process continues. Because of this mechanism of entry, the hepatocyte engraftment is greater in the periportal region, where these intermediate cell reactions predominantly occur in both animals2 and humans.9 It is tempting to suggest that entry to the liver is site specific, either into the canals of Hering7 or as periductal cells.18 Whether the expression by oval cells/ductular reaction cells of c-kit, thy1, and CD34 are markers of cells that have come from the circulation and whether these molecules are functionally important in this process remains to be determined. 414
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One mechanism for recruitment to the liver of circulating cells, as oval cell/ductular reactions, has been preliminarily reported by Petersen’s group.19 They suggest that in severe hepatic injury, surviving hepatocytes produce stromal derived factor-1 (SDF-1) and that the circulating cells, entering the liver as oval cell/ductular reactions, express the receptor for this chemokine, CXCR4. This is the same homing mechanism that has been described for hematopoietic stem cells homing to the bone marrow,20 and may be related to the cells that are functionally isolated by marrow-homing in the Sharkis isolation procedure. A second pattern of engraftment has been noted in the absence of oval cell/ductular reactions. Without injury or with mild injury, the engraftment seems to occur without an identifiable oval cell/ductular intermediate and the periportal predominance is not seen: isolated cells or small clusters appear as though randomly scattered in the hepatic lobule.3, 9 Such distribution suggests that these marrow-derived cells engrafted from the circulation into the liver via an alternative pathway. We may speculate that such isolated cells represent an epithelial equivalent to the short term engrafting progenitor cells from the marrow: they differentiate appropriately into mature cell types, but do not have an open-ended ability to replicate. This question is intriguing and certainly amenable to easy investigation. How cells mobilize from the marrow is also an open question. Do they stream steadily from the marrow or are they mobilized in response to injury? Mobilization of cardiac progenitor cells from the marrow and their subsequent engraftment in ischemic myocardium can be induced by SCF and G-CSF administration, thereby increasing marrow contribution to myocyte repopulation after myocardial infarction.21 It is possible that marrow-mobilizing cytokines are secreted in response to severe liver damage as a signaling pathway to stimulate liver reconstitution from marrow.
ever, closer examination revealed that these cells were actually tetraploid fusion products, with XXXY genotype. An accompanying editorial declared “doubt has been shed on adult stem cell research”, suggesting that the studies of marrow engraftment in the liver and in other organs could be an artifact of such fusion events. That stable fusion events are responsible for the liver findings is very unlikely. Diploidy of hepatocytes in acute, let alone in severe injury, has been well documented for many decades.24 Tetraploidy is only seen in aging livers and then predominates in the centrilobular regions, i.e. not where the most significant engraftment of marrow cells has been documented. This does not rule out the possibility of unstable fusion events, in which the tetraploid fusion product immediately redivides into two diploid cells. But if this occurs it would simply be a mechanism for the demonstrated plasticity, since it still requires that genes or chromosomes which were in a circulating cell population now reside in hepatocytes. Although fusion is unlikely, control experiments that directly address this issue should be included in published studies for the near term, at least. The second point of controversy involves the relative capacity of marrow cells to repopulate liver compared with other cells, in particular with transplanted hepatocytes. This issue is raised by cell transplantation rescue with the murine tyrosinemia model for which hepatocytes are clearly a more dependable and efficient population.12 However, it would again be unwise to over interpret this difference: the comparatively greater rescue capacity of hepatocytes probably relates to the specifics of the model. Above, we surmise that large scale hepatic repopulation by marrow cells is probably a receptor dependent, site-specific process. On the other hand, engraftment of transplanted hepatocytes is dependent on where these large cells get trapped within the sinusoids of the liver.25 One may presume that in the tyrosinemia model there is near complete disruption of the microanatomy upon which large-scale marrow engraftment depends and, at the same time, an increase in circulatory sluggishness, favoring hepatocyte trapping and integration. Thus, the model itself may be the selective factor, not the general capacities of the donor cells. It is not clear whether the data for this model will be directly relevant to the treatment of human liver disease. The murine tyrosinemia model is unlike human tyrosinemia in its morphological and temporal course; only rare, very severe instances of massive hepatic necrosis in humans are a pathologic correlate to the
Controversies Three issues have been raised which lend an air of controversy to this field. The most recent arises from publication of two studies in which male adult stem cells (hematopoietic and neural, respectively) were co-cultured with female embryonic stem cells.22, 23 Both experiments resulted in embryonic-type cells containing male chromosomes, suggesting differentiation of adult cells to an embryonic phenotype. How415
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model. Until more data are obtained using different models of liver disease, and studying human tissues from patients with variable pathology, we cannot reach any conclusions regarding which cell populations may be the best to use in the development of cellular therapeutics. A third area of controversy involves differences between engraftment detected by Y-chromosome staining in gender mismatched models and that detected by expression of a transgene specific to the donor population. With the exception of the Lagasse study in which both Y-chromosome and beta-galactosidase expression demonstrate large-scale repopulation, Y-chromosome analysis seems to give higher estimates of engraftment. One explanation for this is that whereas nearly every visualized male cell contains a Y-chromosome, transgene expression may be variable in hepatocytes, either being unexpressed or expressed at low levels within the time course of the experiments. On the other hand, extreme care must be taken to be sure that overlapping or emperipolesis of circulating monocytes does not lead to false positive interpretation of Y-chromosome signals in hepatocytes that are actually female derived.
References 1. Sell S (2001) Heterogeneity and plasticity of hepatocyte lineage cells. Hepatology 33:738–750 2. Petersen BE, Bowen WC, Patrene KD, Mars MW, Sullivan AK, Murase N, Boggs SS et al. (1999) Bone marrow as a potential source of hepatic oval cells. Science 284:1168–1170 3. Theise ND, Badve S, Saxena R, Henegariu O, Sell S, Crawford JM, Krause DS (2000) Derivation of hepatocytes from bone marrow cells in mice after radiation-induced myeloablation. Hepatology 31:235–240 4. Petersen BE, Goff JP, Greenberger JS, Michalopoulos GK (1998) Hepatic oval cells express the hematopoietic stem cell marker Thy-1 in the rat. Hepatology 27:433–445 5. Omori N, Omori M, Evarts RP, Teramoto T, Miller MJ, Hoang TN, Thorgeirsson SS (1997) Partial cloning of rat CD34 cDNA and expression during stem cell-dependent liver regeneration in the adult rat. Hepatology 26:720–727 6. Baumann U, Crosby HA, Ramani P, Kelly DA, Strain AJ (1999) Expression of the stem cell factor receptor c-kit in normal and diseased pediatric liver: identification of a human hepatic progenitor cell? Hepatology 30:112–117 7. Theise ND, Saxena R, Portmann BC, Thung SN, Yee H, Chiriboga L, Kumar A, Crawford JM (1999) The canals of Hering and hepatic stem cells in humans. Hepatology 30:1425–1433 8. Yavorkovsky L, Lai E, Ilic Z, Sell S (1995) Participation of small intraportal stem cells in the restitutive response of the liver to periportal necrosis induced by allyl alcohol. Hepatology 21:1702–1712 9. Theise ND, Nimmakayalu M, Gardner R, Illei P, Morgan G, Teperman L, Henegariu O, Krause DS (2000) Liver from bone marrow in humans. Hepatology 32:11–16 10. Alison MR, Poulsom R, Jeffery R, Dhillon AP, Quaglia A, Jacob J, Novelli M, Prentice G, Williamson J, Wright NA (2000) Hepatocytes from non-hepatic adult stem cells. Nature 406:257 11. Krause DS, Theise ND, Collector MI, Henegariu O, Hwang S, Gardner R, Neutzel S, Sharkis SJ (2001) Multi-organ, multi-lineage engraftment by a single bone marrow-derived stem cell. Cell 105:369–377 12. Lagasse E, Connors H, Al-Dhalimy M, Reitsma M, Dohse M, Osborne L, Wang X, Finegold M, Weissman IL, Grompe M (2000) Purified hematopoietic stem cells can differentiate into hepatocytes in vivo. Nat Med 6:1229–1234 13. (a) Korbling M, Katz RL, Khanna A, Ruifrok AC, Rondon G, Albitar M, Champlin RE, Estrov Z (2002) Hepatocytes and epithelial cells of donor origin in recipients of peripheral-blood stem cells. New Engl J Med 346:738–746; (b) Mallet VO, Mitchell C, Mezey E, Fabre M, Guidotti J-E, Renia L, Couloumbel L, Kahn A, Gilgenkrantz H (2002) Bone marrow transplantation in mice leads to a minor population of hepatocytes tah can be selectively amplified in vivo. Hepatology 35:799–804 14. Schwartz RE, Reyes M, Koodie L, Jiang Y, Blackstad M, Lund T, Lenvik T, Johnson S, Hu W-S, Verfaillie CM (2002) Multipotent adult progenitor cells from bone marrow differentiate into functional hepatocyte-like cells. J Clin Invest 109:1291–1302 15. Theise ND, Krause DS (2001) Suggestions for a new paradigm of cell differentiative potential. Blood Cells Mol Dis 27:625–631 16. Theise ND, Krause DS (2002) Toward a new paradigm of cell differentiation capacity. Leukemia 16:542–548 17. Reyes M, Lund T, Lenvik T, Aguiar D, Koodie L, Verfaillie CM (2001) Purification and ex vivo expansion of postnatal human marrow mesodermal progenitor cells. Blood 98:2615–2625
Conclusion This exciting field of investigation is in its infancy. Conclusions about the uses to which these discoveries will be put or about the relative physiological importance of the events are premature. The list of questions far exceeds the list of answers. And we may add to that list still more intriguing questions. The liver is capable of hematopoiesis during embryogenesis and also occasionally in adulthood:26 is this truly, as has been tacitly presumed, because of an hematopoietic stem cell ‘rest’ in the hepatic vascular compartment, left over from fetal development, which gets reactivated? Or might the liver’s own epithelial, intraorgan stem cell compartment send cells back into the circulation? If so, perhaps some marrow-derived hepatic progenitors are actually originally hepatic derived. Might other organs also participate in such trafficking? While such possibilities may have been unimaginable a few years ago, they must now be considered serious hypotheses that require investigation. The only certainty is that much work lies ahead and that we have barely begun to examine these exciting possibilities, let alone to exploit their implied therapeutic potential. 416
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22. Terada N, Hamazaki T, Oka M, Hoki M, Mastalerz DM, Nakano Y, Meyer EM, Morel L, Petersen BE, Scott EW (2002) Bone marrow cells adopt the phenotype of other cells by spontaneous cell fusion. Nature 416:542–545 23. Ying QL, Nichols J, Evans EP, Smith AG (2002) Changing potency by spontaneous fusion. Nature 416:545–548 24. Gupta S (2000) Hepatic polyploidy and liver growth control. Semin Cancer Biol 10:161–171 25. Gupta S, Rajvanshi P, Sokhi R, Slehria S, Yam A, Kerr A, Novikoff PM (1999) Entry and integration of transplanted hepatocytes in rat liver plates occur by disruption of hepatic sinusoidal endothelium. Hepatology 29:509–519 26. Cardier JE, Barbera-Guillem E (1997) Extramedullary hematopoiesis in the adult mouse liver is associated with specific hepatic sinusoidal endothelial cells. Hepatology 26:165–175
18. Lee JH, Rim HJ, Sell S (1997) Heterogeneity of the “oval-cell” response in the hamster liver during cholangiocarcinogenesis following Clonorchis sinensis infection and dimethylnitrosamine treatment. J Hepatol 26:1313–1323 19. Petersen BE, Hatch HM, Jorgensen ML, Stolz DB (2001) SDF-1 as a potential homing protein for bone marrow derived liver oval cells. FASEB J 15:A1084 20. Voermans C, van Hennik PB, van Der Schoot CE (2001) Homing of human hematopoietic stem and progenitor cells: new insights, new challenges? J Hematother Stem Cell Res 10:725–738 21. Orlic D, Kajstura J, Chimenti S, Limana F, Jakoniuk I, Quaini F, Nadal-Ginard B, Bodine DM, Leri A, Anversa P (2001) Mobilized bone marrow cells repair the infarcted heart, improving function and survival. Proc Natl Acad Sci USA 98:10344– 10349
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