Sequential Exposure to Cytokines Reflecting Embryogenesis: The Key for In Vitro Differentiation of Adult Bone Marrow Stem Cells into Functional Hepatocyte-Like Cells
,1
* Ordway Research Institute, 150 New Scotland Avenue, Albany, New York 12208
University of California, San Diego, Department of Pharmacology and Center for Molecular Genetics, La Jolla, California 92093-0636
Wadsworth Center, New York State Department of Health, Empire State Plaza, Albany, New York 12201
1 To whom correspondence should be addressed. Fax: (518) 474-0547. E-mail: sells{at}mail.amc.edu.
Received September 26, 2006; accepted September 27, 2006
The paper by Snykers et al. (2006)
in this issue describes the in vitro differentiation of hepatocyte-like cells from mesenchymal stem cells (MSCs) that originate in the bone marrow. Although the research is directed toward the generation of a steady supply of cells for pharmacological research and testing, it could easily have even more direct clinical implications; namely, the cells derived could be used to generate a supply of functional hepatocytes for regeneration therapy following liver failure.
The methodology used in this report has been described and published before, but what sets this apart is the efficient approach to produce the desired cells. (1) Readily available, multipotent mescenchymal bone marrow–derived stem cells are used as a starting source. (2) Growth and differentiation factors are added serially to induce differentiation. (3) Several layers of confirmatory tests are applied to show that cells in the end are hepatocyte like. The results appear promising, but whether or not they are actually an improvement on previous efforts to obtain fully functioning hepatocytes from stem cells in vitro remains to be determined.
SOURCE OF CELLS
The cells used as a starting point for differentiation were isolated from bone marrow of rats in a manner identical to that described previously by one of the same authors (see Jiang et al., 2002a), although the cells are not identified by the original name, multipotent adult progenitor cells (MAPCs). The actual MAPC isolation takes several weeks to complete, from purification of mononuclear cells to culturing on specific expansion medium and final removal of hematopoietic cells, resulting in a population of cells that are coisolates of MSCs. Importantly, they can be purified from bone marrow from a range of species, including mice, rats, humans, and swine (Reyes et al., 2001; Zeng et al., 2006), as well as from tissues other than bone marrow, such as muscle and brain (Jiang et al., 2002b), thus providing a readily available source of starting cells that could be used in an autologous recipient.
Many cell types can be induced to differentiate into cells with features of hepatocytes, accompanied by varying degrees of mature hepatocyte functionality, but it is not certain whether any of these can function as mature hepatocytes. For example, embryonic stem (ES) cells are totipotent and proliferative and would appear to be the best source from which to produce hepatocytes. Many groups have derived hepatocyte-like cells from mouse and human ES cells using a variety of differentiation protocols (reviewed in Lavon and Benvenisty, 2005; Teramoto et al., 2005a). These hepatocyte-like cells derived in vitro from ES could be transplanted into animals and successfully integrated into the liver or could participate in liver repair following injury (Teratani et al., 2005; reviewed in Lavon and Benvenisty, 2005). In some cases, however, teratomas developed in the livers of animals that received hepatocyte-like cells (Chinzei et al., 2002; Teramoto et al., 2005b). Also, native, undifferentiated ES cells can form liver tumors in animals with liver damage (Yamamoto et al., 2003) that can pose another complication if differentiation into hepatocytes remains incomplete. When therapeutic transplantation is the goal, this potential safety issue, along with uneven or unreported efficacy of differentiation, and the difficulty in obtaining and growing human ES cells, may limit their usefulness as a source of liver cells.
Thus, adult bone marrow–derived stem cells may be a better choice to generate hepatocytes. Dozens of reports show that bone marrow–derived stem cells can give rise to functional hepatocyte-like cells in vitro, as well in vivo, following transplantation into liver-damaged animals. A variety of bone marrow–derived cell types and cell preparations have been used from unfractionated bone marrow to fractionated populations of hematopoietic and MSCs. The culture conditions under which cells have been induced to differentiate, as well as the in vivo models used in transplantations vary widely in terms of numbers of cells, liver injury type, species, and cell detection method. The consensus for hematopoietic stem cells is that these cells, although they may give rise to cells having some properties of hepatocyes in vitro, contribute little to hepatocyte formation in vivo, usually, only a few percent, or a fraction of a percent, of total hepatocytes in the liver, or not at all (Kanazawa and Verma, 2003; Wagers et al., 2002; see reviews: Sell, 2005; Thorgeirsson and Grisham, 2006). However, higher numbers have been reported. Up to 7% of hepatocytes were derived from a highly purified subpopulation of bone marrow–derived hematopoietic stem cells and special conditioning (stem cell homing) prior to transplantation (Jang et al., 2004). Interestingly, these cells were also capable of contributing of up to 20% of lung alveolar cells, as well as smaller amounts of cells in other mouse tissues (Krause et al., 2001). Up to 25% of hepatocytes were reported to be derived from transplanted bone marrow cells in chemically injured livers (Terai et al., 2003). On the other hand, failure to generate hepatocytes from various subsets of hematopoetic cells in an in vitro experimental setting has also been reported (Lian et al., 2006).
An issue that further complicates evaluation of the differentiation potential of bone marrow–derived stem cells, but one that is relevant to in vivo studies only, is whether conversion of these cells into hepatocytes is the result of differentiation or of fusion of transplanted cells with recipient hepatocytes. Thus, fusion has been described as a mechanism behind liver repair in an animal model of lethal liver damage (Vassilopoulos et al., 2003; Wang et al., 2003), and the fusing bone marrow cells were identified as mature monocytes/macrophages (Camargo et al., 2004; Willenbring et al., 2004). However, regardless of their participation in fusion, bone marrow and peripheral monocytes appear to be able to generate hepatocyte-like cells by differentiation as well (Jang et al., 2004; Ruhnke et al., 2005; Shi et al., 2005).
Besides hematopoietic stem cells, the other major stem cell population found in bone marrow is the MSCs, of which cells used in the paper by Snykers et al. (2006) in this journal seem to comprise a rare subset. There are fewer published reports on usage of MSCs as a source of hepatocytes than hematopoietic stem cells. Again, the number of hepatocytes produced after transplantation in vivo varies and ranges from a very few cells to up to 16% of liver cells (Fang et al., 2004; Luk et al., 2005; Oyagi et al., 2006; Sato et al., 2005). It is very different to estimate the differentiation plasticity of those cells due to differences in the types of donor cells (from very crude to more specific MSCs preparations) and the models used. The same holds true for the use of MSCs to generate hepatocytes in vitro (Hong et al., 2005; Kang et al., 2005; Lee et al., 2004; Wang et al., 2004). In an extensive and detailed report, Schwartz et al. (2002) demonstrated conversion of MAPC into hepatocyte-like cells in culture with very high efficiency. These MAPCs can also be maintained in undifferentiated state for more than 100 doublings, thus ensuring their almost unlimited supply. When injected into early mouse blastocysts, MAPCs contribute to liver, as well as most other tissues, and they differentiate into CK18+ and albumin+ cells when injected into SCID mice (Jiang et al., 2002). However, as pointed out by Gilgenkrantz (2004), it has not been demonstrated that they can participate in liver regeneration and, thus, have therapeutic potential. In addition, it is still not clear whether MAPCs exist as such in vivo or whether they arise only as a result of specific in vitro culture conditions (Gilgenkrantz, 2004). Thus, the criteria for demonstrating plasticity of these stem cells, that is, differentiation of single cell into multiple cell lineages, functionality of differentiated cells in vitro and in vivo, and robust and persistent engraftment of transplanted cells, as defined by Lakshmipathy and Verfaillie (2005) have not been confirmed.
In conclusion, MAPCs have been shown in a series of papers to have a wide differentiation plasticity. Unlike ES cells or umbilical cord blood cells, they are readily available and suitable for individualized use and treatment purposes. Moreover, their use is not encumbered by the ethical questions associated with ES cells.
METHOD OF DIFFERENTIATION
The method of sequential additions of cytokines to induce hepatocyte differentiation, as opposed to cocktail treatment, more closely resembles natural development of hepatocytes during embryogenesis and thus holds more promise of efficiency. The idea that growth factors act sequentially to induce the optimal expression of a biological process in hepatocellular systems is not new. Sequential signaling by type 1 and type 2 growth factors was first described experimentally for the optimal initiation of DNA synthesis in primary cultures of fetal and adult rat hepatocytes cultured in serum-free medium by Koch et al. (1976; Koch and Leffert, 1979; Leffert and Koch, 1977; for reviews, see Koch and Leffert, 1994; Koch et al. 1990; Leffert and Koch, 1982). The conclusions of the paper by Snykers et al. (2006) would be more compelling had they included discussions of the many controversies and differences of opinion that surround the field of hepatocyte expression in cultures of cells of primary liver cell as well as stem or progenitor cell origin (Koch and Leffert, 2004). In particular, there are many strain, isolation, and culture variables, which do not involve soluble growth factors, but which nevertheless affect in vitro expression of differentiated functions in normal hepatocytes.
The growth factors described for studies of sequential induction of hepatocellular differentiation are neither liver nor hepatocyte specific. MAPCs themselves have been subjected to sequential addition of cytokines and growth factors in an attempt to obtain neuron-like cells in culture, with considerable success (Jiang et al., 2003). The sequential method used by Snykers et al. (2006) is very similar to the method described before by Hamazaki et al. (2001) for in vitro differentiation of ES cells into hepatocytes, except that, acidic fibroblast growth factor has been replaced with FGF-4 and oncostatin M has not been used. A directly comparable system was used by Schwartz et al. (2002), who induced MAPCs to differentiate into hepatocyte-like cells as well, but through the addition of a cocktail of factors. In the paper by Snykers et al. (2006), certain parameters, such as urea production, are much higher than in the paper by Schwartz et al. (2002), but most of the results cannot be compared due to differences in measurement conditions. Another report on methods to provide functional hepatocyte-like cells for drug screening from ES cells (Kulkarni and Khanna, 2006) includes data similar to that the Snykers et al. paper and also includes data showing an elevation of liver function enzymes, serum glutamic-oxaloacetic transaminase (aspartate aminotransferase), serum glutamate pyruvate transaminase (alanine aminotransferase), and lactate dehydrogenase after treatment with CCl4 that is prevented by the antioxidant, N-acetylcysteine.
END RESULT—HEPATOCYTES
Snykers and colleagues confirm the hepatocyte-like nature of the derived cells on several levels: morphology, marker expression, and tests of hepatocyte functions. However, except for albumin, the expression of most of the transcription factors (HNF3ß or HNF1
) and their differentiated functions are also not hepatocyte specific (e.g., CK18 or CYP1A1). ELISA measurements of albumin secretion could have been accompanied by (1) de novo albumin synthesis data, to verify the specificity of the immunostains used (i.e., distinctions between endogenous synthesis and artifactual uptake or adsorption of serum proteins from the culture medium) and (2) Western blots, to verify the expected molecular mass of secreted albumin (viz., 66–68 kDa). Indirect evidence of presumed hepatocyte-specific metabolism (e.g., urea synthesis, which can be performed by nonhepatic cells of intestinal and renal origin that also express arginase) would have benefited from combination with direct experimental evidence, for example, demonstration of de novo biosynthesis of arginine from ornithine, which depends upon hepatocyte-specific expression of ornithine transcarbamylase (Leffert et al., 1977). Support by evidence of the latter type would have made a more compelling case for accuracy of specific hepatocellular differentiation.
The possibility that MSC-derived hepatocytes will function better than primary hepatocytes is doubtful, since problems similar to those encountered by authentic cultured hepatocytes (e.g., loss of differentiated functions over extended time periods; inability to undergo continuous proliferation and clonogenicity) should occur, regardless of the cell source of hepatocytes, in long-term cultures of differentiated MSC-derived hepatocytes. Thus, the question remains: what happens to hepatocyte marker expression and to hepatocytic functions in long-term cultures? Will MSC-derived hepatocytes maintain their hepatocyte-like characteristics?
The practical significance of the directed differentiation reported by Snykers and colleagues is hard to evaluate. The quantitative levels of "functional" expression in MSC-derived hepatocytes were not compared to levels described for in vivo models (e.g., whole animals or perfused rat livers) to gauge expression capacities; in fact, quantitative comparisons cannot be made by the reader since cell numbers per square centimeter per ml of medium at the time of assay were not stated. Would these cells be capable of participating in liver repair following injury? It is a curious fact that in all previously published papers on the use and capabilities of MAPCs, the question of the contribution of these cells to liver repair in vivo has never been addressed experimentally. Clearly, the limited in vitro function reported in the Snykers et al. paper needs to be extended by in vivo studies. Previously published papers have shown already that MAPCs are capable of differentiating into hepatocyte-like cells (Jiang et al., 2002; Schwartz et al., 2002). The paper by Snykers et al. confirms these earlier studies, but their data do not demonstrate that their method is more efficient, which was their goal.
The question as to when a cell grown in vitro can be considered to be a mature hepatocyte (as opposed to an embryonal or fetal one) is a difficult one, and many discussions have been devoted to it (such as the recent extensive review by Hengstler et al., 2005). A more complete testing of mature hepatocyte function could answer the question of whether or not the hepatocyte-like cells derived are good enough for the purpose intended (in this case, drug testing).
In conclusion, although the concept and in vitro demonstration of sequential growth factor signaling are not new, the results of the paper by Snyker et al. provide an additional, potentially important technical paradigm for the directed differentiation of uncommitted stem cells in vitro. Although comprehensive immunofluorescence panels are reported, failure to include unequivocal quantitative biochemical characterization of prominent differentiated hepatocyte functions, such as albumin or arginine biosynthesis, leaves doubts as to the ability of the hepatocyte-like cells to function like mature hepatocytes for in vitro testing of drug metabolism. In addition, factors that might determine the ability of these cells to function after transplantation in vivo, such as growth factor–unrelated aspects of niche-directed signaling (e.g., spatial and surface determinants), and functional investigations of transplanted MAPC-derived "hepatocytes" in animals with compromised liver function were not discussed. Thus, the promise of directed differentiation of MAPCs both in vitro and in vivo remains undemonstrated.
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  Z. Ilic, H. Leffert, and S. Sell Sequential Exposure to Cytokines Reflecting Embryogenesis: The Key for In Vitro Differentiation of Adult Bone Marrow Stem Cells into Functional Hepatocyte-Like Cells Toxicol. Sci., December 1, 2006; 94(2): 235 - 239. [Full Text] [PDF]
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