STEWART SELL. Division of Experimental Pathology, Department of Pathology and Laboratory Medicine, Albany Medical College, Albany, New York, USA

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1 Electron Microscopic Identification of Putative Liver Stem Cells and Intermediate Hepatocytes following Periportal Necrosis Induced in Rats by Allyl Alcohol STEWART SELL Division of Experimental Pathology, Department of Pathology and Laboratory Medicine, Albany Medical College, Albany, New York, USA Key Words. Liver Stem cell Allyl alcohol Injury Repair ABSTRACT The ultrastructural characteristics of the putative liver stem cells that repopulate the necrotic periportal zones after allyl alcohol induced liver injury are described. Periportal liver cell necrosis was induced in adult female Sprague-Dawley rats by i.p. injection with 0.62 mmol/kg of allyl alcohol. Electron microscopic examination of the livers was carried out at 33, 57, 81 and 129 h after injection. After periportal necrosis small nondescript intraportal cells (putative liver stem cells) as well as three types of progenitor cells are seen: type I, immature precursor cells; type II, bile duct-like; and type III, hepatocyte-like, with numerous cells of intermediate type between type I and type III. The periportal necrotic zone (zone I) is reconstituted largely by an increase in hepatocyte-like cells containing mitochondria, lysosomes, lipidfilled vacuoles, rare peroxisomes, prominent endoplasmic reticulum and lateral microvilli (type III cells) with a relatively small number of type I (immature) cells participating. The type III cells display different degrees of differentiation; the less mature are termed restitutive and the more mature transitional hepatocytes to emphasize the probable relationship between these cell types. Immature ductular cells (type II cells) are seen located basally within hyperplastic ducts in the periportal zone. It is postulated that hepatocyte restitution after periportal necrosis is accomplished by proliferation and differentiation of stem cells with both biliary and hepatic potential that specifically differentiate into hepatic cells through restitutive and transitional intermediates. These postulated liver stem cells may be intraportal cells seen h after injury that precede the type I and type III hepatic precursors seen later. Stem Cells 1995;15: INTRODUCTION The location and degree of liver injury may selectively induce restitutive proliferation of different cellular components of the liver. Based on identification of proliferation of hepatocytes following two-thirds partial hepatectomy of rats, it was concluded that the liver could reconstitute itself through proliferation of mature hepatocytes [1-3]. However, there is increasing evidence supporting the existence of reserve liver stem cells, which proliferate in response to severe liver injury induced by chemicals [4]. For example, we previously described proliferation of small null intraportal cells that sequentially acquired markers of differentiation to hepatocytes [5] after periportal liver necrosis induced by allyl alcohol [6-8]. Such cells are seen in an intraportal location during the first h after allyl alcohol-induced liver injury. The progeny of these cells then extend across the necrotic zone and, as they do, acquire differentiation markers that reflect markers seen during maturation of the fetal liver [5]. Electron microscopic characterization of these differentiating cells reveals the three cell types similar to those found in chronic ductular reactions [9] and focal nodular hyperplasia [10] of human liver: type I, small immature or progenitor cells; type II, bile duct-like progenitor cells; and type III, hepatocyte-like progenitor cells. At three days after injury over 90% of the cells restoring the necrotic zone are type III cells. The ultrastructural appearance of these cells is consistent with the conclusion that Correspondence: Dr. Stewart Sell, Department of Pathology and Laboratory Medicine, Albany Medical College, 27 New Scotland Avenue, Albany, NY 12208, USA. Accepted for publication April 2, AlphaMed Press /97/$5.00/0 STEM CELLS 1997;15:

2 Sell 379 liver stem cells differentiate through stages of restitutive and transitional hepatocytes before developing the appearance of mature hepatocytes. In this process the cells do not demonstrate any bile duct-like structures as do the oval cells seen after carcinogenic regimens. On the other hand, proliferation of bile ducts also occurs, leading to redundant ducts in zones of liver injury. Type II, bile duct-like cells, are only seen within bile duct structures after allyl alcohol injury, and proliferation of duct cells does not appear to contribute to the restitution of the liver cords. The results suggest that intraportal liver stem cells proliferate and differentiate directly into intermediate progenitor cell types, termed type I ( immature ) and type III ( restitutive and transitional ) progenitor cells that eventually restore the damaged hepatic cord. METHODS Animals Twelve female Sprague-Dawley rats (weight g) were injected i.p. with 0.62 mmol/kg of allyl alcohol, and three animals each were euthanized 33, 57, 81, and 129 h later [5]; note that the last time of sacrifice was 153 h in the experiments reported in previous experiments). The livers were examined by gross inspection. Tissue blocks from each lobe of the liver were taken separately and fixed in 3% phosphatebuffered glutaraldehyde for one micron sections for electron microscopy. One micron sections from eight blocks from each animal were placed on acid-cleaned glass slides, heat fixed and processed as previously described [11, 12]. The results of the light microscopic autoradiography on thick sections have already been reported [5] and will be presented only for background information in this paper. The one micron sections were stained with a solution of two parts 0.5% toluidine blue to one part 1% sodium borate, and areas showing injury or cellular response were selected for processing for ultrastructural examination. Thin sections from selected areas were cut on an MT-II Ultratome and stained with filtered 5% aqueous uranyl acetate diluted with an equal volume of 95% ethyl alcohol for 20 min in the dark and with Reynold s lead citrate for five min. Dried slides were coated with a thick layer of carbon using an Edwards 306 vacuum coater. The sections were viewed on a JOEL JEM 1200 EX electron microscope. RESULTS Light Microscopic Observations The light microscopic histopathologic changes following allyl alcohol-induced periportal necrosis of the rat liver were described in detail in the preceding paper in this series [5] and will be presented briefly here. The tissue changes were divided into four phases: A) injury, necrotic periportal necrosis and scattered apoptosis in non-necrotic zones 6 to 24 h after injection; B) reaction, focal periportal edema and minimal inflammation 12 to 33 h after injection; C) regeneration, proliferation of small periportal cells h after injection, and D) differentiation, acquisition of cellular markers alpha-fetoprotein, cytokeratin 14/19 (OV-6 [13]), glutathione-s-transferase-p and hepatocyte marker H-4 [13] from h after injection. One Micron Sections Light microscopic pictures of the one micron sections from which the electron-microscopic sections were made are shown in Figure 1. At 33 h (Fig. 1A) small inflammatory cells are intermixed with cellular debris from necrotic hepatocytes. There is a distinct demarcation between the necrotic zone and the margin of the surviving hepatocytes. It is not possible to identify putative liver stem cells at this time. At 57 h (Fig. 1B) the necrotic zone becomes filled with a mixture of small cells. Some surviving hepatocytes at the margin of the injured zone and next to the portal vein have an accumulation of lipid, consistent with sublethal injury. At this time cells without cytoplasmic extensions and with prominent large nuclei without obvious perinuclear chromatin and single large light nucleoli are seen. These may be differentiated from activated lymphocytes which have prominent cytoplasmic extensions and nuclei with peripheral chromatin and small dark nucleoli. The restitutive process is more marked at 81 h (Fig. 1C). There is a mixture of cells of different sizes with generally larger cells containing lipid-filled vacuoles near the margin of surviving hepatocytes. There is enlargement and some redundancy in the bile ducts. At 129 h (Fig. 1D) the zone occupied by small cells is greatly reduced, and the bile ducts have become relatively more prominent with some small cells remaining in between the ducts. At this time some of the small cells appear to be duct cells and inflammatory cells. Electron-Microscopic Findings The restitutive cellular response to allyl alcohol injury is described at the times examined after induction of injury: 33 h, 57 h, 81 h and 129 h. Thirty-Three Hours (Fig. 2) In zones of periportal necrosis there are scattered debris of dying hepatocytes admixed with inflammatory cells (polymorphonuclear cells), red cells and activated lymphocytes (Fig. 2A). The sharp demarcation of the portal plate seen normally is lost. Surviving hepatocytes contain swollen endoplasmic reticulum, dense bodies and lipid vacuoles. In Figure 2B, small cells with oval or distorted nuclei are seen next to a bile duct and extending into the adjacent mixture of fragmenting hepatocytes and inflammatory cells. Higher

3 380 Liver Stem Cells Figure 1. One micron sections of periportal zones after allyl alcohol-induced liver injury. A) 33 h, 40. Necrotic cellular debris (N), inflammatory cells and red cells are present between portal vein (V) and surviving hepatocytes (H). B) 57 h, 40. Small cells between bile ducts (D), hepatic artery (A) and surviving hepatocytes (H). The cells indicated by the small arrow are activated lymphocytes and the cells indicated by the large arrow are putative stem cells (as seen by electron microscopy; see below). C) 81 h, 40. The small cells of variable shapes are type I or III cells, or inflammatory cells located between the ducts (D) and surviving hepatocytes (H). The long thin cells indicated by (f) are most likely fibroblasts. D) 129 h, 40. Most of the small cells are ductular and represent new duct formation. The small cells located between bile ducts (D) and hepatocytes (H) are a mixture of inflammatory cells and type I and III cells. Figure 2. Electron microscopic pictures of periportal liver injury 33 h after allyl alcohol-induced liver injury. The black bar = 5 µm. A) 1,200. Degenerating hepatocytes are mixed with inflammatory cells (lymphocytes, large arrows; polymorphonuclear cells, small arrows) and cellular debris. B) 1,500. Small nondescript cells (arrows) are seen next to a bile duct (D). Clearly identifiable stem cells are not seen in these pictures. magnifications of these cells fail to identify cellular organelles or membrane microvilli that would distinguish their nature. These cells are similar to the periductular oval cells seen during the first one to three days after feeding rats N-2-fluorenylactamide in a choline-deficient diet [11, 12, 14], but cannot be clearly distinguished from lymphocytes. Fifty-Seven Hours (Fig. 3) There is a marked increase in the number and diversity of intraportal cells in injured areas, including small- and intermediate-sized hepatocyte-like cells with increasing numbers of vacuoles and large surviving hepatocytes with cytoplasm filled with lipid-laden vacuoles or dense bodies, as well as a few inflammatory cells and red cells. Small bile ductules and terminal ducts may be identified. Small cells with oval nuclei and abundant cytoplasm containing rough endoplasmic reticulum surround and abut the outside of the basement membrane of a portal venule. These cells are similar to the nondescript oval cells described above. Adjacent to these cells are inflammatory cells and small cells between the portal veins and bile ducts that appear to have some of the characteristics of type I progenitor cells (see below).

4 Sell 381 Figure 3. Electron microscopic picture 57 h after allyl alcoholinduced injury. The bar indicates 5 µm. A) 1,200. The area of injury extends from the portal venule (V) to a surviving hepatocyte with dense bodies (H). There is a bile ductule in the center (D). Clear zones represent fluid. There are polymorphonuclear leukocytes and lymphocytes (arrowheads) between the venule and the ductule, and a variety of cell types is seen between the bile ductule and the hepatocyte, including a terminal duct (TD), hepatocyte cytoplasm containing dense bodies, and small nondescript cells (large arrows), which may be liver stem cells. B) Perivenular cells in portal zone. 2,000. Surrounding the hepatic vein are plump cells with prominent endoplasmic reticulum and oval nuclei with scanty heterochromatin. These cells are seen on both the ductal and tissue side of the vein (picture not shown). Between the venule and the bile duct (D) are inflammatory cells (arrowheads), and nondescript cells (large arrows). Above the bile duct is a pair of cells that resemble transition-duct or Herring-canal cells (TD) and above these another nondescript cell (large arrow). F = Fibroblast. Figure 4. Electron microscopic pictures 81 h after allyl alcohol injury. A) 1,200. Mixture of small type III hepatocyte-like cells. Less mature type III cells are designated restitutive cells (R) and larger more mature cells with lipid vacuoles are termed transition cells (T) in the zone of restitutive proliferation. B) 1,200. Restitutive cell types (R) adjacent to surviving hepatocytes (H). At the bottom of the picture is a mitotic figure (arrow), and just above this is a lymphocyte. Eighty-One Hours (Figs. 4-6) At this time the periportal necrotic zones are filled with a mixture of intermediate hepatocyte progenitor cells. In Figure 4 a series of restitutive cells with oval or slightly indented nuclei are seen extending from the hepatic vein to the edge of the surviving hepatocytes. Some of the cells of different sizes with hepatocytic features as well as surviving hepatocytes contain lipid-filled vacuoles. Inflammatory cells and cellular debris are still present, but much less obvious than at earlier times. The restitutive hepatocytes contain rough endoplasmic reticulum, tonofibrils, liposomes and have lateral microvilli that form poorly defined canaliculi with adjacent cells. A mixture of restitutive hepatocytes along with larger cells containing lipid vacuoles (transitional hepatocytes) is shown in Figure 4B. A mitotic figure is seen near the portal vein at the lower left of 4B and a lymphocyte is located just above it. No putative stem cells without organelles are seen in this figure. While mitotic figures are not common by electron microscopy, proliferating cells are commonly associated with the type I, II and III populations of cells by light microscopic autoradiography [5]. The types of small cells at this time are similar to the three types of undifferentiated progenitor cells in focal nodular hyperplasia of the human liver described by DeVos

5 382 Liver Stem Cells Figure 5. Different types of immature liver cells [9, 10]. A) Type I, small immature progenitor cell (I) is located next to a cell that has more rough endoplasmic reticulum and may be in transition to a type III cell. B) Type II, bile duct-like cells located in duct. These are more differentiated than those described by Roskams et al. [10] and by Novikoff et al. [16] and are only seen within ducts. C) Type III, hepatocyte-like cells. These small cells have some rough endoplasmic reticulum and mitochondria, but are much smaller than mature hepatocytes (4,200x). Some of the characteristics of the different types of progenitor cells are listed in Table 1. and Desmet [9]; Roskams et al., [10] and during cholangiocarcinogenesis in the hamster by Lee et al., [15]. Type I cells (Fig. 5A) are immature periductular or sinusoidal cells with prominent marginal heterochromatin in the nucleus, relatively few mitochondria, scanty rough endoplasmic reticulum, and poorly developed lateral microvilli. Type II is a duct-like intermediate cell. This type of cell resembles the blast-like ductular cell described by Novikoff et al. [16] in carcinogen-induced proliferating bile ductules and is seen only within hypercellular bile ducts [10, 16] (Fig. 5B). Type III cells are hepatocyte-like cells. They are located outside of biliary structures in the liver cord, have rounded or irregular nuclei and less marginal heterochromatin than type I cells. Type III cells have tight junctions with other hepatocyte-like cells and lateral microvilli that form poorly defined canalicular structures. Their cytoplasm contains prominent rough endoplasmic reticulum, a poorly defined Golgi apparatus and moderate numbers of mitochondria (Fig. 5C). A mixture of type III cells is shown in Figure 6. Most of the cells in this field are type III hepatocyte-like cells that have canalicular microvilli, mitochondria, endoplasmic reticulum and lipid vacuoles. One-Hundred Twenty-Nine Hours (Fig. 7) By 129 h little injury remains. Most of the liver does not have any recognizable change. It appears that most of the injured areas have been restored. Figure 7 shows two areas selected for the presence of periportal cells. In Figure 7A fibroblasts may be seen surrounding the bile ducts. The small area between these and the hepatocytes contains restitutive type III hepatocytes, ductular cells and a few lymphocytes. Transitional type III cells are seen in some other areas (not shown). With higher doses of allyl alcohol, necrosis may be more severe and involve the Figure 6. Mixture of type III hepatocyte-like cells in periportal zone of restitutive proliferation 81 h after allyl alcohol-induced injury. The cell marked (I) resembles a type I immature cell but has small mitochondria and lipid droplets indicating differentiation to a type III phenotype. The cells to the left and in the middle represent type III hepatocyte-like cells. The cells to the right of the picture have acquired more cellular organelles and are called transitional hepatocytes (T), the less mature type III cells are termed restitutive (R). entire liver lobule. This degree of injury is followed by fibrosis with scarring (data not shown). However, this was not seen with the doses of allyl alcohol used in this study. What was seen is the restoration of the necrotic zones with hepatocytes separated from bile ducts by a few remaining inflammatory cells or type III cells.

6 Sell 383 Figure 7. Electron microscopic pictures 129 h after allyl alcohol injection. A) 1,500. B) 1,500. In A), fibroblasts surround the small bile duct (D); type III hepatocytelike restitutive cells (R) and a few lymphocytes are seen between the small ducts and hepatocytes (H) in both A) and B). Type III transitional cells are seen in other sections (not shown). Table 1. Electron microscopic features of liver progenitor cells Stem cell Primitive cell Duct-like cell Hepatocyte-like cell Features (Type 0) (Type I) (Type II) (Type III) Restitutive Transitional Location: Intraportal Sinusoidal, periportal Ductular, basal Sinusoidal/ Sinusoidal/ liver cord liver cord Nuclear: Shape Oval Oval Round or oval Round/irregular Round/irregular Heterochromatin Variable marginal Prominent marginal Less prominent Irregular/marginal Irregular/marginal Nucleoli None Rare, marginal Rare, central Marginal More central Cytoplasmic: Tonofibrils None Few present Prominent Prominent Prominent Desmosomes None Join to other type I cells Join to duct cells Join to hepatocytes Join to hepatocytes Mitochondria None Present Rare to moderate Moderate Many Rough endoplasmic reticulum Rare Moderate Moderate Prominent Prominent Golgi None Rudimentary Poorly formed Poorly formed Better formed Lysosomes None Few Very rare Variable Common/many Membrane: Microvilli None Poorly developed Apical Lateral Lateral DISCUSSION The term oval cell, originally proposed by Farber [17], is a descriptive term for a small cell with an oval nucleus that appears after exposure of the liver to certain carcinogenic regimens [17-20]. With time the term oval cell has been applied indiscriminately to small cells with oval nuclei that may have different biologic origins and potential. The oval cells seen in the present study have a different appearance from the oval cells that proliferate during chemical hepatocarcinogenesis in the rat [12, 14]. A classification of different types of liver progenitor cells is given in Table 1. Carcinogen-induced oval cells are first seen as nondescript periductular cells and later as type II-like cells that extend along the spaces of Disse [21, 22], to the central zone of the liver with eventual formation of ductlike structures which maintain connections with the pre-existing bile ducts [23, 24]. As they extend along the hepatic cords, they have desmosomal junctions with both hepatocytes and other oval cells and have ultrastructural features more like ductular-type progenitor cells than hepatocyte-like progenitor cells [10, 25]. Later they are able to differentiate either into hepatocytes [25, 26] or into bile duct cells [27], as well as serve as the progenitors of hepatocellular carcinomas [19, 20, 28-30]. Thus, carcinogen-induced oval cells most resemble type II (duct-like) oval cells but share phenotypic characteristics of biliary cells and hepatocytes [31] and are able to differentiate into either bile duct cells or hepatocytes

7 384 Liver Stem Cells [29, 30, 32]. Largely from studies on carcinogen-induced oval cells, we have concluded that small nondescript intraportal cells are the precursors of the oval cells and thus, the true liver stem cells [4, 20, 28, 33-36]. Small periportal nonparenchymal cells containing alpha-fetoprotein, or mrna for alpha-fetoprotein, have been found after severe liver injury of mice or rats with CCl 4 or galactosamine [37, 38]. These cells also appear to arise from a periductular or ductular cell in the intraportal zone of the liver [39]. These cells have a similar light microscopic appearance as the oval cells that proliferate after exposure of rats to chemical hepatocarcinogens, and it has been suggested that both carcinogen-induced oval cells and small hepatocytes arise from proliferation of duct cells after galactosamine injury [40, 41] as well as during chemical hepatocarcinogenesis [23]. However, on the basis of the present results it seems more likely that the oval cells which proliferate after CCl 4 or galactosamine are similar or the same as those seen after allyl alcohol-induced liver injury, but different from those seen after exposure to chemical carcinogens. Proliferation of type II cells after allyl alcohol injury appears to be limited to ducts. Ductular proliferation and branching occurs (see Figs. 1C and D), but this does not appear to contribute to restitution of the hepatocytes. During chemical hepatocarcinogenesis it is not clear if type II ductular-like cells differentiate into hepatocytes or if stem cells are present among the type II cells that are able to differentiate into hepatocytes. From the results of this study it is hypothesized that there is a small intraportal stem cell that is able to proliferate and differentiate directly into hepatocytes through type I and type III progenitor cells without an intermediate duct-like cell stage. This cell could be a bipolar progenitor cell capable of giving rise to either biliary or hepatic cells or it could be a tissuedetermined hepatic cell committed to hepatic differentiation. Since some hepatocytes immediately adjacent to the portal zone may survive allyl alcohol-induced toxicity, it is possible that these cells could transdifferentiate into cells of a less mature phenotype [42, 43] and serve as a source for the proliferating restitutive cells. However, no evidence for such a transition was evident in this study. We hypothesize that proliferation of small null intraportal cells with oval nuclei ( stem cells ) begins the restitutive process. These cells differentiate into type I progenitor hepatocytes which, in turn, continue to proliferate and differentiate into type III progenitor cells, which then differentiate into mature hepatocytes. This process appears to be distinct from the cellular response involved in periductular oval cells during chemical hepatocarcinogenesis. 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