Cell Competition Leads to a High Level of Normal Liver Reconstitution by Transplanted Fetal Liver Stem/Progenitor Cells
2006; Elsevier BV; Volume: 130; Issue: 2 Linguagem: Inglês
10.1053/j.gastro.2005.10.049
ISSN1528-0012
AutoresMichael Oertel, Anuradha Menthena, Mariana D. Dabeva, David A. Shafritz,
Tópico(s)Pancreatic function and diabetes
ResumoBackground & Aims: A critical property of stem cells is their ability to repopulate an organ or tissue under nonselective conditions. The aims of this study were to determine whether we could obtain reproducible, high levels of liver repopulation by transplanted fetal liver stem/progenitor cells in normal adult liver and the mechanism by which liver replacement occurred. Methods: Wild-type (dipeptidyl peptidase IV [DPPIV+]) embryonic day (ED) 14 fetal liver cells underwent transplantation into DPPIV− mutant F344 rats to follow the fate and differentiation of transplanted cells. To determine the mechanism for repopulation, proliferation and apoptosis of transplanted and host liver cells were also followed. Results: Transplanted ED 14 fetal liver cells proliferated continuously for 6 months, differentiated into mature hepatocytes, and replaced 23.5% of total liver mass. The progeny of transplanted cells were morphologically and functionally indistinguishable from host hepatocytes and expressed unique liver-specific genes commensurate with their location in the hepatic lobule. Repopulation was based on greater proliferative activity of transplanted cells and reduced apoptosis of their progeny compared with host hepatocytes, coupled with increased apoptosis of host hepatocytes immediately adjacent to transplanted cells. This process, referred to as cell-cell competition, has been described previously in Drosophila during wing development. Conclusions: We show for the first time that cell-cell competition, a developmental paradigm, can be used to replace functional organ tissue in an adult mammalian species under nonselective conditions and may serve as a strategy for tissue reconstitution in a wide variety of metabolic and other disorders involving the liver, as well as other organs. Background & Aims: A critical property of stem cells is their ability to repopulate an organ or tissue under nonselective conditions. The aims of this study were to determine whether we could obtain reproducible, high levels of liver repopulation by transplanted fetal liver stem/progenitor cells in normal adult liver and the mechanism by which liver replacement occurred. Methods: Wild-type (dipeptidyl peptidase IV [DPPIV+]) embryonic day (ED) 14 fetal liver cells underwent transplantation into DPPIV− mutant F344 rats to follow the fate and differentiation of transplanted cells. To determine the mechanism for repopulation, proliferation and apoptosis of transplanted and host liver cells were also followed. Results: Transplanted ED 14 fetal liver cells proliferated continuously for 6 months, differentiated into mature hepatocytes, and replaced 23.5% of total liver mass. The progeny of transplanted cells were morphologically and functionally indistinguishable from host hepatocytes and expressed unique liver-specific genes commensurate with their location in the hepatic lobule. Repopulation was based on greater proliferative activity of transplanted cells and reduced apoptosis of their progeny compared with host hepatocytes, coupled with increased apoptosis of host hepatocytes immediately adjacent to transplanted cells. This process, referred to as cell-cell competition, has been described previously in Drosophila during wing development. Conclusions: We show for the first time that cell-cell competition, a developmental paradigm, can be used to replace functional organ tissue in an adult mammalian species under nonselective conditions and may serve as a strategy for tissue reconstitution in a wide variety of metabolic and other disorders involving the liver, as well as other organs. The potential use of stem cells, endogenous or transplanted, to rejuvenate solid organ tissues has generated considerable interest. Because the liver is unique in its ability to regenerate after massive injury,1Michalopoulos G.K. DeFrances M.C. Liver regeneration.Science. 1997; 276: 60-66Crossref PubMed Scopus (2884) Google Scholar this organ is a logical site to study the role of stem cells in tissue reconstitution. However, identification of stem cells in the liver has been problematic because there are no known specific markers for liver stem cells, and, during liver regeneration after acute hepatocyte loss, restoration of liver mass occurs through proliferation of preexisting mature hepatocytes.1Michalopoulos G.K. DeFrances M.C. Liver regeneration.Science. 1997; 276: 60-66Crossref PubMed Scopus (2884) Google Scholar Nonetheless, during extensive liver injury under conditions in which hepatocyte proliferation is blocked by chemical agents, hepatic progenitor cells can be activated to repopulate the liver.2Evarts R.P. Nagy P. Marsden E. Thorgeirsson S.S. A precursor-product relationship exists between oval cells and hepatocytes in rat liver.Carcinogenesis. 1987; 8: 1737-1740Crossref PubMed Scopus (394) Google Scholar, 3Evarts R.P. Nagy P. Nakatsukasa H. Marsden E. Thorgeirsson S.S. In vivo differentiation of rat liver oval cells into hepatocytes.Cancer Res. 1989; 49: 1541-1547PubMed Google Scholar, 4Lemire J.M. Shiojiri N. Fausto N. Oval cell proliferation and the origin of small hepatocytes in liver injury induced by D-galactosamine.Am J Pathol. 1991; 139: 535-552PubMed Google Scholar, 5Dabeva M.D. Shafritz D.A. Activation, proliferation, and differentiation of progenitor cells into hepatocytes in the D-galactosamine model of liver regeneration.Am J Pathol. 1993; 143: 1606-1620PubMed Google Scholar These cells, generally referred to as oval cells,2Evarts R.P. Nagy P. Marsden E. Thorgeirsson S.S. A precursor-product relationship exists between oval cells and hepatocytes in rat liver.Carcinogenesis. 1987; 8: 1737-1740Crossref PubMed Scopus (394) Google Scholar, 3Evarts R.P. Nagy P. Nakatsukasa H. Marsden E. Thorgeirsson S.S. In vivo differentiation of rat liver oval cells into hepatocytes.Cancer Res. 1989; 49: 1541-1547PubMed Google Scholar, 4Lemire J.M. Shiojiri N. Fausto N. Oval cell proliferation and the origin of small hepatocytes in liver injury induced by D-galactosamine.Am J Pathol. 1991; 139: 535-552PubMed Google Scholar, 5Dabeva M.D. Shafritz D.A. Activation, proliferation, and differentiation of progenitor cells into hepatocytes in the D-galactosamine model of liver regeneration.Am J Pathol. 1993; 143: 1606-1620PubMed Google Scholar express genes in both the hepatocytic and bile duct epithelial cell lineages and have been considered to represent a reserve stem cell compartment.6Grisham J.W. Thorgeirsson S.S. Liver stem cells.in: Cotton C.S. Stem cells. Academic Press, London1997: 233-282Crossref Google Scholar, 7Fausto N. Liver regeneration and repair hepatocytes, progenitor cells, and stem cells.Hepatology. 2004; 39: 1477-1487Crossref PubMed Scopus (631) Google Scholar Recent studies suggest that oval cells are derived from undifferentiated epithelial cells residing in the canals of Hering.8Theise N.D. Saxena R. Portmann B.C. Thung S.N. Yee H. Chiriboga L. Kumar A. Crawford J.M. The canals of Hering and hepatic stem cells in humans.Hepatology. 1999; 30: 1425-1433Crossref PubMed Scopus (615) Google Scholar, 9Paku S. Schnur J. Nagy P. Thorgeirsson S.S. Origin and structural evolution of the early proliferating oval cells in rat liver.Am J Pathol. 2001; 158: 1313-1323Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar However, oval cells isolated from D-galactosamine-injured liver or copper-depleted pancreas have only limited ability to repopulate the normal liver.10Dabeva M.D. Hwang S.G. Vasa S.R. Hurston E. Novikoff P.M. Hixson D.C. Gupta S. Shafritz D.A. Differentiation of pancreatic epithelial cells into hepatocytes following transplantation into rat liver.Proc Natl Acad Sci U S A. 1997; 94: 7356-7361Crossref PubMed Scopus (177) Google Scholar To identify cells that might be more efficient in repopulating normal liver, we have taken advantage of the developing rat in which foregut endodermal cells migrate into the septum transversum at ∼embryonic day (ED) 8.5, become tissue specified, and begin to express genes in the hepatic lineages at ∼ED 9.0–9.5, initially α-fetoprotein (AFP) and albumin (Alb) for hepatocytes and subsequently cytokeratin (CK)-19 for cholangiocytes (Figure 1A).11Zaret K. Molecular genetics of early liver development.Annu Rev Physiol. 1996; 58: 231-251Crossref PubMed Scopus (96) Google Scholar These cells proliferate massively and form the liver bud, which we then isolate by microdissection before the cells diverge along the hepatocytic and cholangiocytic lineages, which occurs at ∼ED 16. The ED 14 fetal rat liver contains a subpopulation of bipotent (AFP+/CK-19+) cells (Figure 1B and 1C) that we have used previously to repopulate the normal rat liver (6.6% ± 2.8%), which does not occur to any significant extent with adult hepatocytes (Figure 1D).12Sandhu J.S. Petkov P.M. Dabeva M.D. Shafritz D.A. Stem cell properties and repopulation of the rat liver by fetal liver epithelial progenitor cells.Am J Pathol. 2001; 159: 1323-1334Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar Using a higher number of fetal liver cells for transplantation, we have increased the level of long-term repopulation to 23.5% ± 2.1%, which allowed us to determine the mechanism by which liver reconstitution takes place. Repopulation occurs by cell-cell competition in which more highly proliferative transplanted fetal liver stem/progenitor cells progressively replace less proliferative neighboring host hepatocytes by inducing their apoptosis in a mechanism remarkably similar to that described in Drosophila during wing development.13Moreno E. Basler K. dMyc transforms cells into super-competitors.Cell. 2004; 117: 117-129Abstract Full Text Full Text PDF PubMed Scopus (452) Google Scholar, 14de la Cova C. Abril M. Bellosta P. Gallant P. Johnston L.A. Drosophila myc regulates organ size by inducing cell competition.Cell. 2004; 117: 107-116Abstract Full Text Full Text PDF PubMed Scopus (457) Google Scholar The level of long-term tissue reconstitution obtained far exceeds that observed with any other cell type or cell line studied to date in a normal hepatic environment. This repopulation strategy should be effective for treatment of most inherited metabolic disorders of the liver, as well as diseases of other organs. Chemicals and reagents were from Sigma Chemical Co., St. Louis, MO and DAKO Corp., Carpinteria, CA; collagenase type 1 from Worthington Biochemical Corporation, Lakewood, NJ; cell culture medium and supplements from Gibco BRL Life Technologies, Grand Island, NY; and fetal bovine serum (FBS) from Gemini Bio-Products, Woodland, CA. Pregnant, ED 14 dipeptidyl peptidase IV (DPPIV)+ Fisher (F) 344 rats, purchased from Taconic Farms, German Town, NY, were used to prepare fetal liver cells. DPPIV− F344 rats were provided by the Special Animal Core of the Liver Research Center, Albert Einstein College of Medicine. All animal studies were conducted under protocols approved by the Animal Care Use Committee of the Albert Einstein College of Medicine in accordance with National Institutes of Health (NIH) guidelines. Unfractionated fetal liver stem/progenitor cells (FLSPC) were isolated from ED 14 fetal livers of DPPIV+ pregnant F344 rats, as described previously.12Sandhu J.S. Petkov P.M. Dabeva M.D. Shafritz D.A. Stem cell properties and repopulation of the rat liver by fetal liver epithelial progenitor cells.Am J Pathol. 2001; 159: 1323-1334Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar After two-thirds partial hepatectomy (PH), freshly isolated and unfractionated FLSPC (1–6 × 107 cells) were infused through the portal vein into normal DPPIV− F344 rats. In some experiments, PH was performed 1 day before or 1 day after cell transplantation, and, in others, PH was not performed. Cytospins of unfractionated FLSPC were stained with sheep anti-AFP (Nordic Immunological Laboratories, Tilburg, NL), mouse anti-CK-19 (Novocastra Laboratories Ltd., Newcastle, UK), and rabbit anti-albumin (ICN Biochemicals, Inc., Aurora, OH). Secondary antibodies used in various experiments included Cy 2-conjugated donkey anti-sheep IgG and Cy 3-conjugated donkey anti-mouse IgG (Jackson Immunoresearch Laboratories, West Grove, PA), Cy 3-conjugated donkey anti-rabbit IgG, and Cy 5-conjugated donkey anti-mouse IgG (Jackson). DPPIV expression was determined by enzyme histochemistry in liver cryosections, and liver repopulation was quantified by computerized scanning of liver sections and confirmed by biochemical quantitation of DPPIV enzyme activity in liver homogenates from the same tissue, as previously reported.12Sandhu J.S. Petkov P.M. Dabeva M.D. Shafritz D.A. Stem cell properties and repopulation of the rat liver by fetal liver epithelial progenitor cells.Am J Pathol. 2001; 159: 1323-1334Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar Apoptosis of DPPIV+ cells vs the surrounding parenchyma was detected in situ by end labeling the fragmented DNA of apoptotic cells using a commercially available kit (Promega Corp., Madison, WI) with a modified protocol to allow simultaneous DPPIV detection. Biotinylated nucleotides were incorporated at the 3′-OH DNA ends using the terminal deoxynucleotidyl transferase. Cryosections were stained with mouse anti-CD26 (Biotrend Chemikalien GmbH, Koln, Germany), followed by Cy 2-conjugated Streptavidin (Rockland) and Cy 3-conjugated donkey anti-mouse IgG (Jackson). Cryosections were stained with mouse anti-CD26 (Biotrend) and rabbit anti-caspase-3 or rabbit anti-Bax (Lab Vision Corp., Fremont, CA). Secondary antibodies used were Cy 3-conjugated donkey anti-mouse IgG and Cy 2-conjugated donkey anti-rabbit IgG (Jackson). Cryosections were stained with mouse anti-CD26 (Biotrend) and mouse anti-Ki-67 (BD Biosciences, San Jose, CA) or mouse anti-MMP-2 (Lab Vision) or rabbit anti-UGT1A1 (kindly provided by Dr. J. Roy–Chowdhury, Liver Research Center, Albert Einstein College of Medicine). Secondary antibodies included biotin-conjugated goat anti-mouse IgG2 (Nordic), followed by Cy 2-conjugated Streptavidin (Rockland Inc., Gilbertsville, PA) and TRITC-conjugated rabbit anti-mouse IgG1 (Rockland) or Cy 3-conjugated donkey anti-rabbit IgG (Jackson). Cryosections were stained with mouse anti-CD26 (Biotrend) and rabbit anti-albumin (ICN) or rabbit anti-ASGPR (kindly provided by Dr. R. Stockert, Liver Research Center, Albert Einstein College of Medicine), followed by Cy 3-conjugated donkey anti-mouse IgG and Cy 2-conjugated donkey anti-rabbit IgG (Jackson). Unfixed cryosections were incubated for 20 minutes in substrate reagent: 10 mmol/L D-Glucose-6-phosphate, 270 mmol/L sucrose, 2.4 mmol/L Pb(NO3)2, and 40 mmol/L Tris/maleate buffer, pH 6.5. After fixation, cryosections were immersed in 0.22% (NH4)2S diluted in 0.1 mol/L Tris maleate/0.1 mol/L NaCl, and counterstained with hematoxylin. Cryosections were incubated for 5 minutes in 0.5% periodic acid solution, followed by Schiff’s reagent for 25 minutes. Sections were counterstained with hematoxylin. Data are reported as mean ± SEM. The significance was analyzed by the Student t test or Mann–Whitney rank sum test with SigmaStat 2.01 software (SPSS Scientific, Erkrath, Germany). P values <.05 were considered significant. Previously, we transplanted 1.5–3.0 × 106 ED 14 rat fetal liver cells enriched 3-fold for epithelial progenitors by panning with anti-rbc antibody.12Sandhu J.S. Petkov P.M. Dabeva M.D. Shafritz D.A. Stem cell properties and repopulation of the rat liver by fetal liver epithelial progenitor cells.Am J Pathol. 2001; 159: 1323-1334Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar These preparations contained 2.3–4.5 × 105 fetal liver epithelial cells, and we obtained 6.6% ± 2.8% liver repopulation at 6 months. Subsequently, we discovered that we could transplant much greater numbers of cells and that panning was unnecessary because hematopoietic cells, which represent ∼70% of total cells in ED 14 rat fetal liver, readily pass through the liver sinusoids. In the present study, we transplanted unfractionated ED 14 rat fetal liver cells in increasing numbers, beginning with 1.0 × 107 cells of which 2.0% were AFP+/CK-19+ (equivalent to the number of epithelial cells used previously). As shown in Figure 2A, the number and size of DPPIV+ cell clusters increased progressively as the number of cells undergoing transplantation increased from 1 × 107 (Figure 2A, left panel) to 2.5 × 107 (Figure 2A, middle panel) to 4.0 × 107 (Figure 2A, right panel). To quantitate repopulation precisely over time, we transplanted 4–6 × 107 ED 14 fetal liver cells and determined the number of clusters/cm2; the cluster size; and the percentage of liver replacement at 2, 4, and 6 months after cell transplantation (Table 1). There was substantial proliferation of transplanted cells within 2 months, with many clusters containing up to 100 cells, increasing to 100–500 cells/cluster at 4 months and >500 cells/cluster at 6 months. The number of clusters/cm2 increased continuously over time, and the percentage repopulation also increased throughout time, with greatest increase between 4 and 6 months. At 6 months, we observed 23.5% ± 2.1% replacement of total liver mass by ED 14 fetal liver cells (Table 1). Figure 2B shows entire sections from 2 animals exhibiting uniform and extensive liver repopulation at 6 months after cell transplantation. In periportal areas (Figure 2C), liver repopulation exceeded 50%–60%, and large cell clusters contained mature hepatocytes and bile duct structures undergoing confluence.Table 1Kinetics of Liver Repopulation by Transplanted ED14 Fetal Liver Stem/Progenitor Cells in Normal RatsMonths after cell transplantation2 (n = 3)4 (n = 3)aOne animal in this group was killed 14 weeks after cell transplantation.6 (n = 4)Clusters/cm2148.3 ± 10.2249.7 ± 32.0408.5 ± 49.7Cluster sizebSmall, up to 100 cells/cluster; medium, 100–500 cells/cluster; large, >500 cells/cluster.Predominantly small, some mediumSmall to medium, some largeSmall to largePercentage repopulation2.3 ± 0.1cP = .003 (Student t test).5.2 ± 0.8cP = .003 (Student t test).dP < .001 (Student t test).23.5 ± 2.1dP < .001 (Student t test).NOTE. n = the number of animals in each group. Data are presented as mean ± SEM.a One animal in this group was killed 14 weeks after cell transplantation.b Small, up to 100 cells/cluster; medium, 100–500 cells/cluster; large, >500 cells/cluster.c P = .003 (Student t test).d P < .001 (Student t test). Open table in a new tab NOTE. n = the number of animals in each group. Data are presented as mean ± SEM. From the number of DPPIV+ cell clusters observed on 2-dimensional sections at 6 months (408.5 ± 49.7 clusters/cm2), we estimate that the entire liver contained >100,000 clusters of transplanted cells. Based on our finding that ∼2.0% of the cells are AFP+/CK-19+, we estimate that each animal undergoing transplantation received ∼1.0 × 106 stem/progenitor cells. Assuming a 10% hepatic engraftment efficiency (it is probably much lower), each recipient liver started with ∼1.0 × 105 transplanted stem/progenitor cells. Because the adult rat liver contains ∼5 × 108 hepatocytes, and 23.5% of the regenerated liver mass at 6 months was of donor origin (∼1.2 × 108 cells), we estimate that donor-derived cells increased a minimum of 1000-fold during the repopulation process. An important property of stem cells is that their progeny differentiate to produce normal tissue structure with normal biologic function. At 6 months after fetal liver cell transplantation, the overall liver structure appeared normal histologically (Figure 3A, left panel), except for mild bile duct hyperplasia (Figure 3A, left panel, arrows). On analysis of serial sections (Figure 3A, right panel), transplanted DPPIV+ cells differentiated into hepatocytes and cholangiocytes and became incorporated into the parenchymal plates and bile ducts, respectively, with no evidence of bile duct obstruction or inflammation (serum ALT, bilirubin, and γGT levels were within normal range). Double-label immunohistochemistry showed that transplanted cells expressed DPPIV in a bile canalicular pattern unique to hepatocytes and also expressed albumin (Figure 3B). Using double-label immunohistochemistry, DPPIV and the asialoglycoprotein receptor (ASGPR; another unique hepatocyte-specific protein) were shown to be present on the apical (DPPIV) and basolateral (ASGPR) domains, respectively, of the same cells (Figure 3C). The progeny of transplanted DPPIV+ ED 14 fetal liver cells also expressed UDP-glucuronosyl transferase (UGT1A1), which specifically conjugates bilirubin into mono- and diglucuronides in hepatocytes (Figure 3D). In serial sections (Figure 3E), DPPIV+ cell clusters that were distributed throughout the hepatic parenchyma (Figure 3E, left panel) showed normal lobular gradients in expression of glucose-6-phosphatase (Figure 3, middle panel) and glycogen storage (Figure 3E, right panel). These gradients are mirror images of each other, with glucose-6-phosphatase favoring the periportal region (Figure 3E, middle panel, black arrow), and glycogen storage favoring the central lobular region (Figure 3E, right panel, white arrow). Thus, transplanted fetal liver cells that had differentiated into hepatocytes were both morphologically and functionally indistinguishable from neighboring host hepatocytes at all locations within the hepatic lobule. PH was necessary to obtain liver repopulation by ED 14 fetal liver cells. In its absence, rare, very small clusters of DPPIV+ hepatocytes, bile ducts, or hematopoietic cells were observed (Figure 4A). If PH was performed 1 day before or at the time of cell transplantation, we obtained comparable levels of liver repopulation (Figure 4B). However, if PH was performed 1 day after cell transplantation, only rare DPPIV+ cells were observed. This suggested a marked decrease in ED 14 fetal liver cell engraftment in the absence of PH (Figure 5A vs 5B).Figure 5Engraftment of ED 14 fetal liver stem/progenitor cells. 1.1 × 107 Wt ED 14 rat fetal liver cells underwent transplantation into DPPIV− F344 rats in the presence (A) or absence (B) of two-thirds PH. Animals were killed at 1, 3, or 7 days after cell transplantation, and DPPIV expression in the recipient liver was determined by enzyme histochemistry. (A) ED 14 FLSPC are negative for DPPIV expression at the time of their isolation, and the liver at 1 day after cell transplantation/PH was negative for DPPIV (left). At 3 days after ED 14 fetal liver cell transplantation/PH (middle), small clusters of DPPIV+ cells were visualized, which increased both in size and number at 7 days after cell transplantation (right). (B) In the absence of PH, DPPIV+ cells were not observed at 3 days after cell transplantation (left) and were rare and very small at 7 days after ED 14 fetal liver cell transplantation (right). Original magnification, 200×.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To investigate the mechanism of hepatic replacement by transplanted cells, we measured the proliferative activity of cells in DPPIV+ clusters vs the surrounding parenchyma, using dual-label immunohistochemistry for DPPIV and Ki-67, a nuclear antigen expressed in cycling cells and apoptosis of transplanted cells vs host hepatocytes, using the TUNEL assay. At 2 and 6 months after cell transplantation (Figure 6A, left and right, respectively), a substantial number of cells within DPPIV+ clusters were positive for Ki-67. On quantitative analysis, 3.5% ± 0.8% and 2.8% ± 0.4% of DPPIV+ cells were Ki-67+ at 2 and 6 months, respectively, whereas, in DPPIV− host hepatocytes, this was 0.6% ± 0.1% and 0.4% ± 0.1% at 2 and 6 months, respectively (Table 2). Therefore, the number of actively cycling cells was 6- to 7-fold higher in transplanted cells vs host hepatocytes at both 2 and 6 months after fetal liver cell transplantation, and this difference remained constant.Table 2Proliferative Activity of Cells in DPPIV+ Clusters Versus the Surrounding Parenchyma in Normal Liver Undergoing Transplantation With ED14 Fetal Liver CellsMonths after cell transplantation26DPPIV+ clustersaUsing immunohistochemistry for DPPIV and Ki-67 (see Materials and Methods section for details), 7 or 8 random microscopic fields containing DPPIV+ cell clusters from 2 different liver sections of 1 or 2 animals at 2 and 6 months after fetal liver cell transplantation were examined, and the percentage of proliferating cells was determined. Total cell number analyzed5281222 Ki-67+ cells2234 Percentage3.5 ± 0.8cP = .039 (Student t test).2.8 ± 0.4dP = .006 (Student t test).DPPIV− surrounding parenchymabThree random microscopic fields were chosen and analyzed. Total cell number analyzed43473165 Ki-67+ cells2412 Percentage0.6 ± 0.1cP = .039 (Student t test).0.4 ± 0.1dP = .006 (Student t test).NOTE. Data are presented as mean ± SEM.a Using immunohistochemistry for DPPIV and Ki-67 (see Materials and Methods section for details), 7 or 8 random microscopic fields containing DPPIV+ cell clusters from 2 different liver sections of 1 or 2 animals at 2 and 6 months after fetal liver cell transplantation were examined, and the percentage of proliferating cells was determined.b Three random microscopic fields were chosen and analyzed.c P = .039 (Student t test).d P = .006 (Student t test). Open table in a new tab NOTE. Data are presented as mean ± SEM. During liver regeneration following two-thirds PH, the liver mass (measured as the liver/body weight ratio × 100) returns to normal within 2 to 4 weeks (Figure 7). In the present study, although the number of transplanted liver cells increased progressively between 2 and 6 months, the liver/body weight ratio remained normal (3.1% ± 0.1%). For this to occur, there must be compensatory apoptosis in the host liver. This was determined quantitatively by double-label TUNEL assay/DPPIV immunohistochemistry (Table 3). Apoptosis was rarely observed in transplanted cells and their progeny (DPPIV+) at both 2 and 6 months after cell transplantation (4–5 times lower than in host parenchyma) but was increased in host (DPPIV−) liver at 6 months after cell transplantation, as compared with control rat liver (Table 3). At 2 and 6 months, respectively, apoptotic host hepatocytes were evident at the junctional margin and within 1 or 2 cells distance from transplanted DPPIV+ clusters (Figure 6B, left and right). These results were further supported by immunohistochemical studies showing increased expression of activated caspase-3 and Bax in selected host hepatocytes in direct contact with or immediately adjacent to DPPIV+ transplanted cells (Figure 6C, left and right). The very few apoptotic hepatocytes observed within transplanted cell clusters were randomly distributed (Figure 6B, left and right). These results suggest that apoptosis is increased in host hepatocytes immediately adjacent to clusters of proliferating transplanted cells, and this maintains the host liver mass within normal limits.Table 3Apoptotic Cells in DPPIV+ Clusters Versus the Surrounding Parenchyma in Normal Liver Undergoing Transplantation With ED14 Fetal Liver CellsMonths after cell transplantation2 Exp2 Con6 Exp6 ConDPPIV+ clusters Total cell number analyzed671—4586— Apoptotic cells1—3— Percentage0.06 ± 0.04aP < .001 (Mann–Whitney rank sum test).—0.06 ± 0.04bP < .001 (Mann–Whitney rank sum test).—DPPIV− surrounding parenchyma Total cell number analyzed39,186—23,951— Apoptotic cells90—58— Percentage0.23 ± 0.04aP < .001 (Mann–Whitney rank sum test).cP = .658 (Mann–Whitney rank sum test).—0.27 ± 0.04bP < .001 (Mann–Whitney rank sum test).dP = .011 (Mann–Whitney rank sum test).—Total liver tissue Total cell number analyzed39,85728,17928,53729,446 Apoptotic cells91596145 Percentage0.23 ± 0.040.21 ± 0.03cP = .658 (Mann–Whitney rank sum test).0.22 ± 0.030.16 ± 0.02dP = .011 (Mann–Whitney rank sum test).NOTE. Data were determined quantitatively by the TUNEL assay in conjunction with DPPIV immunohistochemistry (see Materials and Methods section for details) examining 20 random microscopic fields from 2 sections of liver tissue of 2 animals at 2 and 6 months after fetal liver cell transplantation (Exp) and 2 control (nontransplanted) livers (Con), each from DPPIV− F344 rats of similar age. Data are presented as mean ± SEM.a P < .001 (Mann–Whitney rank sum test).b P < .001 (Mann–Whitney rank sum test).c P = .658 (Mann–Whitney rank sum test).d P = .011 (Mann–Whitney rank sum test). Open table in a new tab NOTE. Data were determined quantitatively by the TUNEL assay in conjunction with DPPIV immunohistochemistry (see Materials and Methods section for details) examining 20 random microscopic fields from 2 sections of liver tissue of 2 animals at 2 and 6 months after fetal liver cell transplantation (Exp) and 2 control (nontransplanted) livers (Con), each from DPPIV− F344 rats of similar age. Data are presented as mean ± SEM. To determine the specific distribution of apoptotic hepatocytes, we examined 38 adjacent microscopic fields of a tissue section (∼45,000–50,000 hepatocytes) at 6 months after fetal liver cell transplantation, scoring cells for DPPIV expression and apoptosis (Figure 8). Thirty-three percent of host hepatocytes that were apoptotic were at or within 1 or 2 cells distance from the border with transplanted cells. This percentage accounts for the overall increase in hepatic apoptosis observed in fetal liver cell transplanted rats as compared with nontransplanted control rats of similar age (Table 3). Thus, transplanted fetal liver
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