Epithelial Tissue Chimerism after Human Hematopoietic Cell Transplantation Is a Real Phenomenon
2004; Elsevier BV; Volume: 164; Issue: 4 Linguagem: Inglês
10.1016/s0002-9440(10)63203-8
ISSN1525-2191
AutoresAlexandros Spyridonidis, Annette Schmitt‐Graeff, Tina Tomann, Anne Dwenger, Marie Follo, Dirk Behringer, Jürgen Finke,
Tópico(s)Immunotherapy and Immune Responses
ResumoBone marrow transplantation in animals has been shown to generate epithelial populations, a phenomenon that has also recently been suggested to take place after human hematopoietic cell transplantation. However, reports in humans are not conclusive because they still leave open the possibility that the identified donor-derived cells are not epithelial cells but intraepithelial lymphocytes. Here, we demonstrate that donor-derived CD45+ hematopoietic cells in close contact with epithelial tissue may be falsely characterized as donor-derived epithelial cells if the three-dimensional structure of the tissue is not considered and the hematopoietic markers are not examined. By using a rigorous three-dimensional analysis on single sections of colon biopsies triple stained with donor-specific, epithelial-specific, and hematopoietic-specific markers we demonstrate that chimerism of colon epithelium is a real phenomenon occurring constantly after human hematopoietic cell transplantation. We exclude horizontal DNA transfer or cell fusion as the underlying mechanism of our findings. Tissue damage enhances the engraftment of the donor-derived epithelial cells. The physiological and therapeutical role of the donor-derived epithelial cells after human hematopoietic cell transplantation needs further investigation. However, their identification requires stringent and unequivocal detection systems. Bone marrow transplantation in animals has been shown to generate epithelial populations, a phenomenon that has also recently been suggested to take place after human hematopoietic cell transplantation. However, reports in humans are not conclusive because they still leave open the possibility that the identified donor-derived cells are not epithelial cells but intraepithelial lymphocytes. Here, we demonstrate that donor-derived CD45+ hematopoietic cells in close contact with epithelial tissue may be falsely characterized as donor-derived epithelial cells if the three-dimensional structure of the tissue is not considered and the hematopoietic markers are not examined. By using a rigorous three-dimensional analysis on single sections of colon biopsies triple stained with donor-specific, epithelial-specific, and hematopoietic-specific markers we demonstrate that chimerism of colon epithelium is a real phenomenon occurring constantly after human hematopoietic cell transplantation. We exclude horizontal DNA transfer or cell fusion as the underlying mechanism of our findings. Tissue damage enhances the engraftment of the donor-derived epithelial cells. The physiological and therapeutical role of the donor-derived epithelial cells after human hematopoietic cell transplantation needs further investigation. However, their identification requires stringent and unequivocal detection systems. Hematopoietic cell transplantation (HCT) is a standard term used in clinical practice indicating a procedure in which a cellular graft usually derived from bone marrow (BM) or peripheral blood stem cells reconstitutes the hematopoietic system in a myeloablated host. Allogeneic HCT results in true biological chimeras. Although circulating hematopoietic cells and their tissue derivatives such as Kupffer cells in the liver,1Gale RP Sparkes RS Golde DW Bone marrow origin of hepatic macrophages (Kupffer cells) in humans.Science. 1978; 201: 937-938Crossref PubMed Scopus (168) Google Scholar Langerhans cells in the skin,2Volc-Platzer B Stingl G Wolff K Hinterberg W Schnedl W Cytogenetic identification of allogeneic epidermal Langerhans cells in a bone marrow graft recipient.N Engl J Med. 1984; 310: 1123-1124PubMed Google Scholar and microglial cells in the brain3Unger ER Sung JH Manivel JC Chenggis ML Blazar BR Krivit W Male donor-derived cells in the brains of female sex-mismatched bone marrow transplant recipients: a Y chromosome specific in situ hybridization study.J Neuropathol Exp Neurol. 1993; 42: 460-470Crossref Scopus (161) Google Scholar become donor genotype after transplantation, other cells are believed to remain recipient in origin. However, studies in laboratory animals throughout the last few years indicate that bone marrow transplantation (BMT) generates unexpected populations in vivo, such as liver cells and other epithelial cells.4Petersen BE Bowen WC Patrene KD Mars WM Sullivan AK Murase N Boggs SS Greenberger JS Goff JP Bone marrow as a potential source of hepatic oval cells.Science. 1999; 284: 1168-1170Crossref PubMed Scopus (2181) Google Scholar, 5Theise ND Badve S Saxena R Henegariu O Sell S Crawford JM Krause DS Derivation of hepatocytes from bone marrow cells in mice after radiation-induced myeloablation.Hepatology. 2000; 31: 235-240Crossref PubMed Scopus (896) Google Scholar, 6Krause DS Theise ND Collector MI Henegariu O Hwang S Gardner R Neutzel S Sharkis SJ Multi-organ, multi-lineage engraftment by a single bone marrow-derived stem cell.Cell. 2001; 105: 369-377Abstract Full Text Full Text PDF PubMed Scopus (2464) Google Scholar, 7Lagasse E Connors H Al-Dhalimy M Reitsma M Dohse M Osborne L Wang X Finegold M Weissman IL Grompe M Purified hematopoietic stem cells can differentiate into hepatocytes in vivo.Nat Med. 2000; 6: 1229-1234Crossref PubMed Scopus (2130) Google Scholar, 8Poulsom R Forbes SJ Hodivala-Dilke K Ryan E Wyles S Navaratnarasah S Jeffery R Hunt T Alison M Cook T Pusey C Wright NA Bone marrow contributes to renal parenchymal turnover and regeneration.Pathology. 2001; 195: 229-235Crossref Scopus (581) Google Scholar More recently, genetic marker studies in humans suggested generation after HCT of donor-derived hepatocytes, skin cells, and epithelial cells of the gastrointestinal tract.9Alison MR Poulsom R Jeffery R Dhillon AP Quaglia A Jacob J Novelli M Prentice G Williamson J Wright NA Hepatocytes from non-hepatic adult stem cells.Nature. 2000; 406: 257Crossref PubMed Scopus (954) Google Scholar, 10Theise ND Nimmakayalu M Gardner R Illei PB Morgan G Teperman L Henegariu O Krause DS Liver from bone marrow in humans.Hepatology. 2000; 32: 11-16Crossref PubMed Scopus (1153) Google Scholar, 11Korbling M Katz RL Khanna A Ruifrok AC Rondon G Albitar M Champlin RE Estrov Z Hepatocytes and epithelial cells of donor origin in recipients of peripheral-blood stem cells.N Engl J Med. 2002; 346: 738-746Crossref PubMed Scopus (713) Google Scholar, 12Okamoto R Yajima T Yamazaki M Kanai T Mukai M Okamoto S Ikeda Y Hibi T Inazawa J Watanabe M Damaged epithelia regenerated by bone marrow-derived cells in the human gastrointestinal tract.Nat Med. 2002; 8: 1011-1017Crossref PubMed Scopus (346) Google Scholar However, reports in humans are not conclusive because of methodological limitations that still leave open the possibility that the identified donor-derived cells are not epithelial cells but intraepithelial lymphocytes, and therefore have been treated with skepticism.13Abkowitz JL Can human hematopoietic stem cells become skin, gut, or liver cells?.N Engl J Med. 2002; 346: 770-772Crossref PubMed Scopus (35) Google Scholar, 14DeWitt N Knight J Biologists question adult stem-cell versatility.Nature. 2002; 416: 354Crossref PubMed Scopus (14) Google Scholar, 15Holden C Vogel G Stem cells. Plasticity: time for a reappraisal?.Science. 2002; 296: 2126-2129Crossref PubMed Scopus (84) Google Scholar We sought to find out if human HCT results in generation of donor-derived epithelial cells. Attention has been paid to the technical aspects to be sure that the identified donor-derived cells express epithelial cell markers and are not contaminating hematopoietic cells. Frozen colonic biopsies of eight female patients who underwent a sex-mismatched allogeneic HCT were triple-stained by combining interphase fluorescence in situ hybridization (FISH) with a Y-chromosome-specific probe, immunofluorescent labeling for cytokeratins (CKs) and either TOTO-3 nuclear label or CD45 immunofluorescence. The samples were examined by confocal microscopy and then underwent a rigorous three-dimensional analysis. Here, we demonstrate that donor-derived CD45+ lymphocytes in close contact with epithelial tissue may be falsely characterized as donor-derived epithelial cells if the three-dimensional structure of the tissue is not considered and the hematopoietic markers are not examined. We found that chimerism of colon epithelium is a real phenomenon occurring constantly after human HCT. Three-dimensional X-chromosome enumeration in individual Y+ epithelial cells was performed to examine whether or not fusion is the underlying mechanism of the chimeric events found. Clinical information regarding the HCT recipients, their diagnosis, the history of male childbearing, the type of transplantation (BMT or peripheral blood stem cell transplantation) and the time from transplantation to colon tissue sampling is summarized in Table 1. BM recipients received between 190 to 280 × 108 mononuclear cells and peripheral blood stem cell recipients between 743 to 1010 × 108Poulsom R Forbes SJ Hodivala-Dilke K Ryan E Wyles S Navaratnarasah S Jeffery R Hunt T Alison M Cook T Pusey C Wright NA Bone marrow contributes to renal parenchymal turnover and regeneration.Pathology. 2001; 195: 229-235Crossref Scopus (581) Google Scholar mononuclear cells. In patients showing clinical signs of gastrointestinal graft versus host disease at different time points after transplantation, endoscopic biopsies were taken after informed consent to verify the diagnosis. Snap-frozen biopsies obtained from eight female patients who underwent a sex-mismatched allogeneic HSCT as well as from six female and two male sex-matched transplanted patients were stored at −180°C after informed consent. Other biopsies obtained from the same endoscopy were formalin-fixed and examined in the Pathology Department of the University of Freiburg. Cryostat sections used in this study were examined after informed consent. All patients had received multiple blood transfusions in the past; blood products transfused after transplantation were always leukocyte depleted and irradiated.Table 1Patient Characteristics and Colon Epithelium ChimerismIntraepithelial chimeric events/ 100 cryptsPtRecipient/ DonorMale child-bearingAge at HCT (years)Reason for HSCTType of HSCTDays from HSCT to biopsyPathological diagnosisY+/CD45+Y+/CK+/CD45−Total% of intraepithelial Y+/total epithelial cells% of Y+/CK+/ CD45−/total epithelial cellsSex-mismatched1F/MNo41CMLBM+PBSC420GvHD I3912510.80%0.19%2F/MYes43CMLBM29Intact epithelium1340.06%0.05%3F/MYes29CMLBM43GvHD III3011410.65%0.17%4F/MNo28AMLBM72GvHD I2224460.73%0.38%5F/MYes51CMLPBSC16Intact epithelium1230.05%0.03%6F/MYes38AMLBM/PBSC*BMT followed by PBSCT 5 months later.540†Time after the BMT.GvHD III3319520.82%0.30%7F/MNo22CMLBM41GvHD IV1714310.49%0.22%8F/MNo48AMLPBSC15GvHD I96150.24%0.09%Sex-matched9F/FNo19ALLPBSC19GvHD IY−NA10F/FYes62AMLBM40GvHD IIY−NA11F/FYes34MelPBSC20GvHD IIIY−NA12F/FYes42LyPBSC231GvHD IIIY−NA13F/FYes44CMLPBSC60Intact epitheliumY−NA14F/FYes44ALLPBSC20GvHD IY−NA‡Y-FISH was performed in paraffin-embedded materials.15F/FNo67AMLPBSC51Intact epitheliumY−NA‡Y-FISH was performed in paraffin-embedded materials.16M/M-18AMLPBSC30Intact epithelium3>99%NA17M/M-55LyPBSC45GvHD III23>99%NAEight-μm sections were used for quantification.Y−/+, Y-chromosome negative/positive; NA, epithelial chimerism analysis not applicable; BM, bone marrow; PBSC, peripheral blood stem cells; AML/CML, acute-/chronic-myeloid leukemia; Mel, melanoma; Ly, lymphoma; GvHD, graft versus host disease.* BMT followed by PBSCT 5 months later.† Time after the BMT.‡ Y-FISH was performed in paraffin-embedded materials. Open table in a new tab Eight-μm sections were used for quantification. Y−/+, Y-chromosome negative/positive; NA, epithelial chimerism analysis not applicable; BM, bone marrow; PBSC, peripheral blood stem cells; AML/CML, acute-/chronic-myeloid leukemia; Mel, melanoma; Ly, lymphoma; GvHD, graft versus host disease. Cryostat sections (8 μm except otherwise indicated) were fixed in cold methanol/acetone solution (1:1) for 10 minutes, and air-dried. The sections were pretreated with ribonuclease A (RNase A) (2 mg/ml at 37°C for 45 minutes; Roche Diagnostics, Indianapolis, IN USA) and then processed at 37°C for 30 minutes in preheated 2× standard saline citrate (SSC)/0.1% Nonidet P-40 buffer (Calbiochem, La Jolla, CA), pH 7.0. Serial ethanol dehydration was done (70%, 85%, and 100%; 1.5 minutes each), and the slides were air-dried at room temperature. One μl of the human Y-chromosome-specific probe labeled with rhodamine (CEP Y satellite III Spectrum Orange; Vysis, Bergisch-Gladbach, Germany) was mixed with 7 μl of hybridization buffer (CEP hybridization buffer containing dextran sulfate, formamide, and SSC; Vysis) and 2 μl of purified H2O and applied to the sections. In the case of the combined XY-chromosome stain, a XY mixed probe was used (Vysis). The sections were coverslipped and sealed with rubber cement. The sections and the probe were co-denaturated by placing the slides on the surface of a 73°C prewarmed plate (HYBrite, Vysis) for 3 minutes. Hybridization was performed overnight at 37°C in a humidified box. The next day, the coverslips were carefully removed in 2× SSC/0.3% Nonidet P-40 buffer at room temperature and posthybridization washes were done three times for 2 minutes each in 2× SSC/0.3% Nonidet P-40 preheated buffer at 73°C. For combined Y-chromosome FISH and CK fluorescence immunohistochemistry, sections were then incubated for 30 minutes at room temperature in 1% normal bovine serum (30% albumin bovine solution; Sigma, Germany) diluted in phosphate-buffered saline (BioWhittaker, Germany) to block nonspecific binding. After washing in 2× SSC, the sections were exposed for 3 hours at 37°C to a fluorescein isothiocyanate (FITC)-labeled, monoclonal mouse antibody that detects human CK7 and CK8 (clone CAM 5.2; Becton Dickinson, San Jose, CA). For those sections without CD45 staining, the sections were stained with the nucleic acid dye TOTO-3 as a nuclear counterstain. After a thorough wash in SSC/0.1% Tween 20, the sections were treated with ribonuclease A (see above) and processed with TOTO-3 nucleic acid dye (4 × 10−4 mmol/L, at room temperature, for 10 minutes; Molecular Probes, Leiden, The Netherlands). For the combination of FISH for Y-chromosome and immunofluorescence for CD45 and CK, after posthybridization washes and antigen blocking, sections were first incubated with a CD45 primary antibody (monoclonal mouse anti-human LCA, clones 2B11+ PD7/26, 16 μg/ml, at room temperature for 1 hour; DAKO, Glostrup, Denmark), washed in 2× SSC/0.1% Tween and then exposed to a goat anti-mouse antibody labeled with Alexa Fluor 633 [Alexa Fluor 633 F(ab′)2-fragment of goat anti-mouse IgG (H+L), 10 μg/ml, at room temperature for 1 hour; Molecular Probes]. These steps were followed by the CK staining procedure as described above. After washing in SSC/0.1% Tween 20, slides were mounted with Vecta-shield anti-fade (Vector Laboratories, Burlingame, CA) and the coverslip was added. Negative controls included omission of the Y-probe in FISH, use of isotype-matched control antibodies (IgG1-FITC; Immunotech, Marseille, France) for CK and CD45 staining, and staining of gut samples from female patients who received BM transplants from a female donor. Positive controls consisted of samples obtained from transplanted male BM recipients. The sensitivity of our Y/CK/TOTO or Y/CK/CD45 stain to identify Y signals was tested in control male biopsies and was found to be dependent on the thickness of the section (see Results). By using 8-μm sections the sensitivity was found to be >98% for the Y-signal. Nearly all of the TOTO-stained nuclei in the 20-μm male sections were found to be Y-positive indicating that in these thicker sections our FISH technique did not lose hybridization efficiency. Combined XY FISH analysis (X/Y/CK stain) was tested in 20-μm control male and female sections. Despite the thickness of these sections, expression of the signals was consistent throughout the whole tissue. Individual cells from female and male controls were tested with three-dimensional analysis and found to contain either XX or XY signals, respectively. The adjacent serial sections (8 μm or 4 μm) below and above the section that was used for FISH Y-chromosome analysis (middle section) were fixed with acetone and stained with CD45 (LCA)-specific antibodies (monoclonal antibody, clone 2B11+PD7/26; DAKO-Diagnostica, Hamburg, Germany). Antibody binding was detected at room temperature using affinity-purified rabbit anti-mouse IgG (1:25) for 30 minutes and mouse alkaline phosphatase anti-alkaline phosphatase complex (1:50) for 30 minutes (DAKO). Two percent normal pooled human serum was added to rabbit anti-mouse IgG. Incubations with the secondary antibodies and the alkaline phosphatase anti-alkaline phosphatase complex were repeated once. Bound alkaline phosphatase was demonstrated using naphthol AS-Bi phosphate (Sigma, St. Louis, MO) as a substrate and New Fuchsine (Serva, Heidelberg, Germany) as a coupling reagent in 0.2 mol/L of Tris-HCl buffer, pH 8.5) with 1 mmol/L of levamisole in the reaction mixture to block endogenous enzyme activity. The slides were counterstained with hematoxylin and mounted. Those colonic crypts that were found to contain Y+/CK+ cells in the triple-stained middle section were identified in the CD45-labeled sections and color photographed at various magnifications (×10, ×63, and ×100). Serial frozen sections were stained with CD68-specific antibodies (monoclonal antibody, clone PG-M1; DAKO-Diagnostica) by using the alkaline phosphatase anti-alkaline phosphatase technique as described above. Confocal laser-scanning microscopy was performed using a Zeiss LSM 410 scanning confocal microscope (Zeiss, Jena, Germany). The Y-chromosome rhodamine signal was excited at 543 nm and emission was collected from 590 to 610 nm whereas the CK FITC signal was excited at 488 nm and emission collected from 510 to 525 nm. TOTO-3 and Alexa 633 were excited at 633 nm while emission was collected above 665 nm. For clarity, in those samples in which CD45-Alexa 633 was present, the CK-FITC signal was shown on the blue channel of the microscope. This allowed the signal of the CD45-Alexa 633 to be more easily seen on the relatively brighter green channel. Between 60 to 100 serial confocal images of suspected cells were collected at an interval of from 0.12 to 0.2 μm along the z-axis with the 1.2 numerical aperture ×40 water immersion or the 0.5 numerical aperture ×20 objective. The serial two-dimensional images collected through the thickness of the specimen were then processed to construct three-dimensional projections with the use of a specialized commercial software (Autovisualize 5.5; AutoQuant Imaging). To minimize the possible influence of spherical aberration of the signals on our analysis, three-dimensional co-localization of the Y-chromosome and CK was considered positive only if at least two-thirds of the Y-chromosome signal was surrounded by the CK label. At least five frozen sections from each patient were examined with the Y-chromosome/CK/TOTO-3 or the Y-chromosome/CK/CD45 triple stain. To obtain quantitative data of epithelial cell engraftment, at least 100 large bowel crypts triple stained with Y/CK/CD45 were examined for each patient. In the more than 500 crypts that were carefully examined, we found that every crypt contained between 39 to 73 epitheliocytes (mean, 63 epitheliocytes), regardless of the presence of GvHD. Therefore, examination of 100 crypts results in an evaluation of a mean 6300 epitheliocytes (range, 3900 to 7300 epitheliocytes) in every patient. The degree of epithelial chimerism was presented as the absolute number of Y+/CK+/CD45− cells/100 bowel crypts examined or as the calculated mean percentage of Y+/CK+/CD45− cells/total epitheliocytes by using the formula: Y+/CK+/CD45− cells/total epithelial cells in % = (absolute number of Y+/CK+/CD45− cells/100 bowel crypts × 100)/6300. Clinical information regarding the HCT recipients, their diagnosis, the history of male childbearing, the type of transplantation (BMT or peripheral blood stem cell transplantation), and the time from transplantation to colon tissue sampling is summarized in Table 1. Fresh frozen colon biopsies were in situ hybridized with a human Y-chromosome-specific probe. Male and female patients who had received sex-matched HCT were used as controls. In these samples Y-chromosome was only detected in the male intestine, demonstrating hybridization specificity (Figure 1, Table 1). We never detected any Y-chromosome in biopsies taken from seven sex-matched transplanted women used as controls, despite the fact that five of them had a history of male childbearing and all of them received multiple blood transfusions in the past (Table 1). The FISH Y-signal was localized within the TOTO-3-counterstained nuclei representing true Y-chromosomal material rather than nonspecific debris. Epithelial cells have a diameter of ∼10 to 15 μm. Therefore, thin sections may result in a partial sampling of the cells. We tested the sensitivity of our FISH technique to identify Y-chromosome cells on sections of different thicknesses. In the control 4-μm male thin sections we found the Y-signal in ∼75% of the cells (Figure 1c). However, by using 8-μm sections we could reliably find Y-signal in >98% of the cells (Figure 1b). Therefore, 8-μm sections were used in this study. To detect donor-derived epithelial cells in female sex-mismatched HCT recipients we performed triple staining by combining FISH-Y labeling, immunofluorescent labeling for CKs, and TOTO-3 nuclear counterstain within a single 8-μm colon section. By examining the FISH-Y/CK/TOTO-stained sections with two-dimensional microscopy we identified Y-chromosome-positive (Y+) cells that appeared to be also CK+ in all eight patients examined (Figure 2). However, by using confocal microscopy and a three-dimensional analytical method we found that two-dimensional microscopy may misrepresent critical information about the three-dimensional distribution of the examined markers. Overlapping cells produced artifacts showing in the two-dimensional analysis what appears to be marker co-localization within a single cell, although the markers are actually expressed in different cells. This phenomenon is clearly illustrated in Figure 2, a and b. In the two-dimensional image of a colonic crypt some CK+ cells seem to display the Y-chromosome (Figure 2, a and b; left). However, the collection of serial optical sections through the thickness of the specimen by laser-scanning confocal microscopy and their three-dimensional reconstruction demonstrates a missing co-localization of the CK and Y-chromosome signals in the z-dimension (Figure 2, a and b, right; and Movie 2a, which is published as supporting information on The American Journal of Pathology web site). The Y-chromosome-specific signals could belong to a donor-derived lymphocyte lying above or below the CK+ cell, leading to a misinterpretation of the two-dimensional micrographs. Of a total of 63 chimeric events that were detected with two-dimensional microscopy as intraepithelial, we found that on examination in three-dimensional microscopy 19 of these cases were not truly intraepithelial. Thus, compared to three-dimensional microscopy two-dimensional microscopy of 8-μm sections results in ∼30% false-positive events. The absence of green CK staining in the top or the bottom of the three-dimensional projections shown in Figure 2, c and d, is because of the partial sampling of the cell in the 8-μm tissue sections. In contrast, in Figure 2e the Y-chromosome-positive nucleus is surrounded by a CK-positive cytoskeleton as a result of the sampling of an entire epithelial cell located within the 20-μm tissue section. Thick 20-μm sections were examined from a total of five patients and completely sampled CK+/Y+/TOTO-3 cells were found in each case. By using the FISH-Y/CK/TOTO triple stain and the three-dimensional analysis we found cells that appeared to be donor-derived Y+/CK+ epithelial cells in all eight patients examined (Figure 2; c, d, and e). However, with this stain we could not exclude the possibility that the intraepithelial found Y-chromosome signal was from infiltrating lymphocytes enclosed within the epithelial tissue. We established a triple fluorescence stain by combining FISH Y-chromosome labeling (red) with CK (blue) and CD45 (green) immunofluorescence. Donor-derived lymphocytes stained positive as expected for both the Y-chromosome and CD45. We found that intraepithelial lymphocytes can be easily mistaken as donor-derived epithelial cells if the expression of hematopoietic markers is not examined, as shown in Figure 3a. Staining of CD45 in serial 4-μm sections, as has been used in studies to date, cannot exclude the presence of lymphocytes in the section studied by FISH Y analysis. As is clearly illustrated in Figure 3b, CD45+ cells found in one section (4 μm thick) are not necessarily detectable in the neighboring sections (4 μm each) below and above. For example, the intraepithelial lymphocyte found in the Y/CK/CD45-stained middle section is not detected in the serial sections above or below. In the same vein, other CD45+ cells that were stained in the middle and the one adjacent section are not apparent in the other section. We used the triple FISH/CK/CD45 stain in a single 8-μm section combined with three-dimensional analysis to detect donor-derived epithelial colon cells in the sex-mismatched HCT recipients. As expected, the majority of the intraepithelial chimeric events found were because of Y+/CD45+ intraepithelial lymphocytes (Table 1). However, we could also detect Y+/CK+/CD45− cells in all eight patients (Figure 4 and Figure 3b). In all cases examined, Y+/CK+/CD45− cells were identified as isolated single cells scattered throughout the colonic crypts and not as clusters. At least 100 large bowel crypts were examined for each patient. Up to 24% of the crypts were found to contain single epithelial cells of donor origin (patient 4). Considering that each crypt contains a mean of 63 epitheliocytes, we conclude that a mean of 0.18% of the overall colon epithelial cells were donor-derived, with higher numbers found in the sections with histologically documented tissue damage (mean, 0.22%; six patients) compared to sections with intact epithelium (mean, 0.04%; two patients). Tissue macrophages may down-regulate their CD45-expression and therefore escape their detection as hematopoietic cells. Although CD68-positive macrophages could be identified in the lamina propria, we never detected macrophages within the colon epithelium (data not shown). We examined whether or not the chimeric events found within the gut epithelium are the products of cell fusion. We combined CK staining with FISH for the Y- and the X-chromosome and examined the number of X-chromosomes present within the CK+/Y+ cells. To be sure that we did not underestimate the number of X-chromosomes within a single cell, we used 20-μm sections for these stains to sample whole epithelial cells (diameters, 10 to 15 μm) with their entire nucleus and chromosome content. For correct enumeration of the X signals within a single cell we examined the CK+/Y+ cells in all three dimensions. This was done because examination by confocal microscopy in only a single plane may result in an underestimation of the FISH signals. Individual Y+/CK+ epithelial cells from three female recipients were found to contain only one X-chromosome (Figure 4b). Furthermore, we examined the TOTO-3 nuclei signal in the Y+/CK+/TOTO-3 triple-stained cells in all patients, but we never found any doubled TOTO-3 signal intensity indicative for fusion events. BMT in animals has been shown to generate unexpected populations in vivo, such as liver cells and other epithelial cells.4Petersen BE Bowen WC Patrene KD Mars WM Sullivan AK Murase N Boggs SS Greenberger JS Goff JP Bone marrow as a potential source of hepatic oval cells.Science. 1999; 284: 1168-1170Crossref PubMed Scopus (2181) Google Scholar, 5Theise ND Badve S Saxena R Henegariu O Sell S Crawford JM Krause DS Derivation of hepatocytes from bone marrow cells in mice after radiation-induced myeloablation.Hepatology. 2000; 31: 235-240Crossref PubMed Scopus (896) Google Scholar, 6Krause DS Theise ND Collector MI Henegariu O Hwang S Gardner R Neutzel S Sharkis SJ Multi-organ, multi-lineage engraftment by a single bone marrow-derived stem cell.Cell. 2001; 105: 369-377Abstract Full Text Full Text PDF PubMed Scopus (2464) Google Scholar, 7Lagasse E Connors H Al-Dhalimy M Reitsma M Dohse M Osborne L Wang X Finegold M Weissman IL Grompe M Purified hematopoietic stem cells can differentiate into hepatocytes in vivo.Nat Med. 2000; 6: 1229-1234Crossref PubMed Scopus (2130) Google Scholar, 8Poulsom R Forbes SJ Hodivala-Dilke K Ryan E Wyles S Navaratnarasah S Jeffery R Hunt T Alison M Cook T Pusey C Wright NA Bone marrow contributes to renal parenchymal turnover and regeneration.Pathology. 2001; 195: 229-235Crossref Scopus (581) Google Scholar More recently, Y-chromosome marker studies have suggested that this phenomenon is also taking place after human HCT.9Alison MR Poulsom R Jeffery R Dhillon AP Quaglia A Jacob J Novelli M Prentice G Williamson J Wright NA Hepatocytes from non-hepatic adult stem cells.Nature. 2000; 406: 257Crossref PubMed Scopus (954) Google Scholar, 10Theise ND Nimmakayalu M Gardner R Illei PB Morgan G Teperman L Henegariu O Krause DS Liver from bone marrow in humans.Hepatology. 2000; 32: 11-16Crossref PubMed Scopus (1153) Google Scholar, 11Korbling M Katz RL Khanna A Ruifrok AC Rondon G Albitar M Champlin RE Estrov Z Hepatocytes and epithelial cells of donor origin in recipients of peripheral-blood stem cells.N Engl J Med
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