The aim was to detect conjunctival epithelial cells with donor-derived genome after human hematopoietic stem-cell transplantation (HSCT). Conjunctival biopsies from allotransplanted females were stained for the Y-chromosome, cytokeratin and CD45 followed by a DAPI counterstain and CD68 staining and then evaluated by laser-scanning confocal microscopy and 3D analysis. In five patients, donor-derived epithelial cells identified as Y/DAPI/CK+/CD45−/CD68−/epithelial morphology could be found (57%) with a mean incidence of 5.4% (range 2.42%–7.94%). Our study demonstrates that in addition to the already published results on the liver, colon, lung, skin, and oral mucosa, epithelial cells with donor-derived genome also emerge in the conjunctiva after human HSCT. The biological significance and the underlying mechanism of these findings need further clarification.
HSCT has become a standard treatment of many hematological malignancies. The donor’s HSC engraft, proliferate, and finally reconstitute hematopoiesis in the recipient. The result is the creation of a biological chimera, which describes the presence of tissues of different genetic origin in the same organism; the hematopoietic cells are derived from the donor, whereas the other tissues (e.g., epithelium) are genetically derived from the patient recipient. This unphysiological formation of biological chimeras results in graft-versus-host disease (GVHD) (1) and as recently described in epithelial cells with donor-derived genotype (2). Despite the initial skepticism of the reported observations due to methodological limitations, more recent studies using strict criteria and examinations in isolated single cells have clearly shown that after allogeneic HSCT in humans, epithelial cells containing graft-derived genome may emerge (3–5). Moreover, genome analysis in the fingernails of transplant recipients confirmed the presence of donor-derived genome in tissues not containing blood contaminants (4). After HSCT in humans, donor-derived hepatocytes, cholangiocytes, and epithelial cells of gut, lung, skin, and mucosal tissues have been detected (3, 5–7). But detection of donor-derived epithelial cells varies between different organs and is suggested to be tissue dependent and related to the degree of tissue damage and rate of cell turnover (8–10). Conjunctival epithelium is characterized by a high cell turnover and conjunctival GVHD occurs frequently after HSCT, mainly when systemic immunosuppression is tapered. The aim of this study was to examine whether chimeric cells are also present in the human conjunctiva after HSCT.
Conjunctival tissue specimens were obtained from the temporal bulbar conjunctiva of seven female patients after human leukocyte antigen-matched, sex mismatched HSCT. Time of biopsy was 9 to 40 months after HSCT (mean 20 months). All patients had received GVHD prophylaxis, consistent with methotrexate+cyclosporine. Conjunctival biopsies were taken for diagnostic reasons, after informed consent, and were formalin fixed and paraffin embedded. In all patients, hematoxylin-eosin staining was performed to confirm GVHD (Fig. 1). Then, 3-μm sections were stained by a combination of FISH for the Y-chromosome (CEPY Spectrum Orange DNA probe and CEP Hybridization Buffer, Vysis, Germany), immunofluorescent stain for cytokeratin (Pancytokeratin Clone C-11, fluorescein isothiocyanate-conjugated, Sigma), and immunofluorescent stain for CD45 (leukocyte common antigen, Clone 2B11+PD7/26, Dako, M0701), followed by a goat anti-mouse antibody labeled with Alexa Fluor 647 (goat anti-mouse IgG DACO A-21237) and a 4’6-diamidino-2-phenylindole (DAPI) nuclear counterstain (Sigma, D-9542), as described (11). Z-stacks were obtained from Y/CK/CD45/DAPI-stained slides by laser-scanning confocal microscopy (TCS/SP2/AOBS, Leica-Microsystems, Germany) and evaluated in three-dimensional reconstructions using a commercial software (Autovisualize 5.5, AutoQuant Imaging, USA). Cell overlap was excluded by confocal scan. Epithelial cells containing donor-derived genomes were defined as Y+/CK+/CD45− and contained the Y(+)-signal within the DAPI-stained nucleus. A median of 400 nonoverlapping CK+ epithelial cells with distinct nuclei was counted per section and the amount of cells containing the Y-positive signal in the nucleus was noted. In addition, slides of each patient were stained for CD68 and CD8 as described before (6). Negative controls included omission of the Y-probe in FISH, use of isotype matched control antibodies (IgG1-fluorescein isothiocyanate, Immunotech, Marseille, France) for cytokeratin and CD45 staining, and staining of samples from female patients who received transplants from a female donor. Autofluorescence could be excluded. Positive controls consisted of samples obtained from transplanted male HSCT recipients.
Y-chromosome-positive epithelial cells were detected in five of seven female patients with a mean incidence of 5.4% Y+/CK+/CD45− cells and a range of 2.42% to 7.94% (Table 1). Particular care was taken to detect interspersed CD45-positive cells and tissue macrophages as possible sources for Y+ signals: first, all identified donor-derived epithelial cells were CD45-negative, expressed cytokeratin, exhibited a typical epithelial morphology, and were found exclusively in the epithelial layer (Fig. 2). Second, CD68-positive cells were present in the subepithelial tissue, but absent in the epithelial layers of all patients. CD8-positive cells were present in the subepithelial space and the basal epithelium (see Figure, Supplemental Digital Content 1, http://links.lww.com/A838).
Our work presents evidence for the presence of chimeric epithelial cells in human conjunctiva after HSCT. Six of seven patients had active conjunctival GVHD, identified by histology (hematoxylin-eosin staining) (Table 1). The rate of epithelial chimerism in conjunctiva (5.4%) is in the range described in oral mucosa (1.8%–12.7%) and colon crypts (0.18%) identified with the same methodology (6, 11, 12). It has been reported that FISH tends to overestimate the frequency of epithelial chimerism in thin sections (10). However, in combination with confocal microscopy and 3D analysis, FISH allows to exclude cell overlay, thus rendering an accurate estimate of the real frequency of chimerism. Moreover, we used slides of 3-μm thickness, reducing the possibility of cell overlay even further. Hallberg et al. identified donor cells in the conjunctiva of female patients after sex-mismatched HSCT. In contrast to our study, cells were CK−, Y-chromosome, but expressed smooth muscle actin and CD45, indicating both myofibroblast phenotype and hematopoietic origin (13). Recently, Aguilar et al. (14) could show that myofibroblasts are also present in normal conjunctiva. Chimeric cells found in our study were definitely CK+ and Y-chromosome and did not express CD45. Therefore, we believe that these cells are not myofibroblasts generated from hematopoietic cells but epithelial cells with donor characteristics.
Two unresolved questions remain: the biological significance and the underlying mechanism of these findings. Whether emergence of conjunctival epithelial cells with donor-derived genomes is an incidental by-product of transplantation without ancillary biological significance or whether these hold any clinical, pathologic, or therapeutic relevance remains to be answered by examination of a great number of transplanted patients. Regarding the underlying mechanisms, suggested theories include transdifferentiation of hematopoietic cells, generation of epithelial cells from unknown epithelial precursors or universal stem cells like “multipotent adult progenitor cell” (15), “very small embryonic like cell” (16), or fusion of donor hematopoietic cells with recipient epithelial cells (17). Although these mechanisms have been demonstrated in specific experimental systems, they have never been unambiguously shown to operate in the in vivo generation of epithelial cells containing donor-derived genomes. More recently, Ratajczak et al. (16, 18) proposed microvesicle-mediated horizontal mRNA transfer from donor to recipient cells as a potential underlying mechanism. According to this scenario, HSCs engraft in the tissues, incorporate microvesicles containing tissue-specific mRNA released from the tissue in case of injury, and alter this way their phenotype leading to the observed epithelial chimerism. A similar mechanism, which needs further experimental proof, is horizontal gene transfer (19). Horizontal gene transfer is well described in prokaryotic organisms as a mechanism for functional and phenotypic change to adapt to different environmental condition. In this case, genetic material released from the hematopoietic-derived donor cell will be transferred, incorporated into the nucleus, and eventually expressed by the recipient epithelial cell, ultimately leading to the emergence of epithelial cells with donor-derived genomes.
Although allogeneic HSCT has been part of clinical practice for more than 30 years, development of epithelial cells with donor-derived genotype is only one recently recognized phenomenon caused by the coexistence of two genetically distinct populations in the transplant recipient. The ultimate mechanisms, the clinical consequences, and the extent of the generation of epithelial cells with donor-derived genome after HSCT are still under investigation. By using strict criteria and a rigorous analytical method, we clearly show that besides the already published results on the liver, colon, lung, skin, and oral mucosa, epithelial cells with donor-derived genome also emerge in the conjunctiva after human HSCT.
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Ocular surface; Conjunctiva; Stem cell; Epithelial chimerism
Supplemental Digital Content
© 2009 Lippincott Williams & Wilkins, Inc.