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Original Article

Hepatocytes and Epithelial Cells of Donor Origin in Recipients of Peripheral-Blood Stem Cells

Martin Körbling, M.D., Ruth L. Katz, M.D., Abha Khanna, M.A., Arnout C. Ruifrok, Ph.D., Gabriela Rondon, M.D., Maher Albitar, M.D., Richard E. Champlin, M.D., and Zeev Estrov, M.D.

N Engl J Med 2002; 346:738-746March 7, 2002

Abstract

Background

Bone marrow contains stem cells with the potential to differentiate into mature cells of various organs. We determined whether circulating stem cells have a similar potential.

Methods

Biopsy specimens from the liver, gastrointestinal tract, and skin were obtained from 12 patients who had undergone transplantation of hematopoietic stem cells from peripheral blood (11 patients) or bone marrow (1 patient). Six female patients had received transplants from a male donor. Five had received a sex-matched transplant, and one had received an autologous transplant. Hematopoietic stem-cell engraftment was verified by cytogenetic analysis or restriction-fragment–length polymorphism analysis. The biopsies were studied for the presence of donor-derived epithelial cells or hepatocytes with the use of fluorescence in situ hybridization of interphase nuclei and immunohistochemical staining for cytokeratin, CD45 (leukocyte common antigen), and a hepatocyte-specific antigen.

Results

All six recipients of sex-mismatched transplants showed evidence of complete hematopoietic donor chimerism. XY-positive epithelial cells or hepatocytes accounted for 0 to 7 percent of the cells in histologic sections of the biopsy specimens. These cells were detected in liver tissue as early as day 13 and in skin tissue as late as day 354 after the transplantation of peripheral-blood stem cells. The presence of donor cells in the biopsy specimens did not seem to depend on the intensity of tissue damage induced by graft-versus-host disease.

Conclusions

Circulating stem cells can differentiate into mature hepatocytes and epithelial cells of the skin and gastrointestinal tract.

Media in This Article

Figure 3Donor-Derived Mucosal Cells in the Gastric Cardia in a Female Recipient (Patient 7) of Peripheral-Blood Stem Cells from a Male Donor.
Figure 2Donor-Derived Hepatocytes in the Liver of a Female Recipient (Patient 7) of Peripheral-Blood Stem Cells from a Male Donor.
Article

Pluripotent bone marrow stem cells have the capacity for self-renewal and can differentiate into hematopoietic or mesenchymal1 cell lineages. Studies in laboratory animals and humans indicate that bone marrow stem cells can give rise to hepatic oval cells, hepatocytes, cholangiocytes,2-4 skeletal-muscle cells,5,6 astrocytes, and neurons.7-9 To investigate whether such progenitor cells circulate in the blood, we studied biopsy specimens of skin, liver, and gastrointestinal tract from recipients of peripheral-blood stem cells from HLA-matched, sex-mismatched siblings for the presence of donor-derived epithelial cells and hepatocytes.

Methods

Characteristics of the Donors and Recipients

Eleven patients received high-dose chemotherapy alone or chemotherapy combined with radiotherapy, followed by a transplant of allogeneic peripheral-blood stem cells (in 10 patients) or autologous peripheral-blood stem cells (in 1 patient) for the treatment of hematologic cancers or breast cancer. One patient underwent allogeneic bone marrow transplantation. Both myeloablative and nonmyeloablative10 regimens were used before transplantation. All allogeneic grafts were derived from HLA-matched siblings. The peripheral-blood stem cells were obtained by apheresis after the donor had been treated for four days with recombinant human granulocyte colony-stimulating factor at a dose of 12 μg per kilogram of body weight per day. The total number of CD34+ cells transplanted ranged from 3.9×106 per kilogram of the recipient's body weight to 14.8×106 per kilogram.

Six women received stem cells from a brother; five patients received sex-matched stem cells, and one woman received autologous stem cells. The latter six patients served as controls. Hematopoietic stem-cell engraftment was verified by cytogenetic analysis or restriction-fragment–length polymorphism (RFLP) analysis.11 In three control patients, engraftment was documented on the basis of the recovery of peripheral-blood cells alone.

Tissue Specimens

After stem-cell transplantation, tissue specimens were obtained by a needle or punch biopsy that was performed for diagnostic purposes. All 12 patients gave informed consent for biopsies to be performed for diagnostic purposes. By the time our study began, all patients had died. The retrospective analysis of biopsy specimens was approved by the internal review board of the M.D. Anderson Cancer Center. All biopsies were performed for the purpose of establishing the diagnosis of graft-versus-host disease.

A total of five consecutive sections were obtained from each biopsy specimen. Each section was 4 μm thick, which is approximately half the thickness of a nucleus, with neighboring sections cut 4 μm apart. The sections closest to the center section that was used for fluorescence in situ hybridization were those stained for cytokeratin and CD45 (leukocyte common antigen), followed by those stained with hematoxylin and eosin and with a hepatocyte-specific antigen. This procedure allowed matching fields to be as close to each other as possible.

Immunohistochemistry

Pretreatment of Slides

After removal of paraffin with xylene, tissue sections were rehydrated with graded alcohols (100 percent, 90 percent, and 70 percent ethanol in distilled water) and washed with water and phosphate-buffered saline. Endogenous peroxidase activity was blocked by the application of 0.3 percent hydrogen peroxide in methanol for 15 minutes at room temperature, and the slides were washed in phosphate-buffered saline again. Tissues were then digested with 0.2 percent ficin (Sigma, St. Louis) in distilled water for 15 minutes at room temperature and washed in phosphate-buffered saline.

Staining for Cytokeratin

For staining for cytokeratin, antigen retrieval was performed by incubating the tissue for eight minutes in 0.01 M citrate buffer in a microwave oven. Blocking serum (bovine serum albumin) was applied to the slides for 30 minutes at room temperature, and the slides were then incubated for 60 minutes at room temperature with monoclonal mouse antihuman cytokeratin antibodies (CAM 5.2 [25 μg per milliliter; Becton Dickinson, San Jose, Calif.] at a dilution of 1:5 plus AE1/AE3 [1 mg per milliliter; Boehringer Mannheim, Indianapolis] at a dilution of 1:480).

Staining for CD45

For staining for CD45, antigen retrieval was performed by incubating the tissue for 45 minutes in TRIS-EDTA buffer in a steamer. Blocking serum (bovine serum albumin) was applied to the slides for 30 minutes at room temperature, and the slides were then incubated with monoclonal mouse antibodies against CD45 (clones PD7/26 and 2B11, Dako, Carpinteria, Calif.) at a dilution of 1:300 for 45 minutes. To detect the antigen–antibody reaction, a streptavidin–biotin detection system (Super Sensitive Immunodetection System, Biogenex, San Ramon, Calif.) was used according to the manufacturer's instructions. Sections from tonsils and peripheral-blood smears were used as positive controls.

Staining for Hepatocytes

For staining for hepatocytes, antigen retrieval was performed by incubating the tissue for 45 minutes in TRIS-EDTA buffer in the steamer. Blocking serum (bovine serum albumin) was applied to the slides for 30 minutes at room temperature, and then slides were incubated with a monoclonal mouse IgG antihuman hepatocyte antibody (clone OCH1E5, Dako) at a dilution of 1:50 for 60 minutes. To detect the antigen–antibody reaction, we used a streptavidin–biotin detection system (Super Sensitive Immunodetection System, Biogenex) according to the manufacturer's instructions.

Fluorescence in Situ Hybridization

Paraffin-embedded slides were deparaffinized by baking in an oven overnight at 56°C and then clearing in xylene three times, for 10 minutes each, for a total of 30 minutes; they were then dehydrated and air-dried. Slides were pretreated in 0.2 N hydrochloric acid for 20 minutes, washed with water, and rinsed in 2× saline sodium citrate (SSC) (1× SSC is 0.15 M sodium chloride and 0.015 M sodium citrate) for 3 minutes at room temperature. Slides were then incubated in 1 M sodium thiocyanate in distilled water at 80°C for 30 minutes, washed with water, washed with 2× SSC for 3 minutes, and air-dried. Tissue was digested with 1.5 μg of proteinase K (Sigma) per milliliter in 0.2 N hydrochloric acid, pH 2.0, at 37°C for 1 hour, washed with water, and then rinsed in 2× SSC for 3 minutes, air-dried, and fixed in Carnoy's solution (methanol and acetic acid in a 3:1 ratio) for 10 minutes. Slides were then denatured with 70 percent formamide in 2× SSC at 73°C for five minutes and rinsed with 70 percent ethanol for three minutes, dehydrated, and air-dried. The mixture of probes for the X and Y chromosomes (Vysis, Downers Grove, Ill.) was denatured at 74°C for five minutes and applied to the denatured tissue. The slides were covered with a coverslip, sealed with rubber cement, and incubated in a humid chamber overnight at 37°C for hybridization. After 16 hours of hybridization, slides were washed in 0.4× SSC containing 0.3 percent Nonidet P-40 for two minutes at 73°C, transferred to 2× SSC containing 0.1 percent Nonidet P-40 for one minute at room temperature, and drained. Slides were then counterstained with 10 μl of 4',6-diamidine-2-phenylidole dihydrochloride (DAPI, Boehringer Mannheim) at a concentration of 14 μg per milliliter of VectaShield mounting medium (Vector Laboratories, Burlingame, Calif.), and a coverslip was applied.

Quantification of XY-Positive, Donor-Derived Nonlymphohematopoietic Cells

The slides were scanned at a magnification of 100 under a fluorescence microscope (Leica, Wetzlar, Germany) equipped with an epi-illumination system, a 100-W mercury lamp, and a set of filters, including DAPI single-bandpass (DAPI counterstain), Spectrum Orange single-bandpass, Spectrum Green single-bandpass, and Red/Green dual-bandpass filters (all from Vysis). A total of 200 nonoverlapping cells and nuclei with distinct cells were counted, and the Y-positive (red) and X-positive (green) signals were identified. The percentage of cells that were XY-positive or XX-positive was less than 100 percent because of the truncation of nuclei during sectioning and incomplete hybridization. The stringent criteria used in counting positive signals led to an underestimation of the percentages of XX- or XY-positive cells in cases of female-to-female or male-to-male transplantation. Fields were matched to the corresponding fields in photomicrographs of the variously stained slides according to the location and architecture of the tissue on the slide. Matching microscopic fields were either 4 μm apart (in the slides stained with antibodies against cytokeratin or CD45) or 8 μm apart (in the slides stained with hematoxylin and eosin or the anti-hepatocyte antibody). The slides stained with hematoxylin and eosin and with antibody against CD45 were carefully evaluated to exclude the presence of lymphocytes, monocytes, and granulocytes, and XY-positive epithelial cells were identified with the use of only those cells that could reliably be classified on the basis of their staining properties.

Staining for Cytokeratin and Fluorescence in Situ Hybridization

Slides were prepared for staining for cytokeratin as described above. They were then washed in 1× phosphate-buffered saline for 5 minutes, and Texas Red–conjugated donkey antimouse IgG (Jackson ImmunoResearch Laboratories, West Grove, Pa.) at a dilution of 1:200 was applied for 60 minutes. After they had been washed in phosphate-buffered saline for five minutes, the slides were counterstained with 10 μl of DAPI at a concentration of 14 μg per milliliter of VectaShield antifade solution (Vector Laboratories). After they were stained for cytokeratin, the sections were systematically scanned and photographed at a magnification of 63 with the use of a calibrated and automated motorized stage. Next, the slides were washed in phosphate-buffered saline for five minutes and prepared for fluorescence in situ hybridization as outlined above. The slides were then scanned for XY-positive cells; pictures were taken at a magnification of 63 and were matched with the stored cytokeratin images.

Results

Characteristics of Patients and Transplantations

The characteristics of the donors and recipients, the type of regimen used before transplantation, and the quantity of CD34+ cells transfused are presented in Table 1Table 1Characteristics of the Donors and the Recipients..

Hematopoietic Chimerism after Allogeneic Stem-Cell Transplantation

Complete hematopoietic chimerism was demonstrated by RFLP analysis in four of the six recipients of sex-mismatched stem cells (Patients 7, 8, 10, and 12) and by cytogenetic analysis of bone marrow cells in all six (Table 2Table 2Incidence of Donor Chimerism and Biopsy Reports.). In three of the six control patients, donor chimerism was documented by RFLP analysis.

Donor-Derived Epithelial Cells and Hepatocytes in Recipients of Sex-Mismatched Stem Cells

We studied biopsy specimens of skin, liver, and gastrointestinal tract for the presence of donor-derived epithelial cells and hepatocytes. Whereas a DNA probe specific for the centromeres detected X and Y chromosomes in 35 to 75 percent of cells in biopsy specimens from the three male patients who received an allograft from a male donor (Patients 4, 5, and 6) (Table 3Table 3Percentage of XY-Positive Cells in Various Tissue Biopsy Specimens from Six Control Recipients of Sex-Matched Stem-Cell Allografts or Autograft and Six Recipients of Sex-Mismatched Stem-Cell Allografts.), they did not detect XY-positive cells in any biopsy specimens from women who received a stem-cell transplant from a sister (Patients 1 and 2) or the woman who received her own cells (Patient 3). By contrast, XY-positive cells were present in biopsy specimens of the skin, liver, or gastrointestinal tract from the five female recipients of peripheral-blood stem cells from male donors (Patients 7, 8, 10, 11, and 12) and in the female recipient of a bone marrow allograft from her brother (Patient 9).

In epidermal tissue of the skin, donor-derived cells were located in the deep layer of Malpighi (the stratum spinosum of the stratum germinativum), close to the dermal–epidermal junction and the stratum granulosum (Figure 1Figure 1Donor-Derived Epidermal Cells in the Skin of a Female Recipient (Patient 10) of Peripheral-Blood Stem Cells from a Male Donor.). In the liver, XY-positive hepatocytes were distinguished by large, round nuclei and abundant granular cytoplasm (Figure 2Figure 2Donor-Derived Hepatocytes in the Liver of a Female Recipient (Patient 7) of Peripheral-Blood Stem Cells from a Male Donor.). In the glandular epithelium of the gastric cardia, cells containing the Y chromosome were found in the foveolae or tubular pits of the superficial glandular layer, which is composed of mucus-containing cells lining the foveolae (Figure 3Figure 3Donor-Derived Mucosal Cells in the Gastric Cardia in a Female Recipient (Patient 7) of Peripheral-Blood Stem Cells from a Male Donor.). The organ specificity of these cells was indicated by their location, staining for cytokeratins and hepatocytes (in the liver), and the absence of CD45.

We also analyzed slides from biopsy specimens of all three organs after they had been stained with anti-cytokeratin antibodies and examined by fluorescence in situ hybridization with probes for the X and Y chromosomes. XY-positive signals in cytokeratin-positive cells would indicate the epithelial character of donor-derived cells. As demonstrated in epidermal cells in skin (Figure 1E and Figure 1F), hepatocytes (Figure 2E and Figure 2F) and mucosal cells of the gastrointestinal tract (Figure 3E and Figure 3F), XY-positive signals were detectable in cytokeratin-positive cells.

The frequency of XY-positive cells in biopsy specimens from female recipients of grafts from male donors ranged from 0 to 7 percent (Table 3). XY-positive cells were detected in liver tissue as early as 13 days after transplantation (in Patient 8) and were seen in skin tissue 354 days after transplantation of peripheral-blood stem cells (in Patient 10) and 867 days after transplantation of bone marrow (in Patient 9). The biopsy reports and the patients' clinical status at the time of biopsy did not suggest that donor-cell engraftment was more likely in tissues injured by graft-versus-host disease than in other tissues (as shown by the results for Patients 7, 10, 11, and 12 in Table 2).

Discussion

Circulating blood is known to contain stem cells that can completely restore hematopoiesis after ablation of the bone marrow.12,13 Recently, mesenchymal stem cells with a capacity for self-renewal and the potential to differentiate into bone, cartilage, fat, tendon, muscle, or marrow stroma have been identified in human bone marrow.1,14 Whether such stem cells circulate in the blood is unsettled.15-17 A stem cell in rat bone marrow has been found to differentiate into the epithelial lineage that generates hepatic oval cells,2 and in mice with a metabolic defect that impairs liver function, the infusion of purified hematopoietic stem cells can restore both hematopoiesis and liver function.18 Progenitors in mouse bone marrow have also been shown to be myogenic and can induce muscle regeneration.5,6

The existence of stem cells with multiple differentiating capabilities19 was conclusively demonstrated by Krause et al.,20 who showed that a single bone marrow stem cell not only can restore hematopoiesis in mice that have received otherwise lethal doses of radiation but also can differentiate into mature epithelial cells of the skin, lungs, and gastrointestinal tract. Moreover, human progenitor cells transplanted into fetal sheep have been reported to differentiate into hematopoietic cells and hepatocytes.21 There is also evidence that human kidney,22 liver, and muscle cells23 can transform into blood-forming cells. Moreover, two groups have reported the presence of donor-derived hepatocytes and cholangiocytes in recipients of sex-mismatched bone marrow transplants.3,4

Our findings indicate that human blood contains stem cells that can differentiate into cells of the liver, gastrointestinal tract, and skin. The origin of these stem cells and the way in which they generate hepatocytes and epithelial cells are unknown. It is possible that multiple lineage-restricted stem cells in the circulating blood can differentiate independently into their corresponding mature tissue. Alternatively, primitive adult multipotent stem cells may give rise to differentiated, lineage-restricted stem cells that can generate mature cells. It is also possible that stem cells that are committed to differentiation primarily along a particular pathway (e.g., hematopoiesis) can switch to another lineage under the influence of signals of the local microenvironment. Beltrami and coworkers24 have postulated that circulating stem cells differentiate into dividing myocytes that repair necrotic myocardium after infarction in humans. It is also conceivable that, in addition to mobilizing hematopoietic stem cells, recombinant human granulocyte colony-stimulating factor mobilizes clonogenic cells of epithelial origin into the peripheral blood.25

Technically, our studies of thin tissue sections are not infallible. Nonhematopoietic tissue may harbor a few lymphohematopoietic cells that standard histologic staining techniques fail to detect. In our study, we used a restricted number of consecutive tissue sections and used stringent criteria in the enumeration of XY-positive cells. Furthermore, to ensure that the X- and Y-chromosome signals on fluorescence in situ hybridization were indeed detected in cytokeratin-positive cells, we used sequential staining of the same tissue sections. This procedure, however, exposed the tissue to rough conditions, which may have led us to underestimate the numbers of donor-derived cells in tissue sections.

Tissue damage caused by radiation, chemotherapy, or graft-versus-host disease, among other causes, is believed to be responsible for the homing of peripheral-blood stem cells and their differentiation into various solid-organ–specific tissues.19 Our results indicate a rather uniform pattern of engraftment of donor-derived hepatocytes and epithelial cells irrespective of the presence or absence of tissue damage caused by graft-versus-host disease. In conclusion, our findings suggest the existence of a population of circulating stem cells with the capacity to differentiate into epithelial cells and hepatocytes. The physiologic role of these cells is currently unknown.

We are indebted to Drs. S. Giralt, I. Khouri, K. vanBesien, R. Mehra, D. Przepiorka, J. Gajewski, and D. Claxton of the University of Texas M.D. Anderson Cancer Center Bone Marrow Transplant Service for their clinical contributions; to Drs. H. Zang and F. Jiang for their technical expertise; and to Ms. K. Suilleabhain for editing the manuscript.

Source Information

From the Departments of Blood and Marrow Transplantation (M.K., G.R., R.E.C.), Pathology (R.L.K., A.K., A.C.R.), Hematopathology (M.A.), and Bioimmunotherapy (Z.E.), University of Texas M.D. Anderson Cancer Center, Houston.

Address reprint requests to Dr. Körbling at the University of Texas M.D. Anderson Cancer Center, Department of Blood and Marrow Transplantation, Box 423, 1515 Holcombe Blvd., Houston, TX 77030, or at .

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