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Human cord blood- and bone marrow-derived CD34⁺ cells regenerate gastrointestinal epithelial cells

FUMIHIKO ISHIKAWA¹, MASAKI YASUKAWA^*, SHURO YOSHIDA, KEI-ICHIRO NAKAMURA, YOSHIHISA NAGATOSHI, TAKAAKI KANEMARU^¶, KAZUYA SHIMODA, SHINJI SHIMODA, TOSHIHIRO MIYAMOTO, JUN OKAMURA, LEONARD D. SHULTZ and MINE HARADA

Department of Medicine and Biosystemic Science, Kyushu University Graduate School of Medicine, Fukuoka, Japan;
^* First Department of Internal Medicine, Ehime University School of Medicine, Shigenobu, Japan;
Second Department of Anatomy, Kurume University, Kurume, Japan;
Section of Pediatrics, National Kyushu Cancer Center, Fukuoka, Japan;
^¶ Morphology Core, Kyushu University, Fukuoka, Japan; and
The Jackson Laboratory, Bar Harbor, Maine, USA

¹ Correspondence: Kyushu University Graduate School of Medicine, Department of Medicine and Biosystemic Science, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. E-mail: f_ishika{at}intmed1.med.kyushu-u.ac.jp

SPECIFIC AIMS

In the present study, we aimed to clarify if human cord blood and bone marrow contained progenitor cells that would generate gastrointestinal epithelial cells in clinical and experimental transplantation settings.

PRINCIPAL FINDINGS

1. Three-dimensional analysis excludes the possibility of cell overlay
We analyzed gastrointestinal specimens derived from sex-mismatched female recipients of cord blood (CB), bone marrow (BM), and mobilized peripheral blood (MPB) transplantations to determine whether each hematopoietic cell source contained progenitor cells to generate gastrointestinal epithelial cells. To rule out the possibility of cell overlay using FISH analysis, we performed detailed analysis on signals for a human Y chromosome probe using laser scanning confocal microscopy (Fig. 1 ). Fluorescent and Nomarksy images were obtained from intestinal specimens, which were subjected to FISH analysis using a Spectrum green-conjugated Y chromosome probe and nuclear staining with DAPI (Fig. 1A-E ). To clarify the localization of signals for chromosomes inside or outside of nuclei, we obtained 12 serial images from different levels at 0.4 µm intervals (Fig. 1F ). X-Z imaging reconstructed from serial X-Y images demonstrated that the FITC-labeled signal for the human Y chromosome was localized inside the nucleus (Fig. 1G ).

View larger version (116K): [in this window] [in a new window]   Figure 1. Localization of signal for Y chromosome in the nucleus. In sex-mismatched allogeneic transplantation, donor-derived intestinal epithelial cells were examined with FISH analyses. A) Low-magnified image showed that a coronal section of crypt contained a donor-derived cell (arrow) identified with the Spectrum green-conjugated Y chromosome probe. B) The existence of the male-derived cell was confirmed with Spectrum green-conjugated Y chromosome probe in a higher magnified view. C) Nomarsky imaging revealed the detailed morphology of the cells. D) Nuclei of the cells were stained with DAPI. E) Images of panels B–D merged. F) Signals for Y chromosome and nuclei staining are shown in twelve serial images obtained from different levels at 0.4 µm interval. G) The location of signal for human X chromosome was determined at the same level of nucleus by X-Z image.

2. Generation of gastrointestinal epithelial cells in human allogeneic recipients
To further identify the types of donor-derived cells, we performed FISH analysis and immunostaining on the same specimen, not using serial sections of the specimen. We obtained four types of information from each specimen; the nature of nuclei, contour of the cells, antigen expression, and origin of the cells. The FISH and immunofluorescence analyses for different portions of specimens demonstrated that the incidence of donor-derived epithelial cells was between 0.4% and 1.9%. In the present study using specimens derived from pediatric and juvenile recipients without experience of pregnancy, fetal-derived microchimerism can be excluded as a mechanism for the presence of Y chromosome⁺ cells. The three sources of stem cells, CB-, BM-, and MPB, contained stem/progenitor cells, which could give rise to epithelial cells in allogeneic recipients.

3. Human CB- and BM-derived CD34⁺ cells give rise to epithelial cells in NOD/SCID/ß2M^null mice
Next, we examined the capacity of "purified" human progenitor cells in an experimental transplantation setting. We transplanted 1 x 10⁵ human CB- or BM-derived CD34⁺ progenitor cells into newborn NOD/SCID/ß2M^null mice, which exhibited extremely low activity of NK cells as well as complete lack of mature B cells and T cells. At 3 months post-transplantation, high levels of engraftment by human CD45⁺ cells and multilineage reconstitution of human progenitor cells (CD34⁺), myeloid lineage cells (CD33⁺), B-lineage cells (CD19⁺), and T-lineage cells (CD3⁺) were observed in the xenogeneic host BM. High levels of hematopoietic engraftment by human cells is considered essential to support a relatively low incidence of generation of hematopoietic tissue-derived epithelial cells. Similar to the analysis of clinical specimens, the gastrointestinal tissues of recipient mice were analyzed for the presence of human cells by performing FISH analysis and immunofluorescent studies of the same specimens. The incidence of human CD34⁺ cell-derived epithelial cells in xenogeneic intestinal or gastric tissue was 0.23–0.58% in CB recipients and 0.15–0.30% in BM recipients.

4. The mechanism for the generation of human CB-derived intestinal epithelial cells
We examined the possibility of cell fusion as a mechanism for the generation of CB- or BM-derived epithelial cells. When we examined the presence of human cells in mesenteric lymphoid nodes, the vast majority of cells were dually positive for human X chromosome and human CD45 (Fig. 2 A), showing that human CD34⁺ progenitor cells could effectively reconstitute lymphoid tissues of the recipient intestine. As no cytokeratin⁺ epithelial cells were stained with anti-human CD45 antibody, we performed double FISH analyses using human and murine chromosome probes and immunostaining for cytokeratin on the same specimen. Nomarsky imaging demonstrated that a human CB-derived human X chromosome⁺ cell was identified in organized sequence of epithelial cells in villi (Fig. 2B ). In Fig. 2C , a human X chromosome⁺ cell was positively stained with anti-cytokeratin antibody, suggesting that the human cell was an epithelial cell. We tested whether human X chromosome⁺ epithelial cells with donor chromosomes were generated due to the fusion of donor-derived progenitors and recipient-derived epithelial cells. Although the majority of epithelial cells had murine centromeres, simultaneous FISH analyses using species-specific probes demonstrated that the nucleus of the human X chromosome⁺ cell was not labeled with the mouse centromere probe (Fig. 2D ), indicating that the human chromosome⁺ epithelial cell was not generated by fusion of human CD34⁺ cells and murine epithelial cells. X-Z imaging reconstructed from 10 slices of 0.3 µm serial X-Y images confirmed that the dot indicating the presence of human X chromosome was located inside the nucleus of the epithelial cell (Fig. 2E ) and not was an artifact due to cell overlay. Such human X chromosome⁺ cells were identified both along villi and at the bottom of crypt (Fig. 2F ), a candidate location of intestinal stem cells.

View larger version (68K): [in this window] [in a new window]   Figure 2. Human X chromosome-positive epithelial cell was not generated by the fusion between human cell and murine epithelial cell. Human CB-derived CD34+ cells were transplanted into newborn NOD/SCID/ß2Mnull recipients. At 3 months post-transplantation, in vivo generation of human intestinal epithelial and lymphoid cells was examined by laser confocal microscopy. A) Lymphoid tissue derived from a recipient intestine was subjected to immunostaining with anti-human CD45 antibody (green) and FISH analysis with human X chromosome probe (red). The majority of the cells in lymphoid tissue of the recipient mice were human hematopoietic cells as judged by the positivity of human X chromosome and human CD45. B–E) After FISH analyses and immunofluorescent studies were performed on the same specimen derived from recipient intestine, four-color (FITC, Spectrum orange, Cy5, DAPI) analyses were executed with laser scanning confocal microscopy along with Nomarsky imaging. B) Nomarsky image showed that human cell identified with anti-human X chromosome probe (red) was identified in organized sequence of villi. C) The cell with human X chromosome was confirmed as an epithelial cell by positive staining with Cy5-conjugated anti-cytokeratin antibody. D) The majority of the cells in the same specimen were murine cells, which were identified with FITC-conjugated mouse pan-centromeric probe. The cell identified with human X chromosome probe did not possess any mouse centromeres in the nucleus. Bar represents 10 µm. E) The hybridization signal for human X chromosome was located inside nucleus as judged by X-Z image reconstructed from serial X-Y images. (F) Such human cells were also identified at the bottom of crypt.

CONCLUSIONS AND SIGNIFICANCE

We have used clinical and experimental transplantation settings in order to examine the capacity of human hematopoietic progenitor cells to generate gastrointestinal epithelial cells in allogeneic and xenogeneic recipients.

In terms of technical issue, we successfully combined FISH analysis with immunofluorescent studies to identify donor chromosome⁺ cytokeratin⁺ cells. The simultaneous FISH and immunofluorescent studies of the same specimens, not using serial sections, enabled the identification of donor-derived epithelial cells accurately. One of the criticisms against previous FISH studies was that a tiny dot indicating the presence of donor chromosome could be localized at a different level of the nucleus due to cell overlay. Both in allogeneic and xenogeneic transplantations, X-Z images, reconstructed from serial X-Y images captured by laser scanning confocal microscopy, clearly demonstrated that the hybridization signal for human chromosome existed inside the nucleus, which supported the specificity of FISH analyses in the present study. Our analyses of pediatric and juvenile recipients demonstrated that Y chromosome⁺ epithelial cells of gastrointestinal tract were derived from transplanted hematopoietic tissue-derived cells, not reminiscent of fetus-derived cells.

Considering the limited availability of clinical specimens and the controversy regarding the generation of BM-derived epithelial cells in clinical cases, we developed experimental xenotransplantation assays, in which purified human progenitor cells could be analyzed for their capacity. We here identified that human CB- and BM-derived CD34⁺ cells were capable of generating epithelial cells in vivo. Although the capacities of CB and BM did not differ significantly in the present study, CB could be used as precious source for allogeneic and autologous progenitor cells as well as BM in the future regenerative medicine.

Molecular mechanism underlying stem cell plasticity has yet to be understood. Cell fusion between donor-derived stem cells and mature cells of recipient origin may account for seemingly donor stem cell-derived progeny. As hepatocytes and myocytes are known to fuse in physiological condition, the mechanism for the generation of CB- and BM-derived gastrointestinal epithelial cells needs to be clarified. Sex chromosome painting in the FISH analysis of clinical specimens cannot determine the possibility of cell fusion as thin sections do not include the whole chromosomes. Xenogeneic assay enabled us to evaluate the possibility of cell fusion by using human chromosome probe and mouse centromere probe on the same specimens. Double FISH analyses demonstrated that the cells positive for human chromosome and cytokeratin were not labeled with anti-mouse centromere probe. Altogether, it is concluded that fusion between stem cells and epithelial cells is not the only mechanism at least in intestine and that human purified progenitor cells can regenerate intestinal epithelial cells across allogeneic and xenogeneic histocompatibility barrier.

View larger version (26K): [in this window] [in a new window]   Figure 3. Schematic diagram.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2396fje;

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