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Increase of Bone Marrow–Derived Secretory Lineage Epithelial Cells During Regeneration in the Human Intestine

2005; Elsevier BV; Volume: 128; Issue: 7 Linguagem: Inglês

10.1053/j.gastro.2005.03.085

1528-0012

Tomoko Matsumoto, Ryuichi Okamoto, Tomoharu Yajima, Takehiko Mori, Shinichiro Okamoto, Yasuo Ikeda, Makio Mukai, Motomi Yamazaki, Shigeru Oshima, Kiichiro Tsuchiya, Tetsuya Nakamura, Takanori Kanai∥, Hideyuki Okano, Johji Inazawa, Toshifumi Hibi∥, Mamoru Watanabe,

Liver physiology and pathology

Background & Aims: We have previously reported that bone marrow (BM)-derived cells contribute to the regeneration of the human intestinal epithelium. To analyze further how these cells arise, proliferate, and differentiate as epithelial cells, histologic analysis was conducted using endoscopic specimens. Methods: Thirty biopsy specimens from 14 female, sex-mismatched BM-transplantation recipients were examined. BM-derived cells were identified by fluorescent in situ hybridization (FISH) for the Y chromosome and immunohistochemistry. Multicolor FISH was used to exclude cell fusion. These cells were further analyzed for various differentiation or proliferation markers. Results: No evidence of cell fusion was detected. BM-derived cells did not distribute within the crypt as stem cells and rarely expressed Musashi-1. However, BM-derived epithelial cells frequently expressed Ki-67, and some of these cells appeared as pairs of adjacent cells. These cells also expressed markers of all 4 lineages of terminally differentiated cells. During regeneration following graft-vs-host disease, the number of BM-derived cells was substantially increased within Ki-67–positive cells. Interestingly, the number of cells expressing markers for secretory lineage cells was significantly increased within BM-derived cells. This change was unique for BM-derived cells, resulting in a significantly increased proportion of BM-derived cells among secretory lineage cells. Conclusions: BM-derived epithelial cells arise via a mechanism other than cell fusion and rarely give rise to stem cells. However, a small proportion of these cells express proliferation markers, and a majority reside as terminally differentiated cells. During regeneration BM-derived cells increase as secretory lineage cells, thereby contributing to restore epithelial functions. Background & Aims: We have previously reported that bone marrow (BM)-derived cells contribute to the regeneration of the human intestinal epithelium. To analyze further how these cells arise, proliferate, and differentiate as epithelial cells, histologic analysis was conducted using endoscopic specimens. Methods: Thirty biopsy specimens from 14 female, sex-mismatched BM-transplantation recipients were examined. BM-derived cells were identified by fluorescent in situ hybridization (FISH) for the Y chromosome and immunohistochemistry. Multicolor FISH was used to exclude cell fusion. These cells were further analyzed for various differentiation or proliferation markers. Results: No evidence of cell fusion was detected. BM-derived cells did not distribute within the crypt as stem cells and rarely expressed Musashi-1. However, BM-derived epithelial cells frequently expressed Ki-67, and some of these cells appeared as pairs of adjacent cells. These cells also expressed markers of all 4 lineages of terminally differentiated cells. During regeneration following graft-vs-host disease, the number of BM-derived cells was substantially increased within Ki-67–positive cells. Interestingly, the number of cells expressing markers for secretory lineage cells was significantly increased within BM-derived cells. This change was unique for BM-derived cells, resulting in a significantly increased proportion of BM-derived cells among secretory lineage cells. Conclusions: BM-derived epithelial cells arise via a mechanism other than cell fusion and rarely give rise to stem cells. However, a small proportion of these cells express proliferation markers, and a majority reside as terminally differentiated cells. During regeneration BM-derived cells increase as secretory lineage cells, thereby contributing to restore epithelial functions. The gastrointestinal (GI) epithelial cells arise from the intestinal stem cells residing in the lower part of the crypt. The intestinal stem cell provides daughter cells, which proliferate and in turn give rise to the 4 main lineages of terminally differentiated cells, namely, absorptive cells, goblet cells, enteroendocrine cells, and Paneth cells.1Cheng H. Leblond C.P. Origin, differentiation and renewal of the four main epithelial cell types in the mouse small intestine. V. Unitarian theory of the origin of the four epithelial cell types.Am J Anat. 1974; 141: 537-561Crossref PubMed Scopus (1177) Google Scholar, 2Marshman E. Booth C. Potten C.S. 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Stem cells the intestinal stem cell as a paradigm.Carcinogenesis. 2000; 21: 469-476Crossref PubMed Scopus (279) Google Scholar Recent studies have drawn attention to the importance of Wnt signaling in the regulation of the differentiation of immature intestinal epithelial cells.5Pinto D. Gregorieff A. Begthel H. Clevers H. Canonical Wnt signals are essential for homeostasis of the intestinal epithelium.Genes Dev. 2003; 17: 1709-1713Crossref PubMed Scopus (833) Google Scholar, 6Okamoto R. Watanabe M. Molecular and clinical basis for the regeneration of human gastrointestinal epithelia.J Gastroenterol. 2004; 39: 1-6Crossref PubMed Scopus (76) Google Scholar Also, a stepwise differentiation model of intestinal epithelial cells has been proposed, in which differentiation is regulated by a series of transcription factors downstream of Notch signaling.7Yang Q. Bermingham N.A. Finegold M.J. Zoghbi H.Y. Requirement of Math1 for secretory cell lineage commitment in the mouse intestine.Science. 2001; 294: 2155-2158Crossref PubMed Scopus (768) Google Scholar, 8van Den Brink G.R. de Santa Barbara P. Roberts D.J. Development Epithelial cell differentiation—a Mather of choice.Science. 2001; 294: 2115-2116Crossref PubMed Scopus (47) Google Scholar In this model, goblet cells, enteroendocrine cells, and Paneth cells arise from a shared progenitor cell expressing Math1 and are categorized as secretory lineage cells. In contrast, absorptive cells arise from a distinct progenitor cell expressing Hes1. In the previous studies, we demonstrated that bone marrow (BM)-derived cells contribute to the regeneration of damaged intestinal epithelium as epithelial cells.9Okamoto 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 (351) Google Scholar, 10Okamoto R. Watanabe M. Prospects for regeneration of gastrointestinal epithelia using bone-marrow cells.Trends Mol Med. 2003; 9: 286-290Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar This suggested that BM-derived cells could be a potential source for epithelial tissue regeneration. However, little is known about how these cells arise or whether they could proliferate and differentiate into functional, tissue-specific, terminally differentiated cells. Several studies suggest cell fusion between BM cells and tissue-specific cells as one mechanism by which BM-derived nonhematopoietic cells arise,11Ying Q.L. Nichols J. Evans E.P. Smith A.G. Changing potency by spontaneous fusion.Nature. 2002; 416: 545-548Crossref PubMed Scopus (1382) Google Scholar, 12Terada N. Hamazaki T. Oka M. Hoki M. Mastalerz D.M. 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Fusion of bone-marrow-derived cells with Purkinje neurons, cardiomyocytes and hepatocytes.Nature. 2003; 425: 968-973Crossref PubMed Scopus (1455) Google Scholar whereas other studies suggest other mechanisms, including transdifferentiation of BM cells into tissue-specific stem cells.16Tran S.D. Pillemer S.R. Dutra A. Barrett A.J. Brownstein M.J. Key S. Pak E. Leakan R.A. Kingman A. Yamada K.M. Baum B.J. Mezey E. Differentiation of human bone marrow-derived cells into buccal epithelial cells in vivo a molecular analytical study.Lancet. 2003; 361: 1084-1088Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 17Ianus A. Holz G.G. Theise N.D. Hussain M.A. In vivo derivation of glucose-competent pancreatic endocrine cells from bone marrow without evidence of cell fusion.J Clin Invest. 2003; 111: 843-850Crossref PubMed Scopus (699) Google Scholar, 18Ferrari G. Cusella-De Angelis G. Coletta M. Paolucci E. Stornaiuolo A. Cossu G. Mavilio F. 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Bone marrow-derived progenitor cells are important for lung repair after lipopolysaccharide-induced lung injury.J Immunol. 2004; 172: 1266-1272PubMed Google Scholar suggesting that BM-derived cells could possibly express a tissue-specific function within the organ of residence. In the present study, we demonstrate that BM-derived epithelial cells observed in the human intestine arise via a mechanism other than cell fusion and rarely give rise to intestinal stem cells. Our results show that a small number of these cells express markers of proliferation, but a majority express markers of functional, terminally differentiated epithelial cells within the human intestinal epithelium. During regeneration following epithelial damage, BM-derived epithelial cells increased as secretory lineage cells, thereby supporting both the regeneration and the essential functions of the intestinal epithelium. These results not only provide further support for the use of BM-derived cells to regenerate human intestinal epithelium but also suggest the existence of a unique regulatory system exclusive for BM-derived cells, which changes their differentiation pattern at the site of intestinal inflammation. We studied total of 14 female recipients who had received sex-mismatched bone marrow transplantation (BMT). All patients were subjected to allogenic BMT for the treatment of severe hematologic disorders: acute myeloblastic leukemia, aplastic anemia, and acute lymphoblastic leukemia. Three patients developed GI inflammation because of graft-vs-host disease (GVHD). Control of Y-fluorescent in situ hybridization (FISH) staining was provided from 2 males and 2 females (not undergoing transplantation). The details of the patients are summarized in Table 1.Table 1Characteristics of PatientsCaseReason for TransplantationaALL, acute lymphoblastic leukemia; MDS, myelodysplastic syndromes; AML, acute myelogenous leukemia; NHL, non-Hodgkin lymphoma; MM, multiple myeloma.Time from BMT to sampling (days)Location of samplesbE, esophagus; S, stomach; D, duodenum; C, colon.Histologic diagnosis1ALL26E,S, DAcute GVHD51E,S, DAcute GVHD77E,S, DAcute GVHD2MDS26E,S, Dn.p161E,S, DChronic GVHD381Cn.p3Aplastic anemia45E,S, Dn.p182E,S, Dn.p288E,S, Dn.p4AML32E,S, Dn.p5AML21E,S, Dn.p6ALL40E,S, DGastric polyp7AML30E,S, Dn.p8NHL33E,S, Dn.p9AML39E,S, Dn.p10AML23E,S, Dn.p11AML39E,S, Dn.p12MDS27E,S, DAcute GVHD96S, DAcute GVHD13MM27E,S, DEsophagitis63E,S, DEsophageal ulcer77E,S, DEsophageal granulation14MDS28E,S, Dn.p.n.p., no particular finding.a ALL, acute lymphoblastic leukemia; MDS, myelodysplastic syndromes; AML, acute myelogenous leukemia; NHL, non-Hodgkin lymphoma; MM, multiple myeloma.b E, esophagus; S, stomach; D, duodenum; C, colon. Open table in a new tab n.p., no particular finding. All samples were taken at the Keio University Hospital. We obtained written informed consent from each patient in the formal style after explaining the nature and possible consequences of the studies. The ethics committees of Keio University and Tokyo Medical and Dental University both approved this study. We took endoscopic specimens of the GI tract because patients developed clinical symptoms such as nausea, vomiting, abdominal pain, or diarrhea and were suspected of chronic GVHD, acute GVHD, or other intestinal inflammation. A total of 30 biopsy specimens obtained from 14 female patients were examined and analyzed retrospectively. Details of the specimens examined are also summarized in Table 1. FISH for chromosomes 1, 18, and Y using formalin-fixed, paraffin-embedded biopsy specimens has already been described.9Okamoto 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 (351) Google Scholar Paraffin-embedded tissue samples were cut into either 6-μm- or 3-μm-thick serial sections and subjected to FISH analysis. The specific DNA probes used were as follows: clone pUC 1.7724Cooke H.J. Hindley J. Cloning of human satellite III DNA different components are on different chromosomes.Nucleic Acids Res. 1979; 6: 3177-3197Crossref PubMed Scopus (466) Google Scholar for chromosome 1 (purchased from HSRBB, Osaka, Japan), clone L 1.8425Devilee P. Slagboom P. Cornelisse C.J. Pearson P.L. Sequence heterogeneity within the human alphoid repetitive DNA family.Nucleic Acids Res. 1986; 14: 2059-2073Crossref PubMed Scopus (71) Google Scholar for chromosome 18, and clone pHY1026Nakahori Y. Mitani K. Yamada M. Nakagome Y. A human Y-chromosome specific repeated DNA family (DYZ1) consists of a tandem array of pentanucleotides.Nucleic Acids Res. 1986; 14: 7569-7580Crossref PubMed Scopus (180) Google Scholar for the Y chromosome. The probes were labeled by nick translation either with biotin-dUTP or DIG-dUTP (Roche Diagnostics, Tokyo, Japan) and detected by incubation with either avidin-FITC or anti-DIG-rhodamine (Roche Diagnostics, Indianapolis, IN). FISH images were captured using a Nikon epifluorescence microscope (Eclipse 800, Tokyo, Japan) coupled to a Sensys CCD camera and analyzed with QUIPS image software (Vysis, Downers Grove, IL). One section per course of endoscopy, containing 1–3 biopsy specimens of the GI tract, was used for the subsequent FISH analysis. Formalin-fixed, paraffin-embedded biopsy specimens were used unless otherwise mentioned. For anti-Musashi-1 antibody (Ab), cryosections prefixed in 4% paraformaldehyde were used. Immunohistochemistry using anti-human cytokeratin Ab (AE1/AE3, DAKO-USA, Carpinteria, CA), anti-human LCA (CD45) Ab (DAKO, Glostrup, Denmark), anti-human chromgranin A Ab (DAKO), anti-human CD10 Ab (clone 56C6, Serotec, United Kingdom), anti-human Ki-67 antigen Ab (ZYMED, San Francisco, CA), and anti-Musashi-1 Ab (14H1) was done as described elsewhere.9Okamoto 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 (351) Google Scholar Briefly, paraffin-embedded tissue samples were cut into 6-μm- or 3-μm-thick serial sections, placed on coated slides, and deparaffinized through a series of xylene and ethanol. Slides were then incubated with primary antibodies at 4°C overnight, followed by biotin-conjugated anti-mouse IgG antibody (E0433, DAKO-USA), biotin-conjugated anti-rabbit IgG antibody (E0435, DAKO-USA), or biotin-conjugated anti-rat IgG antibody (BA4000, Vectastain, Burlingame, CA). The following steps were done using the standard ABC method (Elite ABC kit, Vectastain). Diaminobenzidine tetrahydrochloride (DAB) was used as the substrate for the peroxidase reaction (Vectastain). All slides were counterstained with hematoxylin and observed under a microscope (CH40, Olympus, Tokyo, Japan). Y-FISH-positive epithelial cells were confirmed and quantified as described elsewhere.9Okamoto 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 (351) Google Scholar Y-FISH–positive, CD45-positive cells did not always show complete lack of cytokeratin staining because of its too intense staining. Therefore, all CD45-positive cells were counted as intraepithelial lymphocytes throughout the study, following exactly the same criteria used in our previous report.9Okamoto 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 (351) Google Scholar Expression of lineage-specific markers or Ki-67 antigen within the Y-FISH-positive epithelial cells was determined using serial sections. For detection of BM-derived enteroendocrine cells, immunohistochemical staining of chromogranin A was used as a specific marker. For detection of BM-derived goblet cells, alcian blue staining and Y-FISH were performed using a single section. Paneth cells were identified after H&E staining by their eosinophilic granules. Thus, for detection of BM-derived Paneth cells, H&E staining and Y-FISH were performed using a single section. BM-derived absorptive cells were determined as cells with negative staining for lineage markers of the other 3 lineages, negative staining for Musashi-1 or Ki-67 by immunohistochemical analysis and columnar shaped morphology consistent with the differentiated absorptive cells, and positive staining for CD10 in the serial section with Y-FISH. Regenerative epithelium in the sections was identified by the microscopic features of the epithelial cells, such as dense cytoplasm, nuclear swelling, or hyperchromatin. The results were expressed as the mean ± standard error of mean (SE). Groups of data were compared by the Mann–Whitney U test. P values less than .05 were considered statistically significant. We have previously demonstrated the presence of BM-derived epithelial cells in every part of the human GI tract.9Okamoto 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 (351) Google Scholar In the present study, we attempted to characterize further the BM-derived epithelial cells. For this purpose, we refined our experimental procedure of Y-FISH analysis in this study. In our previous report, we used 6-μm-thick sections to identify BM-derived epithelial cells. We confirmed that Y chromosomes were also clearly detectable in 3-μm-thick sections (Figure 1A). We also confirmed that Y chromosome-positive epithelial cells within a female BMT recipient were clearly distinguishable from Y chromosome-positive lymphocytes by the analysis of 3-μm-thick serial sections (Figure 1B). Therefore, we used 3-μm-thick sections throughout this study to obtain strict and definite characterization of the BM-derived cells. The results obtained from 3-μm-thick serial sections were further confirmed by double staining with Y-FISH and specific markers in single sections, as previously described. A total of 330 BM-derived cells were analyzed by this method, and 239 cells (72.4%) were identified as BM-derived epithelial cells and the remainder as BM-derived intraepithelial lymphocytes. We then examined whether BM-derived epithelial cells detected by the present method arise via the mechanism of cell fusion. Cells generated by cell fusion in vivo are reported to form polyploid cells named heterokaryons.13Wang X. Willenbring H. Akkari Y. Torimaru Y. Foster M. Al-Dhalimy M. Lagasse E. Finegold M. Olson S. Grompe M. Cell fusion is the principal source of bone-marrow-derived hepatocytes.Nature. 2003; 422: 897-901Crossref PubMed Scopus (1476) Google Scholar, 14Vassilopoulos G. Wang P.R. Russell D.W. Transplanted bone marrow regenerates liver by cell fusion.Nature. 2003; 422: 901-904Crossref PubMed Scopus (1200) Google Scholar, 27Weimann J.M. Johansson C.B. Trejo A. Blau H.M. Stable reprogrammed heterokaryons form spontaneously in Purkinje neurons after bone marrow transplant.Nat Cell Biol. 2003; 5: 959-966Crossref PubMed Scopus (407) Google Scholar To reveal the ploidity of BM-derived epithelial cells, we performed multicolor FISH using specific DNA probes for chromosomes 1, 18, and Y. If BM-derived epithelial cells are generated by cell fusion, they will be aneuploid, and, therefore, probes for a single somatic chromosome will be targeted against at least 4 potential target chromosomes within the nucleus of these cells. Thus, we first examined the sensitivity of our FISH method by detecting 4 potential target chromosomes within a single nucleus (Figure 2A). Using probes for chromosomes 1 and 18, we confirmed that our FISH method could detect at least 3 fluorescent signals from 4 potential target chromosomes in a single nucleus at a sensitivity of around 70% (Table 2). Under the same experimental conditions, we examined tissue samples from normal male, normal female, and female BMT recipients, using specific probes for chromosomes 1 and Y (Figure 2B). Results of multicolor FISH in normal male or female samples showed that up to 2 discrete signals were detected in each nucleus of epithelial cells, whether or not a signal of the Y chromosome was present (Table 2). Surprisingly, results of multicolor FISH using tissue samples from BMT recipients showed that Y-FISH-positive as well as Y-FISH-negative epithelial cells observed in female BMT recipient tissue showed no more than 2 discrete signals within a single nucleus (Figure 2C). Analysis of 177 Y chromosome–positive and 421 Y chromosome–negative epithelial cells revealed no significant difference between the 2 groups in the number of green signals seen in a nucleus (1.06 ± 0.713 vs 1.13 ± 0.703 signals/nucleus, respectively, P > .1, Table 2). These results suggest that BM-derived epithelial cells are euploid cells and are not heterokaryons generated by cell fusion. However, because of the limitation of this method, we cannot totally exclude the possibility of cell fusion followed by cytoreductive division.Table 2Results of Multi color FISHNumber of FITC-positive chromosomes in a single nucleusNormal female epithelial cellsNormal male epithelial cellsFemale BMT recipient Y chromosome positive epithelial cellsFemale BMT recipient Y Chromosome negative epithelial cellsChromosome 1 (FITC)Chromosome 1 (FITC)Chromosome 1 (FITC)Chromosome 1 (FITC)Chromosome 1 (FITC)Chromosome 18 (FITC)Y (rhodamine)Y (rhodamine)Y (rhodamine)Y (rhodamine)012/578 (2.1%)44/298 (14.8%)27/220 (12.3%)40/177 (22.6%)79/421 (18.8%)138/578 (6.5%)129/298 (43.3%)86/220 (39.0%)87/177 (49.2%)206/421 (48.9%)2132/578 (22.8%)125/298 (41.9%)107/220 (48.6%)50/177 (28.2%)136/421 (32.3%)3241/578 (41.6%)0/298 (0.0%)0/220 (0.0%)0/177 (0.0%)0/421 (0.0%)4 or more155/578 (26.8%)0/298 (0.0%)0/220 (0.0%)0/177 (0.0%)0/421 (0.0%)Average ± SD2.84 ± 0.9611.27 ± 0.7031.36 ± 0.6921.06 ± 0.7131.13 ± 0.703NOTE. Table reflects examined cell population by probe fluorescence. Values indicate number of cells positive for the indicated number of fluorescent signals/total cells examined.FITC, fluorescein-isothiocyanate. Open table in a new tab NOTE. Table reflects examined cell population by probe fluorescence. Values indicate number of cells positive for the indicated number of fluorescent signals/total cells examined. FITC, fluorescein-isothiocyanate. First, we attempted to determine whether BM-derived epithelial cells reside within the intestinal epithelium as intestinal stem cells. Cells arising from a single intestinal stem cell form continuous, clustered columns of daughter cells along the crypt-villus axis.3Booth C. Potten C.S. Gut instincts thoughts on intestinal epithelial stem cells.J Clin Invest. 2000; 105: 1493-1499Crossref PubMed Scopus (299) Google Scholar If BM-derived epithelial cells reside in the intestinal epithelium as intestinal stem cells, one would expect to see a single crypt of cells all expressing the Y chromosome marker. By our Y-FISH staining, this is easily demonstrated in a male crypt but never in a female crypt (Figure 3A, left panel). However, crypts of female BMT recipients showed patchy and rarely clustered distributions of Y-FISH–positive cells, even when the population of BM-derived cells was increased because of epithelial regeneration following GVHD (Figure 3A, right panel). Another distinctive feature of intestinal stem cells is their long lifetime; they remain within the crypt at least for several years.28Bjerknes M. Cheng H. Clonal analysis of mouse intestinal epithelial progenitors.Gastroenterology. 1999; 116: 7-14Abstract Full Text Full Text PDF PubMed Scopus (354) Google Scholar, 29Wright N.A. Stem cell repertoire in the intestine.in: Potten C.S. Stem cells. 1st ed. Academic Press, London2000: 315-330Google Scholar Therefore, we next attempted to examine whether any BM-derived epithelial cells remain within the intestinal crypt for an extended period. One of our female BMT recipients underwent BMT twice: the first time from a male donor and the second time from a female donor. If any BM-derived epithelial cells do remain within the intestinal crypt for a period of years, Y-FISH–positive epithelial cells should be detected long after the second BMT in this patient. Therefore, we examined GI biopsy specimens taken from this patient at different time points following the first BMT by Y-FISH (Figure 3B). At 45 days after the first BMT, Y-FISH–positive epithelial cells were detected within the small intestinal tissue. However, Y-FISH–positive epithelial cells could not be detected after the second BMT (182 days after first BMT). This suggests that BM-derived epithelial cells do not remain within the crypt for longer than 182 days and therefore lack one of the essential features of intestinal stem cells. We further sought to examine whether any of the BM-derived cells express a specific molecular marker for intestinal stem cells, although no definitive markers have been confirmed. One candidate for such a marker is Musashi-1, an RNA-binding protein initially identified in neural stem cells.30Sakakibara S. Imai T. Hamaguchi K. Okabe M. Aruga J. Nakajima K. Yasutomi D. Nagata T. Kurihara Y. Uesugi S. Miyata T. Ogawa M. Mikoshiba K. Okano H. Mouse-Musashi-1, a neural RNA-binding protein highly enriched in the mammalian CNS stem cell.Dev Biol. 1996; 176: 230-242Crossref PubMed Scopus (483) Google Scholar Recent studies have demonstrated that Musashi-1 is expressed exclusively in the stem cell region of the murine small intestine.31Kayahara T. Sawada M. Takaishi S. Fukui H. Seno H. Fukuzawa H. Suzuki K. Hiai H. Kageyama R. Okano H. Chiba T. Candidate markers for stem and early progenitor cells, Musashi-1 and Hes1, are expressed in crypt base columnar cells of mouse small intestine.FEBS Lett. 2003; 535: 131-135Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar, 32Potten C.S. Booth C. Tudor G.L. Booth D. Brady G. Hurley P. Ashton G. Clarke