For personal use. Only reproduce with permission from The Lancet Publishing Group. ARTICLES 1084 THE LANCET • Vol 361 • March 29, 2003 • www.thelancet.com Summary Background Adult bone marrow-derived (BMD) cells could be used to repair damaged organs and tissues, but the intrinsic plasticity of these cells has been questioned by results of in- vitro studies suggesting that such cells might fuse with other cells giving the appearance of differentiation. We aimed to determine whether fusion events are important in vivo. Methods To test whether BMD cells can colonise an epithelial tissue and differentiate there without fusion, we did in-situ hybridisation with Y and X chromosome probes labelled with 35-sulphur or digoxigenin, or labelled fluorescently. We did immunohistochemistry with anticytokeratin 13 along with fluorescence in-situ hybridisation to identify Y-chromosome positive buccal epithelial cells in cheek scrapings obtained from five females who had received either a bone-marrow transplant or an allogeneic mobilised peripheral-blood progenitor-cell transplant (enriched in CD34+ cells) from male donors. Findings When examined 4–6 years after male-to-female marrow-cell transplantation, all female recipients had Y-chromosome-positive buccal cells (0·8–12·7%). In more than 9700 cells studied, we detected only one XXXY-positive cell (0·01%) and one XXY cell (0·01%), both of which could have arisen when an XY cell fused with an XX cell. Interpretation Male BMD cells migrate into the cheek and differentiate into epithelial cells, an occurrence that does not depend on fusion of BMD cells to recipient cells. This finding might be an example of transdifferentiation of haemopoietic or stromal progenitor cells. Plasticity of BMD cells could be useful in regenerative medicine. Lancet 2003; 361: 1084–88 Introduction Evidence from studies in recipients of bone-marrow transplants shows that bone marrow-derived (BMD) cells can differentiate into cells other than blood. This occurrence is of importance because such cells could be used to regenerate organs after disease or trauma. 1–5 However, the plasticity of BMD cells has been questioned by results of studies suggesting that stem cells might fuse with other cells and give the appearance of differentiation. 6,7 Furthermore, caution is needed in interpretation of data purporting to show that non- haemopoietic cells are derived from donor BMD cells. 8–10 Tissues, especially when affected by graft-versus-host disease (GVHD), might contain infiltrating donor haemopoietic or lymphoid cells. In histological sections, non-haemopoietic tissue cells might be confounded with haemopoietic cells because of cell overlap (attributable to the thickness of the sections) or loss of haemopoietic lineage-specific markers. We aimed to establish whether BMD cells differentiate into cells of another tissue lineage, and to assess whether this event is attributable to fusion. To avoid contamination of our samples by haemopoietic inflammatory cells, we did our studies on healthy transplant recipients without oral GVHD, several years after bone-marrow transplantation. Methods Patients Bone-marrow stem-cell transplant recipients residing within the USA, who each had received an allogeneic transplant from a male sibling donor through protocols from the National Heart, Lung, and Blood Institute, were invited to participate in the study. All those who agreed to participate were well, in haematological remission, with full donor lymphohaemopoietic engraftment and no active oral GVHD. The transplants these patients had received consisted of at least 310 6 CD34+ cells/kg and a total of 110 7 –310 8 CD3+ lymphocytes/kg given within 3 months of the stem cells. After the transplant, all patients received only irradiated blood products. We also recruited healthy volunteers as controls. This study was approved by the Institutional Review Board of the National Institute of Dental and Craniofacial Research (NIDCR), National Institutes of Health (NIH). Procedures We obtained cheek scrapings from all women to look for Y-chromosome-positive buccal epithelial cells. Study of buccal cells has the advantage of providing a non- invasively obtained, pure single-cell population of mature nucleated cells with a characteristic morphology and phenotype (anticytokeratin 13+). Cheek scrapings spread on glass slides were independently examined by two pathologists. We also gathered blood samples from these women. Differentiation of human bone marrow-derived cells into buccal epithelial cells in vivo: a molecular analytical study Simon D Tran, Stanley R Pillemer, Amalia Dutra, A John Barrett, Michael J Brownstein, Sharon Key, Evgenia Pak, Rose Anne Leakan, Albert Kingman, Kenneth M Yamada, Bruce J Baum, Eva Mezey National Institute of Dental and Craniofacial Research, Gene Therapy and Therapeutics Branch (S D Tran DMD, S R Pillemer MD, R A Leakan RN, B J Baum DMD); Craniofacial Developmental Biology and Regeneration Branch (K M Yamada MD); Biostatistics Core (A Kingman PhD); National Human Genome Research Institute, Cytogenetic Core (A Dutra PhD, E Pak MD); National Heart Lung and Blood Institute, Hematology Branch (A J Barrett MD); National Institute of Mental Health, Laboratory of Genetics (M J Brownstein MD); National Institute of Neurological Disorders and Stroke, Basic Neuroscience Programme (S Key BS, E Mezey MD); National Institutes of Health, Bethesda, MD, USA Correspondence to: Dr Simon Tran, National Institutes of Health/NIDCR, 10 Center Drive, Room 1N113, MSC 1190, Bethesda MD 20892-1190, USA (e-mail: stran@dir.nidcr.nih.gov)