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Related Collections Hematopoiesis and Stem Cells Transplantation Cell Cycle Brief Reports Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  Blood, 15 March 2001, Vol. 97, No. 6, pp. 1876-1878 BRIEF REPORT Replicative stress after allogeneic bone marrow transplantation: changes in cycling of CD34+CD90+ and CD34+CD90 hematopoietic progenitors Ian Thornley, D. Robert Sutherland, Rakash Nayar, Lillian Sung, Melvin H. Freedman, and Hans A. Messner From the Division of Hematology/Oncology, The Hospital for Sick Children, and the Department of Medical Oncology and Hematology, University Health Network, University of Toronto, Ontario, Canada.     Abstract Top Abstract Introduction Study design Results and discussion References To further characterize hematopoietic "replicative stress" induced by bone marrow transplantation (BMT), the cell-cycle status of CD90+/ subsets of marrow CD34+ cells obtained 2 to 6 months after transplantation from 11 fully chimeric recipients was examined. Cycling profiles, derived by flow cytometry after staining with Hoechst 33342 and pyronin Y, were compared with those of 14 healthy marrow donors. Primitive CD34+CD90+ cells represented a smaller proportion of CD34+ cells in recipients (10% ± 4% versus 19.6% ± 5.3% in donors; P < .0001) and were more mitotically active, with the proportion of cells in S/G2/M nearly 4-fold higher than in donors (15.6% ± 3% and 4.4% ± 1.6%, respectively; P < .0001). By comparison, there was a modest increase in the proportion of CD34+CD90 progenitors in S/G2/M after BMT (10.9% ± 1% vs 9.6% ± 2% in donors; P = .04). Replicative stress after BMT is borne predominantly by cells in a diminished CD34+CD90+ population. (Blood. 2001;97:1876-1878) © 2001 by The American Society of Hematology.     Introduction Top Abstract Introduction Study design Results and discussion References The typical marrow graft contains only a small fraction of the donor's hematopoietic cells, yet is required to sustain hematopoiesis for the lifetime of the recipient. Peripheral blood cell counts in the recipient usually return to the normal range within weeks of bone marrow transplantation (BMT). However, recipients have profound deficits in hematopoietic progenitors (determined by functional assays and absolute CD34+ cell enumeration) persisting for more than 10 years after BMT.1-5 The demonstration of accelerated telomere shortening in leukocytes of BMT recipients,6-8 reflecting an increase in the mitotic rate of bone marrow progenitors and/or stem cells, suggests that hematopoietic reconstitution imposes a "replicative stress" upon the graft. The distribution of this stress within the hematopoietic hierarchy is unknown and is of considerable importance. An increase in the proliferative rate of lineage-committed progenitors (LCPs) may have little consequence for the host, but an increase at the level of the stem cell could lead, through critical telomere shortening or other mechanisms, to cytogenetic instability and the emergence of clonal disorders.9 The majority of human hematopoietic stem/progenitor cells express the CD34 antigen.10 In vitro assays,11-13 a recent clinical trial,14 and experiments in immunodeficient mice11,13 and preimmune sheep15 have strongly suggested that most primitive hematopoietic progenitors (PHPs) in humans, and virtually all CD34+ repopulating cells, are contained within the CD90+ subset. Although markedly enriched for PHPs, the CD34+CD90+ population is heterogeneous and contains some LCPs. The vast majority of LCPs, however, reside in the larger CD34+CD90 subset.11,12 To establish which progenitors respond to the increased demands on hematopoiesis after BMT, we compared the cell-cycle status of CD34+CD90+ and CD34+CD90 marrow cells from engrafted recipients with values obtained in healthy bone marrow donors.     Study design Top Abstract Introduction Study design Results and discussion References Subjects Eleven recipients of matched sibling donor BMT (median age, 51 years; range, 42-58 years) and 14 healthy marrow donors (median age, 48 years; range, 36-61 years) consented to participate in this ethically approved study between September 1999 and March 2000. Among these 25 subjects were 8 donor/recipient pairs. Indications for BMT were non-Hodgkin lymphoma in 4 patients (B, F, H, J), chronic myeloid leukemia in 4 (E, G, I, K), acute myeloid leukemia in 2 (A, C), and chronic lymphocytic leukemia in 1 (D). Bone marrow transplants Our institutional transplantation protocol has been previously published.16 We conditioned 9 patients with busulphan and cyclophosphamide, and 2 with radiation-based regimens. All recipients received unmodified bone marrow grafts containing a median of 3.0 × 108 (range, 1.0-3.8) nucleated cells per kilogram recipient weight. Graft versus host disease prophylaxis consisted of short-course methotrexate and cyclosporine A. Hematopoietic cytokines were not administered to any patient after transplantation or to donors before harvest. In all patients, neutrophil counts recovered to greater than 0.5 × 109/L before day 28. Chimerism studies Peripheral blood leukocyte DNA was extracted, and amplification of 8 microsatellite regions performed by the polymerase chain reaction, as described.17 Following gel electrophoresis, hematopoietic chimerism was determined by comparing the size of amplified bands from the recipient at the time of bone marrow aspiration after BMT with the size of bands from donor and recipient samples before BMT. The test allows detection of admixtures of donor and recipient DNA down to the level of 5% to 10%. Cell-cycle analysis of hematopoietic progenitors We obtained 5 to 10 mL of bone marrow from 14 donors undergoing bone marrow harvest and from 11 recipients at either 2 months (n = 3) or 6 months (n = 7) after BMT or at both these times (n = 1). CD34+ cells were selected from the low-density mononuclear cell fraction by means of Minimacs CD34 separation columns (Miltenyi Biotec, Auburn, CA).18 CD34+-enriched samples were stained with CD34FITC, CD45PE/CY5, and CD90PE, and the CD90+/ percentages of CD34+ cells were determined by flow cytometry. CD34+CD90+ and CD34+CD90 cells were sorted on a FACSVantage cell sorter (Becton Dickinson Biosciences, San Jose, CA) equipped with Enterprise (Coherent, Santa Clara, CA) and Hene (Spectra-Physics 127, Mountain View, CA) lasers, as previously described.19,20 Sort purity exceeded 94%, and viability 90%, in all cases. Sorted CD34+CD90+ and CD34+CD90 cells were incubated with 1 µg/mL of Hoechst 33342 (stains DNA) and 1 µg/mL pyronin Y (stains RNA), and their cell-cycle status was ascertained by flow cytometry (Figure 1).21-23 A mean of 910 events (range, 300-2126) were gated for CD34+CD90+ cells. The continuous staining pattern observed with pyronin Y prohibits absolute discrimination between CD34+ cells in G0 and G1. To achieve consistent fluorescence-2 (FL2) channel settings, the instrument was calibrated by means of Calibrite beads (BDIS, San Jose, CA). Thereafter, delineation of G0 and G1 subsets was accomplished with the discriminator set at a mean FL2 channel setting of 160 (vertical axis, Figure 1). Single-parameter histograms of Hoechst staining were used to confirm discrimination between cells in G0/G1 and those in S/G2/M. View larger version (53K): [in this window] [in a new window]   Figure 1. Cell-cycle analysis of CD34+CD90+ and CD34+CD90 progenitors from BMT donor (donor 4) at time of bone marrow harvest, and fully chimeric matched recipient (recipient D) 6 months after BMT. (A) Flow cytometric analysis of donor CD34+CD90+ cells, after staining with Hoechst 33342 and pyronin Y. Analysis of the cellular uptake of these vital dyes allows the discrimination of G0 (2N DNA + low levels of RNA), G1 (2N DNA + higher levels of RNA), and S/G2/M (greater than 2N DNA + higher levels of RNA) phases of the cell cycle. (B) Donor CD34+CD90 cells. (C) Analysis of recipient's CD34+CD90+ cells 6 months after BMT. (D) Recipient CD34+CD90 cells. Statistical analysis Means and standard deviations were calculated. Univariate analyses were performed by means of a 2-tailed independent t test. A paired 2-tailed t test was applied to data from the 8 matched donor/recipient pairs. Analyses were performed by means of the SAS statistical program (SAS-PC, Version 8.0; SAS Institute, Cary, NC).     Results and discussion Top Abstract Introduction Study design Results and discussion References At the time of bone marrow aspiration after BMT, all recipients were complete hematopoietic chimeras. Their median hemoglobin concentration was 104 g/L (range, 93-127 g/L), their white blood cell count 4.3 × 109/L (range, 2.7-9.3 × 109/L), and their platelet count 134 × 109/L (range, 10-264 × 109/L). One recipient was platelet-transfusion-dependent 2 months after BMT. The proportion of CD34+ marrow cells expressing the CD90 antigen was reduced in these recipients (10% ± 4% compared with 19.6% ± 5.3% in donors; P < .0001). This diminished CD34+ CD90+ population showed marked changes in cell-cycle status after BMT (Tables 1 and 2). Specifically, 15.6% ± 3% were in S/G2/M compared with 4.4% ± 1.6% of donor cells (P < .0001), and 43.8% ± 13% were in G0 compared with 71.3% ± 12.3% of donor cells (P < .0001). Progenitors tested 2 months after BMT yielded results similar to those assessed at 6 months. When changes in CD90 expression by CD34+ cells and cycling status of CD34+CD90+ progenitors were examined within the 8 matched transplant pairs, the significance of these changes was retained (P < .001 for each analysis).                                View this table: [in this window] [in a new window]   Table 1. CD90 expression by bone marrow CD34+ cells, and cycling status of CD34+CD90+ and CD34+CD90 progenitors in bone marrow transplantation donors                                View this table: [in this window] [in a new window]   Table 2. CD90 expression by bone marrow CD34+ cells, and cycling status of CD34+CD90+ and CD34+CD90 progenitors in bone marrow transplantation recipients There was a slight increase in the proportion of CD34+CD90 cells in S/G2/M after BMT (10.9% ± 1% compared with 9.6% ± 2% in donors; P = .04), but the significance of this difference must be regarded as questionable owing to the number of univariate analyses performed. There was no change in the proportion of CD34+CD90 progenitors in G0 (Table 1). The increase in proliferation of CD34+CD90+ cells after BMT was such that the proportion of these cells in S/G2/M exceeded the proportion of CD34+CD90 cells in the same phases (P = .003)a reversal of the pattern seen in steady-state bone marrow. We offer 3 methodologic caveats. First, phenotypic designations of "primitivity," such as the one we have used, are limited by inevitable omissions. For example, very rare primitive CD34Lin cells are excluded from the CD34+CD90+ population. Second, we have compared phenotypically, but not necessarily functionally, identical cells in steady-state and posttransplant marrow. The functional properties of the CD34+CD90+ population may differ in BMT recipients, in whom functional constituents may be depleted to varying extents. Finally, the method we used for cell-cycle analysis allows precise definition of cells in S/G2/M, but the absolute distinction of cells in G0 and G1 on the basis of differential RNA content24 is somewhat arbitrary. The significance of our reported G0 proportions, derived in a consistent manner in these experiments, lies in the difference between donor and recipient values. We have determined proportions, not absolute numbers, of CD90+/ subsets of CD34+ cells in the marrow of study subjects. The weight of existing evidence suggests that absolute numbers of both subsets are diminished for prolonged periods after transplantation.1-5 Selleri et al5 examined the bone marrow of BMT donors and recipients and found 3- to 4-fold fewer CD34+ cells in the marrow of recipients as late as 8 years after BMT. The accentuated decline in the CD90+ subset reported here suggests that this deficit is most pronounced in primitive progenitors, and it anticipates the finding that post-BMT reductions in long-term-culture-initiating cells are more profound, and prolonged, than reductions in LCPs.4,5 Our results imply that early hematopoietic reconstitution after BMT is associated with a compensatory increase in mitotic rate in a population of CD34+CD90+ progenitors. The accelerated proliferation of these cells is the most likely cause of the striking telomere shortening observed in leukocytes of BMT recipients,6-8 though direct correlation must be sought in future studies. Further characterization of these "stressed" CD34+CD90+ cells is required before the consequences of the increase in their mitotic rate may be inferred and tested.     Acknowledgments We thank Robert Wynn (Royal Children's Hospital, Manchester, United Kingdom) for many useful discussions and Leslie Steele, Tracy Stockley, and Peter Ray for assessment of hematopoietic chimerism.     Footnotes Supported by a 1999 Young Investigator Award from the American Society of Clinical Oncology (I.T.), and by a Seed Grant from The Hospital for Sick Children Research Institute. Submitted June 27, 2000; accepted November 1, 2000. The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734. Reprints: Hans A. Messner, Department of Medical Oncology and Hematology, Rm 5107, 610 University Ave, Toronto, ON M5G 2M9, Canada; e-mail: hans.messner{at}uhn.on.ca.     References Top Abstract Introduction Study design Results and discussion References 1. Messner HA, Curtis JE, Minden MD, et al. Clonogenic hemopoietic precursors in bone marrow transplantation. Blood. 1987;70:1425-1432[Abstract/Free Full Text]. 2. Ma DD, Varga DE, Biggs JC. Haemopoietic reconstitution after allogeneic bone marrow transplantation in man: recovery of haemopoietic progenitors (CFU-Mix, BFU-E and CFU-GM). Br J Haematol. 1987;65:5-10[Medline] [Order article via Infotrieve]. 3. Novitzky N, Mohammed R. Alterations in the progenitor cell population follow recovery from myeloablative therapy and bone marrow transplantation. Exp Hematol. 1997;25:471-477[Medline] [Order article via Infotrieve]. 4. Podesta M, Piaggio G, Frassoni F, et al. Deficient reconstitution of early progenitors after allogeneic bone marrow transplantation. Bone Marrow Transplant. 1997;19:1011-1017[CrossRef][Medline] [Order article via Infotrieve]. 5. Selleri C, Maciejewski JP, De Rosa G, et al. Long-lasting decrease of marrow and circulating long-term culture initiating cells after allogeneic bone marrow transplant. Bone Marrow Transplant. 1999;23:1029-1037[CrossRef][Medline] [Order article via Infotrieve]. 6. Notaro R, Cimmino A, Tabarini D, Rotoli B, Luzzatto L. In vivo telomere dynamics of human hematopoietic stem cells. Proc Natl Acad Sci U S A. 1997;94:13782-13785[Abstract/Free Full Text]. 7. Wynn RF, Cross MA, Hatton C, et al. Accelerated telomere shortening in young recipients of allogeneic bone-marrow transplants. Lancet. 1998;351:178-181[CrossRef][Medline] [Order article via Infotrieve]. 8. Wynn R, Thornley I, Freedman M, Saunders EF. Telomere shortening in leucocyte subsets of long-term survivors of allogeneic bone marrow transplantation. Br J Haematol. 1999;105:997-1001[CrossRef][Medline] [Order article via Infotrieve]. 9. Bonnet D, Dick JE. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat Med. 1997;3:730-737[CrossRef][Medline] [Order article via Infotrieve]. 10. Civin CI, Trischmann MJ, Fackler MJ, et al. Report on the CD34 cluster workshop. In: Knapp W, ed. Leucocyte Typing IV. Oxford United Kingdom: Oxford University Press; 1990:818-825. 11. Baum CM, Weissman IL, Tsukamoto AS, Buckle AM, Peault B. Isolation of a candidate human hematopoietic stem-cell population. Proc Natl Acad Sci U S A. 1992;89:2804-2808[Abstract/Free Full Text]. 12. Craig W, Kay R, Cutler RL, Lansdorp PM. Expression of Thy-1 on human hematopoietic progenitor cells. J Exp Med. 1993;177:1331-1342[Abstract/Free Full Text]. 13. Murray L, Chen B, Galy A, et al. Enrichment of human hematopoietic stem cell activity in the CD34+Thy-1+Lin subpopulation from mobilized peripheral blood. Blood. 1995;85:368-378[Abstract/Free Full Text]. 14. Negrin RS, Atkinson K, Leemhuis T, et al. Transplantation of highly purified CD34+Thy-1+ hematopoietic stem cells in patients with metastatic breast cancer. Biol Blood Marrow Transplant. 2000;6:262-271[CrossRef][Medline] [Order article via Infotrieve]. 15. Sutherland DR, Yeo EL, Stewart AK, et al. Identification of CD34+ subsets following glycoprotease selection: engraftment of CD34+/Thy-1+/Lin stem cells in fetal sheep. Exp Hematol. 1996;24:795-806[Medline] [Order article via Infotrieve]. 16. Tutschka PJ, Copelan EA, Klein JP. Bone marrow transplantation for leukemia following a new busulfan and cyclophosphamide regimen. Blood. 1987;70:1382-1388[Abstract/Free Full Text]. 17. Lins AM, Micka KA, Sprecher CJ, et al. Development and population study of an eight-locus short tandem repeat (STR) multiplex system. J Forensic Sci. 1998;43:1168-1180[Medline] [Order article via Infotrieve]. 18. Dror Y, Freedman MH. Shwachman-Diamond syndrome: an inherited preleukemic bone marrow failure disorder with aberrant hematopoietic progenitors and faulty marrow microenvironment. Blood. 1999;94:3048-3054[Abstract/Free Full Text]. 19. Sutherland DR, Anderson L, Keeney M, Nayar R, Chin-Yee I. The ISHAGE guidelines for CD34+ cell determination by flow cytometry. International Society of Hematotherapy and Graft Engineering. J Hematother. 1996;5:213-226[Medline] [Order article via Infotrieve]. 20. Gratama JW, Keeney M, Sutherland DR. Enumeration of CD34+ hematopoietic stem and pro-genitor cells. Curr Protocols Cytometry. 1999;6.4.1-6.4.22. 21. Shapiro HM. Flow cytometric estimation of DNA and RNA content in intact cells stained with Hoechst 33342 and pyronin Y. Cytometry. 1981;2:143-150[CrossRef][Medline] [Order article via Infotrieve]. 22. Gothot A, Pyatt R, McMahel J, Rice S, Srour EF. Functional heterogeneity of human CD34+ cells isolated in subcompartments of the G0/G1 phase of the cell cycle. Blood. 1997;90:4384-4393[Abstract/Free Full Text]. 23. Holyoake T, Jiang X, Eaves C, Eaves A. Isolation of a highly quiescent subpopulation of primitive leukemic cells in chronic myeloid leukemia. Blood. 1999;94:2056-2064[Abstract/Free Full Text]. 24. Darzynkiewicz Z, Traganos F, Sharpless T, Melamed MR. Lymphocyte stimulation: a rapid multiparameter analysis. Proc Natl Acad Sci U S A. 1976;73:2881-2884[Abstract/Free Full Text]. © 2001 by The American Society of Hematology.   CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this? This article has been cited by other articles: R. Bhatia, K. Van Heijzen, A. Palmer, A. Komiya, M. L. Slovak, K. L. Chang, H. Fung, A. Krishnan, A. Molina, A. Nademanee, et al. Longitudinal Assessment of Hematopoietic Abnormalities After Autologous Hematopoietic Cell Transplantation for Lymphoma J. Clin. 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[Abstract] [Full Text] [PDF] This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Thornley, I. Articles by Messner, H. A. Search for Related Content PubMed PubMed Citation Articles by Thornley, I. Articles by Messner, H. A. Related Collections Hematopoiesis and Stem Cells Transplantation Cell Cycle Brief Reports Social Bookmarking                   What's this?

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