Is It Time to Revisit Our Current Hematopoietic Progenitor Cell Quantification Methods in the Clinic?
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Bone Marrow Transplantation (2012) 47, 1391 --1396 & 2012 Macmillan Publishers Limited All rights reserved 0268-3369/12 www.nature.com/bmt REVIEW Is it time to revisit our current hematopoietic progenitor cell quantification methods in the clinic? M Beksac1 and F Preffer2 In the clinical practice of hematopoietic SCT, the minimum numbers of cells required for a successful engraftment are defined on the basis of their CD45 and CD34 expression profiles. However, the quantity of earlier progenitors or CD34-positive cells at different differentiation stages within stem cell grafts is not generally taken into consideration. During the last decade, various teams have quantified the number of cells expressing various combinations of CD34, CD38, CD133, CD90 co-expression and/or aldehyde dehydrogenase functional capacity using flow cytometry. Some of these studies resulted in the greater appreciation that combinations of these Ags were associated with varied myeloid, erythroid and platelet engraftment rates whereas others showed that the relative absence or presence of these markers could define cells responsible for either short- or long-term engraftment. These findings were also extended to differences between progenitor cell populations found within BM vs peripheral or cord-blood grafts. Cells harvested from donors are also generally frozen and stored; thawed cells have variable levels of viability and functional capacity based on the time tested post thaw, which also can be assessed by flow cytometry. Finally, flow cytometry has the potential for analysis of cells carrying a mesenchymal stem cell phenotype, which may be quiescent within some of the stem cell products. This review will address the need for stem cell subpopulation quantification and summarize existing published data to identify some Ags and functional characteristics that can be applicable to daily clinical practice. Bone Marrow Transplantation (2012) 47, 1391--1396; doi:10.1038/bmt.2011.240; published online 5 December 2011 Keywords: hematopoietic SCT; CD34; CD133; aldehyde dehydrogenase; flow cytometry INTRODUCTION additionally contain CD34À cells as well as early progenitors of 4 It is now almost 50 years since allogeneic BMT (alloBMT) was myelopoiesis. This review will focus not only on the different introduced to the field of clinical hematology.1 Over this time subpopulations of HSCs that are quantified by current routine flow period alloBMT has earned the title of a ‘standard treatment’ cytometric methods and their impact on transplantation kinetics, modality for an expanding number of hematological diseases. but also on the differences between traditional HSCs and earlier Developing cytotherapeutic techniques and utilization of recom- progenitor cells that are committed to generate cells of myeloid binant hematopoietic growth factors has made both allogeneic- lineage. and autologous-mobilized PBSC transplantation possible. The mobilization and engraftment kinetics have been investigated Do CD34 counts always predict neutrophil and platelet extensively in experimental and clinical models. These studies engraftment successfully? have led to certain guidelines regarding the optimal number of The existing recommendations about the minimum number of hematopoietic stem cells (HSC) to be collected and infused HSCs to be used for transplantation have been acquired following their thawing. These counts are based on the numbers predominantly from retrospective data.3,5,6 There are reports of cells expressing CD34 (a surface sialomucin) on CD45dim þ demonstrating faster neutrophil or platelet engraftment with 2 mononuclear cells. Although initially beset by varied preparative increasing doses of HSCs in the autologous setting.5,7,8 techniques, several workshops and international collaborations The numbers of HSC to be transplanted during BMT is calculated have resulted in a consensus on methodology and commercia- by dividing the total number of cells infused to the actual body lized standard platforms for worldwide application. Organizations weight. As patients may develop weight loss resulting from bone such as the European Group for Blood & Marrow Transplantation resorbtion, and/or fat or muscle loss, investigators aimed to (EBMT) and International Society of Hematotherapy and Graft analyze the impact of actual body weight or ideal body weight on Engineering (ISHAGE) recommend that laboratories have external engraftment. These investigators were able to show correlation quality controls so as to be able to compare results across national between engraftment speed and HSC/ideal body weight, but 3 6 borders. Use of a minimum of 2 Â 10 cells/kg for autologous and not with HSC/actual body weight. Thus, Singh et al.9 have higher numbers of cells for allogeneic/unrelated PBSC transplan- recommended use of ideal body weight for this purpose. tation are recommended in the EBMT handbook. However, these Furthermore, Allan et al.10 have drawn attention not solely to recommendations have opened up new discussions and areas in the count of harvested cells, but also the numbers of CD34 þ cells need of further clarification. In addition to CD34 þ cells, sources of recovered post thaw and subsequently infused, as critical. When HSC such as BM, growth factor-mobilized PB and cord blood (CB) cell numbers o2 Â 106,2--5Â 106 or 45 Â 106 per kg were 1Ankara University School of Medicine, Department of Hematology, Ankara, Turkey and 2Department of Pathology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA. Correspondence: Dr M Beksac, Ankara University School of Medicine, Department of Hematology, Cebeci Yerles¸kesi, Dikimevi 06620, Ankara, Turkey. E-mail: [email protected] Received and accepted 31 October 2011; published online 5 December 2011 Hematopoietic progenitor cell quantification methods M Beksac and F Preffer 1392 compared, decreased HSC counts were found to influence platelet Table 1. MoAbs recognizing stem cell populations19,20 but not neutrophil-recovery speed. A decrease in CD34 þ cell 6 counts from 5 to 3 Â 10 per kg after thawing did not prevent Ag Other names Differentiated Stem/progenitor rapid engraftment. However, values that were between 2.9 and 1.7 cell expression expression led to a delay in engraftment. The actual mononuclear cell subpopulations lost during cryopreservation and thawing proce- CD9 P24, DRAP-1, Leukocytes, NSC dures are unknown, and have not been sufficiently studied. MRP1 endothelial, There is accumulating evidence suggesting that more complex epithelial mechanisms are involved in the mobilization of stem cells, in that CD34 8G12, 581, Endothelial HSC, EPC specific mobilization protocols impact the HSC content of the QBEnd10 CD73 Ecto-50 Leukocyte MSC resulting grafts. For example, use of chemotherapeutic agents in nucleotidase subsets, addition to myeloid growth factors augment the myelosupression, endothelial, resulting in an amplified rebound and surge of HSC into the epithelial circulation. However, these cells do not result in a more rapid CD81 TAPA-1 Lymphocytes, NSC recovery for the patient. It remains perplexing that we are able to endothelial, mobilize increased numbers of HSC with fewer apheresis epithelial procedures, but still not achieve more rapid engraftment. The CD90 Thy1 Endothelial MSC, HSC cellular and molecular mechanisms underlying these observations CD105 Endoglin, Endothelial, MSC with CD73+, remain unclear. It was suggested that the cytotoxic damage by SH2 macrophage/ CD16+, CD90+ monocyte CD29+ CD34À, chemotherapy on MSC may lead to mobilization of numerically CD45-- more HSCs, but that these have inherently poorer functional CD117 c-kit, steel, Breast epithelial, Myeloid, HSC capacity and do not increase the kinetics of engraftment. Lazarus SCF renal tubule, et al.11 were the first to obtain MSCs from patient BM grafts melanocytes whereas they were not able to repeat these with PBSC from normal CD133 AC133, Endothelium, HSC, EPC, NSC donors or patients. It is postulated that the CD34 þ cells mobilized Prominin-1 epithelial following G-CSF, or G-CSF and chemotherapy have different CD135 FMS-like Macrophage/ Human marrow adhesion phenotypes and repopulating potentials as well.12,13 kinase-3 monocytes CD34+ and Our current knowledge of basic stem-cell biology in the (FLT3) or STK- dendritic 1, or Flk-2 precursors, murine research setting has provided most of the insights into the short-term HSC mechanisms of stem-cell activity and has thus influenced the CD146 MUC-18, Mel- Activated T cells, EPC protocols used in clinical practice. Such investigations have helped CAM, MCAM endothelial define that HSCs are generally quiescent cells that are capable of CD150 SLAMf1; T, B, dendritic, Distinguishes HSC asymmetric replication with the capacity to either self-renew or used with endothelial from multipotent develop into more differentiated progenitors, responding to need. CD244 and and B-cell Self-renewal is the process by which a stem cell divides and CD48 progenitors produces a daughter cell with identical potential and capacity. The Aldefluor HSC/MSC second hallmark characteristic of a stem cell is its capacity to Side HSC population repopulate a given system via a variety of downstream multi- potent progenitors.14 Abbreviations: EPC ¼ endothelial progenitor cells; HSC ¼ hematopoietic In addition to HSCs, BM appears to be the origin of additional stem cells; NSC ¼ neural stem cells. stem cells such as MSC that are also capable of differentiating to progenitors of mature cell populations of hematopoiesis, as well