Is It Time to Revisit Our Current Hematopoietic Progenitor Cell Quantification Methods in the Clinic?

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 multipotent 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 as many other tissues such as cartilage, muscle and bone.15 --18 There is a need for analysis and quantification of HSC subpopula- CD133 (AC133) is a transmembrane cell surface marker that tions and earlier myeloid progenitors with marrow-repopulating belongs to the prominin family of pentaspan membrane proteins, capacity. In the absence of information on the number of earlier and is expressed on hematopoietic stem and progenitor cells.33,34 progenitors transplanted in clinical practice, many questions Jamie-Perez et al.28 have quantified CD133 þ HSC within PB and regarding engraftment failure and delay will remain unsolved. grafts following stem-cell mobilization in the autologous setting. They were able to demonstrate a significant correlation between CD133 þ and CD34 þ HSCs in the PB before collection and in the HSC subpopulations that have been defined and associated with final product for grafting (r ¼ 0.62 and 0.64; Po0.01). There was a clinical outcome stronger correlation between CD34 þ cells and CD133 þ cells in With the increasing number of BM and peripheral stem cell the leukapheresis product (r ¼ 0.94) compared with pre-collection transplants being performed, the need to more carefully define PB (r ¼ 0.33; Po0.01). However, time to recovery did not correlate hematopoietic subpopulations began in earnest about 10 years with the number of CD133 þ HSCs infused. ago, and was facilitated greatly by the use of combinations of With an aim to analyze the human hematopoietic precursors in MoAbs recognizing surface structures on stem and progenitor cell all stem-cell sources Arber et al.32 initiated a study on 26 patients. subpopulations. As the repertoire of available Ab reagents These investigators demonstrated the presence of distinct expanded, far more exquisite characterization became possible; distributions of defined HSC and progenitor cell subpopulations some of these reagents are summarized in Table 1. HSCs are within the lineage negative (linÀ) CD34 þ compartment. On the relatively rare within normal BM, being present at levels in the basis of published data the most frequently observed subpopula- range of 1 in 104 -- 1 0 5 cells.21 Although these are relatively low tions are summarized in Table 3. Investigators found all these frequency cell types, current cytometric technology permits their subpopulations’ cell counts to be variable in distribution and detailed immunophenotypic analysis. Flow cytometry is a very numbers, depending on both the source of stem cells (BM, mobi- powerful investigative tool that enables researchers to character- lized peripheral or CB) and the chronological age of the donor. ize subpopulations based on their immunophenotype or func- Moreover, they were able to show that platelet and reticulocyte tional properties.20 Table 2 summarizes the impact of flow engraftment post transplant was correlated with the presence of cytometric stem cell-marker expression on engraftment kinetics. the common myeloid progenitor and megakaryocyte--erythroid

Bone Marrow Transplantation (2012) 1391 --1396 & 2012 Macmillan Publishers Limited Hematopoietic progenitor cell quantiﬁcation methods M Beksac and F Preffer 1393 Table 2. The frequency and engraftment potential of HSC subpopulations in clinical practice

Cell type Frequency in Cell dose PNL engraftment Plt engraftment

Total CD34+5,7,10 1/200 (of MNC, BM) 2--8 Â 106/kg Yes Yes (month 3,6,9) CD34+38À22 --24 1/40 Unknown Yes (month 6,9) Yes 1/1000 (of MNC, BM) No CD34+90+22,25,26 1/1000 25--80 Â 104/kg Yes Yes (month 3,6,9,12a) no CD34+CD110+27 Yes CD34À/low24 1/24000 Unknown Unknown Unknown CD133+28 Same as CD34 Same as CD34+ Similar to CD34 Similar to CD34 CD34+133+22 Not reported Not reported Yes (month 3) Yes(month 3,6,9) SSC low/ALDH bright29 --31 1.2% (BM) 5.4 Â 106/kg (PNL) Yes Yes 7.22 Â 106/kg (Plt) Yes Yes No No Lin--CD34+CD38ÀIL3Ra low 73.20 (17.8--86.2) % (PBSC) 44.4 Â 106/kg Similar to CD34 Similar to CD34 CD45RA-- (CMPs)32 35.2 (20.4--42.3) % (BM) (PNL, Plt and erythroid) 3.2 (1.3--16.1) % (CB)

Abbreviations: CMP ¼ common myeloid progenitors; MNC ¼ mononuclear cell; PNL ¼ polymorphonuclear cells; SSC ¼ side light scatter. aAlso enhanced erythroid engraftment on month 12.

Table 3. Flow cytometric markers used to define the most frequently express ALDH and CD133 are also potent stem cells with high observed stem cell subpopulations12,26,32,35 engraftment capacity with 65% of CD34 þ CD133 þ cells expressing additionally high levels of ALDH.17 In contrast, cells with low Cell type Lin-- subpopulation ALDH have been shown to exert a healing effect following an immunophenotype ischemia-induced skin necrosis model suggesting endothelial progenitor cell capacity. HSC Hematopoietic stem cell CD34+/CD38À Quantitation of stem cells based on their functional properties is CMP Common myeloid CD34+/CD38+/IL3Ra an exciting approach and theoretically more relevant than relying progenitor low/CD45RA solely on the numbers of immunophenotyped cells harvested, GMP Granulocyte-macrophage CD38+/IL3Ra low/ and subsequently thawed and infused. As stem cells posses progenitor CD45RA+ p-glycoprotein ABC transporters to protect them from environ- MEP Megakaryocyte-- CD38+/IL3RaÀ/CD45RAÀ erythroid progenitor mental toxins and threats, high efflux of dyes such as Hoechst CLP Common lymphoid CD10+/CD19À 33342 or Rhodamine 123 have been used successfully progenitor to characterize cell function. Following this approach investigators Pro-B cells CD10+CD19+ have identified side populations in the circulation of normal T and NK progenitors CD10À/CD7+/CD45RA+ human blood based on initial studies that detected the highest MSC Mesenchymal stem cell CD90+/CD105+/ALDH+/ hematopoietic repopulation potential in side population cells CD45À/CD34À/CD133À/ obtained from murine BM.40,41 Unfortunately, the impact of the CD38À/HLAÀDRÀ number of cells belonging to the side population within a STRC-M Early myeloid short-term CD34+/CD38+/CD45À/ graft has not yet been fully investigated in human patients. The repopulating ALDHÀ/CD133À/CD90+ human linÀ BM mononuclear cell population, LinÀCD34 þ Late myeloid HSC CD34+/CD38À/ low CD45dim+/ALDH+a/ CD38 /À cells also generally express ALDH activity, but they CD133+/CD90+ are especially enriched within the combined side population/ ALDHbright þ subpopulation.42,43 Abbreviations: ALDH ¼ Aldehyde dehydrogenase; STRC-M ¼ short term a There is growing evidence that the selection of stem cells might repopulating cells-myeloid. Bright positive cells have the highest be improved based on their ALDH expression. Human BM repopulation capacity compared with dim positive cells. mononuclear cells, sorted on the basis of their low side light scatter (SSClow) ALDH bright þ , make up 1.2% of all nucleated cells. Fallon et al.29 were the first to analyze the relationship progenitor compartments, and that an improved understanding between the levels of ALDH-expressing cell counts in the graft and resulting engraftment. Interestingly, they were able to detect a of these defined compartments could have beneficial effects on low future cellular-based therapies. correlation between engraftment and SSC ALDH bright þ , but not CD34 þ cell numbers in fresh autologous PBSC transplants. Recently, Gunduz et al.31 quantified the SSClow ALDH bright þ Functional assessment of HSCs using aldehyde dehydrogenase cells in PB and autologous grafts, and were able to define (ALDH) and dyes thresholds for engraftment (Table 2). Lioznov et al.30 investigated Stem cells produce the detoxifying enzyme ALDH, which the relationship between engraftment following allogeneic distinguishes them from more differentiated cells. ALDH-produ- transplantation and the number of cells expressing ALDH activity. cing cells can be detected by flow cytometry, with those co- Surprisingly, they found that ALDH activity not always correlated expressing CD34 and ALDH activity identifying those cells with with CD34 counts, but rather could better predict both colony- maximum repopulating potential.30,36 --39 Within the ALDH-expres- forming potential and post-transplant donor chimerism. Moreover, sing subpopulation, HSCs are enriched within the ALDH bright þ the CD34 counts were influenced by storage of PB or BM stem segment although ALDH bright þ -expressing cells do not cells at 41C or room temperature, and correlated less with colony- necessarily repopulate immunodeficient mice.36 Cells that co- forming unit (CFU) compared with ALDH activity. Continuously

& 2012 Macmillan Publishers Limited Bone Marrow Transplantation (2012) 1391 --1396 Hematopoietic progenitor cell quantification methods M Beksac and F Preffer 1394 increasing use of cryopreserved stem cell products, namely CB or Nevertheless, MSC are observed in 1--20 Â 10À5 of BM cells. unrelated products that are subject to long transportation hours, Staining patterns using multiple Ags have revealed multiple favors the need to use either ALDH activity or colony forming populations some of which are summarized in Table 3. When assays to measure the remaining stem cell functions in the CD34 þ /CD90 þ , CD34 þ /AC133 þ , CD34 þ /CD38À and product infused. Certainly, ALDH activity quantification by flow CD34 þ /HLA-DR-- cells were analyzed, a strong correlation cytometry can be accomplished reliably and faster than CFU tests. between the total numbers of CD34 þ 90 þ cells, and stable and Although flow cytometry is the gold standard for measuring efficient platelet and neutrophil engraftment was observed. ALDH activity, such information is yet to attain a level of utility to CD34 þ /90 þ cells were better predictors of engraftment than suggest its implementation into clinical practice.44 ALDH iso- the other subpopulations analyzed.26 The number of CD34 þ / enzymes are being defined and among many, ALDH1A1 and CD90 þ cells constitute approximately one-fourth of the total ALDH31 are the ones most effective on self-protection, differ- CD34 þ cells and are generally fewer than the CD34 þ /CD133 þ entiation and cellular expansion of stem cells. ALDH activity is cells, but more highly represented than the CD34 þ /CD38-- cells. not only exclusive to HSCs but also can be expressed by CD34 þ /CD90 þ cells are mobilized into blood at a maximum cancer stem cells and progenitors of tissues with regenerative level a few days earlier than peak mobilization of the total CD34 þ potential.44 Results from preclinical trials conducted by Aldagen cells.12 Inc. (Durham, NC, USA) suggest the angiogenic potential of Sumikuma et al.27 have shown a stronger impact of ALDH-bright cell population in human CB. As ALDH has a cell CD34 þ CD90 þ linÀ cells than total CD34 þ cells in predicting cycle-control role on HSCs, inhibition of this enzyme by stable long-term platelet engraftment; 80 Â 104 cells/kg was the pharmacological agents (DEAB) or molecular targeting (siRNA) minimal number required to achieve this goal. Three of five has been shown to induce stem cell expansion and engraftment subsequent studies analyzing the threshold value for in experimental models.44 CD34 þ CD90 þ linÀ cells shared these results.22,26 However, as seen in Table 2 neither the threshold nor the phenotype of cells to Functional assessment of HSCs by colony forming assays predict platelet engraftment was found to be consistent among these trials. Of note, this population must not be confused with Another approach to measure the functional repopulating MSCs, which are CD34ÀCD90 þ LinÀ. potential of stem cell sources were initially determined by in vitro The incidence of GVHD is observed less frequently when donor CFU cultures: CFU-granulocyte-macrophage (GM), CFU-erythroid cells are obtained from BM instead of PB. Although the infusion of (E) or CFU-granulocyte-erythrocyte-macrophage-megakaryocyte lymphocytes following BMT has been postulated to be responsible (GEMM). These tests are standardized and quantitative, but for this effect, the role of functionally active marrow-resident MSC relatively cumbersome and time consuming. Using CFU cell- may also contribute to this observation. Increased clinical culture techniques, subpopulations of stem cells within a graft experience with in vitro-generated MSC use may provide evidence were defined on the basis of their short term repopulating for their efficacious use in the treatment of extensive GVHD.51 MSC characteristics (STRCs) or long term repopulating characteristics. also may contribute to engraftment when co-transplanted with With the advent of MoAb use, additional immunophenotypically CB. Moreover, there is growing evidence on a wider differentiation separable sub-types of human STRC were defined. As also seen in potential of BM over PB. The surface markers expressed on MSCs Table 3, CD34 þ CD38 þ co-expression correlates exclusively with are not specific and can be shared by progenitors of other myeloid progeny developing rapidly post transplant, lasting 3--5 lineages, such as endothelial progenitors. The International weeks (myeloid-restricted STRC). CD34 þ CD38-- produced large Society for Cellular Therapy has defined the criteria for the numbers of B lymphoid and myeloid progeny following an initial definition of MSCs. These include the expression of CD73, CD90, 3--4 week delay, lasting for the first 2--4 months post transplant. A 35 CD105 but lacking CD45, CD34, CD11b, CD14, CD49a, CD79, CD19 third population was lymphoid-restricted STRC. These stem cells and HLA-DR.52 enumerated by in vitro short-term cultures were shown to Another source of stem cells for both in vitro and in vivo studies influence engraftment in the early 1990s. Later, long-term cell is CB. The CD34 þ and MSC content of CB has been extensively cultures became available and led to estimation of stem cell investigated.36,44,54 Recently, an updated guideline on the numbers responsible from long-term repopulation. These long- minimum number of CD34 þ cells within CB units acceptable term cultures are now being widely used in experimental stem 5 54 for transplantation was established as 1.5 Â 10 CD34 þ per kg. cell studies, but correlation with clinical parameters is lacking. As increasing numbers of CB transplants are being utilized for The immunophenotypic quantification of these long term elderly patients, such a guideline was highly desirable. On the repopulating characteristics or STRCs may replace such cumber- basis of the 20 years of clinical experience in this field for CB some techniques in future. transplantation, one log fewer CD34 þ cells/kg than utilized from the BM was found to be adequate. However, as previously Progenitors of hematopoiesis involved in mobilization and indicated, CD34 enumeration does not measure the functional engraftment and proliferative capacity of stem and progenitor cells within CB. The adrenergic system regulates hematopoiesis through axonal Attempts to quantify earlier progenitors that possess high control with neuronal cells shown to grow from the BM under proliferative and functional capacity have led to the application certain in vitro conditions.45 --47 In addition, BM-derived MSC can of ALDH quantification in CB transplantation. Christ et al.35 have differentiate to all mesodermal lineages. Furthermore, BM is demonstrated subpopulations of CD34, CD133 and ALDH expres- composed of both cellular compartments and fatty tissue, which sing stem cells in human CB. HSC and most myeloid-restricted are all potential important sources of stem cells, and are located in STRC as well as myeloid and lymphoid-restricted STRC are also the proximity of bone matrix, which is postulated to be the ‘niche’ ALDH þ , suggesting that selection of ALDH þ cells for clinical within which stem and progenitor cells interact with osteoblasts transplants will not necessarily have an adverse effect on myeloid to influence either the self-renewal or differentiation of stem recovery with ALDH activity serving as the best predictor for long- cells.17,48 --50 term engraftment. One-fourth of CD34 þ cells and two-thirds of Although the impact of phenotypic HSC compartments within CD133 þ cells were also ALDH þ . In this study, CD133 was not mobilized PB on post-transplant engraftment has been analyzed expressed on most of the STRCs and did not posses a potential considerably, there are not many data regarding the repopulating for either defining STRCs or predicting engraftment. Finally, potential of these cells. However, it has been shown that BM is these investigators suggest ALDH þ human CB cells to be richer in earlier progenitors (for example, MSC) compared with PB. responsible for long term and ALDH-- cells for short-term

Bone Marrow Transplantation (2012) 1391 --1396 & 2012 Macmillan Publishers Limited Hematopoietic progenitor cell quantification methods M Beksac and F Preffer 1395 repopulation in this experimental model. These data favor the use as routine treatments. However, our knowledge of hematopoiesis of ALDH-activity measurement as a valuable tool for quantification and tools to define stem cell subsets both phenotypically and of CB stem cells. functionally are ready to be implemented to clinical practice; it is a propitious time to introduce additional characterization with MoAbs to CD90, CD133 and possibly CD105 into our current panel Phenotypic similarities between the stem cells of cancer, of HSC quantification. With antibodies against ALDH, functional embryogenesis and hematopoiesis HSC quantification is gaining importance. The viability of thawed The phenotypic markers of normal and cancer stem cells share cells should be uniformly measured by flow cytometry as this many common features. The developing molecular technologies measure is capable of influencing the engraftment fate. The carry the potential to capture such cells before they are authors, based on current evidence recommend enumeration of differentiated or lost under unnatural in vitro conditions. ALDH is viable and functional stem cell subsets before the administration among those properties shared by both early progenitors and of these cells in the clinic. Thus, with the greater collaborative cancer cells. Higher activity of ALDH is correlated with increased effort between cytometrists and transplant physicians we hope to tumorigenicity, resistance, metastasis potential and reduced 17,44 be able to unveil problems related to engraftment kinetics and survival. Likewise, embryonic stem cells express markers that improve success rates. are shared by differentiated cells. In contrast, these very immature cells possess transcription factors (OCT 4, SOX2 and NANOG) that have been identified to be specific for embryonic stem cells. These molecular markers can be measured by quantitative PCR enabling CONFLICT OF INTEREST quantification of embryonic stem cells too. In addition, simulta- The authors declare no conflict of interest. neous immunophenotyping with a surface embryonic stem cell marker (SSEA-1) increases the specificity and sensitivity of these methods. Recently, it has been demonstrated in a murine model REFERENCES that a surface T-cell Ig mucin (TIM3) was present on human Lin-- 1 Thomas ED, Lochte Jr HL, Lu WC, Ferrebee JW. Intravenous infusion of bone CD34 þ CD38ÀCD90À leukemia stem cells, but not on the normal marrow in patients receiving radiation and chemotherapy. 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