Multi-Color Immune-Phenotyping of CD34 Subsets Reveals Unexpected Differences Between Various Stem Cell Sources

ORIGINAL ARTICLE Multi-color immune-phenotyping of CD34 subsets reveals unexpected differences between various stem cell sources

J Dmytrus1, S Matthes-Martin2, H Pichler2, N Worel3, R Geyeregger1, N Frank1, C Frech1 and G Fritsch1

Flow cytometric routine CD34 analysis enumerates hematopoietic stem and progenitor cells irrespective of their subpopulations although this might predict engraftment dynamics and immune reconstitution. We established a multi-color CD34 assay containing CD133, CD45RA, CD10, CD38 and CD33. We examined PBSC, donor bone marrow (BMd) and BM of patients 1 year after allografting (BM1y) regarding their CD34 subset composition, which differed significantly amongst those materials: the early CD45RA−CD133+ CD38low subpopulations were significantly more frequent in PBSC than in BMd, and very low in BM1y. Vice versa, clearly more committed CD34 stages prevailed in BM, particularly in BM1y where the proportion of multi-lymphoid and CD38++ B-lymphoid precursors was highest (mean 59%). CD33 was expressed at different intensity on CD45RA±CD133± subsets allowing discrimination of earlier from more committed myeloid precursors. Compared with conventional CD34+ cell enumeration, the presented multi- color phenotyping is a qualitative approach defining different CD34 subtypes in any CD34 source. Its potential impact to predict engraftment kinetics and immune reconstitution has to be evaluated in future studies.

Bone Marrow Transplantation (2016) 51, 1093–1100; doi:10.1038/bmt.2016.88; published online 4 April 2016

INTRODUCTION CD133+CD38lowCD10−), EMP remain CD45RA− but lose CD133 − − /low + − Hematopoietic stem and progenitor cells (HSPC) were originally expression (CD45RA CD133 CD38 CD10 ). LMPP harbor described by simple flow cytometric methods using only CD34 to the potential to create neutrophil and macrophage progenitors characterize them.1,2 Since 1996 the two-color ISHAGE protocol (GMP) as well as CD10 upregulating multi-lymphoid + + focusing on CD45 and CD34 has served as the standard for CD34+ precursors (MLP). Among others, these MLP create CD34 CD19 cell enumeration.3 Addition of 7-AAD to define cell viability, and B-lymphoid progenitors (BLP), which downregulated CD133 + − ++ + the use of beads for standardized quantification expanded the (CD45RA CD133 CD38 CD10 ). Based on the definition of these test to a single-platform three-color analysis tool to enumerate subsets, we established a multi-color approach to define and CD34+ cell numbers in bone marrow (BM), blood or cord blood.4,5 enumerate CD34 subpopulations in the three different HSPC In the clinical setting, these analyses have not changed and sources. CD34, CD45, 7-AAD and scatter properties were used to + address only CD34+ cells as such. However, one important issue define viable CD34 cells, which were then further classified using concerning repopulation and engraftment after hematopoietic CD45RA, AC133, CD7, CD10, CD19, CD3, CD33 and CD38. stem cell transplantation is to define distinct subsets of CD34+ cells.6–8 Markers like CD45RA and CD38 were suggested in 9–12 research experiments. Expression studies of CD34 with CD10, MATERIALS AND METHODS CD38, CD45RA or CD90 led to the discovery of functional Specimen collection HSPC-subpopulations and to the presentation of the classic model of the human hematopoietic lineage tree.13,14 Some studies All cell specimens used in this study were obtained for routine CD34 enumeration. Donor BM samples (BMd, n = 31) were from healthy correlated the co-expression of CD38 and CD71 with neutrophil 6,15 allogeneic BM donors aged from 2 to 48 (median 26) years. Patient BM and platelet recovery. CD133/Prominin-1 was included only specimens (BM1y, n = 21) were from biopsies routinely drawn for clinical recently into a panel of markers to define different CD34 examination 1 year after allografting. Six of these were paired samples, that 16 17 subsets. Based on these studies, Görgens et al. suggested a is, we analyzed both BMd and BM1y from these patients. The BM recipients revision of the most recent model of human hematopoiesis, the (11 males, 10 females) had been diagnosed with AML (4), ALL (6), MDS (4), composite model (Figure 1), originally proposed by the Jacobsen CML (2), RCC (2), SCID (1), SAA (1) and Hyper IG E syndrome (1). Their group.14,18 According to this, multi-potent progenitors (MPP) median age was 10 years (range 0.8–18). Conditioning regimens were represent early CD34 developmental stages enriched within myeloablative (n = 11) and reduced intensity (n = 10). Fifteen BM recipients the CD45RA−CD133+CD38lowCD10− cell fraction most of which had received antithymocyte globulin. PBSC samples (n = 35) were from 19 autologous stem cell collections from adult patients (22 males, 13 females) dividing asymmetrically. Tracking CD133 segregation in with multiple myeloma (n = 19) and Non-Hodgkin's lymphoma (n = 16), functional single cell analysis, the authors showed that MPP with a median age of 54 years (range 22–73). The mobilization regimen create pairs of daughter cells, the lymphoid-primed multi-potent comprised Cht (chemotherapy) and hGF (hematopoietic growth factors) progenitors (LMPP) and the erythro-myeloid progenitors (EMP). n = 13), Cht+hGF and plerixafor (n = 6), hGF alone (n = 5) or hGF and Whereas LMPP upregulate CD45RA and remain CD133+ (CD45RA+ plerixafor (n = 11). Informed consent was obtained from all patients and

1Children's Cancer Research Institute, Vienna, Austria; 2St. Anna Children's Hospital, Department of Pediatrics, Medical University, Vienna, Austria and 3Department for Blood Group Serology and Transfusion Medicine, Medical University, Vienna, Austria. Correspondence: Dr G Fritsch, Clinical Cell Biology and FACS Core Unit; Children's Cancer Research Institute, Zimmermannplatz 10, Vienna 1090, Austria. E-mail: [email protected] Received 28 October 2015; revised 19 February 2016; accepted 25 February 2016; published online 4 April 2016 Multi-color analysis deﬁnes distinct CD34 subsets J Dmytrus et al 1094

CD45RA- + CD133 MPP Multi- potent CD38low progenitors CD10-

CD45RA+ CD45RA- CD133+ CD133-/low Lympho- LMPP EMP Erythro- myeloid myeloid + progenitors CD38low CD38 progenitors - CD10- GMP CD10 CD38+ CD10- Granulocytes Multi- MLP and lymphoid macrophages CD38+ EoBP MEP progenitors progenitors CD10+

CD45RA+ CD133- BLP Late GMP Eosinophils B-lymphoid CD38++ Megakaryocytes + basophils progenitors CD10+ CD38 erythrocytes CD19+ CD10-

B-lymphocytes Neutrophils monocytes Figure 1. Model of the human hematopoietic tree, adapted from the recent version by Görgens et al.17 Only CD34+ cell stages are depicted. The CD45RA−CD133+CD38lowCD10− multi-potent progenitors (MPP, upper) either remain CD45RA− and downregulate CD133 (middle right) to form cells of the erythro-myeloid lineage (EMP), or they acquire CD45RA and become CD133+ lympho-myeloid progenitors (LMPP, middle left) that also comprise neutrophil and monocyte precursors (GMP). Upon downregulation of CD133 (lower left), these form late GMP and, in case of CD10 acquisition, cells of the B-lymphoid lineage (BLP).

the extended staining experiments had been approved by the local ethical According to the ISHAGE guidelines,4 viable and true HSPC were committee. determined by their positivity for CD34, their weak expression of CD45, their typical position in the lympho-monocytic area of the FSC/SSC dot plot and their negativity for 7-AAD. To define subpopulations, CD34+ cells were Cell processing and immune staining − + first divided into an earlier CD45RA and a more committed CD45RA cell Blood cell counts were obtained from a Sysmex KX-21N (Sysmex fraction. These were then separately depicted in a CD133 vs CD10 contour Corporation, Kobe, Japan). If necessary, cells were diluted with Dulbecco's plot (Figure 2, Supplementary Figure 1D). The resulting subpopulations 6 PBS (Carlsbad, CA, USA) and the WBC adjusted to 5–15 × 10 cells/mL prior were examined for their expression of CD38, CD33, CD10 and CD7. Beads to immune staining. All monoclonal antibodies were used at pretested were double-gated in two different dot plots (APC vs SSC and APC-Cy7 vs concentrations and after respective compensation. Isotype controls and FITC) to exclude false-positive events, and the following formula was used fluorescence-minus-one analyses were used to define gating and to calculate the number of target cells/μL: compensation. total number ofbeads per tube events of target population sample dilution factor x x number of beads counted sample volumeðÞμL 1 Single platform protocol To calculate absolute cell numbers in donor BM, an additional dilution In total, 100 μL of the cell sample were reverse-pipetted into a Trucount factor of 1.1 was considered to compensate for the anticoagulant added. tube (BD Biosciences, San Jose, CA, USA). After adding the monoclonal antibodies cocktail, cells were mixed and incubated light-shielded at room temperature for 20 min. RBCs were lysed by adding 2 ml of ammonium Statistical analyses chloride working solution (BD Biosciences) for 10 min before samples were Differences in cell counts (both absolute and relative) between the three analyzed on the flow cytometer. The monoclonal antibody cocktail HSPC sources were assessed with the unpaired one-sided Wilcoxon contained the stem cell enumeration kit (BD Biosciences) comprising signed-rank test. A P-value o0.05 was considered statistically significant CD45 FITC, CD34 PE and 7-AAD, as well as the following monoclonal (*o0.05, **o0.01, ***o0.001). Mean values (± s.d.) are provided in the antibody: AC133-1 APC, CD7 PeCF594, CD10 BV421, CD19 APC-Cy7, CD38 text. In depicted Box-and-Whisker plots, boxes range from first to third PE-Cy7, CD45RA BV510, CD3 PerCPeFl710, and CD33 APC-R700. For quartile (containing 50% of data points). The median value is indicated by detailed information please see Supplementary Table 1. a thick horizontal line. Whiskers extend to the most extreme data point, which is o1.5 times the interquartile range ( = box height) away from the box, and indicate the range that contains 95% of data points in a normally Flow cytometry distributed sample. R version 3.2.0 (2 015-04-16) was used for all statistical Acquisition of 150 000 CD45+events was done on a FACS Fortessa analyses. (BD Biosciences) equipped with four solid state lasers with excitation wave lengths (nm) of 488, 405, 561 and 640. The FACSDiVa 6 software (BD Biosciences) was used for cell acquisition and data evaluation. RESULTS For quality control of the instrument’s performance, CS&T beads Enumeration of total HSPC irrespective of CD34 subtypes revealed (BD Biosciences) were used at least weekly. significant differences between the three cell sources (Figure 3a). Results were expressed as absolute numbers (viable CD34/μL) and Gating strategy as relative values (viable CD34+ cells as percentage of viable WBC). + Viable WBC were defined by their CD45 expression, negativity for 7-AAD, The mean percentage ( ± s.d.) of total CD34 cells was highest in and typical position in the forward- and side scatter (FSC/SSC) dot plot. BMd (2.1% ±2), followed by BM1y (1.1% ± 0.6) and by autologous

105 PBSC BLP 104 MLP 103 105 late CD45RA+ LMPP+GMP GMP 0 104

-472 2 0 103 104 105 103 -807 CD45RA- 5 102 10 104 2 3 4 5 3

CD45RA 10 10 10 10 10

EMP 0 MPP CD34 1 -472 0 103 104 105 -807

105 5 BMd BLP 104 MLP 3 5 10 10 late CD45RA+ LMPP+GMP GMP 0 104 2 -472 0 103 104 105 103 -807 CD45RA- 5 102 10 104 2 3 4 5 3

CD45RA 10 10 10 10 10 EMP 0 MPP CD34 1 -472 0 103 104 105 -807

105 BM1y BLP 104 MLP 3 5 10 10 late CD45RA+ LMPP+GMP GMP 0 104 -472 0 103 104 105 103 -807 CD45RA- 5 102 10 104 2 3 4 5 3 CD45RA 10 10 10 10 10 EMP MPP CD34 0 -472 103 104 105 -807 0 CD10

CD133 Figure 2. Multi-color CD34 subtype analysis: representative examples of autologous PBSC (top), donor bone marrow (BMd, middle) and patient bone marrow 1 year after allogeneic transplantation (BM1y, bottom). Only viable and true CD34+ events are depicted (see Supplementary Figure 1 for the gating strategy). Cells were ﬁrst separated into fractions negative or positive for CD45RA (larger plots). The proportion of earlier CD45RA− progenitors was generally higher among PBSC than BMd and, particularly, BM1y cells. All CD34+CD45RA+ or CD34+CD45RA− events are shown in CD133 vs CD10 contour plots, where they form the CD45RA−CD133+CD10− MPP, the CD45RA+CD133+ CD10− LMPP, the CD45RA−CD133−CD10− EMP, the CD45RA+CD133−CD10− late GMP, the CD45RA+CD133+CD10+ MLP and CD45RA+CD133− CD10+ BLP.

PBSC (0.8% ± 0.9). In terms of absolute CD34 numbers, the highest the pediatric patients had been allografted with BMd, only few values were obtained in PBSC (1402/μL ± 1049), followed by BMd PBSC samples had been obtained and analyzed from healthy (794/μL ± 753) and by BM1y (255/μL ± 253). The majority of PBSC mobilized donors, but their comparison revealed a similar samples described in the present work was from adult patients distribution of CD34 subtypes in patient and donor PBSC (data diagnosed with different lymphoid malignancies. Since most of not shown).

a Total CD34 absolute Total CD34 relative

5 *** ** 4000 ** 4 ** 3000 3

2000 2 % of WBC CD34 cells/µl 1000 1

0 0 PBSC BMd BM1y PBSC BMd BM1y

b 2000 *** *** *** ns *** *** *** ** ns ***

1500

*** *** *** ** ns 1000 Cells/µl

500

0 MPP EMP LMPP+GMP Late GMP MLP+BLP

*** *** *** *** *** 80 **** ns *** *** ***** *** ns ***

40 % of CD34+

0 MPP EMP LMPP+GMP Late GMP MLP+BLP Figure 3. Comparison of median absolute and relative values of (a) total CD34+ cells and (b) CD34 subpopulations in autologous PBSC (n = 35), donor bone marrow (BMd, n = 31) and patient bone marrow one year after allogeneic transplantation (BM1y, n = 21). For total CD34+ cells (a), absolute numbers (left) were significantly higher in PBSC (open circles) than in BMd (gray), and higher in BMd than in BM1y (black), whereas relative values (right) were highest in BMd and lowest in PBSC. Regarding the CD34 subgroups (b), absolute numbers (upper graph) of MPP, EMP and LMPP were significantly higher in PBSC than in BMd, and higher in BMd than in BM1y. Relative values (lower graph) of MPP and EMP were also significantly more frequent in PBSC than in BMd, and more frequent in BMd than in BM1y, and LMPP fractions were higher in BMd than in BM1y. This was in contrast to late GMP, which were clearly higher in BMd than PBSC, and particularly to the more mature MLP and BLP subsets, which were much more frequent in BMd and BM1y than in PBSC, showing the highest proportions (mean 59%) in BM1y.

CD34 subtyping was always started with CD45RA, as this marker PBSC. Analogous results were obtained for absolute MPP numbers, allows definition of two distinct subgroups in all materials, which were significantly higher in PBSC (608/μL; ± 475) than in separating earlier CD45RA− from more committed CD45RA+ HSPC BMd (138/μL ± 156; Po0.001), and in BMd than in BM1y (5/μL±6; (Figure 2, Supplementary Figure 1). Both subpopulations were Po0.001). then further evaluated in CD133/CD10 contour plots where they MPP differentiate to either LMPP (CD45RA+CD133+) or EMP formed at least six distinct CD34 subpopulations. Between the (CD45RA−CD133− /low). Both subsets showed a higher CD38 three cell sources analyzed, we observed considerable differences expression than MPP supporting their higher differentiation regarding the composition of CD34 subsets, both for absolute cell (Figure 4). As shown in Figure 3b, the mean frequency of LMPP numbers and relative values (Figure 3b). The mean cell proportion was similar between PBSC and BMd (25.6% ± 11.1, and (in % CD34+ cells ± s.d.) of the early MPP cells was significantly 23.7% ± 10.1; ns), but significantly lower in BM1y (16.3% ± 8.3) higher in PBSC (42% ± 13.7) than in BMd (16% ± 8; Po0.001). In than in BMd (Po0.01). Absolute LMPP cell numbers differed BM1y, their frequency was only 2.5% ± 1.8, which was significantly significantly between PBSC (383/μL ± 412) and BMd (170/μL ± 158; lower than in BMd (Po0.001) and roughly 17-fold less than in Po0.01), and between BMd and BM1y (33/μL ± 28; Po0.001).

Bone Marrow Transplantation (2016) 1093 – 1100 © 2016 Macmillan Publishers Limited, part of Springer Nature. Multi-color analysis defines distinct CD34 subsets J Dmytrus et al 1097 BM1y (171/μL ± 191; ns). Out of the 31 BMd and 21 BM1y CD34 subset MFI specimens analyzed, six were paired samples, that is, we examined BMd and BM1y pairs from the same patients. The results were virtually identical to those obtained from the whole groups: MPP 18.191 The median proportions of the CD34 subsets for BMd/BM1y were 12.1%/1.8% (MPP), 22%/17.4% (LMPP), 20.1%/10% (EMP), 5.5%/9.1% (late GMP) and 34%/65.2% (MLP and BLP). The expression intensity of distinct markers often correlates LMPP 46.974 with differentiation as shown for CD38 (see below). Such differences were also observed for CD33 and CD133. Expression of CD133 on MPP and LMPP was generally higher in PBSC than in BMd and BM1y (not depicted), suggesting that these cell stages EMP 66.165 are more differentiated in BM than in PBSC. Nevertheless, it was possible to distinguish the different CD133+/ − subpopulations in all cell samples (Figure 2). The myeloid marker CD33 was expressed in all CD34 subfractions, although it was weaker on Late GMP 71.427 MPP in PBSC with high CD133 expression than on MPP in BM with weaker CD133 expression (not depicted). Distinct CD10+ (and CD19+) HSPC subsets were detectable among both the CD45RA+ and the CD133− progenitors, and the CD33 expression was clearly higher in BM1y than PBSC (Figure 5). In all cell sources, BLP 96.670 a potential co-expression of CD7 as a marker of T- and NK-cell progenitors was only seen on 0–2.8% of CD34+ cells (see CD38 Supplementary Figure 1 D3). As non-specific staining could not Figure 4. Rise of CD38 mean fluorescence intensity (MFI) values be excluded, this subtype was not pursued any further. on HSPC with increasing differentiation of CD34+ subsets. We used the PE-Cy7-labeled H7 clone of CD38 in all Representative bone marrow sample from a patient 1 one year experiments performed. Virtually, all CD34+ cells were positive − + after allogeneic transplantation (BM1y) depicting CD45RA CD133 for this Ab, albeit at clearly different intensity (Figure 4). MPP CD10− multi-potent progenitors (MPP), CD45RA+CD133+CD10− − showed the lowest expression intensity, followed by LMPP and lymphoid-primed multi-potent progenitors (LMPP), CD45RA + − − + − EMP. The intensity was higher among CD45RA late myeloid CD133 CD10 erythro-myeloid precursors (EMP), CD45RA CD133 + + + CD10− late granulocyte monocyte progenitors (late GMP) and precursors, and highest among the CD45RA CD10 CD19 BLP. CD45RA+CD133−CD10+ B-lymphoid progenitors (BLP). See This differential CD38 expression was generally observed in all Supplementary Figure 1 for the gating strategy. specimens examined although the differences were not always as clear as depicted in the BM1y sample shown in Figure 4 (see also Supplementary Figure 2). Owing to the considerable overlap This may suggest a higher proportion of neutrophil progenitors in between the different CD34 subgroups, CD38 was never used as favor of BLP in the LMPP subfraction of PBSC compared with BMd. first-line Ab for subgroup discrimination. EMP that give rise to erythrocytes, megakaryocytes and granulocytes other than neutrophils, showed results comparable to those obtained for LMPP. Their mean frequency was similar in DISCUSSION PBSC (24.5% ± 8.7) and BMd (19.5% ± 6.5; Po0.05) but differed Based on previously presented data,13,14,17–22 we extended our significantly between BMd and BM1y (11.9% ± 7.3; Po0.001). recently described five-color CD34 enumeration protocol23 to an Absolute EMP numbers differed clearly and were 331/μL ± 261 for 11-color single-platform analysis tool, which we used to describe at PBSC vs 162/μL ± 176 for BMd (Po0.001), and 24/μL ± 25 for BM1y least six distinct CD34 subpopulations differing in terms of vs BMd (Po0.001). In terms of LMPP and EMP, BM1y thus maturation und lineage commitment. We have thus reenacted mediates an impression of exhaustion when compared with BMd. and translated into clinical routine application what the above Late myeloid progenitors (late GMP) with a CD45RA+CD133 − authors described in their extensive functional analyses, and we CD33+CD10− phenotype differed mainly with regard to relative show that the composition of the CD34 subsets differs substantially values, which were significantly lower in PBSC (4% ± 3.1) than in between the three CD34 sources examined. We assume that BMd (8.5% ± 4.1; Po0.001), but similar between BMd and BM1y routine application of the presented protocol will allow high from (11.1% ± 6.5; ns). In terms of absolute values, they were 51/μL±62 medium or poor quality transplant materials to be distinguished in PBSC vs 72/μL ± 91 in BMd (ns), and slightly lower in BM1y and may thus predict engraftment characteristics as addressed by (23/μL ± 20) than in BMd (Po0.01). Owing to the low frequency of several authors,6–8,15,24 but this will have to be proven in future the CD133+ MLP, this CD34 subset was evaluated together with studies. The present results show clearly that PBSC contain the CD133− BLP. These cells were hardly detectable in PBSC significantly higher proportions of MPP, LMPP and EMP than BMd. (5% ± 7.9) but clearly present in BMd (31.9% ± 15.1; Po0.001). This may be the reason for the more rapid sustained engraftment In BM1y, they represented the largest CD34 subfraction after infusion of PBSC instead of BM.25,26 In BM1y, these earlier CD34 (58.5% ± 17.6), which was significantly higher than in BMd subtypes are again significantly lower than in BMd. In contrast, the (Po0.001). more committed CD34 stages, that is, MLP and particularly BLP, are Despite the rather low proportions of the CD133dim MLP in the significantly higher in BM1y than in BMd, whereas they are hardly BM samples, it has to be noted that this CD34 subset, when present in PBSC. Our data may be biased by the fact that the PBSC expressed as percentage of the MLP/BLP fraction, represented a were from autologous patients with a median age of 54 years, clearly lower median cell proportion (P = 1.66e-09; one-sided whereas the BMd specimens were from younger and healthy unpaired Wilcoxon rank sum test) in BM1y (3.6%) than in BMd donors (median age 26 years). However, one can assume that PBSC (11.9%). A representative example is depicted in Figure 2. In terms from younger and healthy donors (or from cord blood samples) of absolute CD10+ stem cell numbers, significant differences were display a more immature rather than a more committed phenotype observed between PBSC (43/μL ± 83) and BMd (255/μL ± 302; thus making the difference between PBSC and BM early CD34 Po0.001), whereas the values were similar between BMd and subtypes rather larger than smaller.

105 105

104 104

PBSC 103 103

102 102

0 0

3 4 5 3 4 5 -472 0 10 10 10 -472 0 10 10 10

105 105

104 104

BM1y 103 103

102 102 CD33

0 0

3 4 5 3 4 5 -472 0 10 10 10 -472 0 10 10 10

CD34+ CD133+ CD34+ CD133-

105 105

104 104 BM1y 103 103

102 102

0 0

3 4 5 3 4 5 -472 0 10 10 10 -472 0 10 10 10 CD10 Figure 5. Multi-color CD34 subtype analysis: CD10 vs CD33 expression of CD45RA and CD133 subsets in PBSC and BM1y. Only viable and true CD34+ events are depicted (see Supplementary Figure 1 for the gating strategy). The CD10 vs CD33 dot plots depict all CD34+ cells positive or negative for CD45RA or CD133 (as indicated). CD10+ (and CD19+) B-lymphoid progenitors are present in the CD34+CD45RA+ and CD34+ CD133− HSPC subsets. Expression of CD33 is higher in BM1y than in PBSC, and higher among CD45RA+ than CD45RA− precursors. Similar results were obtained for 10 CD10/CD33 analyses performed.

In normal BM, B-cell precursors may represent a The differences described between PBSC and BMd in terms of considerable CD34 subfraction, which in our BMd CD34 subset composition conﬁrm an earlier observation of higher cohort, accounted for median 32%. These cells represent proportions of CD45RA− progenitors in PBSC than in BM. Cell predominantly (~90%) the more mature CD133−CD10+ sorting and culture in semisolid medium revealed that the CD19+ BLP, but only few MLP still exhibiting a weak CD133 CD45RA− vs CD45RA+ cells formed compact vs dispersed colonies expression. During mobilization, such rather committed originating from earlier and more committed myeloid progenitors, precursors hardly appear in the circulation, which is in contrast respectively.10 A differential mobilization is also supported by the to MPP and EMP of which high proportions enter the blood observation that mobilized PBSC are not or hardly in G2S stream. Reasons for the differential mobilization of the various phase,28,29 which is in contrast to BMd CD34+ cells and, CD34 subsets from the BM into the circulation may be that the particularly, to BM1y cells.30 In 1997, Anderlini & Körbling assumed earlier CD34 subsets show a better stimulation response to the a more primitive phenotype of CD34+ cells contained in PBSC myeloid growth factor G-CSF than late GMP and B-lineage compared with BM.25 Furthermore, they postulated a faster progenitors. However, it can also not be excluded that the earlier engraftment after PBSC transplantation compared with BM progenitors are less adherent to the BM niche because they allografting as described before.26 This was later conﬁrmed by prevail not only in mobilized PBSC but also in non-mobilized different authors, who reported no differences in both overall and blood or in cord blood.18,27 event-free survival between PBSC and BM, but observed a clearly

Bone Marrow Transplantation (2016) 1093 – 1100 © 2016 Macmillan Publishers Limited, part of Springer Nature. Multi-color analysis defines distinct CD34 subsets J Dmytrus et al 1099 faster neutrophil and platelet engraftment after infusion of cell source. This approach may provide a solid basis for future – PBSC.31 33 The most likely reason for this is the higher number studies to determine the impact of different CD34 subsets in the of LMPP and EMP giving rise to neutrophil- and platelet-forming graft as well as in post-transplant BM on engraftment kinetics and 17 cells, respectively. It is also noteworthy that the GMP which form immune reconstitution. Whether or not the analysis will allow monocytes and neutrophils, are contained in the LMPP cohort, prediction of engraftment kinetics on a routine basis remains to fi which, in BM, contains signi cantly more B-lineage progenitors. be examined. The relative fraction of neutrophil progenitors should therefore be clearly larger in PBSC than in BM although similar frequencies of LMPP (which can give rise to both neutrophil and B-lineage CONFLICT OF INTEREST precursors) were found in these two stem cell sources. The authors declare no conflict of interest. CD38 was used by several authors to distinguish earlier from more committed CD34 cell stages.24,34,35 Despite a relatively high expression on all HSPC subsets, its mean fluorescence intensity ACKNOWLEDGEMENTS values were clearly lower on CD133+ HSPC subtypes than on more We would like to thank Daniela Scharner, Dijana Trbojevic and Elke Zipperer for their committed CD34 stages. However, owing to the substantial excellent commitment and input when establishing the described flow cytometric overlap between the different CD34 subtypes, CD38 could never assay, and for their continuous support with cell preparation and data acquisition and be used to unambiguously separate early from more committed evaluation. Dieter Printz is particularly acknowledged for his technical support in all progenitors. It was therefore important that CD34 subtyping was issues of flow cytometry. started with CD45RA and followed by CD133 and CD10. When the resulting subsets were then analyzed for their CD38 mean fluorescence intensity, the gradational increase became obvious, REFERENCES ranging from the lowest expression among the young MPP, over 1 Siena S, Bregni M, Brando B, Belli N, Ravagnani F, Gandola L et al. Flow cytometry LMPP and EMP to the highest among BLP. for clinical estimation of circulating hematopoietic progenitors for autologous One year after allogeneic BM transplantation, MPP accounted transplantation in cancer patients. Blood 1991; 77:400–409. + for only 2.5% of CD34 cells in BM1y, which is ~ 6 times and 17 2 Siena S, Bregni M, Brando B, Ravagnani F, Bonadonna G, Gianni AM. Circulation of times lower than in BMd and PBSC, respectively. In contrast, MLP CD34+ hematopoietic stem cells in the peripheral blood of high-dose and BLP in particular represented almost 2/3 of CD34+ cells in cyclophosphamide-treated patients: enhancement by intravenous recombinant BM1y, which is significantly more than in BMd. One possible human granulocyte-macrophage colony-stimulating factor. Blood 1989; 74: explanation for the marked relative and absolute loss of MPP may 1905–1914. ‘ ’ 3 Sutherland DR, Anderson L, Keeney M, Nayar R, Chin-Yee I. The ISHAGE guidelines be the replicative stress . 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