Mobilized Peripheral Blood Stem Cells Provide Rapid Reconstitution but Impaired Long-Term Engraftment in a Mouse Model

ORIGINAL ARTICLE Mobilized peripheral blood stem cells provide rapid reconstitution but impaired long-term engraftment in a mouse model

JSG Yeoh, A Ausema, P Wierenga, G de Haan and R van Os

Department of Cell Biology, Section Stem Cell Biology, University Medical Centre Groningen, Groningen, The Netherlands

In this study, we use competitive repopulation to compare become the primary source of hematopoietic stem cells for the quality and frequency of stem cells isolated from clinical transplantation over the past 10 years.4 mobilized blood with stem cells isolated from bone marrow Although multiple hematopoietic growth factors are (BM) in a mouse model. LinÀSca-1 þ c-Kit þ (LSK) cells capable of inducing mobilization of hematopoietic pro- were harvested from control BM and peripheral blood of genitors, granulocyte colony-stimulating factor (G-CSF) is mice following granulocyte colony-stimulating factor (G- at present the most used mobilizing molecule in clinical CSF) administration. LSK cells were used because oftheir protocols.5 The ability of G-CSF to mobilize BM resemblance to human CD34 þ cells. We confirmed that precursors and long-term repopulating cells was initially transplantation ofphenotypically defined mobilized per- shown in preclinical studies. Molineux et al.6 observed a ipheral blood (MPB) stem cells results in rapid recovery of marked increase in the colony-forming unit spleen pool in blood counts. However, in vitro results indicated that LSK the peripheral blood of mice treated with repeated doses of cells purified from MPB had lower cobblestone area- G-CSF. In addition, it was observed that G-CSF alone and forming cell day 35 activity compared to BM. Addition- in combination with stem cell factor or interleukin-7 ally, evaluation ofchimerism after co-transplantation of mobilizes hematopoietic precursors capable of both radio- LSK cells purified from blood and BM revealed that MPB protection and generating sustained lymphohematopoiesis stem cells contained 25-fold less repopulation potential in transplanted recipients.7 In vitro data from human compared to BM stem cells. Competitive repopulating patients appear consistent with the concept that the quality unit frequency analysis showed that freshly isolated MPB of human mobilized peripheral blood (MPB) progenitor LSK cells have 8.8-fold fewer cells with long-term cells is at least equivalent to that corresponding to BM repopulating ability compared to BM LSK cells. Second- grafts.8,9 ary transplantation showed no further decline in contribu- Surprisingly, despite the prevalent use of hematopoietic tion ofhematopoiesis relative to BM. We conclude that stem cell mobilization in clinical transplantation, few the reduced frequency of stem cells within the LSK reports exist describing the competitive repopulating population ofMPB, rather than poorer quality, causes the quality of mobilized stem cells compared to BM stem cells reduced repopulation potential. following G-CSF treatment. Most available reports only Bone Marrow Transplantation (2007) 39, 401–409. outlined the differences in the kinetics and efficiency of doi:10.1038/sj.bmt.1705601; published online 12 February 2007 engraftment, in homing properties and in cell cycle profiles Keywords: granulocyte colony-stimulating factor; mobi- between mobilized blood stem cells and those isolated from lized peripheral blood stem cells; bone marrow stem cells; resting BM.10–13 competitive repopulating unit; long-term engraftment In view of the increased use of peripheral blood stem cells in clinical transplant settings, it is of relevance to investigate long-term functioning of stem cells isolated from different sources. In this study, we directly assessed the function of MPB stem cells compared to control BM Introduction stem cells when co-transplanted in a single recipient mouse in an in vivo competitive repopulation assay. We show a The initial source of hematopoietic cells used for trans- 1 reduced frequency of repopulating stem cells in purified plantation was bone marrow (BM). However, owing to the G-CSF-MPB LinÀSca-1 þ c-Kit þ (LSK) stem cells, which faster regeneration of both circulating neutrophils (9–11 2,3 caused a 25-fold reduction in repopulation potential days) and platelets, peripheral blood stem cells have compared with BM LSK cells.

Correspondence: Dr R van Os, Department of Cell Biology, Section Materials and methods Stem Cell Biology, University Medical Centre Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands. E-mail: [email protected] Mice Received 14 September 2006; revised 29 November 2006; accepted 5 Female C57BL/6 (B6), C57BL/6.SJL (CD45.1), (C57BL/ January 2007; published online 12 February 2007 6 Â C57BL6.SJL) F1 (CD45.1/2) or C57BL/6-Tg(ACTB- MPB stem cells have impaired long-term engraftment JSG Yeoh et al 402 EGFP)10sb/J transgenic GFP (GFP) mice were used as national IBL 637 137Cs-source, 20–24 h before transplant donors, competitors or recipients of blood and marrow ation. For competitive repopulation determination, stem cells depending on the experimental model. CD45.1 unfractionated cells or MPB LSK cells, were mixed with and transgenic GFP mice were originally obtained from the competitor cells (unfractionated or LSK BM cells) and Jackson Laboratory (Bar Harbor, ME, USA) and bred in intravenously transplanted into recipient mice. Each our local animal facility. Wild-type female B6 mice were transplant group consisted of 8–10 recipients. Following purchased from Harlan (Horst, The Netherlands) and transplantation, blood samples (60 ml) were taken monthly maintained under clean conventional conditions in the to determine donor chimerism. Levels of chimerism animal facilities of University Medical Centre Groningen were determined by detecting the presence of GFP þ or (The Netherlands). Mice were fed ad libitum with food CD45.1 þ and CD45.2 þ cells in transplanted mice. To pellets and acidified tap water (pH ¼ 2.8). All animal detect CD45.1 þ and CD45.2 þ cells, cells were stained with procedures were approved by the local animal ethics anti-CD45.2 (FITC) and anti-CD45.1 (PE) antibodies (BD committee of the University Medical Centre Groningen. Pharmingen, San Diego, CA, USA) for 30 min and analyzed on a flow cytometer (fluorescence-activated cell Mobilization and harvesting of stem cells sorter (FACS) Calibur; Becton Dickinson Biosciences, San BM cells were harvested by flushing the femoral shaft with Jose, CA, USA). In addition, the competitive repopulating a-Modified Eagle’s Medium (GibcoBRL, Invitrogen, CA, index (CRI) was determined. CRI is a relative measure of USA) supplemented with 2% fetal calf serum (FCS; the competitive ability of test cells to that of fresh BM cells. GibcoBRL, Invitrogen, CA, USA). Pegylated G-CSF The CRI was calculated by taking the ratio of white blood (Neulasta) (Amgen, Thousand Oaks, CA, USA) (250 mg/ cell (WBC) derived from mobilized blood cells to BM cells kg/mouse) was used to mobilize stem cells.14 Two doses of in the circulation and dividing it by the ratio of mobilized G-CSF were subcutaneously administered to donor mice at blood cells to BM cells transplanted. A CRI value of one day À6 and day À3 before stem cell harvest. MPB cells indicates by definition that MPB cells and BM cells have were harvested by cardiac puncture. Approximately 1 ml of equal competitive ability. blood was collected and was diluted with 4 ml of Iscove’s modified Dulbecco’s modified Eagle’s medium (IMDM; CRU assay GibcoBRL, Paisley, Scotland) supplemented with 5% FCS B6-recipient mice were transplanted with a series of diluted (GibcoBRL, Invitrogen, CA, USA) and heparin (25 IU) CD45.1 LSK cells (1200, 600 and 300) from mobilized (Leo Pharma, Breda, Netherlands). The collected blood cell blood and control BM and with a fixed number of B6 suspension (5 ml) was centrifuged over an equal volume of competitor cells (5 Â 105). Twelve weeks after transplant- Lympholyte-M (Cedarlane Laboratories Ltd, Hornby, ation, donor cell contribution in the peripheral blood was Canada) at 400 Â g for 30 min at room temperature. After determined. Recipients with a contribution of X5% in both centrifugation, the mononuclear cells within the opaque myeloid and lymphoid lineages were considered to be interface layer were isolated and washed in IMDM/5% positive. FCS for 5 min at 2000 r.p.m. at 41C. Alternatively, red To evaluate and quantify the repopulating potential of

blood cells were lysed using ammonium chloride (NH4Cl) mobilized blood LSK cells and control BM LSK cells, the without prior density separation. Nucleated cells were frequency of competitive repopulation units (CRU) was measured on a Coulter Counter Model Z2 (Coulter calculated. CRU frequencies per 1000 LSK were calculated Electronics, Hialeah, FL, USA). from the resultant percentage positive recipients by limiting dilution analysis procedures which uses Poisson statistics.16 Isolation of LSK cells BM and MPB cells were stained as described previously15 CAFC assays with biotinylated lineage-speciﬁc Mouse Lineage Panel CAFC assays were performed as described17–19 to assess the antibodies, containing anti-CD45R, anti-CD11b, anti- number of hematopoietic progenitor cells (CAFC day 7) or TER119, anti-Gr-1 and anti-CD3e (BD Pharmingen, San more primitive stem cells (CAFC day 35) in MPB Diego, CA, USA), ﬂuorescein isothiocyanate (FITC)-anti- stem cells. Sca-1 and APC-anti-c-kit (BD Pharmingen, San Diego, CA, USA). Biotinylated antibodies were visualized with Secondary transplantations streptavidin–phycoerythrin (PE) (Pharmingen, San Diego, In one of the competitive repopulation experiments, in CA, USA). After antibody staining, cells were sorted by a which recipients were transplanted in different ratios with MoFlow cell sorter (DakoCytomation, Fort Collins, CO, CD45.1 MPB LSK cells and CD45.2 BM LSK competitor À þ þ USA). LSK and Lin non-Sca-1 c-Kit cells were sorted cells, mice were killed for secondary transplantation. BM and used in transplantation assays or in in vitro cobblestone cells from B6 primary chimeric recipients were isolated on area-forming cell (CAFC) assays. the basis of CD45 isoforms. CD45.1 BM cells represent cells derived from MPB LSK population (CD45.1 MPB Long-term competitive repopulation ability LSK-derived BM) and CD45.2 (B6) BM cells represent Female B6, CD45.1 or transgenic GFP mice were used as cells derived from BM LSK cells (CD45.2 BM LSK-derived donors for competitor cells. Female B6 mice were used as BM). Female CD45.1-recipient mice were transplanted recipients in all experiments. Recipient mice were irradiated with CD45.2 BM LSK-derived BM cells whereas B6- with 9.5 Gy g-rays (0.7026 Gy/min) in a CIS Biointer- recipient mice were transplanted with CD45.1 MPB LSK-

Bone Marrow Transplantation MPB stem cells have impaired long-term engraftment JSG Yeoh et al 403 derived BM cells. Recipient mice were transplanted in three a 10000 5 ratios (1:1, 1:2 and 1:4) with a ﬁxed number (4 Â 10 )of cells CD45.1 control BM 6 1000 CD45.1/2 (F1) competitors. Each transplant group con- GFP control BM sisted of ﬁve recipients and blood samples (60 ml) were 100 MPB taken on a monthly basis to determine donor chimerism and CRI. 10

CAFC frequency/10 1 735 Statistical analysis Days P-values were calculated using the Mann–Whitney test or b MPB + CD45.1 control BM the Student’s t-test (assuming unequal variances of the two 2 MPB + GFP untreated BM variables) and were employed to determine the statistical 1.8 CD45.1 control BM + GFP control BM 1.6 significance between mobilized blood and normal BM 1.4 (Po0.05). Quantification of CAFC at days 7 and 35 was 1.2 20 1 performed by using maximum likelihood ratio method. index 0.8 The Poisson-based limiting dilution analysis calculation 0.6 P < 0.05 0.4 was used with a 95% confidence interval (CI) to determine 0.2 significant differences at Po0.05. Competitive repopulation 0 0 5101520 25 30 Weeks after transplant Figure 1 Unfractionated MPB cells have reduced repopulation ability in Results comparison with BM cells. Unfractionated monunuclear cells from MPB were obtained from B6 mice following treatment with two doses of Peg G- Unfractionated MPB cells have reduced repopulation CSF 6 and 3 days before isolation. Control unfractionated BM cells were ability compared to unfractionated BM cells collected from CD45.1 and GFP-transgenic mice and transplanted in equal numbers. (a) Analysis of CAFC day 7 and day 35 activity for CD45.1 BM, To compare the repopulation ability of unfractionated BM GFP BM and MPB. (b) CRI analysis of mice transplanted with B6 and unfractionated MPB cells, blood cells were extracted MPB þ CD45.1 control BM, B6 MPB þ GFP control BM and CD45.1 from donor B6 mice following treatment with two doses of control BM þ GFP control BM. All mice were transplanted with 2 Â 106 6 G-CSF. Untreated BM cells were obtained from GFP-Tg donor cells and 2 Â 10 competitor cells. Average chimerism levels at 20 weeks were 42718% (GFP BM þ CD45.1 BM), 20719% (B6 or CD45.1 mice. Clonogenic activity of the two cell sources MPB þ CD45.1 BM) and 16721% (B6 MPB þ GFP BM). All chimerism was measured by seeding in limiting dilutions in a CAFC levels were converted into CRI values as indicated in Materials and assay. At day 7, cells from each source displayed high methods. CAFC activity, with average values of B1400 progenitors/ 106 cells (95% CIs: 908–2294). No significant differences were observed between BM and peripheral blood BM cells may underestimate the difference in repopulating (Figure 1a). Although BM cells isolated from either ability. CD45.1 or GFP Tg mice displayed a high CAFC day 35 activity of 10.5 (95% CI: 6.6–16.8)/106 and 16 (95% CI: 10– 24)/106, respectively, CAFC day 35 frequency in MPB was MPB cells have a lower frequency of LSK cells only 1.8 (95% CI: 1–3.3)/106 (Figure 1a). These results We next determined whether the poor repopulation ability indicate that unfractionated MPB cells had significantly of unfractionated MPB cells is reflected by the size of the lower primitive stem cell activity compared to BM cells. phenotypically defined stem cell population, LSK. To test To verify the in vitro results, three groups of lethally this possibility we measured LSK frequencies in BM cells irradiated mice were transplanted in a competitive repopu- and MPB. Typical FACS plots for isolating LSK cells from lation assay. Donor chimerism levels of MPB cells were BM and MPB cells are shown in Figure 2a and b. MPB used to calculate CRI post transplant (Figure 1b) and were differs from BM cells, as they contain fewer c-Kit þ cells B20% (data not shown). As expected, a CRI value of 1 and more Sca-1Àc-KitÀ cells (Figure 2b). This finding is was maintained throughout the 7-month transplant period consistent with that of Levesque et al.,21 who reported that for recipient mice receiving CD45.1 BM mixed with GFP mobilization with G-CSF results in a downregulation of BM, indicating equal competitive repopulating ability of c-Kit on mouse hematopoietic progenitor cells in vivo.On both sources (Figure 1b). However, mice transplanted with average, BM cells have a LSK frequency of 0.1770.1%, MPB in competition with either CD45.1 BM or GFP BM significantly different to that of MPB cells, which have exhibited significantly lower average CRI levels (0.3370.41 a three- to four-fold lower frequency of 0.0570.04% and 0.4170.29, respectively) at 5 months after transplant (Po0.05) (Table 1). The lower frequency in mobilized (Figure 1b). blood may explain the lower repopulation potential of Both in vitro CAFC results and in vivo competitive unfractionated mobilized blood. repopulation assays suggest that unfractionated MPB cells To compare mobilized blood and BM LSK on a per cell have lower repopulation ability compared to unfractio- basis, LSK cells were isolated from BM and MPB and nated BM cells. In addition, no apparent differences were placed in limiting dilution in a CAFC assay to assess observed between different donor mouse strains. The fact clonogenic activity. CAFC day 7 frequency of 250/103 LSK that we used the mononuclear cell fraction from mobilized cells (153–402; 95% CI) was recorded for control BM LSK blood (which is enriched for stem cells) and unseparated cells, whilst MPB LSK cells had a 2.4-fold higher CAFC

Bone Marrow Transplantation MPB stem cells have impaired long-term engraftment JSG Yeoh et al 404 a BL/6 control BM

104 104

3 3 10 10 0.15% 102 102 Lin c-Kit 101 101 5% 100 100 0 1 2 3 0 1 2 3 10 10 10 10 10 10 10 10 10000 Sca-1 Control BM 1000 MPB LSK cells

3 100

10 b BL/6 Mobilized PB 4 4 1 10 10 CAFC /10 735 Days 3 3 10 10 0.04% * 102 102 SSC c-Kit 101 101 3.3% 100 100 100 101 102 103 100 101 102 103 FSC Sca-1

c BL/6 Mobilized PB Lin- non Sca-1+c-Kit+ 10000 104 104 1000 3 3 10 10 cells 3 100 102 102 SSC c-Kit 10 1 1 10 10 1 87% nd 0 0 10 10 CAFC /10 0.1 100 101 102 103 100 101 102 103 735 FSC Sca-1 Days Figure 2 LSK contains the majority of cells with long-term repopulating ability in both BM and MPB. Characteristic FACS plot of LinÀ and Sca-1 þ c-Kit þ sorts for (a)B6BMand(b) MPB cells and comparison in limiting dilution analysis using the CAFC assay. (c) FACS plot of the LinÀ non-Sca-1 þ c-Kit þ sort for MPB cells placed in CAFC assay. *Indicates signiﬁcant difference (see Table 1 for details).

Table 1 Average LSK frequencies in bone marrow and G-CSF fractionated into non-Sca-1 þ c-Kit þ cells and placed in mobilized blood limiting dilution in a CAFC assay (Figure 2c). As shown in Frequency LSK Total LSK cells/2 CAFC day Figure 2c, only some day 7 CAFC activity was detected for À þ þ (%) femurs or per ml blood 35/106 Lin non-Sca-1 c-Kit cells, but no CAFC day 35. This demonstrates that cells outside the LSK gate do not have Control BM 0.1770.1 84837743361 1253779695 clonogenic activity. A summary of LSK frequencies and 7 a 7 7 MPB 0.05 0.04 8968 6377 2150 1064 CAFC day 35 frequencies and a calculation of their total Abbreviations: BM ¼ bone marrow; CAFC ¼ cobblestone area-forming pool size is shown in Table 1. As we did not splenectomize cell; G-CSF ¼ granulocyte colony-stimulating factor; LSK ¼ LinÀSca-1+ our mice, a considerable number of LSK cells may have c-Kit+; MPB ¼ mobilized peripheral blood. been present in the spleen. However, we aimed at Average frequency, total LSK cells and CAFC day 35 frequency in control comparing the phenotypically deﬁned cell population BM (n ¼ 5) and MPB (n ¼ 6). (LSK) from mobilized blood with the same population in aIndicates that LSK frequency is signiﬁcantly (Po0.05) lower compared to normal BM. steady-state BM.

MPB stem cells promote accelerated hematological activity of (600/103 LSK cells (308–1160; 95% CI)) (Figure reconstitution 2a and b). CAFC day 35 frequency for MPB LSK cells was To directly compare the rates of hematopoietic recovery of 3.0/103 LSK cells (1–8; 95% CI), similar to control BM peripheral blood cell values after transplant, 1250 LSK cells LSK cells with a CAFC activity of 6.7/103 (0.9–48; 95% CI) were isolated from BM or MPB and transplanted into two (Figure 2a and b). Thus, these data suggest that, compared groups of lethally irradiated B6 mice. Circulating blood to BM, MPB LSK cells contain somewhat more progenitor cells were counted on days 7 and 10 and then once a week cells, but slightly fewer stem cells. to 5 weeks after transplant. WBC counts of recipients It is well known that in normal BM all stem cell activity transplanted with MPB stem cells increased at day 10 and is contained in the LSK fraction.22,23 To verify that in day 14, reaching normal values of B8 Â 106/ml. At days mobilized blood, stem cell activity is also restricted to 21 and 35, WBC counts were similar to those of animals the LSK population, LinÀ cells from MPB were further transplanted with BM LSK cells (Figure 3). Consistent with

Bone Marrow Transplantation MPB stem cells have impaired long-term engraftment JSG Yeoh et al 405 14 a 30 1250 MPB LSK 750 MPB LSK + 750 control BM LSK 12 25 1250 control BM LSK 1000 MPB LSK + 1000 control BM LSK 10 20 /ml 6 8 15

6 10 % Donor chimerism WBC x 10 4 5

2 0 0 5 10 15 20 25 30 0 Weeks after transplant 0 7 14 21 28 35 42 b Days after transplant 0.6

Figure 3 MPB LSK cells promote faster WBC recovery. Donor mice 1000 MPB LSK + 1000 control BM LSK 0.5 were treated with two doses of Peg-G-CSF 6 and 3 days before donor cell 750 MPB LSK + 750 control BM LSK isolation. Two groups of lethally irradiated mice were transplanted with 250 MPB LSK + 500 control BM LSK 0.4 1250 LSK cells from control BM (n ¼ 10) or 1250 LSK cells from MPB 1600 MPB LSK + 400 control BM LSK (n ¼ 6). Following transplant, blood samples were analyzed for WBC 0.3 counts (WBC Â 106/ml) from 7 days to 12 weeks after transplant.

0.2 the results in Figure 2a and b (showing the presence of 0.1

many progenitors/short-term repopulating cells in blood), Competitive repopulation index 0 these data indicate that transplants with MPB stem cells 0 5 10 15 20 25 30 results in faster recovery of WBC number compared to Weeks after transplant control LSK BM cells. c 0.25

Repopulation ability of peripheral blood stem cells 0.2 To further examine and directly compare the repopulation ability of MPB stem cells to BM stem cells, LSK cells from 0.15 BM and MPB were tested in a competitive long-term repopulation assay in different groups of mice. Recipients 0.1 were transplanted with various ratios of LSK cells from blood and LSK cells from BM. A chimerism level of 50% 0.05 would be expected if both sources contained the same Competitive repopulation index number of stem cells and performed equally well. However, 0 the contribution of cells derived from transplanted MPB 0 5 10 15 20 25 30 was extremely low, with values of 2.673 and 5.674% at Weeks after transplant 28 weeks when transplanted in a 1:1 ratio (Figure 4a). The Figure 4 MPB stem cells have reduced repopulation potential compared CRI of peripheral blood stem cells for each individual to BM stem cells. MPB stem cells were obtained from donor mice treated mouse is plotted in Figure 4b. The average CRI with two doses of Peg G-CSF. Both control BM and MPB were sorted for 7 LSK cells. (a) Donor chimerism of MPB stem cells transplanted in a 1:1 (0.04 0.04) for all transplanted mice indicate that purified ratio with competitor BM cells. (b) Lethally irradiated recipients were MPB stem cells have a severely reduced repopulation transplanted with 1600 LSK MPB þ 400 LSK (n ¼ 5), 1000 LSK potential (25-fold) compared to BM stem cells (Figure 4c). MPB þ 1000 LSK control BM (n ¼ 6), 750 LSK MPB þ 750 LSK control Recipient mice were either female B6 or female CD45.1/2 BM (n ¼ 10) and 250 LSK MPB þ 500 LSK BM (n ¼ 4). Chimerism data was converted into a CRI. CRI values for each individual mouse is shown mice. against weeks post transplant. (c) Average CRI for all transplanted mice (n ¼ 25). Mobilized blood stem cells have a reduced frequency of long-term repopulating stem cells To determine whether the inefficiency of MPB stem cells to LSK cells. Analysis of the frequency of CRU 12 weeks after promote long-term repopulation was due to a decline in transplant revealed that control BM LSK cells are more quality of blood stem cells or that mobilized blood stem competitive in reconstituting the recipient (17 of 18 ¼ 94% cells contained fewer long-term reconstituting stem cells, we contained X5% of engraftment) (data not shown) resulting quantified the frequency of long-term repopulating stem in a significantly (Po0.01) higher frequency of CRU (6.6 cells in mobilized blood and BM stem cells. Mice were CRU/1000 LSK; 1.6–15; 95% CI versus 0.75 CRU/1000 transplanted with CD45.1-mobilized blood or control BM LSK; 0.35–2.8; 95% CI for mobilized blood LSK) LSK cells in limiting dilution (1200, 600 and 300) with a (Figure 5). Similar differences were observed at 38 weeks fixed number (5 Â 105) of B6 competitors BM cells. The after transplantation. A threshold level of chimerism of 2% repopulation ability of both mobilized blood and control resulted in 4.7 (95% CI: 1.8–12.8) CRU per 1000 MPB BM LSK cells was assessed by calculating the CRU/1000 LSK and 35.2 (95% CI: 17.1–72.5) per 1000 BM LSK. This

Bone Marrow Transplantation MPB stem cells have impaired long-term engraftment JSG Yeoh et al 406 indicates that MPB stem cells have a reduced number of were killed 37 weeks after transplant. Donor CD45.1 long-term repopulating cells compared to normal BM. mobilized blood LSK-derived BM cells and CD45.2 BM Taken together, these results indicate that reduced repopula- LSK-derived BM cells were selected, isolated by FACS tion potential is due to lower frequency of stem cells rather sorting and transplanted in limiting dilutions into second- than an impaired function of mobilized blood LSK cells. ary recipients with 4 Â 105 CD45.1/2 competitor cells, respectively (Figure 6a). Average CRI values of recipients receiving CD45.1 blood LSK cells or CD45.2 BM LSK Engrafted MPB stem cells do not exhaust faster than cells are shown in Figure 6b. BM cells originating BM stem cells from LSK blood cells displayed similarly low CRI Recipient mice (CD45.1/2) previously transplanted with a levels (B0.1470.1) as BM derived from BM LSK cells mixture of mobilized blood LSK cells and BM LSK cells (B0.170.1). This indicates that the quality of stem cells from mobilized blood-derived donor did not decrease after 20 transplantation and that exhaustion of initially engrafted 6.6 stem cells is similar for stem cells from both sources. 15

10 Reduced repopulation potential of LSK MPB stem cells in a non-competitive setting 5 CRU/1000 LSK 0.75 To model a more clinically relevant protocol, recipient mice 0 were transplanted with 1250 LSK from pooled BM or 1250 MPB LSK BM LSK LSK from pooled MPB cells without competitor cells Figure 5 Reduced frequency of long-term repopulating stem cells in (Figure 7). Donor chimerism for mice transplanted with mobilized blood. Frequency of CRU/1000 LSK cells with 95% confidence purified BM stem cells displayed little variation, with limits of recipient mice transplanted in limiting dilution (1200, 600, 300) average donor chimerism of 8778.8% 20 weeks after with CD45.1 MPB LSK cells and control bone marrow (BM) LSK cells 5 transplant. In contrast, donor chimerism of mice trans- with 5 Â 10 B6 BM cells. CRU frequency of 0.75 CRU/1000 LSK (0.35– 7 2.8; 95% confidence limit) was observed for MPB LSK group. Control BM planted with MPB LSK cells varied widely (44 39%), with LSK group contained a significant (Po0.01) 8.8-fold higher CRU half of the recipients exhibiting high chimerism levels frequency (6.6 CRU/1000 LSK; 1.6–15; 95% confidence limit). (8074.2%) whereas the other half was below 10%. This

a 1000 CD45.1 MPB LSK + 104 1000 CD45.2 control BM LSK 3% CD45.1 MPB LSK derived BM 103 9Gy 37 weeks, kill 102

Sort BM for CD45.1 PE 86% and CD45.2 CD45.1/2 recipient 101 CD45.2 BM LSK derived BM

100 100 101 102 103 104 FITC

CD45.1 MPB LSK derived-BM + 4x105 CD45.1/2 and 9Gy CD45.2 BM LSK derived-BM + 4x105 CD45.1/2

CD45.1 or B6 recipient

CD45.1 MPB LSK derived-BM cells

CD45.2 BM LSK derived-BM cells b 0.4

0.3

0.2 CRI

0.1

0 0 5 10 15 20 Weeks after transplant

Figure 6 Similar long-term functioning of engrafted mobilized blood and BM stem cells. (a) Primary recipients were transplanted with CD45.1 MPB LSK and CD45.2 BM LSK cells. Thirty-seven weeks after transplant, recipient mice were killed and their BM was isolated and the CD45.1 and CD45.2 fractions were separated. CD45.1 MPB LSK-derived BM and CD45.2 BM LSK-derived BM cells were isolated separated by FACS sorting and transplanted in different ratios (1:1, 1:2 and 1:4) with 4 Â 105 CD45.1/2 F1 competitor cells into lethally irradiated CD45.1 or B6 secondary recipients. (b) Average CRI values of secondary recipients receiving CD45.1 mobilized blood LSK-derived BM cells or CD45.2 BM LSK-derived BM cells.

Bone Marrow Transplantation MPB stem cells have impaired long-term engraftment JSG Yeoh et al 407 a 1250 LSK control BM cells as mobilized blood stem cells have a 8.8-fold decrease in CRU compared to BM stem cells (Figure 5). 100 To compensate for the possible differences in stem cell

80 frequency our experiments were performed using a phenotypically defined stem cell population, LSK. 60 Although LSK cells are not a pure stem cell population, in steady-state BM they contain all cells with long-term þ 40 repopulating ability, very much like CD34 cells in humans.22,23 The overall LSK cell number in MPB was 20 markedly lower than in the BM pool (Table 1). As inter- % Donor chimerism strain variations in stem cell mobilization are reported,24 it 0 is not unlikely that in other mouse strains G-CSF induced 0 5 10 15 20 25 mobilization would lead to higher numbers of LSK cells in Weeks after transplant the circulation. In mobilized blood, fewer c-Kit þ cells were observed. These data are consistent with a downregulation b 1250 LSK MBP of c-Kit on mouse hematopoietic progenitor cells in vivo 100 resulting from proteolytic cleavage as previously re- 21 80 ported. G-CSF administration also causes a change in cell surface marker expression of CD34.25 Importantly, we 60 found no in vitro stem cell activity in cells negative for either c-Kit or Sca-1, documenting that also in mobilized 40 blood all stem cells are contained in the LSK population. Purified stem cells with the same phenotype do not 20 always have the same functional properties. One in five % Donor chimerism Thy-1lowSca-1 þ LinÀ c-Kit þ cells isolated from young BM 0 provides long-term reconstitution following transplant- 0 510152025 26,27 Weeks after transplant ation into irradiated mice. Poorer repopulating efficien- cies were reported for stem cells isolated from other Figure 7 MPB stem cells have reduced long-term repopulation ability in sources. Only one in 10 BM stem cells from old mice a non-competitive setting. (a) Lethally irradiated B6 recipients (n ¼ 7) were engraft and one in 75 cyclophosphamide/G-CSF mobilized transplanted with 1250 LSK from CD45.1 BM. Donor chimerism levels are low þ À þ shown for each recipient. (b) Lethally irradiated B6 recipients (n ¼ 6) spleen Thy-1 Sca-1 Lin c-Kit cells provided long-term 28,29 transplanted with 1250 LSK purified from CD45.1 MPB cells. Donor multilineage engraftment. Purification of mobilized chimerism levels are shown for individual recipients. spleen stem cells using signalling lymphocytic activation molecule family markers, CD150 þ CD48À resulted in a Btwo-fold reduced long-term engrafting capacity when compared to the same population in young BM stem suggests that the frequency of cells that contribute to cells.30 This suggests that fewer ‘true’ stem cells are present long-term repopulation is at limiting dilution; 1250 MPB in the Thy-1lowSca-1 þ LinÀc-Kit þ stem cell population in LSK cells contain very few (0, 1 or 2) stem cells. This is mobilized blood. Our CRU data also indicate that LSK consistent with our CRU data (0.75 CRU/100 LSK). cells from blood are inherently worse than LSK from BM. Reducing the dose of BM LSK to 625 led to more variation In our study, transplantation of purified stem cells from in chimerism but overall levels were still higher (52734%) peripheral blood displayed a rapid increase in recovery of than those observed in some recipients transplanted with WBC counts demonstrating an increase in short-term 1250 MPB LSK. engraftment, consistent with clinical and experimental data.2,3,31,32 The observed consistency between clinical and experimental data validates the use of LSK cells in the murine system as a model to study the quality of MPB stem Discussion cells. Murine LSK, much like human CD34 þ cells, are predominantly progenitor cells but contain the vast In the present study, we exploited a murine model to majority of long-term repopulating stem cells. evaluate the quality of MPB stem cells in in vitro and in vivo The observed reduction in the frequency of stem cells assays. Our data demonstrated that unfractionated MPB rather than a faster deterioration of blood stem cells stem cells have a five-fold reduction in CAFC day 35 suggest that recipients should receive higher cell dose of activity and a five-fold reduction in stem cell repopulation mobilized blood stem cells to compensate for the decrease potential compared to unfractionated control BM cells in repopulation potential. In clinical studies, patients (Figure 1). Mobilized blood stem cells, selected on basis of receiving MPB typically receive a much higher CD34 þ cell LSK expression, displayed an even greater fold reduction dose than the BM group.31,33 Currently, 15–20 Â 104 CFU- (25-fold) in long-term repopulation potential when trans- GM/kg or 2–2.5 Â 106 CD34 þ cells/kg is generally the planted in competition with normal BM stem cells agreed minimum threshold below which rapid hemato- (Figure 4). Limiting dilution analysis showed that this poietic reconstitution may not occur.4,34 However, our data was a result of fewer stem cells rather than less potent stem cautions the use of the frequency of a phenotypically

Bone Marrow Transplantation MPB stem cells have impaired long-term engraftment JSG Yeoh et al 408 defined population such as CD34 þ in humans or LSK in 7 Yan XQ, Hartley C, McElroy P, Chang A, McCrea C, mice, as the only parameter for decisions about a minimum McNiece I. Peripheral blood progenitor cells mobilized by cell dose. Although the number of harvested mobilized recombinant human granulocyte colony-stimulating factor CD34 þ cells in humans is usually sufficient, our results plus recombinant rat stem cell factor contain long-term warrant careful analysis of the long-term repopulation engrafting cells capable of cellular proliferation for more than Blood potential of these cells. The presence of fewer long-term two years as shown by serial transplantation in mice. 1995; 85: 2303–2307. repopulating cells within this population will increase the 8 Demuynck H, Pettengell R, de Campos E, Dexter TM, Testa risk of regrowth of host (malignant) hematopoiesis as NG. The capacity of peripheral blood stem cells mobilised with shown in our non competitive transplants (Figure 7). chemotherapy plus G-CSF to repopulate irradiated marrow Interestingly, a Bseven-fold difference in CRI was stroma in vitro is similar to that of bone marrow. Eur J Cancer observed between transplants with unfractionated MPB 1992; 28: 381–386. cells (CRI of 0.3170.1) (Figure 1) and transplants with 9 Pettengell R, Luft T, Henschler R, Hows JM, Dexter TM, mobilized purified blood stem cells (CRI of 0.0470.04) Ryder D et al. Direct comparison by limiting dilution analysis (Figure 4). This could be caused by the difference in cells of long-term culture-initiating cells in human bone marrow, used (mononuclear cells for MPB and unseparated (480% umbilical cord blood, and blood stem cells. Blood 1994; 84: granuloid) cells from BM, or it may suggest a potential role 3653–3659. 10 Molineux G, McCrea C, Yan XQ, Kerzic P, McNiece I. Flt-3 of accessory cells to facilitate engraftment of MPB cells. It þ À ligand synergizes with granulocyte colony-stimulating factor to was reported that a CD8 /TCR cell population from the increase neutrophil numbers and to mobilize peripheral blood donor BM facilitates engraftment of purified allogeneic BM stem cells with long-term repopulating potential. Blood 1997; stem cells.35 MPB LSK cells may be more dependent on 89: 3998–4004. accessory cells than BM stem cells. Thus, it is possible that 11 Wagers AJ, Allsopp RC, Weissman IL. Changes in integrin during the purification of LSK cells from MPB, accessory expression are associated with altered homing properties of cells facilitating engraftment were lost, leading to a further Lin(-/lo)Thy1.1(lo)Sca-1(+)c-kit(+) hematopoietic stem cells decrease in repopulation of purified MPB stem cells. following mobilization by cyclophosphamide/granulocyte In summary, although MPB stem cells promote faster colony-stimulating factor. Exp Hematol 2002; 30: 176–185. hematological recovery, our competitive and non-competi- 12 Szilvassy SJ, Meyerrose TE, Ragland PL, Grimes B. Differ- ential homing and engraftment properties of hematopoietic tive transplant models suggest that their lower stem cell progenitor cells from murine bone marrow, mobilized peri- frequency affects the long-term repopulation potential of pheral blood, and fetal liver. Blood 2001; 98: 2108–2115. blood LSK stem cells in mice. 13 Uchida N, He D, Friera AM, Reitsma M, Sasaki D, Chen B et al. The unexpected G0/G1 cell cycle status of mobilized hematopoietic stem cells from peripheral blood. Blood 1997; 89: 465–472. Acknowledgements 14 de Haan G, Ausema A, Wilkens M, Molineux G, Dontje B. Efficient mobilization of haematopoietic progenitors after a We thank Geert Mesander and Henk Moes for their assistance single injection of pegylated recombinant human granulocyte with cell sorting and the staff of our animal facility for taking colony-stimulating factor in mouse strains with distinct care of the mice. No conflicts of interest exists between authors. marrow-cell pool sizes. Br J Haematol 2000; 110: 638–646. This work was supported by Grants from the: European 15 de Haan G, Szilvassy SJ, Meyerrose TE, Dontje B, Grimes B, Union (EU-LSHC-CT-2004–503436) and the Ubbo Emmius Van Zant G. Distinct functional properties of highly purified Foundation. hematopoietic stem cells from mouse strains differing in stem cell numbers. Blood 2000; 96: 1374–1379. 16 Szilvassy SJ, Humphries RK, Lansdorp PM, Eaves AC, Eaves CJ. Quantitative assay for totipotent reconstituting hemato- References poietic stem cells by a competitive repopulation strategy. 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