Hematopoietic Stem Cell Yield Research Articles

The Combination of AMD3100 + G-CSF Restores the Ineffective Hematopoietic Stem Cell Mobilization with G-CSF Alone in a Thalassemic Mouse Model.

Abstract 3234Poster Board III-171Gene therapy has been recently postulated as a realistic therapeutic potential for thalassemia and the mobilized autologous hematopoietic stem cells (HSCs) may represent the preferable source of stem cells for genetic modification due to the higher yield of HSCs compared to conventional bone marrow (bm) harvest. We have previously shown (manuscript under revision) that G-CSF mobilization in the HBBth-3 thalassemic mouse model is less efficient compared to normal C57Bl6 strain, mainly due to increased trapping of hematopoietic stem (Lin-sca-1+ckit+–LSK) and progenitor cells (CFU-GM) in the enlarged thalassemic spleen. The novel mobilizer, AMD3100 (plerixafor, mozobil), has been shown to reversibly bind to CXCR4 and inhibit the interaction between SDF-1 and CXCR4 within the bm microenvironment, resulting in the egress of CD34+ cells into the circulation of healthy donors and cancer patients. The addition of AMD to G-CSF results in even greater increases in circulating CD34+cells. We explored in the current study whether AMD alone or in combination with G-CSF improves the mobilization efficiency of thalassemic mice. C57 and HBBth-3 mice received G-CSF-alone at 250microgr/kgX7 days, AMD-alone at 5mg/kgX3 days or the combination of two with AMD administered in the evening of days 5-7 of G-CSF administration. Hematopoietic tissues (blood, bm, spleen) were collected and the absolute LSK and CFU-GM numbers were calculated based on their frequency within tissues (by FCM and clonogenic assays) in relation to the individual cell count per tissue. AMD-alone didn't significantly affect the HSC yield as compared to G-CSF mobilization in thal mice (LSK/μl blood: 103±85 vs 69±26 p=ns), although it significantly increased the circulating Colony Forming Cells (CFU-GM/ml blood: 1205±533 vs 330±87, p=0,05). In contrast, the AMD+G-CSF combination significantly improved the mobilization efficiency of HBBth-3 mice over the G-CSF-treated group (LSK cells/μl blood: 224±104 vs 69±26 p=0,04, CFU-GM/ml blood: 1671±984 vs 330±87 p=0,05, respectively) at levels comparable to normal mice treated with G-CSF (LSK cells/ μl blood: 241±167, CFU-GM/ml blood: 1235±1140, respectively). AMD induced a “detachment” of stem cells from the bm because reduced numbers of bm LSK cells were counted in the AMD-alone group as compared to the untreated group (LSK/2 femurs×103: 692±429 vs 1687±1016, respectively, p=0,05). This was in contrast to the marrow hyperplasia caused by G-CSF over the steady-state condition (LSK/2 femurs×103: 2684±1743 vs 1687±1016 p=0,02 / CFU-GM/2femurs:111.841±15.391 vs 76.774±31.728 p=0,01). Consequently, the combination of AMD+G-CSF resulted in increased numbers of circulating stem and progenitor cells without inducing marrow hyperplasia as compared to steady-state condition (LSK/2femurs×103: 1681±862 vs 1686±1017, p=ns / CFU-GM/2femurs: 76.774±31.728 vs 82.905±26.277, p=ns). AMD, also in contrast to G-CSF, did not cause increased trapping of stem and progenitor cells in the spleen compared to the untreated condition (LSK cells/spleen×103: 4738±2970 vs 8303±4166 p=ns / CFU-GM/spleen:146.269±93.174 vs 98.518±25.549, p=ns). However, the combination of AMD+G-CSF still resulted in splenic sequestration of progenitor cells (CFU-GM/spleen: 412.176±157.417 vs 98.518±25.549, p=0,0003) but not of LSK cells (LSK cells/spleen×103: 10.200±7.260 vs 8.300±4.166 p=ns). Overall, the combination of AMD3100+G-CSF seems to restore the less efficient mobilization in a thalassemic mouse model. This combination may prove beneficial in a GT setting for obtaining the high numbers of HSCs needed for genetic correction. In addition, the combination of AMD3100+G-CSF, by avoiding the marrow hyperplasia induced by G-CSF alone, indicates a better safety profile because it will not further burden the hyperplastic –due to the increased erythroid demand and the intramarrow destruction of erythroblasts-thalassemic bone marrow. DisclosuresNo relevant conflicts of interest to declare.

Improving stem cell mobilization strategies: future directions

Autologous hematopoietic SCT (auto-HSCT) provides hematopoietic support after high-dose chemotherapy and is the standard of care for patients with multiple myeloma (MM) or chemosensitive relapsed high- or intermediate-grade non-Hodgkin's lymphoma (NHL). However, yields of hematopoietic stem cells vary greatly between patients, and the optimal strategy to mobilize hematopoietic stem cells into peripheral blood for collection has not been defined. Current mobilization strategies consist of cytokines alone or in combination with chemotherapeutic agents. Cytokine-only mobilization regimens are well tolerated, but their utility is limited by suboptimal PBSC yields. When a myelosuppressive chemotherapeutic agent is added to a cytokine mobilization regimen, PBSC collections improve two- to five-fold. This benefit is tempered by increased toxicity, morbidity and resource utilization. All current regimens fail to mobilize sufficient hematopoietic stem cells to proceed to transplantation in 5-30% of patients, necessitating additional mobilization attempts or precluding transplantation, which may negatively affect patient outcomes and survival. Improved strategies to mobilize stem cells would increase the availability of auto-HSCT and optimize engraftment and outcomes in patients with MM or NHL. Novel agents used in conjunction with cytokines have the potential to increase PBSC collections without introducing additional morbidity, thereby improving patient outcomes.

Mobilized Hematopoietic Stem Cell Yield Depends on Species-Specific Circadian Timing

Hematopoietic stem and progenitor cell (HSPC) recovery after mobilization remains one of the major limiting factors to perform successful bone marrow transplantation (BMT) procedures since up to 60% of cancer patients fail to mobilize enough HSPC for BMT. We have recently shown that under steady-state conditions, HSPC are released from the bone marrow (BM) in a circadian manner, peaking in the circulation 5 hours after onset of light (Zeitgeber time 5, ZT5) and reaching a nadir at ZT13. The molecular clock controls local norepinephrine activity in the BM from sympathetic nerve fibers, which downregulates CXCL12 production by BM stromal cells via the β3 adrenergic receptor (Nature 2008; 452:442). Here, we show that HSPC circadian rhythms could be exploited to enhance HSPC mobilization. Mice mobilized with granulocyte colony-stimulating factor (G-CSF, 250μg/kg administered daily for 4 days) have significantly more circulating colony-forming progenitors (CFU-C) at ZT5 (2598 ± 236 CFU-C/ml of blood) than at ZT13 (1604 ± 188 CFU-C/ml of blood; p<0.01). The stem cell–enriched Lin−Sca1+c-kit+ (LSK) cell recovery was also increased at ZT5 (1.4-fold more; p<0.05). To test whether circadian variations also affected rapid mobilizers, we treated mice with the CXCR4 antagonist AMD3100 (5mg/kg, 1 hour before blood collection). We found that CFU-C recovery was significantly higher at ZT5 (517 ± 79 CFU-C/ml of blood) than ZT13 (316 ± 50 CFU-C/ml of blood; p<0.05). LSK cells elicited by AMD3100 were also more abundant at ZT5 than at ZT13 (1.8-fold; p<0.05). These results suggested that synchronization of blood collection with circadian rhythms can produce greater HSPC recovery. In contrast to G-CSF-induced mobilization, which triggers CXCL12 downregulation in the bone marrow, AMD3100 results cannot be explained by changes in CXCL12 levels as the compound antagonizes CXCR4 on HSPC. Analysis of CXCR4 expression on LSK cells revealed higher levels at ZT13 than ZT5 (1.5-fold increase; p<0.05). Moreover, CXCR4 oscillations were dependent on the molecular clock as they disappeared in mice lacking the clock gene Bmal-1. These results suggest that the coordinated expression of CXCL12 in the microenviroment and CXCR4 on HSPC regulate HPSC rhythms. To determine whether similar HSPC rhythms take place in humans, we determined the number of CD34+ cells or the more primitive CD34+CD38− cells in the blood of 9 healthy human subjects at 8:00AM and 8:00PM. Both CD34+ cells (2554 ± 242 cells/ml of blood at 8:00AM vs. 5887 ± 508 cells/ml of blood at 8:00PM; p<0.001) and CD34+CD38− cells (217 ± 42 cells/ml of blood at 8:00AM vs. 497 ± 53 cells/ml of blood at 8:00PM; p<0.001) were more abundant in the early night. Analysis of CFU-C progenitor cells in the blood of the same donors showed that these cells were also increased in human peripheral blood at 8:00 PM (143 ± 12 CFU-C/ml of blood) when compared with 8:00 AM (73 ± 12 CFU-C/ml of blood; p<0.001). These results thus demonstrate that HSPC circadian rhythms take place in humans and that they are phase-shifted when compared to mice, as predicted from the nocturnal behavior of mice. To determine whether human HSPC rhythms could be used to enhance mobilization yield, we performed a retrospective data analysis of mobilization efficiency in 82 healthy donors that underwent G-CSF-induced mobilization for allogeneic bone marrow transplantation between 2000 and 2006 at the Mount Sinai Medical Center. In these donors, aphereses were performed in the morning and in the early afternoon. Hence, we divided the donors in two groups according to the collection half time: those between 10:00AM and 12:30PM and those between 12:30PM and 15:00PM. The mobilization yield (expressed as number of CD34+ cells per ml of blood processed per kg of donor weight) was significantly higher in patients mobilized in the afternoon (0.35 ± 0.02 CD34+ cells/mL/kg vs. 0.55 ± 0.05 CD34+ cells/mL/kg; p < 0.001). These results indicate that circulating human HSPC fluctuate in a phase-shifted circadian rhythm compared to that of the mouse. In both species, mobilization yield may be enhanced by synchronizing the collection time with the optimal circadian time. Although this issue needs to be tested in a prospective clinical trial, these data indicate that a simple change in the apheresis time may have a significant clinical impact.

Isolation and Analysis of Hematopoietic Stem Cells from the Placenta

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. All HSCs emerge during embryonic development, after which their pool size is maintained by self-renewing cell divisions. Identifying the anatomical origin of HSCs and the critical developmental events regulating the process of HSC development has been complicated as many anatomical sites participate during fetal hematopoiesis. Recently, we identified the placenta as a major hematopoietic organ where HSCs are generated and expanded in unique microenvironmental niches (Gekas, et al 2005, Rhodes, et al 2008). Consequently, the placenta is an important source of HSCs during their emergence and initial expansion. In this article, we show dissection techniques for the isolation of murine placenta from E10.5 and E12.5 embryos, corresponding to the developmental stages of initiation of HSCs and the peak in the size of the HSC pool in the placenta, respectively. In addition, we present an optimized protocol for enzymatic and mechanical dissociation of placental tissue into single-cell suspension for use in flow cytometry or functional assays. We have found that use of collagenase for single-cell suspension of placenta gives sufficient yields of HSCs. An important factor affecting HSC yield from the placenta is the degree of mechanical dissociation prior to, and duration of, enzymatic treatment. We also provide a protocol for the preparation of fixed-frozen placental tissue sections for the visualization of developing HSCs by immunohistochemistry in their precise cellular niches. As hematopoietic specific antigens are not preserved during preparation of paraffin embedded sections, we routinely use fixed frozen sections for localizing placental HSCs and progenitors.

Isolation and Analysis of Hematopoietic Stem Cells from the Placenta

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. All HSCs emerge during embryonic development, after which their pool size is maintained by self-renewing cell divisions. Identifying the anatomical origin of HSCs and the critical developmental events regulating the process of HSC development has been complicated as many anatomical sites participate during fetal hematopoiesis. Recently, we identified the placenta as a major hematopoietic organ where HSCs are generated and expanded in unique microenvironmental niches (Gekas, et al 2005, Rhodes, et al 2008). Consequently, the placenta is an important source of HSCs during their emergence and initial expansion. In this article, we show dissection techniques for the isolation of murine placenta from E10.5 and E12.5 embryos, corresponding to the developmental stages of initiation of HSCs and the peak in the size of the HSC pool in the placenta, respectively. In addition, we present an optimized protocol for enzymatic and mechanical dissociation of placental tissue into single-cell suspension for use in flow cytometry or functional assays. We have found that use of collagenase for single-cell suspension of placenta gives sufficient yields of HSCs. An important factor affecting HSC yield from the placenta is the degree of mechanical dissociation prior to, and duration of, enzymatic treatment. We also provide a protocol for the preparation of fixed-frozen placental tissue sections for the visualization of developing HSCs by immunohistochemistry in their precise cellular niches. As hematopoietic specific antigens are not preserved during preparation of paraffin embedded sections, we routinely use fixed frozen sections for localizing placental HSCs and progenitors.

Kinetics of Stem Cell and Lymphoid Subset Mobilization in Response to Intravenous (IV) AMD3100 in Mouse and Man.

Background: Mobilization of hematopoietic stem cells (HSCs) in mice and man can be induced using G-CSF (G) or by the use of chemokines and chemokine receptor antagonists. Recent preclinical and clinical data using the bicyclam AMD3100 (A) suggests that the combination of G+A results in significantly improved yields of HSCs compared to G alone in both mice and man (Blood. 2005; 106:1867). In humans, optimal mobilization of HSCs occurs 6–9 h after subcutaneous (s.c.) A. These mobilization kinetics necessitate the somewhat undesirable administration of A the evening before the first day of apheresis.Methods: We assessed the kinetics of HSC mobilization in mice and man after IV or s.c. administration of A. Human donors were initially mobilized with increasing doses of IV A (80, 160, 240 or 320 μg/kg). After 4 days of drug clearance, the same donors were then mobilized with a single s.c. dose of A (240 μg/kg) and collected cells were used as a source of HSCs for HLA-matched sibling recipients undergoing single dose (550 cGy) TBI and cytoxan allogeneic HSC transplantation (Devine et. al. Blood . 2006; 108:53).Results: Peak mobilization of mouse CFU-GM occurred 3 h after s.c A (5 mg/kg; 15–18-fold; n=5) and 0.5–1 h after IV A (1–3 μg/kg; 10–12-fold; n=3). Identical CFU-GM kinetics were observed after mice were first given G (250 μg/kg) for 4 days then administered either s.c. A (250–500 fold in 3 h; n=3) or IV A (170–350 fold n=3). To date, 6 normal HLA-matched sib donors have been treated with 80 μg/kg (n=3) or 160 μg/kg (n=3) IV A over 30 minutes followed 4 days later by 240 μg/kg s.c. A. Peak mobilization of CD34+ cells occurred 1–4 h after 80 μg/kg IV A, 4–6 h after 160 μg/kg IV A, and 9 h after s.c. A. The more rapid kinetics of CD34 mobilization after IV A is in contrast to the kinetics of s.c. A in autologous transplant patients receiving both G and A (J Clin. Oncol. 2004; 22:1095) and in normal volunteers receiving A alone (∼9 hours; Blood . 2003; 102:2728). This data suggests that IV A not only mobilizes HSCs from mouse and man more quickly than s.c. A, but also, that the peak of mobilization may be enhanced (6.4-fold at 160 uμg/kg IV vs. 5.2-fold for 240 μg/kg s.c.) and prolonged (5.7 fold at 160 μg/kg IV vs. 3.8 fold for 240 μg/kg s.c. at 6 h post-A) after IV A. IV A also induced: (i.) a more rapid and greater peak of CD19+ B cell mobilization (6-fold at 1 h and 6.7-fold at 4 h) than s.c. A (2.2-fold at 6 h), (ii.) a modest increase in CD4+ and CD8+ CD3+ T cell mobilization (3.2-fold vs. 1.1-fold for sc A at 6h) and (iii.) no significant changes in Treg, NK, CMV-specific CD8, and γδ T cells. To identify genes that are differentially expressed following G or A mobilization, we performed RNA profiling analyses using Affymatrix U133+2 arrays and RNA isolated from purified (>95%) CD34+ HSCs obtained from 5 individual normal donors mobilized sequentially with A and G. Of note, CXCR4, CCR9, ALCAM, ICAM, and CEECAM1 were expressed more abundantly in all A mobilized CD34+ cells while mucin, Integrin α 6, Integrin α 2b, and CEACAM1 were more abundantly expressed in all G-mobilized CD34+ cells.Conclusions: IV A results in more rapid and prolonged mobilization of mouse HSC and human CD34+ cells compared to s.c. A. IV A was not associated with any adverse events in the 6 donors mobilized at the first two IV dose levels. The enhanced and more rapid mobilization of CD34+ cells by IV A may have significant applications for the optimal collection of peripheral blood HSC products.

Re: Toward a Worldwide Standard for CD34+ Enumeration

Journal of HematotherapyVol. 6, No. 2 Re: Toward a Worldwide Standard for CD34+ EnumerationD.R. Sutherland, L. Anderson, M. Keeney, R. Nayar, and I. Chin-YeeD.R. SutherlandSearch for more papers by this author, L. AndersonSearch for more papers by this author, M. KeeneySearch for more papers by this author, R. NayarSearch for more papers by this author, and I. Chin-YeeSearch for more papers by this authorPublished Online:27 Mar 2009https://doi.org/10.1089/scd.1.1997.6.85AboutSectionsPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail FiguresReferencesRelatedDetailsCited byCirculating CD34+ hematopoietic stem/progenitor cells paralleled with level of viremia in patients chronically infected with hepatitis B virus20 February 2018 | Regenerative Medicine Research, Vol. 6Standardized Flow Cytometry Assays for Enumerating CD34 + Hematopoietic Stem Cells27 January 2016Safety and efficacy of hematopoietic stem cells mobilization in patients with multiple sclerosis2 September 2015 | Hematology, Vol. 21, No. 1More than Just Quality ControlClinics in Laboratory Medicine, Vol. 27, No. 3Enumeration of CD 34 + Hematopoietic Stem and Progenitor Cells15 August 2003 | Current Protocols in Cytometry, Vol. 25, No. 1Validation of the single-platform ISHAGE method for CD34+ hematopoietic stem and progenitor cell enumeration in an international multicenter studyCytotherapy, Vol. 5, No. 1Flow cytometric enumeration and immunophenotyping of hematopoietic stem and progenitor cellsSeminars in Hematology, Vol. 38, No. 2Single- versus dual-platform assays for human CD34+ cell enumerationCytometry, Vol. 38, No. 6Comparison of volumetric capillary cytometry with standard flow cytometry for routine enumeration of CD34+ cellsTransfusion, Vol. 39, No. 8The Absolute Number of Circulating CD34+ Cells as the Best Predictor of Peripheral Hematopoietic Stem Cell Yield Rita Fontao-Wendel, Arlete Lazar, Silvia Melges, Cristina Altobeli, and Silvano Wendel9 July 2004 | Journal of Hematotherapy, Vol. 8, No. 3Primordial role of CD34 + 38 − cells in early and late trilineage haemopoietic engraftment after autologous blood cell transplantation4 January 2002 | British Journal of Haematology, Vol. 103, No. 2Role of the CD34+38− cells in posttransplant hematopoietic recovery4 June 2009 | STEM CELLS, Vol. 16, No. S2Quality assessment of CD34 + stem cell enumeration: experience of the United Kingdom National External Quality Assessment Scheme (UK NEQAS) using a unique stable whole blood preparation25 December 2001 | British Journal of Haematology, Vol. 102, No. 2Tumor Evaluation and Hematotherapy: What Is the Situation in 1998?27 March 2009 | Journal of Hematotherapy, Vol. 7, No. 2A Convergence of Methods for a Worldwide Standard for CD34+ Cell Enumeration Genald Marti, Hans Johnsen, and Stefan Serke27 March 2009 | Journal of Hematotherapy, Vol. 7, No. 2Not All Healthy Donors Mobilize Hematopoietic Progenitor Cells Sufficiently After G-CSF Administration to Allow for Subsequent CD34 Purification of the Leukapheresis Product Mette Holm and Peter Hokland27 March 2009 | Journal of Hematotherapy, Vol. 7, No. 2Letter to the Editor: Will CD34+ Standardization Solve All Problems Related to Cell Therapy?27 March 2009 | Journal of Hematotherapy, Vol. 6, No. 5Current status of CD34+ cell analysis by flow cytometry: The ISHAGE guidelinesClinical Immunology Newsletter, Vol. 17, No. 2-3Quality Control in Flow Cytometry Volume 6Issue 2Apr 1997 To cite this article:D.R. Sutherland, L. Anderson, M. Keeney, R. Nayar, and I. Chin-Yee.Re: Toward a Worldwide Standard for CD34+ Enumeration.Journal of Hematotherapy.Apr 1997.85-89.http://doi.org/10.1089/scd.1.1997.6.85Published in Volume: 6 Issue 2: March 27, 2009PDF download

The effect of steroids and filtration leukapheresis on circulating hematopoietic progenitor cells.

Filtration leukapheresis (FL) in which donors are pretreated with steroids, induces a rapid neutropenia followed by neutrophilia. To investigate whether these phenomena are associated with changes in circulating progenitor cells (CFU-GM and BFU-E), 5 donors who underwent FL following steroid administration were compared with a control group who received steroids alone. Steroids alone caused an initial reduction in both circulating lymphocytes and committed progenitor cells followed by a rebound above baseline. Among donors undergoing FL, the changes in lymphocyte counts were identical to the steroid controls and the same early suppression of progenitor cells also occurred, but the rebound was blunted. Thus, the neutrophilia that occurs as a result of the FL procedure is not associated with an increase in circulating progenitor cells; and hence, the procedure does not appear to be a useful adjunct for increasing the yield of hematopoietic stem cells from the peripheral blood.