Blood Reviews (2000) 14, 205–218 © 2000 Harcourt Publishers Ltd doi: 10.1054/ blre.2000.0138, available online at http://www.idealibrary.com on
Mobilization of hematopoietic stem cells
S. Fu, J. Liesveld
Hematopoietic stem cell transplantation has been extensively exploited as a therapeutic and research modality and has revolutionized current patient care. At present, more and more medical centers use peripheral blood progenitor cells for transplantation by mobilizing hematopoietic stem cells from bone marrow to peripheral blood because of potential advantages of peripheral blood stem cell transplantation over bone-marrow transplantation. Different effective mobilization regimens have been developed recently with chemotherapeutic agents, hematopoietic growth factors or their combination. This article reviews current developments related to hematopoietic stem cell mobilization including the biology of hematopoietic stem cells, strategies for mobilization, management for mobilization failure, mechanisms of mobilization, and side effects during mobilization. Finally, the Initiation-Amplification-Emigration-Adaptation Model is proposed to help aid understanding of the mechanisms of hematopoietic stem cell mobilization and to stimulate development of novel and optimal mobilization strategies for patient care. © 2000 Harcourt Publishers Ltd aspirations from the posterior iliac crests and in some cases from the anterior crests, while the peripheral blood progenitor cells can be collected by leukapheresis after mobilization with chemotherapy and/or hematopoietic growth factors.2,3 Hematopoietic stemcell mobilization refers to a process of expanding circulating hematopoietic stem cell numbers. Potential advantages of peripheral blood progenitor cell transplantation include the avoidance of marrow harvest and anesthesia and the ability to expand the pool of eligible patients to include those with marrow involvement with tumor or prior pelvic radiation therapy, which frequently precludes marrow harvest.4 Another advantage of using peripheral blood progenitor cells compared to marrow has been enhanced engraftment of myeloid cells and platelets, resulting in significantly decreased medical support such as blood product use, antibiotic use and length of hospitalization.5,6 At present, more and more medical centers use peripheral blood progenitor cells for transplantation by mobilizing hematopoietic stem cells with chemotherapeutic agents and/or hematopoietic growth
INTRODUCTION The development of successful hematopoietic stem-cell transplantation as a therapeutic and research modality has revolutionized patient care in hematology and oncology, and has provided a curative method for some diseases including cancers and genetic diseases. Hematopoietic stem cells can be obtained from bone marrow, peripheral blood and umbilical cord blood, and from autologous, syngeneic and allogeneic donors.1 The marrow harvest is obtained under general or spinal anesthesia via multiple needle punctures and
Siqing Fu MD, PhD, Jane Liesveld MD, University of Rochester Medical Center, Department of Internal Medicine Hematology/Oncology, Blood and Marrow Transplant Program, 601 Elmwood Avenue, Box 610, Rochester, NY 14642, USA. Correspondence to: Jane Liesveld MD, University of Rochester Medical Center, Department of Internal Medicine, Hematology/ Oncology, Blood and Marrow Transplant Program, 601 Elmwood Avenue, Box 610, Rochester, NY 14642, USA. Tel.: +1 (716) 275 4099; Fax: +1 (716) 473 4314; E-mail: [email protected]
factors.7 Success of mobilization is a prerequisite for success of transplantation. Thus, review of the known and unknown areas of mobilization has potential impact for understanding and maximizing the process.
THE BIOLOGY OF HEMATOPOIETIC STEM CELLS A hematopoietic stem cell must have two characteristics:8 the ability to differentiate to all hematopoietic lineages and the ability to maintain hematopoiesis over a life-span by a self-replication process.9 After hematopoietic stem cells were found to circulate routinely in the peripheral blood, initial studies using animal transplant models had positive results with these peripheral progenitor cells to establish sustained hematopoietic function.10 In the mid-1980s, investigators reported successful autologous peripheral blood stem cell autografts for patients with different diseases from several different medical centers working independently. Then, transplantation of allogeneic bloodderived cells was documented to result in sustained hematopoietic function of donor origin. The above clinical trials provided evidence for functional hematopoietic stem cells in the human circulation. There is the possibility that two general hematopoietic stem cell compartments exist in the bone marrow, one that frequently cycles and is the primary source of blood production over time, and another that rarely cycles but represents the major source of engraftment post-transplant.11 Only long-term engraftment of all blood lineages after bone marrow transplantation can truly test hematopoietic stem cell function. The invitro assays including CFU-C (a colony-forming unit culture assay), LTCIC (a long-term culture-initiating cell assay) and CAFC (a cobblestone area forming cell assay) provide supplemental tools to test hematopoietic stem cell function. Both LTCIC and CAFC assays are labor-intensive. The CFU-C assay is not reliably predicative of engraftment potential. Thus, phenotypic analysis of cell-surface antigens by flow cytometry has become a promising method to assess the quality of a potential transplant product if hematopoietic stem cells have specific antigen phenotype on the cellular surface.12 The human hematopoietic stem cell phenotype includes expression of CD34 and CD90 (Thy-1) antigens in the absence of a series of other antigens that mark differentiation lineage such as CD38.13,14 These CD34+CD90+CD38- hematopoietic cells which constitute 0.05% to 0.1% of human marrow and circulating hematopoietic cells are capable of giving rise to long-term repopulating hematopoietic cells of multiple lineages. CD34 antigen may function as an
adhesion molecule, based on homology to selectin and functional studies, and Thy-1 antigen may be involved in triggering signal-transduction cascades.15 In human postnatal hematopoietic tissues, 0.1 to 0.5% of CD34+ cells expressed VEGFR2 (vascular endothelial growth factor receptor 2 or KDR receptor).16 Primitive uncommitted hematopoietic stem cells are restricted to the CD34+KDR+ cell fraction. Another characteristic phenotype such as expression of MDR1 (gp170, multiple drug resistance phenotype) has been successfully used to define a primitive subset of CD34+ cells and even to predict the existence of hematopoietic stem cells that are CD34–, which may represent precursors of the CD34+ stem cells with better engraftment over long periods of time.17 New evidence from aging donors suggested even in the non-transplant setting of human hematopoiesis, the ability of the hematopoietic stem cell to self-replicate is not unlimited. The studies on the kinetics of cell division among hematopoietic stem cells suggested hematopoietic stem cells were quiescent with respect to the cell cycle and few of them were in cycle at any given time. These cycling hematopoietic stem cells failed to engraft the nucleated cell lineages in irradiated recipient animals when transplanted intravenously. Hematopoietic stem cells mobilized from the marrow by chemotherapy and/or hematopoietic growth factors were believed to be largely out of cell cycle when collected from the peripheral blood by leukapheresis.18 Thus, control of the cell cycle and expression of specific molecules are keys to understanding hematopoietic stem-cell biology and transplantation. As noted above, the CD34+ peripheral blood cells represent a pool of hematopoietic progenitor cells which include a small percentage of hematopoietic stem cells. To find a parameter to predict the quality of the collected CD34+ peripheral blood cells for engraftment, we examined the use of the ratio of CFU-GM vs CD34+ cells as a index to correlate with prompt reconstitution of hematopoietic lineages. Before transplantation, the infused number of CD34+ cells per kg and the infused number of CFU-GM per kg from all the studied patients at University of Rochester in 1999 were identified. The ratio of CFU-GM vs CD34+ cells was calculated. The recipient patients, most of whom received more than 2 ×106 CD34+ cells/kg (90%) or more than 5 ×106 CD34+ cells/kg (40%), were monitored with daily complete blood cell count until patients achieved 50 000 platelets/µl. Linear regression analyses were conducted to determine whether the ratio of CFU-GM vs CD34+ cells as a parameter for the quality of the collected CD34+ cells correlated with prompt reconstitution of platelets. Data as shown in Figure 1 supported significantly that the ratio of CFU-GM vs CD34+ cells could be applied to predict
Mobilization of hematopoietic stem cells
prompt reconstitution of platelets (P=0.031 and P=0.024 for days to reach platelet 20,000 per microliter and 50,000 per microliter, respectively). Further analyses showed that the average day to reach a platelet count of 20 000 per microliter was day 20 if the ratio was less than 1.4 and day 26 if the ratio was more than 1.4 with P-value <0.04, and the average days to reach platelet 50 000 per microliter was day 22 if the ratio was less than 1.4 and day 31 if the ratio was more than 1.4 with P-values <0.03. Therefore, successful engraftment is determined not only by the quantity, but also by the quality of mobilized peripheral blood progenitor cells.
STRATEGIES FOR HEMATOPOIETIC STEM CELL MOBILIZATION (TABLE 1) Autologous and allogeneic peripheral blood progenitor cell transplants have been increasingly performed for an effective curative treatment modality for a variety of disorders, including malignancy, aplasia, immunodeficiency and genetic disorders and are rapidly replacing conventional bone-marrow transplantation, not only due to decreased risks and greater convenience for the donor, but also because transplant with peripheral blood progenitor cells results in more rapid engraftment with less medical support.1,19–22 Collection of enough hematopoietic stem cells for transplantation from peripheral blood during steadystate marrow function usually requires leukapheresis from more than 40 L of blood. Thus, mobilization of hematopoietic stem cells from marrow to peripheral blood before leukapheresis is desirable. The number of circulating peripheral blood progenitor cells is greatly increased during the recovery phase following chemotherapy with myelosuppressive, but not myeloablative, agents such as cyclophosphamide.23 Hematopoietic growth factors were also found to significantly increase the number of peripheral blood progenitor cells. When chemotherapy and hematopoietic growth factors are combined, a synergistic effect resulting in increased peripheral blood progenitor cells occurs. Therefore, substantially larger numbers of peripheral blood progenitor cells can be collected following mobilization with hematopoietic growth factors, chemotherapy or their combination. By the use of the CD34 cell surface marker as a means of identifying the number of primitive hematopoietic stem cells contained in a peripheral blood progenitor cell collection, most reports suggested that a target number of 5 ×106 CD34+ cells/kg be infused for sustained neutrophil, platelet, and red blood cell recovery.24,25 Thus, the goal of peripheral blood progenitor cell mobilization is to collect at least 5 ×106 CD34+ cells/kg after leukapheresis, and after positive and
Table 1 Strategies for stem-cell mobilization 1. Use of myelosuppressive but not myeloablative chemotherapy 2. Use of hematopoietic growth factors 3. Use of chemotherapy and hematopoietic growth factors in combination Single Growth Factors Combination chemotherapy chemotherapy Regimens agents (Utilized with G-CSF) Cyclophosphamide G-CSF ICE Paclitaxel GM-CSF TEC SCF CP Flt-3 ligand DICEP The above are examples of regimens that have been used for progenitor cell mobilization. See text for definitions of abbreviations and acronyms.
negative stem-cell selection, if such procedures are to be performed. Although 5 ×106 CD34+ cells/kg is an optimal goal for mobilization, successful transplantation with as few as 1 ×106 mobilized CD34+ cells/kg have been demonstrated in several clinical trials26 and most centers routinely perform transplantation with 2 ×106 CD34+ cells/kg with acceptable engraftment success. In contrast to the typical bone-marrow collection performed in an operating room during general anesthesia, peripheral blood progenitor cell collection is accomplished by using a leukapheresis device in an outpatient setting without anesthesia.27–29 First,placement of central venous catheters is needed for leukapheresis, administration of high-dose therapy and medical support of patients during recovery after transplant.30 Then mobilization is achieved with chemotherapeutic agents and/or hematopoietic growth factors. Finally, the leukapheresis device is programmed to collect cells with characteristics of low-density monocytes or lymphocytes. Instructions for collecting peripheral blood progenitor cells are available from manufacturers. Each leukapheresis procedure requires about 2.5 to 4 h to process 8 to 15 L of blood, which can be repeated daily as needed.31 Usually two or three standard-volume leukapheresis procedures are required to harvest a sufficient number of hematopoietic stem cells.32 These peripheral blood progenitor cells can be further manipulated for negative and positive selection to meet the patient’s need.33,34 The utilization of chemotherapeutic agents in mobilization Mobilization originated from an observation made in the 1970s that circulating hematopoietic stem cells increased following chemotherapy with myelosuppressive agents. Administration of myelosuppressive agents produces a mobilization of hematopoietic stem cells from marrow to peripheral blood about 2 weeks
later as recovery from the nadir of peripheral cytopenia starts.35,36 Cyclophosphamide and paclitaxel were used frequently, among many chemotherapeutic agents.37–39 Mobilization by chemotherapeutic agents alone was used widely during the 1980s and early 1990s and has been replaced with hematopoietic growth factor-induced or hematopoietic growth factor plus chemotherapeutic agent-induced mobilization.40 The utilization of hematopoietic growth factors in mobilization The initial fear of using hematopoietic growth factors as mobilizing agents was that administration of hematopoietic growth factors to mobilize hematopoietic stem cells might stimulate circulating hematopoietic stem cells to differentiate from the uncommitted to the committed stage so that fewer primitive hematopoietic stem cells could be collected.41 The utilization of hematopoietic growth factors in mobilization has been used widely after first reports in 1988 of the mobilizing effects of G-CSF (granulocyte colony-stimulating factor) and GMCSF (granulocyte-macrophage colony-stimulating factor) and a later report of long-term hematopoietic reconstitution in patients transplanted with only hematopoietic growth factor mobilized peripheral blood progenitor cells.42 Several hematopoietic growth factors have been used as mobilizing agents to procure autologous as well as allogeneic peripheral blood progenitor cells.43 The kinetics of hematopoietic stem cell mobilization are similar with different hematopoietic growth factors, with peak levels of peripheral blood progenitor cells achieved after 5 to 10 days of hematopoietic growth factor treatment.44,45 A broad spectrum of hematopoietic cells are mobilized, including primitive hematopoietic stem cells, megakaryocytic, erythroid and myeloid progenitors as well as mature neutrophils. However, the efficacy of mobilization is not similar among different hematopoietic growth factors, ranging from a fivefold to a 500-fold increase of peripheral blood progenitor cells over baseline.46,47 G-CSF is the most commonly used hematopoietic growth factor for mobilization among others, including GM-CSF, SCF (stem cell factor, c-kit ligand), flt-3 ligand, IL1 (interleukin 1), IL3(interleukin 3), IL7 (interleukin 7), IL11 (interleukin 11), and IL12 (interleukin 12).48–55 In addition, G-CSF has been shown to act synergistically with other hematopoietic growth factors to induce hematopoietic stem cell mobilization. The maximum mobilization effect occurs on about the fifth day of G- CSF administration. Escalation of G-CSF doses up to 16 µg/kg/12 h is well tolerated and results in the collection of a higher number of peripheral blood progenitor
cells in healthy donors.56 Ninety-one percent of GCSF-stimulated normal donors produce more than 2 ×106 CD34+ cell/kg by a single leukapheresis. The combination of sequential and concurrent G-CSF with GM-CSF has been reported to be more effective than mobilization with G-CSF alone.57–59 The utilization of chemotherapy agents and hematopoietic growth factors in mobilization Although hematopoietic growth factors such as GCSF are generally used in more and more mobilization protocols, the optimal schedules and regimen for priming hematopoietic stem cells still remain to be established.60 Daily administration of hematopoietic growth factors following administration of mobilization doses of chemotherapy provides more effective mobilization of hematopoietic stem cells than either chemotherapy or hematopoietic growth factor treatment alone.61,62 This combination of chemotherapy and hematopoietic growth factors generates a maximum mobilization effect about 7 to 14 days later, which can last for several days during leukapheresis.63 Usually this correlates with rebound of the white blood cell count after nadir. In addition, their combination can be used for their anti-tumor effect, to avoid tumor cell contamination in the collections and to maximize hematopoietic stem-cell mobilization for autologous transplantation.64 An ideal regimen for autologous transplantation should be highly active against the target tumor cells as well as be capable of mobilizing hematopoietic stem cells for collection.65 Among many regimens, single chemotherapeutic agent such as cyclophosphamide66 or paclitaxel67,68 can be used to combine with G-CSF to mobilize hematopoietic stem cells. Multicyclic and dose-intensive chemotherapy in combination with hematopoietic growth factors may allow development of optimal regimens which are safe and well tolerated, highly active against metastatic tumor cells and capable of excellent hematopoietic stem-cell mobilization.69 Successful reports on mobilization induced by the combination strategy were published in chronic myelogenous leukemia with the mini-ICE regimen (idarabine, cytarabine and etoposide plus G-CSF),70 in lymphoma with the IVE regimen (ifosfamide, etoposide and epirubicin plus G-CSF),71,72 in metastatic breast cancer with the TEC regimen (paclitaxel, etoposide and cyclophosphamide plus G-CSF)73 or the CP regimen (cyclophosphamide and paclitaxel plus G-CSF),37 in lung cancer and lymphoma with the ICE regimen (ifosfamide, carboplatin and etoposide plus G- CSF),74 in some hematologic and solid malignancies with the DICEP regimen (dose-intensive cyclophosphamide, etoposide and cisplatin plus G-CSF),75,76 and many others.
Mobilization of hematopoietic stem cells
MANAGEMENT FOR MOBILIZATION FAILURE (TABLE 2) It is clear that previous chemotherapy and radiotherapy can impair the mobilization of sufficient hematopoietic stem cells to proceed with transplantation, especially with previous exposure to alkylating agents such as chlorambucil and purine analogues.77,78 Successful mobilization of hematopoietic stem cells is determined by successful engraftment.79,80 With infusion of adequate peripheral blood progenitor cells, neutrophil recovery will usually occur about 9–10 days post-transplant in almost all patients, and platelet recovery will occur about 10–11 days posttransplant, of whom 5–35% will experience delays in platelet engraftment.81 Therefore, prompt engraftment of platelets can be an index for successful mobilization, which is correlated with the CD34+ cells transplanted. Several retrospective studies document a minimum safe dose of 1 ×106 CD34+ cells/kg, especially according to the ideal body weight. Below this level, up to 80% of patients have delayed platelet engraftment and experience a significant risk of severe hemorrhage. It appears that the optimal CD34+ cell dose for prompt platelet engraftment is 5 ×106 CD34+ cells/kg, with more than 85% of patients having prompt platelet engraftment by day 14 post-transplant. Using the number of collected CD34+ cells/kg after mobilization and leukaphereses as a criterion, three groups of patients can be described as follows (See also Table 3): the non-mobilizable patient who does not accumulate the minimum cell dose of 1 ×106 CD34+ cells/kg after repeated leukaphereses, the poorly mobilizable patient who can reach the minimum cell dose but cannot accumulate the optimal cell dose of 5 ×106 CD34+ cells/kg after repeated leukaphereses, and the mobilizable patient who can easily accumulate the optimal cell dose of 5 ×106 CD34+ cells/kg in less than 5 leukaphereses.82 Several factors are associated with the inability to mobilize and collect 5 ×106 CD34+ cells/kg.83,84 Multivariate analyses have demonstrated that the number of chemotherapy cycles (>6 cycles), regardless of previous radiotherapy, and prior use of alkylating agents are associated with poor hematopoietic stem cell mobilization.85 Other factors include type of malignancy, age greater than 60 years, bone marrow metastases, prior radiotherapy to marrow-producing
Table 2 Donor classification based on mobilization potential 1. The non-mobilizable patient <1 × 106 CD34+ cells/kg* 2. The poorly mobilizable patient >1 ×106 CD34+ cells/kg 3. The mobilizable patient ≥5 ×106 CD34+ cells/kg *Amount able to be collected in ≥5 leukaphereses.
Table 3 Factors associated with poor mobilization 1. 2. 3. 4. 5. 6.
Numbers of previous chemotherapy cycles Prior use of alkylating agents Type of malignancy Age > 60 years Prior radiotherapy to marrow-producing sites Inadequate mobilization regimen
sites, and the mobilization regimen.86 Certain chemotherapeutic agents such as melphalan, nitrosoureas, procarbazine, nitrogen mustard, fludarabine and platinum compounds appear to be associated with poor mobilization capacity.87,88 It is apparent that the alternative approach for patients with mobilization failure is to find an HLAmatched donor for allogeneic transplantation or to expand ex vivo the mobilized or marrow hematopoietic stem cells. Currently only few medical centers can handle this expensive strategy of ex vivo hematopoietic stem cell manipulation, which is beyond the scope of this review. If the matched donor is not available, several other strategies can be tried for this group of patients in spite of data indicating that currently available strategies do not ensure that patients with mobilization failure will achieve adequate CD34+ cell/kg for transplantation. An apparent strategy for hematopoietic stem-cell collection is to perform a bonemarrow harvest, which may be of some benefit in selected patients. Recent studies suggest that bone marrow harvesting is of limited value, especially in the non-mobilizable patient group.84 Thus, when fewer than 1 ×106 CD34+ cells/kg are mobilized, a different approach instead of bone marrow harvesting should be considered. The choices left are remobilization with the same regimen in escalated doses, mobilization with a different regimen such as multi-drug, muli-cyclic and dose-intensive chemotherapy and its combination with hematopoietic growth factors, or the combination of several hematopoietic growth factors (Table 4). Remobilization with the same regimen generally produces the same poor mobilization until the complete hematopoietic recovery from prior chemotherapy.89 Mobilization with a hematopoietic growth factor alone cannot be achieved for 4–5 weeks after the chemotherapy has been completed. Several remobilization strategies were evaluated recently.90,91 A higher dose of cyclophosphamide at 7 g/m2 instead of 4 g/m2 for the second mobilization produces a significant increase in peripheral blood progenitor cells. Higher doses of cyclophosphamide are not well tolerated, however, due to organ toxicity. Remobilization with escalated doses of G-CSF alone from 5 µg/kg/day to 32 µg/kg/day is effective in at least one-third of patients with mobilization failure. In one study, 96% of patients mobilized with a higher dose of G-CSF at
Alternative mobilization strategies
1. For selected patients, find a suitable allogeneic donor. 2. Marrow harvest 3. Escalated doses of the same mobilization regimen; e.g., higher doses of G-CSF 4. Mobilization with a different regimen Chemotherapy plus growth factor where growth factors alone had been utilized. Growth factor combinations (G-CSF + GM-CSF) (G-CSF + SCF) (G-CSF + Flt-3 ligand)
32 µ/kg/day achieved 2 ×106 CD34+ cells/kg in three collections, compared with 83% mobilized with GCSF at 10 µg/kg/day.92 Obviously, this benefit with escalating doses of chemotherapeutic agents and/or hematopoietic growth factors must be balanced against the associated morbidity and costs.73 Mobilization with a different regimen may produce better results.93,94 For patients initially mobilized with GM-CSF plus IL3, the switch to cyclophosphamide and GM-CSF mobilizes a significantly higher peripheral blood progenitor cells, 1.5 vs 4.1 ×106 CD34+ cells/kg. The most commonly utilized chemotherapeutic agent cyclophosphamide at 4–7 g/m2 produces a 5 to 30-fold increase in the number of CD34+ cells/kg in the peripheral blood. The addition of paclitaxel and/or etoposide produces better results than cyclophosphamide alone, as does the addition of GM-CSF, which results in a two-to five-fold greater increase in the number of CD34+ cells/kg after mobilization and leukapheresis. A randomized phase II trial done with poorly mobilizable patients revealed that the total CD34+ cell collection was significantly higher in the group mobilized with G-CSF and SCF or G-CSF and ftl-3 ligand than that with G-CSF alone.95–99 Forty-four percent of patients in the combination group reached the target dose of 5 ×106 CD34+ cells/kg and only 15% did not reach the minimum dose of 1 ×106 CD34+ cells/kg, compared with 17 and 26% in the G- CSF alone group respectively. Therefore, a combination of multiple chemotherapeutic agents68,100 such as paclitaxel, cyclophosphamide, etoposide, anthracyclines, ifosfamide, cisplatin, carboplatin and ara-C, with hematopoietic growth factors appears to be most effective in mobilizing hematopoietic stem cells.
MECHANISMS OF HEMATOPOIETIC STEM CELL MOBILIZATION (TABLE 5) The in vivo studies on the transition of hematopoietic stem cells during embryonic development from both embryonic and extraembryonic sites to fetal liver and finally to bone marrow, and the homing and trafficking
Table 5 Proposed mechanistic influences on mobilization 1. Alteration in adhesion receptor density or affinity 2. Changes related to chemokine or cytokine influences examples: IL-8, G-CSF 3. Release of metalloproteinases 4. Activation of intracellular signal transduction pathways 5. Alteration of proliferation and cell cycle status 6. Possible role of increased blood flow/crowding
of hematopoietic stem cells post transplantation provide us much information to help understand mechanisms of hematopoietic stem-cell mobilization. Although one or more common pathways are believed to be involved in mobilization and the individual parts remain unlinked, several mechanisms are obviously associated based on current limited in vivo and in vitro studies. Below is a summary of the previous studies in the area of mobilization and a description of the proposed initiation-amplification-emigration and adaptation model for mobilization.
The characteristics of circulating hematopoietic stem cells in steady or mobilized state The human bone marrow can be divided into two compartments: vascular and extravascular. Hematopoiesis occurs almost exclusively in the extravascular compartment. Exchange between these two compartments takes place at the level of sinusoids, which form the blood–bone-marrow barrier with a layer of endothelium.101 Thus, mobilization of hematopoietic stem cells takes place when these cells leave the stromal support system and migrate through the endothelial layer into the vascular compartment. Changes in the surface antigen profile, expression of adhesion molecules, proliferative state and responsiveness to hematopoietic growth factors and chemokines are associated with this process.102,103 By utilizing the CD34 antigen as a marker, most circulating hematopoietic progenitor cells in steady state and mobilized peripheral blood progenitor cells are found to be in either G0/G1 or in a prolonged G1 phase. They are ready for cycling soon after exposure to hematopoietic cytokines in vitro, in contrast to bone-marrow hematopoietic progenitor cells which have a longer latency and a higher percentage of S phase progenitors. Changes of surface antigen and adhesion molecule expression are different between these two groups of circulating hematopoietic stem cells.104 For example, an increased L-selectin level and a decreased VLA-4 (the β1 integrin CD29 and the α4 integrin CD49d, very late antigen 4) level are noted in circulating mobilized primitive CD34+ cells,105 Expression of C-kit (CD117, the stem cell factor
Mobilization of hematopoietic stem cells
receptor) and CXCR4 (the receptor for stromal cell derived factor-1) expression decrease in both circulating primitive CD34+ cells in steady state and circulaing mobilized primitive CD34+ cells.106 The role of hematopoietic growth factors and other chemokines Hematopoietic growth factors and chemokines are demonstrated in many clinical trials as effective and safe mobilizing agents for both autologous and allogeneic transplantation. The kinetics of mobilization range from a few minutes with some chemokines to several days with some hematopoietic growth factors. Cross-talk between hematopoietic stem cells, marrow microenvironment and other systems via hematopoietic growth factors and chemokines plays a major role in mobilization. The importance of hematopoietic growth factors and chemokines in mobilization is that they are functioning not only as a initiation trigger, but also as highly activated transmitters for this complicated cross-talking network, functioning directly as chemoattractants in emigration and supporting survival of the mobilized peripheral blood progenitor cells as well.107 Because of its potency and lack of serious toxicity, G-CSF is the most commonly used mobilizing agent.108 The role of G-CSF in mobilization has been intensively explored recently. Obviously, G-CSF can work as an initiation trigger, primarily targeting myeloid cells as well as endothelial cells. Administration of G-CSF significantly increases the percentage and absolute number of CD34+ cells in the peripheral blood,110 which have similar expression patterns of CD13, CD33, CD38, HLA-DR and CD90 antigens, but decrease expression of c-kit (CD117), VLA4 (CD49d-CD29), VLA5 (CD49e-CD29: α5β1 integrins, very late antigen 5), LFA1 (CD11a-CD18: the αLβ2 integrins, leukocyte function-associated antigen 1), LFA3 (CD58, leukocyte function-associated antigen 3) and L-selectin (CD62L) compared with their circulating counterparts in steady state; decreased responsiveness to SDF1 (stromal cell derived factor 1)111 induced chemotaxis through a transendothelial migration assay due to decreased expression of CXCR4; decreased adhesion capacity to preformed stroma; and decreased percentage of CD34+ cells in active cell cycle. The data from G-CSF receptor knock-out mice demonstrate that the presence of G-CSF receptors is required for hematopoietic stem-cell mobilization with G-CSF, cyclophosphamide, and IL8 but not with hematopoietic growth factors flt-3 and IL-12.112 The chemokine IL8 is the best characterized member of the ELR+ (Glu-Leu-Arg motif) and C-X-C
(Cys-X-Cys motif) chemokines and has capacity for mobilizing hematopoietic stem cells up to 100-fold within 1 h.113 After a single administration of IL8, neutropenia is observed within 5 min and is followed by neutrophilia after several hours, associated with increases in several circulating hematopoietic growth factors. Multiple factors including GM-CSF can induce IL8 production. At least three mechanisms are involved in the IL8 induced mobilization. Firstly, it requires the intact G-CSF system shown as above. Secondly, it can be completely blocked by pretreatment with the anti-LFA1 antibody, suggesting the role of the β2 integrins in this pathway. Finally, IL8 can stimulate the rapid release of metalloproteinases such as gelatinase B113 which, in turn, cleave the marrow extracellular matrix and mobilize hematopoietic through the damaged blood–bone-marrow barrier.114 The role of the adhesion molecule system The hematopoietic stem cells are surrounded by stromal cells such as fibroblasts, adipocytes, macrophages and endothelial cells, and other hematopoietic progenitor cells. There are intensive cell–cell and cell–matrix interactions through the adhesion molecule system which includes several distinct superfamilies: integrins, immunoglobulins, selectins and proteoglycan molecules.114 Integrins are heterodimeric membrane glycoproteins composed of α and β subunits, which not only mediate cell–cell interactions, but also serve as signal transducers influencing many aspects of cell behavior such as cell migration, proliferation, differentiation and apoptosis. Integrins are greatly influenced in their actions by signals from coexpressed hematopoietic growth factor receptors or other adhesion molecules. This system is very complicated due to the same type of receptors or ligands expressed on both hematopoietic stem cells and stromal cells. Disturbance in adhesive interactions between hematopoietic stem cells and their marrow microenvironment by mobilizing agents results in release of hematopoietic stem cells into the peripheral blood. Integrins VLA4 and LFA1 as well as L-selectin are believed to be involved in the mobilization pathway.105 Decreased expression of VLA4 on mobilized hematopoietic stem cells by chemotherapy and/or hematopoietic growth factors plays a major role in mobiliztion.115 Treatment with anti-VLA4 monoclonal antibodies as well as antibodies to VLA4-binding receptor VCAM1 (CD106, vascular cell adhesion molecule 1) can induce mobilization, which is independent of GCSF, IL3 and IL7 receptors.116 A simple de-adhesion step in the VLA4 system is not sufficient to elicit
mobilization of hematopoietic stem cells into peripheral blood, since an antibody of the same isotype that binds VLA4 but does not block its function, has no impact on mobilization. The intact functional cellular signal transduction pathway is necessary for this pathway to function. The role of extracellular matrix and metalloproteinases The emigration of hematopoietic stem cells through an endothelial layer of blood–bone-marrow barrier is thought to occur in sinusoids near interendothelial cell junctions and in regions where the endothelial cell basement membrane extracellular matrix is thinned or absent. The marrow extracellular matrix is composed of collagens, glycoproteins, glycosaminoglycans and matrix metalloproteinases,114,117 including gelatinases, collagenases and stromelysins. Damage of this blood–bone-marrow barrier and destruction of more mature hematopoietic progenitor cells by chemotherapeutic agents are supposed to be involved in the process of hematopoietic stem-cell mobilization. IL8 is chemotactic for neutrophils and induces the release of metalloproteinases and stimulates transendothelial emigration. Metalloproteinases as a group of enzymes, such as gelatinases, collagenases and stromelysins, degrade marrow extracellular matrix during the induction of mobilization. The role of intracellular signal transduction pathways Anti-VLA4 antibody without functional blocking activity does not mobilize hematopoietic stem cells. The roles of hematopoietic growth factors and adhesion molecules in mobilization require interaction with their functional receptors. The Janus kinasesignal transducer and activator of transcription (JakStat) pathway is widely used by multiple hematopoietic growth factor and chemokine families.118 Mouse knockouts, especially Stat 4 and Stat5ab knockouts, are expected to generate definitive information to further explore mechanisms of hematopoietic stem-cell mobilization. Negative regulation of the Jak-Stat pathway by tyrosine phosphatases, such as SHP1, is important as well. Deregulation of the Jak-Stat pathway in hematopoietic malignancies may provide indirect information on the involvement of this pathway in mobilization. As expected, anti-VLA4 antibody can induce mobilization in S1/S1d mice (lack expression of membrane-bound kit ligand in the marrow microenvironment with normal kit receptor on hematopoietic cells), but not in W/Wv mice (lack expression of kit receptor on hematopoietic cells with intact marrow
microenvironment). However, anti-VCAM1 antibody cannot induce mobilization in both S1/S1d and W/Wv mice, because a functional c-kit receptor is required for either anti-VLA4 or antiVCAM1 antibody induced mobilization, which does not occur in S1/S1d mice with an abnormal marrow microenvironment response and in W/Wv mice with unresponsive hematopoietic stem cells. Further experiments strengthen this idea that a functional kit receptor and its signal transduction pathway are important in the mobilization process. The tyrosine phosphatase SHP1 is a downstream negative effector of the c-kit receptor signal transduction pathway. Lack of SHP1 expression in me/mev mice results in augmented and prolonged activation of kit receptor phosphorylation, which, in turn, can result in remarkable hematopoietic disorders, including autoimmunity, massive expansion of myeloid cells and splenomegaly. The unexplained splenomegaly may be due to the increased migratory capacity of hematopoietic stem cells in these me/mev mice. It is not clear that downmodulation of the c-kit receptors on mobilized peripheral blood progenitor cells found with different mobilizing agents plays a role to induce mobilization or just reflects a negative feedback of the c-kit receptor signal transduction pathway activated by these mobilizing agents through receptor-mediated endocytosis. This is reflective of the dilemma presented by most proposed mechanisms of mobilization, that is whether the observed changes represent a causative influence on mobilization or a consequence of the mobilization treatment. The role of hematopoietic stem-cell proliferation, differentiation and apoptosis A wide spectrum of hematopoietic progenitor cells can be mobilized from uncommitted stem cells to committed hematopoietic progenitor cells. The more efficient the mobilization, the more primitive hematopoietic progenitor cells are mobilized. Ex vivo hematopoietic stem-cell-expansion strategies from either marrow or peripheral blood in non- and poorlymobilizable patients are often unsuccessful, indicating that hematopoietic stem cells with intrinsic defects in proliferation resulting from prior chemotherapy and/or radiotherapy cannot be mobilized. Hematopoietic stem-cell proliferation before emigration is believed to occur and plays an important role in this process since most of the mobilized CD34+ cells in peripheral blood are not in active cell cycle.119,120 Administration of G-CSF causes increased angiogenesis and marrow blood flow and stimulates a significant increase in the colonogenic capacity of all the progenitors evaluated.121 Profound proliferation within marrow can cause reorganization of existing
Mobilization of hematopoietic stem cells
The initiation-amplification-emigration-adaptation model of mobilization It is of great interest to establish the relationship among different mechanisms involved in hematopoietic stem cell mobilization and to define the role and sequence of different factors in a common pathway to induce mobilization. The role of cyclophosphamide locates upstream to the G-CSF system, while the cellular protein tyrosine kinase pathway locates downstream to the VLA4 system. The relationship between the VLA4 system and the G-CSF system in mobilization is not clear at present, but can be defined if the expression of VLA4 can be knocked out. Based on the previous observations of in-vivo and in-vitro studies, and synthesis of several postulates, the initiation-amplificationemigration-adaptation model as shown in Figure 1 may represent a common pathway involved in the process of hematopoietic stem cell mobilization. In the initiation phase, mobilizing agents act on their primary targets to initiate a network of reactions to induce mobilization. Thus, an indirect effect of mobilizing agents on hematopoietic stem cells is more important than its direct effect in this initiation phase. In the
CFU-GM/CD34vs Platelet 20,000
6 5 4 3 2 1 0 0
Days to reach platelet 20,000
CFU-GM/CD34vs Platelet 50,000
6 CFU-GM/CD34 ratio
pools of hematopoietic progenitor cells and remodel marrow blood flow and anatomic structure. Increased proliferation and overcrowding, per se, cannot lead to emigration automatically. In flt3 ligand treated mice, proliferation within bone marrow peaks early at day 3, but peak emigration is present much later. In cyclophosphamide treated G-CSF receptor knockout mice, no mobilization response is noted, but an extensive proliferation within marrow is present. An increase of CD34+/CD90+CD13+ cells and a decrease of CD34+/CD90+/CD13- and CD34+/CD90-/CD13- cells is found during G-CSF administration, indicating a progressive differentiation toward committed cells from more primitive uncommitted cells. But the absolute number of mobilized primitive uncommitted cells is also significantly increased. Therefore, the optimal mobilizing regimens prevent further differentiation and maximize hematopoietic stem-cell collection. Although there are few studies on the anti-apoptosis of hematopoietic stem cells involved in mobilization, it is apparent that this mechanism plays a important role to keep the mobilized peripheral blood progenitor cells alive in a new environment.122,123 Compared with peripheral blood progenitor cells in steady state, G-CSF mobilized peripheral blood progenitor cells are significantly less apoptotic.124 Chemotherapeutic agents, hematopoietic growth factors, chemokines, adhesion molecules and extracellular matrix associated with mobilization are all involved in the process of apoptosis.
5 4 3 2 1 0 0
Days to reach platelet 50,000 Fig. 1 The ratio of CFU-GM/CD34+ can be used as a parameter to predict prompt reconstitution of platelet (P=0.031 and P=0.024 for days to reach platelet 20 000/µl and 50 000/µl respectively).
amplification phase, cross-talk among hematopoietic stem cells, marrow microenvironment, and systemic status via hematopoietic growth factors, chemokines, intensive cell–cell and cell–matrix interactions, and their intercellular signal transduction pathways are involved. This may result in proliferation and phenotypic changes in hematopoietic stem cells for the next emigration step. In the emigration phase, the changed mobilizable hematopoietic stem cells lose their responses to stromal chemoattractants, demonstrate decreased adhesive affinity to the marrow microenvironment and, therefore, exit into blood through an endothelial layer. Obviously, damage to this layer may facilitate, but not necessarily result in shift of hematopoietic stem cells into peripheral blood if there are no coincident changes of hematopoietic stem cells and marrow microenvironment. In the adaptation phase, the modification of the mobilized cellular context and surface expression profile provide them a better chance to survive in the peripheral blood. Prevention of apoptosis by alternation of their proliferative status to G0/G1 phase, and inhibition of hematopoietic stem-cell homing by downregulating
1. Initiation: The direct and indirect effects mobilizer and cytokines
Mobilization 2. Amplification: Cell—cell and cell—matrix cross-talk, intracellular signal pathway, phenotype change and proliferation
4 Mobilizer Cytokines 1
3 3. Emigration: Changes of adhesive affinity and responses to chemoattractants
4. Adaptation: Phenotype changes to prevent apoptosis and homing
Fig. 2 Scheme of the initiation-amplification-emigration-adaptation model for hematopoietic stem cell mobilization.
surface antigen expression, may be very important in this phase.125 Although this mobilization model lacks solid support from systemic experimental data, it may help us better understand mechanisms of mobilization at the early stage and develop more sophisticated studies to shed more light on the pathways responsible for hematopoietic stem-cell mobilization. SIDE EFFECTS DURING MOBILIZATION Complications of hematopoietic stem-cell mobilization include those due to the procedures and those due to administration of chemotherapeutic agents and/or hematopoietic growth factors.126,127 The side effects of procedures related to central venous catheter placement and leukapheresis include line-related infection and leukapheresis-induced thrombocytopenia and coagulation abnormality with anticoagulants during collection.128 Tumor cells or abnormal hematopoietic stem cells from bone marrow can be co-mobilized to contaminate mobilized peripheral blood progenitor cells, which may require further purging.129,131 The side effects caused by chemotherapeutic agent administration depend on the dosage and the type of drug used, which may result in a disturbed immune system, infection, secondary malignancy, pulmonary, cardiovascular, hematologic, GI, endocrine, fertility, ophthalmologic, CNS and psychosocial consequences.132 The side effects caused by hematopoietic growth factor administration
include bone pain, headache, fatigue, arthralgia, myalgia, low-grade fever, nausea, cough, dyspnea, local skin rash, reduced platelet aggregation, hypercoagulable status by increasing levels of FVIII:C and thrombin generation, and abnormal serum chemistry with elevated levels of C-reactive protein, LDH, alkaline phosphatase, sodium and uric acid, and slightly decreased levels of albumin, total bilirubin, BUN and potassium.133 In general, the side effects caused by the above mobilizing agents are well tolerated and rarely fatal. These side effects can sometimes be lessened by coadministration of NSAIDs, narcotics, antihistamines and/or steroids. A large number of neutrophils mobilized with G-CSF appear to be functional without significant toxicity for at least 48 h following infusion of reconstituted cryopreserved preserve products, which provides a possible means to fight against infection, reduce or eliminate post-transplant neutropenia and help rapid platelet recovery because G-CSF mobilized neutrophil products are highly contaminated with platelets. CONCLUSIONS Since the 1980s, the peripheral blood progenitor cell transplantation has almost supplanted conventional bone-marrow transplantation because of more rapid hematopoietic recovery, less morbidity, decreased medical support and lower total costs. If a dose of 5
Mobilization of hematopoietic stem cells
×106 CD34+ cells/kg is transplanted, prompt and sustained hematopoietic recovery including neutrophils and platelets will occur in most of the transplanted patients. Although the studies on mobilization mechanisms remain at the early stage, practical mobilization regimens with chemotherapeutic agents and/or hematopoietic growth factors have been developed for current patient care, which are generally safe, well tolerated and effective.1,134 One emphasis of clinical studies has been to identify optimal mobilization regimens that can maximize hematopoietic stem-cell collection with the least toxicity. A better understanding of the mechanisms that regulate hematopoietic stem-cell mobilization may lead to the design of novel and optimal mobilization strategies addressing simplicity, efficacy and cost.135
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