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Hematopoietic Stem Cell Mobilization

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Hematopoietic Stem Cell Mobilization

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    1. Hematopoietic Stem Cell Mobilization Armin Ghobadi, MD Fellow, Hematology & Oncology Washington University in St Louis 10/28/2011 1

    2. Disclosure I have NO financial relationships to disclose 2

    3. Overview History Stem cell niche Pathways/receptors involving stem cell mobilizaion and homing GCSF GCSF + Chemotherapy CXCR-4 Inhibition Plerixafor (Mozobil) Other agents VLA-4 inhibition Summary References 3

    4. History 1957: Thomas et al. IV infusion of bone marrow in patients receiving radiation and chemotherapy ( N. Engl. J. Med. 257, 491496 (1957)) 1958: Bond et al. DNA synthesizing cells in peripheral blood of normal human beings (Science 1958;128:202-203) 1961: Bierman et al. Leukapheresis in Men (Brit. J. haematol., 7:51-63, 1961) 1962: Goodman et al. Evidence for stem cells in the peripheral blood of mice (Blood 1962;19:702-714) 1971: McCredie et al. Cells capable of colony formation in the peripheral blood of man (Science 1971;171:293-4) 1979/1980: Hershko et al and Abrams et al. Failed PBSCT attempts (Lancet 1979;1(8123):945-947 and Blood 1980;56:516-520) 1981: Goldman et al. First successful autologous PBSCT (N Engl J Med 1981;305:700) 1985: Nicola et al. Human G-CSF was introduced (Nature 314:625, 1985) 1988: Dhrsen et al. G-CSF increases PB progenitor cells up to 100 fold (Blood 1988;72(6):2074-2081) 1989: Kessinger et al: First allogeneic PBSCT. (Bone Marrow Transplant.1989;4(6):643-646.) 1991: Filgrastim was approved for chemotherapy induced neuropenia 1992: Sheriden et al. PBSCT using G-CSF as a mobilizing agent 2008/2009: DiPersio et al. 2 randomized phase 3 trials confirmed AMD3100 (Plerixafor) safety and efficacy in NHL and MM (Blood 2009;113(23):5720-5726 and J Clin Oncol 2009;27(28):4767-4773) AMD3100 (Plerixafor) was FDA-approved for HSPC mobilization in combination with G-CSF 4 the term blood stem cell as a transplantable cell population was introduced in 1962 The Nobel Prize in Physiology or Medicine 1990 E. Donnall Thomas 1971: McCredie et al: first attempt at M. D. Anderson Cancer Center of collecting colony-forming stem cells by continuous-flow apheresis was successful Micklem HS, Anderson N, Ross E. Limited potential of circulating haemopoietic stem cells. Nature 1975;256:41-3. ---1979/1980: Two clinical blood stem-cell transplantation attempts among identical twins, one in 1979 at the University of California at Los Angeles12 and the other in 1980 at the NIH,13 resulted in failed or incomplete lymphohematopoietic reconstitution. In the University of California at Los Angeles case the leukapheresis product was administered to the twin patient as 6 separate white blood cell transfusions during the course of 14 days. The total amount of mononuclear cells transfused was 5.8 1010/kg recipient body weight. More than 2 months after transfusion, no platelet or granulocyte engraftment was documented. One may speculate that each individual white blood cell transfusion did not contain a sufficient amount of hematopoietic progenitor cells to enable engraftment. In the NIH case, the leukapheresis-derived mononuclear cells were administered as daily infusions during the course of 8 days. No recovery of granulocytes, monocytes, or platelets was documented at 8 weeks after transfusion. The authors attributed engraftment failure to the low number of clonogenic hematopoietic progenitor cells transfused being only one-eighth the quantity used in BM transplantation. Subsequently, both patients were rescued with their twin's marrow cells. It should be noted that, at that time, stem-cell collection by apheresis was performed at steady state without any growth factor priming 1981: in CML using leukopheresis and cryopreservationthe term blood stem cell as a transplantable cell population was introduced in 1962 The Nobel Prize in Physiology or Medicine 1990 E. Donnall Thomas 1971: McCredie et al: first attempt at M. D. Anderson Cancer Center of collecting colony-forming stem cells by continuous-flow apheresis was successful Micklem HS, Anderson N, Ross E. Limited potential of circulating haemopoietic stem cells. Nature 1975;256:41-3. ---1979/1980: Two clinical blood stem-cell transplantation attempts among identical twins, one in 1979 at the University of California at Los Angeles12 and the other in 1980 at the NIH,13 resulted in failed or incomplete lymphohematopoietic reconstitution. In the University of California at Los Angeles case the leukapheresis product was administered to the twin patient as 6 separate white blood cell transfusions during the course of 14 days. The total amount of mononuclear cells transfused was 5.8 1010/kg recipient body weight. More than 2 months after transfusion, no platelet or granulocyte engraftment was documented. One may speculate that each individual white blood cell transfusion did not contain a sufficient amount of hematopoietic progenitor cells to enable engraftment. In the NIH case, the leukapheresis-derived mononuclear cells were administered as daily infusions during the course of 8 days. No recovery of granulocytes, monocytes, or platelets was documented at 8 weeks after transfusion. The authors attributed engraftment failure to the low number of clonogenic hematopoietic progenitor cells transfused being only one-eighth the quantity used in BM transplantation. Subsequently, both patients were rescued with their twin's marrow cells. It should be noted that, at that time, stem-cell collection by apheresis was performed at steady state without any growth factor priming 1981: in CML using leukopheresis and cryopreservation

    5. Allo-HSCT source - Adults 5

    6. Allo-HSCT Source - Pediatrics 6

    7. Auto-HSCT source 7

    8. HSC Niche Sinusoidal endothelial cells 1. VCAM-1 2. CXCL12 8 --Maintenance of HSCs and regulation of their self-renewal and differentiation in vivo is thought to depend on their specific microenvironment, which has been historically called the haematopoieticinductive microenvironment10 or stem-cell niche --Models of HSPC mobilization by G-CSF and inhibitors of CXCR4 and VLA-4. (a) Bone marrow environment at baseline. HSPCs in the endosteal niche are likely in close contact with osteoblasts and nestin? MSCs, both of which express numerous HSPC retention factors, including CXCL12, VCAM-1 and SCF. The perivascular niche is more distant from the endosteum and includes both nestin? MSCs and CXCL12 abundant reticular cells. Cells of the monocytic lineage support the maintenance of osteoblasts and MSCs. b-Adrenergic nerve cells of the sympathetic nervous system regulate MSC proliferation and induce circadian oscillations of CXCL12 expression. (b) Model of G-CSF-induced HSPC mobilization. Cells of the monocytic lineage express the receptor for G-CSF and provide factors that support the survival of MSCs and osteoblasts. Upon 45 days of stimulation with G-CSF, the monocytes/macrophages disappear, leading to the loss of osteoblast lineage cells and reduced expression of CXCL12, VCAM-1 and SCF on MSCs. Reduced expression of these important HSPC retention factors is also observed following G-CSF signaling through b-adrenergic nerve cells. The net effect of these signaling cascades is the disruption of HSPC retention interactions and mobilization of HSPCs into the peripheral blood. (c, d) Model of HSPC mobilization by inhibitors of CXCR4 or VLA-4. Targeted disruption of the interaction of CXCR4 or VLA-4 with their ligands results in the rapid (within hours) and reversible mobilization of HSPCs into the peripheral circulation. An additive or synergistic affect on HSPC mobilization is observed when a CXCR4 inhibitor is combined with a VLA-4 antagonist, G-CSF or their combination. When used alone, inhibitors of CXCR4 and VLA-4 mobilize fewer HSPCs than G-CSF. ----------------------------------------------------- Support for the hypothesis that one cell type is present at various sites within the marrow niche was obtained by a recent landmark study by Mndez-Ferrer et al. (2010). This group identified a stromal nestin-expressing MSC population (nestin+ MSC) that is closely associated with putative HSCs. Nestin+ MSCs are strictly perivascular and are typically found in more central areas of the marrow, but they are also present in the immediate vicinity of the endosteum, albeit at lower frequency. Moreover, nestin+ MSCs are tightly associated with adrenergic nerve fibers of the sympathetic nervous system (SNS) that regulate HSC mobilization and are responsible for the circadian oscillations in circulating HSC numbers (Katayama et al., 2006; Mndez-Ferrer et al., 2008). Strikingly, these MSCs express higher levels of HSC maintenance factor transcripts, including CXCL12, stem cell factor (SCF), angiopoietin-1 (Ang-1), IL-7, vascular cell adhesion molecule 1 (VCAM1), and osteopontin (OPN), compared with any other stromal cell type including OBs. Depletion of nestin+ MSCs by inducible expression of diphtheria toxin receptor (DTR) in nestin-expressing cells caused the mobilization of ~50% of HSCs to the spleen. In addition, the homing of transplanted progenitor cells into MSC-depleted recipients was reduced by 90%.Moreover, treatment with the HSC-mobilizing factor granulocyte colony-stimulating factor (G-CSF) decreased the expression of CXCL12 and other factors required for HSC maintenance and retention by nestin+ MSCs. These cells also seem to mediate the capacity of parathyroid hormone to increase HSC numbers, which had previously been correlated with an increase in putative niche OBs (Calvi et al., 2003; Adams et al., 2007). Parathyroid hormone directly stimulated proliferation of nestin+ MSCs while simultaneously promoting their differentiation into OBs (Mndez-Ferrer et al., 2010). Collectively, these data support the hypothesis that nestin+ MSCs are a functional component of the bone marrow HSC niche.Most interestingly, nestin+ MSCs show several similarities to recently identified mesenchymal progenitors (Sugiyama et al., 2006; Omatsu et al., 2010). These bipotent adipoosteogenic progenitors were identified in a mouse strain in which GFP was expressed from the endogenous CXCL12 locus (Sugiyama et al., 2006; Omatsu et al., 2010). Because of their high CXCL12 expression and their long cellular processes, these cells were named CXCL12-abundant reticular (CAR) cells. The majority of putative HSCs are found in close proximity to CAR cells by immunohistochemistry, and like nestin+ MSCs, CAR cells are predominantly found in the more central areas of the marrow, with some also located close to vessels near the endosteum (Fig. 1). Although CAR cells are more abundant than nestin+ MSCs, they too are tightly associated with sinusoidal endothelium and have a similar morphology to vascular pericytes. These data are in agreement with studies in humans, suggesting that virtually all MSC activity is found within the larger pericyte population that associates closely with the vascular system in the entire body (Crisan et al., 2008).Similar to nestin+ MSCs, CAR cells express HSC maintenance proteins such as CXCL12 and SCF. Induced depletion of CAR cells using a DTR approach causes a partial loss of HSC activity associated with a decrease in HSC cycling, suggesting that CAR cells promote HSC cycling and self-renewal. Even though this phenomenon was not observed after depletion of nestin+ MSCs, it seems likely that CAR cells and nestin+ MSCs represent two highly overlapping CXCL12-expressing cell populations. Because nestin+ MSCs are approximately four times less abundant than CAR cells, contain all colony-forming-unit fibroblast activity within the marrow, harbor high self-renewal activity in vitro and in vivo, and are capable of multilineage differentiation into bone, cartilage, and fat, it appears that nestin+ MSCs may represent a more primitive population compared with CAR cells and may even be a CAR subpopulation. As it remains unclear whether nestin+ MSCs are homogeneous and whether they all express high levels of CXCL12, it is possible that some nestin+ MSCs may not be CAR cells. Although future studies will need to dissect the cellular relationship between these two niche cell populations, there is now convincing evidence that perivascular mesenchymal stem/progenitor cells are critical inhabitants of the HSC niche. (Edinger et al. JEM vol. 208 no. 3 421-428 ) ----SNS promotes egress by reducing the expression of key retention factors by the niche cell (Mndez-Ferrer et al., 2010b), and in contrast, BM Macrophage promotes the expression of these genes and HSC/progenitor retention in the BM.--Maintenance of HSCs and regulation of their self-renewal and differentiation in vivo is thought to depend on their specific microenvironment, which has been historically called the haematopoieticinductive microenvironment10 or stem-cell niche --Models of HSPC mobilization by G-CSF and inhibitors of CXCR4 and VLA-4. (a) Bone marrow environment at baseline. HSPCs in the endosteal niche are likely in close contact with osteoblasts and nestin? MSCs, both of which express numerous HSPC retention factors, including CXCL12, VCAM-1 and SCF. The perivascular niche is more distant from the endosteum and includes both nestin? MSCs and CXCL12 abundant reticular cells. Cells of the monocytic lineage support the maintenance of osteoblasts and MSCs. b-Adrenergic nerve cells of the sympathetic nervous system regulate MSC proliferation and induce circadian oscillations of CXCL12 expression. (b) Model of G-CSF-induced HSPC mobilization. Cells of the monocytic lineage express the receptor for G-CSF and provide factors that support the survival of MSCs and osteoblasts. Upon 45 days of stimulation with G-CSF, the monocytes/macrophages disappear, leading to the loss of osteoblast lineage cells and reduced expression of CXCL12, VCAM-1 and SCF on MSCs. Reduced expression of these important HSPC retention factors is also observed following G-CSF signaling through b-adrenergic nerve cells. The net effect of these signaling cascades is the disruption of HSPC retention interactions and mobilization of HSPCs into the peripheral blood. (c, d) Model of HSPC mobilization by inhibitors of CXCR4 or VLA-4. Targeted disruption of the interaction of CXCR4 or VLA-4 with their ligands results in the rapid (within hours) and reversible mobilization of HSPCs into the peripheral circulation. An additive or synergistic affect on HSPC mobilization is observed when a CXCR4 inhibitor is combined with a VLA-4 antagonist, G-CSF or their combination. When used alone, inhibitors of CXCR4 and VLA-4 mobilize fewer HSPCs than G-CSF. ----------------------------------------------------- Support for the hypothesis that one cell type is present at various sites within the marrow niche was obtained by a recent landmark study by Mndez-Ferrer et al. (2010). This group identified a stromal nestin-expressing MSC population (nestin+ MSC) that is closely associated with putative HSCs. Nestin+ MSCs are strictly perivascular and are typically found in more central areas of the marrow, but they are also present in the immediate vicinity of the endosteum, albeit at lower frequency. Moreover, nestin+ MSCs are tightly associated with adrenergic nerve fibers of the sympathetic nervous system (SNS) that regulate HSC mobilization and are responsible for the circadian oscillations in circulating HSC numbers (Katayama et al., 2006; Mndez-Ferrer et al., 2008). Strikingly, these MSCs express higher levels of HSC maintenance factor transcripts, including CXCL12, stem cell factor (SCF), angiopoietin-1 (Ang-1), IL-7, vascular cell adhesion molecule 1 (VCAM1), and osteopontin (OPN), compared with any other stromal cell type including OBs. Depletion of nestin+ MSCs by inducible expression of diphtheria toxin receptor (DTR) in nestin-expressing cells caused the mobilization of ~50% of HSCs to the spleen. In addition, the homing of transplanted progenitor cells into MSC-depleted recipients was reduced by 90%.Moreover, treatment with the HSC-mobilizing factor granulocyte colony-stimulating factor (G-CSF) decreased the expression of CXCL12 and other factors required for HSC maintenance and retention by nestin+ MSCs. These cells also seem to mediate the capacity of parathyroid hormone to increase HSC numbers, which had previously been correlated with an increase in putative niche OBs (Calvi et al., 2003; Adams et al., 2007). Parathyroid hormone directly stimulated proliferation of nestin+ MSCs while simultaneously promoting their differentiation into OBs (Mndez-Ferrer et al., 2010). Collectively, these data support the hypothesis that nestin+ MSCs are a functional component of the bone marrow HSC niche.Most interestingly, nestin+ MSCs show several similarities to recently identified mesenchymal progenitors (Sugiyama et al., 2006; Omatsu et al., 2010). These bipotent adipoosteogenic progenitors were identified in a mouse strain in which GFP was expressed from the endogenous CXCL12 locus (Sugiyama et al., 2006; Omatsu et al., 2010). Because of their high CXCL12 expression and their long cellular processes, these cells were named CXCL12-abundant reticular (CAR) cells. The majority of putative HSCs are found in close proximity to CAR cells by immunohistochemistry, and like nestin+ MSCs, CAR cells are predominantly found in the more central areas of the marrow, with some also located close to vessels near the endosteum (Fig. 1). Although CAR cells are more abundant than nestin+ MSCs, they too are tightly associated with sinusoidal endothelium and have a similar morphology to vascular pericytes. These data are in agreement with studies in humans, suggesting that virtually all MSC activity is found within the larger pericyte population that associates closely with the vascular system in the entire body (Crisan et al., 2008).Similar to nestin+ MSCs, CAR cells express HSC maintenance proteins such as CXCL12 and SCF. Induced depletion of CAR cells using a DTR approach causes a partial loss of HSC activity associated with a decrease in HSC cycling, suggesting that CAR cells promote HSC cycling and self-renewal. Even though this phenomenon was not observed after depletion of nestin+ MSCs, it seems likely that CAR cells and nestin+ MSCs represent two highly overlapping CXCL12-expressing cell populations. Because nestin+ MSCs are approximately four times less abundant than CAR cells, contain all colony-forming-unit fibroblast activity within the marrow, harbor high self-renewal activity in vitro and in vivo, and are capable of multilineage differentiation into bone, cartilage, and fat, it appears that nestin+ MSCs may represent a more primitive population compared with CAR cells and may even be a CAR subpopulation. As it remains unclear whether nestin+ MSCs are homogeneous and whether they all express high levels of CXCL12, it is possible that some nestin+ MSCs may not be CAR cells. Although future studies will need to dissect the cellular relationship between these two niche cell populations, there is now convincing evidence that perivascular mesenchymal stem/progenitor cells are critical inhabitants of the HSC niche. (Edinger et al. JEM vol. 208 no. 3 421-428 ) ----SNS promotes egress by reducing the expression of key retention factors by the niche cell (Mndez-Ferrer et al., 2010b), and in contrast, BM Macrophage promotes the expression of these genes and HSC/progenitor retention in the BM.

    9. Endosteal Niche-Stem Cell Synapse 9 A model of the endosteal nichestem-cell synapse. Schematic diagram of the endosteal nichestem-cell synapse showing putative ligandreceptor interactions and adhesion molecules, as well as some of the intracellular pathways that are activated following signalling. ANG1, angiopoietin-1; BMI1, polycomb repressor; BMP, bone morphogenetic protein; BMPR1A, BMP receptor 1A; CSL, CBF1 suppressor of Hairless and LAG1; CXCL12, CXCchemokine ligand 12; CXCR4, CXC-chemokine receptor 4; FAK, focal adhesion kinase; HOXB4, homeobox B4; HSC, haematopoietic stem cell; ICAM1, intercellular adhesion molecule 1; LFA1, lymphocyte function-associated antigen 1; LRP, low-density-lipoprotein-receptor-related protein; MAPK, mitogen-activated protein kinase; OPN, osteopontin; PI3K, phosphatidylinositol-3 kinase; PLC, phospholipase C; PKC, protein kinase C; PPR, PTH/PTH-related protein receptor; PTH, parathyroid hormone; SCF, stem-cell factor; SMADS, mothers against decapentaplegic-related homologue; SNO, spindle-shaped N-cadherin-expressing osteoblast; TIE2, tyrosine kinase receptor 2; VCAM1, vascular cell-adhesion molecule 1; VLA4, very late antigen 4; ? denotes molecules and/or interactions for which only indirect or contradictory evidence is available.A model of the endosteal nichestem-cell synapse. Schematic diagram of the endosteal nichestem-cell synapse showing putative ligandreceptor interactions and adhesion molecules, as well as some of the intracellular pathways that are activated following signalling. ANG1, angiopoietin-1; BMI1, polycomb repressor; BMP, bone morphogenetic protein; BMPR1A, BMP receptor 1A; CSL, CBF1 suppressor of Hairless and LAG1; CXCL12, CXCchemokine ligand 12; CXCR4, CXC-chemokine receptor 4; FAK, focal adhesion kinase; HOXB4, homeobox B4; HSC, haematopoietic stem cell; ICAM1, intercellular adhesion molecule 1; LFA1, lymphocyte function-associated antigen 1; LRP, low-density-lipoprotein-receptor-related protein; MAPK, mitogen-activated protein kinase; OPN, osteopontin; PI3K, phosphatidylinositol-3 kinase; PLC, phospholipase C; PKC, protein kinase C; PPR, PTH/PTH-related protein receptor; PTH, parathyroid hormone; SCF, stem-cell factor; SMADS, mothers against decapentaplegic-related homologue; SNO, spindle-shaped N-cadherin-expressing osteoblast; TIE2, tyrosine kinase receptor 2; VCAM1, vascular cell-adhesion molecule 1; VLA4, very late antigen 4; ? denotes molecules and/or interactions for which only indirect or contradictory evidence is available.

    10. G-CSF/Filgrastim-model of HSPC mobilization 10 Models of HSPC mobilization by G-CSF and inhibitors of CXCR4 and VLA-4. (a) Bone marrow environment at baseline. HSPCs in the endosteal niche are likely in close contact with osteoblasts and nestin? MSCs, both of which express numerous HSPC retention factors, including CXCL12, VCAM-1 and SCF. The perivascular niche is more distant from the endosteum and includes both nestin? MSCs and CXCL12 abundant reticular cells. Cells of the monocytic lineage support the maintenance of osteoblasts and MSCs. b-Adrenergic nerve cells of the sympathetic nervous system regulate MSC proliferation and induce circadian oscillations of CXCL12 expression. (b) Model of G-CSF-induced HSPC mobilization. Cells of the monocytic lineage express the receptor for G-CSF and provide factors that support the survival of MSCs and osteoblasts. Upon 45 days of stimulation with G-CSF, the monocytes/macrophages disappear, leading to the loss of osteoblast lineage cells and reduced expression of CXCL12, VCAM-1 and SCF on MSCs. Reduced expression of these important HSPC retention factors is also observed following G-CSF signaling through b-adrenergic nerve cells. The net effect of these signaling cascades is the disruption of HSPC retention interactions and mobilization of HSPCs into the peripheral blood. (c, d) Model of HSPC mobilization by inhibitors of CXCR4 or VLA-4. Targeted disruption of the interaction of CXCR4 or VLA-4 with their ligands results in the rapid (within hours) and reversible mobilization of HSPCs into the peripheral circulation. An additive or synergistic affect on HSPC mobilization is observed when a CXCR4 inhibitor is combined with a VLA-4 antagonist, G-CSF or their combination. When used alone, inhibitors of CXCR4 and VLA-4 mobilize fewer HSPCs than G-CSF. -- Models of HSPC mobilization by G-CSF and inhibitors of CXCR4 and VLA-4. (a) Bone marrow environment at baseline. HSPCs in the endosteal niche are likely in close contact with osteoblasts and nestin? MSCs, both of which express numerous HSPC retention factors, including CXCL12, VCAM-1 and SCF. The perivascular niche is more distant from the endosteum and includes both nestin? MSCs and CXCL12 abundant reticular cells. Cells of the monocytic lineage support the maintenance of osteoblasts and MSCs. b-Adrenergic nerve cells of the sympathetic nervous system regulate MSC proliferation and induce circadian oscillations of CXCL12 expression. (b) Model of G-CSF-induced HSPC mobilization. Cells of the monocytic lineage express the receptor for G-CSF and provide factors that support the survival of MSCs and osteoblasts. Upon 45 days of stimulation with G-CSF, the monocytes/macrophages disappear, leading to the loss of osteoblast lineage cells and reduced expression of CXCL12, VCAM-1 and SCF on MSCs. Reduced expression of these important HSPC retention factors is also observed following G-CSF signaling through b-adrenergic nerve cells. The net effect of these signaling cascades is the disruption of HSPC retention interactions and mobilization of HSPCs into the peripheral blood. (c, d) Model of HSPC mobilization by inhibitors of CXCR4 or VLA-4. Targeted disruption of the interaction of CXCR4 or VLA-4 with their ligands results in the rapid (within hours) and reversible mobilization of HSPCs into the peripheral circulation. An additive or synergistic affect on HSPC mobilization is observed when a CXCR4 inhibitor is combined with a VLA-4 antagonist, G-CSF or their combination. When used alone, inhibitors of CXCR4 and VLA-4 mobilize fewer HSPCs than G-CSF. --

    11. The most common mobilization strategies Allo-HSCT: G-CSF Auto-HSCT: G-CSG GM-CSF Chemomobilization (Cytoxan + G-CSF) Pleraxifor + G-CSF for NHL and MM 11

    12. G-CSF 10 mcg/kg day or 5 mcg/kg BID Start leukaphaeresis on day 5 Minimum number of CD34+ cells: 2 x 106 /kg Success rate: 70-95% Side effects: Pain, headaches, arthralgias, malaise, fatigue, insomnia, and nausea Transient increase in ALP, ALT, LDH, Na and transient decrease in potasium Increased spleen size in length (in 95% of pts) (mean increase,13%) but 10 days after G-CSF administration, spleen size returns to baseline. Several cases of spontaneous splenic rupture after G-CSF administration to healthy subjects have been reported. 12

    13. --- 2408 unrelated PBSC donors prospectively evaluated by the National Marrow Donor Program (NMDP) between 1999 and 2004. Incidence of bone pain. Pain symptoms were evaluated before administration of filgrastim each day and at each follow-up after donation. (A) Percentage of PBSC donors who experienced bone pain. (B) Site of bone pain frequency on Day 4. (C) Frequency of highest severity of bone pain during mobilization and collection. ---Pain decreased significantly after filgrastim administration was stopped, but was still reported in 24% and 6% of donors at 2 days and 1 week, respectively, after the final apheresis collection.--- 2408 unrelated PBSC donors prospectively evaluated by the National Marrow Donor Program (NMDP) between 1999 and 2004. Incidence of bone pain. Pain symptoms were evaluated before administration of filgrastim each day and at each follow-up after donation. (A) Percentage of PBSC donors who experienced bone pain. (B) Site of bone pain frequency on Day 4. (C) Frequency of highest severity of bone pain during mobilization and collection. ---Pain decreased significantly after filgrastim administration was stopped, but was still reported in 24% and 6% of donors at 2 days and 1 week, respectively, after the final apheresis collection.

    14. Assessment of donor symptoms using the Abbreviated CALGB Toxicity Criteria. Abbreviated CALGB Toxicity Criteria were evaluated before administration of filgrastim each day. (A) Frequency of highest CALGB score reported by PBSC donors during mobilization and collection. (B) Frequency of highest CALGB score across all symptoms reported by PBSC donors during mobilization and collection. --Symptoms disappeared almost completely by 1 week after donation, with the exception of slight persisting increases in fatigue, headache, and myalgia (1 week after donation vs baseline: 7.9% vs 0.8%, 4.9% vs 2.5%, and 3.7% vs 2.1%, respectively). Rates of headache and myalgia returned to baseline by the 1-month assessment, but a few donors continued to report fatigue (3.1% at 1 month vs a baseline of 0.8%) Assessment of donor symptoms using the Abbreviated CALGB Toxicity Criteria. Abbreviated CALGB Toxicity Criteria were evaluated before administration of filgrastim each day. (A) Frequency of highest CALGB score reported by PBSC donors during mobilization and collection. (B) Frequency of highest CALGB score across all symptoms reported by PBSC donors during mobilization and collection. --Symptoms disappeared almost completely by 1 week after donation, with the exception of slight persisting increases in fatigue, headache, and myalgia (1 week after donation vs baseline: 7.9% vs 0.8%, 4.9% vs 2.5%, and 3.7% vs 2.1%, respectively). Rates of headache and myalgia returned to baseline by the 1-month assessment, but a few donors continued to report fatigue (3.1% at 1 month vs a baseline of 0.8%)

    15. Box and whiskers plot of blood counts showing the maximum, upper quartile, median, lower quartile, and minimum values obtained from PBSC donors on the first day of injection (Day 1), before and after donation (Day 5 before and Day 5 after), and during follow-up after donation. (A) Donor white blood cell counts. (B) Donor platelet counts. (C) Donor hemoglobin levels, by sex. ----Although nearly a third of donors experienced a WBCs above 50 109/L, less than 1% of donors exceeded a higher risk level of 75 109/L. No episodes of thrombosis or stroke were reported. After 2 days of collection, nearly 40% of donors had platelet counts below 100 109/L, with approximately 2% of donors going below 50 109/L and a single donor going below 20 109/L. Males and females experienced similar declines in hematocrit values accompanying apheresis, but female donors often declined to values below 30% (5% and 9% occurrence after the first and second apheresis procedure, respectively). Very rarely, donors persisted with low platelet or hemoglobin levels at 1 month after donation-Box and whiskers plot of blood counts showing the maximum, upper quartile, median, lower quartile, and minimum values obtained from PBSC donors on the first day of injection (Day 1), before and after donation (Day 5 before and Day 5 after), and during follow-up after donation. (A) Donor white blood cell counts. (B) Donor platelet counts. (C) Donor hemoglobin levels, by sex. ----Although nearly a third of donors experienced a WBCs above 50 109/L, less than 1% of donors exceeded a higher risk level of 75 109/L. No episodes of thrombosis or stroke were reported. After 2 days of collection, nearly 40% of donors had platelet counts below 100 109/L, with approximately 2% of donors going below 50 109/L and a single donor going below 20 109/L. Males and females experienced similar declines in hematocrit values accompanying apheresis, but female donors often declined to values below 30% (5% and 9% occurrence after the first and second apheresis procedure, respectively). Very rarely, donors persisted with low platelet or hemoglobin levels at 1 month after donation-

    16. G-CSF mobilized vs. BM composition Healthy adult donors: G-CSF mobilized : 31 donors BM: 26 donors 16 ---Previous studies by others have demonstrated that human CD34? cells can be divided into at least three distinct subsets based on their cell surface expression of CD45RA and CD123 (IL-3Ra): (i) CD34? CD45RACD123?/ cells containing common myeloid progenitors, megakaryocyte/erythrocyte progenitors and more primitive HSPCs; (ii) CD34?CD45RA?CD123?/ cells containing more committed granulocyte/macrophage and lymphoid progenitors; and (iii) CD34dimCD45RA?CD123hi cells.98,99---Previous studies by others have demonstrated that human CD34? cells can be divided into at least three distinct subsets based on their cell surface expression of CD45RA and CD123 (IL-3Ra): (i) CD34? CD45RACD123?/ cells containing common myeloid progenitors, megakaryocyte/erythrocyte progenitors and more primitive HSPCs; (ii) CD34?CD45RA?CD123?/ cells containing more committed granulocyte/macrophage and lymphoid progenitors; and (iii) CD34dimCD45RA?CD123hi cells.98,99

    17. G-CSF mobilized vs. BM composition 17 BM: minimum of 2 108 nucleated cells/kg recipient PB: minimum 4 x 106 BM: minimum of 2 108 nucleated cells/kg recipient PB: minimum 4 x 106

    18. PBSCT compared with BMT PLT engraftment: shorter (~ 5 days) Neutrophil engraftment: shorter (~ 5 days) Hospital stay: shorter Rate of grade II-IV aGVHD: the same Rate of chronic GVHD: higher (~ 10% higher) NRM: the same Relapse rate: the same/trend toward lower rate OS: the same BMT CTN Study 0201: A Phase III Randomized Multicenter Trial Comparing G-CSF Mobilized Peripheral Blood Stem Cell with Marrow Transplantation from HLA Compatible Unrelated Donors met its target accrual of 550 patients and closed enrollment on September 9, 2009 18

    19. Hard to mobilize patients G-CSF non responders: 5-30% Strategies to manage non-responders: Dose escalation of G-CSF (12.550?g/kg per day), Addition of another cytokine such as GM-CSF, Addition of chemotherapy Harvesting the BM 19

    20. Chemo-mobilization Cyclophosphamide(typically 2-6 gm/m(2)) plus G-CSF 10 microg/kg on days 3-14 Leukapheresis: After white count recovery (usually days 12-15) Drawbacks: higher toxicities, increased risk for secondary malignances, impairment of fertility, cardiac toxicity, hemorrhagic cystitis, anaphylactic reactions and higher cost 20 In general, patients who fail initial mobilization are more likely to fail remobilization regardless of the remobilization regimen.17, 25 Furthermore, additional mobilization efforts often result in poorer patient outcomes and increased resource utilization. For these reasons, novel mobilization agents that are less toxic, more rapid and increase the yield of collected CD34+ cells for transplantation are needed.In general, patients who fail initial mobilization are more likely to fail remobilization regardless of the remobilization regimen.17, 25 Furthermore, additional mobilization efforts often result in poorer patient outcomes and increased resource utilization. For these reasons, novel mobilization agents that are less toxic, more rapid and increase the yield of collected CD34+ cells for transplantation are needed.

    21. CXCR4/CXCL12 axis Sinusoidal endothelial cells 1. VCAM-1 2. CXCL12 21 -- CXCL12 or stromal cell-derived factor 1a (SDF-1a) is a chemokine that is constitutively produced at high levels in the BM by stromal cells such as osteoblasts, endothelial cells, Nestin+ MSC, and CAR. -- It is a potent chemoattractant for HSPCs and has been shown to regulate cell adhesion, survival and cell-cycle status --- CXCR4 is a member of the large family of seven-transmembrane domain receptors coupled to heterotrimeric Gi proteins and functions as a co-receptor for human immunodeficiency virus type 1 (HIV-1) cell entry -- The binding of CXCR4 to CXCL12 results in the activation of multiple signal-transduction pathways ultimately triggering chemotaxis -- CXCL12 or stromal cell-derived factor 1a (SDF-1a) is a chemokine that is constitutively produced at high levels in the BM by stromal cells such as osteoblasts, endothelial cells, Nestin+ MSC, and CAR. -- It is a potent chemoattractant for HSPCs and has been shown to regulate cell adhesion, survival and cell-cycle status --- CXCR4 is a member of the large family of seven-transmembrane domain receptors coupled to heterotrimeric Gi proteins and functions as a co-receptor for human immunodeficiency virus type 1 (HIV-1) cell entry -- The binding of CXCR4 to CXCL12 results in the activation of multiple signal-transduction pathways ultimately triggering chemotaxis

    22. Genetic alteration of CXCR4/CXCL12 axis CXCR4-/- and CXCL12-/- embryos: die perinatally reduced myelopoiesis in fetal liver and nearly complete loss of myelopoiesis in the BM CXCR4-/- hematopoietic chimeras: Fetal liver cells from 12- to 15-dpc CXCR4 +/- intercrosses Transplanted to lethally irradiated mice 22

    23. AMD-3100/Plerixafor Reversible inhibitor of CXCR4 Originally tested clinically as an agent for the treatment of HIV infection by blocking the HIV entry into CD4+ T cells Accidental finding: leukocytosis after a single intravenous dose of plerixafor A single subcutaneous dose of plerixafor at 160240?g/kg: 6- to 10-fold increase in CD34+ cell Testing of plerixafor as an HIV drug was abandoned: lack of anti-viral effect asymptomatic PVC in two patients 23

    24. AMD-3100/Plerixafor-Phase I Patients: MM and NHL Dosage: 160 g/kg (n = 6) 240 g/kg (n = 7) 24 --Total WBC count and peripheral blood CD34+ cell count observed in patients receiving AMD3100 at 160 g/kg or 240 g/kg at baseline and 4, 6, and 24 hours (WBC count only at 24 hours) after the dose. Each value represents the mean SE (n = 13). --Total WBC count and peripheral blood CD34+ cell count observed in patients receiving AMD3100 at 160 g/kg or 240 g/kg at baseline and 4, 6, and 24 hours (WBC count only at 24 hours) after the dose. Each value represents the mean SE (n = 13).

    25. AMD-3100/Plerixafor-Phase 2 25 patients with MM or NHL in their first or second CR or PR G-CSF alone ? 2-week washout period ? AMD3100+G-CSF G-CSF: 10?g/kg/day and pheresis was begun on day 4 G-CSF+AMD3100: 160240?g/kg/day beginning on day 4 followed 6?h later by apheresis Results: 9 of 25 patients who failed to yield the minimum CD34+ cells per kg after four leukaphereses with G-CSF alone All 9 patients successfully remobilized with AMD3100+G-CSF 25

    26. AMD-3100/Plerixafor-Phase III in NHL Design: phase III, multicenter, randomized (1:1), double-blind, placebo-controlled Subjects: Patients NHL requiring an Auto-SCT in first or second CR or PR Study Arms: G-CSF + AMD3100: 150 G-CSF + Placebo: 148 Primary endpoint: percentage of patients who collected = 5 106 CD34+ cells/kg in < 4 apheresis Secondary endpoints: Percantage of patients able to mobilize = 2 106 CD34+ cells/kg in = 4 apheresis the number of apheresis days required to reach = 5 106 CD34+ cells/kg; neutrophil and platelet engraftment engraftment at 100 days, 6 months, and 12 months after transplantation 26

    27. (A) Kaplan-Meier estimate of proportion of patients reaching = 5 106 CD34+ cells/kg. The median number of apheresis days required to achieve = 5 106 CD34+ cells/kg was 3 days in the plerixafor group, but the median time was not estimable in the placebo group, as less than 50% of patients reached the target within 4 apheresis days. (B) Kaplan-Meier estimate of proportion of patients reaching = 2 106 CD34+ cells/kg. Plerixafor-treated patients collected = 2 106 CD34+ cells after a median of 1 day of apheresis while placebo-treated patients required a median of 3 days of apheresis. G-CSF, granulocyte colony-stimulating factor; HR, hazard ratio.(A) Kaplan-Meier estimate of proportion of patients reaching = 5 106 CD34+ cells/kg. The median number of apheresis days required to achieve = 5 106 CD34+ cells/kg was 3 days in the plerixafor group, but the median time was not estimable in the placebo group, as less than 50% of patients reached the target within 4 apheresis days. (B) Kaplan-Meier estimate of proportion of patients reaching = 2 106 CD34+ cells/kg. Plerixafor-treated patients collected = 2 106 CD34+ cells after a median of 1 day of apheresis while placebo-treated patients required a median of 3 days of apheresis. G-CSF, granulocyte colony-stimulating factor; HR, hazard ratio.

    28. Plerixafor-Phase III in MM Design: phase III, multicenter, randomized (1:1), double-blind, placebo-controlled Subjects: Patients NHL requiring an Auto-SCT in first or second CR or PR Study Arms: G-CSF + AMD3100: 148 G-CSF + Placebo: 154 Primary endpoint: percentage of patients who collected = 6 106 CD34+ cells/kg in < 2 apheresis Secondary endpoints: Percantage of patients able to mobilize = 6 106 CD34+ cells/kg in = 4 apheresis neutrophil and platelet engraftment, and graft failure 28

    29. Kinetics of CD34/kg collection. (A) Kaplan-Meier estimate of proportion of patients reaching 6 106 or more CD34+ cells/kg. (B) Median CD34+ cells collected on each apheresis day.Kinetics of CD34/kg collection. (A) Kaplan-Meier estimate of proportion of patients reaching 6 106 or more CD34+ cells/kg. (B) Median CD34+ cells collected on each apheresis day.

    30. Plerixafor in Allo-HSCT 25 HLA-identical sibling donor/recipient pairs plerixafor 240?g/kg subcutaneously and underwent leukapheresis 4?h later first eight patients ? a 10-day washout period ? G-CSF mobilized backup Median WBC count: baseline level: 6.76 109/L 4 hours: 18.8 109/L Median PB CD34+ cells: (median 8-fold increase) baseline: 2/L 4 hours:16/L Mobilization of >2 106 CD34+ cells/kg recipient: After a single leukapheresis: 2/3 After 2 leukapheresis: 100% 30 --If the minimum CD34 cell dose was not achieved after the first day of mobilization with AMD3100, the donor was asked to return in 2 days to repeat the identical process on day 3. --If the minimum CD34 cell dose was not achieved after the first day of mobilization with AMD3100, the donor was asked to return in 2 days to repeat the identical process on day 3.

    31. Plerixafor in Allo-HSCT 31 -- The cumulative incidence of grade 2 to 4 acute GVHD was 35% -- The cumulative incidence of chronic GVHD at 1 year was 33% -- The cumulative incidence of grade 2 to 4 acute GVHD was 35% -- The cumulative incidence of chronic GVHD at 1 year was 33%

    32. IV Plerixafor Phase I allogeneic transplant trial: IV plerixafor:80, 160, 240, 320, 400 or 480?g/kg After 4 days of drug clearance, SQ 240?g/kg followed 4?h later by leukapheresis Peak numbers of circulating CD34+ cells: 46?h after IV dosing 320?g/kg: maximum eightfold increase in circulating CD34+ cells at 4?h after Phase II study: 28 sibling donors IV plerixafor : 320?g/kg followed 4?h later by leukapheresis leukapheresis yield of 2.0 106/kg CD34+ cells/kg: One leukapheresis: 74% Two leukopheresis: 95% 32 -- The pharmacokinetics of subcutaneous plerixafor requires that it be administered the night before leukapheresis, so that the morning collection would correspond to the peak of the circulating HSPCs. Such administration is associated with inconvenience and additional cost. -- Peak numbers of circulating CD34+ cells: 46?h after IV dosing (vs 69?h after subcutaneous dosing) -- The pharmacokinetics of subcutaneous plerixafor requires that it be administered the night before leukapheresis, so that the morning collection would correspond to the peak of the circulating HSPCs. Such administration is associated with inconvenience and additional cost. -- Peak numbers of circulating CD34+ cells: 46?h after IV dosing (vs 69?h after subcutaneous dosing)

    33. Continuous vs. Bolus Plerixafor 33

    34. Alternative drugs in development targeting the CXCL12/CXCR4 axis POL6326 (Polyphor, Allschwil, Switzerland) BKT-140 (4F-benzoyl-TN14003; Biokine Therapeutics, Rehovot, Israel) NOX-A12 (NOXXON Pharma, Berlin, Germany) TG-0054 (Taigen Biotechnology, Taipei, Taiwan) MDX-1338 (BMS-936564; Medarex/Bristol-Myers Squibb, New York, NY, USA) CXCR4 pepducins (Anchor Therapeutics, Cambridge, MA, USA) ALX-0651 (Ablynx, Zwijnaarde, Belgium) AMD070 (Genzyme Corp., Cambridge, MA, USA) GSK812397 (GlaxoSmithKline, Brentford, England) KRH-3955 (Kureha Chemical Industries, Nishiki, Japan) FC131 is a cyclopentapeptide (c(Gly1-D-Tyr2-Arg3-Arg4-Nal5) WZ811 (N1,N4-di-2-pyridinyl-1,4-benzenedimethanamine) 34

    35. VLA-4/VCAM-1 Axis VLA-4 (Very late antigen 4) (a41): an integrin receptor most leukocytes (monocytes, lymphocytes, eosinophils, basophils) and CD34+ hematopoietic precursor cells VCAM-1 (vascular cell adhesion molecule 1) or CD106: Endothelial cells including sinusiodal endothelial cells in vascular niche, CAR cells, Nestin+ MSC, and osteoblasts Lack of a4 or 1 integrin: embryonic lethal due to non-hematological defects Cre/loxP system of conditional a4 ablation: eightfold increase in the number of circulating HSPCs (CFU-Cs) 35

    36. VLA4/VCAM-1 Axis and VLA-4 Inhibition Sinusoidal endothelial cells 1. VCAM-1 2. CXCL12 36 Models of HSPC mobilization by G-CSF and inhibitors of CXCR4 and VLA-4. (a) Bone marrow environment at baseline. HSPCs in the endosteal niche are likely in close contact with osteoblasts and nestin? MSCs, both of which express numerous HSPC retention factors, including CXCL12, VCAM-1 and SCF. The perivascular niche is more distant from the endosteum and includes both nestin? MSCs and CXCL12 abundant reticular cells. Cells of the monocytic lineage support the maintenance of osteoblasts and MSCs. b-Adrenergic nerve cells of the sympathetic nervous system regulate MSC proliferation and induce circadian oscillations of CXCL12 expression. (b) Model of G-CSF-induced HSPC mobilization. Cells of the monocytic lineage express the receptor for G-CSF and provide factors that support the survival of MSCs and osteoblasts. Upon 45 days of stimulation with G-CSF, the monocytes/macrophages disappear, leading to the loss of osteoblast lineage cells and reduced expression of CXCL12, VCAM-1 and SCF on MSCs. Reduced expression of these important HSPC retention factors is also observed following G-CSF signaling through b-adrenergic nerve cells. The net effect of these signaling cascades is the disruption of HSPC retention interactions and mobilization of HSPCs into the peripheral blood. (c, d) Model of HSPC mobilization by inhibitors of CXCR4 or VLA-4. Targeted disruption of the interaction of CXCR4 or VLA-4 with their ligands results in the rapid (within hours) and reversible mobilization of HSPCs into the peripheral circulation. An additive or synergistic affect on HSPC mobilization is observed when a CXCR4 inhibitor is combined with a VLA-4 antagonist, G-CSF or their combination. When used alone, inhibitors of CXCR4 and VLA-4 mobilize fewer HSPCs than G-CSF. ----------------------------------------------------- Support for the hypothesis that one cell type is present at various sites within the marrow niche was obtained by a recent landmark study by Mndez-Ferrer et al. (2010). This group identified a stromal nestin-expressing MSC population (nestin+ MSC) that is closely associated with putative HSCs. Nestin+ MSCs are strictly perivascular and are typically found in more central areas of the marrow, but they are also present in the immediate vicinity of the endosteum, albeit at lower frequency. Moreover, nestin+ MSCs are tightly associated with adrenergic nerve fibers of the sympathetic nervous system (SNS) that regulate HSC mobilization and are responsible for the circadian oscillations in circulating HSC numbers (Katayama et al., 2006; Mndez-Ferrer et al., 2008). Strikingly, these MSCs express higher levels of HSC maintenance factor transcripts, including CXCL12, stem cell factor (SCF), angiopoietin-1 (Ang-1), IL-7, vascular cell adhesion molecule 1 (VCAM1), and osteopontin (OPN), compared with any other stromal cell type including OBs. Depletion of nestin+ MSCs by inducible expression of diphtheria toxin receptor (DTR) in nestin-expressing cells caused the mobilization of ~50% of HSCs to the spleen. In addition, the homing of transplanted progenitor cells into MSC-depleted recipients was reduced by 90%.Moreover, treatment with the HSC-mobilizing factor granulocyte colony-stimulating factor (G-CSF) decreased the expression of CXCL12 and other factors required for HSC maintenance and retention by nestin+ MSCs. These cells also seem to mediate the capacity of parathyroid hormone to increase HSC numbers, which had previously been correlated with an increase in putative niche OBs (Calvi et al., 2003; Adams et al., 2007). Parathyroid hormone directly stimulated proliferation of nestin+ MSCs while simultaneously promoting their differentiation into OBs (Mndez-Ferrer et al., 2010). Collectively, these data support the hypothesis that nestin+ MSCs are a functional component of the bone marrow HSC niche.Most interestingly, nestin+ MSCs show several similarities to recently identified mesenchymal progenitors (Sugiyama et al., 2006; Omatsu et al., 2010). These bipotent adipoosteogenic progenitors were identified in a mouse strain in which GFP was expressed from the endogenous CXCL12 locus (Sugiyama et al., 2006; Omatsu et al., 2010). Because of their high CXCL12 expression and their long cellular processes, these cells were named CXCL12-abundant reticular (CAR) cells. The majority of putative HSCs are found in close proximity to CAR cells by immunohistochemistry, and like nestin+ MSCs, CAR cells are predominantly found in the more central areas of the marrow, with some also located close to vessels near the endosteum (Fig. 1). Although CAR cells are more abundant than nestin+ MSCs, they too are tightly associated with sinusoidal endothelium and have a similar morphology to vascular pericytes. These data are in agreement with studies in humans, suggesting that virtually all MSC activity is found within the larger pericyte population that associates closely with the vascular system in the entire body (Crisan et al., 2008).Similar to nestin+ MSCs, CAR cells express HSC maintenance proteins such as CXCL12 and SCF. Induced depletion of CAR cells using a DTR approach causes a partial loss of HSC activity associated with a decrease in HSC cycling, suggesting that CAR cells promote HSC cycling and self-renewal. Even though this phenomenon was not observed after depletion of nestin+ MSCs, it seems likely that CAR cells and nestin+ MSCs represent two highly overlapping CXCL12-expressing cell populations. Because nestin+ MSCs are approximately four times less abundant than CAR cells, contain all colony-forming-unit fibroblast activity within the marrow, harbor high self-renewal activity in vitro and in vivo, and are capable of multilineage differentiation into bone, cartilage, and fat, it appears that nestin+ MSCs may represent a more primitive population compared with CAR cells and may even be a CAR subpopulation. As it remains unclear whether nestin+ MSCs are homogeneous and whether they all express high levels of CXCL12, it is possible that some nestin+ MSCs may not be CAR cells. Although future studies will need to dissect the cellular relationship between these two niche cell populations, there is now convincing evidence that perivascular mesenchymal stem/progenitor cells are critical inhabitants of the HSC niche. (Edinger et al. JEM vol. 208 no. 3 421-428 ) ----SNS promotes egress by reducing the expression of key retention factors by the niche cell (Mndez-Ferrer et al., 2010b), and in contrast, BM Macrophage promotes the expression of these genes and HSC/progenitor retention in the BM.Models of HSPC mobilization by G-CSF and inhibitors of CXCR4 and VLA-4. (a) Bone marrow environment at baseline. HSPCs in the endosteal niche are likely in close contact with osteoblasts and nestin? MSCs, both of which express numerous HSPC retention factors, including CXCL12, VCAM-1 and SCF. The perivascular niche is more distant from the endosteum and includes both nestin? MSCs and CXCL12 abundant reticular cells. Cells of the monocytic lineage support the maintenance of osteoblasts and MSCs. b-Adrenergic nerve cells of the sympathetic nervous system regulate MSC proliferation and induce circadian oscillations of CXCL12 expression. (b) Model of G-CSF-induced HSPC mobilization. Cells of the monocytic lineage express the receptor for G-CSF and provide factors that support the survival of MSCs and osteoblasts. Upon 45 days of stimulation with G-CSF, the monocytes/macrophages disappear, leading to the loss of osteoblast lineage cells and reduced expression of CXCL12, VCAM-1 and SCF on MSCs. Reduced expression of these important HSPC retention factors is also observed following G-CSF signaling through b-adrenergic nerve cells. The net effect of these signaling cascades is the disruption of HSPC retention interactions and mobilization of HSPCs into the peripheral blood. (c, d) Model of HSPC mobilization by inhibitors of CXCR4 or VLA-4. Targeted disruption of the interaction of CXCR4 or VLA-4 with their ligands results in the rapid (within hours) and reversible mobilization of HSPCs into the peripheral circulation. An additive or synergistic affect on HSPC mobilization is observed when a CXCR4 inhibitor is combined with a VLA-4 antagonist, G-CSF or their combination. When used alone, inhibitors of CXCR4 and VLA-4 mobilize fewer HSPCs than G-CSF. ----------------------------------------------------- Support for the hypothesis that one cell type is present at various sites within the marrow niche was obtained by a recent landmark study by Mndez-Ferrer et al. (2010). This group identified a stromal nestin-expressing MSC population (nestin+ MSC) that is closely associated with putative HSCs. Nestin+ MSCs are strictly perivascular and are typically found in more central areas of the marrow, but they are also present in the immediate vicinity of the endosteum, albeit at lower frequency. Moreover, nestin+ MSCs are tightly associated with adrenergic nerve fibers of the sympathetic nervous system (SNS) that regulate HSC mobilization and are responsible for the circadian oscillations in circulating HSC numbers (Katayama et al., 2006; Mndez-Ferrer et al., 2008). Strikingly, these MSCs express higher levels of HSC maintenance factor transcripts, including CXCL12, stem cell factor (SCF), angiopoietin-1 (Ang-1), IL-7, vascular cell adhesion molecule 1 (VCAM1), and osteopontin (OPN), compared with any other stromal cell type including OBs. Depletion of nestin+ MSCs by inducible expression of diphtheria toxin receptor (DTR) in nestin-expressing cells caused the mobilization of ~50% of HSCs to the spleen. In addition, the homing of transplanted progenitor cells into MSC-depleted recipients was reduced by 90%.Moreover, treatment with the HSC-mobilizing factor granulocyte colony-stimulating factor (G-CSF) decreased the expression of CXCL12 and other factors required for HSC maintenance and retention by nestin+ MSCs. These cells also seem to mediate the capacity of parathyroid hormone to increase HSC numbers, which had previously been correlated with an increase in putative niche OBs (Calvi et al., 2003; Adams et al., 2007). Parathyroid hormone directly stimulated proliferation of nestin+ MSCs while simultaneously promoting their differentiation into OBs (Mndez-Ferrer et al., 2010). Collectively, these data support the hypothesis that nestin+ MSCs are a functional component of the bone marrow HSC niche.Most interestingly, nestin+ MSCs show several similarities to recently identified mesenchymal progenitors (Sugiyama et al., 2006; Omatsu et al., 2010). These bipotent adipoosteogenic progenitors were identified in a mouse strain in which GFP was expressed from the endogenous CXCL12 locus (Sugiyama et al., 2006; Omatsu et al., 2010). Because of their high CXCL12 expression and their long cellular processes, these cells were named CXCL12-abundant reticular (CAR) cells. The majority of putative HSCs are found in close proximity to CAR cells by immunohistochemistry, and like nestin+ MSCs, CAR cells are predominantly found in the more central areas of the marrow, with some also located close to vessels near the endosteum (Fig. 1). Although CAR cells are more abundant than nestin+ MSCs, they too are tightly associated with sinusoidal endothelium and have a similar morphology to vascular pericytes. These data are in agreement with studies in humans, suggesting that virtually all MSC activity is found within the larger pericyte population that associates closely with the vascular system in the entire body (Crisan et al., 2008).Similar to nestin+ MSCs, CAR cells express HSC maintenance proteins such as CXCL12 and SCF. Induced depletion of CAR cells using a DTR approach causes a partial loss of HSC activity associated with a decrease in HSC cycling, suggesting that CAR cells promote HSC cycling and self-renewal. Even though this phenomenon was not observed after depletion of nestin+ MSCs, it seems likely that CAR cells and nestin+ MSCs represent two highly overlapping CXCL12-expressing cell populations. Because nestin+ MSCs are approximately four times less abundant than CAR cells, contain all colony-forming-unit fibroblast activity within the marrow, harbor high self-renewal activity in vitro and in vivo, and are capable of multilineage differentiation into bone, cartilage, and fat, it appears that nestin+ MSCs may represent a more primitive population compared with CAR cells and may even be a CAR subpopulation. As it remains unclear whether nestin+ MSCs are homogeneous and whether they all express high levels of CXCL12, it is possible that some nestin+ MSCs may not be CAR cells. Although future studies will need to dissect the cellular relationship between these two niche cell populations, there is now convincing evidence that perivascular mesenchymal stem/progenitor cells are critical inhabitants of the HSC niche. (Edinger et al. JEM vol. 208 no. 3 421-428 ) ----SNS promotes egress by reducing the expression of key retention factors by the niche cell (Mndez-Ferrer et al., 2010b), and in contrast, BM Macrophage promotes the expression of these genes and HSC/progenitor retention in the BM.

    37. BIO5192, a small molecule inhibitor of VLA-4 37 BIO 5192: Peripheral blood CFU-GM levels peaked 0.51?h after BIO5192 administration and returned to baseline by 6?h. === Here we show that interruption of the VCAM-1/VLA-4 axis with a small molecule inhibitor of VLA-4, BIO5192, results in a 30-fold increase in mobilization of murine hematopoietic stem and progenitors (HSPCs) over basal levels. An additive affect on HSPC mobilization (3-fold) was observedwhenplerixafor (AMD3100), a small molecule inhibitor of the CXCR-4/SDF-1 axis, was combined with BIO5192. Furthermore, the combination of granulocyte colony-stimulating factor (G-CSF), BIO5192, and plerixafor enhanced mobilization by 17-fold compared with G-CSF alone. HSPCs mobilized by BIO5192 or the combination of BIO5192 and plerixafor mobilized long-term repopulating cells, which successfully engraft and expand in a multilineage fashion in secondary transplantation recipients. Splenectomy resulted in a dramatic enhancement of G-CSFinduced mobilization while decreasing both plerixafor- and BIO5192- induced mobilization of HSPCs. These data provide evidence for the utility of small molecule inhibitors of VLA-4 either alone or in combination with G-CSF or AMD3100 for mobilization of hematopoietic stem and progenitor cells. (Blood. 2009;114:1340-1343) =========== Natalizumab (Tysabri; Biogen/Idec, Cambridge, MA, USA) is a recombinant humanized neutralizing IgG4 monoclonal antibody that binds to the a4-subunit of the a41 (VLA-4) and a47 integrins. Overall, the number of circulating CD34+ cells per l blood increased three- to fivefold during the first 72?h after treatment and remained elevated at these levels (810 CD34+ cells per l) for at least a month after natalizumab injection. Somewhat surprisingly, repeated administration of the antibody failed to mobilize additional CD34+ cells. === As BIO5192 will not be clinically developed, we have performed identical studies with similar results using the small-molecule VLA-4 antagonist firategrast, which is currently in clinical development for the treatment of MS by GlaxoSmithKline (data not shown).BIO 5192: Peripheral blood CFU-GM levels peaked 0.51?h after BIO5192 administration and returned to baseline by 6?h. === Here we show that interruption of the VCAM-1/VLA-4 axis with a small molecule inhibitor of VLA-4, BIO5192, results in a 30-fold increase in mobilization of murine hematopoietic stem and progenitors (HSPCs) over basal levels. An additive affect on HSPC mobilization (3-fold) was observedwhenplerixafor (AMD3100), a small molecule inhibitor of the CXCR-4/SDF-1 axis, was combined with BIO5192. Furthermore, the combination of granulocyte colony-stimulating factor (G-CSF), BIO5192, and plerixafor enhanced mobilization by 17-fold compared with G-CSF alone. HSPCs mobilized by BIO5192 or the combination of BIO5192 and plerixafor mobilized long-term repopulating cells, which successfully engraft and expand in a multilineage fashion in secondary transplantation recipients. Splenectomy resulted in a dramatic enhancement of G-CSFinduced mobilization while decreasing both plerixafor- and BIO5192- induced mobilization of HSPCs. These data provide evidence for the utility of small molecule inhibitors of VLA-4 either alone or in combination with G-CSF or AMD3100 for mobilization of hematopoietic stem and progenitor cells. (Blood. 2009;114:1340-1343) =========== Natalizumab (Tysabri; Biogen/Idec, Cambridge, MA, USA) is a recombinant humanized neutralizing IgG4 monoclonal antibody that binds to the a4-subunit of the a41 (VLA-4) and a47 integrins. Overall, the number of circulating CD34+ cells per l blood increased three- to fivefold during the first 72?h after treatment and remained elevated at these levels (810 CD34+ cells per l) for at least a month after natalizumab injection. Somewhat surprisingly, repeated administration of the antibody failed to mobilize additional CD34+ cells. === As BIO5192 will not be clinically developed, we have performed identical studies with similar results using the small-molecule VLA-4 antagonist firategrast, which is currently in clinical development for the treatment of MS by GlaxoSmithKline (data not shown).

    38. Summary PBSC is the main source of stem cell for HSCT G-CSF has 5-30% failure rate in mobilization Strategies to manage hard to mobilize patients: Dose escalation of G-CSF (12.550?g/kg per day), Addition of another cytokine such as GM-CSF, Addition of chemotherapy Harvesting the BM CXCR4 and VLA-4 are the dominant receptors regulating HSPC migration and retention within the BM Plerixafor in combination with G-CSF, is FDA approved for HSPC mobilization in patients with NHL and MM VLA-4 inhibitors either alone or in combination with G-CSF or plerixafor increase mobilization yield. Dual inhibitor approach may ultimately provide a more efficient method to collect HSC in a single day 38

    39. References 1. www.NMPD.org 2. Iskra Pusic and John F. DiPersio. Current Opinion in Hematology 2010,17:319326 3. Wilson et al. Bone-marrow haematopoietic-stem-cell niches. Nat Rev Immunol 2006; 6: 93106 4. MP Rettig, G Ansstas and JF DiPersio. Leukemia. 2011 Sep 2. doi: 10.1038/leu.2011.197. [Epub ahead of print] 5. Christopher M et al. J Exp Med 2011 208:251-260 6. McCullough et al.. Transfusion 2008;48(9):2008-2025 7. Pulsipher M A et al. Blood 2009;113:3604-3611 8. Hassan et al. Transplant Immunology, 1996; 4: 319-323 9. Blais et al. J Clin Oncol 2000;18(3):537-546 10. Powles et al. Lancet 2000;355(9211):1231-1237 11. Ringden et al. J Clin Oncol 2002;20(24):4655-4664 12. Bensinger et al. N Engl J Med 2001;344(3):175-181 13. Kos et alJ Clin Oncol. 2000 May;18(9):1824-30 14. De Clercq E. The bicyclam AMD3100 story. Nat Rev Drug Discov 2003; 2: 581587 15. Devine S M et al. JCO 2004;22:1095-1102 16. Flomenberg et al. Blood 2005; 106: 18671874 17. DiPersio J F et al. JCO 2009;27:4767-4773 18. DiPersio J F et al. Blood 2009;113:5720-5726 19. Devine S M et al. . Blood 2008; 112: 990998 20. Bonig H et al. Experimental Hematology 2009;37:402415 21. Scott LM et al. Mol Cell Biol 2003; 23: 93499360 22. Ramirez P et al. Blood. 2009;114:1340-1343 39

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