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Neuropeptide Y Regulates the Hematopoietic Stem Cell Microenvironment and Prevents Nerve Injury in the Bone Marrow
22nd Annual ISCT Meeting
that health care professionals should be more involved in educating their patients of the subject matter. 68 NEUROPEPTIDE Y REGULATES THE HEMATOPOIETIC STEM CELL MICROENVIRONMENT AND PREVENTS NERVE INJURY IN THE BONE MARROW H. Jin1, J. Bae2 1 College of Veterinary Medicine, Kyungpook National University, Daegu, Republic of Korea, 2School of Medicine, Kyungpook National University, Daegu, Republic of Korea Many reports have revealed the importance of the sympathetic nervous system (SNS) in the control of the bone marrow environment. However, the specific role of neuropeptide Y (NPY) in this process has not been systematically studied. Here we show that NPY deficient mice have significantly reduced hematopoietic stem cell (HSC) numbers and impaired regeneration in bone marrow due to apoptotic destruction of SNS fibers and/or endothelial cells. Furthermore, pharmacological elevation of NPY prevented bone marrow impairments in a mouse model of chemotherapy-induced SNS injury, while NPY injection into conditional knockout mice lacking the Y1 receptor in macrophages did not relieve bone marrow dysfunction. These results indicate that NPY promotes neuroprotection and restores bone marrow dysfunction from chemotherapy-induced SNS injury through the Y1 receptor in macrophages. They also reveal a new role of NPY as a regulator of the bone marrow microenvironment, and highlight the potential therapeutic value of this neuropeptide. 69 CORD BLOOD-DERIVED CD34+ EX VIVO CELL EXPANSION IN AN AUTOMATED HOLLOW FIBER BIOREACTOR M. Jones, B. Vang, T. Startz, N. Frank, D. Hill, B. Nankervis, K. Nguyen Innovation & Development, Terumo BCT, Inc, Lakewood, Colorado, United States Cord blood-derived progenitor cell populations have been used to reconstitute the immune system for both autologous and allogeneic transplantation in order to treat an array of hematologic malignancies and congenital disorders. The challenge has been to provide a sufficient number of hematopoietic cells that can effectively support long-term engraftment. As a result, method development has been undertaken for the ex vivo expansion of cord blood-derived CD34+ cells in an automated, closed hollow fiber bioreactor under dynamic coculture conditions with hMSCs in order to meet this objective. In our initial Quantum System expansion study over 7-days, we expanded the HSC subpopulations from cord blood without the use of positive selection for CD34+ cells using a serum-free medium formulated with a limited cytokine supplement of rInsulin, rh Flt-3L, rh SCF, and rh TPO. At the conclusion of the cell expansion, RBCs were lysed with an ammonium chloride buffer and harvest samples were stained for flow cytometry analysis. The results suggest that the CD34+38+/− population were expanded by 37.5%. In addition, we observed that the CD34+133+ cell population were expanded from undetectable levels to 1.1% of the total harvest sample during the same time period. The significance of this ex vivo cell expansion approach is based on the development of the CD34+133+ surface biomarker expression profile of HSCs that correlates to successful platelet engraftment. Additional studies are planned for refinement and confirmatory purposes. 70 POOR MOBILIZING LYMPHOMA PATIENTS SUCCESSFULLY HARVESTED AFTER ADDITION OF PLERIXAFOR DURING STEM CELL MOBILIZATION HAVE SIMILAR SURVIVAL AS GOOD MOBILIZERS WHEN GIVEN HIGH DOSE THERAPY WITH AUTOLOGOUS STEM CELL SUPPORT D. Josefsen1, Y. Fløisand2, H. Holte3, G. Kvalheim1 1 Department of Cellular Therapy, Oslo University Hospital, Oslo, Norway, 2 Department of Hematology, Oslo University Hospital, Oslo, Norway, 3 Department of Oncology, Oslo University Hospital, Oslo, Norway High dose therapy followed by autologous stem cell support (HDT) is a curative treatment option in selected lymphoma patients. However, in
S29
15–20% of the lymphoma patients insufficient numbers of CD34+ cells are harvested, characterized as poor mobilizers. Furthermore, previous findings have shown that poor mobilizing lymphoma patients given HDT have a less favorable prognosis than good mobilizers. In recent years, new mobilizing agents including plerixafor, a CXCR4 antagonist, have been developed. Our previous findings have shown that with a low concentration of CD34+ cells (4–10 cells/μL) and a total white cell count >4 × 109/L, addition of plerixafor resulted in successful stem cell harvesting in these poor mobilizing patients. In the present study, we have mobilized 37 poor-mobilizing lymphoma patients with addition of plerixafor from January 2010 to December 2014. Thirty four patients underwent successful stem cell harvest and received high dose therapy. The patients were followed up after reinfusion of autologous stem cells with regard to short term and long term engraftment as well as relapse and death. The day prior to harvest, the level of leukocytes was 12.6 × 109 cells/L (median; range 3.3–65.2), and the CD34+ concentration was 5.7 × 106/L (median; range 1.9–12.4). Following plerixafor injection, the concentration of CD34+ cells increased to 23.4 × 106/L (median; range 5.6–65.6). The patients were then harvested (median: 3.9 × 106 CD34+ cells/kg; range: 1.5–7.3) with 1–2 days of apheresis. In the 34 lymphoma patients receiving high dose therapy followed by autologous stem cell support we observed that time to short term engraftment, defined by neutrophils >0.5 × 109/L and thrombocytes >20 × 109/L were 12 days (median; range 8–19) and 14 days (median; range 10–100) respectively. Furthermore, we examined the thrombocyte levels at day 100 after reinfusion with a median level of 132 × 109/L (range 27–398). Moreover, in contrast to previous findings we have observed durable responses with event free survival of 56% and overall survival of 67% (Median observation time without relapse: 19.5 months, range 0–64 months). In conclusion, our findings show that addition of plerixafor gives a successful mobilization of hematopoietic stem cells in poor mobilizing patients resulting in a quick and sustained engraftment as well as durable responses and similar prognosis as for good mobilizing lymphoma patients after high dose therapy with autologous stem cell support.
71 SCALABLE, CONTROLLED AND COST EFFECTIVE PRESENTATION OF SURFACE IMMOBILISED FACTORS FOR HAEMATOPOIETIC CELL EXPANSION R. Moore, M. Worrallo, K. Glen, R. Thomas University of Loughborough, Nottingham, United Kingdom Human umbilical cord blood (CB) has long been established as a source of stem and progenitor cells for bone marrow (BM) transplantation. Although CB transplants have a number of advantages over other stem cell sources, the relatively low cell dose in a CB collection has limited their therapeutic potential. In order to overcome this disadvantage a number of novel technologies have been reported for proliferating haematopoietic progenitors whilst maintaining a cell phenotype and population balance that is therapeutically useful for rapid repopulation. To date the most promising of these technologies have sought to recreate the BM niche in order for progenitor cells to retain their repopulating ability during ex vivo expansion. Of the molecular components identified within the BM niche, Notch family members have been shown to play a key role in hematopoiesis, influencing cell fate decisions in vivo. The induction of Notch signalling in vitro using immobilised Delta1 ligand enhanced the generation of CD34+ cells and cells capable of NOD/SCID. This technology has demonstrated powerful proof of principle, but as of yet has not been developed into a well-controlled scalable system. It has been previously demonstrated that Notch signalling is density dependent and requires an adherent form of the protein in order to successfully induce signalling. We have developed a straightforward immobilisation technology which offers a high degree of control of protein presentation. Using this technology we have established conditions for the immobilisation of Delta1 and various other growth factors important in hematopoiesis, including: SCF, FLT-3, TPO and GM-CSF. Immobilised growth factors have been shown to act as an alternative to soluble growth factors in scalable systems (stirred tanks), with certain immobilised factors significantly out-performing soluble factors in vitro. We have also determined that Notch immobilised on our bead technology can
that health care professionals should be more involved in educating their patients of the subject matter. 68 NEUROPEPTIDE Y REGULATES THE HEMATOPOIETIC STEM CELL MICROENVIRONMENT AND PREVENTS NERVE INJURY IN THE BONE MARROW H. Jin1, J. Bae2 1 College of Veterinary Medicine, Kyungpook National University, Daegu, Republic of Korea, 2School of Medicine, Kyungpook National University, Daegu, Republic of Korea Many reports have revealed the importance of the sympathetic nervous system (SNS) in the control of the bone marrow environment. However, the specific role of neuropeptide Y (NPY) in this process has not been systematically studied. Here we show that NPY deficient mice have significantly reduced hematopoietic stem cell (HSC) numbers and impaired regeneration in bone marrow due to apoptotic destruction of SNS fibers and/or endothelial cells. Furthermore, pharmacological elevation of NPY prevented bone marrow impairments in a mouse model of chemotherapy-induced SNS injury, while NPY injection into conditional knockout mice lacking the Y1 receptor in macrophages did not relieve bone marrow dysfunction. These results indicate that NPY promotes neuroprotection and restores bone marrow dysfunction from chemotherapy-induced SNS injury through the Y1 receptor in macrophages. They also reveal a new role of NPY as a regulator of the bone marrow microenvironment, and highlight the potential therapeutic value of this neuropeptide. 69 CORD BLOOD-DERIVED CD34+ EX VIVO CELL EXPANSION IN AN AUTOMATED HOLLOW FIBER BIOREACTOR M. Jones, B. Vang, T. Startz, N. Frank, D. Hill, B. Nankervis, K. Nguyen Innovation & Development, Terumo BCT, Inc, Lakewood, Colorado, United States Cord blood-derived progenitor cell populations have been used to reconstitute the immune system for both autologous and allogeneic transplantation in order to treat an array of hematologic malignancies and congenital disorders. The challenge has been to provide a sufficient number of hematopoietic cells that can effectively support long-term engraftment. As a result, method development has been undertaken for the ex vivo expansion of cord blood-derived CD34+ cells in an automated, closed hollow fiber bioreactor under dynamic coculture conditions with hMSCs in order to meet this objective. In our initial Quantum System expansion study over 7-days, we expanded the HSC subpopulations from cord blood without the use of positive selection for CD34+ cells using a serum-free medium formulated with a limited cytokine supplement of rInsulin, rh Flt-3L, rh SCF, and rh TPO. At the conclusion of the cell expansion, RBCs were lysed with an ammonium chloride buffer and harvest samples were stained for flow cytometry analysis. The results suggest that the CD34+38+/− population were expanded by 37.5%. In addition, we observed that the CD34+133+ cell population were expanded from undetectable levels to 1.1% of the total harvest sample during the same time period. The significance of this ex vivo cell expansion approach is based on the development of the CD34+133+ surface biomarker expression profile of HSCs that correlates to successful platelet engraftment. Additional studies are planned for refinement and confirmatory purposes. 70 POOR MOBILIZING LYMPHOMA PATIENTS SUCCESSFULLY HARVESTED AFTER ADDITION OF PLERIXAFOR DURING STEM CELL MOBILIZATION HAVE SIMILAR SURVIVAL AS GOOD MOBILIZERS WHEN GIVEN HIGH DOSE THERAPY WITH AUTOLOGOUS STEM CELL SUPPORT D. Josefsen1, Y. Fløisand2, H. Holte3, G. Kvalheim1 1 Department of Cellular Therapy, Oslo University Hospital, Oslo, Norway, 2 Department of Hematology, Oslo University Hospital, Oslo, Norway, 3 Department of Oncology, Oslo University Hospital, Oslo, Norway High dose therapy followed by autologous stem cell support (HDT) is a curative treatment option in selected lymphoma patients. However, in
S29
15–20% of the lymphoma patients insufficient numbers of CD34+ cells are harvested, characterized as poor mobilizers. Furthermore, previous findings have shown that poor mobilizing lymphoma patients given HDT have a less favorable prognosis than good mobilizers. In recent years, new mobilizing agents including plerixafor, a CXCR4 antagonist, have been developed. Our previous findings have shown that with a low concentration of CD34+ cells (4–10 cells/μL) and a total white cell count >4 × 109/L, addition of plerixafor resulted in successful stem cell harvesting in these poor mobilizing patients. In the present study, we have mobilized 37 poor-mobilizing lymphoma patients with addition of plerixafor from January 2010 to December 2014. Thirty four patients underwent successful stem cell harvest and received high dose therapy. The patients were followed up after reinfusion of autologous stem cells with regard to short term and long term engraftment as well as relapse and death. The day prior to harvest, the level of leukocytes was 12.6 × 109 cells/L (median; range 3.3–65.2), and the CD34+ concentration was 5.7 × 106/L (median; range 1.9–12.4). Following plerixafor injection, the concentration of CD34+ cells increased to 23.4 × 106/L (median; range 5.6–65.6). The patients were then harvested (median: 3.9 × 106 CD34+ cells/kg; range: 1.5–7.3) with 1–2 days of apheresis. In the 34 lymphoma patients receiving high dose therapy followed by autologous stem cell support we observed that time to short term engraftment, defined by neutrophils >0.5 × 109/L and thrombocytes >20 × 109/L were 12 days (median; range 8–19) and 14 days (median; range 10–100) respectively. Furthermore, we examined the thrombocyte levels at day 100 after reinfusion with a median level of 132 × 109/L (range 27–398). Moreover, in contrast to previous findings we have observed durable responses with event free survival of 56% and overall survival of 67% (Median observation time without relapse: 19.5 months, range 0–64 months). In conclusion, our findings show that addition of plerixafor gives a successful mobilization of hematopoietic stem cells in poor mobilizing patients resulting in a quick and sustained engraftment as well as durable responses and similar prognosis as for good mobilizing lymphoma patients after high dose therapy with autologous stem cell support.
71 SCALABLE, CONTROLLED AND COST EFFECTIVE PRESENTATION OF SURFACE IMMOBILISED FACTORS FOR HAEMATOPOIETIC CELL EXPANSION R. Moore, M. Worrallo, K. Glen, R. Thomas University of Loughborough, Nottingham, United Kingdom Human umbilical cord blood (CB) has long been established as a source of stem and progenitor cells for bone marrow (BM) transplantation. Although CB transplants have a number of advantages over other stem cell sources, the relatively low cell dose in a CB collection has limited their therapeutic potential. In order to overcome this disadvantage a number of novel technologies have been reported for proliferating haematopoietic progenitors whilst maintaining a cell phenotype and population balance that is therapeutically useful for rapid repopulation. To date the most promising of these technologies have sought to recreate the BM niche in order for progenitor cells to retain their repopulating ability during ex vivo expansion. Of the molecular components identified within the BM niche, Notch family members have been shown to play a key role in hematopoiesis, influencing cell fate decisions in vivo. The induction of Notch signalling in vitro using immobilised Delta1 ligand enhanced the generation of CD34+ cells and cells capable of NOD/SCID. This technology has demonstrated powerful proof of principle, but as of yet has not been developed into a well-controlled scalable system. It has been previously demonstrated that Notch signalling is density dependent and requires an adherent form of the protein in order to successfully induce signalling. We have developed a straightforward immobilisation technology which offers a high degree of control of protein presentation. Using this technology we have established conditions for the immobilisation of Delta1 and various other growth factors important in hematopoiesis, including: SCF, FLT-3, TPO and GM-CSF. Immobilised growth factors have been shown to act as an alternative to soluble growth factors in scalable systems (stirred tanks), with certain immobilised factors significantly out-performing soluble factors in vitro. We have also determined that Notch immobilised on our bead technology can