The Use Of Tevagrastim (Biosimilar Filgrastim XMO2) For Hematopoietic Stem Cell Mobilization In HLA Matched Sibling Donors For Allogeneic Stem Cell Transplantation To AML/MDS Patients

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3275-3275 ◽  
Author(s):  
Ivetta Danylesko ◽  
Rina Sareli ◽  
Nira Bloom-Varda ◽  
Ronit Yerushalmi ◽  
Noga Shem-Tov ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Side Effects ◽  
Conventional Therapy ◽  
Post Transplantation ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Grade Ii ◽  
Historical Controls ◽  
Biosimilar Filgrastim

Abstract Introduction Human recombinant G-CSF Filgrastim (Neupogen) has been widely used for the mobilization of CD34+ hematopoietic stem cells (HSC) from healthy donors. The experience with biosimilar G-CSF agents is limited and the only publication, to our knowledge, is the recent study by Schmitt M and co-authors (BMT 2013; 48, 922–925). This study involved 11donors and pts with various disease categories. The Biosimilar G-CSF was found to be comparable in efficacy and safety to Neupogen. Developing new cost effective mobilizing reagents if important. We, therefore, initiated a prospective study assessing Tevagrastim (biosimilar Filgrastim XMO2) for mobilization of CD34+ PB HSC in healthy sibling donors (MRD) for transplantation in pts with AML/MDS (NCT01542944). Materials and methods 24 pts with AML or high-risk MDS undergoing allo-SCT from MRD were investigated. The study was approved by the National Regulatory Authorities and both patients and donors signed an informed consent. The donors, median age 46 years (range, 25–64), F- 14; M- 10 received Tevagrastim in a standard dose of 10 μg/kg BW s.c. BID for 4 days. On the morning of the 5th day they underwent conventional leukapheresis. The target yields of CD 34 cell was 5 × 106 CD34+ cells/kg BW of the recipient. If one leukapheresis was insufficient a second was performed and, the last dose of Tevagrastim was administered on the evening of the 5th day. The conditioning was myeloablative Bu/Cy (n=10), reduced toxicity Flu/Treo (n=7), Flu/Bu4 (n=3) or RIC Flu/Bu2 (n=4). The study parameters were: a CD34+ cell count,both absolute numbers and the CD34+ cells per kg BW of the recipient, the number of leukapheresis procedures, the number of CD3+ T lymphocytes in the graft, post transplantation engraftment including the WBC, the neutrophils and the platelets, as well as side effects. Follow up was 100 days post allo-SCT. Results Efficacy 77-1982 × 106 (median 749 × 106) CD34+ were collected. The number of CD34+ cell per kg BW of the pts was 0.93-35.4 × 106 (median 10.2 × 106). Collections contained 144-709 × 108 (median 299 × 108) CD3+ T-cells, 1.74-11.6 ×108 (median 4.4 ×108) per kg BW of the pts. The mean number of leukapheresis procedures was 1.3. Engraftment was: ANC >0.5× 109/L and >1× 109/L within a median of 13 days (range, 10–21) and 13.5 days (range, 10–22), respectively. PLT reached counts of >20× 109/L and >50× 109/L within a median of 16 days (range, 12–33) and 17 days (range, 12–33) from allo-SCT, respectively. The median days of isolation was 10 (range, 6-21). As for blood support, the median number of PC and PLT transfusions was 5 (range, 2-20) and 21 (range, 0-180), respectively. 18/22 (81.8%) pts achieved full donor chimerism at 1 month after transplantation (2 pts are too early to evaluate). Safety Overall Tevagrastim was found to be safe with minimal transient side effects. Neither allergic reactions nor severe adverse events were observed in the donors. 12/24 donors reported transient arthralgias and 2 developed flu-like syndrome while receiving Tevagrastim. As for transplantation related toxicity in the 24 pts transplanted with Tevagrastim mobilized HSC grafts, side effects were not different than those we observed in historical controls. The main side effects were mucositis (n-15, grade II -9, grade III-IV- 6), infection complications (n-20) and fluid retention (n-8). One pt suffered from VOD (Grade-I) that resolved with conventional therapy. Five pts developed acute GVHD (grade II-III) that responded to conventional therapy. In total TRM was 1/24 at d 100. 2 pts died from leukemia progression. Conclusions Our study with 24 AML/MDS pts, indicates that the G-CSF biosimilar XM02, Tevagrastim is safe and efficient for stem cells mobilization in HLA matched normal sibling donors. The CD34 yield and post transplantation engraftment are similar to those achieved with the human recombinant G-CSF Filgrastim (Neupogen). We have not seen significant differences in the graft CD34+ and CD3+ T lymphocytes cell count, the number of leukapheresis procedures and the regeneration of WBC, neutrophils and platelets in comparison with our historical controls. All patients promptly engrafted, and the donors developed only expected side effects like arthralgias and flu-like syndrome. Neither graft rejection nor side effects occurred more frequently than expected from the standard G-CSF. Disclosures: Nagler: Teva : Consultancy, Honoraria, Research Funding.

scholarly journals Use of Eltrombopag in Improving Poor Graft Function after Allogeneic Hematopoietic Stem Cell Transplantation

2018 ◽  
Vol 11 (1) ◽  
pp. 191-195 ◽  
Author(s):  
Samip Master ◽  
Ashish Dwary ◽  
Richard Mansour ◽  
Glenn M. Mills ◽  
Nebu Koshy
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Side Effects ◽  
Hematopoietic Stem Cell Transplantation ◽  
Graft Function ◽  
Young Female ◽  
Post Transplantation ◽  
Hematopoietic Stem ◽  
Post Transplant ◽  
Poor Graft Function

Eltrombopag is a thrombopoietin agonist and has been used in aplastic anemia and post-transplantation thrombocytopenia. The c-MPL receptor is present on hematopoietic stem cells. There are no reports of eltrombopag utilization for improving poor graft function in the post-transplant setting. Here were report a case of a young female with post-transplant poor graft function as evident from the low absolute neutrophil count, anemia, and thrombocytopenia on day 60. Eltrombopag was started on day 72 and resulted in improvement in all 3 cell lines. The counts continued to be stable even after eltrombopag was discontinued. The patient tolerated the drug without significant side effects for 1 year.

scholarly journals A Stemness Screen Reveals C3ORF54/INKA1 As a Gate-Keeper of Human Stem Cell Latency

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 325-325
Author(s):  
Kerstin B. Kaufmann ◽  
Laura Garcia Prat ◽  
Shin-Ichiro Takayanagi ◽  
Jessica McLeod ◽  
Olga I. Gan ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Fold Increase ◽  
Steady Increase ◽  
Post Transplantation ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract The controversy generated from recent murine studies as to whether hematopoietic stem cells (HSC) contribute to steady-state hematopoiesis emphasizes how limited our knowledge is of the mechanisms governing HSC self-renewal, activation and latency; a problem most acute in the study of human HSC and leukemia stem cells (LSC). Many hallmark stem cell properties are shared by HSC and LSC and therefore a better understanding of stemness regulation is crucial to improved HSC therapies and leukemia treatments targeting LSC. Our previous work on LSC subsets from >80 AML patient samples revealed that HSC and LSC share a transcriptional network that represent the core elements of stemness (Eppert, Nature Med 2011; Ng, Nature 2016). Hence, to identify the key regulators of LSC/HSC self-renewal and persistence we selected 64 candidate genes based on expression in functionally validated LSC vs. non-LSC fractions and assessed their potential to enhance self-renewal in a competitive in vivo screen. Here, we transduced cord blood CD34+CD38- cells with 64 barcoded lentiviral vectors to assemble 16 pools, each consisting of 8 individual gene-transduced populations, for transplantation into NSG mice. Strikingly, individual overexpression (OE) of 5 high scoring candidates revealed delayed repopulation kinetics of human HSC/progenitor cells (HSPC): gene-marking of human CD45+ and lin-CD34+ cells was reduced relative to input and control at 4w post transplantation, whereas by 20w engraftment of marked cells reached or exceeded input levels. For one of these candidates, C3ORF54/INKA1, we found that OE did not alter lineage composition neither in in vitro nor in vivo assays but increased the proportion of primitive CD34+ cells at 20w in vivo; moreover, secondary transplantation revealed a 4.5-fold increase in HSC frequency. Of note, serial transplantation from earlier time points (2w, 4w) revealed superior engraftment and hence greater self-renewal capacity upon INKA1-OE. Since we observed a 4-fold increase of phenotypic multipotent progenitors (MPP) relative to HSC within the CD34+ compartment (20w) we assessed whether INKA1-OE acts selectively on either cell population. The observation of latency in engraftment was recapitulated with sorted INKA1-OE HSC but not MPP. Likewise, liquid culture of HSPC and CFU-C assays on sorted HSC showed an initial delay in activation and colony formation upon INKA1-OE that was completely restored by extended culture and secondary CFU-C, respectively. INKA1-OE MPP showed a slight increase in total colony count in primary CFU-C and increased CDK6 levels in contrast to reduced CDK6 levels in INKA1-OE HSC emphasizing opposing effects of INKA1 on cell cycle entry and progression in either population. Taken together, this suggests that INKA1-OE preserves self-renewal capacity by retaining HSC preferentially in a latent state, however, upon transition to MPP leads to enhanced activation. Whilst INKA1 has been described as an inhibitor of p21(Cdc42/Rac)-activated kinase 4 (PAK4), no role for PAK4 is described in hematopoiesis. Nonetheless, its regulator Cdc42 is implicated in aging of murine HSPC by affecting H4K16 acetylation (H4K16ac) levels and polarity and has recently been described to regulate AML cell polarity and division symmetry. In our experiments immunostaining of HSPC subsets cultured in vitro and from xenografts indicates that INKA1-OE differentially affects epigenetics of these subsets linking H4K16ac to the regulation of stem cell latency. In AML, transcriptional upregulation of INKA1 in LSC vs. non-LSC fractions and at relapse in paired diagnosis-relapse analysis (Shlush, Nature 2017) implicates INKA1 as a regulator of LSC self-renewal and persistence. Indeed, INKA1-OE in cells derived from a primary human AML sample (8227) with a phenotypic and functional hierarchy (Lechman, Cancer Cell 2016) revealed a strong latency phenotype: In vitro and in vivo we observed label retention along with a steady increase in percentage of CD34+ cells, transient differentiation block, reduced growth rate, G0 accumulation and global reduction of H4K16ac. In summary, our data implicates INKA1 as a gate-keeper of stem cell latency in normal human hematopoiesis and leukemia. Studying the detailed pathways involved will shed light upon the mechanisms involved in HSC activation and latency induction and will help to harness these for novel therapeutic approaches. Disclosures Takayanagi: Kyowa Hakko Kirin Co., Ltd.: Employment.

scholarly journals Outcome of Severe Aplastic Anemia Post Allogenic Stem Cell Transplant with Mycophenolate and Calcineurin Inhibitor for Graft Versus Host Disease Prophylaxis

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4879-4879
Author(s):  
Omar Albanyan ◽  
Hyejeong Jang ◽  
Seongho Kim ◽  
Andrew Kin ◽  
Asif Alavi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Conditioning Regimen ◽  
Unrelated Donor ◽  
Cell Transplant ◽  
Advisory Committees ◽  
Hematopoietic Stem ◽  
Gvhd Prophylaxis ◽  
Grade Ii ◽  
Matched Donor

Abstract Introduction: Severe aplastic anemia (SAA) is a rare hematopoietic stem cell disorder characterized by hypocellular marrow and pancytopenia. Multiple factors play an important role in treatment approach include age, comorbidities, degree of pancytopenia and availability of stem cell donor to either immunosuppression irrespective (IST) or allogenic hematopoietic stem cell transplant (alloSCT). The use of nonmyeloablative conditioning regimen has improved the outcomes, however the choice for post-transplant GVHD prophylaxis remain a topic of debate. The use of mycophenolate mofetil (MMF) has been used as an alternative for methotrexate (MTX) as has shown to be associated with lower incidence of mucositis and shorter time to engraftment. Methods: We retrospectively evaluated consecutive adult patients with SAA who underwent alloSCT at Karmanoc Cancer Institute. All patients received fludarabine, cyclophosphamide and antithymocyte globulin for conditioning regimen with calcineurin inhibitors (CNI) and MMF for GVHD prophylaxis. MMF was started at day -3 at 15 mg/kg three times daily and stopped at day +30 in the absence of active GVHD. The primary objectives were to estimate cumulative incidence of acute (aGVHD) and chronic GVHD (cGVHD) and overall survival (OS). Secondary objectives were to evaluate time to engraftment, days of hospitalization and incidence of mucositis. Results: From January 2005 and May 2019, 33 patients with SAA underwent alloSCT. Patient characteristics are detailed in Table 1. Median age was 36 years (range, 18-71). Twenty-seven patients received bone marrow stem cells (82%) and six patients received peripheral blood stem cells (18%). Thirty patients (91%) received 8/8 HLA matched donor and three patients (9%) received 7/8 HLA matched donor. Sixteen patients (48%) received stem cells from sibling donor and 17 patients (52%) received stem cells from unrelated donor. Thirteen patients (39%) had received IST prior to alloSCT, and 20 patients (61%) received upfront alloSCT. For GVHD prophylaxis all patients received MMF and CNI (tacrolimus=32, and cyclosporine=1). Median time from diagnosis to transplant was 15.8 months for patients who received IST prior to alloSCT and 2 months for patients who received upfront alloSCT. Median time to platelet engraftment was 13.5 days and neutrophil engraftment was 12 days, while one patient experienced graft failure. The median number of days for hospital stay were 25 days. Four patients (11%) developed grade I-II mucositis, no grade III-IV mucositis was observed in the first 30 days and 6 patients had CMV reactivation. The 100-day cumulative incidence rate of grade II-IV aGVHD was 21.2% (95% CI, 9.2 - 36.5), grade III-IV aGVHD was 9.1% (2.3-21.9) and 1-year CIR of cGVHD was 21.2% (95% CI, 9.2-36.5). Comparing patients who received IST prior to alloSCT versus upfront alloSCT, the 100-day CIR of grade II-IV aGVHD was 30.8% (95% CI, 8.2 - 56.5) and 15% (95% CI, 3.6 - 34.0), respectively, (Gray p=0.26) and the 3-year CIR of cGVHD was 39.6% (95% CI, 13.1 - 65.5) and 27.8% (95% CI, 9.2 - 50.3), respectively, (Gray p=0.37). Comparing patients who received alloSCT from related versus unrelated donor, 100-day CIR of II-IV aGVHD was 12.5% (95% CI, 1.9 - 33.6) and 29.4% (95% CI, 10.2 - 51.9), respectively, (Gray p=0.26), and the 3-year CIR of cGVHD was 34.2% (95% CI, 11.4 - 58.9) and 29.4% (95% CI, 10.1 - 52.0), respectively (Gray p=0.90). Median follow up of surviving patient was 5 years (95% CI, 3.1-6.8). Three-year OS was 87% (95% CI, 75.7- 99.9) and median OS was not reached. Six patients died by the time of the analysis, one patient died from graft failure (86 days after transplant from 8/8 HLA MUD), two patients died due infectious complications (808 days and 1637 days after transplant), three patients died due to multiorgan failure (215, 297 and 1097 days after transplant). Conclusion: Our data with use of CNI and MMF for GVHD prophylaxis for SAA following alloSCT with nonmyeloablative conditioning regimen showed that the rate of mucositis was low, engraftment time was rapid, and hospitalization was short, while OS, rates of acute and chronic GVDH were comparable to previously published rates with CNI and MTX-based GVHD prophylaxis. Figure 1 Figure 1. Disclosures Modi: Genentech: Research Funding; Seagen: Membership on an entity's Board of Directors or advisory committees; MorphoSys: Membership on an entity's Board of Directors or advisory committees. Deol: Kite, a Gilead Company: Consultancy.

scholarly journals Very Small Embryonic-Like Stem Cells, Endothelial Progenitor Cells, and Different Monocyte Subsets Are Effectively Mobilized in Acute Lymphoblastic Leukemia Patients after G-CSF Treatment

2018 ◽  
Vol 2018 ◽  
pp. 1-8 ◽  
Author(s):  
Andrzej Eljaszewicz ◽  
Lukasz Bolkun ◽  
Kamil Grubczak ◽  
Malgorzata Rusak ◽  
Tomasz Wasiluk ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Acute Lymphoblastic Leukemia ◽  
Progenitor Cells ◽  
Endothelial Progenitor Cells ◽  
Lymphoblastic Leukemia ◽  
Monocyte Subsets ◽  
Endothelial Progenitor ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Background. Acute lymphoblastic leukemia (ALL) is a malignant disease of lymphoid progenitor cells. ALL chemotherapy is associated with numerous side effects including neutropenia that is routinely prevented by the administration of growth factors such as granulocyte colony-stimulating factor (G-CSF). To date, the effects of G-CSF treatment on the level of mobilization of different stem and progenitor cells in ALL patients subjected to clinically effective chemotherapy have not been fully elucidated. Therefore, in this study we aimed to assess the effect of administration of G-CSF to ALL patients on mobilization of other than hematopoietic stem cell (HSCs) subsets, namely, very small embryonic-like stem cells (VSELs), endothelial progenitor cells (EPCs), and different monocyte subsets. Methods. We used multicolor flow cytometry to quantitate numbers of CD34+ cells, hematopoietic stem cells (HSCs), VSELs, EPCs, and different monocyte subsets in the peripheral blood of ALL patients and normal age-matched blood donors. Results. We showed that ALL patients following chemotherapy, when compared to healthy donors, presented with significantly lower numbers of CD34+ cells, HSCs, VSELs, and CD14+ monocytes, but not EPCs. Moreover, we found that G-CSF administration induced effective mobilization of all the abovementioned progenitor and stem cell subsets with high regenerative and proangiogenic potential. Conclusion. These findings contribute to better understanding the beneficial clinical effect of G-CSF administration in ALL patients following successful chemotherapy.

Isolation and characterization of human CD34−Lin− and CD34+Lin− hematopoietic stem cells using cell surface markers AC133 and CD7

Blood ◽  
2000 ◽  
Vol 95 (9) ◽  
pp. 2813-2820 ◽  
Author(s):  
Lisa Gallacher ◽  
Barbara Murdoch ◽  
Dongmei M. Wu ◽  
Francis N. Karanu ◽  
Mike Keeney ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Surface Markers ◽  
Cell Surface Markers ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Cd34 Cells

Recent evidence indicates that human hematopoietic stem cell properties can be found among cells lacking CD34 and lineage commitment markers (CD34−Lin−). A major barrier in the further characterization of human CD34− stem cells is the inability to detect this population using in vitro assays because these cells only demonstrate hematopoietic activity in vivo. Using cell surface markers AC133 and CD7, subfractions were isolated within CD34−CD38−Lin− and CD34+CD38−Lin− cells derived from human cord blood. Although the majority of CD34−CD38−Lin− cells lack AC133 and express CD7, an extremely rare population of AC133+CD7− cells was identified at a frequency of 0.2%. Surprisingly, these AC133+CD7− cells were highly enriched for progenitor activity at a frequency equivalent to purified fractions of CD34+ stem cells, and they were the only subset among the CD34−CD38−Lin− population capable of giving rise to CD34+ cells in defined liquid cultures. Human cells were detected in the bone marrow of non-obese/severe combined immunodeficiency (NOD/SCID) mice 8 weeks after transplantation of ex vivo–cultured AC133+CD7− cells isolated from the CD34−CD38−Lin− population, whereas 400-fold greater numbers of the AC133−CD7− subset had no engraftment ability. These studies provide novel insights into the hierarchical relationship of the human stem cell compartment by identifying a rare population of primitive human CD34− cells that are detectable after transplantation in vivo, enriched for in vitro clonogenic capacity, and capable of differentiation into CD34+ cells.

scholarly journals AMD3100 mobilizes hematopoietic stem cells with long-term repopulating capacity in nonhuman primates

Blood ◽  
2006 ◽  
Vol 107 (9) ◽  
pp. 3772-3778 ◽  
Author(s):  
André Larochelle ◽  
Allen Krouse ◽  
Mark Metzger ◽  
Donald Orlic ◽  
Robert E. Donahue ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Genetic Manipulation ◽  
Retroviral Vectors ◽  
Lymphoid Cells ◽  
Alternative Source ◽  
Hematopoietic Stem ◽  
Cd34 Cells

AMD3100, a bicyclam antagonist of the chemokine receptor CXCR4, has been shown to induce rapid mobilization of CD34+ hematopoietic cells in mice, dogs, and humans, offering an alternative to G-CSF mobilization of peripheral-blood hematopoietic stem cells. In this study, AMD3100-mobilized CD34+ cells were phenotypically analyzed, marked with NeoR-containing retroviral vectors, and subsequently transplanted into myeloablated rhesus macaques. We show engraftment of transduced AMD3100-mobilized CD34+ cells with NeoR gene marked myeloid and lymphoid cells up to 32 months after transplantation, demonstrating the ability of AMD3100 to mobilize true long-term repopulating hematopoietic stem cells. More AMD3100-mobilized CD34+ cells are in the G1 phase of the cell cycle and more cells express CXCR4 and VLA-4 compared with G-CSF-mobilized CD34+ cells. In vivo gene marking levels obtained with AMD3100-mobilized CD34+ cells were better than those obtained using CD34+ cells mobilized with G-CSF alone. Overall, these results indicate that AMD3100 mobilizes a population of hematopoietic stem cells with intrinsic characteristics different from those of hematopoietic stem cells mobilized with G-CSF, suggesting fundamental differences in the mechanism of AMD3100-mediated and G-CSF-mediated hematopoietic stem cell mobilization. Thus, AMD3100-mobilized CD34+ cells represent an alternative source of hematopoietic stem cells for clinical stem cell transplantation and genetic manipulation with integrating retroviral vectors.

Delayed Platelet Recovery Following Cord Blood Transplantation: Insights from a Mouse Model.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1727-1727
Author(s):  
Tao Du ◽  
Camelia Iancu-Rubin ◽  
George F. Atweh ◽  
Rona Singer Weinberg
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Cord Blood ◽  
Lower Number ◽  
Post Transplantation ◽  
Hematopoietic Stem ◽  
Platelet Recovery ◽  
Platelet Counts ◽  
Polyploid Cells

Abstract Umbilical cord blood (CB) is an important source of hematopoietic stem cells for stem cell transplantation and is being used with increasing frequency. A major concern related to clinical CB transplantation is the long delay in platelet recovery. Megakaryopoiesis is characterized by the acquisition of lineage-specific markers (e.g. CD41) during the early stages that is followed by polyploidization (DNA content > 4N) during the later stages of megakaryopoiesis. Using a newborn blood (NB) model of CB transplantation that was previously developed in our laboratory, we asked whether the delay in platelet recovery is a result of a decrease in the rate of megakaryocyte production or a delay in their maturation. C57BL/6 mice were transplanted with either murine adult bone marrow (BM) cells or murine new-born blood (NB) cells following lethal irradiation. We had previously shown that the concentration of Lin-Sca-1+c-Kit+ stem cells in murine adult BM was approximately 3 times higher than that in NB. To correct for this difference in stem cell concentration, irradiated mice were transplanted with either 0.5 ×106 BM cells or 2×106 NB cells. Platelet counts at 2 and 4 weeks were lower in mice transplanted with NB cells than in mice transplanted with BM cells (Table 1). Interestingly, the platelet counts became comparable in NB and BM recipients at 8 weeks post-transplantation. We compared the ability of BM cells from both NB and BM recipients to form CFU-Meg colonies in methylcellulose. At 2 and 4 weeks post-transplantation, BM cultures derived from NB recipients generated fewer CFU-Meg colonies than cultures from BM recipients (Table 1). Interestingly, after 8 weeks, the numbers of CFU-Meg from both stem cell sources were similar. We also compared the ability of BM cells from NB and BM recipients to differentiate into megakaryocytes in liquid culture, using CD41 expression and polyploidy as markers of megakaryocytic maturation. At 2 weeks post-transplantation, cultures from NB recipients generated 13% CD41+ cells whereas cultures from BM recipients generated 22% CD41+ cells. However, by 4 weeks post-transplantation, the numbers of CD41+ cells were similar in cultures derived from NB recipients and BM recipients (Table 1). Moreover, at 2 and 4 weeks post-transplantation, there were fewer polyploid cells in liquid cultures from NB recipients compared to BM recipients (Table 1). The lower number of polyploid cells was commensurate with the lower number of CD41+ cells. This suggests that the rate of maturation of megakaryocyte is similar in NB and BM. In conclusion, our studies show that CB stem cells generate megakaryocytic progenitors at a slower rate than BM stem cells and that the delay in platelet recovery is not a result of a delay in the maturation of megakaryocytic progenitors. Thus, in order to increase the rate of platelet recovery following CB transplantation, the focus should be on enhancing the rate of production of megakaryocytic progenitors from hematopoietic stem cells. Table 1. Platelet(×103/μl) CFU-Meg(Colonies/1 × 105 Cells) CD41(%) Ploidy(% >4N DNAContent) normal BM(n=5) 1200±120 35.5±5 55±5 27±3 2 weeks NBT-2M(n=5) 190± 38 10±2 13±2 7.5±1 BMT-0.5M(n=5) 400±50 17±4 22±1 11±2 4 weeks NBT-2M(n=5) 550±59 18±2 26±2 14.5±2 BMT-0.5M(n=5) 730±110 23±3 27±1 18±3 8 weeks NBT-2M(n=5) 930±115 39±7 N/A N/A BMT-0.5M(n=5) 970±130 40±4 N/A N/A

Purified T Depleted Peripheral Blood and Bone Marrow Cd34 Transplantation from Haploidentical Mother to Child with Thalassemia.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3106-3106
Author(s):  
Pietro Sodani ◽  
Buket Erer ◽  
Javid Gaziev ◽  
Paola Polchi ◽  
Andrea Roveda ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
T Cell ◽  
Peripheral Blood ◽  
Therapeutic Option ◽  
B Cell Lymphoma ◽  
Marrow Transplant ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Approximately 60% of thalassemic patients can not apply to “gene therapy today” which the insertion of one allogenic HLA identical stem cell into the empty bone marrow as the vector of the normal gene for beta globin chain synthesis. We studied the use of the haploidentical mother as the donor of hematopoietic stem cells assuming that the immuno-tollerance established during the pregnancy will help to bypass the HLA disparity and allow the hemopoietic allogeneic reconstitution in the thalassemic recipient of the transplant. We have employed a new preparative regimen for the transplant in fourteen thalassemic children aged 3 to 12 years (median age 5 years) using T cell depleted peripheral blood stem cell (PBSCTs) plus bone marrow (BM) stem cells. All patients received hydroxyurea (OHU) 60 mg/kg and azathioprine 3 mg/kg from day -59 until day-11, fludarabine (FLU) 30 mg/m 2 from day -17 to day -11, busulphan (BU) 14 mg/kg starting on day -10, and cyclophosphamide(CY) 200mg/kg, Thiotepa 10 mg/kg and ATG Sangstat 2.5 mg/kg, followed by a CD34 + t cell depleted (CliniMacs system), granulocyte colony stimulating factor (G-csf) mobilized PBSC from their HLA haploidentical mother. The purity of CD34+ cells after MACS sorting was 98–99%, the average number of transplanted CD34+ cells was 15, 4 x 10 6/kg and the average number of infused T lymphocytes from BM was 1,8 x 10 5/Kg.The patients received cyclosporin after transplant for graft versus host disease(GVHD) prophylaxis during the first two months after the bone marrow transplantation. Results. Thirteen patients are alive. Four patients rejected the transplant and are alive with thalassemia One patients died six months after bone marrow transplant for central nervous system diffuse large B cell lymphoma EBV related. Nine patients are alive disease free with a median follow up of 30 months (range12–47). None of the seven patients showed AGVHD and CGVHD. This preliminary study suggest that the transplantation of megadose of haploidentical CD34+ cell from the mother is a realistic therapeutic option for those thalassemic patients without genotipically or phenotipically HLA identical donor.

Long-Term Reconstitution of Transduced Rhesus CD34+ Cells Mobilized by G-CSF and Plerixafor.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1449-1449
Author(s):  
Naoya Uchida ◽  
Aylin Bonifacino ◽  
Allen E Krouse ◽  
Sandra D Price ◽  
Ross M Fasano ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Blood Cells ◽  
Transgene Expression ◽  
Hematopoietic Stem ◽  
Expression Levels ◽  
Cd34 Cells ◽  
One Year

Abstract Abstract 1449 Granulocyte colony-stimulating factor (G-CSF) in combination with plerixafor (AMD3100) produces significant mobilization of peripheral blood stem cells in the rhesus macaque model. The CD34+ cell population mobilized possesses a unique gene expression profile, suggesting a different proportion of progenitor/stem cells. To evaluate whether these CD34+ cells can stably reconstitute blood cells, we performed hematopoietic stem cell transplantation using G-CSF and plerixafor-mobilized rhesus CD34+ cells that were transduced with chimeric HIV1-based lentiviral vector including the SIV-capsid (χHIV vector). In our experiments, G-CSF and plerixafor mobilization (N=3) yielded a 2-fold higher CD34+ cell number, compared to that observed for G-CSF and stem cell factor (SCF) combination (N=5) (8.6 ± 1.8 × 107 vs. 3.6 ± 0.5 × 107, p<0.01). Transduction rates with χHIV vector, however, were 4-fold lower in G-CSF and plerixafor-mobilized CD34+ cells, compared to G-CSF and SCF (13 ± 4% vs. 57 ± 5%, p<0.01). CD123+ (IL3 receptor) rates were higher in CD34+ cells mobilized by G-CSF and plerixafor (16.4%) or plerixafor alone (21.3%), when compared to G-CSF alone (2.6%). To determine their repopulating ability, G-CSF and plerixafor-mobilized CD34+ cells were transduced with EGFP-expressing χHIV vector at MOI 50 and transplanted into lethally-irradiated rhesus macaques (N=3). Blood counts and transgene expression levels were followed for more than one year. Animals transplanted with G-CSF and plerixafor-mobilized cells showed engraftment of all lineages and earlier recovery of lymphocytes, compared to animals who received G-CSF and SCF-mobilized grafts (1200 ± 300/μl vs. 3300 ± 900/μl on day 30, p<0.05). One month after transplantation, there was a transient development of a skin rash, cold agglutinin reaction, and IgG and IgM type plasma paraproteins in one of the three animals transplanted with G-CSF and plerixafor cells. This animal had the most rapid lymphocyte recovery. These data suggested that G-CSF and plerixafor-mobilized CD34+ cells contained an increased amount of early lymphoid progenitor cells, compared to those arising from the G-CSF and SCF mobilization. One year after transplantation, transgene expression levels were 2–5% in the first animal, 2–5% in the second animal, and 5–10% in the third animal in all lineage cells. These data indicated G-CSF and plerixafor-mobilized CD34+ cells could stably reconstitute peripheral blood in the rhesus macaque. Next, we evaluated the correlation of transgene expression levels between in vitro bulk CD34+ cells and lymphocytes at one month, three months, and six months post-transplantation. At one and three months after transplantation, data from G-CSF and plerixafor mobilization showed higher ratio of %EGFP in lymphocytes to that of in vitro CD34+ cells when compared to that of G-CSF and SCF mobilization. At six months after transplantation the ratios were similar. These results again suggest that G-CSF and plerixafor-mobilized CD34+ cells might include a larger proportion of early lymphoid progenitor cells when compared to G-CSF and SCF mobilization. In summary, G-CSF and plerixafor mobilization increased CD34+ cell numbers. G-CSF and plerixafor-mobilized CD34+ cells contained an increased number of lymphoid progenitor cells and a hematopoietic stem cell population that was capable of reconstituting blood cells as demonstrated by earlier lymphoid recovery and stable multilineage transgene expression in vivo, respectively. Our findings should impact the development of new clinical mobilization protocols. Disclosures: No relevant conflicts of interest to declare.

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