Quantity Of Hematopoietic Stem Cells Research Articles

Hematopoietic progenitor cell count as a potential quantitative marker in apheresis products during allogeneic stem cell transplantation.

The quality and quantity of hematopoietic stem cells in apheresis products are essential to the success of peripheral blood hematopoietic stem cell transplantation (PB-HSCT). While the flow cytometry measurement of CD34+ cells as a golden standard for stem cell count is labor and cost-intensive, hematopoietic progenitor cell number evaluated by XN Sysmex series automated hematology analyzers (XN-HPC) is suggested as a surrogate marker. We evaluated the correlation and consistency of XN-HPC and CD34+ cell count in apheresis samples from both allogeneic donors and autologous patients during PB-HSCT. Good correlation and consistency were observed between XN-HPC and CD34+ cell counts in harvests collected from healthy donors (R = .852) rather than autologous patients (R = .375). Subgroup analysis showed that the correlation was especially poor when autologous patients used plerixafor as an additional mobilizer or were diagnosed with multiple myeloma (MM). In the setting of allogeneic transplantation, the correlation coefficients were even better in samples from non-first-round apheresis (R = .951), with high white blood cell (WBC) counts (R = .941), or having successful engraftment within 2 weeks (R = .895). ROC analysis suggested that an optimal XN-HPC count of 1127 × 106 /L best predicted a sufficient yield of CD34+ stem cells, with diagnostic sensitivity and specificity being 92% and 72%, respectively (AUC = 0.852). XN-HPC is a sufficient quantitative marker for stem cell assessment of harvest yield in allogeneic but not autologous HSCT.

Constructing a Machine Learning-Based Rating Model to Predict the Outcome of GCSF Mobilization of Hematopoietic Stem Cells in Peripheral Blood

Objective Peripheral blood stem cells serve as the primary source of hematopoietic stem cells for both autologous and allogeneic stem cell transplantation. Granulocyte colony-stimulating factor (G-CSF) stands as the most commonly employed mobilizing agent in clinical practice. While the majority of donors receiving G-CSF mobilization demonstrate high mobilization efficiency, there remains a subset of donors who fail to yield a sufficient quantity of hematopoietic stem cells. Inefficient mobilization not only leads to wastage of valuable resources but also significantly impacts transplant outcomes for recipients. Therefore, addressing the challenges associated with predicting mobilization outcomes, optimizing donor selection strategies, and further enhancing mobilization approaches is of paramount importance. Methods In this study, we conducted a retrospective analysis of mobilization data from 1008 donors who underwent hematopoietic stem cell mobilization using G-CSF at the Bone Marrow Transplantation Center, the First Affiliated Hospital, Zhejiang University School of Medicine, over the past 5 years. We meticulously examined the correlation between mobilization outcomes (quantified by the quantity of collected CD34+ cells) and various factors, including mobilizing agent usage, demographic data, complete blood count, and blood biochemistry. Furthermore, we harnessed the power of machine learning algorithms, such as linear regression, decision tree, and AdaBoost, to develop a robust scoring model capable of predicting mobilization outcomes. The primary objective of this model is to furnish a predictive tool for assessing mobilization outcomes with accuracy and precision. Results (1) Prior to receiving G-CSF, several factors exhibited a significant positive correlation with the quantity of CD34+ cells in the collection: donor's body weight (R=0.37, p<0.001), peripheral blood leukocytes (R=0.24, p<0.001), hemoglobin (R=0.23, p<0.001), monocytes (R=0.22, p<0.001), lymphocytes (R=0.18, p<0.001), platelets (R=0.18, p<0.001), red blood cells (R=0.12, p<0.001), neutrophils (R=0.15, p<0.001), hematocrit (R=0.21, p<0.001), mean corpuscular-hemoglobin concentration (R=0.15, p<0.001), low-density lipoprotein (R=0.11, p<0.01), albumin (R=0.09, p<0.05), ferritin (R=0.19, p<0.001), triglycerides (R=0.17, p<0.001), creatinine (R=0.17, p<0.001), and uric acid (R=0.29, p<0.001). (2) Age exhibited a significant negative correlation with the quantity of collected CD34+ cells in the collection (R=-0.11, p<0.001). (3) When the total dose of G-CSF administration was fixed, mobilization with twice-daily dosing yielded superior results compared to once-daily dosing (collected CD34+ cell count: 1268/μl vs. 1044/μl, p<0.001). (4) The ratio of G-CSF dosage to body weight demonstrated a significant negative correlation with the quantity of CD34+ cells (R=-0.09, p<0.01). Mobilization with a ratio of 6-7 μg/kg/day showed better results compared to a ratio of 7-12 μg/kg/day (p=0.07). (5) A scoring model was developed using machine learning methods to predict the mobilization outcomes of donors. Validation on the test dataset exhibited a high predictive ability of the model (AUC>0.7). Conclusion This study provides valuable insights for selecting hematopoietic stem cell donors and serves as a reference for future strategies aimed at improving mobilization outcomes. The identified factors, such as donor weight, peripheral blood parameters, G-CSF dosing regimen, and the ratio of dosage to body weight, can be considered when selecting donors and optimizing mobilization protocols. By incorporating machine learning techniques, a scoring model was developed, demonstrating a high predictive ability that can assist in forecasting mobilization outcomes for potential donors. These findings contribute to the optimization of donor selection and mobilization strategies.

Human Hematopoietic Stem Cells Have Altered Mitochondrial Activity after Stem Cell Transplantation

Hematopoietic stem cells (HSCs) give rise to the entire blood system. With their high regenerative potential, they are sometimes the only curative option for many malignancies. The success of HSCT depends on both the quantity of HSCs and on their ability to produce life-long blood. However, their regenerative potential is not unlimited. Transplanted HSCs sustain injury that alters their subsequent ability to generate blood cells. This leads to ineffective hematopoiesis and can ultimately lead to graft failure. Injury to HSCs is also a risk factor for secondary neoplasms, including leukemia. Our goal is to elucidate the mechanisms behind post-transplant HSC decline. Mitochondria are critically important for HSC function (Filippi Exp Hematol 2021). During regeneration of the bone marrow, HSCs become activated and undergo mitochondrial reprogramming, including reorganization of the mitochondrial network and enhanced mitochondrial activity (Filippi et al Blood 2019, Ito et al Nat Rev Mol Cell Biol 2014). However, after transplantation, HSCs accumulate mitochondria that are abnormal in organization and activity. These abnormal mitochondria lead to decline of HSC function and ineffective hematopoiesis (Hinge et al Cell Stem Cell 2020). While these findings have been replicated in murine models, it has not been studied within the human system. Our hypothesis is that after transplantation, human HSCs have altered mitochondria leading to ineffective hematopoiesis. We examined changes in HSC function using post-transplant samples of patient's bone marrow and a xenotransplant system (human CD34+ peripheral blood stem cells transplanted into sublethally irradiated immunodeficient NOD/SCID/IL-2 receptor-γ null mice). We first measured mitochondrial membrane potential using tetramethyl rhodamine ethyl ester (TMRE) dye and analyzed via flow cytometry. Three to six months after transplant in the xenotransplant model, we found that the mitochondrial membrane potential of huCD34+CD38-CD90+ HSCs was similar to the non-transplanted HSCs. However, huCD34+CD38+ progenitors exhibited a drastic 3-fold increase in mitochondrial membrane potential in comparison to the non-transplanted, huCD34+CD38+ progenitor cells. The mitochondrial membrane potential of CD34+CD38+ progenitors from transplanted bone marrow patient samples was also higher than non-transplanted CD34+CD38+bone marrow control cells. To understand if alterations in mitochondrial activity affect HSC differentiation, we placed xenotransplanted HSCs into in vitro culture. Post-transplanted huCD34+ cells failed to expand within 6 days of in vitro culture (serum-free culture with self-renewing cytokines, SCF+TPO+FLT3) compared to huCD34+ control. In subsequent differentiation assays, post-transplanted huCD34+ cultures gave rise to myeloid cells only. This contrasts with control huCD34+ cultures that produced myeloid, erythroid, and megakaryocytic cells. Interestingly, if the post-transplanted HSCs were given serum and cytokines for differentiation after only 3 days in self-renewing conditions, the cultured cells expanded similarly to non-transplanted HSCs and generated myeloid, erythroid, and megakaryocytic cells. This suggests that transplanted HSCs have decreased proliferation and differentiation ability in comparison to non-transplanted HSCs and respond differently to cytokines in vitro. This could account for ineffective hematopoiesis observed in some post-transplant patients. In conclusion, this study indicates that both mitochondrial activity and in-vitro growth change in HSCs after transplantation, despite re-establishing quiescence. This indicates that human hematopoietic stem and progenitor cells incur injury after transplantation. These findings are clinically important as the changes in mitochondrial activity in HSCs may play a role in post-transplant hematopoietic stem cell decline. Future single cell RNA sequencing analyses will provide mechanistic information regarding post-transplant changes in metabolism and potential targets for improving HSC function in transplant patients.

Optimization of methods for collecting peripheral hematopoietic stem cells in children with cancer: literature review

Autologous hematopoietic stem cell transplantation (auto-HSCT) is a standard for the treatment of oncological, hematologic, and also some immune diseases, ensuring the restoration of blood counts after high-dose chemotherapy. In children, the success of mobilization and collection of hematopoietic stem cells (HSCs) is especially important. Mobilization schemes for children are decided on an individual basis, which requires the development and implementation of recommendations for improving the efficiency of mobilization and collection of HSCs. Mobilization schemes include the use of granulocyte colony-stimulating factor in the form of monotherapy or in combination with CXCR4 antagonists. These schemes are ineffective in some children, which requires re-mobilization or rejection of transplantation, which negatively affects the prognosis. When preparing a patient for HSCs collection, it is necessary to take into account all previous therapy, the patient’s age, weight and height indicators, and general somatic state. Harvesting the required amount of HSCs will allow for high-dose therapy followed by auto-HSCT, and thereby increase the effectiveness of treatment. It is necessary to optimize the protocol for mobilization of HSCs with a large bias for pediatric patients, which will clearly define the criteria for mobilization, give indications for this procedure and determine the criteria for technical collection, which will allow to obtain the optimal number of CD34+ cells, which will ensure the success of the treatment.

Metabolic Regulations in Hematopoietic Stem Cells.

One of the bottlenecks of the treatments for malignant hematopoietic disorders is the unavailability of sufficient amount of hematopoietic stem cells (HSCs). HSCs areconsidered to be originated from the aorta-gonad-mesonephros and gradually migrates into fetal liver and resides in a unique microenvironment/niche of bone marrow. Although many intrinsic and extrinsic factors (niche components) are reported to be involved in the origination, maturation, migration, and localization of HSCs at different developmental stages, the detailed molecular mechanisms still remain largely unknown. Previous studies have shown that intrinsic metabolic networks may be critical for the cell fate determinations of HSCs. For example, HSCs mainly utilize glycolysis as the main energy sources; oxidative phosphorylation is required for the homeostasis of HSCs; lipid or amino acid metabolisms may also sustain HSC stemness. Mechanistically, lots of regulatory pathways, such as MEIS1/HIF1A and PI3K/AKT/mTOR signaling, are found to fine-tune the different nutrient metabolisms and cell fate commitments of HSCs. However, more efforts are required for the optimization and establishment of precise metabolic techniques specific for the HSCs with relatively rare cell frequency and understanding of the basic metabolic properties and their underlying regulatory mechanisms of different nutrients (such as glucose) during the different developmental stages of HSCs.

Pegylated granulocyte-colony stimulating factor versus non-pegylated granulocyte-colony stimulating factor for peripheral blood stem cell mobilization: A systematic review and meta-analysis.

Granulocyte-colony stimulating factor (G-CSF) mobilizes and increases the amount of hematopoietic stem cells in peripheral blood, enabling its harvest by few apheresis procedures. The pegylated G-CSF has longer half-life and is given once only, which is more comfortable for patients, whereas the non-pegylated requires multiple daily injection because of its short half-life. We summarized results of randomized trials comparing the efficacy and safety of pegylated and non-pegylated G-CSF for peripheral blood stem cell mobilization. We searched the Cochrane CENTRAL, MEDLINE, EMBASE, and two conference proceedings. Two authors made the selection, extracted data and evaluated methodological quality using GRADE independently. We used random-effects model for meta-analysis. We found 3956 records and retrieved 47 full texts. We included eight randomized trials with a total number of 554 randomized and 532 analyzed subjects. The meta-analysis included five trials because not all trials reported the same outcomes. Pooling data from two studies shows no evidence for a difference in the successful mobilization rate (CD34+ cell ≥ 2 × 106 /kg collected) between pegfilgrastim 6 mg (early administration) and filgrastim 5 µg/kg/day (147 participants; risk ratio (RR) 0.87, 95% confidence interval (95%CI) 0.67-1.11; P = .26). Pooling data from three studies shows no difference in the incidence of adverse events between pegylated and non-pegylated G-CSF (170 participants; RR 0.86, 95%CI 0.34-2.17; P = .75). No difference found on the quantity of CD34+ cells collected, number of apheresis procedure in successful mobilization, level of peak PB CD34+ cells achieved, and day of neutrophil and platelet engraftment.

Novel cell population data from a haematology analyzer can predict timing and efficiency of stem cell transplantation

Autologous hematopoietic stem cell transplantation (AHSCT) is a necessary component for many oncohematological diseases treatment. For a successful result of AHSCT a sufficient quantity of hematopoietic stem cells (HSCs) is needed. It has been proposed that morphological changes of myeloid cells could reflect the processes of bone marrow stimulation and may provide useful information to predict the stimulation efficiency and expected outcome of CD34(+) stem cells. The Beckman Coulter Cellular Analysis System DxH800 performs Flow Cytometric Digital Morphology analysis of leukocytes. All leukocyte cellular measurements can be reported as numerical values called Cell Population Data (CPD), which are able to detect morphological changes in the cell size and distribution of neutrophils. Our findings suggest that the changes in neutrophil CPD were detectable 2-4days before the observed increase in CD34(+) count in the peripheral blood and can potentially improve the management of patients. There was also a good correlation between MN-V-NE and ImmNeIndex with the CD34(+) count suggesting they can be used as a surrogate for the CD34(+) count (r=0.67 and 0.65 p<0.005 respectively).

Top-down motif engineering of a cytokine receptor for directing ex vivo expansion of hematopoietic stem cells

The technique to expand hematopoietic stem cells (HSCs) ex vivo is eagerly anticipated to secure an enough amount of HSCs for clinical applications. Previously we developed a scFv-thrombopoietin receptor (c-Mpl) chimera, named S-Mpl, which can transduce a proliferation signal in HSCs in response to a cognate antigen. However, a remaining concern of the S-Mpl chimera may be the magnitude of the cellular expansion level driven by this molecule, which was significantly less than that mediated by endogenous wild-type c-Mpl. In this study, we engineered a tyrosine motif located in the intracellular domain of S-Mpl based on a top-down approach in order to change the signaling properties of the chimera. The truncated mutant (trunc.) and an amino-acid substitution mutant (Q to L) of S-Mpl were constructed to investigate the ability of these mutants to expand HSCs. The result showed that the truncated and Q to L mutants gave higher and considerably lower number of the cells than unmodified S-Mpl, respectively. The proliferation level through the truncated mutant was even higher than that of non-transduced HSCs with the stimulation of a native cytokine, thrombopoietin. Moreover, we analyzed the signaling properties of the S-Mpl mutants in detail using a pro-B cell line Ba/F3. The data indicated that the STAT3 and STAT5 activation levels through the truncated mutant increased, whereas activation of the Q to L mutant was inhibited by a negative regulator of intracellular signaling, SHP-1. This is the first demonstration that a non-natural artificial mutant of a cytokine receptor is effective for ex vivo expansion of hematopoietic cells compared with a native cytokine receptor.

High Correlation of Pre-Apheresis Peripheral Blood CD34+ Cell Counts with Final Apheresis Product CD34+ Cell Count by Using a More Accurate Determination of Patient Blood Volume

Abstract 2996The final apheresis product CD34+ cell count per Kg of recipient weight is used to determine if additional apheresis procedures are necessary to collect the targeted amount of hematopoietic stem cells for autologous peripheral blood stem cell transplantation. Flow cytometric analysis of CD34+ cells can take several hours. A more timely technique to predict apheresis product CD34+ cell counts during apheresis may help determine if further administration of cytokines is necessary or if apheresis catheters can be removed, and overall improve the efficiency of patient (pt) care. We performed a retrospective review of all pts undergoing autologous peripheral blood stem cell mobilization with granulocyte-colony stimulating factor (G-CSF) alone, G-CSF and plerixafor, or with chemotherapy followed by G-CSF, from July 2010 through May 2011 who underwent peripheral blood stem cell collection with apheresis on the COBE Spectra cell separator. Linear regression models were used to formulate the calculation of pt blood volume based on the pre-apheresis CD34+ cell count per micro liter of blood, the final apheresis product CD34+ cell counts, and the amount of blood processed during the apheresis procedure. This calculated blood volume is expressed by the formula BV = 82.5(patients weight in Kg) + 793. We then prospectively evaluated the next consecutive pts who underwent stem cell mobilization and apheresis in June and July 2011. Twenty-seven apheresis collections were done on 26 pts. Fourteen pts were female, and 12 pts were male. Seventeen pts were diagnosed with myeloma, 6 pts with NHL, and 3 pts with other diseases. Twenty pts were mobilized with G-CSF (10 ug/kg daily) with apheresis to begin on day 5. Fifteen of the 20 pts required plerixafor on day 4 because of low peripheral blood CD34+ cell counts (< 10/ul). Six pts were mobilized with chemotherapy followed by G-CSF 10 ug/kg daily until peripheral CD34 cell counts recovered greater than 10/ul and then apheresis was started. Each pt had their blood volume calculated according to the formula above and the peripheral blood CD 34+ cell count was measured on the first and second day of apheresis. The peripheral blood CD 34+ cell count/ul was multiplied by 1000 and this product was multiplied by the calculated blood volume and then divided by the pts weight [(PBCD34+ cell count × 1000) × BV]/Kg to determine the predicted apheresis product CD34+ cell count, which was then compared to the actual apheresis product final CD34+ cell count. On the first day of collection the mean for the predicted product CD34+ cell count was 4.98 × 106 +/− 3.1 × 106, and the actual apheresis product CD34+ cell count was 4.61 × 106 +/− 2.90 × 106 (Pearson correlation r value of 0.913 and a p value <0.001)(see figure). Nineteen collections were evaluable on the second day of collection with the mean for the predicted product CD34 + cell count of 2.08 × 106 +/− 1.64 × 106, and the mean for the actual apheresis product CD34+ cell count of 2.29 × 106 +/− 0.768 × 106 (Pearson correlation r value of 0.620 with a p value of 0.005). There was no significant difference in the correlation between patients mobilized with G-CSF alone, G-CSF and plerixafor or after chemotherapy and G-CSF. In conclusion, a more accurate determination of patient blood volume allowed for a high degree of correlation on the first day of peripheral blood stem cell collection on the COBE Spectra machine between the predicted product CD34+ cell count and the actual apheresis product CD34+ cell count. An accurate prediction of the final apheresis product CD34+ cell count may allow for less cytokine administration, quicker removal of apheresis catheters, and more efficient disposition of patients undergoing peripheral blood stem cell collection. [Display omitted] Disclosures:Shaughnessy:Otsuka: Honoraria, Speakers Bureau; Millenium: Honoraria, Speakers Bureau; Genzyme: Consultancy, Honoraria, Research Funding, Speakers Bureau.

Cell Population Data As a Predictor of Stem Cell Aphaeresis Efficiency

Abstract 2991 Introduction:Autologous Hematopoietic Stem Cell Transplantation (AHSCT) is a necessary component of treatment for many oncohaemathological diseases. For success of AHSCT a sufficient quantity of Hematopoietic Stem Cells (HSC) are needed. The estimation of the quantity of HSC post aphaeresis is vital. The procedure of aphaeresis is time consuming and expensive but the CD34+ count (equivalent to HSC) measured by traditional flow cytometry, in peripheral blood can predict the quality of the aphaeresis product but is much more expensive than a complete blood count. It has been proposed that morphological changes of myeloid cells detected by some hematological analyzers could reflect the processes of bone marrow stimulation and may provide useful information for the prediction of the efficiency of stimulation and expected outcome of CD34+ stem cells.Materials and methods: Nine patients, 7 with multiple myeloma and 2 with Hodgkins disease were studied after informed consent: 8 female, 1 male, age range 21 to 59. Patients received the standard dose G-CSF, protocol-driven chemotherapy +G-CSF (dose=5 mcg/kg/day). Initial mobilization typically processed 10–12 liters of blood. The Beckman Coulter Cellular Analysis System DxH800 performs Flow Cytometric Digital Morphology analysis of leukocytes with measurement of cell volume (impedance), internal complexity and nucleo/cytoplasm ratio (cell conductivity in the radio-frequency current) and granularity (measurement of 5 angles of light scatter). All these measurements (Mean and Standard Deviations (SD)) are reported as numerical values, called Cell Population Data (CPD). Analysis of CD34+ stem cells was performed on FC500 Flow Cytometer with Stem Kit (Beckman Coulter) using the single-platform ISHAGE protocol. All patients were followed-up daily, starting from the day before G-CSF administration. Results:For patients, responding to therapy (8 from 9 included in the study) an increase in Ne CPD - Neutrophil Mean Volume (NeMV), and Neutrophil SD Volume (NeSDV) was seen from 2 to 4 days before the increase in CD34+ count in peripheral blood. The calculated parameter, Ne Immaturity Index (ImmNeIndex), using the formula (NeMV × NeSDV)/100 was introduced for the analysis. Patients that responded to the stimulation had an increase in NeMV greater than 15%, increase in NeSDV more than 60% and increase in ImmNeIndex more that 85% compared to the values pre- treatment. There was significant correlation between CD34+ and NeMV (correlation coefficient = 0.491, significance level P=0.0011, n=41), CD34+ and NeSDV (correlation coefficient = 0.418, significance level P=0.0065, n=41) and CD34+ and Imm Ne Index (correlation coefficient = 0.489, significance level P=0.0012, n=41). Patients were classified into 3 groups according to %CD34+ cells in peripheral blood at the end of one cycle of stimulation with NeMV, NeSDV and ImmNeIndex (Table 1), and this estimation can give information for the best time for harvest of CD34+ cells after aphaeresis.Table 1Ratio before treatment/after treatment%CD34+ cells in PB at the end of one cycle of stimulationImm Ne IndexNeMVNeSDV>0.52.28–2.921.26–1.471.87–2.370.2–0.51.9–2.031.14–1.241.64–1.77<0.21.131.041.09ROC curve analysis with MedCalc Software (Mariakerke, Belgium) showed that patients with % CD34+ cells higher than 0.2% in peripheral blood can be detected with NeMV (AUC 0. 903, cut-off 162, sensitivity 93%, specificity 89%) and Imm Ne Index (AUC 0.847 cut-off >41.43, sensitivity 93%, specificity 70%). Discussion:Cell Population Data available from Beckman Coulter Cellular Analysis System DxH800 are able to detect morphological changes in the cell size of neutrophils and anisocytosis of neutrophil population. These morphological features follow the stimulation of the bone marrow activity and the production of immature cells, including CD34+ cells. Changes in CPD were detectable 2–4 days before the observed increase in CD34+ count in the peripheral blood and can improve the management of patients. These parameters available on the routine haematological analyser without any additional cost or time can be used as additional criteria in the laboratory to make the decision about timing of aphaeresis for patients after the G-CSF stimulation. Disclosures:No relevant conflicts of interest to declare.

Stem Cell Mobilization in Poor Mobilizers Using Plerixafor and G-CSF with or without Chemotherapy: A European Perspective

Abstract 2993High-dose chemotherapy followed by autologous stem cell transplantation is an approved therapeutic intervention in relapsed Hodgkin-lymphoma (HL) and Non-Hodgkin lymphoma (NHL). In multiple myeloma (MM) it remains standard of care in first remission. Unfortunately, a significant portion of patients fail to mobilize and collect a sufficient amount of hematopoietic stem cells, being considered as “poor-mobilizers”.The effectiveness of the hematopoietic stem cell mobilizing agent plerixafor was evaluated in nationwide compassionate use programs in 13 European countries and reported to the European Consortium of Stem Cell Mobilization (ECOSM). Here we describe the mobilization success of 580 proven poor-mobilizers (304 male, 276 female) with NHL, HL and MM in Europe between May 2008 and August 2009. Furthermore, we analyzed the mobilization of stem cells in major NHL subgroups.All patients received plerixafor plus granulocyte colony-stimulating factor in standard doses with or without chemotherapy. Two-hundred seventy patients with NHL (138 male, 132 female) with a median age of 56 years (range 12 – 75 years) and a median of two prior chemotherapy regimens (range 0 – 10) were enrolled. Median cell yield was 2.56 × 10 ^6 CD34+ cells/kg BW (range 0 – 17.37). The general accepted minimum of 2.0 × 10 ^6 CD34+ cells / kg bodyweight (BW) for transplantation was reached by 175 patients (64.8%) in a median of two apheresis sessions (range 1 – 4). Thirty-four patients (12.6%) yielded more than 5.0 × 10 ^6 CD34+ cells/kg BW. There were no significant differences in in stem cell harvests regarding number of prior mobilization attempts or number of prior chemotherapeutic regimens, as well as in comparing patients with diffuse large B cell lymphoma (n=28), follicular lymphoma (n=15), and mantle cell lymphoma (n=24), respectively.Fifty-four HL patients (24 male, 30 female) with a median age of 36 years (range 19 – 76) and a median of three prior lines of therapy (range 1 – 5) were enrolled. Median cell yield was 3.14 × 10^6 CD34 cells/kg BW (range 0 – 32.6). Forty-four patients (81.5%) collected the minimum of 2.0 × 10^6 CD34+ cells/kg BW in a median of two apheresis sessions (range 1 – 4). Twelve patients (22.2%) collected more than 5.0 × 10 ^6 CD34+ cells/kg BW.A total of 256 patients (148 male, 108 female) with a median age of 60 years (range 28 – 76) diagnosed with MM were enrolled. Patients had received a median of two prior lines of treatment and collected a median of 3.60 × 10 ^6 CD34+ cells/kg BW (range 0 – 15.27) in a median of two apheresis sessions (range 1 – 5). The minimum of 2.0 × 10 ^6 CD34+ cells/kg BW was collected by 209 patients (81.6%). Eighty-two patients (32.0%) yielded more than 5.0 × 10 ^6 CD34+ cells/kg BW allowing tandem transplantation.Overall, the CD34+ cell yield was significantly higher in MM patients than in NHL patients (p <0.0001) and also significantly higher in HL patients than in NHL patients (p =0.013). CD34+ cell yield was not statistically significant between MM patients and HL patients. Furthermore, the number of patients collecting the minimum of 2.0 × 10 ^6 CD34+ cells/kg BW was significantly higher in MM patients compared to NHL patients (p <0.0001) and also significantly higher in HL compared to NHL patients (p =0.017). Analyzing the mobilization strategies and collection success of individual countries demonstrated only minor variations compared to the global results. Chemomobilization and steady state mobilization are used in most countries; however, there is a clear preference for chemotherapy combined with G-CSF/plerixafor in the Czech Republic, Germany, Hungary, Italy and Poland. The data emphasize the role of plerixafor in patients who failed prior mobilization attempts, but the development of improved strategies in poor mobilizers especially with NHL is required. Disclosures:Duarte:Genzyme: Membership on an entity’s Board of Directors or advisory committees. Kröger:Genzyme: Membership on an entity’s Board of Directors or advisory committees. Mohty:Genzyme: Honoraria, Membership on an entity’s Board of Directors or advisory committees. Hübel:Genzyme: Membership on an entity’s Board of Directors or advisory committees.

Role of Cohesin-Resolving Protease, Separase, In Hematopoiesis.

AbstractAbstract 1593 Introduction:Cohesin is an evolutionarily conserved protein complex that forms during the replication of sister chromatids. It is a multi-protein complex that consists of four proteins, Smc1, Smc3, Rad21, and Scc3. Resolution of sister chromatid cohesion at the onset of anaphase depends on Separase, an endopeptidase that separates sister chromatids by cleaving cohesion Rad21. A recent study suggests a new role of Cohesin proteins in gene expression and development with implications in hematopoiesis. Our data indicates that cohesin-resolving protease Separase may play a critical role in hematopoiesis. HYPOTHESIS: We hypothesize that Separase plays a role in hematopoiesis by increasing the quantity of hematopoietic stem cells (HSC). METHODS: Our experimental approach was to isolate murine long-term HSC from WT mice and mice with one mutated copy of Separase (i.e. Separase heterozygotes). In addition, in vivo competitive long term repopulation assays were used assess the function of HSC in Separase heterozyotes. RESULTS: Separase heterozygote have increased HSC numbers (p<0.05) as compared to WT mice. In addition, an improved engraftment in a competitive repopulation assay (p < 0.001) was seen in the Separase heterozyotes. Analysis of the engrafted cells demonstrated no difference between the wild type and Separase heterozygote animals, indicating the increased engraftment may be due to unique features in the primitive hematopoietic stem cells. CONCLUSION: Investigation of the mechanism for improved HSC engraftment in Separase heterozygote mice will significantly contribute to our understanding of marrow engraftment and function. Elucidating the mechanisms of hematopoietic dysregulation will provide insights into the development of life-threatening disorders such as leukemia and, in the setting of bone marrow transplant, engraftment failure. Disclosures:No relevant conflicts of interest to declare.

Preliminary findings on the use of pulsatile machine reperfusion of a placenta to improve the cord blood collection yield including primitive hematopoietic stem cell fractions

Umbilical cord blood (CB) use is limited in most adults since the mean amount of hematopoietic stem cells (HSCs) collected is generally insufficient to engraft successfully. This study demonstrates for the first time the feasibility of pulsatile machine placenta reperfusion (PMPR), which significantly improves CB collection quantity and quality compared to standard collection methods. PMPR was performed on eight delivered placentas up to 39 hours after venipuncture-based collection. PMPR was performed on average for 26 (20-30) minutes, 17 hours after delivery (6.25-39), using perfusate approved for donated organ reperfusion. Both PMPR and conventional collection-derived CB cells were analyzed using immunophenotyping and colony assays. The combination of PMPR and the conventional venipuncture method yielded a mean of 1.5-, 4.9-, 11-, 7.5-, 7.5-, and 7.7-fold increased number of total mononuclear, CD34+, CD34+/CD38-, CD133+, CD133+/CD34+, and CD133+/CD34- cells, respectively, compared to conventional venipuncture alone. CB cells obtained by PMPR alone generally demonstrated a significant increase in percentages of primitive HSC phenotype with equivalent cell viability between PMPR and venipuncture. This article demonstrates for the first time that CB can be collected up to 39 hours after delivery with PMPR while maximizing CB collection including HSCs with primitive phenotypes.

Transplantation of Ex Vivo Expanded Lin− Cells Selected from Autologous PBSC Grafts in Patients with Diagnosis of Lymphoma.

Background: Hematopoietic stem cells are able to regenerate hematopoiesis in all of its lineages. They are clinically used in transplantation of bone marrow or peripheral blood stem cells (PBSC) in patients with diagnosis of leukemia or lymphoma. While amount of hematopoietic stem cells is critical for the long-term engraftment, the amount of progenitor and precursor cells can influence time to engraftment, and it is critical for duration of neutropenia after transplantation. The methods of expansion of hematopoietic stem cells could reduce time to engraftment and decrease the risk of early post-transplant complications.Methods: Authors analyzed expansion of enriched hematopoietic stem cells (HSC), selected by immunomagnetic separation of Lin− cells from PBSC, in the culture of serum-free medium in vitro with all combinations of 5 cytokines (SCF, Flt-3-L, IL-3, IL-6, TPO) with and without G-CSF. Cell counts, morphology, immunophenotyping, and CFU-GM and CFU-Meg cultures were performed. Clinical transplantation protocol based on these results was developed. 10 patients with diagnosis of non-Hodgkin's or Hodgkin's lymphoma indicated for autologous transplantation, who signed the informed consent, were enrolled into the protocol. Except of standard PBSC graft, additional cells were collected, Lin− cells were selected by immunomagnetic separation, frozen and stored at Tissue bank. At day -14, Lin− cells were thawed and expanded in culture of serum-free medium with cytokines SCF, Flt-3-L, IL-3, IL-6 and G-CSF. Patients received high-dose chemotherapy regimen BEAM from day −7 to day −2. Progenitor cells expanded ex vivo in culture from Lin− cells were infused at day 0, following transplantation of PBSC. Patients were monitored, blood counts were performed, side effects were observed, and times to engraftment in granulocytes and platelets were calculated.Results: In experiments, the highest number of CFU-GM colonies was observed at day +14 with cytokine combination SCF+IL-3+Flt-3-L+ IL-6. The highest number of CFU-Meg colonies was observed in cytokine combination SCF+IL-3+TPO. G-CSF increased count and maturation of cells. Number of total cells grew 200 to 350 times at day +14. In the clinical protocol, 2 patients were excluded for technical reasons and 1 patient was excluded because of disease progression. 7 patients completed the protocol. The clinical procedure was free of serious adverse effects in all patients. Patients received doses from 5•107 to 3•109 cells. The group of patients was compared with historical controls - 142 patients treated at our department with autologous PBSC transplantation after BEAM regimen for diagnosis of lymphoma. Control group received higher average dose of CD34+ cells than experimental group: 7.7•106 / kg versus 6.6•106 / kg. Engraftment in granulocytes > 1000 / μl was shortened from 11 to 7.3 days in experimental group. Engraftment in platelets was not changed significantly (12 versus 11 days). Duration of neutropenia was shortened from 8 to 5.6 days. In patients, who received higher doses of infused cells > 2•109, average engraftment in granulocytes occurred at 6.3 days and duration of neutropenia was 5 days.Conclusions: HSC can be enriched from PBSC grafts, cultured and expanded ex vivo, and safely used in the cellular therapy protocols. The procedure resulted in significant shortening of critical period of neutropenia.

Comparative Study of Chimerism Dynamics between Allogenic Bone Marrow Transplantation and Nonmyeloablative Stem Cell Transplantation.

Objective To explore whether the dynamics and outcome of chimerism are different between allogenic bone marrow transplantation (allo-BMT) and nonmyeloablative stem cell transplantation (NST) these two kinds of transplant strategies in order to define factors that significantly influence the early engraftment of donor stem cells.Methods A retrospective comparative study was performed between 20 patients undergoing BMT and 18 patients receiving NST. There are several differences between NST and standard allo-BMT, such as conditioning regimens, the source and the quantity of hematopoietic stem cells and the protocal of GVHD prophylaxis. But component of two groups patients were similar. Sequential monitoring of chimerism were performed in 38 patients in different periods post transplantation using multiple short tandem repeat(STR) amplification by fluorescence labeling polymerase chain reaction(PCR) combined with a capillary electrophoresis.ResultsThe median age of recipient, median quantity of MNC, CD34+ cells and T lymphocyte cells of NST were significantly higher compared with the BMT. The median time for absolute neutrophils recovery of NST(without G-CSF) was similar to that of BMT. While compared to BMT, NST lead to faster engraftment of platelets.The establishment of full donor chimerism (FDC) of NST was much more earlier than that of BMT (1 month VS 3 months, p=0.01). In the early post-transplant period(up to day 30), there was an apparent increase in the incidence of full donor chimerism (FDC) following NST compared with BMT(38.9% VS 20%, p=0.019). Whereas a significantly lower incidence of mixed chimerism(MC) occurred in recipients of NST compare to BMT.(61.2% VS 80%, p=0.02). It suggest that in the early stage after transplantation, the engraftment of donor cells of NST was more rapid than that of BMT. The major relevant factor causing full donor chimerism formation seems to be the high quantity of donor CD34+ cells and T lymphocyte cells.Fludarabine-based reduced-intensity conditioning didn't result in a significant delay of donor cell engraftment compared to standard conditioning regimen.The incidence of chronic GVHD was much higher in group of NST than that of BMT group(80% VS 50%, p=0.007).This finding was related to that patients of NST group received the higher doses of CD34+ cells. Conclusion Factors that significantly influence the early kinetics of donor chimerism and donor cell engraftment after transplantation were the quantity of stem cell and T lymphocyte cell of grafts.

Increased Engraftment after Double Cord Blood Transplantation in NOD/SCID Mice.

Umbilical cord blood (UCB) is considered as an attractive alternative source of hematopoietic stem cells for allogeneic stem cell transplantations in patients who lack HLA-matched donors. However, the low cell dose adversely affects the speed of hematopoietic recovery and therefore limits the application of UCB transplantation in adults. Although ex-vivo expansion of cord blood cells has been explored as a strategy to increase the cell dose, compromised engraftment potential of expanded cells has been demonstrated. Another approach to overcome cell dose limitations is transplantation of multiple, unrelated UCB units. To investigate the effect of multiple cord transplantation on engraftment, NOD/SCID mice were transplanted with human hematopoietic progenitor cells (CD34+) derived from two UCB units with HLA disparity. During the first six weeks after transplantation the number of human platelets in peripheral blood was quantified by flow cytometry. Six weeks after transplantation, the mice were sacrificed and the percentage and donor origin of human CD45+ cells in blood, and in bone marrow was determined by flow cytometry. Transplantation of CD34+ cells derived from two UCB donors resulted in significantly higher number of human platelets in peripheral blood than transplantation of CD34+ cells from either donor alone, ranging from 3.92x106/ml to 10.29x106/ml (mean 6.4x106 ± 2.55x106/ml) and 0.11x106/ml to 3.12.106/ml (mean 1.42x106 ± 1.17x106/ml), respectively. Furthermore, the overall human cell engraftment level in bone marrow after double cord blood transplantation ranged from 7.01% to 64.34% (mean 29.6 ± 21.5%) a nearly 7-fold increase compared to single cord blood transplantation ranging from 0.27% to 13.5% (mean 4.6 ± 3.8%) Although consistently higher engraftment levels were reached after double cord blood transplantation, two different patterns were observed: in 2 out of 4 experiments cells from one donor predominated the engraftment (ratio 3:1), while in two other experiments the two units contributed equally to BM engraftment. The mechanism underlying these effects are <S>is</S> not yet clear. It is not very likely that the single donor predominance results from an unequal amount of hematopoietic stem cells in the cord blood units because each cord blood showed comparable levels of engraftment as a single unit. Alternatively, the unequal engraftment may result from an immunological competition or a graft versus graft stimulatory effect between the cords during the engraftment process and further studies are required to determine if the contribution of both units is dependent on the degree of HLA matching between the two cords. Taken together, these results demonstrate that double cord blood transplantation may represent a means of achieving increased engraftment, making multiple cord blood transplantation a promising strategy to improve the outcome of UCB transplantation. Studies are underway to unravel the mechanisms underlying the enhanced engraftment.

Impact of stem cell dose on hematopoietic recovery in autologous blood stem cell recipients.

Hematopoietic recovery rates following high-dose chemotherapy in autologous blood stem cell recipients have been shown, in part, to be dependent on the source and quantity of hematopoietic stem cells infused. Mobilized blood stem cell quantity (identified by the surface expression of the CD34 antigen) has been demonstrated in multiple studies to be the strongest predictor of days to hematopoietic recovery (ie platelets and neutrophils) in autologous blood stem cell recipients. Additionally, data from four large studies confirm that prompt and sustained hematopoietic recovery will occur in the majority of patients treated with high-dose chemotherapy if a stem cell dose > or = 5 x 10(6)/kg is administered. However, multiple aphereses are needed in the majority of patients to achieve these optimal stem cell doses. Problems associated with multiple aphereses procedures include hypocalcemia, anemia, thrombocytopenia, infection, thrombosis, increased costs, and malignant cell contamination of the apheresis product. Recent data indicate that less differentiated (eg CD33 ) stem and progenitor cells result in both early and sustained hematopoietic recovery in bone marrow and blood stem cell recipients. Future trials using new growth factors such as stem cell factor (which has been shown to increase CD34+/CD33 cell mobilization), as well as improvements in purging strategies are needed to ensure prompt, sustained, and malignant-cell-free engraftment for the majority of autologous blood stem cell recipients.

Cyclophosphamide, Doxorubicin, Vincristine, Prednisone Dose Intensification With Granulocyte Colony-Stimulating Factor Markedly Depletes Stem Cell Reserve for Autologous Bone Marrow Transplantation

Hematopoietic growth factors allow dose escalation of chemotherapy. This approach may potentially reduce the quality and quantity of hematopoietic stem cells. The capacity of stem cells recovered after dose intensification to support myeloablative therapy is unknown. In patients with previously untreated advanced follicular lymphoma, trilineage hematopoietic engraftment was compared in two sequential trials of induction therapy (standard dose cyclophosphamide, doxorubicin, vincristine, prednisone [CHOP] without growth factors or dose intensification CHOP supported by granulocyte colony-stimulating factor [G-CSF ]) followed by identical myeloablative therapy and autologous stem cell support. Neutrophil, platelet, and red blood cell (RBC) engraftment were compared on days 100, 180, and 360 after stem cell reinfusion. Despite similar patient characteristics including reinfusion of comparable numbers of marrow mononuclear cells, after stem cell transplantation, a highly significant prolongation of neutrophil and platelet engraftment was seen in patients who received high dose CHOP and G-CSF in comparison to standard dose CHOP. These findings suggest that dose intensified chemotherapy and G-CSF recruited stem cells into a proliferative phase and that G-CSF allowed retreatment at a time when stem cells were susceptible to damage by cytotoxic therapy. Such inadequate hematologic engraftment after myeloablative therapy might be avoided by either shortening the time that growth factor support is administered, lengthening the interval between cycles, or attempting to repetitively harvest additional stem cells either from the marrow or peripheral blood. Therefore, intensification of chemotherapy with growth factor support must be used with caution if stem cells are to be used to support myeloablative therapy.

Cyclophosphamide, Doxorubicin, Vincristine, Prednisone Dose Intensification With Granulocyte Colony-Stimulating Factor Markedly Depletes Stem Cell Reserve for Autologous Bone Marrow Transplantation

AbstractHematopoietic growth factors allow dose escalation of chemotherapy. This approach may potentially reduce the quality and quantity of hematopoietic stem cells. The capacity of stem cells recovered after dose intensification to support myeloablative therapy is unknown. In patients with previously untreated advanced follicular lymphoma, trilineage hematopoietic engraftment was compared in two sequential trials of induction therapy (standard dose cyclophosphamide, doxorubicin, vincristine, prednisone [CHOP] without growth factors or dose intensification CHOP supported by granulocyte colony-stimulating factor [G-CSF ]) followed by identical myeloablative therapy and autologous stem cell support. Neutrophil, platelet, and red blood cell (RBC) engraftment were compared on days 100, 180, and 360 after stem cell reinfusion. Despite similar patient characteristics including reinfusion of comparable numbers of marrow mononuclear cells, after stem cell transplantation, a highly significant prolongation of neutrophil and platelet engraftment was seen in patients who received high dose CHOP and G-CSF in comparison to standard dose CHOP. These findings suggest that dose intensified chemotherapy and G-CSF recruited stem cells into a proliferative phase and that G-CSF allowed retreatment at a time when stem cells were susceptible to damage by cytotoxic therapy. Such inadequate hematologic engraftment after myeloablative therapy might be avoided by either shortening the time that growth factor support is administered, lengthening the interval between cycles, or attempting to repetitively harvest additional stem cells either from the marrow or peripheral blood. Therefore, intensification of chemotherapy with growth factor support must be used with caution if stem cells are to be used to support myeloablative therapy.