Transplantation Of CD34 Research Articles

Local Transplantation of Granulocyte Colony Stimulating Factor-Mobilized CD34+ Cells for Patients With Femoral and Tibial Nonunion: Pilot Clinical Trial

Most bone fractures typically heal, although a significant proportion (5%-10%) of fractures fail to heal, resulting in delayed union or persistent nonunion. Some preclinical evidence shows the therapeutic potential of peripheral blood CD34(+) cells, a hematopoietic/endothelial progenitor cell-enriched population, for bone fracture healing; however, clinical outcome following transplantation of CD34(+) cells in patients with fracture has never been reported. We report a phase I/IIa clinical trial regarding transplantation of autologous, granulocyte colony stimulating factor-mobilized CD34(+) cells with atelocollagen scaffold for patients with femoral or tibial fracture nonunion (n = 7). The primary endpoint of this study is radiological fracture healing (union) by evaluating anteroposterior and lateral views at week 12 following cell therapy. For the safety evaluation, incidence, severity, and outcome of all adverse events were recorded. Radiological fracture healing at week 12 was achieved in five of seven cases (71.4%), which was greater than the threshold (18.1%) predefined by the historical outcome of the standard of care. The interval between cell transplantation and union, the secondary endpoint, was 12.6 ± 5.4 weeks (range, 8-24 weeks) for clinical healing and 16.1 ± 10.2 weeks (range, 8-36 weeks) for radiological healing. Neither deaths nor life-threatening adverse events were observed during the 1-year follow-up after the cell therapy. These results suggest feasibility, safety, and potential effectiveness of CD34(+) cell therapy in patients with nonunion.

Dissecting Stem Cell Proliferation and Differentiation In Association With The Central Cell Cycle Regulator APC/CCdh1In Vitro and In Vivo

IntroductionHematopoietic stem cells (HSCs) and multipotent progenitor cells continuously maintain hematopoiesis by self-renewal and differentiation. The stem cell fate is tightly connected with the cell cycle, where the major regulator anaphase-promoting complex or cyclosome (APC/C) with its co-activators Cdc20 and Cdh1 marks cell cycle regulatory proteins, such as cyclin A and B, for proteasomal degradation and thus controls their activity. Known targets of Cdh1 are involved in regulation of self-renewal and granulopoiesis. This raises the hypothesis that Cdh1 may be a critical mediator of HSC proliferation, self-renewal and differentiation. MethodsCD34+ cells were collected from peripheral blood (PB) of G-CSF mobilized donors and cultured in the presence of different cytokines. To analyze cell division and self-renewal versus differentiation, CFSE staining was used in combination with CD34 detection. The knockdown (kd) of Cdh1 was achieved by lentiviral delivery of specific shRNA into target cells. ResultsIn vitro cultivation of CD34+ cells under conditions resulting in either self-renewal (SCF, TPO, Flt3-l) or differentiation/granulopoiesis (SCF, G-CSF) showed impressive downregulation of Cdh1 during culture. A high Cdh1 expression in CD34+ cells and lower expression in myeloid cells (CD41a+, CD15+, Glycophorin A+) reflects the situation we found in vivo in bone marrow (BM) subsets. Western blotting also revealed inactivation of Cdh1 by its specific inhibitor Emi1 which stabilized the ubiquitin ligase Skp2 and promoted cell cycle entry and proliferation by degrading the Cdk inhibitor p27. In addition, the APC/CCdh1 target cyclin B was upregulated. These data indicate that initial Cdh1 downregulation is required to promote cell cycle entry and proliferation of CD34+ HSCs under conditions mediating both self-renewal as well as differentiation. When cultured under self-renewal conditions, CD34+ cells showed diminished proliferation with cells residing in lower generations, whereas during granulopoiesis, cells accumulated within higher generations. These experiments also revealed a more rapid decrease of CD34+ cells in granulopoiesis after three cell divisions in contrast to a moderate decline under self-renewal conditions. We also found a decreased colony-forming ability in cells divided more than twice during granulopoiesis, which correlates with their lower CD34 expression. This is consistent with more symmetric divisions into CD34+ daughter cells under self-renewal conditions and gradual commitment during granulopoiesis. Our current experiments extent these analyses to immunofluorescence of Numb distribution in individual cells to elucidate the impact of Cdh1 on symmetric/asymmetric cell division. We could already show that Cdh1-kd led to expansion of CD34+ HSCs in vitro. To further validate our results in vivo, we have established a NOD/SCID/IL-2Rγ chain-/- (NSG) xenotransplant mouse model. Human CD34+ cells depleted of Cdh1 engrafted to a much higher degree in the murine BM 8 and 12 weeks after injection as shown by higher frequencies of engrafted human CD45+ cells. Moreover, we also found an increased frequency of human CD19+ B cells after transplantation of CD34+ Cdh1-kd cells. Further analyses of the contributing subsets to the pool of CD45+ human cells are ongoing. These results suggest an enhanced in vivo repopulation capacity of human CD34+ HSCs in NSG mice when Cdh1 is depleted. ConclusionsAPC/CCdh1 mediates cell cycle entry and proliferation during self-renewal and differentiation in CD34+ HSCs in vitro and improves engraftment capacity in vivo. Disclosures:No relevant conflicts of interest to declare.

Magnetic targeting of human peripheral blood CD133+ cells for skeletal muscle regeneration.

Skeletal muscle injuries often leave lasting functional damage or pain. Muscle injuries are routinely treated conservatively, but the most effective treatment to promote the repair of injured muscles has not yet been established. Our previous report demonstrated that human peripheral blood-derived CD133(+) cell transplantation to rat skeletal muscle injury models inhibited fibrosis and enhanced myogenesis after injury. However, the acquisition of a sufficient number of cells remains the limitation for clinical application, as the CD133(+) population is rare in human blood. In this study, we applied a magnetic cell targeting system to accumulate transplanted cells in the muscle injury site and to enhance the regenerative effects of CD133(+) cell transplantation, focusing on the fact that CD133(+) cells are labeled with a magnetic bead for isolation. For the magnetic cell targeting, the magnet field generator was set up to adjust the peak of the magnetic gradient to the injury site of the tibialis anterior muscle, and 1×10(4) human peripheral blood CD133(+) cells were locally injected into the injury site. This cell number is 10% of that used in the previous study. In another group, the same number of CD133(+) cells was injected without magnetic force. The CD133(+) cells transplanted with the magnetic force were more accumulated in the muscle injury site compared with the CD133(+) cells transplanted without the magnetic force. In addition, the transplantation of CD133(+) cells under the magnetic control inhibited fibrous scar formation and promoted angiogenesis and myogenesis, and also upregulated the mRNA expression of myogenic transcription factors, including Pax7, MyoD1 and Myogenin. However, the transplantation of CD133(+) cells without the magnetic force failed to demonstrate these effects. Thus, our magnetic cell targeting system enables transplantation of a limited number of CD133(+) cells to promote the repair of skeletal muscle injury.

Cytometry in stem cell research and therapy

recent Nobel Prize in medicine was awarded to two stemcell researchers, John Gurdon and Shinya Yamanaka, for theirachievements in stem cell research and reprogramming ofsomatic cells. Flow cytometry is by nature the ideal tool toidentify, characterize, and isolate stem and progenitor cells forresearch and potential clinical use (1). The major strength offlow cytometry is its ability to rapidly perform highlymultiplexed quantitative measurements on single cells withina heterogeneous cell population. However, when the cell typeof interest is extremely rare, as most stem and progenitor cellsare, several sources of artifact must be addressed. The impor-tance of flow cytometry as a driving force for stem cellresearch was demonstrated in a focused issue of the journalexactly three years ago (1). This current focus issue of Cytome-try A is devoted to the topic of stem cells due to numerouscurrent innovations and discoveries.Applications of stem cells include several disciplines,from embryogenesis, adult tissue maintenance, and repair,and more recently, cancer as well as for toxicity screening anddisease modeling. All of these topics are represented in thisissue, with special emphasis on the role of analytic and pre-parative flow cytometry in the elucidation of stem cell pheno-type and function, and best laboratory practices as they applyto flow cytometry. Image and flow cytometry together withcell sorting have revolutionized the study of stem cell biologyand the implications of these cells and their progeny indevelopmental biology, tissue engineering, and cellulartherapy. The number of parameters and the speed of theirsimultaneous measurements in single cells has continued toincrease with advances in hardware, reagents, and analyticalsoftware (2).

Long-term effects of stem cell transplantation in heart failure

Background: We investigated long-term effects of intracoronary transplantation of CD34+ cells in patients with dilated cardiomyopathy (DCM). Methods: Of 110 DCM patients, 55 were randomized to CD34+ cell transplantation (SC) group, and 55 patients received no cell therapy (controls). In the SC group, peripheral CD34+cells were mobilized by G-CSF and collected via apheresis. Patients underwent myocardial scintigraphy and CD34+ cells were injected in the artery supplying the segments with reduced viability. Patients were followed for 5 years. Results: At baseline, the 2 groups did not differ in age, gender, left ventricular ejection fraction (LVEF), or NT-proBNP levels. At 5 years, stem cell therapy was associated with an increase in LVEF (from 24.3 ± 6.5 % to 30.0 ± 5.1 %; P = 0.02), an increase in 6-minute walk distance (from 344 ± 90 m to 477 ± 130 m; P < 0.001), and a decrease in NT-proBNP (from 2322 ± 1234 pg/mL to 1011 ± 893 pg/mL; P < 0.01). During followup, 27 (25 %) patients died and 9 (8 %) underwent heart transplantation. Of the 27 deaths, 13 were attributed to pump failure, and 14 to sudden cardiac death. Total mortality was lower in SC group (8/55 [14 %]) than in controls (19/55 [35 %]) (P = 0.01). The same was true of pump failure (3/55 [5 %] vs. 10/55 [18 %], P = 0.03), but not of sudden cardiac death (5/55 [9 %] vs. 9/55 [16 %], P = 0.39). SC therapy was an independent predictor of outcome on multivariable analysis (P = 0.04). Conclusions: Intracoronary stem cell transplantation may be associated with improved ventricular remodeling, exercise tolerance, and longterm survival in patients with DCM.

CD99 Identifies Disease Stem Cells in Acute Myeloid Leukemia and the Myelodysplastic Syndromes

AbstractAbstract 210The myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) are initiated and sustained by self-renewing stem cells. Our data indicate that HSCs (Lin-CD34+CD38-CD90+CD45RA-) from MDS patients exhibit widespread gene dysregulation (3,258 differentially expressed genes, FDR<0.1), including 25 cell surface protein transcripts. As these proteins can be easily measured by flow cytometry, we hypothesized that they may represent promising biomarkers.We used flow cytometry to assess the expression of 25 candidate disease markers on HSCs in bone marrow (BM) samples from patients with therapy related MDS (n=26). Nine surface markers were upregulated on MDS HSCs as compared with cord blood HSCs in at least 50% of samples. The most frequently upregulated surface marker was CD99 (85%). CD99 was also upregulated on HSCs in 27/37 (73%) de novo MDS cases. To determine whether CD99 upregulation was specific to MDS, we analyzed 78 paired diagnosis/relapse AML specimens encompassing all non-M3 subtypes of AML and a variety of cytogenetic subgroups. CD99 was found to be frequently overexpressed both at diagnosis (81%) and at relapse (83%). Notably, the mean intensity of CD99 expression was significantly higher at relapse (p=0.007). This suggests that CD99 is selected for during relapse and may represent a high risk AML marker. To investigate whether CD99 is upregulated in other myeloid disorders, we measured CD99 in CML samples (n=12). CD99 was decreased in chronic phase CML HSCs (7/10) but increased in blast crisis CML HSCs (2/2), suggesting that it may be a marker of leukemic transformation. Nevertheless, its overexpression in low-risk MDS suggests a distinct role in MDS biology.In CD34+ AMLs, CD99 expression was significantly higher in the stem cell enriched CD34+CD38- fraction compared with bulk blasts (p=0.003). In most samples (91%), CD99 high cells within the CD34+CD38- fraction exhibited a CD90-CD45RA+ “LMPP-like” immunophenotype, previously shown to be enriched for leukemia-initiating cell activity, whereas CD99 low cells exhibited a CD45RA- phenotype resembling normal HSCs and MPPs. Thus, CD99 may be preferentially expressed at higher levels on leukemic stem cells (LSC). We therefore used CD99 expression levels to prospectively purify potential LSCs (high CD99) from residual normal HSCs (low CD99) by sorting CD34+CD38- cells from three primary AML specimens into complete methylcellulose. CD99 low cells generated more myeloid colonies than CD99 high cells (mean 33 and 12, respectively, p=0.02). PCR-based assays revealed that molecular abnormalities associated with the prospectively sorted leukemias were present in CD99 high derived colonies, but absent in CD99 low derived colonies (two Flt3-ITD+ and one AML1-ETO+ samples tested). Transplantation of CD34+CD38-CD99 low cells from a Flt3-ITD+ AML sample into sublethally irradiated NOD/SCID/IL2-R null mice (NSG) (n=2) led to lympho-myeloid engraftment at 12 weeks (2/2 transplanted mice), with Flt3-ITD absent in the engrafted cells. Together, these data indicate that CD99 expression can distinguish LSCs from residual normal HSCs.To study the function of CD99 in AML, we measured CD99 expression on 14 AML cell lines, identifying 12 with high levels, including HL60 and MOLM13. Using a lentiviral vector to introduce a CD99 shRNA into HL60 cells, we achieved an 8.2-fold reduction in CD99 surface expression. Cells expressing the CD99 shRNA exhibited decreased growth in vitro compared with vector control (p=0.05). When these cells were transplanted into NSG mice, animals transplanted with CD99 shRNA expressing cells demonstrated a significant improvement in survival compared with vector controls (median OS 26d vs. 22d, p=0.04, log-rank). Conversely, NSG mice transplanted with MOLM13 cells overexpressing CD99 (4.7-fold increased surface expression) demonstrated a significantly impaired survival as compared with vector controls (median OS 11d vs. 28d, p=0.05, log-rank).In summary, our results establish CD99 as a cell surface protein that is highly expressed in MDS HSCs, AML blasts, and LSCs. Using CD99 expression to prospectively isolate residual normal HSCs from LSCs may provide a useful tool for the performance of purged autologous stem cell transplants. Furthermore, CD99 expression appears to promote leukemic cell growth, particularly in vivo. Further studies are needed to fully establish the role of CD99 in the biology of MDS and AML. Disclosures:No relevant conflicts of interest to declare.

Cryopreservation of allogeneic PBSC from related and unrelated donors is associated with delayed platelet engraftment but has no impact on survival

Cryopreservation of PBSC for allo-SCT offers potential advantages; however, its impact on engraftment and outcomes remains unclear. A total of 76 allo-SCT performed using cryopreserved PBSC from HLA identical related (n=57) and unrelated donors (n=19) were compared with 123 fresh PBSC allo-SCT. Median neutrophil engraftment was on day 12 for both cryopreserved and fresh PBSC; in multivariate analysis, there was a slight but significant delay in neutrophil engraftment after the median date (hazard ratio (HR)=1.44, P=0.003). Platelet engraftment was significantly delayed in cryopreserved PBSC recipients (median time 19 vs 14 days). In multivariate analysis cryopreservation (HR=1.85, P<0.001), earlier date of transplant and lower CD34+ cell dose were associated with delayed platelet engraftment. Two-year OS and relapse and 1-year TRM rates did not differ significantly. Acute GVHD incidence was comparable, and extensive chronic GVHD at 1 year was higher in cryopreserved PBSC recipients (40.3 vs. 28.3%), but not significantly so (P=0.13). Cryopreservation of related and unrelated donor allogeneic PBSC is safe and effective where its benefits outweigh the risks of delayed platelet engraftment; its impact on chronic GVHD incidence requires further assessment.

The functional duality of HoxB4 in hematopoietic reconstituting cells

Transplantation of CD34⁺ hematopoietic reconstituting cells (HRC) is an important treatment modality. The cells needed for clinical hematopoietic reconstitution after myeloablation most commonly derive from bone marrow which have been mobilized into the peripheral blood. The number of CD34⁺ cells in mobilized blood samples is used to indicate the appropriateness of transplantation although it does not evaluate the two necessary functions for successful transplantation: long-term reconstitution mediated by cells with self-renewing proliferation and short-term hematopoietic differentiation mediated by progenitor cells. Using a novel high-resolution immunophenotyping technology on a flow cytometric platform, we have assessed uniformly mobilized CD34⁺ cells for expression levels of 16 molecules previously associated with HRC function and sought correlations of these expression data with functional short-term assays for colony formation that do predict successful transplantation. We found that colony-forming units were significantly correlated with HoxB4 expression, which was explained by the number of CD34⁺ cells in the samples. However, analysis of colony-forming units normalized to the CD34⁺ cell count revealed a significant negative correlation with HoxB4 expression. Thus, increased levels of HoxB4 enhance CD34⁺ cell number via self-renewing expansion but concomitantly depreciate the capacity for short-term differentiation per cell. Our findings demonstrate the translation of experimental findings into a clinical setting and suggest that the expression level of HoxB4 in CD34⁺ cells can be used as a measure of a sample's appropriateness for transplantation.

Functional Characterization of TPO‐Expanded CD34 + Cord Blood Cells Identifies CD34 − CD61 − Cells as Platelet‐Producing Cells Early After Transplantation in NOD/SCID Mice and rCD34 + Cells as CAFC Colony‐Forming Cells

Transplantation of thrombopoietin (TPO)-expanded cord blood CD34(+) cells accelerates human platelet recovery in NOD/SCID mice. It is unknown which subpopulations of the TPO-expanded cells mediate accelerated platelet recovery and bone marrow (BM) engraftment. In this study, the contribution of these subpopulations to human platelet appearance in the blood and BM engraftment was studied in NOD/SCID mice. Following transplantation of CD34(-) /CD61(-)/lineage(-) cells (Lin(-)), human platelets were detected in the blood of recipient mice from day 4. Both time to platelet recovery and blood platelet counts at 6 weeks after transplantation showed Lin(-) dose dependence. The Lin(-) population was virtually negative for lineage marker expression and lacked CD42b expression but was heterogeneous with regard to CD36 and CD38 expression, reflecting a population in transit but not fully committed toward the megakaryocyte (MK) lineage. Although no definitive phenotype could be established of the cells generating prompt platelet production and cells generating platelets 6 weeks after transplantation, this relatively heterogeneous Lin(-) population is prerequisite to accelerate platelet recovery in vivo. The interval to platelet recovery after transplantation of the CD34(+) cells remaining after expansion (rCD34(+)) was similar to mice transplanted with nonexpanded CD34(+) cells, although the total platelet counts and the engraftment levels in the BM were lower. Cobblestone area-forming cell colony-forming cells resided mostly in the rCD34(+) population. The pro-MK CD61(+) cells did not contribute to human platelet recovery or engraftment in the BM. Our study shows that not all expanded cells appear critical for transplantation. These data support that functional characterization of the expanded cell populations is warranted to make future expansion protocols suitable for clinical application.

Five-year follow-up of the local autologous transplantation of CD133+ enriched bone marrow cells in patients with myocardial infarction.

The implantation of a CD133+ bone marrow cell population into an ischemic myocardium has emerged as a promising therapeutic modality for myocardial regeneration and restoration of ventricular contractility. While previous studies have documented the short-term safety and efficacy of CD133+ cell transplantation in patients with acute myocardial infarction, there are few reports of long-term follow-up results. Here, we present the results of long-term follow-up of our acute myocardial infarction patients who were treated with intramyocardial injection of CD133+ cells after coronary bypass graft. After five years, 13 patients in the cell transplantation group and 5 patients in the control group underwent safety and efficacy investigations by New York Heart Association classification and two-dimensional echocardiography (2D echo). During the five-year study period, no major cardiac adverse events were reported among patients who received CD133+ stem cells. Regarding efficiency, we observed no statistically significant treatment effects for the echocardiographic parameters [left ventricular end-diastolic and end-systolic volumes, and resting ejection fraction] measured during the follow-up period. However, detailed analysis of regional wall motion revealed an improvement in the Wall Motion Score Index from baseline to the six month follow-up, which was maintained during the follow-up period. Taken together, the long-term results of the present study indicate that transplantation of CD133+ is a safe and feasible procedure; however, we could not show any major benefits in our patients. Thus, this issue needs to be addressed by conducting other studies with more patients.

Efficacy and long-term evaluation of intramyocardial injection of autologous CD34-enriched PBMSC in old myocardial infarction

Aims: We have shown that autologous transplant of CD34+-enriched peripheral-blood mononuclear cells (PBMSC) could restore depressed myocardial function, and sustain adequate myocardial function 12 months after surgery in patients with old (>one year-old) myocardial infarction. Our aim is to report the long-term morbidity and mortality efficacy of this procedure. Methods and results: Seventy patients with an old anteroseptal myocardial infarction were followed for 2 to 7 years, 35 had a revascularization procedure and received an intra-myocardial injection of autologous CD34+-enriched PBMSC (8 × 108 mononuclear cells/ml including 3 × 107 CD34+ cells/ml)(Group A). Group B patients only had the revascularization. Abnormal pre-surgical values of LVEF (33.2% ± 4.8%), LVDV (178 ± 13.7 ml), LVSV (120 ± 16 m), LVDD (58.9 ± 3.84 mm), E and A waves without contractility in infarction area in group A patients improved to approximate normal values (50% ± 3% for LVEF; 90 ± 9.3 ml for LVDV; 80 ± 9.9 ml for LVSV; 55.3 ± 3 mm for LVDD; 5.2 ± 0.5 cm/s for E wave and 4.18 ± 0.3 cm/s for A wave) 1 year after the procedure and have remained unaltered for all the follow-up period. All the patients remain alive. Only seven patients have been readmitted to the hospital for non-myocardial related events. Group B only 11 patients continued alive to 5 years after surgery and LEVF never increased more than 6%, all of them with many hospitalizations (n ≥ 10) by heart failure events. Conclusion: Intramyocardial injection of CD34+ highly enriched PBSC represent an encouraging alternative for patients with severely scarred and dysfunctional myocardium.

Abstract P019: Exosomes from Human CD34+ Cells: Critical Mediators of Proangiogenic Paracrine Effect of EPCs

Local transplantation of human CD34+ hematopoietic stem cells has been shown to promote neovascularization in pre-clinical studies in models of myocardial and limb ischemia. In early phase clinical trials, transplantation of CD34+ cells has been associated with reduced angina, improved exercise time and reduced amputation rates. Several studies have suggested that paracrine effects by these pro-angiogenic cells mediate the effects induced by cell transplantation. We hypothesized that CD34+ cells secrete exosomes (Exo), which mediate at least a part of the therapeutic function of the cells. Methods and Results: We isolated Exo from the conditioned media of adult human peripheral blood (PB) CD34+ cells. The angiogenic and therapeutic potency of CD34+ Exo was compared with the intact CD34+ cells and also with PB mononuclear cell (MNC) Exo. Exo from both CD34+ cells and MNC are 50–90nm in size, have cup shaped morphology, and carry known Exo-marker proteins such as CD63, TSG101 and Annexin V as shown by electron microscopy, Western blot and flow cytometry. Compared to CD34+ cells or MNC Exo, CD34+ Exo significantly induces in vitro angiogenic activities such as viability, proliferation and tube formation of HUVECs on matrigel- in a dose dependent manner. In vivo, CD34+ Exo stimulated significant neovascularization in mouse corneal angiogenesis assay (14±4 mm v MNC Exo, 4±1 mm, p&lt;0.01) and incorporation of endothelial (CD31+) cells in mouse matrigel-plug assay (6±1.7% v CD34+ cells, 2±0.8%, p&lt;0.01). Finally, in a mouse model of hind limb ischemia (HLI), CD34+ Exo significantly improved perfusion (ratio: 1.01±0.04 v 0.57±0.1, P&lt;0.05), increased capillary density (1.8±0.3/HPF v 0.9±0.1/HPF, p&lt;0.001) and prevented ischemic leg amputation (16% v 100%), as compared with MNC Exo. Conclusions: These data demonstrate that CD34+ Exo induce angiogenic activity and ischemic tissue repair in the absence of CD34+ cells, and suggest that Exo represent important mediators of the therapeutic effects associated with CD34+ cell therapy. We speculate that Exo derived from CD34+ cells may represent a significant component of the paracrine effect of progenitor-cell transplantation for therapeutic angiogenesis.

Kinetics and Apoptotic Profile of Circulating Endothelial Cells In Patients Undergoing Autologous Stem Cell Transplantation

Abstract 2961 Objectives:Tumor growth and progression are influenced by endothelial cells proliferation and differentiation. In patients with hematological malignancies circulating endothelial cells (CEC) are elevated at the diagnosis and decrease after successful chemotherapy. The other spectrum of interest is evaluation of CEC in the context of their influence on regeneration of hematopoiesis from damage caused by high dose chemotherapy and stem cells transplantation. The aim of our study was to assess the kinetics of CEC and their subsets (mature, progenitor and apoptotic CEC) in patients undergoing autologous hematopoietic stem cells transplantation. Additionally we analyzed the relationship between the number of CEC subsets and clinical course in the post-transplant period and time to engraftment. Methods: CEC were measured in peripheral blood of 39 patients (18 males and 21females, median age 54; range 26–69) scheduled for high dose chemotherapy with autologous stem cell transplantation. The group consisted of 29 multiple myeloma patients and 10 lymphoma patients. All of the patients were in best response to previous treatment before transplantation, 20 in CR and 19 in PR. Conditioning treatment in patients with myeloma was Melphalan 200 mg/m2, while in lymphoma patients BEAM. Evaluation of CEC: Blood samples were collected at different time points: 1) at baseline - directly before conditioning; 2) a day after finishing of chemotherapy; 3) at the day “0” just before transplantation of CD34+ HSC and 4) four times after transplantation :1hour and 1 day after HSC transfusion, at the time of the nadir in peripheral blood and at the day of neutrophil engraftment). CEC were evaluated by 4 colour flow-cytometry according to the procedure described previously. Circulating progenitor cells (CEPC) were defined as negative for hematopoietic marker CD45 and positive for the endothelial cell markers CD 34, CD31 and the endothelial progenitor marker CD133. CD105 and CD106 positive CEC were classified as activated CEC (aCEC). Expression of CD36 characterized CEC originating from microvessels. Apoptotic CEC (apoCEC) were defined as CD146 positive and Annexin V positive. Results: The median (Me) CEC number at the baseline was 9, 5/μL and dropped significantly 24 h after chemotherapy (Me 8, 3/μL; p=0, 002). At day “0” before infusion of HSC, the count of CEC continued to decrease (Me 6, 6/ μL, p<0, 0001 when compared to baseline) and remained stable shortly after transplantation (1h and 1day after transplantation Me 6, 2/μL p=0, 0001 and 6, 8/μL, p=0, 0002 respectively). At nadir of WBC count, the nadir of CEC (Me 4, 6/μL) was also observed. CEC number increased at the engraftment period, but did not reach pretreatment values (Me 5, 3/μL, p0, 0001). The number of aCEC and CEPC followed CEC kinetics. In contrast, the count of apoptotic CEC 24h after conditioning (Me 4, 15/μL) was significantly enhanced in comparison to baseline values (Me 3, 1/μL; p=0, 0005) and drop down 1 day after transplantation to pretreatment values (Me 3, 6/μL; p=0, 4), and then continued to decrease (WBC nadir - Me 2, 1/μL, engraftment Me 1, 85 /μL).For the purpose of the study CEC and their subsets were evaluated as dichotomous variables using median value as cut-off to divide patients into low- or high- expressers groups. Patients with low (<Me) expression apoCEC at baseline had significantly shorter time for neutrophil engraftment (Me 13; range11–18 days) than high expressers: (Me15, 5; range 11–21days, p=0, 028). Additionally we observed significant correlation between the higher number of CEPC at 1 h after transplantation with shorter time to neutrophil engraftment (Spearman rank coefficient r= −0, 49, p=0, 008). Similar correlation between the increased count of endothelial cells originating from microvessels measured 1h after transplantation and shorter time to neutrophil engraftment was also observed (r= −0, 39, p=0, 04). Conclusions: Low count of apoptotic CEC at baseline correlates with shorter time to engraftment which may suggest that intact bone marrow microenviroment takes part in effective engraftment. Additionally, our results indicate that progenitor CEC and CEC derived from microvessels could support bone marrow regeneration after HSCT. The role of CEC and their subsets in the context of hematopoietic recovery after high dose chemotherapy and autoHSCT needs further evaluation. Disclosures:No relevant conflicts of interest to declare.

Rapid and Sustained Engraftment of a Single Allogeneic Ex-Vivo Expanded Cord Blood Unit (CBU) After Reduced Intensity Conditioning (RIC) in Adults. Preliminary Results of a Prospective Trial

Abstract 486CBU is a widely used source of stem cells for allogeneic transplantation (SCT). Engraftment rate and speed of a single CBU in adults remains unsatisfactory. Transplantation of 2 CBU may overcome this problem to the expense of an increased incidence of GVHD. Until now, attempts at using ex-vivo expanded CBU have been unsuccessful to promote long term engraftment of the expanded product. We report the results achieved in the first 8 Pts included in a PCT of transplantation of a single ex-vivo expanded allogeneic CBU. Eudract 2008–006665–81, Clinicaltrials.gov NCT 01034449. Methods:Adults patients with an indication for SCT and unable to tolerate MAC (age>45, comorbidities, previous high-dose therapy) were included after inform consent if no Id sibling, no MUD 9 (C or DQ mismatch accepted) to 10/10 HLA matches and no CBU fulfilling the HLA matching (≥ 4/6) and richness (≥ 3 to 4 × 107 TNC/kg before thawing) criteria were available. RIC consisted of Flu (40 mg/m2/d × 5d), Cyclophosphamide (50 mg/kg × 1d) ICT 2 Gy. GVHD prevention consisted of MMF (d-3 to d28) and CSA from d-3. Graft engineering: 1 CBU with > 2 TNC/kg < 3 and 4 to 6 HLA compatibilities was thawed, CD34+ cells were selected through magnetic device (Miltenyi) and submitted to ex-vivo expansion in SF medium ( HPO1-Macopharma) supplemented with SCF, Flt3l, G-CSF and TPO during 12 days, starting d-12 of the transplantation (Ivanovic, Cell Transplant 2011) CD34 neg cells were cryopreserved. On d0, expanded cells were washed and resuspended in HSA 4% and upon viability and sterility were injected to the pt. Cd34 neg cells were thawed and injected to the pt 3 h later. Results:From 03/2010 to 06./2011 8 pts have been included, med age 55y.o. (26–64) with AL: 3, Hodgkin’s: 2, MDS: 3. Pts had received 1 to 3 lines of Tx (med:2). For 1 pt the expanded product was contaminated and this pt then received a back-up unmanipulated CBU. He engrafted correctly and is AW at 14 m with full donor chimerism. For the 7 other pts, the ex-vivo median fold expansion of CD34+ cells and TNC was 39 (29–75) and 390 (127–526) respectively, leading to a graft that contained 1.3 to 13 × 106 CD34+ cells/kg (med: 2 × 106/kg). The CD34 neg counterpart contained 3 × 106 CD3+/kg (1–5) and 0,9 × 106 CD19+ cells/kg (0,3–1,5). At d42, 6/7 pts who received the expanded graft engrafted with ≥99% donors cells. A 2d RIC was performed followed by a double CBU transplant in the patient who did not engraft. That 2d graft again failed to engraft. However the patient remains alive at 9m. For the 6 pts who engrafted with the expanded product the time to reach 500, 1000 PMN’s and 20 000 plts/mm3 was 7d (6–19), 8d (6–21) and 24d (0–39) respectively. The chimerism on WBC and CD3+cells (evaluated on d 15, 42, 60, 100, 180, 365) remains full donor up to 1 year + after transplant (1y+: 2 pts; 6m+: 1pt; 180d+: 1 pt; 60d+: 1pt) or to relapse (at 1y) in the one pt who relapsed. Five pts experienced an AGVHd (grade III-IV: 1 pt). With a median FU of 10m (2 to 18m) 7 pts are alive, 6 wo disease. One pt died 1y after transplant from relapse. Conclusion:Ex-vivo expansion of a single CBU is feasible and reproducible. Transplantation of the expanded product together with the CD34 neg counterpart of the same CBU produces rapid, complete and sustained donor engraftment after RIC in adults. Disclosures:No relevant conflicts of interest to declare.

Immune mechanisms underlying the beneficial effects of autologous hematopoietic stem cell transplantation in multiple sclerosis.

A recent phase I/II clinical trial drew serious attention to the therapeutic potential of autologous hematopoietic stem cell transplantation (AHSCT) in multiple sclerosis. However, questions were raised as to whether these beneficial effects should be attributed to the newly reconstituted immune system per se, or to the lymphoablative conditioning regimen-induced immunosuppression, given that T-cell depleting combinational drug therapies were used in the study. We discuss here the possibility that both AHSCT and T-cell depleting therapies may re-program alternatively the immune system, and why transplantation of CD34+ hematopoietic stem cells may offer AHSCT a possible advantage regarding long-term remission.

Reduced intensity conditioning with combined haploidentical and cord blood transplantation results in rapid engrafment and durable remissions.

6525 Background: Transplantation of CD34+ stem cells from a haploidentical family member plus a cord blood (UCB) unit is a promising approach for patients (pts) who lack HLA donors that facilitates early engraftment while preserving long-term cord blood reconstitution. Methods: We prospectively investigated this approach in combination with reduced intensity conditioning consisting of fludarabine, melphalan and ATG in 39 patients with high-risk hematological malignancies from January 29, 2007 to November 12, 2010. Median age was 48 yrs (20-69); 24 (62%) had active disease at transplant. 12 pts belonged to ethnic minorities. Results: The median haploidentical donor CD34+ dose was 3.08 x 106/kg (1.0-11); CD3+ cells were 0.8 x 104/kg (0.03-2.4). Median UCB nucleated cell dose was 1.55 x 107/kg (range, 0.86-5.23); CD34+ cells were 0.06 x 106/kg (0.02-0.75). 37 (95%) pts had neutrophil recovery with a median time to ANC >500/mL of 11 days (9-43). 27 (69%) pts became platelet transfusion independent at median time of 19.5 days (10-132). In the majority of pts, early haploidentical engraftment was replaced by durable engraftment of UCB by 100 days. However, 5 pts had persistent haploidentical only chimerism, while 4 pts had delayed engraftment with only UCB. Acute GVHD grade II-IV occurred in 10 pts (26%) with only 2 Grade III GVHD (5%). Day 100 mortality rate was 21%. With a median follow up for survivors of 298 days (89-1133), 19 pts (49%) are currently alive. 8 pts died from disease relapse, 4 from infection, and 3 from graft failure. For the entire cohort 1 yr OS was 47% and PFS 38%. For pts in CR at SCT 1 yr PFS was 63% and OS 70%. For pts with active disease at SCT 1 yr OS and PFS were 37%. Conclusions: Combined haploidentical donor and UCB transplantation in the large majority of patients results in rapid neutrophil engraftment due to the haplo-identical progenitor cells. Durable engraftment is provided by the UCB graft with a low rate of GVHD and excellent long term disease control, even in patients with active disease at transplant.

Effectiveness and cost analysis of “just‐in‐time” salvage plerixafor administration in autologous transplant patients with poor stem cell mobilization kinetics

Plerixafor is a recently Food and Drug Administration (FDA)-approved CXCR4 antagonist, which is combined with granulocyte-colony-stimulating factor (G-CSF) to facilitate stem cell mobilization of lymphoma and myeloma patients. To evaluate the effectiveness and the related costs of a "just-in-time" strategy of plerixafor administration, we performed a retrospective cohort study comparing 148 consecutive lymphoma and myeloma patients in whom mobilization was attempted during 2008 before the Food and Drug Administration (FDA) approval of plerixafor with 188 consecutive patients mobilized during 2009 after FDA approval. Plerixafor was administered to 64 of 188 patients considered to be at risk for mobilization failure due to either their medical history ("high risk," n = 23) or the occurrence of peripheral blood CD34+ count of fewer than 15 × 10(6) cells/L with a white blood cell count of greater than 10 × 10(9) cells/L after at least 5 days of G-CSF administration (just-in-time, n = 41). The success rates of collecting a minimum transplant CD34+ cell dose (≥2 × 10(6) cells/kg) or target cell dose (≥5 × 10(6) lymphoma or ≥10 × 10(6) CD34+ cells/kg myeloma) in the just-in-time patients compared favorably with the 36 poor mobilizers collected with G-CSF alone: 93% versus 72% and 42% versus 22%, respectively. The use of plerixafor in selected high-risk patients and poor mobilizers did not increase the total charges associated with stem cell collection when compared with poor mobilizers treated with G-CSF alone. The targeted use of plerixafor increased the overall success rate of mobilizing a minimum number of CD34+ cells from 93% to 98% in patients with hematologic malignancies scheduled for autotransplant and increased the overall charges associated with stem cell collection in all patients by an average of 17%.

Development of human CD4+FoxP3+ regulatory T cells in human stem cell factor–, granulocyte-macrophage colony-stimulating factor–, and interleukin-3–expressing NOD-SCID IL2Rγnull humanized mice

AbstractHuman hematolymphoid mice have become valuable tools for the study of human hematopoiesis and uniquely human pathogens in vivo. Recent improvements in xenorecipient strains allow for long-term reconstitution with a human immune system. However, certain hematopoietic lineages, for example, the myeloid lineage, are underrepresented, possibly because of the limited cross-reactivity of murine and human cytokines. Therefore, we created a nonobese diabetic/severe combined immunodeficiency/interleukin-2 receptor-γ–null (NOD-SCID IL2Rγnull) mouse strain that expressed human stem cell factor, granulocyte-macrophage colony-stimulating factor, and interleukin-3, termed NSG-SGM3. Transplantation of CD34+ human hematopoietic stem cells into NSG-SGM3 mice led to robust human hematopoietic reconstitution in blood, spleen, bone marrow, and liver. Human myeloid cell frequencies, specifically, myeloid dendritic cells, were elevated in the bone marrow of humanized NSG-SGM3 mice compared with nontransgenic NSG recipients. Most significant, however, was the increase in the CD4+FoxP3+ regulatory T-cell population in all compartments analyzed. These CD4+FoxP3+ regulatory T cells were functional, as evidenced by their ability to suppress T-cell proliferation. In conclusion, humanized NSG-SGM3 mice might serve as a useful model to study human regulatory T-cell development in vivo, but this unexpected lineage skewing also highlights the importance of adequate spatiotemporal expression of human cytokines for future xenorecipient strain development.

Chronic Granulomatous Disease: Lessons from a Rare Disorder

Chronic granulomatous disease (CGD) is a rare primary immunodeficiency with X-linked or autosomal recessive inheritance involving defects in genes encoding phox proteins, which are the subunits of the phagocyte NADPH oxidase. This results in failure to produce superoxide anion and downstream antimicrobial oxidant metabolites and to activate antimicrobial proteases. Affected patients are susceptible to severe, life-threatening bacterial and fungal infections and excessive inflammation characterized by granulomatous enteritis resembling Crohn's disease and genitourinary obstruction. Early diagnosis of CGD and rapid treatment of infections are critical. Prophylaxis with antibacterial and mold-active antifungal agents and the administration of interferon-γ has significantly improved the natural history of CGD. Currently, the only cure is allogeneic hematopoietic cell transplant (HCT), although there remains controversy as to which patients with CGD should get a transplant. Allele-based HLA typing of alternative donors, improved supportive care measures, and use of reduced toxicity conditioning have resulted in event-free survival (EFS) of at least 80% even with an unrelated donor and even better when the patient has no active infections/inflammation. Gene correction of CGD would eliminate the risks of graft-versus-host disease (GVHD) and the immunoablative chemotherapy required for allogeneic HCT. Based on gene therapy trials in patients with SCID-X1, ADA-SCID, and the early experience with CGD, it is clear that at least some degree of myeloablation will be necessary for CGD as there is no inherent selective growth advantage for gene-corrected cells. Current efforts for gene therapy focus on use of lentivector constructs, which are thought to be safer from the standpoint of insertional mutagenesis and more efficient in transducing hematopoietic stem cells (HSCs).

Prevention of osteonecrosis by intravenous administration of human peripheral blood-derived CD34-positive cells in a rat osteonecrosis model

Aseptic idiopathic osteonecrosis of the femoral head is a painful disorder of the hip that can lead to collapse of the femoral head and the need for total hip replacement following joint destruction. Treatment of this disease still remains a clinical challenge. Adult human circulating CD34(+) cells have been demonstrated to contribute to vasculogenesis and osteogenesis in immunodeficient rat non-union models in vivo. We hypothesized and proved that the transplantation of CD34(+) cells could have a role for improvement of osteonecrosis by promoting vasculogenesis and osteogenesis. Vascular deprivation-induced femoral head necrosis was developed in immunodeficient rats and we then administered human G-CSF mobilized CD34(+) cells intravenously. At 4 weeks after administration, the structure of the femoral head and neck were evaluated histologically and morphometrically with haematoxylin and eosin (H&E) staining and micro-CT imaging. Microangiography was carried out for macroscopic evaluation of neovascularization, and the contribution of human cells to vasculogenesis and osteogenesis was evaluated by immunofluorescent staining with human-specific antibodies. Our treatment resulted in an obvious improvement of osteonecrosis after CD34(+) cell administration and demonstrated the differentiation potential of CD34(+) cells into endothelial cells and osteoblasts. In conclusion, this new therapeutic approach using circulating cell fraction could be a promising cell-based therapy for early-stage osteonecrosis of the hip.