Enumeration of viable CD34+ cells by flow cytometry in blood, bone marrow and cord blood: results of a study of the novel BD™ stem cell enumeration kit
Abstract Previously, we demonstrated that BMI1 acts as a stem cell maintenance factor for human stem/progenitor cells. Here, we report that BMI1 collaborates with BCR-ABL in inducing leukemogenic transformation of human cord blood (CB) CD34+ cells. BMI1 and BCR-ABL were co-expressed into CB CD34+ cells (further referred as B/B cells) using a retroviral approach and cells were transplanted into NOD-SCID mice. In two out of five mice we observed leukemia within 4 months after transplantation. Chimerism levels reached 80–90% in the bone marrow and peripheral blood and morphological analysis revealed the appearance of primitive blast-like human hematopoietic cells with features that recapitulate human lymphoid leukemia. The mice were lethargic, with splenomegaly and infiltration of leukemic cells in the spleen, liver and the bone marrow and immunophenotypical analyses revealed that the cells expressed CD34 and CD19. To further understand the mechanisms underlying the leukemic transformation we performed ex-vivo long-term cultures on bone marrow stroma. We observed that the double transduced B/B cells had a strong proliferative advantage and elevated self-renewal potential as compared to controls. Expanding cultures could be maintained for over 20 weeks and Cobblestone Area Forming Cells (CAFCs) could be harvested and replated to initiate new expanding cocultures. Stem cell frequencies were determined in Long-Term Culture-Initiating Cell (LTC-IC) assays and frequencies were enhanced over 100-fold as compared to controls. Depending on the MS5 co-culture conditions, both myeloid as well as lymphoid long-term cultures could be established, indicating that extrinsic factors might dictate the lineage fate of transformed cells. To determine the necessity of a bone marrow microenvironment, we performed stroma-free liquid cultures and observed that the B/B cells were capable of expanding over 23 weeks, BMI1 cells were able to grow for 16 weeks and, importantly, BCR-ABL cells were not able to propagate long-term in stromain-dependent cultures. Thus, these data suggest that BCR-ABL cells are still dependent on cues from the bone marrow microenvironment for long-term self-renewal, and that co-expression of the intrinsic stem cell regulator BMI1 might alleviate this necessity of BCR-ABL+ cells for a microenvironment. Experiments in which B/B-transduced cells were sorted into HSC, CMP, GMP and MEP populations indicated that long-term self-renewal and expansion could particularly be imposed on the HSC population, and much less efficiently on progenitor subpopulations. In order to study whether the B/B-leukemic stem cells could be targeted by Imatinib, we applied a short pulse of Imatinib to expanding MS5 cocultures for 7 days. While the vast majority of cells in all cultures did not survive, in the B/B-transduced group a population of immature cells remained that was capable of re-initiating proliferative cultures of self-renewing CAFCs with very high frequencies (1/96 as determined by LTC-IC assays). Finally, we asked whether retroviral introduction of BMI1 in BCR-ABL+ CD34+ cells isolated from CML patients in chronic phase that expressed low endogenous BMI1 levels would affect long-term growth and self-renewal. Upon overexpression of BMI1 we observed increased proliferation capacity of the BMI1 transduced CML cells, and cultures could be maintained for much longer periods than control-transduced cultures. In conclusion, our data indicate that BMI1 collaborates with BCR-ABL in leukemic transformation, and our human-based system should provide a useful model to study the pathology of leukemias and test new drug entities.
Abstract The regulation of cell cycle activity, differentiation and self-renewal of stem cells are dependent on accurate processing of intrinsic and extrinsic signals. Traditionally, signaling pathway activation has been detected by immunobloting using phospho-specific antibodies. However, detection of signal transduction in rare cells within heterogeneous populations, such as hematopoietic stem and progenitor cells (HSC) has been difficult to achieve. In a recently reported approach to visualize signaling in selected single c-Kit+ Sca-1+ Lin− (KSL) bone marrow cells, cells were sorted onto glas slides by flow cytometry and signaling was detected by confocal fluorescence microscopy, a very time consuming method that thus restricts the number of cells that can be analysed simultaneously. Moreover it permits only qualitative, but not quantitative signaling evaluation (Yamazaki et al., EMBO J. 2006). Here, we report a new protocol allowing quantitative measurement of signaling activity in large numbers of defined murine and human hematopoietic cells. The cells are stained with established surface markers and then phospho-specific antibodies are used to detect the levels of active intracellular signaling molecules. Signals are quantified by flow cytometry fluorescence measurement. Importantly, the protocol developed in our laboratory enables preservation of surface marker staining identifying the cells of interest inspite the fixation and permeabilization procedures necessary for intracellular signaling detection. This applies also for antigens previously reported to be particularly vulnerable to standard fixation and permeabilization approaches (e.g. the murine stem cell markers c-Kit and Sca1). Thus, our protocol provides an easy and reliable method for quantifying the activation degree of several intracellular signaling pathways on single cell level in defined hematopoietic (stem) cells within the heterogeous bone marrow (BM) compartment. Using cytokines known to exert a biological effect on HSCs, we have examined the susceptibility of KSL murine BM cells and human BM CD34+ cells to cytokine-induced signaling. We have performed extensive dosage titration and time course analysis for multiple cytokines (SCF, TPO, Flt-3, IL-3, IL-6, Ang-1, SDF-1α, TGF-β, and BMP-4) and signaling pathways (ERK, Akt, p38MAPK, Jak-Stat, TGF-β/BMP-Smad) in murine KSL BM cells. The activation intensity and the duration of signal activity as measured by the expression of corresponding phosphorylated proteins were cytokine specific. The obtained results can be used as a platform to explore signaling alterations in distinct compartments of the hematopoietic system, and may provide mechanistical insights for observed bone marrow defects (e.g impaired ERK signaling pathway has been detected as a possible cause of hematopoietic defects in Caspase-3 mutant murine HSCs, Janzen et al, Cell Stem Cell 2008). Furthermore, we could show that the technique is also applicable to human BM cells and that the human hematopoietic stem cell marker CD34 is also preserved by our fixation and permeabilization protocol. Preliminary results suggest that cytokines induce similar signaling activation in human CD34+BM cells collected from healthy donors. As observed in mouse KSL BM cells, stimulation of human CD34+cells with human stem cell factor (hSCF) induced activation of the ERK but not the Akt pathway. Ongoing experiments analyse the stimulatory effects of other cytokines such as thrombopoietin (TPO) and fms-related tyrosine kinase 3 (Flt-3) and their corresponding pathways. Moreover, comparative studies are underway analyzing cross-reactivity between mouse and human cytokines, aiming to provide insights into cytokine-induced biases in commonly used xenotransplantation models.
Abstract 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.
Abstract Background and objectives: Normal human bone marrow (BM), cord blood (CB) and mobilized peripheral blood (MPB) are the most commonly used sources for allogeneic hematopoietic stem cell transplantation (HSCT). The aim of this study was to detect the expression of CXCR4 on CD34+ cells and to assess the distribution of lymphocyte subsets in each type allograft. Methods: CD34+ cells were separated from BM (n=30), CB (n=30) and MPB (n=30) by the CD34 MultiSort Kit immunomagnetic bead system. The expression of CXCR4 on CD34+cells was assayed by double color flow cytometry. The lymphocyte subsets in each type of allograft were detected by three-color flow cytometry. The groups of monoclonal antibodies were used as the following: CXCR4-PE/CD34−Pecy5, CD8−FITC/CD4−R-PE/CD3−TC, CD45RA-FITC/CD45RO-PE/CD4−Pecy5, CD45RA-FITC/CD45RO-PE/CD8−Pecy5, and CD3−FITC/CD16+56-PE. Isotype-specific antibodies were used as controls. Results: The expression of CXCR4 of cord blood and mobilized peripheral blood CD34+ cells was lower than that of bone marrow cells (BM 40.21%±6.72%, CB 20.93%±3.96%, MPB 20.93%±3.96%, P <0.05). The difference between cord blood and mobilized peripheral blood was not significant (P>0.05). The CD3+CD8low and CD3+CD4−CD8low subsets were higher in BM than that of CB and MPB (BM 8.61%±1.40%, CB 3.31%±0.88%, MPB 5.11%±0.76%,P<0.01). The relative frequencies of the naïve CD45RA+ CD45RO− phenotype among CD4+ and CD8high T cells were highest in CB, and it was higher in MPB than in BM grafts (BM 28.09%±4.52%, 41.86 %±3.31%; CB83.83%±12.24%, 86.69%±6.12%; MPB 43.58%±4.54%, 57.64%±4.77%, P<0.01). Naïve T cells (CD45RA+ CD45RO−) were mobilized preferentially compared to memory T cells (CD45RA− CD45RO+)(P <0.01); The relative frequencies of NKT (CD3+CD16+56+) among lymphocytes were lower in CB than that in BM and MPB (CB 0.77±0.19, BM4.15±1.10, MPB 4.13±0.84, P<0.01). Conclusion: BM, CB and MPB allografts differ widely in cellular makeup of CD34+ cells and lymphocyte subsets, which are associated with the distinct characteristics after allogeneic HSCT from different allogeneic hematological sources.
Abstract Background and Objectives: Due to platelet shortage, megakaryocytes have been regarded as an effective substitute to the alleviation of frequent thrombocytopenia after stem cell transplantation. However, ex vivo expansion of megakaryoblasts and their subsequent differentiation into mature megakaryocytes for clinical applications remains a challenge. Here, we describe the development of a two-stage culture system for producing megakaryocytes from cord blood CD34+ cells. Design and Methods: Firstly, we expanded CD34+ hematopoietic progenitor/stem cells for 6 days in a serum-free culture system (IMDM basal medium with the addition of biotin, putrescine, insulin, human serum albumin, selenium, and some other nutrients) supplemented with stem cell factor (SCF), Flt-3 ligand (FL), thrombopoietin (TPO), interleukin 3 (IL-3), low density lipoprotein (LDL), StemRegenin 1 (SR1), and DMSO. CD34+ cells expansion was monitored by flow cytometric analysis of cell surface markers coupled with cell counting. Subsequently, these expanded cells were induced toward the megakaryocytic lineage for additional 7 days in the same serum-free medium as above supplemented with SCF, TPO, IL-3, IL-6, IL-11, granulocyte-macrophage colony-stimulating factor (GM-CSF), and LDL. Megakaryocytes were detected by flow cytometry using antibodies against specific cell surface markers including CD41a and CD42b. Differentiated megakaryocytes were also confirmed by morphological criteria such as cell size and DNA polyploidy. To functionally evaluate induced megakaryocytes, these cells were transplanted into sublethally irradiated NOD/SCID mice. Viability and cell being of these mice were monitored after injection. Mice of the negative control group (n=3) were injected with saline. In the experimental group (n=6), each mouse was injected with 1.0×107 cells from the second stage of culture. Results: After the first stage culture, proliferation folds of total cells and CD34+ cells were 85.65±7.03 and 62.91±4.36, respectively. The calculated yield from each CD34+ cell was between 1.0×104 to 1.5×104 CD41+ megakaryocytes with a purity of CD41+ and CD42+ cells reaching 93.7%±2.8% and 80.3%±5.8%, respectively. Differentiated cells were morphologically discernible as they were much larger than starting CD34+ cells with apparent lobular nuclei and numerous α-granules. In addition, about 32.67%±7.43% of induced megakaryocytes exhibited 4N or larger DNA content (4N 17.6%±4.12%; 8N 10.93%±2.48%; >8N 3.23%±1.34%). In mouse studies, samples collected from the negative control group contained no cells positive for human CD41a and CD42b markers. In the experimental group, human platelets were detected in mouse peripheral blood 3 days post-transplantation. At day 14 post-transplantation, the percentage of platelets derived from injected human megakaryocytes reached 13.6%±6.2%. Human megakaryocytes were also detected in mouse bone marrow 7 days post-transplantation, peaking at day 14 (~2.38% of total bone marrow megakaryocytes). Conclusions: We have established a stem cell expansion and differentiation platform that can be adapted to large-scale production of mature megakaryocytes from umbilical cord blood cells. Significantly, induced megakaryocytes are capable of engrafting in mouse bone marrow and producing platelets after transplantation into irradiated NOD/SCID mice. Therefore, our experimental platform is capable of producing a sufficient number of functional megakaryocytes for various clinical applications in the future. Disclosures Qin: Biopharmagen corp: Employment. Jiang:Biopharmagen.corp: Employment. Ren:Biopharmagen corp: Employment. Jiang:Biopharmagen.corp: Employment.
Enumeration of viable CD34+ cells in cord blood using a novel stem cell enumeration kit
Objective To assess the detection performance of the hematopoietic stem cell enumeration kit developed by BD Biosciences. Methods Cord blood samples were prepared using a hematopoietic stem cell enumeration kit developed by BD Biosciences and Stem-Kit reagents from Beckman Coulter. CD34+ cells were enumerated using a BD FACSCanto instrument and FACSDiva software. Results A total of 519 samples were analyzed in this study. The hematopoietic stem cell enumeration kit developed by BD Biosciences yielded absolute counts of CD34-positive cells that were on average 8.7% lower than Beckman Coulter Stem-Kit reagents (range: −5.7% to−14.7%). The BD Biosciences kit yielded relative counts that were on average 9.9% higher compared with Beckman Coulter Stem-Kit reagents (range: −2.1% to +13.8%). The intraclass correlation coefficients for absolute and relative counts of CD34-positive cells were 0.9967 (95% confidence interval [CI]: 0.9961–0.9972) and 0.9512 (95% CI: 0.9423–0.9587) for the BD Biosciences and Beckman Coulter kits, respectively. Conclusions The hematopoietic stem cell enumeration kit developed by BD Biosciences can be used to enumerate CD34-positive stem cells from cord blood samples.
Abstract Introduction:Micropartices (MPs) are small vesicles covered by a bilayered membrane which can serve as cell-to-cell shuttles for bioactive molecules such as lipids, growth factors, microRNAs. Umbilical Cord Blood Transplant Units (UCB) has been established as a source of Hematopoietic Stem Cells (HSC) in allogeneic transplantation. Although that UCB has unique advantages of easy procurement, low risk of transmitting infections, absence of risk to donors, immediate availability, greater tolerance of HLA disparity and lower-than-expected incidence of severe graft-versus-host disease monitoring of certain parameters on UCB transplantation remains a challenging issue. MicroRNAs (miRNAs) are short non-coding RNA chains that regulate gene expression displaying important regulatory roles in many cellular processes, including differentiation, cell replication and regeneration. We have reported herein monitoring of miRNAs containing CD34+ microparticles (MPs) as a candidate parameter of the UCBs. Methods: Cord blood units (CBUs) (n=37) were processed using the Sepax automated method (Biosafe). After processing, cryopreservation was performed using 10% DMSO in a controlled rate freezer (IceCube, Sy-lab). Adjusted variables of the CBUs included their net volume, the absolute number of CD34+ per graft and their viability as determined by flow cytometry, the number of leukocytes as recorded with a hematology analyzer and the absolute number of erythroblasts as estimated by optical microscope after Giemsa staining were assessed. Mononuclear cells were seeded in semisolid cultures in the presence of a cocktail of growth factors for colony forming unit growth. The MPs were isolated after centrifugation of the plasma and their number was determined after incubation with Annexin V (AnnV+) and CD34 antibody by flow cytometry. Electron microscope photographs were obtained, after the appropriate preparation for TEM. CD34+ specific MPs as well as CD34+ cells isolation with immunomagnetic beads has been performed through MiniMACS columns (Miltenyi). Total RNA, including miRNA, was extracted from CD34+ and MPs using the miRNeasy Micro kit and miRNeasy Serum/Plasma kit (Qiagen), respectively. Samples' concentration was assessed by Qubit fluorimeter (Thermo Fisher scientific) and cDNA synthesis was achieved using miScript II RT kit (Qiagen). Quantitative Real Time PCR was performed to assess the presence of selected miRNAs in CD34+ and MPs in five independent donor samples (miScript SYBR Green PCR kit, Qiagen). Statistical analysis was done using t-test, Pearson's and Spearman's depending on the normality of the distribution of variables. Results: The AnnV+/CD34+MPs during post-processing (51,38±37,87) decreased compared to the number of pre-processing AnnV+/CD34+MPs (57,7±51,2). On univariate analysis found that the number of AnnV+/CD34+MPs (pre-processing vs. post-processing) has statistically significant mean positive correlation (p<0,000, Spearman's rho =0,696) whereas the number of post-processing CD34 cell per unit was not statistically significant. The number of post-processing AnnV+/CD34+MPs has positive correlation to the number of BFUe pre- and post-processing (p<0,037;r=0,344 and p<0,003;r=0,474 respectively). The post-processing AnnV+/CD34+MPs has positive correlation to the number of CFU-GM pre- and post-processing (p<0,015;r=0,396 and p<0,036;r=0,347 respectively). Furthermore molecular analysis of post-processing CD34+ cells and MPs was performed for the presence of microRNA 106b, 221, 224, 517c, 518a, 519d, 520h. miRNAs 517c and 520h were detected in low levels. miRNAs 106b, 221, 224, 518a, 519d were very low or undetectable. Interestingly a different profile was observed in cell lines suggesting a line specificity of the selected miRNAs. Conclusions: Post-processing stem cell derived MPs were correlated with CFU progenitor cells (BFUe and CFU-GM). Molecular analysis confirmed the identification of line-specific miRNAs in CD34+ cells and MPs from post- processed cord blood samples. Thus miRNAs in stem cell-derived MPs could be candidate biomarkers in cord blood units. Disclosures No relevant conflicts of interest to declare.
Abstract Human umbilical cord blood has become a very valuable source for hematopoietic transplantation. Our group was able to show that CB contains non-hematopoietic stem cells, which were called unrestricted somatic stem cells (USSCs) with a multipotent differentiation potential. These cells have the potential to differentiate into different germ layers (Kögler et al. 2004, Kögler et al. 2005, Kögler et al. 2006, Sensken et al. 2007, Greschat et al. 2008, Ghodsizad A et al. 2008, Trapp et al. 2008). Some studies have now reported a presumably embryonic like nature of cord blood cells. However, Nanog and Oct 4 harbours potential pitfalls for data misinterpretation due to pseudogenes and alternative spliced variants (Liedtke et al. 2007, Liedtke et al. 2008). The related data based on the stem cell markers Nanog and Oct4 concerning these results remain questionable. Therefore, we evaluated the embryonic-like nature of MNCs (n=7), USSC (n=7), CD34+ cells (n=7) derived from cord blood, MNCs from peripheral blood (n=3), MNCs (n=7) and MSCs (n=3) from bone marrow. Using RT-PCR, quantitative RT-PCR and immunohistochemistry, we studied the expression of the pluripotency markers Oct4, Nanog, Sox2 as well as the transcription factors Klf4 and cMyc utilized for the induction of pluripotent stem cells from adult human fibroblasts. The expression level of the transcription factors Klf4 and cMyc was nearly equal in all USSC cell lines and BM MSCs. We neither detected expression of Oct4, Nanog and Sox2 in all tested USSC cell lines nor in MNCs and CD34+ cells from cord blood nor in MSCs, MNCs and CD34+ cells from bone marrow. To increase the sensitivity of our method we performed quantitative Oct4 PCRs. This method revealed that USSCs reach the same Oct4 expression level as human dermal fibroblasts. These results are also supported by the inactive status of the telomerase. As a positive control we used the embryonic carcinoma cell line nTERA-2 showing a high expression of Oct4, Nanog and Sox2. In addition we were able to show that the markers SSEA1, SSEA3 and SSEA4 cannot be used as markers of an embryonic-like phenotype. SSEA-1 recognizes the CD15 epitope, SSEA-4 cross-reacts with an adult MSC subpopulation and SSEA3 was always negative applying the correct isotype controls. However, cord blood does not have to contain embryonic like cells, but it contains neonatal cells as USSC expressing Sox17. For hematopoietic stem cells (HSC) it had already been shown that the transcription factor Sox 17 is required to maintain fetal and neonatal HSC and distinguishes their transcriptional regulation from adult HSCs (Kim et al. 2007). Our results indicate that USSCs and cord blood are neonatal cells without expression of typical embryonic stem cell markers. In more than 10.000 unrelated Cord blood transplants performed so far, no tumor formation associated with an Oct4 positive cell/teratoma formation was observed. Therefore the embryonic-like nature of cord blood cells must be reconsidered.
Abstract Background We have previously identified very primitive human cord blood (CB)-derived CD34-negative (CD34-) severe combined immunodeficiency (SCID)- repopulating cells (SRCs) using the intra-bone marrow injection (IBMI) method (Blood 2003:101;2924). A series of our studies suggests that the identified CD34- SRCs are a distinct class of primitive hematopoietic stem cell (HSC) and that they are at the apex of human HSC hierarchy. Recently, we developed a high-resolution purification method for primitive CD34- SRCs using 18 lineage (Lin)-specific antibodies, which can enrich CD34- SRC at 1/1,000 level (Exp Hematol 2011: 39:203). In the present study, we tried to identify the positive marker of CD34- SRCs in order to further purify and characterize the CD34- SRCs (HSCs). Materials and Methods First, we extensively analyzed candidate positive markers, including known HSC markers and various adhesion molecules by FACS using highly purified CB-derived 18Lin-CD34+/- cells. Finally, we identified CD133 as a positive marker of human CB-derived CD34- SRCs. Then, CB-derived 18Lin- CD34+/-CD133+/- cells were sorted by FACS, and hematopoietic stem/progenitor cell (HSPC) capacities of these four fractions of cells were extensively investigated. HSPC capacities were evaluated using (1) colony-forming cell (CFC) assays, (2) measurement of maintenance/production of CD34+ cell capacities in co-cultures with human bone marrow-derived mesenchymal stromal cells (BM-MSCs) (Blood 2010:24:162), (3) SRC activities using NOG mice, (4) limiting dilution analyses (LDA) to determine the SRC frequency in the 18Lin-CD34-CD133+ fractions, and (5) comparison of gene expression profiles between 18Lin-CD34+/-CD133+/- cells by real-time RT-PCR. Results Seventy-five percent of 18Lin-CD34+ and 13.5% of 18Lin-CD34- cells highly expressed CD133. In the CFC assays, the plating efficiencies of 18Lin-CD34+CD133+, CD34+CD133-, CD34-CD133+ and CD34-CD133- cells were 57%, 65%, 39% and 19%, respectively. Interestingly, most of 18Lin-CD34-CD133+/- cells formed erythroid-bursts (71% and 73%) and erythro/megakaryocytes-containing mixed colonies (25% and 27%). On the contrary, they formed few granulocyte/macrophage colonies (4.2% and 0%). Then, we co-cultured these four fractions of cells with human BM-MSCs. One thousand of 18Lin-CD34+/-CD133+/- cells were seeded into each well and cells were co-cultured for 7 days in the presence of SCF+TPO+FL+IL-3+IL-6 +G-CSF. Both the 18Lin-CD34-CD133+/- cells produced CD34+ cells. However, the percentage and absolute number of CD34+ cells produced from 18Lin-CD34-CD133+ cells (31.7 % and 3.2 x 104 cells) were greater than those of 18Lin-CD34-CD133- cells (13.2 % and 0.4 x 104 cells). In addition, both the 18Lin-CD34- CD133+/- cells generated higher percentages (13.5 % and 11.5%) of CD41+ cells compared to those of the 18Lin- CD34+CD133+/- (1.8% and 4.2%) cells. Collectively, 18Lin-CD34+/-CD133+/- cells showed different in vitro lineage differentiation potentials. Then, these four fractions of cells were transplanted into NOG mice by IBMI. We performed primary and secondary transplantations for up to 36 weeks. In the results, all of the mice received 18Lin-CD34+CD133+ cells (n = 5) or 18Lin-CD34-CD133+ cells (n = 9) showed primary and secondary human CD45+ cell repopulations. However, neither 18Lin-CD34+CD133- cells nor 18Lin-CD34-CD133- cells showed human cell repopulations (n = 6 in each group). These results clearly demonstrated that the CD133 expression clearly segregated SRC activities in the 18Lin-CD34+/- cells. Moreover, LDA demonstrated that the frequency of SRCs in the 18Lin-CD34-CD133+ fraction was 1/142. Interestingly, HSC self-renewal maintenance genes, such as Notch1, HoxB4, HoxA9, and Bmi-1, were highly expressed in both 18Lin-CD34+/-CD133+ cells. Conclusion These results clearly demonstrated that CD133 is a positive marker of human CB-derived CD34- SRCs (HSCs). Furthermore, CD133 segregated SRC activities of 18Lin-CD34- as well as 18Lin-CD34+ cells in its positive fractions. More importantly, these findings suggest that number of CD133+ cells in cord blood units is a more appropriate marker to detect/predict HSC potentials in cord blood stem cell transplantation in comparison to currently used CD34+ cell numbers. Disclosures: No relevant conflicts of interest to declare.
Introduction Cell-derived microparticles (MPs), small membrane vesicles, are considered as markers of cell activation, as well as apoptosis and recently was shown to participate in important normal biological functions and pathological conditions associated mainly with endothelial injury, thrombosis and inflammation. Recently platelet MPs have been detected in patients undergoing allogeneic stem cell transplantation. Umbilical cord blood is an alternative stem cell source for transplantation where the presence of MPs has not been extensively studied. The aim of this study is to quantify the MPs of CD34+ cells in cord blood units and to investigate their relationship with different parameters. Methods Cord blood units (CBUs) (n=41) were processed using the Sepax automated method (Biosafe). After processing, cryopreservation was performed using 10% DMSO in a controled rate freezer (IceCube, Sy-lab). Units were thawed after two weeks. Adjusted variables of the CBUs included their net volume, the absolute number of CD34+ per graft and their viability as determined by flow cytometry, the number of leukocytes as recorded with an hematology analyzer and the absolute number of erythroblasts as estimated by optical microscope after Giemsa staining. Mononuclear cells were seeded in semisolid cultures in the presence of a cocktail of growth factors for colony forming unit granulocyte/macrophage (CFU-GM), burst forming unit erythroid (BFU-E) and Colony-Forming Unit-Granulocyte, Erythrocyte, Monocyte/macrophage, Megakaryocyte (CFU-GEMM) colony growth. The MPs were isolated after centrifugation of the plasma and their number was determined after incubation with Annexin V and CD34 by flow cytometry. Electron microscope photographs were obtained, after the proceedings for TEM. Statistical analysis was done using t-test, Pearson's and Spearman's depending on the normality of the distribution of variables. Results The mean number of AnnV+/CD34+MPs increased during post-processing (76,7±15,9) compared to the number of pre-processing AnnV+/CD34+MPs (73±22 ) and decreased during post-thawing (52,4 ±16,7). On univariate analysis found that the number of AnnV+/CD34+MPs (pre-processing vs. post-processing) has statistically significant mean positive correlation (p<0,012, Pearson's rho =0,823) whereas the number of MPs post-thawing was not statistically significant. Pre-processing AnnV+/CD34+MPs has statistically significant correlation with the absolute number of pre-processing CD34+ cell per unit (p<0,0001, Spearman's rho =0,835), post-processing (p<0,0001, Spearman's rho =0,814) and post-thawing CD34+ cells per unit (p<0,0001, Spearman's rho =0,993). Correlation of post-processing CFUs and AnnV+/CD34+MPs showed that the number of AnnV+/CD34+MPs has statistically significant mean negative correlation to the number of CFU-GEMM (p<0,028, Spearman's rho = -0,348). Conclusions These data confirm the identification of stem cell-derived MPs from cord blood samples. Moreover, the number of pre- and post- processing AnnV+/CD34+MPs was statistically correlated. In addition, the absolute number of CD34 (pre-, post- processing and post-thawing) was associated with the AnnV+/CD34+MPs pre- processing. Thus it remains to be shown if the stem cell derived MPs could be a useful parameter to predict the quality of the cord blood unit. Disclosures: No relevant conflicts of interest to declare.