Effect of dimethyl sulfoxide on post-thaw viability assessment of CD45+ and CD34+ cells of umbilical cord blood and mobilized peripheral blood

BACKGROUND: The effect of dimethyl sulfoxide (Me2SO) on enumeration of post-thaw CD45+ and CD34+ cells of umbilical cord blood (HPC-C) and mobilized peripheral blood (HPC-A) has not been systematically studied. METHODS: Cells from leukapheresis products from multiple myeloma patients and umbilical cord blood cells were suspended in 1, 2, 5, or 10% Me2SO for 20 min at 22 degrees C. Cells suspended in Me2SO were then immediately assessed or assessed following removal of Me2SO. In other samples, cells were suspended in 10% Me2SO, cooled slowly to -60 degrees C, stored at -150 degrees C for 48 h, then thawed. The thawed cells in 10% Me2SO were diluted to 1, 2, 5, or 10% Me2SO, held for 20 min at 22 degrees C and then immediately assessed or assessed after the removal of Me2SO. CD34+ cell viability was determined using a single platform flow cytometric absolute CD34+ cell count technique incorporating 7-AAD. RESULTS: The results indicate that after cryopreservation neither recovery of CD34+ cells nor viability of CD45+ and CD34+ cells from both post-thaw HPC-A and HPC-C were a function of the concentration of Me2SO. Without cryopreservation, when Me2SO is present recovery and viability of HPC-C CD34+ cells exposed to 10% Me2SO but not CD45+ cells were significantly decreased. Removing Me2SO by centrifugation significantly decreased the viability and recovery of CD34+ cells in both HPC-A and HPC-C before and after cryopreservation. DISCUSSION: To reflect the actual number of CD45+ cells and CD34+ cells infused into a patient, these results indicate that removal of Me2SO for assessment of CD34+ cell viability should only be performed if the HPC are infused after washing to remove Me2SO.     

Enumeration of CD34+ cells in cord blood: a variation on a single-platform flow cytometric method based on the ISHAGE gating strategy

Single-platform flow cytometric absolute cell counting protocols provide increased robustness for CD34+ cell enumeration by limiting potential sources of imprecision. However, samples with any cellular fragmentation or debris, such as cord blood samples, provide challenges for these assays. We describe a simple, robust absolute CD34+ cell counting protocol, suitable for cord blood, using TRUCOUNT absolute count tubes (BD Biosciences, San Jose, CA) and a modified ISHAGE (International Society for Hematotherapy and Graft Engineering) gating strategy. An advantage of TRUCOUNT tubes is that each tube is supplied with a known number of lyophilized fluorescent beads. The method includes no-wash fixative-free ammonium chloride red blood cell lysis and the viability dye, 7-amino actinomycin D, to exclude dead cells. The threshold was set on CD45 expression in the FL1 channel and an exclusion gate in the forward scatter channel reduced debris. No manual adjustment of the gating regions was required, even for samples in less than optimal condition. Comparison of the TRUCOUNT-ISHAGE protocol with the original dual-platform ISHAGE assay (n = 30) and the single-platform ISHAGE protocol using Flow-Count Fluorospheres (Beckman Coulter, Fullerton, CA; n = 22) showed high correlation (R(2) = 0.949 and 0.989, respectively) and no significant difference or bias for samples ranging from 22 to 600 CD34+ cells per microliter. Results are presented that demonstrate the detrimental effect of a fixative-containing lysis reagent when used in a lyse-and-wash procedure. The TRUCOUNT-ISHAGE protocol combines the attributes of TRUCOUNT tubes and the ISHAGE gating strategy to provide a single-platform protocol capable of achieving readily standardization of CD34+ cell enumeration.     

Comparison of single and dual platform methodologies for the estimation of CD34+ hematopoietic progenitor cells: correlation with colony assay

In this study three assays for the enumeration of CD34+ progenitors were compared: 1) a modified version of the Milan protocol, used in the standard dual-platform format; 2) a dual-platform version of the ISHAGE protocol; 3) the ProCOUNT software version 2.0/ProCOUNT kit. The assays were compared to validate the accuracy of CD34+ cell counts in mobilized peripheral blood (PB), apheresis products (AP), and cord blood (CB). The ProCOUNT protocol uses reference beads for absolute CD34+ cell counting, whereas CD34 counts by other techniques are derived from a separate leukocyte count performed by a hematology analyzer. A good correlation between the ISHAGE and ProCOUNT methods was obtained for estimation of CD34+ counts in PB (n=42 samples analyzed) and AP (n=35)--except for samples having a leukocyte count >25 x 10(9)/L or a CD34 count <0.0025 x 10(9)/L)--while a suboptimal correlation between the methods was observed for CB (n=30). The ProCOUNT system proved to be effective in reducing the variability in CD34+ cell counting and appeared to be useful for intralaboratory methodology standardization. The main disadvantage of the ProCOUNT assay was its inability to calculate CD34 counts in leukopenic samples and in CB samples showing a high erythroblast count. As far as the correlation with hematopoietic colonies is concerned, data collected from apheresis samples showed a good correlation between the three flow cytometry methods and colony-forming unit granulocyte-macrophage (CFU-GM) counts, confirming the value of the flow cytometric test as a real-time, truly predictive test to measure the hematopoietic potential of the graft. In summary, all methods are suitable for enumeration of most PB samples, while the single-platform methodology should be preferred for the analysis of AP and CB. We also found the dual-platform format of the ISHAGE method precise and accurate for the estimation of CD34+ cells from CB samples. Based on these data it can be concluded that the single-platform flow cytometry assay format should be the preferred approach for CD34+ stem cell enumeration in different types of samples.     

CD34+VEGFR‐3+ progenitor cells have a potential to differentiate towards lymphatic endothelial cells

Endothelial progenitor cells (EPCs) play an important role in postnatal neovascularization. However, it is poorly understood whether EPCs contribute to lymphangiogenesis. Here, we assessed differentiation of a novel population of EPCs towards lymphatic endothelial cells and their lymphatic formation. CD34⁺VEGFR‐3⁺ EPCs were isolated from mononuclear cells of human cord blood by fluorescence‐activated cell sorting. These cells expressed CD133 and displayed the phenotype of the endothelial cells. Cell colonies appeared at 7–10 days after incubation. The cells of the colonies grew rapidly and could be repeatedly subcultured. After induction with VEGF‐C for 2 weeks, CD34⁺VEGFR‐3⁺ EPCs could differentiate into lymphatic endothelial cells expressing specific markers 5′‐nucleotidase, LYVE‐1 and Prox‐1. The cells also expressed hyaluronan receptor CD44. The differentiated cells had properties of proliferation, migration and formation of lymphatic capillary‐like structures in three‐dimensional collagen gel and Matrigel. VEGF‐C enhanced VEGFR‐3 mRNA expression. After interfering with VEGFR‐3 siRNA, the effects of VEGF‐C were diminished. These results demonstrate that there is a population of CD34⁺VEGFR‐3⁺ EPCs with lymphatic potential in human cord blood. VEGF‐C/VEGFR‐3 signalling pathway mediates differentiation of CD34⁺VEGFR‐3⁺ EPCs towards lymphatic endothelial cells and lymphangiogenesis. Cord blood‐derived CD34⁺VEGFR‐3⁺ EPCs may be a reliable source in transplantation therapy for lymphatic regenerative diseases.     

Dimethyl sulfoxide-free cryopreservation solutions for hematopoietic stem cell grafts

Background aimsThe use of effective methods for the cryopreservation of hematopoietic stem cells (HSCs) is vital to retain the maximum engraftment activity of cord blood units (CBUs). Current protocols entail the use of dimethyl sulfoxide (DMSO) as intracellular cryoprotective agent (CPA) and dextran and plasma proteins as extracellular CPAs, but DMSO is known to be cytotoxic, and its infusion in patients is associated with mild to moderate side effects. However, new, commercially available, DMSO-free cryopreservation solutions have been developed, but their capacity to protect HSCs remains poorly investigated.MethodsHerein the authors compared the capacity of four DMSO-free freezing media to cryopreserve cord blood (CB) HSCs: CryoProtectPureSTEM (CPP-STEM), CryoScarless (CSL), CryoNovo P24 (CN) and Pentaisomaltose (PIM). Clinical-grade DMSO/dextran solution was used as control.ResultsOf the four cryopreservation solutions tested, the best post-thaw cell viability, recovery of viable CD45+ and CD34+ cells and potency were achieved with CPP-STEM, which was equal or superior to that seen with the control DMSO. CSL provided the second best post-thaw results followed by PIM, whereas CN was associated with modest viability and potency. Further work with CPP-STEM revealed that CB CD34-enriched HSCs and progenitors cryopreserved with CPP-STEM maintained high viability and growth expansion activity. In line with this, a pilot transplantation assay confirmed that CPP-STEM-protected CB grafts supported normal short- and long-term engraftment kinetics.ConclusionsThe authors’ results suggest that new, valuable alternatives to DMSO are now available for the cryopreservation of HSCs and grafts, including CBUs.     

Improved immunomagnetic enrichment of CD34(+) cells from umbilical cord blood using the CliniMACS cell separation system

Background aimsCD34+ enrichment from cord blood units (CBU) is used increasingly in clinical applications involving ex vivo expansion. The CliniMACS instrument from Miltenyi Biotec is a current good manufacturing practice (cGMP) immunomagnetic selection system primarily designed for processing larger numbers of cells: a standard tubing set (TS) can process a maximum of 60 billion cells, while the larger capacity tubing set (LS) will handle 120 billion cells. In comparison, most CBU contain only 1–2 billion cells, raising a question regarding the optimal tubing set for CBU CD34+ enrichment. We compared CD34+ cell recovery and overall viability after CliniMACS processing of fresh CBU with either TS or LS.MethodsForty-six freshly collected CBU (≤ 36 h) were processed for CD34+ enrichment; 22 consecutive units were selected using TS and a subsequent 24 processed with LS. Cell counts and immunophenotyping were performed pre- and post-selection to assess total nucleated cells (TNC), viability and CD34+ cell content.ResultsTwo-sample t-tests of mean CD34+ recovery and viability revealed significant differences in favor of LS (CD34+ recovery, LS = 56%, TS = 45%, P = 0.003; viability, LS = 74%, TS = 59%, P = 0.011). Stepwise linear regression, considering pre-processing unit age, viability, TNC and CD34+ purity, demonstrated statistically significant correlations only with the tubing set used and age of unit.ConclusionsFor CD34+ enrichment from fresh CBU, LS provided higher post-selection viability and more efficient recovery. In this case, a lower maximum TNC specification of TS was not predictive of better performance. The same may hold for smaller scale enrichment of other cell types with the CliniMACS instrument.     

Recovery of CD45−/Lin−/SSEA-4+ very small embryonic-like stem cells by cord blood bank standard operating procedures

Background aimsVery small embryonic-like (VSEL) stem cells are a rare cell population present in bone marrow, cord blood and other tissues that displays a distinct small cell size and the ability to give rise to cells of the three germ layers. VSEL stem cells were reported to be discarded in the red blood cell fraction by Ficoll-Paque density gradient centrifugation during the processing of bone marrow and cord blood specimens. However, most cord blood banks do not include density gradient centrifugation in their procedures while red blood cells are removed by Hespan sedimentation following the Cord Blood Transplantation Study cord blood bank standard operating procedures (COBLT SOP). To clarify the retention of VSEL stem cells, we investigated the recovery of VSEL stem cells following COBLT SOP guidelines.MethodsThe recovery of CD45⁻/Lin⁻/SSEA-4⁺ VSEL stem cells of umbilical cord blood was examined by flow cytometry before and after COBLT SOP processing, and relative expression of pluripotent genes was analyzed by quantitative polymerase chain reaction.ResultsCD45⁻/Lin⁻/SSEA-4⁺ VSEL stem cells were mostly recovered in the final products following COBLT SOP guidelines. The expression of pluripotent genes could be maintained at >80% in products after hetastarch (Hespan; B. Braun Medical Inc., Irvine, CA, USA) processing.ConclusionsThe rare sub-population of CD45⁻/Lin⁻/SSEA-4⁺ VSEL stem cells survived after Hespan sedimentation. This finding suggests that umbilical cord blood units cryopreserved by COBLT SOP in cord blood banks should retain most VSEL stem cells present in the un-processed specimens.     

Elevation of Survivin Levels by Hematopoietic Growth Factors Occurs in Quiescent CD34+ Hematopoietic Stem and Progenitor Cells Before Cell Cycle Entry

Survivin is a member of the inhibitor of apoptosis protein (IAP) family that is over-expressed during G2/M phase in most cancer cells. In contrast, we previously reported that Survivin is expressed throughout the cell cycle in normal CD34+ hematopoietic stem and progenitor cells stimulated by the combination of Thrombopoietin (Tpo), Stem Cell Factor (SCF) and Flt3 ligand (FL). In order to address whether Survivin expression is specifically up-regulated by hematopoietic growth factors before cell cycle entry, we isolated quiescent CD34+ cells and investigated Survivin expression in response to growth factor stimulation. Survivin is up-regulated in CD34+ cells with 2N DNA content following growth factor addition, suggesting it becomes elevated during G0/G1. Survivin is barely detectable in freshly isolated umbilical cord blood (UCB) Ki-67negative and Cyclin Dnegative CD34+ cells, however incubation with Tpo, SCF and FL for 20 hrs results in up-regulation without entry of cells into cell cycle. Culture of G0 CD34+ cells isolated based on Hoechst 33342/PyroninY staining with Tpo, SCF and FL for 48 hrs, results in significantly elevated Survivin mRNA and protein levels. Moreover, labeling of fresh G0 CD34+ cells with 5-(and 6-) carboxyfluorescein diacetate succinimidyl ester (CFSE) before culture with growth factors for up to 72 hrs, revealed that Survivin expression was elevated in CFSEbright G0 CD34+ cells, indicating that up-regulation occurred before entry into G1. These results suggest that up-regulation of Survivin expression in CD34+ cells is an early event in cell cycle entry that is regulated by hematopoietic growth factors and does not simply reflect cell cycle progression and cell division.

Key Words:

Survivin, Cord blood, CD34+ cells, Cell cycle     

Pre-Transplantation Serum Parathyroid Hormone Influences the Number of Mobilized CD34+ Hematopoietic Stem Cells in Autologous Hematopoietic Stem Cell Transplantation

Background:Parathyroid hormone (PTH) is a calcium homeostasis regulator and can affect bone marrow niche. PTH leads to the bone marrow stem cell niche expansion as well as the induction of stem cell mobilization from the bone marrow into peripheral blood. In this study, we evaluated the association between pre- transplantation serum PTH levels and the number of circulating CD34+ cells along with the platelets/white blood cells (Plt/WBC) engraftment in patients who underwent autologous Hematopoietic Stem Cell Transplantation.Methods:Subjects for the study were 100 patients who received autologous hematopoietic stem cell transplantation (auto-HSCT), retrospectively. Serum levels of PTH, calcium, phosphorus, and alkaline phosphatase were measured before mobilization. Their impacts were measured on the number of mobilized CD34+ hematopoietic stem cells, and Plt/WBC engraftment.Results:High levels of serum PTH (> 63.10 pg/mL) was significantly associated with higher number of CD34+ cells in peripheral blood after granulocyte- colony stimulating factor (G-CSF)-induced mobilization (p= 0.079*). Serum calcium at low levels were associated with higher number of circulating CD34+ cells post mobilization. Pre- transplantation serum levels of phosphorus and alkaline phosphatase on CD34+ numbers were not statistically significant. Serum Plt/WBC engraftment was not improved in presence of high levels of serum PTH.Conclusion:We suggested that serum PTH levels before transplantation could be influential in raising the number of circulating CD34+ hematopoietic stem cell after mobilization.Key Words: Auto-HSCT, CD34+ Cell, Pre- transplant PTH     

Expansion of CD34+ cells from human umbilical cord blood by FL and/or TPO gene transfected human marrow stromal cell lines

To elucidate the effect of gene transfected marrow stromal cell on expansion of human cord blood CD34⁺ cells, a culture system was established in which FL and TPO genes were transfected into human stromal cell line HFCL. To establish gene transfected stromal cells co-culture system, cord blood CD34⁺ cells were purified by using a magnetic beads sorting system. The number of all cells and the number of CD34⁺ cells and CFC (CFU-GM and BFU-E) were counted in different culture systems. The results showed that in all 8 culture systems, SCF+IL-3+HFT manifested the most potent combination, with the number of total nucleated cells increasing by (893.3 ±52.1)-fold, total progenitor cells (CFC) by (74.5 ±5.2)-fold and CD34⁺ cells by 15.7-fold. Maximal expansions of CFC and CD34⁺ cells were observed at the end of the second week of culture. Within 14 days of culture, (78.1 ± 5.5)-fold and (57.0 ± 19.7)-fold increases in CFU-GM and BFU-E were obtained. Moreover, generation of LTC-IC from amplified CD34⁺ cells within 28 days was found only in two combinations, i.e. SCF+IL-3+FL+TPO and SCF+IL-3+HFT, and there was no significant difference between these two groups statistically. These results suggest that human umbilical cord blood CD34⁺ cells can be extensively expandedex vivo by using gene transfected stromal cells along with cytokines.     

Predictive analytics and cord blood banking: toward utilization-based unit selection

Background aimsTotal nucleated cell (TNC) and CD34+ cell doses are considered among the most important parameters when assessing the suitability of a human leukocyte antigen-matched cord blood unit (CBU) for allogeneic hematopoietic stem cell transplantation (HSCT). Cord blood banks therefore frequently select CBUs for cryopreservation based on pre-process TNC content. However, cell loss during processing can lead to a significant quantity of CBUs that do not meet desired post-process quality criteria, and such grafts are less likely to be selected by transplant centers for HSCT. Here the authors present a multi-parameter linear regression (MLR) model capable of identifying CBUs that would process poorly, despite meeting established pre-process TNC and CD34+ quality thresholds.MethodsHistorically processed CBUs were graded from A+ to D depending on post-process cell content, and the utilization rate of each grade category was examined. Eight pre-process predictors of post-process cell content were used to train the MLR model, including red blood cell (RBC) content; CBU volume; age of CBU when received; and TNC constituent cell subsets. The selection efficacy of this model was then compared to that of methods conventionally used to select CBUs for processing, with receiver operating characteristic (ROC) and mean inventory quality analysis forming the basis of assessment.ResultsWithin the Anthony Nolan Cell Therapy Centre, CBUs graded ‘D’ accounted for 37% of processing expenditures despite providing only 11% of grafts shipped for HSCT. The MLR model significantly improved pre-process identification of 'D' grade CBUs relative to thresholds based primarily on CD34+ cell content (P < 0.0001) and TNC content (P < 0.0001). At a comparable financial investment, this translated to a banked graft inventory of significantly higher quality than that produced by CD34+ (+8.8% mean increase, P = 0.007) and TNC (+9.9% mean increase, P = 0.010) selection methods.ConclusionsA predictive modelling approach to pre-process CBU selection is a simple and effective means to increase graft inventory quality and potentially future graft utilization, at no additional financial investment.     

Systematic investigation of oxygen and growth factors in clinically valid ex vivo expansion of cord blood CD34(+) hematopoietic progenitor cells

Background aimsCord blood is considered to be a superior source of hematopoietic stem and progenitor cells for transplantation, but clinical use is limited primarily because of the low numbers of cells harvested. Ex vivo expansion has the potential to provide a safe, effective means of increasing cell numbers. However, an absence of consensus regarding optimum expansion conditions prevents standard implementation. Many studies lack clinical applicability, or have failed to investigate the combinational effects of different parameters.MethodsThis is the first study to characterize systematically the effect of growth factor combinations across multiple oxygen levels on the ex vivo expansion of cord blood CD34⁺ hematopoietic cells utilizing clinically approvable reagents and methodologies throughout.ResultsOptimal fold expansion, as assessed both phenotypically and functionally, was greatest with thrombopoietin, stem cell factor, Flt-3 ligand and interleukin-6 at an oxygen level of 10%. With these conditions, serial expansion showed continual target population expansion and consistently higher expression levels of self-renewal associated genes.ConclusionsThis study has identified optimized fold expansion conditions, with the potential for direct clinical translation to increase transplantable cell dose and as a baseline methodology against which future factors can be tested.     

Optimal umbilical cord blood collection,processing and cryopreservation methods for sustained public cord blood banking

Background aimsUmbilical cord blood is an established source of stem cells in patients with hematologic malignancies who do not have HLA-compatible matched related or unrelated donors. The success of an umbilical cord blood transplant depends on the dose of total nucleated and CD34+ cells infused. Therefore, collecting, banking and listing high-quality cord blood units with high total nucleated and CD34+ cell dose are essential.MethodsHere the authors describe their cord blood bank's novel collection technique, which involves both in utero and ex utero collection of a single cord blood unit. The authors also evaluated maternal, neonatal and collection parameters that may impact the cell dose.ResultsMaternal gestational age and race, and neonatal weight and sex correlated with the total nucleated cell dose.ConclusionsThe optimized collection of umbilical cord blood is critical for its use as a source of stem cells for transplantation.     

逆病毒载体介导双耐药基因在脐血CD34^＋细胞中的表达和抗性研究

To explore whether human umbilical cord blood CD34+ cells transduced with human aldehyde dehydrogenase class-1 (ALDH1) and multidrug resistance gene (MDR1) increase resistance to 4-Hyaroxycyclophosphophamide (4-HC) and P-Glycoprotein Effluxed Drugs, a bicistronic Retroviral vector G1Na-ALDH1-IRES-MDR1 was constructed. The vector was transduced into the packaging cell lines GP + E86 and PA317 by LipofectAMINE. Using the medium containing VCR and 4-HC for cloning selection and pingponging supernatant infection between ecotropic producer clone and amphotropic producer clone, we obtained high titer amphotropic PA317 producer clone with the highest titer up to 5.6 x 10(5) CFU/ml. Cord blood CD34+ cells were transfectced repeatedly with supernatant of retrovirus containing human ALDH1 and MDR1cDNA under stimulation of hemopoietie growth factors. PCR, RT-PCR, Southern blot, Northern blot, FACS and MTT method analyses show that dual drug resistance genes have been integrated into the genomic DNA of cord blood CD34+ cells and expressed efficiently. The transgenes recipient cells confered 4- to 7.2-folds stronger resistance to cyclophospsphamede and P-Glycoprotein Effluxes drug in comparison with the nontransduced cells. This study provided a foundation for the application of combination chemotherapy in tumor clinical trial.     

Placental/umbilical cord blood-derived mesenchymal stem cell-like stromal cells support hematopoietic recovery of X-irradiated human CD34+ cells

AimsThe potential of human mesenchymal stem cell-like stroma prepared from placental/umbilical cord blood for hematopoietic regeneration by X-irradiated hematopoietic stem cells is herein assessed.Main methodsPlacental/umbilical cord blood-derived mesenchymal stem cell-like stromal cells were applied to a regenerative ex vivo expansion of X-irradiated human CD34⁺ cells in a serum-free liquid culture supplemented with a combination of interleukine-3 plus stem cell factor plus thrombopoietin.Key findingsThe total number of cells and of lineage-committed myeloid hematopoietic progenitor cells generated in the co-culture of both non-irradiated and X-irradiated cells with stromal cells was significantly higher than those in the stroma-free culture. In addition, the number of CD34⁺ cells and CD34⁺/CD38^? cells, immature hematopoietic stem/progenitor cells also increased more than the stroma-free culture. The stromal cells produced various types of cytokines, although there was little difference between the co-cultures of non-irradiated and X-irradiated cells with stromal cells. Furthermore, when X-irradiated cells came in contact with stromal cells for 16 h before cytokine stimulation, a similar degree of hematopoiesis was observed, thus suggesting the critical role of cell-to-cell interaction.SignificanceThe present results showed the potential efficacy of human mesenchymal stem cell-like stroma for hematopoietic regeneration from irradiated hematopoietic stem/progenitor cells.     

Cord blood derived CD4+ CD25(high) T cells become functional regulatory T cells upon antigen encounter

Background: Upon antigen exposure, cord blood derived T cells respond to ubiquitous environmental antigens by high proliferation. To date it remains unclear whether these “excessive” responses relate to different regulatory properties of the putative T regulatory cell (Treg) compartment or even expansion of the Treg compartment itself.Methods: Cord blood (>37 week of gestation) and peripheral blood (healthy controls) were obtained and different Treg cell subsets were isolated. The suppressive potential of Treg populations after antigen exposure was evaluated via functional inhibition assays ([³H]thymidine incorporation assay and CFSE staining) with or without allergen stimulation. The frequency and markers of CD4⁺CD25^highFoxP3⁺ T cells were characterized by mRNA analysis and flow cytometry.Results: Cord blood derived CD4⁺CD25^high cells did not show substantial suppressor capacity upon TCR activation, in contrast to CD4⁺CD25^high cells freshly purified from adult blood. This could not be explained by a lower frequency of FoxP3⁺CD4⁺CD25^highcells or FOXP3 mRNA expression. However, after antigen-specific stimulation in vitro, these cells showed strong proliferation and expansion and gained potent suppressive properties. The efficiency of their suppressive capacity can be enhanced in the presence of endotoxins. If T-cells were sorted according to their CD127 expression, a tiny subset of Treg cells (CD4⁺CD25⁺CD127^low) is highly suppressive even without prior antigen exposure.Conclusion: Cord blood harbors a very small subset of CD4⁺CD25^high Treg cells that requires antigen-stimulation to show expansion and become functional suppressive Tregs.     

CD133 immunomagnetic separation: effectiveness of the method for CD133(+) isolation from umbilical cord blood

Background aimsUmbilical cord blood (UCB) is a rich source of stem cells, the characterization and isolation of which requires specific stem cell markers and reliable and reproducible protocols.MethodsWe assessed CD133 isolation in 39 UCB samples, using a commercial immunomagnetic cell-sorting protocol, and, because of its non-reproducibility, we applied optimized protocols in an effort to improve it. These included extra-labeling of the selected CD133⁺ subpopulation and indirect labeling using anti-phycoerythrin (PE) microbeads, goat anti-mouse IgG microbeads or a combination of both. The CD34 isolation was used as a control.ResultsThe mononuclear cell fraction expressed 0.53 ± 0.06% CD133. The corresponding value for CD34 was 1.64 ± 0.15%. Following the manufacturer's instructions, the CD34 isolation resulted in a population expressing 93 ± 1.25% CD34 while, after the corresponding process, CD133⁺ expression ranged from 10% to 85% (median 60%). The optimized isolation protocols did not result in improved CD133⁺ yield. The variation in the purity of the CD133 population cannot be attributed to the different clones of CD133 used, because they do not cross-block, while other factors such as glycosylation, which could possibly interfere, do not apply in normal hematopoietic stem cells (HSC).ConclusionsCD34 isolation by the immunomagnetic method results in highly pure CD34⁺ population, while the efficient and reproducible yield of a pure CD133⁺ population is not feasible. Therefore quantification of the positive cells should follow each isolation procedure in order to confirm the number of CD133⁺ cells.     

Both CD34+38+ and CD34+38- cells home specifically to the bone marrow of NOD/LtSZ scid/scid mice but show different kinetics in expansion

Human hemopoietic stem cells (HSC) have been shown to engraft, differentiate, and proliferate in the hemopoietic tissues of sublethally irradiated NOD/LtSZ scid/scid (NOD/SCID) mice. We used this model to study homing, survival, and expansion of human HSC populations from different sources or phenotype. We observed that CD34+ cells homed specifically to bone marrow (BM) and spleen, but by 3 days after injection, survived only in the BM. These BM-homed CD34+ cells proliferated intensively and gave rise to a 12-fold, 5.5-fold, and 4-fold expansion in 3 days for umbilical cord blood, adult mobilized peripheral blood, and adult BM-derived cells, respectively. By injection of purified subpopulations, it was demonstrated that both CD34+38+ and CD34+38- umbilical cord blood HSC homed to the BM and expanded. Importantly, kinetics of expansion were different: CD34+38+ cells started to increase in cell number from day 3 onwards, and by 4 wk after injection, virtually all CD34+ cells had disappeared. In contrast, CD34+38- cells remained quiescent during the first week and started to expand intensively from the third week on. In this paper, we have shown that homing, survival, and expansion of stem cells are three independent phenomena important in the early phase of BM engraftment and that kinetics of engraftment differ between CD34+38+ and CD34+38- cells.     

Comparison of the Efficacy of Cord Blood Mononuclear Cells (MNCs) and CD34+ Cells for the Treatment of Neonatal Mice with Cerebral Palsy

To compare the efficacy of cord blood mononuclear cells (MNCs) and CD34+ cells for the treatment of neonatal mice models with cerebral palsy (CP). CP model in neonatal mice was established by the ligation of carotid artery. Mice were randomly designated into MNCs group, CD34+ group, model group and control group (30 mice per group). MNCs and CD34+ cells were isolated from human umbilical cord blood. MNCs were transplanted into mice in the MNCs group and CD34+ cells into mice in the CD34+ group through the jugular vein, respectively. The body weight, histopathology, apoptosis-related gene expression, learning and memory, and motor function of mice in the four groups were compared. Compared with control group, the body weight of mice in model group was significantly lower (P < 0.05). In addition, the right hemisphere was significantly liquefied and voids were found in model mice, in which degeneration and necrosis were found by HE staining. Real-time quantitative fluorescent PCR showed elevated levels of apoptosis-related gene expression and learning and memory function, and motor function were significantly decreased (P < 0.05) in model mice. In the MNCs group and CD34+ group, the weight of mice was significantly increased compared with the model group (P < 0.05). Moreover, neither liquefaction and voids in the hemispheres of mice were found in these two groups, nor degeneration and necrosis of cell. Meanwhile, levels of apoptosis-related gene expression were significantly lower than that of the model group (P < 0.05). Compared with the MNCs group, the expression of apoptotic gene TNF-α and CD40 was significantly lower (P < 0.05). Learning and memory function, and motor function of mice in the MNCs group and CD34+ group were significantly improved than the model group (P < 0.05), and the CD34+ group produced greater improvement than the MNCs group (P < 0.05). MNCs and CD34+ cells can reduce the degree of injury in the neonatal mice with CP. In addition, treatment with MNCs and CD34+ cells suppressed apoptotic gene expression and restored memory and motor function. The efficacy of CD34+ cells after separation and purification was more significant for the treatment of mice with CP.