Autologous transplantation: the viable transplanted CD34+ cell dose measured post-thaw does not predict engraftment kinetics better than the total CD34+ cell dose measured pre-freeze in patients that receive more than 2x10(6) CD34+ cells/kg

BACKGROUND: The aim of the study was to investigate whether the number of viable CD34+ cells in cryopreserved PBPC autografts is a better predictor of engraftment than the total CD34+ cell number determined before freezing. METHODS: A total of 119 patients was treated with autotransplantation for various malignant disorders during the period 1996-2002. All patients were reinfused with at least 2x10(6)/kg total CD34 cells analyzed before programmed freezing in 10% DMSO. The total CD34 cell number determined before freezing was compared with the number of viable cells determined after cryopreservation for 51 of these patients. The number of viable cells was determined by a flow cytometric analysis including triple staining with anti-CD34, anti-CD45 and the viability marker 7-actinomycin D (7-AAD). RESULTS: Simple linear regression analyses showed that both the total transplanted CD34 cell dose measured before freezing and the viable CD34 cell dose determined after cryopreservation were significantly correlated with neutrophil and platelet engraftment. In a multiple regression model the prediction of engraftment was not improved when the transplanted viable CD34 cell dose was included as a variable in addition to the total CD34 cell dose measured immediately after collection. DISCUSSION: Routine estimation of viable CD34 cells after cryopreservation of PBPC autografts is not necessary as long as the total CD34 cell dose is determined before freezing and the patients are reinfused with at least 2x10(6) cells/kg body weight.     

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.     

IL-3 increases production of B lymphoid progenitors from human CD34+CD38- cells

The effect of IL-3 on the B lymphoid potential of human hemopoietic stem cells is controversial. Murine studies suggest that B cell differentiation from uncommitted progenitors is completely prevented after short-term exposure to IL-3. We studied B lymphopoiesis after IL-3 stimulation of uncommitted human CD34+CD38- cells, using the stromal cell line S17 to assay the B lymphoid potential of stimulated cells. In contrast to the murine studies, production of CD19+ B cells from human CD34+CD38- cells was significantly increased by a 3-day exposure to IL-3 (p < 0.001). IL-3, however, did not increase B lymphopoiesis from more mature progenitors (CD34+CD38+ cells) or from committed CD34-CD19+ B cells. B cell production was increased whether CD34+CD38- cells were stimulated with IL-3 during cocultivation on S17 stroma, on fibronectin, or in suspension. IL-3Ralpha expression was studied in CD34+ populations by RT-PCR and FACS. High IL-3Ralpha protein expression was largely restricted to myeloid progenitors. CD34+CD38- cells had low to undetectable levels of IL-3Ralpha by FACS. IL-3-responsive B lymphopoiesis was specifically found in CD34+ cells with low or undetectable IL-3Ralpha protein expression. IL-3 acted directly on progenitor cells; single cell analysis showed that short-term exposure of CD34+CD38- cells to IL-3 increased the subsequent cloning efficiency of B lymphoid and B lymphomyeloid progenitors. We conclude that short-term exposure to IL-3 significantly increases human B cell production by inducing proliferation and/or maintaining the survival of primitive human progenitors with B lymphoid potential.     

Importance of CD34+ cell subsets in autologous PBSC transplantation: the mulhouse experience using CD34+CD38- cells as predictive tool for hematopoietic engraftment

Over the years, various biological parameters have been proposed for predicting rapidity and long term maintenance of hematopoietic engraftment after peripheral blood stem cell transplantation (PBSCT). Determination of the graft content in CFU-GM was the only one available until the end of the eighties. But, for technical reasons, and also because it does not actually evaluate the self-renewal potential of the cell products reinfused, it has now been commonly replaced by the determination of CD34+ cell amounts, which are known to contain the pluripotent hematopoietic stem cells. However, a frequent discrepancy still exists between the number of CD34+ cells reinfused and the engraftment efficiency. We have recently demonstrated a higher accuracy of the numbers of CD34+38- cells contained in graft products to predict rapidity of trilineage engraftment, which has further been confirmed by other investigators. Furthermore, we and others, have proposed a threshold dose of 5 x 10(4) CD34+38- cells/kg b.w. below which the trilineage engraftment kinetics are significantly slower and unpredictible. This "cut-off" value also appears to be a realistic clinical tool to decide if hematopoietic growth factor(s) must be administered or not after PBSCT. Indeed, when for example, rh-G-CSF administration after transplant of CD34+38- amounts < 5 x 10(4) kg has indisputable positive effects on the rapidity of neutrophil engraftment, length of hospitalization and posttransplant costs, enough to make it fully justified in this situation, it is absolutely not the case when it is administered after reinfusion of CD34+38- cell amounts > 5 x 10(4) /kg. In this case, posttransplant rh-G-CSF administration could even result in a decrease in stem cells with self-renewal potential of the graft, which should still raise more concerns for its indiscriminate and costly use.     

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.     

High frequency of CD34+CD38-/low immature leukemia cells is correlated with unfavorable prognosis in acute myeloid leukemia

AIM To evaluate the importance of the CD34+CD38-cell population when compared to the CD34+CD38+/low and CD34+CD38+/high leukemic cell sub-populations and to determine its correlations with leukemia characteristics and known prognostic factors, as well as with response to therapy and survival.METHODS Two hundred bone marrow samples were obtained at diagnosis from 200 consecutive patients with newly diagnosed acute myeloid leukemia(AML) were studied between September 2008 and December 2010 at our Institution(Hematology Department, Lyon, France). The CD34/CD38 cell profile was analyzed by multiparameter flowcytometry approach using 8 C panels and FACS CANTO and Diva software(BD Bioscience).RESULTS We analyzed CD34 and CD38 expression in bone marrow samples of 200 AML patients at diagnosis, and investigated the prognostic value of the most immature CD34+CD38-population. Using a cut-off value of 1% of CD34+CD38-from total "bulk leukemic cells" we found that a high( 1%) level of CD34+CD38-blasts at diagnosis was correlated with advanced age, adverse cytogenetics as well as with a lower rate of complete response after induction and shorter disease-free survival. In a multivariate analysis considering age, leukocytosis, the % of CD34+ blasts cells and the standardized cytogenetic and molecular risk subgroups, a percentage of CD34+CD38-leukemic cells 1% was an independent predictor of DFS [HR = 2.8(1.02-7.73), P = 0.04] and OS [HR = 2.65(1.09-6.43), P = 0.03].CONCLUSION Taken together, these results show that a CD34/CD38 "backbone" for leukemic cell analysis by multicolour flowcytometry at diagnosis provides useful prognostic information.     

Mobilization of CD34~+CD38~- hematopoietic stem cells after priming in acute myeloid leukemia

AIM: To evaluate quantitatively and qualitatively the different CD34+cell subsets after priming by chemotherapy granulocyte colony-stimulating factor(± G-CSF)in patients with acute myeloid leukemia.METHODS: Peripheral blood and bone marrow sampleswere harvested in 8 acute myeloid leukemia patients during and after induction chemotherapy. The CD34/CD38 cell profile was analyzed by multi-parameter flow cytometry. Adhesion profile was made using CXC chemokine receptor 4(CXCR4)(CD184), VLA-4(CD49d/CD29) and CD47.RESULTS: Chemotherapy ± G-CSF mobilized immature cells(CD34+CD38 population), while the more mature cells(CD34+CD38lowand CD34+CD38+populations) decreased progressively after treatment. Circulating CD34+cells tended to be more sensitive to chemotherapy after priming with G-CSF. CD34+cell mobilization was correlated with a gradual increase in CXCR4 and CD47expression, suggesting a role in cell protection and the capacity of homing back to the marrow.CONCLUSION: Chemotherapy ± G-CSF mobilizes into the circulation CD34+bone marrow cells, of which, the immature CD34+CD38-cell population. Further manipulations of these interactions may be a means with which to control the trafficking of leukemia stem cells to improve patients’ outcomes.     

CD34(+) CD38(-) and CD34(+) HLA-DR(-) cells in BM stem cell grafts correlate with short-term engraftment but have no influence on long-term hematopoietic reconstitution after autologous transplantation

BACKGROUND: Prior studies have demonstrated that relatively immature hematopoietic stem cells, including CD34(+) CD38(-) and CD34(+) HLA-DR(-) subsets, correlate with short-term hematopoietic reconstruction (SHR) after transplantation. The aim of this study was to investigate whether these immature CD34(+) subsets also correlate with long-term hematopoietic reconstitution (LHR) in recipients of ABMT. METHODS: We examined stem cell grafts from 58 patients with B-cell lymphoma or CLL who underwent ABMT after myeloablative conditioning. We determined whether total mononuclear cell dose (MNC), colony-forming unit-granulocyte-monocyte (CFU-GM), CD34(+) cell dose and CD34(+) cell subsets (CD34(+) CD38(-) and CD34(+) HLA-DR(-) were associated with SHR and/or LHR. Time to neutrophil engraftment (TNE) and time to platelet engraftment (TPE) were used to measure SHR, while platelet counts at day 100 and 1 year post-ABMT were used as indicators for LHR. RESULTS AND DISCUSSION: CD34(+) cell dose and CD34(+) cell subsets were significantly associated with SHR. However, at day 100 and 1 year post-transplant only total CD34(+) cell dose was associated with LHR. The association of total CD34(+) cell dose with LHR persisted after adjusting for age, sex and disease. None of the CD34(+) cell subsets analyzed showed evidence of significant association with LHR.     

Cryopreservation and thawing of hematopoietic stem cell CD34-induced apoptosis through caspase pathway activation: Key role of granulocytes

IntroductionCell damage inescapably occurs during both the freezing and the thawing graft processes for autologous hematopoietic stem cell (HSC) transplantation. To estimate HSC injury, a quality control is performed including: (i) CD34⁺ quantification; (ii) percentage of CD34⁺ viability and (iii) evaluation of HSC functional ability to form colony forming unit–granulocyte macrophage (CFU-GM). Apoptosis involves complex pathways such as caspase enzymes. Here, we assess the extent of apoptosis that is caspase-dependent before and after cryoconservation of CD34⁺, using a Fluorescent Labeled Inhibitor of CAspases (FLICA).MethodsCaspase pathway activation status was evaluated in 46 patients (multiple myeloma [n = 24], lymphoma [n = 22]), by flow cytometry, using a 7-aminoactinomycin-D (7AAD)/FLICA staining test, in CD34⁺, CD3⁺, CD14⁺ and CD56⁺ cells. Viable 7AAD^?/FLICA⁺ cells were then correlated with various parameters.ResultsWe showed a significant caspase pathway activation, with 23% CD34⁺/7AAD^?/FLICA⁺ cells after thawing, compared with the 2% described in fresh CD34⁺ cells (P < 0.0001). Moreover, caspase pathway was significantly activated in thawing CD3⁺, CD56⁺ and CD14⁺ cells. We also report a significant correlation between the rate of CD34⁺/7AAD^?/FLICA⁺ cells and post-thawing granulocytes count (P = 0.042) and their potential to be differentiated into CFU-GM (P = 0.004).DiscussionOur results show substantial cell death, induced by the increase of caspase pathway activation, secondary to the thawing process, and across all study cell types. This observation may affect the immune response quality during recipient aplasia, without detecting a clinical impact. Moreover, caspase pathway activation through CD3⁺ and CD56⁺ subpopulations could modify the therapeutic result of donor lymphocytes infusion (DLI).     

Proteomic Profiling Identifies Distinct Protein Patterns in Acute Myelogenous Leukemia CD34+CD38- Stem-Like Cells

Acute myeloid leukemia (AML) is believed to arise from leukemic stem-like cells (LSC) making understanding the biological differences between LSC and normal stem cells (HSC) or common myeloid progenitors (CMP) crucial to understanding AML biology. To determine if protein expression patterns were different in LSC compared to other AML and CD34+ populations, we measured the expression of 121 proteins by Reverse Phase Protein Arrays (RPPA) in 5 purified fractions from AML marrow and blood samples: Bulk (CD3/CD19 depleted), CD34-, CD34+(CMP), CD34+CD38+ and CD34+CD38-(LSC). LSC protein expression differed markedly from Bulk (n=31 cases, 93/121 proteins) and CD34+ cells (n= 30 cases, 88/121 proteins) with 54 proteins being significantly different (31 higher, 23 lower) in LSC than in either Bulk or CD34+ cells. Sixty-seven proteins differed significantly between CD34+ and Bulk blasts (n=69 cases). Protein expression patterns in LSC and CD34+ differed markedly from normal CD34+ cells. LSC were distinct from CD34+ and Bulk cells by principal component and by protein signaling network analysis which confirmed individual protein analysis. Potential targetable submodules in LSC included the proteins PU.1(SP1), P27, Mcl1, HIF1α, cMET, P53, Yap, and phospho-Stats 1, 5 and 6. Protein expression and activation in LSC differs markedly from other blast populations suggesting that studies of AML biology should be performed in LSC.     

Cryopreservation of umbilical cord blood: 2. Tolerance of CD34(+) cells to multimolar dimethyl sulphoxide and the effect of cooling rate on recovery after freezing and thawing

Cryopreservation protocols for umbilical cord blood have been based on methods established for bone marrow (BM) and peripheral blood stem cells (PBSC). The a priori assumption that these methods are optimal for progenitor cells from UCB has not been investigated systematically. Optimal cryopreservation protocols utilising penetrating cryoprotectants require that a number of major factors are controlled: osmotic damage during the addition and removal of the cryoprotectant; chemical toxicity of the cryoprotectant to the target cell and the interrelationship between cryoprotectant concentration and cooling rate. We have established addition and elution protocols that prevent osmotic damage and have used these to investigate the effect of multimolar concentrations of Me(2)SO on membrane integrity and functional recovery. We have investigated the effect of freezing and thawing over a range of cooling rates and cryoprotectant concentrations. CD34(+) cells tolerate up to 60 min exposure to 25% w/w (3.2M) Me(2)SO at +2 degrees C with no significant loss in clonogenic capacity. Exposure at +20 degrees C for a similar period of time induced significant damage. CD34(+) cells showed an optimal cooling range between 1 degrees C and 2.5 degrees C/min. At or above 1 degrees C/min, increasing the Me(2)SO concentration above 10% w/w provided little extra protection. At the lowest cooling rate tested (0.1 degrees C/min), increasing the Me(2)SO concentration had a statistically significant beneficial effect on functional recovery of progenitor cells. Our findings support the conclusion that optimal recovery of CD34(+) cells requires serial addition of Me(2)SO, slow cooling at rates between 1 degrees C and 2.5 degrees C/min and serial elution of the cryoprotectant after thawing. A concentration of 10% w/w Me(2)SO is optimal. At this concentration, equilibration temperature is unlikely to be of practical importance with regard to chemical toxicity.     

Increased DNA single-strand break joining activity in UV-irradiated CD34+ versus CD34- bone marrow cells

The kinetics of UV-irradiation-induced (254 nm) DNA single-strand breaks (SSBs) were studied in single human hematopoietic cells using alkaline comet assay. Three cell populations were investigated: (i) Bone marrow mononuclear cells (BMMNCs) isolated by density gradient centrifugation, (ii) CD34- cells, and (iii) CD34+ cells. The two latter populations were purified from BMMNCs by negative and positive selection, respectively, using anti-CD34 immunobeads. SSBs were induced faster by 10 and 50 J/m2 than by 2 J/m2 and those caused by 2 J/m2 were joined faster that those caused by 10 or 50 J/m2. During the first 1.5 h after irradiation with a dose of 10 J/m2, CD34+ cells joined SSBs faster than did BMMNCs. The superior joining capacity of CD34+ cells was further substantiated with a higher UV dose. The comet lengths, indicating the extent of DNA repair, among 8/8 study subjects were shorter in CD34+ than in CD34- cells when assessed 24 h after a dose of 50 J/m2. Overall, the comet lengths at 24 h after irradiation were: CD34+ cells; 39+/-12 *m, and CD34- cells; 65+/-18 *m (8 subjects, 50 cells measured from each donor, mean+/-S.D.; p=0.0087, Mann-Whitney U-test). These results strongly suggest that nucleotide excision repair, the major mechanism responsible for the repair of UV-irradiation-induced DNA lesions in mammalian cells, is increased in CD34+ cells compared with CD34- cells and with BMMNCs. These results may have implications in stem cell purging, clinical chemotherapy and carcinogenesis.     

人造血干／祖细胞多态性的研究：Ⅷ.人正常骨髓CD34^＋造血细胞体视学的特征

人ＣＤ３４＋造血细胞是具有高度自我更新,多向分化及重建长期造血与免疫学功能的独特体细胞。为系统探索ＣＤ３４＋造血细胞的形态,细胞化学及超微结构特征,新近我们设计组合并建立了ＣＩＭＳ－１００ＦＡＣＳ４４０无菌二次分选术,可使所获ＣＤ３４＋５造血的纯度达１００％。     

A French single-center experience on allogeneic stem cell transplant cryopreservation during severe acute respiratory syndrome coronavirus 2 pandemic

Background aimsAllogeneic hematopoietic stem cell transplantation (allo-SCT) is a curative treatment for chemo-resistant hematological malignancies. Because of transport restriction imposed by the coronavirus disease 2019 pandemic, regulatory bodies and societies recommended graft cryopreservation before recipient conditioning. However, the freezing and thawing processes, including washing steps, might impair CD34+ cell recovery and viability, thereby impacting the recipient engraftment. Over 1 year (between March 2020 and May 2021), we aimed to analyze the results of frozen/thawed peripheral blood stem cell allografts in terms of stem cell quality and clinical outcomes.MethodsTransplant quality was evaluated by comparing total nucleated cells (TNCs), CD34+ cells and colony-forming unit–granulocyte/macrophage (CFU-GM)/kg numbers as well as TNC and CD34+ cell viabilities before and after thawing. Intrinsic biological parameters such as granulocyte, platelet and CD34+ cell concentrations were analyzed, as they might be responsible for a quality loss. The impact of the CD34+ cell richness of the graft on TNC and CD34 yields was evaluated by designing three groups of transplants based on their CD34 /kg value at collection: >8 × 10 ⁶/kg, between 6 and 8 × 10⁶/kg and <6 × 10⁶/kg. The consequences of cryopreservation were compared in the fresh and thawed group by evaluating the main transplant outcomes.ResultsOver 1 year, 76 recipients were included in the study; 57 patients received a thawed and 19 patients a fresh allo-SCT. None received allo-SCT from a severe acute respiratory syndrome coronavirus 2–positive donor. The freezing of 57 transplants led to the storage of 309 bags, for a mean storage time (between freezing and thawing) of 14 days. For the fresh transplant group, only 41 bags were stored for potential future donor lymphocyte infusions. Regarding the graft characteristics at collection, median number of cryopreserved TNC and CD34+ cells/kg were greater than those for fresh infusions. After thawing, median yields were 74.0%, 69.0% and 48.0% for TNC, CD34+ cells and CFU-GM, respectively. The median TNC dose/kg obtained after thawing was 5.8 × 10⁸, with a median viability of 76%. The median CD34+ cells/kg was 5 × 10⁶, with a median viability of 87%. In the fresh transplant group, the median TNC/kg was 5.9 × 10⁸/kg, and the median CD34+ cells/kg and CFU-GM/kg were 6 × 10⁶/kg and 276.5 × 10⁴/kg, respectively. Sixty-one percent of the thawed transplants were out of specifications regarding the CD34+ cells/ kg requested cell dose (6 × 10⁶/kg) and 85% of them would have had this dose if their hematopoietic stem cell transplant had been infused fresh. Regarding fresh grafts, 15.8% contained less than 6 × 10⁶ CD34+ cells /kg and came from peripheral blood stem cells that did not reach 6 × 10⁶ CD34+ cells /kg at collection. Regarding the factor that impaired CD34 and TNC yield after thawing, no significant impact of the granulocyte count, the platelet count or the CD34+ cells concentration/µL was observed. However, grafts containing more than 8 × 10 ⁶/kg at collection showed a significantly lower TNC and CD34 yield.ConclusionsTransplant outcomes (engraftment, graft-versus-host disease, infections, relapse or death) were not significantly different between the two groups.     

CD34+ cells homing: quantitative expression of adhesion molecules and adhesion of CD34+ cells to endothelial cells exposed to shear stress

To investigate the function of the main adhesion receptors (CD62L, CD49d, CD49e, CD11b and CD18) on CD34+ cells during homing, their expression was quantified by flow cytometry using calibration beads. CD34+ cells were isolated from bone-marrow (BM), cord blood (CB) or peripheral blood (PB) from patients with myeloma. As this process might mimic the mature leukocyte migration, we also observed the effect of exposing endothelial cells to shear stress (7 dyn/cm(2)) on the adhesion of CB CD34+ cells.The proportion of CD34+/CD62L+ cells was greater in PB than in BM (p<0.05). Likewise, we found a significantly greater expression of CD62L receptor on PB cells compared to BM cells (p<0.05) and on BM cells compared to CB cells (p<0.05). The proportions of CD34+/CD49d+ cells and CD34+/CD49e+ cells were significantly higher in the BM and CB than in PB. However, no significant difference in CD49d or CD49e antigen densities was observed. The beta_2 integrins (CD11b and CD18) receptors are also implicated in CD34+ cells homing to BM. No significant variation in CD34+/CD11b+ and CD34+/CD18+ cells frequency was noted. However quantitative analysis revealed that CD18 was more strongly expressed on BM cells than on PB and CB cells.The adhesion assay showed that fluid flow may favour a firm adhesion of CB CD34+ cells to endothelial cells whereas static conditions just allowed CD34+ cells sedimentation.In conclusion, quantitative expression of the main receptors on CD34+ cells indicates that the three main sources of CD34+ cells currently used for transplantation have neither the same phenotype nor the same number of antigenic sites for a receptor. So, we hypothesize that migrational capacity of these cells might be different. Moreover, it seems that shear stress could favor adhesion of CD34+ cells to endothelial cells.     

A non-BRICHOS surfactant protein c mutation disrupts epithelial cell function and intercellular signaling

Background

Cryopreservation is the only widely applicable method of storing vital cells for nearly unlimited periods of time. Successful cryopreservation is essential for reproductive medicine, stem cell research, cord blood storage and related biomedical areas. The methods currently used to retrieve a specific cell or a group of individual cells with specific biological properties after cryopreservation are quite complicated and inefficient.

Results

The present study suggests a new approach in cryopreservation, utilizing the Individual Cell-based Cryo-Chip (i3C). The i3C is made of materials having appropriate durability for cryopreservation conditions. The core of this approach is an array of picowells, each picowell designed to maintain an individual cell during the severe conditions of the freezing - thawing cycle and accompanying treatments. More than 97% of cells were found to retain their position in the picowells throughout the entire freezing - thawing cycle and medium exchange. Thus the comparison between pre-freezing and post-thawing data can be achieved at an individual cell resolution. The intactness of cells undergoing slow freezing and thawing, while residing in the i3C, was found to be similar to that obtained with micro-vials. However, in a fast freezing protocol, the i3C was found to be far superior.

Conclusions

The results of the present study offer new opportunities for cryopreservation. Using the present methodology, the cryopreservation of individual identifiable cells, and their observation and retrieval, at an individual cell resolution become possible for the first time. This approach facilitates the correlation between cell characteristics before and after the freezing - thawing cycle. Thus, it is expected to significantly enhance current cryopreservation procedures for successful regenerative and reproductive medicine.     

HLA-DR+ CD38+ CD4+ T lymphocytes have elevated CCR5 expression and produce the majority of R5-tropic HIV-1 RNA in vivo

Percentages of activated T cells correlate with HIV-1 disease progression, but the underlying mechanisms are not fully understood. We hypothesized that HLA-DR(+) CD38(+) (DR(+) 38(+)) CD4(+) T cells produce the majority of HIV-1 due to elevated expression of CCR5 and CXCR4. In phytohemagglutinin (PHA)-stimulated CD8-depleted peripheral blood mononuclear cells (PBMC) infected with HIV-1 green fluorescent protein (GFP) reporter viruses, DR(-) 38(+) T cells constituted the majority of CCR5 (R5)-tropic (median, 62%) and CXCR4 (X4)-tropic HIV-1-producing cells (median, 61%), although cell surface CCR5 and CXCR4 were not elevated in this subset of cells. In lymph nodes from untreated individuals infected with R5-tropic HIV-1, percentages of CCR5(+) cells were elevated in DR(+) 38(+) CD4(+) T cells (median, 36.4%) compared to other CD4(+) T-cell subsets (median values of 5.7% for DR(-) 38(-) cells, 19.4% for DR(+) 38(-) cells, and 7.6% for DR(-) 38(+) cells; n = 18; P < 0.001). In sorted CD8(-) lymph node T cells, median HIV-1 RNA copies/10(5) cells was higher for DR(+) 38(+) cells (1.8 × 10(6)) than for DR(-) 38(-) (0.007 × 10(6)), DR(-) 38(+) (0.064 × 10(6)), and DR(+) 38(-) (0.18 × 10(6)) subsets (n = 8; P < 0.001 for all). After adjusting for percentages of subsets, a median of 87% of viral RNA was harbored by DR(+) 38(+) cells. Percentages of CCR5(+) CD4(+) T cells and concentrations of CCR5 molecules among subsets predicted HIV-1 RNA levels among CD8(-) DR/38 subsets (P < 0.001 for both). Median HIV-1 DNA copies/10(5) cells was higher in DR(+) 38(+) cells (5,360) than in the DR(-) 38(-) (906), DR(-) 38(+) (814), and DR(+) 38(-) (1,984) subsets (n = 7; P ≤ 0.031). Thus, DR(+) 38(+) CD4(+) T cells in lymph nodes have elevated CCR5 expression, are highly susceptible to infection with R5-tropic virus, and produce the majority of R5-tropic HIV-1. PBMC assays failed to recapitulate in vivo findings, suggesting limited utility. Strategies to reduce numbers of DR(+) 38(+) CD4(+) T cells may substantially inhibit HIV-1 replication.     

Macrophage-inflammatory protein-1alpha receptor expression on normal and chronic myeloid leukemia CD34+ cells

We have assessed expression of MIP-1alpha binding sites on the surface of CD34+ cells from normal bone marrow (NBM) and chronic myeloid leukemia (CML) peripheral blood. This study has highlighted a small subpopulation of CD34+ (15.7 +/- 6.2% in NBM and 9 +/- 4% in CML), which has specific macrophage-inflammatory protein-1alpha (MIP-1alpha) cell surface binding sites. Further phenotypic characterization of the receptor-bearing cells has shown that they do not express the Thy-1 Ag, suggesting that they are committed progenitor cells rather than CD34+ Thy+ stem cells. However, more than 80% of methanol-fixed CD34+ cells do bind MIP-1alpha, suggesting that these cells may possess a pool of internal receptors, although we were unable to induce cell surface expression by cytokine stimulation. The percentage of these CD34+, MIP-1alpha-R+ cells present in the CD34 compartment of NBM is significantly higher than in CML, implicating lack of binding sites as part of the mechanism for the loss of response to this chemokine seen in CML. Specific Ab to the MIP-1alpha receptor implicated in HIV infection, CCR5, revealed that very few CD34+ cells expressed these receptors and that expression was confined to the CD34+ Thy- progenitor population. Data presented in this work suggest that active binding sites for the stem cell growth inhibitor MIP-1alpha are not constitutively expressed on the surface of most resting primitive multipotent cells, and that these cells are not potential targets for HIV-1 infection through CCR5.     

CD34+ stem cells in chronic myeloproliferative disorders

Contrasting the wealth of information that is available about various biological and therapeutic aspects of human CD34+ stem cells, little data exist concerning their quantity and dynamics as well as their mutual relationships with other hematopoietic constituents in the bone marrow of patients with chronic myeloproliferative disorders. In comparison with a control group frequency of progenitors is significantly increased in chronic myeloid leukemia (CML). Following different therapeutic modalities their quantity reflects therapeutic efficacy (responder and non-responder patients) and therefore exerts a predictive value regarding acceleration and blastic crisis. The significant correlations between fiber content and number of these precursors elucidates the complex interactions between stroma and progenitor cell differentiation and maturation. Following allogeneic bone marrow transplantation there is a rapid recovery of the CD34+ stem cell population in the first month. A higher number of these cells is related with graft size, an earlier independence for platelet transfusion and a more extended regeneration of erythro- and megakaryopoiesis. The slight increase in reticulin fibers in these patients may be associated with the complex and so far ill-defined pathomechanism of homing (adherence to the fibrous matrix). In idiopathic myelofibrosis (IMF) an increased number of CD34+ stem cells is found predominantly in the early (prefibrotic or mild fibrotic) hypercellular stages and probably indicates a higher proliferative activity of the precursor cell pool. According to sequential biopsies most patients with early IMF that later evolved into an overt fibrosclerotic stage usually display a reduction of progenitor cells during the development of myelofibrosis. The unequal distribution of CD34+ stem cells in the bone marrow versus spleen in IMF (advanced fibrosclerotic stage) is in support of the currently discussed hypothesis of splenic filtration and concentration of precursor cells as an essential feature of myeloid metaplasia. Regarding prognosis in CML a higher amount of CD34+ stem cells is significantly associated with an unfavorable survival and thus confirms the assumed implication of an accelerated phase of disease at onset. On the other hand, in polycythemia vera (PV) and IMF a low number of progenitors is probably due to a decreased proliferation rate (reduced hematopoietic turnover index) and therefore reflects a reduction in the regenerative capacity of hematopoiesis. For this reason, a presumptive defect in the recovery of normal and clonally transformed stem cells is speculated to add to the worsening of prognosis by causing the well-known bone marrow insufficiency in terminal stage PV and IMF.     

Mobilization of CD34+-Progenitor Cells in Patients with Severe Trauma

Circulating CD34+ progenitor cells () gained importance in the field of regenerative medicine due to their potential to home in on injury sites and differentiate into cells of both endothelial and osteogenic lineages. In this study, we analyzed the mobilization kinetics and the numbers of CD34+, CD31+, CD45+, and CD133+ cells in twenty polytrauma patients (n = 13 male, n = 7 female, mean age 46.5±17.2 years, mean injury severity score (ISS) 35.8±12.5 points). In addition, the endothelial differentiation capacity of enriched CD34+cells was assessed by analyzing DiI-ac-LDL/lectin uptake, the expression of endothelial markers, and the morphological characteristics of these cells in Matrigel and spheroid cultures. We found that on days 1, 3, and 7 after a major trauma, the number of CD34+cells increased from 6- up to 12-fold (p<0.0001) over the number of CD34+cells from a control population of healthy, age-matched volunteers. The numbers of CD31+ cells were consistently higher on days 1 (1.4-fold, p<0.01) and 7 (1.3-fold, p<0.01), whereas the numbers of CD133+ cell did not change during the time course of investigation. Expression of endothelial marker molecules in CD34+cells was significantly induced in the polytrauma patients. In addition, we show that the CD34+ cell levels in severely injured patients were not correlated with clinical parameters, such as the ISS score, the acute physiology and chronic health evaluation II score (APACHE II), as well as the sequential organ failure assessment score (SOFA-2). Our results clearly indicate that pro-angiogenic cells are systemically mobilized after polytrauma and that their numbers are sufficient for the development of novel therapeutic models in regenerative medicine.