Analytical performance verification protocols and specifications of CD34 +cell enumeration by flow cytometry / 中华检验医学杂志

Objective:To investigate the analytical performance verification protocols and performance specifications of CD34+cell enumeration by flow cytometry for clinical laboratories.Methods:According to international guidelines and National Health Standard of China, we designed the performance verification protocols of CD34 +cell enumeration (including percent count and absolute count) by flow cytometry. Four quality assessment materials, three leukapheresis products and three samples of peripheral blood were selected to verify the precision, linearity, carryover, trueness and accuracy of FACSCanto Ⅱ measurement system, and the assessment criterion was set according to the detection technologies of clinical laboratories. Results:The CVs of intra-run precision of percent count and absolute count were 2.5% to 8.9% and 3.0% to 9.0%; the CVs of inter-run precision were 2.8% to 10.5% and 3.8% to 9.9%, respectively. The slopes of linearity regression equation of low range (3.6/μl to 123.6/μl) and high range (113.2/μl to 1196.3/μl) were 0.993 2 and 0.965 2, and R2 were 0.999 6 and 0.993 9, and the biases were -8.67% to 0.22%. The carryover of percent and absolute count were 0.07% and 0.00%. When percent count≤0.2% or absolute count≤20/μl, the absolute biases of trueness were in the range of ±0.006% or ±0.5/μl, and the absolute biases of accuracy were in the range of ±0.02% or ±0.9/μl; when percent count>0.2% or absolute count>20/μl, the relative biases of trueness were in the range of ±5.65%, and the relative biases of accuracy were in the range of ±8.19%. The verification results met the assessment criterion set in this study. Conclusions:The performance verification protocols and assessment criterion formulated in this study not only conform to the recommendations of domestic and foreign guidelines, but also conform to state of the detection technologies of native clinical laboratories, which can be taken as a reference of performance verification for clinical laboratories.

Effect of hydrogen-rich water on the CD34 expression in lesion boundary brain tissue of rats with traumatic brain injury / 中华危重病急救医学

Objective To observe the effect of hydrogen-rich water on the CD34 expression and angiogenesis in lesion boundary brain tissue of rats with traumatic brain injury (TBI).Methods A total of 54 adult male SpragueDawley (SD) rats were divided into three groups by random number table:namely sham-operated group (sham group),trauma group (TBI group),and trauma + hydrogen-rich water group (TBI+HW group),the rats in each group were subdivided into 1,3 and 7 days subgroups according to the time points after trauma,with 6 rats in each subgroup.The TBI model was reproduced by using a modified Feency method for free fall impact,and the rats in sham group were not given brain impact after craniotomy.The rats in TBI+HW group were given intraperitoneal injection of hydrogen-rich water (5 mL/kg) after TBI model reproduction,and then once a day until being sacrificed,and the rats in sham group and TBI group were given the same amount of normal saline.The neurological severity scores (NSS) for neurologic deficits were calculated at corresponding time points,and then the rats were sacrificed for brain tissue at 3 mm around lesion boundary.After hematoxylin-eosin (HE) staining,the pathological changes in lesion boundary brain tissue were observed under light microscope.The expression of CD34+ cells was observed by immunohistochemical analysis,which markers were used to count the newborn blood capillary sprouts around the traumatic brain tissue.The protein expression of CD34was determined by Western Blot.Results NSS scores at all time points in sham group were 0.NSS scores in TBI and TBI+HW groups showed a decreased tendency with time prolongation after TBI,which showed more significant in TBI+HW group,NSS scores at 3 days and 7 days were significantly lower than those of TBI group (3 day:8.67 ± 0.52 vs.11.56 ± 1.94,7 days:7.33±0.52 vs.8.17±0.98,both P ＜ 0.05).Under light microscope,the brain tissue of rats in sham group was normal.After injury,pathological changes in lesion boundary brain tissue in TBI group were characterized by obvious hemorrhagic necrosis,severe brain edema,a large number of degeneration and necrosis of nerve cells and inflammatory cell infiltration,and the pathological changes were more obvious at 3 days.The edema area in TBI+HW group was slightly smaller than that of TBI group,and the surrounding edema was slightly reduced.It was shown by immunohistochemistry that only a very small number of neoformative capillaries were found in sham group.The number of neoformative capillaries in lesion boundary brain tissue was gradually increased with time prolongation in TBI group.The number of neoformative capillaries in TBI+HW group was more significantly,which was significantly higher than that of TBI group at 3 days and 7 days after injury (cells/HP:10.59 ± 1.88 vs.8.61 ± 1.22 at 3 days,23.20 ± 3.16 vs.17.01 ± 2.64 at 7 days,both P ＜ 0.05).It was shown by Western Blot that the expression of CD34 protein at all time points in TBI group was significantly increased as compared with that of sham group.The expression of CD34 protein at 1 day and 3 days in TBI+HW group was slightly increased as compared with that of TBI group without significant difference,but it was significantly up-regulated at 7 days after injury,which was significantly higher than that of TBI group (gray value:1.36 ± 0.36 vs.0.74±0.08,P ＜ 0.05).Conclusion Hydrogen-rich water promote CD34+ cells home to the site of injured tissue in rats with TBI,is involved in angiogenesis,and improve clinical outcomes during brain functional recovery.

Clinical Applicability of Newly Developed Image-based Cell Counter for Counting CD34+ Cells: Comparison with Flow Cytometric Analysis / 임상소아혈액종양

BACKGROUND: Flow cytometric analysis is the standard method for enumerating CD34+ stem cells in hematopoietic stem cell transplantation. However, it has some limitations such as expensive instrumentation, high reagent costs, and discrepancies between technicians and laboratories. We compared counts of total nucleated cells (TNCs) and CD34+ cells counts obtained from a flow cytometer with a newly-developed image-based microscopic cell counter (ADAM II) to evaluate the possibility of clinical application of the ADAM II.METHODS: We used 18 samples of circulating peripheral blood (PB) and waste tube fractions of peripheral blood stem cells (PBSCs) harvested by apheresis after G-CSF mobilization from adult volunteer donors. We assessed the reproducibility and linearity of the new procedure and compared the numbers of TNCs and viable CD34+ cells determined with the ADAM II and two different flow cytometers (FACSCalibur, FACSCanto II).RESULTS: Numbers of viable CD34+ cells determined with the ADAM II were accurate over the expected range; the intra-assay coefficient of variation was ≤19.8%. Linearity was also satisfactory (R²=0.99). TNC counts obtained with the ADAM II were highly correlated with those obtained with the FACSCalibur (R²>0.9841, P<0.0001) and FACSCanto II (R²>0.9620, P<0.0001), as were the numbers of viable CD34+ cells obtained with the ADAM II and the FACSCalibur and FACSCanto II (R²>0.9911, P<0.0001 and R²>0.9791, P<0.0001), respectively.CONCLUSION: The newly developed image-based microscopic cell counter (ADAM II) appears to be suitable for enumerating TNCs and viable CD34+ cells.

Clinical Applicability of Newly Developed Image-based Cell Counter for Counting CD34+ Cells: Comparison with Flow Cytometric Analysis / 임상소아혈액종양

BACKGROUND: Flow cytometric analysis is the standard method for enumerating CD34+ stem cells in hematopoietic stem cell transplantation. However, it has some limitations such as expensive instrumentation, high reagent costs, and discrepancies between technicians and laboratories. We compared counts of total nucleated cells (TNCs) and CD34+ cells counts obtained from a flow cytometer with a newly-developed image-based microscopic cell counter (ADAM II) to evaluate the possibility of clinical application of the ADAM II. METHODS: We used 18 samples of circulating peripheral blood (PB) and waste tube fractions of peripheral blood stem cells (PBSCs) harvested by apheresis after G-CSF mobilization from adult volunteer donors. We assessed the reproducibility and linearity of the new procedure and compared the numbers of TNCs and viable CD34+ cells determined with the ADAM II and two different flow cytometers (FACSCalibur, FACSCanto II). RESULTS: Numbers of viable CD34+ cells determined with the ADAM II were accurate over the expected range; the intra-assay coefficient of variation was ≤19.8%. Linearity was also satisfactory (R²=0.99). TNC counts obtained with the ADAM II were highly correlated with those obtained with the FACSCalibur (R²>0.9841, P0.9620, P0.9911, P0.9791, P<0.0001), respectively. CONCLUSION: The newly developed image-based microscopic cell counter (ADAM II) appears to be suitable for enumerating TNCs and viable CD34+ cells.

In-Vitro Stem Cell Derived Red Blood Cells for Transfusion: Are We There Yet?

To date, the use of red blood cells (RBCs) produced from stem cells in vitro has not proved practical for routine transfusion. However, the perpetual and widespread shortage of blood products, problems related to transfusion-transmitted infections, and new emerging pathogens elicit an increasing demand for artificial blood. Worldwide efforts to achieve the goal of RBC production through stem cell research have received vast attention; however, problems with large-scale production and cost effectiveness have yet to prove practical usefulness. Some progress has been made, though, as cord blood stem cells and embryonic stem cells have shown an ability to differentiate and proliferate, and induced pluripotent stem cells have been shown to be an unlimited source for RBC production. However, transfusion of stem cell-derived RBCs still presents a number of challenges to overcome. This paper will summarize an up to date account of research and advances in stem cell-derived RBCs, delineate our laboratory protocol in producing RBCs from cord blood, and introduce the technological developments and limitations to current RBC production practices.

Successful Selective CD34+ Cell Infusion after Late Graft Failure of Hematopoietic Stem Cell Transplantation in Two Cases of Severe Aplastic Anemia / 임상소아혈액종양

Matched sibling bone marrow transplantation (BMT) in severe aplastic anemia (SAA) has been known as the treatment of choice in children and young adults. To overcome graft failure, second stem cell transplantation showed good results in previous studies. Here we report two cases of aplastic anemia patients with late graft failure and resulted in successful complete recovery after selective CD34+ cell boost infusion. The patients previously underwent allogeneic BMT from HLA-matched sibling donors and the engraftment was achieved although their CBC started to decrease respectively 3 months and 11 months after transplantation. Both patients received selective CD34+ cell infusion without additional conditioning therapy. Their CBC showed significant improvement and they are doing well without transfusion or complications. From this study we suggest that selected CD34+ cell boost treatment can be a promising curative treatment for late graft failure after matched sibling BMT in SAA patients.

Successful Selective CD34+ Cell Infusion after Late Graft Failure of Hematopoietic Stem Cell Transplantation in Two Cases of Severe Aplastic Anemia / 임상소아혈액종양

Matched sibling bone marrow transplantation (BMT) in severe aplastic anemia (SAA) has been known as the treatment of choice in children and young adults. To overcome graft failure, second stem cell transplantation showed good results in previous studies. Here we report two cases of aplastic anemia patients with late graft failure and resulted in successful complete recovery after selective CD34+ cell boost infusion. The patients previously underwent allogeneic BMT from HLA-matched sibling donors and the engraftment was achieved although their CBC started to decrease respectively 3 months and 11 months after transplantation. Both patients received selective CD34+ cell infusion without additional conditioning therapy. Their CBC showed significant improvement and they are doing well without transfusion or complications. From this study we suggest that selected CD34+ cell boost treatment can be a promising curative treatment for late graft failure after matched sibling BMT in SAA patients.

Successful Selective CD34+ Cell Infusion after Late Graft Failure of Hematopoietic Stem Cell Transplantation in Two Cases of Severe Aplastic Anemia / 임상소아혈액종양

Matched sibling bone marrow transplantation (BMT) in severe aplastic anemia (SAA) has been known as the treatment of choice in children and young adults. To overcome graft failure, second stem cell transplantation showed good results in previous studies. Here we report two cases of aplastic anemia patients with late graft failure and resulted in successful complete recovery after selective CD34+ cell boost infusion. The patients previously underwent allogeneic BMT from HLA-matched sibling donors and the engraftment was achieved although their CBC started to decrease respectively 3 months and 11 months after transplantation. Both patients received selective CD34+ cell infusion without additional conditioning therapy. Their CBC showed significant improvement and they are doing well without transfusion or complications. From this study we suggest that selected CD34+ cell boost treatment can be a promising curative treatment for late graft failure after matched sibling BMT in SAA patients.

Cutoff Value of PB CD34+ Cells for Optimization of PBSC Collection / 대한수혈학회지

BACKGROUND: Peripheral hematopoietic stem cell mobilization is increasing due to its advantages. For successful engraftment, obtaining sufficient stem cells is prerequisite. The number of CD34+ cells of collected blood are widely used to predict the engraftment potential. To determine the optimal point for collection of peripheral blood stem cell (PBSC), enumeration of the number of CD34+ cells in peripheral blood (PB) is known to be helpful. The purpose of this study is to analyze cutoff value of CD34+ cells in PB. METHODS: We analyzed 407 cases of autologous PBSC collection and 107 cases of allogenic PBSC collection during 2004~2009 in Pusan National University Hospital. Complete blood count, HPC fraction and number, CD34+ cells in PB and product of PBSC collection were analyzed. RESULTS: The each number of mononuclear cells and HPC in PB showed a strong correlation with CD34+ cells in PB. A strong correlation between the number of circulation CD34+ cells in PB on the day of collection and the number of collected CD34+ cells was found. The ROC curve revealed that the cutoff point having the optimal sensitivity and specificity at 8.5/uL for target CD34+ cells > or =1.0x10(6)/kg, 10.5/uL for target CD34+ cells > or =1.5x10(6)/kg and 13.5/uL for target CD34+ cells > or =2.0x10(6)/kg in this study. CONCLUSION: To obtain a sufficient yield of CD34+ cells during PBSC collection, determination of cut off point for each target CD34+ cells//kg is helpful to decide the collection.

Comparison between clinical results of peripheral blood stem cell transplantation from HLA-matched sibling donors mobilized with different regimens / 中华器官移植杂志

Objective To retrospectively review and compare the clinical results of allogeneic peripheral blood stem cell transplantation (allo-PBSCT) from HLA- matched sibling donors mobilized with different regimens. Methods Seventy-one patients with hematological malignant diseases received allo-PBSCT from HLA-matched sibling donors in our department. Among them, 24 received allografts mobilized with G-CSF (group G), and the remaining (47 cases) were mobilized with G-CSF and GM-CSF (group G+ M). CD34+ subsets and T cell subsets in the allografts were analyzed, and the time of hematopoietic reconstitution and the incidence of graft versus host diseases (GVHD) were compared. The adverse effects on the donors after mobilization were also observed. Results The enough targeted CD34+ cells in all donors were harvested by 1-3 aphereses. Ninety-six h after mobilization, WBC counts of the donors were significantly higher in group G than in group G + M [(49. 6± 19. 5) 109/L vs (25.4 ± 10. 4) 109/L, P＜0. 05]. Analysis of the CD34+ subsets showed that the percentage of cells with the CD34+/CD38- phenotype was significantly higher in group G + M than in group G [(37. 7 ± 5. 7) % vs (31.4 ± 4. 5) %, P＜0. 05]. There was no significant difference in T cells and subsets of grafts. There was no significant difference in the number of total CD34+ cells and CD34+ CD38- cells, and infusion of T cells between two groups. The days required for the recovery of neutrophils and platelets was inversely correlated with the infused CD34+ and CD34+ /CD38- cell number. There was no significant difference in incidence of acute and chronic GVHD between two recipient groups. Seventeen cases and 10 eases among 71 eases died of relapses of primarydiseases, and complications of transplantation such as severe GVHD and infections respectively.Fourteen cases in group G (58.3 %) and 31 cases in group G+ M (66.0 %) survived. The most common adverse events in the donors were bone pain and fever, which mostly occurred 36 h after mobilization and could be relieved by non-steroidal anti-inflammatory drugs. Conclusion Two mobilization regimens showed equivalent clinical results. But the combined regimen of G-CSF and GM-CSF demonstrated a significantly greater mobilization of cells with the CD34+/CD38- phenotype.Meanwhile in allogeneic PBSCT, a greater number of total CD34+ cells and CD34+ CD38- cells infused may be associated with faster hematopoietic reconstitution of recipients.

Factors Influencing Peripheral Blood Stem Cell Collection / 종양간호학회지

PURPOSE: Peripheral blood stem cell transplantation (PBSCT) has been widely used. The optimal time for collection is a critical factor to obtain proper counts of CD34 cell by peripheral blood stem cell collection (PBSC). The purpose of this study was to identify the factors influencing peripheral blood stem cell collection in order to figure out the more effective timing for PBSC. METHOD: The subjects of this study were 189 patients undergoing 3 leukapheresis from January 28, 2005 to December 31, 2006. Group's characteristics, checkup opinion of pre-peripheral blood on the day of harvest & outcome of PBSC were analyzed and evaluated using SAS statistics program after grouping patients as below; group 1-CD34 cell counts or =4x10(6)/kg (n=63). RESULTS: Based on outcome of peripheral blood stem cell according to diagnosis, acute myelocytic leukemia (AML) was 65.5% at Group 1, Lymphoma was 21.7% at Group 2 and multiple myeloma (MM) was 70.8% at Group 3. There were significant differences in CD34 cell counts according to diagnosis (p=0.00004). Type of cytokine mobilization according to diagnosis, Lenograsim was using 62.5% of MM & 38.2% of AML and filgrastim is using 22.0% of AML only. Circular peripheral blood CD34 cell counts prior to harvest was 258.1/microliter at Group 3 which was much higher comparing to Group 1 (10.5/microliter) and Group 2 (39.9/microliter) (p<0.001). TNC counts of collected peripheral blood stem cell was 15.36x10(6)/kg at Group 3 microliter and it's much higher than Group 2 (13.16x10(6)/kg) and Group 1 (12.36x10(6)/kg) (p=0.083). There was no significant difference in MNC counts inbetween 3 groups. CONCLUSIONS: Circular peripheral blood CD34+ cell counts prior to harvest was much higher at Group 3 than Group 1 and Group 2. Therefore, the number of CD34+ cells on the day of harvest can be used as an accurate predictor for peripheral blood stem cell.

Reconstitution of Human Immune Cells with Co-transplantation of Fetal Liver/Thymus Tissues and Cultured Umbilical Cord Blood-derived Hematopoietic Stem Cells in Rag2(-/-)gamma(c)(-/-) Mice

PURPOSE: Many researchers have tried to develop animal models that mimic the human immune system, e.g. a humanized mouse model, to improve the engraftment of hematopoietic stem cells and develop human immune cells in an animal model. This study evaluated the feasibility of the cultured human umbilical cord blood (hUCB)-derived CD34(+) cells for cell expansion, in Rag2(-/-)gamma(c)(-/-) mice, and establish co-transplantation with human fetal thymus/liver tissue (Thy/Liv) under the kidney capsule. METHODS: Co-transplantation of hUCB-derived CD34(+) cells with Thy/Liv was performed. The hUCB-derived CD34(+) cells were prepared by freshly thawing (G1) and culturing for 7 days with two types of cytokine combinations (G2, G3). The CD45(+) cell populations were measured at 6, 8, 10 and 16 weeks in the peripheral blood. The splenocytes were cultured with mitogenic stimuli (PHA -L or IL-2) at 20 weeks post- transplantation, and the proliferation of human immune cells was evaluated. RESULTS: There were no significant differences in the human CD45(+) cell populations at 6, 8, 10 and 16 weeks post-transplantation between the groups. In the cultured splenocytes at 20 weeks post-transplant with PHA-L or IL-2, there was remarkable expansion of CD3(+) cells in the three groups. Although no CD19(+) cells were detected in the spleen, human Ig G was detected in the sera of these mice. CONCLUSION: The cultured and expanded hUCB-derived cells with cytokine combinations might be a feasible cell source in humanized mouse modeling. In addition, human immune cells can be reconstituted from the co-transplantation of Thy/Liv and cultured hUCB-derived CD34(+) cells.

Enforced Expression of BMI-1 in Postnatal Human CD34+ Cells Promotes Erythroid Differentiation / 대한혈액학회지

BACKGROUND: The Polycomb-group gene Bmi-1 is known to be a molecular regulator of self-renewal of normal and leukemic stem cells and be involved in various aspects of cellular proliferation, differentiation, and survival. METHODS: This study evaluated the effects of overexpression of Bmi-1 on human cord blood CD34+ cells. Bmi-1 was introduced into CD34+ cells through lentivirus transduction. Bmi-1 expressing CD34+ cells were applied to colony forming assay, stromal co-culture, and cytokine-stimulatied culture. RESULTS: Ectopic expression of Bmi-1 resulted in the increased number of erythroid colonies in primary and secondary colony forming assay in an erythropoietin dependent manner. In stromal co-culture, Bmi-1-expressing postnatal hematopoietic stem cells seemed to lose the ability of self-renewal, as determined by week 5 cobblestone area-forming cell assay and by week 5 secondary colony assay. In cytokine-stimulated suspension culture of Bmi-1-transduced CD34+ cells, we observed increased erythropoiesis marked by Glycophorin A expression. CONCLUSION: Our data suggest that ectopic expression of Bmi-1 in human hematopoietic stem/progenitor cells may result in the differentiation to the erythroid lineage rather than promoting self-renewal.

Cord blood-derived CD34(+) cells promotes functional recovery in transient middle cerebral artery occlusion model of rat / 대한산부인과학회지

OBJECTIVE: This study was designated to determine the effect of cord blood cell transplantation in ischemic injury model. METHODS: In this study, we administered human umbilical cord blood (hUCB)-derived CD34(+) cells into the lateral ventricle or directly into the striatum and assessed cell migration in mice with cryoinjury and behavioral recovery in rats with transient middle cerebral artery occlusion (MCAo). CD34(+) cells were isolated by magnetic cell sorting using CD34-microbeads and labeled with CM-Dil. RESULTS: When CD34(+) cells were injected into mice brain with cryoinjury, cells were migrated into a injury site after one week of injection. Similarly, injected CD34(+) cells were migrated into the periphery of infarcted area in rats with transient MCAo. When spontaneous activity was measured using a modified neurological severity score (mNSS), it was found that functional recovery was significantly higher when CD34(+) human umbilical cord blood cell (hUCBC) was transplanted 24 hours after stroke compared with phosphate buffered saline (PBS)-injected or CD34(-) transplanted, stroked animals (P<0.05). Although only small portion of transplanted cells were differentiated into neural lineages, CD34(+) hUCBC transplantation increased Brdu incorporation and recruitment of doublecortin (DCX) (+) cells in ischemic boundary zone. CONCLUSION: These results suggest that hUCBC transplantation may be an effective treatment for brain injuries, such as stroke, or neurodegenerative disorders by promoting endogenous repair process of the brain.

The Correlation between G-CSF Dosage and the Number of Collected CD34+ Cells in Pediatric Patients Undergoing Peripheral Blood Stem Cell Mobilization / 대한혈액학회지

BACKGROUND: In high risk malignancies of pediatric patients, many investigators have explored the use of stronger myeloablative regimens with autologous peripheral blood stem cell rescue such as the Tandem protocol. As the collection of peripheral blood stem cells in children have many risks, it is important to achieve the maximum number of CD34+ cells per leukapheresis. We analyzed the use of different doses of granulocyte-colony stimulating factor during a mobilization for an increased number of CD34+ cells. METHODS: A retrospective chart review was performed for 31 patients undergoing autologous peripheral blood stem cell mobilization. All patients had received specific chemotherapies. At the nadir, each patient was injected with 5 microgram/kg/day G-CSF. When the level of white blood cells reached more than 1,000/microliter, patients were divided according to the different dose of G-CSF that was given per day: Group I, 5 microgram/kg, Group II, 10 microgram/kg, Group III, 15 microgram/kg. After 2~3 days, leukapheresis was performed, and then the number of CD34+ cells and other cells were counted. RESULTS: In group III, the number of collected CD34+ cells was 19.86+/-14.45x10(6)/kg and significantly higher than in the other two groups. Also, group III had significantly higher numbers of total nucleated cell, mononucleated cells, white blood cells, though the absolute neutrophil count compared with the other groups. There were no serious adverse effects associated with the higher G-CSF doses employed. CONCLUSION: At nadir, a pediatric patient received 5 microgram/kg G-CSF daily until the WBC count reached more than 1,000/ microliter. If the patient is injected with 15 microgram/kg/day of G-CSF, we can achieve a more sufficient CD34+ cell yield with one leukapheresis, safely.

Expression of Fas/FasL in Children with Aplastic Anemia and Its Clinical Significance / 实用儿科临床杂志

Objective To investigate the expression of Fas/FasL in children with aplastic anemia(AA) and its clinical significance.To explore the pathogenesis of AA.Methods CD34+ cell counts and the expression of Fas/FasL in 12 children with severe AA(SAA),18 with chronic AA(CAA)and 10 normal children were detected by flow cytometry combining monocolon antibody.Results The expression of Fas in children with AA were significantly higher than that in control group(P0.05).Conclusions Fas/FasL systeam take part in inducing CD34+ cell apoptosis.This may contribute to understanding the decrease number of stem cells and bone marrow failure.

Effect of LIF gene modified ECV-304 cells on the ex vivo culture of HSC/HPC in cord blood / 临床检验杂志

Objective To study the effect of the ECV-304 cells modified with LIF gene on the ex vivo culture of HSC/HPC in cord blood.Methods The ECV-304 cells were infected by Eukaryotic Expression plasmid pcDNA3.0LIF,and the positive ECV-304 cells were obtained by selected with G418.These cells were used to co-culture with CD34+ cells of cord blood.The phenotype of CD34+ and CD34+ CD54+、CD34+ CD62L+ primitive progenitors was detected by flow cytometry.Results The LIF gene can express in ECV-304 cells steadily.ECV-304 cells modified with LIF gene can improve expansion of CB CD34+、CD34+CD54+ and CD34+ CD62L+ cells while sustaining the expression of homing-related adhesion molecule.Conclusion The ECV-304 cells modified with LIF gene can not only significantly expand CB hematopoietic progenitor cells ex vivo,but the expanded CD34+ cells may well retain their homing ability.

Plating Efficiency of Single CD34+ Cell from Bone Marrow, Peripheral Blood and Cord Blood / 대한혈액학회지

BACKGROUND: Although bone marrow (BM) CD34+cells and peripheral blood (PB) CD34+ cells are developmentally and functionally related, the recent data suggested that they may have different functional and clinical capabilities. Moreover, they have differential gene expression underlying the functional distinctions of primary human CD34+ hematopoietic stem and progenitor cells from BM and PB. The aim of this study was to investigate the plating efficiency of single CD34+ progenitor cell from BM, PB and umbilical cord blood (UCB). METHODS: After sorting, single CD34+ cells were cultured in individual wells of 96-well plates in serum-free medium containing selected hematopoietic growth factors, with or without G-CSF. Plating efficiency was microscopically determined by the presence of clusters of viable cells:[the number of positive (cells were present) wells/total wells]x100. CD34+ cell-derived colonies were classified according to the cell number per well. RESULTS: Although there was some variation of plating efficiency of CD34+ cells among six normal BMs, six PBs and five UCBs, overall average plating efficiency of single CD34+ cells from BM, PB and UCB was 30% (30.0+/-11.7, mean+/-SD), 79% (78.6+/-11.7) and 45% (45.3+/-9.3) respectively. As expected, the colony size was increased in the presence of G-CSF. CONCLUSION: The results of this study clearly showed that the different ex vivo expansion of single CD34+ progenitor cells from BM, PB and UCB. These might be an important data for understanding stem cell expansion in vivo and designing clinical application.

Expansion and Differentiation of Dendritic Cells in Clinical Scale from Human Cord CD34+ Progenitor Cells

Dendritic cells are the most potent antigen presenting cells and appear to be the only cell type capable of limiting a primary T-cell dependent immune response. Recently, progress in the understanding of DCs biology has been relatively fast and systems using CD34+ stem cells stimulated with GM-CSF and tumor necrosis factor-alpha(TNF-alpha have been described. These systems have been further modified by others to increase the diversity and the yield. Indeed, several studies have shown distinct clinical responses after vaccination with tumor antigen- loaded, autologous DC. Despite this progress, the total number of DC available for immunotherapy remains limited. In vitro human DC can be generated from human CD34+ bone marrow and peripheral blood progenitor cells after culture with different cytokine combinations or from peripheral blood CD14+ monocytes when grow in the presence of GM-CSF and IL-4. Here, we have explored another source of DC precursors, human CD34+ cord blood cells, which in contrast to monocytic precursors, expand when cultured in the presence of GM-CSF and TNF-alpha The CD34+ cells were purified using MACS and expanded in culture with cytokine mixtures (SCF, Flt-3 TPO, IL-3, and IL-6). The CD34+ cells (4.0 +/-1.8 x105) isolated from cord blood cultured for 1, 2, 3, and 4 weeks resulted in a mean increase of total cell number of 41.5 +/-26.2 x105 (10-fold), 143.8 +/-78.9 x105 (36 fold), 197.5 +/-145.5 x105 (49-fold), 241.5 +/-167.4 x105 (60-fold), respectively. The precursor cells progressively lose most of the CD34 expression in culture and are over 95% positive for CD38 and low expression for CD3/CD19 indicating that all precursors are from myeloid origin. The percentage of CD14 positive precursors was significantly increased according to the expansion duration. The CD1a expression of expanded DC precursors was all negative (0.15-0.57%). The CD1a expression, which were in immature DCs, was high (28-78%), and CD40, CD80, CD11c and HLA-DR was positive after expanded precursor DCs were cultured for 1 weeks using GM-CSF and IL-4. The immature DC derived from all precursor culture conditions were negative for CD83. TNF-alphaactivated DCs derived from the four precursor culture condition according to the day of culture were used as stimulator cells in allogenenic MLR. When the total DC population was used, the expanded DCs for 2 weeks induced a slightly but reproducibly stronger MLR than those for 4 weeks. In this study, we show the sequential culture method after expansion is particularly appropriate for immunotherapeutical approaches, because relatively large numbers of DC can from cord blood be generated to overcome the limitation of cell count, which are needed for repetitive vaccination.

Clinical Outcomes according to Transplanted CD34+ Cell Dose in Allogeneic Peripheral Blood Stem Cell Transplantation / 대한혈액학회지

BACKGROUND: Recently, allogeneic peripheral blood stem cell transplantation (Allo-PBSCT) instead of bone marrow transplantation (BMT) has been widely used with the benefits of an earlier recovery of blood cells and a lower incidence of graft failure because of higher CD34+ cell dose. However, recent studies suggested that the higher dose of CD34+ cells might be related to the lower survival and the higher morbidity due to chronic graft versus host disease (cGVHD). We have analyzed the impact of transplanted CD34+ cell dose on clinical outcomes in unmanipulated allo-PBSCT from HLA identical siblings. METHODS: Thirty-one consecutive adult patients with hematological diseases, who survived until at least day 90 after allo-PBSCT and were evaluable for cGVHD, were included. Peripheral blood stem cells were collected from HLA-matched sibling donors mobilized with G-CSF and/or GM-CSF. The patients were classified into a "low" or "high" CD34+ cell dose group based on whether they received less or more than a median CD34+ cell dose of 11.17X10(6)/kg, respectively. RESULTS: The median CD34+ cell dose was 11.17X10(6)/kg (range, 4.12-58.80X10(6)/kg). Acute GVHD (grade II-IV) appeared in 24 patients (77.4%) and extensive cGVHD in 14 patients (45.2%). During the follow-up (median: 340 days, range: 111-1263 days), relapses were observed in 12 patients (38.7%) and 19 patients are still alive. There was a significant difference in the incidence of extensive cGVHD (20.0% vs 68.8%, P=0.011) and relapse (60.0% vs 18.8%, P=0.029) between low and high CD34+ cell dose groups, but no difference of the incidence of acute GVHD or the days of engraftments between the two groups. The estimated survival rate was significantly different between the two groups (3 year survival rate, 31.5% vs 79.8%, P=0.022) and the patients with extensive cGVHD showed a higher survival rate than those without extensive cGVHD (55.6% vs 12.5%, P=0.013). CONCLUSION: Better survival rate was observed in high CD34+ cell dose group for alloPBSCT, while a higher incidence of extensive cGVHD was noted. The optimal dose of CD34+ cells need to be determined to minimize the morbidity related to cGVHD and to improve survival.