Comparison of Hematopoietic Stem Cell Containing Fractions between Cryopreserved-thawed Cord Blood and Mobilized Peripheral Blood / 대한소아혈액종양학회지

PURPOSE: Discordance with expectation there are a few differences in hematopoietic stem cells according to their source. The purpose of this study is to compare the cryopreservative potential of hematopoiectic stem cell containing fractions between cord blood (CB) and mobilized peripheral blood (mPB) during long term cryopreservation. METHODS: Nineteen CB and seven mPB were frozen with a programed freezer and stored in liquid phase of nitrogen from 1 to 4 years. After thawing the viability of mononuclear cells (MNCs) and the recovery rate of MNC, CD34 positive cell, colony form unit-granulocyte/monocyte was measured by CD34 flow cytometry and colony formation in semisolid methycellulose culture. CD34 positive cell was purified from cryopreserved-thawed or fresh mPB using magnetic associated cell sorting (MACS) CD34 selection kit. RESULTS: Though there is no difference in the viability and the recovery rate of CFU-GM between cryopreserved-thawed CB and mPB, the recovery rate of MNCs and CD34 positive cells is much higher in CB. Cell aggregation during the thawing process of long term cryopreserved mPB was effectively prevented by using high viscosity thawing buffer like as 10% dextran and 20% human serum albumin. We can also purify the CD34 positive cells from the long term cryopreserved mPB in the purity of more than 90%. CONCLUSION: Contrary to mPB long term cryopreserved CB can maintain the hematopoietic stem cell fraction without considerable loss, so it can be clinically used for hematopoietic stem cell transplantation.

Ex vivo Expansion and Clonal Maintenance of CD34+ Selected Cells from Cord Blood and Peripheral Blood / 소아과

PURPOSE: Because of the unavailability of marrow transplantation, umbilical cord blood (CB) is increasingly being used. We evaluated the potential of ex vivo expansion and clonality in CD34+ cells separated from cord blood source and mobilized peripheral blood (PB) in a serum-free media. METHODS: The CD34+ cells, selected from CB and mobilized PB, were expanded with hematopoietic growth factors. They were then cultured for burst-forming units of erythrocytes (BFU-E), colony-forming units of granulocytes and monocytes (CFU-GM) and colony-forming units of megakaryocytes (CFU-Mk) at culture days 0, day 4, day 7, and day 14 with various growth factors. RESULTS: The CB-selected CD34+ cells showed significantly higher total cell expansion than those from the PB at day 7 (2 fold increase than PB). The CB-selected CD34+ cells produced more BFU-E colonies than did the PB on culture at days 7 and at day 14. Also, the CB-selected CD34+ cells produced more CFU-Mk colonies than did the PB on culture at day 4 and at day 7. CONCLUSION: The ex vivo expansion of the CB cells may be promising in producing total cellular expansion, CFU-Mk and BFU-E compared with PB for 7 to 14 days. The growth factors combination including megakaryocyte growth and development, flt3-ligand and interleukin-3 showed more expansion in the view of total cells and clonal maintenance compared with less combination.

CD34 Analysis according to ISHAGE Guideline / 대한혈액학회지

BACKGROUND: Flow cytometric analysis for CD34 has been widely used for hematopoietic stem cell enumeration. The procedure is simple and rapid for clinical use but the lack of standardization resulted in great intralaboratory variations. In 1995, a guideline for CD34 analysis was established by International Society of Hematotherapy and Gene Engineering (ISHAGE) for reliable testing. We performed CD34 analysis using the ISHAGE guideline in umbilical cord blood (UCB), mobilized peripheral blood (MPB) and leukapheresis product (LP) and compared the results with those of in-house method. METHODS: CD34 analyses were performed in thirty units each of UCB, MPB and LP according to the ISHAGE guideline and in-house method and the results were analyzed by the t-test. Both methods used CD45FITC/CD34PE and its isotype controls. In ISHAGE guideline, among CD34+/ CD45+ cells, only those with low forward scattering, low to intermediate side scattering and low to intermediate CD45 fluorescent intensity were identified as stem cells, and the percentage of those cells among CD45+ cells was calculated. In in-house method, cells expressing both CD34 and CD45 antigens were selected by isotype control and the percentage of CD34+/CD45+ cells among CD45+ cells were calculated. RESULTS: Significant differences were observed in the percentages of CD34+ cells in UCB, MPB and LP between ISHAGE guideline (0.25%, 0.42%, 0.80%) and in-house method (0.40%, 0.55%, 1.20%) (P<0.001). So were the CD34+ cell counts : mean values of CD34+ cells in microliter of UCB, MPB and LP were 20, 40, 1,392 by ISHAGE guideline, and 35, 62, 2,079 by in-house method (P<0.001). CONCLUSION: ISHAGE guideline for CD34 enumaration was considered as a simple, rapid and reliable method for clinical setting and to have economic benefits because no additionalmonoclonal antibodies were required.

Immunophenotypes of umbilical cord blood cells and GM-CSF mobilized peripheral blood cells / 대한임상병리학회지

BACKGROUDS: Different clinical outcomes of umbilical cord blood (CB) transplantation and GM-CSF mobilized peripheral blood stem cell (LB) transplantation stimulated our interests in immune characteristics of various stem cell sources so that we investigated immunophenotypes of mononuclear cells of twenty-six CB and ten LB cases and twenty normal adult peripheral blood (AB). METHODS: Two-color direct immunofluorescence analysis was performed using FACScan (Becton-Dickinson, USA) and monoclonal antibodies were CD34FITC/CD33PE, CD34FITC/CD45ROPE, CD34PE/CD45RAFITC, CD34PE/CD38FITC, CD4PE/CD45RAFITC, CD4FITC/CD45ROPE, CD8PE/CD45RAFITC, CD8PE/CD11bFITC, CD8PE/CD57FITC, CD8FITC/CD38PE (Serotec), and Simulset IMK lymphocyte kit (Becton-Dickinson). RESULTS: CD34+ cell frequencies of CB and LB were four times higher than that of AB. CD34+CD38- cell frequency was higher in CB, 41.5+/-28.1% compared to 20.1+/-15.8% in LB (P=0.0391) and frequencies of CD34+CD33+, CD34+CD45RA-, and CD34+CD45RO+ showed no difference. LB showed higher frequency of CD3+ and CD8+ cells and lower frequency of CD4+ and CD19+ cells compared to that of CB and AB. CD4+CD45RA+ na ve cell frequency was higher in CB (29.3%) compared to 2.3% of LB. CD4+CD45RO+ memory T lymphocyte frequency (8.5%) and CD8+CD57+ cell frequency (1.0%) were lowest in CB. CD8+CD45RA+ and CD8+CD11+ cell frequencies showed no difference. No statistically significant differences in CD34+ cell and its subsets and lymphocytes and their subsets were observed between normal vaginal delivered umbilical cord blood (VD) and Cesarean sectioned umbilical cord blood (CS), except CD8+CD11+ cell frequency was higher in VD (11+/-7.6%) compared to that (4.0+/-2.3%) in CS (P=0.0081). CONCLUSIONS: CB showed higher frequency of immature hematopoietic stem cell precursors and na ve lymphocytes compared to that of LB and AB and LB showed higher frequency of T lymphocytes and lower frequency of B lymphocytes compared to those of CB and AB, which might be the possible causes of the different clinical outcome. Study on the standard immunophenotypic values in various hematopoietic stem cell products using sufficient number of subjects is necessary for the guideline of clinical utilization.