Establishment of Human Bone Marrow-Derived Mesenchymal Stem Cell Lines with SV40 Large T Antigen and Their Differentiation to Chondrocytes / 대한소아혈액종양학회지

PURPOSE: Mesenchymal stem cells (MSC) can be isolated from bone marrow (BM) and when systemically administrated to different species, they undergo site-specific differentiation. In this study, we isolated MSC from human BM and generated a continuously growing colony of cell lines (SNU-hMSC) with SV40 large T antigen. The purposes of this study are to identify whether SNU-hMSC have the characteristics of MSC and their possibility of chondrogenic differentiation. METHODS: MSC were mobilized from BM and cultured in DMEM-LG media for 2 weeks. We obtained SNU-hMSC, by introducing a viral vector of SV40 large T antigen and culturing it in the selected media for 6 months. We identified specific cell markers of MSC via FACS analysis and analyzed expression of cytokines, chemokines and receptors by RT-PCR. To stimulate the proliferation of the cells, we processed the media with FGF, BMP-2 and IL-6. The each medium's cell counts were counted in day 7 and day 14. To differentiate SNU-hMSC, they were cultured in chondrogenic media. After 2 weeks, chondrogenic differentiation was evaluated with safranin-O staining and the expression of COMP, aggrecan and SOX-9. RESULTS: SNU-hMSC exhibited MSC markers. When the IL-6, BMP-2 and FGF were added to each medium, the cell numbers were significantly increased as compared with control. In the study of differentiation, SNU-hMSC exhibited strong safranin-O staining, and chondrogenic gene expression was observed. CONCLUSION: SNU-hMSC expressed markers and cytokines identical with MSC. SNU-hMSC maintained multipotency of differentiation.

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.

The Effect of Stromal Cells and Hematopoietic Growth Factors on Proliferation and Differentiation of Megakaryocytic Progenitors from Umbilical Cord Blood / 대한소아혈액종양학회지

PURPOSE: CD34+ cells from umbilical cord blood (UCB) were cultured with stromal cells and growth factors, and cell proliferation and megakaryocytic differentiation were observed. The purposes of this study are to find out the optimal culture condition for the megakaryocytic differentiation of CD34+ cells from UCB. METHODS: CD34+ cells of mobilized peripheral blood (PB) and UCB were cultured in IMDM at a concentration of 1x105 cells/mL for 11 days. Thrombopoietin (TPO) 5 ng/mL and 50 ng/mL, flt-3 ligand (FL) 50 ng/mL and stem cell factor (SCF) 50 ng/mL were used as growth factors. Stromal cells cultured from bone marrow (BM) mononuclear cells appeared as a single layer of fusiform adherent cells with expression of SH-2 and ASMA. RESULTS: When PB CD34+ cells were cultured with growth factors only, the cell number increased in TPO 5 ng/mL and 50 ng/mL, TPO and FL, TPO, FL and SCF group increased upto mean 2.0 (in TPO 5 ng/mL), 2.9 (in TPO 50 ng/mL), 2.4 (TPO and FL), 2.8 (TPO, FL and SCF) fold, and the expression of CD61a were mean 11.5%, 12.5%, 13.2% and 17.0%, respectively. When the stromal cells were added to the growth factors, the cell number increased upto mean 4.6, 4.9, 9.2 and 68.5 fold and CD61a were expressed in mean 43.1%, 48.4%, 35.6%, and 6.2% of cultured cells. For UCB CD34+ cells, the cell number were increased upto 7.5, 7.6, 8.2 and 23.6 fold with growth factors and stromal cells. Expression of CD61a were mean 26.0%, 34.3%, 31.5% and 23.5%, respectively. CONCLUSION: Stromal cell enhanced the cellular proliferation and megakaryocytic differentiation of UCB CD34+ cells. The combination of TPO, FL and SCF increased total cell number upto the highest value but the proportion of the CD61a+ cells were relatively low. The combination of TPO and FL induced both cellular proliferation and megakaryocytic differentiation in a large amount.

Myeloablative Treatment Supported by Autologous Stem Cell Infusion with Neuroblastoma

Bcr-abl antisense oligodeoxynucleotides (AS-ODNs) have provided evidence of an antileukemia effect when tested in vitro against Philadelphia-positive cells. In order to investigate the efficacy of AS-ODNs as purging agents in chronic myeloid leukemia (CML) patients, K562 cells, a human CML cell line, were treated in vitro with various types of AS-ODNs and interferon-alpha. Cells were treated in vitro for 0 and 36 hr with 40 microgram/mL of AS-ODNs, respectively, and incubated at 37 degrees C for 36 hr. Cytotoxic effects were measured by counting the number of viable cells as well as by MTT test. Clonogenic activities were evaluated by methylcellulose culture for 2 weeks. The effects of purging agents on the rearrangement of bcrabl gene were evaluated by RT-PCR. AS-ODNs inhibited the proliferation of K562 cells with time in cell count assay and MTT test. AS-ODNs were superior to INF-alpha in inhibiting clonogenic activity (recovery rate; 26.3% vs 64.0%). After incubation with bcr-abl AS-ODNs primers and mRNA isolated from K562 cells, positive bands were abolished, especially of b3a2 type and phosphorothioate type. Our results suggest that AS-ODNs mediated purging may be one of the efficient methods and that autograft may be an alternative treatment for allograft in high-risk group patients of CML if they do not have a stem cell donor.

Proliferation and Differentiation of Erythroid Progenitors from Cord Blood CD34 (+) Cells Using Extracellular Matrix and Stromal Cells / 대한소아혈액종양학회지

PURPOSE: This study was performed to induce proliferation and differentiation of erythroid progenitors from cord blood CD34 cells using extracellular matrix (ECM) and stromal cells. METHODS: Cord blood mononuclear cells were separated using Ficoll-Hypaque and CD34 cells were purified by Mini-MACS column. Cells were cultured in IMDM medium including 10% FBS and several cytokines (EPO, flt3-ligand, SCF and TPO) upto 14 days. ECM like laminin, collagen IV, fibrinogen and fibronectin were used alone or in combination. Stromal cells were derived from BM mononuclear cells for 4 weeks of culture and irradiated before use. On day 14 of stromal coculture of cord blood CD34 cells, flow cytometric analysis were done with fluorescence isothiocyanate-conjugated (FITC) antihuman glycophorin A antibody for erythroid cells. RESULTS: ECM-treated group showed 16.3~31.3 fold increase of the cell numbers on day 14 and there were no difference from cytokines-treated group. Stromal cells induced great amount of fold increase compared with ECM-treated group on day 7 (25.4 fold vs. 2.2~5.4 fold). The cell numbers increased upto 16.4 fold on day 7, 92.8 fold on day 10, and 198.4 fold on day 14 with stromal cells. Erythroid progenitors expressing glycophorin A increased from 2.78% on day 0 to 21.57% on day 7 and 43.87% on day 14. CONCLUSION: Stromal coculture of cord blood CD34 cells induced marked proliferation and differentiation of erythroid cells compared with cytokines or ECM-treated group. Efficient in vitro erythroid culture might have implications for gene therapy in RBC defects or developing blood substitutes for transfusions.

Cord Blood RBC Depletion Using 10% Pentastarch and 6% Hydroxyethylstarch / 대한소아혈액종양학회지

PURPOSE: For separation of RBC from cord blood, it is important to minimize RBC contamination without significant loss of nucleated cells using sedimentation agent that is safe for human use. This study was performed to investigate the possibility of replacing 6% hydroxyethylstarch (HES) with 10% pentastarch (PS) which is a lower molecular weight hetastarch-analog that is cleared from the circulation rapidly. METHODS: After dilution of cord blood till hematocrit 25%, PS or HES were added by the ratio of 7:1 and 5:1 respectively. Sedimentation was performed for 2 hours by gravity. RESULTS: PS was used in 14 cases with volume of 72.4+/-22.3 mL (45~126 mL) and HES in 8 cases with volume of 58.4+/-8.0 mL (50~70 mL). Sedimentation rate has reached at plateau by 90 minutes in PS group and it was slightly faster than in HES group. Recovery rate of nucleated cells and residual RBC were 82.9+/-10.7%, 7.6+/-5.4% in PS group, and 84.0+/-4.7%, 10.7+/-2.3% in HES group. There were no significant differences between the two groups (P=0.657, 0.219). Cell viabilities were high in both groups; 92+/-3% before separation and 97+/-2% in PS group and 98+/-3% in HES group. CD34+ cells were 0.75+/-0.28% before separation and 0.64+/-0.21% in PS group and 0.60+/-0.30% in HES group (P=0.690). CFU-GM after 2 week culture were 27.4+/-20.0 per 1 105 mononuclear cells in PS group and 22.9+/-8.6 in HES group (P=0.856). CONCLUSION: These results demonstrated that PS has similar efficacy to HES for separation of RBC from umbilical cord blood. Considering its rapid clearance and faster sedimentation rate, PS can replace HES for RBC separation in cord blood banking.

The Effect of Overnight Storage of Cord Bloods on Cell Viability, Live CD34 Cell Fraction, and Clonogenic Potential under Different Storage Conditions / 대한소아혈액종양학회지

PURPOSE: In children, at least two or more stem cell mobilization processes are needed in autologous peripheral blood stem cell transplantation to prevent delayed engraftment. And to decrease the risk of tumor cell contaminations, the use of CD34 positive cell selcetion is in increasing tendency. The first leukapheresis product is stored overnight and undergoes CD34 positive selection process mixed with the next day leukapheresis product to save the costs. We intended to find out the optimal overnight storage condition that might minimize the loss of stem cell components. METHODS: RBC (red blood cell)- depleted human umbilical cord bloods (UCB) were used as the source of stem cells because of their easy availability. UCB were processed by isolating the mononuclear cell (MNC) layer using Ficoll-Paque to make the nature similar to leukapheresis products. Fifteen individual UCB were analyzed by several parameters (MNC count and viability, live CD34 positive cell fraction, clonogenic potential) at fresh conditions and under four different overnight storage conditions (room temperatiure (RT), room temperature with autoplasma (AP), 4degrees C, 4degrees C with autoplasma). Analysis of variance, Kruskal-Wallis test, and Wilcoxon signed rank test were used for statistical analyses. RESULTS: Though MNC counts were statistically not different between each conditions (P=0.07), the best recovery (mean 86.9%) was observed at 4degrees C with AP but without statistical significance. MNC viability decreased at RT with or without AP (P 0.05). Live CD34 positive cell fractions were significantly decreased under all four different storage conditions compared with fresh ones. However, the samples stored at 4degrees C showed less prominent decreases in live CD34 positive cell fractions than those of RT conditions irrespective of the presence of AP (P=0.0001). CONCLUSION: It seems that 4degrees C condition is superior to RT when short term storage of stem cell products is mandatory. The addition of AP seemed to be advantageous but without statistical significance. The overnight storage of stem cell products at 4degrees C seems to be mandatory because it offers relatively high recovery and less loss of stem cell components. Although the effect of AP was statistically not significant, the role of AP should be studied further because there was a tendency of higher recovery of stem cells in the presence of AP.

Expression of WT1 Gene in Childhood Acute Leukemia / 대한소아혈액종양학회지

PURPOSE: Recently Wilms tumor gene (WT1) transcripts have been detected in leukemia regardless of the disease subtype and the specific DNA markers suggesting that WT1 gene might be a useful panleukemic marker for monitoring minimal residual disease (MRD). This study was performed to investigate the expression of WT1 gene by a quantitative methods and to find the prognostic value of WT1 gene in childhood acute leukemia. METHODS: From the mononuclear cells isolated from bone marrow aspirates and peripheral bloods of 22 childhood acute and chronic leukemia patients, mRNA were extracted for the reverse transcriptase-polymerase chain reactions (RT-PCR). Relative levels of WT1 gene expression was calculated by using the value in K562 cell line to be 1.00 as a positive control. RESULTS: The sensitivity of detection of MRD with WT1 primers was 10 4 and comparable to that of bcr/abl expression in K562 cells and a patient with CML in blast crisis. WT1 gene expression was detected in 17 of 22 (77%) patients; 9/10 of acute lymphoblastic leukemia (ALL), 6/10 acute myelogenous leukemia (AML), 1 acute mixed lineage leukemia (AMLL) and 1 chronic myelogenous leukemia (CML) in blast crisis. In 4 AML patients who received autologous peripheral blood stem cell transplantation (PBSCT), two patients relapsed after reappearance of WT1 gene expression in bone marrow aspirates and the remaining two were in complete remission without expression of WT1 gene. CONCLUSION: These results show that WT1 gene expression is frequently noted in childhood acute leukemia and can be a useful sensitive marker for the detection of MRD comparable to bcr/abl transcripts. WT1 gene can be used as a panleukemic marker for the MRD monitoring for the evaluation of the remission status and in predicting early relapse in children with acute leukemia in the molecular levels. It may also be a useful tool for the detection of leukemic cell contamination in the process of peripheral blood stem cell transplantation.