Hong Hoe Koo

Mesenchymal stem cells feeder layer from human umbilical cord blood for ex vivo expanded growth and proliferation of hematopoietic progenitor cells.

Ex vivo expansion of hematopoietic stem cells was suggested as the best way of overcoming problems caused by limited hematopoietic cell number for cord blood transplantation. In this study, we quantified and characterized an ex vivo expansion capacity of umbilical cord blood (UCB)-derived mesenchymal stem cells (MSCs) as a cell feeder layer for support of UCB-derived committed hematopoietic progenitor cells (HPCs) in the absence or presence of recombinant cytokines. The UCB-derived MSCs used in the study differentiated into osteoblast, chondrocytes, and adipocytes under proper conditions. Frequencies in colony forming unit-granulocyte, macrophage, colony forming unit-granulocyte, erythrocyte, macrophage, megakaryocyte, burst forming unit-erythrocyte, and colony forming unit-erythrocyte increased to 3.46-, 9.85-, 3.64-, and 2.03-folds, respectively, only in culture supplemented by UCB-derived MSCs as a cell feeder layer without recombinant cytokines (culture condition C). Identified expansion kinetics in all kinds of committed HPCs showed plateaus at 7 culture days, suggesting some consumable components were required for the expansion. Physiological importance and different roles for different committed HPCs of UCB-derived MSCs as a cell feeder layer were revealed by a distinguished expansion capacity for colony forming unit-megakaryocyte. The preferred maintenance of CD33−CD34+ in culture condition C was also identified. The presence of cobblestone-like areas as hematopoietic microenvironment and various cell feeder layer-originated hematopoietic cytokines including interleukin-1β and granulocyte, macrophage-colony stimulating factor were suggested as underlying mechanisms for the identified expansion capacity. The present numeric and biological information about intrinsic expansion capacity for UCB-derived committed HPCs will increase further biological and clinical applications of UCB-derived MSCs.

Targeting survivin overcomes drug resistance in acute lymphoblastic leukemia

Relapse of drug-resistant acute lymphoblastic leukemia (ALL) has been associated with increased expression of survivin/BIRC5, an inhibitor of apoptosis protein, suggesting a survival advantage for ALL cells. In the present study, we report that inhibition of survivin in patient-derived ALL can eradicate leukemia. Targeting survivin with shRNA in combination with chemotherapy resulted in no detectable minimal residual disease in a xenograft model of primary ALL. Similarly, pharmacologic knock-down of survivin using EZN-3042, a novel locked nucleic acid antisense oligonucleotide, in combination with chemotherapy eliminated drug-resistant ALL cells. These findings show the importance of survivin expression in drug resistance and demonstrate that survivin inhibition may represent a powerful approach to overcoming drug resistance and preventing relapse in patients with ALL.

The impact of post-thaw colony-forming units-granulocyte/macrophage on engraftment following unrelated cord blood transplantation in pediatric recipients

We retrospectively reviewed the engraftment kinetics following unrelated cord blood transplantation (CBT) in association with the post-thaw colony-forming units-granulocyte/macrophage (CFU-GM) number along with the numbers of total nucleated cells (TNC), CD34+ cells and CD3+ cells. A total of 71 cord blood units prepared for 53 patients (double-unit CBT in 18 patients) were evaluated. Either the number of infused CFU-GM or CD34+ cells was significantly lower in patients who failed to achieve engraftment (P=0.028 and 0.005, respectively). Post-thaw CFU-GM, TNC and CD34+ cells correlated with the speed of neutrophil engraftment (P=0.004, 0.037 and 0.004, respectively), whereas only CFU-GM showed a significant correlation with platelet engraftment (r=−0.385, P=0.024). In double-unit transplants, the number of CFU-GM was the only significant factor predicting engraftment of the predominating unit (P=0.006). We conclude that the post-thaw CFU-GM number is a reliable predictor of rapid engraftment after CBT as well as of the predominating unit in double-unit transplants. Thus, it would be important to perform post-thaw CFU-GM assay on cryopreserved aliquots from several candidate cord blood units in advance before CBT to avoid selecting the unit that might possess a low clonogenic potential.

Hepatic veno-occlusive disease in children after hematopoietic stem cell transplantation: incidence, risk factors, and outcome

Four hundred and sixty-seven hematopoietic stem cell transplantations (HSCTs) (217 autologous and 250 allogeneic HSCT) were performed in 374 children at four pediatric HSCT centers in Korea from January 2005 to December 2007. Among 467 transplants, veno-occlusive disease (VOD) developed in 72 transplants (15.4%) at a median of 10 days after HSCT. Multivariate analysis showed that BU or TBI-containing regimen (P=0.002), VOD prophylaxis without lipo-prostaglandin E1 (PGE1) (P=0.012), number of previous HSCT (P=0.014), and pretransplant serum ferritin (P=0.018) were independent risk factors for developing VOD. Mean serum ferritin levels were significantly higher in HSCT with VOD (2109.6±2842.5 ng/ml) than in HSCT without VOD (1315.9±1094.4 ng/ml) (P<0.001). The relative risk of death within 100 days of HSCT in transplants with VOD compared with transplants without VOD was 3.39 (confidence interval: 1.78–6.45). Our results suggest that lipo-PGE1 might have a protective effect against the development of VOD, and pretransplant serum ferritin could act as a risk factor for VOD. A larger prospective study is needed to confirm a possible role of lipo-PGE1 and iron chelation therapy in reducing the incidence of VOD.

Integrin alpha4 blockade sensitizes drug resistant pre-B acute lymphoblastic leukemia to chemotherapy

Bone marrow (BM) provides chemoprotection for acute lymphoblastic leukemia (ALL) cells, contributing to lack of efficacy of current therapies. Integrin alpha4 (alpha4) mediates stromal adhesion of normal and malignant B-cell precursors, and according to gene expression analyses from 207 children with minimal residual disease, is highly associated with poorest outcome. We tested whether interference with alpha4-mediated stromal adhesion might be a new ALL treatment. Two models of leukemia were used, one genetic (conditional alpha4 ablation of BCR-ABL1 [p210(+)] leukemia) and one pharmacological (anti-functional alpha4 antibody treatment of primary ALL). Conditional deletion of alpha4 sensitized leukemia cell to nilotinib. Adhesion of primary pre-B ALL cells was alpha4-dependent; alpha4 blockade sensitized primary ALL cells toward chemotherapy. Chemotherapy combined with Natalizumab prolonged survival of NOD/SCID recipients of primary ALL, suggesting adjuvant alpha4 inhibition as a novel strategy for pre-B ALL.

Tandem high-dose chemotherapy and autologous stem cell rescue in patients over 1 year of age with stage 4 neuroblastoma.

From June 1997 to August 2005, 52 consecutive newly diagnosed stage 4 neuroblastoma patients over 1 year of age were assigned to receive tandem high-dose chemotherapy and autologous stem cell rescue (HDCT/ASCR) as consolidation therapy. Fifty of the 52 patients underwent a first HDCT/ASCR and 44 patients underwent a second HDCT/ASCR. Eight patients (15.4%) died from treatment-related toxicity (seven during the second HDCT/ASCR). Total body irradiation (TBI) in the first HDCT/ASCR and a shorter interval (<12 weeks) between the first and second HDCT/ASCR were associated with a higher rate of treatment-related death in the second HDCT/ASCR (P=0.032 and 0.095, respectively). The tumor relapsed or progressed in 11 patients, and 33 patients remained event free with a median follow-up of 53 months (range 19–117) from diagnosis. The 5-year event-free survival (EFS) (±95% confidence interval) for all 52 patients was 62.1±13.7%. The application of TBI and local radiotherapy, and a longer interval between the first and second HDCT/ASCR were independently associated with a better EFS (P=0.026, 0.007 and 0.020, respectively). However, further studies will be needed to decrease the toxic death rate in the second HDCT/ASCR while reducing the relapse rate.

Stem and progenitor cells in human umbilical cord blood

Both stem cells and progenitor cells are present in umbilical cord blood (UCB) at a high frequency, making these cells a major target population for experimental and clinical studies. As the use of autologous or allogeneic hematopoietic stem cell transplantation in the treatment of various diseases has grown rapidly in recent years, the concept of UCB banking for future use has drawn increasing interest. Stem and progenitor cells derived from UCB offer multiple advantages over adult stem cells, such as their immaturity (which may play a significant role in reducing rejection after transplantation into a mismatched host) and ability to produce large quantities of homogeneous tissue or cells. These cells can also differentiate across tissue lineage boundaries into neural, cardiac, epithelial, hepatic, and dermal tissues. Human UCB provides an alternative cell source that is ethically acceptable and widely supported by the public. This paper summarizes the characteristics of human UCB-derived stem and progenitor cells and their potential therapeutic use for tissue and cell regeneration.

Loss of heterozygosity analysis of chromosome 17p13.1-13.3 and its correlation with clinical outcome in medulloblastomas.

Cytogenetic and molecular genetic studies have shown that deletions on the short arm of chromosome 17 distal to p53 locus are the most common genetic events in medulloblastoma. We examined the occurrences and frequencies of allelic deletions on chromosome 17p13.1–13.3 by loss of heterozygosity (LOH) analysis to investigate the possible involvement of 17p13.1–13.3 in medulloblastoma development. We also performed survival analysis to determine whether LOH analysis of 17p13.1–13.3 can be used to predict prognosis in medulloblastoma.Loss of heterozygosity was analyzed by polymerase chain reaction on chromosome 17p13.1–13.3 using three microsatellite markers, TP53 on 17p13.1, D17S796 on 17p13.1–13.2, and D17S1574 on 17p13.3, in 17 medulloblastoma DNAs extracted either from archival tissue or fresh frozen tissue specimens.Allelic deletions were detected in five of 17 informative cases (29%) on TP53, eight of 17 informative cases (47%) on D17S796, and four of 17 informative cases (24%) on D17S1574. Overall, nine of 17 cases (53%) showed LOH on chromosome 17p13.1–13.3. The 5-year progression free survival (PFS) and 5-year overall survival rates were identical (59%). The 5-year PFS for nine medulloblastoma patients with LOH on 17p13.1–13.3 was 56%, and the 5-year PFS for eight medulloblastoma patients without LOH on 17p13.1–13.3 was 63%. In our survival analysis, we did not find a significant association between survival and LOH on 17p13.1–13.3.Our results support the notion that deletions of chromosome 17p13.1–13.3 may be involved in the pathogenesis of medulloblastoma. From survival analysis, we conclude that LOH on chromosome 17p13.1–13.3 may not be a significant predictor of prognosis in medulloblastoma.

Co-transplantation of third-party umbilical cord blood-derived MSCs promotes engraftment in children undergoing unrelated umbilical cord blood transplantation

Success of umbilical cord blood transplantation (UCBT) has been limited by a high rate of graft failure and delayed hematological recovery. It has been postulated that MSCs have hematopoiesis-supportive properties. Therefore, to overcome the limitation of UCBT, third-party UCB-derived MSCs were co-transplanted in recipients receiving unrelated UCBT. Seven patients received UCB and third-party UCB–MSCs. Hematopoietic recovery and transplantation outcomes were compared with historic controls. There was no acute toxicity associated with the infusion of MSCs. The median day to neutrophil engraftment was 19 days in patients, as compared with 24 days in controls (P=0.03). The median day of platelet engraftment was 47 days and 57 days in patients and controls, respectively (P=0.26). In addition, there was no engraftment failure in the MSC group. The incidence of acute and chronic GVHD was comparable between the two groups. However, veno-occlusive disease and TRM did not occur in the MSC group. Third-party UCB–MSCs infusion was safe and feasible. MSCs may also enhance the engraftment of UCBT and prevent rejection. In addition, MSCs may have a role in decreasing TRM. Randomized, controlled trials are required to confirm these results and longer follow-up will determine the effects of MSCs on the risk of relapse.

Clinical features and treatment outcomes of Langerhans cell histiocytosis: a nationwide survey from Korea histiocytosis working party.

A nationwide survey was conducted to clarify the clinical features and outcomes of Korean children with Langerhans cell histiocytosis (LCH). Korea Histiocytosis Working Party analyzed the data of 603 patients who were diagnosed with LCH between 1986 and 2010 from 28 institutions in Korea. Median age at diagnosis was 65 months (range, 0 to 276 mo). Bone was the most frequently affected organ (79.6%) followed by skin (19.2%). Initially, 419 patients (69.5%) had single-system involvement (SS), 85 (14.1%) with multisystem (MS) disease without risk organ involvement (MS-RO−), and 99 (16.4%) multisystem disease with risk organ involvement (MS-RO+). The 5-year overall survival (OS) rates in the SS, MS-RO−, and MS-RO+ groups were 99.8%, 98.4%, and 77.0%, respectively (P<0.001), and the 5-year reactivation rates were 17.9%, 33.5%, and 34.3%, respectively (P<0.001). The OS rate was lower in patients with RO involvement (P=0.025) and lack of response to initial treatment (P=0.001). MS involvement (P=0.036) was an independent risk factor for reactivation. Permanent consequences were documented in 99 patients (16.4%). Reactivation of disease, MS involvement, and age at diagnosis ⩽2 years were associated with higher incidence of permanent consequences. This study emphasized that further efforts are required to improve survival of MS-RO+ patients and reduce reactivation in younger patients with MS involvement.