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Hypoxia is known to regulate the expression of genes involved in the migration of various cell types. Although many studies have shown that hypoxia increases cell migration, it still remains unclear whether hypoxia could modulate the stromal cell derived factor-1 (SDF-1)-dependent migration of leukemic cell. Herein, we demonstrated that the SDF-1-dependent migration of HL-60, was reduced under hypoxia with no comparable decrease of CXC-type chemokine receptor CXCR4, a cognate receptor for SDF-1. Furthermore, we showed that migration toward SDF-1 was reduced by inactivation of either serine/threonine kinase Akt or extracellular signal regulated kinase Erk, which was confirmed by selective pathway inhibitor LY294002 and PD98059. In our results, phosphorylation of Erk was increased under hypoxia, but phosphorylation of Akt was attenuated on the contrary. These results led us to conclusion that hypoxia could inhibit the SDF-1-dependent migration of HL-60 via blocking of Akt activation.

It has been suggested that epigenetic regulation plays an important role in maintaining the stemness and lineage differentiation of hematopoietic stem cells (HSCs), 5-aza-deoxycytidine (aza-D) and Trichostatin A (TSA) being candidate additives for HSC ex vivo expansion. Although they have potent activity to maintain the stemness, they can also cause serious cell death. This study examined the effects of mesenchymal stem cells (MSCs) on the maintenance of CD34+ cells driven by aza-D and TSA in culture with the combined cytokines of thrombopoietin, flt-3 ligand, stem cell factor, interleukin-3, and interleukin-6. In cultures without MSCs, although aza-D and TSA retained the CD34 frequency 4 to 8 times more than in the cytokines alone, a large portion of cells underwent apoptotic cell death. Consequently, CD34+ cell expansion could not be achieved in any condition without MSCs. In cultures with MSCs, the total cell number was higher in aza-D or TSA than in any conditions in the cultures without MSCs. The CD34 frequency was also similar to the level in the cultures in aza-D or TSA without the MSCs. These results suggest that a co-culture of CD34+ cells with the MSCs might not simply deliver the proliferation signals but also stemness and survival signals, and overlap the action of epigenetic regulators.

It has been reported that human mesenchymal stem cells (MSCs) can transfer mitochondria to the cells with severely compromised mitochondrial function. We tested whether the reported intercellular mitochondrial transfer could be replicated in different types of cells or under different experimental conditions, and tried to elucidate possible mechanism. Using biochemical selection methods, we found exponentially growing cells in restrictive media (uridine(-) and bromodeoxyuridine [BrdU](+)) during the coculture of MSCs (uridine-independent and BrdU-sensitive) and 143B-derived cells with severe mitochondrial dysfunction induced by either long-term ethidium bromide treatment or short-term rhodamine 6G (R6G) treatment (uridine-dependent but BrdU-resistant). The exponentially growing cells had nuclear DNA fingerprint patterns identical to 143B, and a sequence of mitochondrial DNA (mtDNA) identical to the MSCs. Since R6G causes rapid and irreversible damage to mitochondria without the removal of mtDNA, the mitochondrial function appears to be restored through a direct transfer of mitochondria rather than mtDNA alone. Conditioned media, which were prepared by treating mtDNA-less 143B rho(0) cells under uridine-free condition, induced increased chemotaxis in MSC, which was also supported by transcriptome analysis. Cytochalasin B, an inhibitor of chemotaxis and cytoskeletal assembly, blocked mitochondrial transfer phenomenon in the above condition. However, we could not find any evidence of mitochondrial transfer to the cells harboring human pathogenic mtDNA mutations (A3243G mutation or 4,977 bp deletion). Thus, the mitochondrial transfer is limited to the condition of a near total absence of mitochondrial function. Elucidation of the mechanism of mitochondrial transfer will help us create a potential cell therapy-based mitochondrial restoration or mitochondrial gene therapy for human diseases caused by mitochondrial dysfunction.

We report here central nervous system (CNS) recurrence in neuroblastoma (NBL) after CD34(+) peripheral blood stem cell transplantation (PBSCT). Fifteen stage 4 NBL patients underwent CD34(+) transplantation with myeloablative chemotherapy consisting of carboplatin, etoposide, and melphalan. There were three primary site recurrences and five distant metastases including four brain metastases (two isolated CNS recurrences) at 4-7 months after CD34(+) transplantation. Three of four patients died of CNS progressive disease at 2, 8, and 9 months after recurrence and the remaining single patient was lost to follow-up. CNS recurrence in NBL is fatal and requires identification of risk factors and more effective treatment strategies.

Dendritic cells (DCs) are the most potent antigen-presenting cells in terms of initiating primary T-cell-dependent immune responses. We devised a 2-step culture method for obtaining sufficient numbers of functional DCs from umbilical cord blood (CB) CD34+ cells. In the first step, CB CD34+ cells were expanded by stimulation with early-acting cytokines such as stem cell factor (SCF), flt3 ligand (FL), and thrombopoietin (TPO) to amplify the hematopoietic progenitor cells. In the second step, granulocyte-macrophage colony-stimulating factor and interleukin 4 were added, and incubation was continued for another 5 days to induce differentiation of the expanded cells into DCs. During the first step of culturing with TPO, SCF, and FL, the total numbers of nucleated cells gradually increased, peaking at 4 weeks (245.3-fold). During the second step, expression of CD1a, CD83, and CD86 increased. Electron microscopic findings showed that these cells had cytosolic expansion to form dendrites and major histocompatibility complex class II compartments, which are characteristic of DCs. Functional analyses revealed that these cells had phagocytic activity and were capable of stimulating allogeneic T-cells in vitro.

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.

The cure rate of pediatric acute lymphoblastic leukemia (ALL) has dramatically improved after the introduction of maintenance therapy. But many Korean patients could not tolerate the planned dose of methotrexate (MTX) and 6-mercaptopurine (MP) from Western protocols. Recently, tailored therapy based on individual pharmacogenetic background is introduced to improve the survival with the reduction of complication. To establish the basis for the tailored therapy, genes associated with MTX and MP metabolism were analyzed in Korean children with ALL. The dose percent to the planned dose of MTX (20 mg/m2) and MP (50 mg/m2) at final maintenance cycle were analyzed with the 7 polymorphism site (MTHFR 677 C>T, MTHFR 1298 A>C, RFC 80 G>A, TYMS enhancer repeat, TPMT 238 G>C, TPMT 460 G>A, TPMT 719 A>G) in 96 ALL patient using TotalPlex PCR method (except TYMS). The median dose percent of MTX and MP of all patients were 57.5% and 49%, respectively. Only 31/96 and 22/96 patients tolerated more than 75% of planned MTX and MP doses, respectively. Variant TPMT was found in 6 patients (one *1/*3B and five *1/*3C). The mean dose percent of MP in patients with variant TPMT (33.2%) was lower than that of others (53.5%), significantly (P=0.04). MTHFR 677, MTHFR 1298, RFC 80, and TYMS enhancer repeat were not associated with the dose percent of MTX. But the dose percent of MTX was correlated with the dose percent of MP significantly (R2=0.64, P=0.00). The 5 year event free survival of all patients was 93.7%. Although 16/96 patients received less than 25% of full MP dose, there was no event in these patients. Dose reduction of MP is recommended for the patients with 3 major variant types of TPMT but many others with wild type could not tolerate the planned dose of MP. Study of novel polymorphism is necessary for the basis of tailored therapy in Korean pediatric ALL patients.

Cord blood transplantation (CBT) is a promising alternative means of allogeneic stem cell transplantation. However, limited cell doses may compromise outcome. To enhance engraftment, CBT has been conducted using two units with promising results. Our preliminary results showed the dominance of one unit which occurred early after CBT (Bone Marrow Transplant. 38:197). However, little is known about the mechanism of engraftment and factors influencing the dominancy. To improve the outcomes, CBT was performed with 2 partially HLA-matched units in pediatric acute leukemia with the analysis of factors that affect the determination of dominancy. The median age and body weight of patients (M:11, F:4) were 13 years (3–17) and 45.0 kg (10.5–62.9), respectively. Patients received various conditioning regimens according to the disease status. The median number of the infused nucleated cells by the sum of 2 units after thawing was 5.28×107/kg (0.72–15.09×107/kg). Engraftment achieved in 14/15 Patients. The median number of days required for ANC of more than 0.5×109 or 1.0×109/l were 18 days (13–31 days) and 21 days (14–34 days), respectively. Spontaneous platelet recovery to more than 20×109/l required a median 50 days (19–93 days). The early engraftment kinetics reveals that dominancy of one of two units determined from the day of engraftment (ANC > 0.5×109/l). The numbers of total nucleated cells, CD34+ cells, CD3+ cells, CFU-GM, order of infusion, and donor-recipient HLA disparities of the UCB units were viewed as factors that potentially influence dominancy. Among them infused CFU-GM per patient body weight was associated with the dominancy (P = 0.017). Interestingly, CFU-GM could not be found from each cord blood units in patient failed engraftment. Multiple factors associated with outcomes of CBT could influence the determination of dominancy, such as those factors that affect the recipient environment (underlying disease, previous treatment, conditioning regimen, use of ATG and the method of GVHD prophylaxis), the characteristics of each UCB unit and HLA disparity. For this reason, the selection of the two units requires consideration of the above factors. However optimal selection guidelines that take into account influencing factors are unavailable. CFU-GM was associated with the dominancy in our preliminary result but this kind of data is hard to get before CBT practically. Introducing the analysis of new parameters such as CFU-GM before freezing could help the selection of optimal cord blood unit and improve the outcomes with the consideration of cost-benefit.