Biomedical subjects

Il-Hoan Oh

Publications and source records attributed to Il-Hoan Oh.

10 recordsLinked to original sources

PubMed2006

Early apoptosis in CD34+ cells as a potential heterogeneity in quality of cryopreserved umbilical cord blood.

The increased use of umbilical cord blood (UCB) raises issues regarding the quality of cryopreserved UCB. This study investigated whether early apoptosis of CD34+ cells is part of the functional heterogeneity of cryopreserved UCB. Annexin V binding of CD34+ PI(-) cells showed wide variations in both fresh and cryopreserved UCBs, with greater variation among units frozen for > 5 years. Xenotransplantation of sorted cells into non-obese diabetic severe combined immunodeficient mice demonstrated that the Annexin V assay identified most repopulating activities in UCB units. Thus, early apoptosis of CD34+ cells could influence the outcome of transplantation using cryopreserved UCB.

Animals↗

PubMed2006

Unique effects of Stat3 on the early phase of hematopoietic stem cell regeneration.

Self-renewal of hematopoietic stem cells (HSCs) is key to their reconstituting ability, but the signaling pathways that regulate this process remain poorly understood. Here we show that transduction of adult mouse bone marrow cells with a constitutively activated form of Stat3 (Stat3-C) increased their regenerative activity in lethally irradiated recipients. Conversely, transduction of these cells with a dominant-negative form of Stat3 suppressed their regenerative activity. Serial transplantation and clonal tracking of the HSC progeny regenerated in vivo from STAT3-C-transduced HSCs demonstrated that the major effect of forced expression of STAT3-C was to enhance HSC self-renewal during the initial phase of hematopoietic recovery. This acquired potential for enhanced self-renewal divisions then became latent, but was reactivated when the cells were transferred to new irradiated recipients. Increased levels of activated STAT3 were also found to be associated with greater preservation of primitive hematopoietic cells in short-term cultures. These results indicate a novel biphasic regulation of HSC self-renewal in vivo in which activated STAT3 promotes HSC self-renewal under stimulated, but not homeostatic, conditions. STAT3 may thus be an important regulator of hematopoietic regeneration and a novel target for HSC engineering.

Animals↗

PubMed2004

Differential modulation of Myb family genes by Ets-2.

The myb family of genes encodes highly homologous nuclear transcription factors that play distinct roles in the development of breast, germ cells and hematoid organs. While the mechanisms associated with the regulation of these genes remain unknown, the transactivation of c-Myb was previously shown to be upregulated by transcriptional cooperation with Ets-2. The present study examines the transactivation potential of the myb gene family in cooperation with Ets-2. A-Myb and c-Myb showed similar transcriptional cooperation with Ets-2, but not with Ets-1. Interestingly, B-Myb showed no cooperative activity with Ets-2 or Ets-1. Additionally, deletion mutants of A-Myb or c-Myb, where the C-terminal negative regulatory domain was deleted, did not abrogate their ability to cooperate with Ets-2. However, the deletion mutant of B-Myb, where the C-terminal positive regulatory domain was deleted, restored its ability to cooperate with Ets-2. Furthermore, studies using a series of 'domain-swapped' mutants between c-Myb and B-Myb revealed that the C-terminus of B-Myb, which is responsible for the protein's transactivation potential, blocks transcriptional cooperation with Ets-2. These results suggest that the myb gene family can be differentially modulated by Ets-2, and that the C-terminus is the domain that regulates this activity.

Animals↗

PubMed2004

Expression of an anti-sickling beta-globin in human erythroblasts derived from retrovirally transduced primitive normal and sickle cell disease hematopoietic cells.

OBJECTIVE: Recent improvements in human beta-globin vector design have fueled interest in gene therapy approaches to the treatment of human thalassemia and sickle cell disease (SCD). The present study was undertaken to determine whether human beta-globin mRNA and protein could be obtained in the erythroid progeny of more primitive human target cells transduced with a retrovirus containing murine stem cell virus long terminal repeats, a phosphoglycerate kinase promoter driving the expression of a green fluorescence protein (GFP) cDNA, and an anti-sickling beta-globin (beta87(+)) gene under the control of an HS2, HS3, HS4 enhancer cassette. MATERIALS AND METHODS: A two-step pseudotyping strategy was devised to obtain useful preparations of this virus. Primitive cells present in normal human cord blood (CB) and adult SCD patients' blood samples were infected and the level of gene transfer (% GFP(+) cells) and erythroid-specific beta87(+)-globin expression assessed. RESULTS: Analysis of the proportion of infected cells that became GFP(+) showed that this virus transduced approximately 50% of initial CD34(+) CB and SCD cells and up to 23% of cells able to regenerate both lymphoid and myeloid cells in sublethally irradiated primary and secondary NOD/SCID mice. beta87(+)-globin transcripts were readily detected in erythroblasts generated from primitive transduced CB cells and SCD progenitors. Evidence of beta87(+)-derived protein in transduced CB cell-derived erythroblasts also was obtained. CONCLUSION: These findings demonstrate that retroviral vector-based gene transfer approaches can be used to achieve human beta-globin protein expression in the erythroid progeny of transplantable human precursors.

Anemia, Sickle Cell↗

PubMed2003

Cotransplantation of third-party mesenchymal stromal cells can alleviate single-donor predominance and increase engraftment from double cord transplantation.

Although the infusion of umbilical cord blood (UCB) from multiple donors can be a strategy to overcome the cell dose limitation frequently encountered in UCB transplantation, clinical trials have revealed that cells from one donor dominate engraftment. To investigate the origin of and the factors influencing this inequality, we performed mixed transplantation of 2 UCB units with varying degrees of HLA disparities into NOD/SCID mice and determined donor origins by polymerase chain reaction-sequence-specific oligonucleotide probe (PCR-SSOP) or real-time quantitative (RQ)-PCR for human short tandem repeats (STRs). When total mononuclear cells from 2 units were transplanted as a mixture, cells from one donor predominated (ratio, 81:19), despite comparable overall engraftment when infused as single units, and no augmentation in overall engraftment was observed when compared with the single-unit controls. However, lineage depletion or cotransplantation of mesenchymal stromal cells (MSCs) expanded from third-party bone marrow resulted in more balanced coengraftment. Direct comparison of double UCB transplantation in the presence or absence of MSCs showed that the reduced deviation in the donor ratio (1.8:1 vs. 2.8:1) correlated with a higher overall level of engraftment with MSC cotransplantation. These results indicate that third-party MSCs can be used to alleviate donor deviation and to facilitate engraftment of multidonor UCB.

Animals↗

PubMed2002

Expression of a human beta-globin transgene in erythroid cells derived from retrovirally transduced transplantable human fetal liver and cord blood cells.

Transfer of therapeutic genes to human hematopoietic stem cells (HSCs) using complex vectors at clinically relevant efficiencies remains a major challenge. Recently we described a stable retroviral vector that sustains long-term expression of green fluorescent protein (GFP) and a human beta-globin gene in the erythroid progeny of transduced murine HSCs. We now report the efficient transduction of primitive human CD34(+) fetal liver or cord blood cells with this vector and expression of the beta-globin transgene in the erythroid progeny of these human cells for at least 2 months. After growth factor prestimulation and then a 2- to 3-day exposure to the virus, 35% to 55% GFP(+) progeny were seen in assays of transduced colony-forming cells, primitive erythroid precursors that generate large numbers of glycophorin A(+) cells in 3-week suspension cultures, and 6-week long-term culture-initiating cells. In immunodeficient mice injected with unselected infected cells, 5% to 15% of the human cells regenerated in the marrow (including the erythroid cells) were GFP(+) 3 and 6 weeks after transplantation. Importantly, the numbers of GFP(+) human lymphoid and either granulopoietic or erythroid cells in individual mice 6 weeks after transplantation were significantly correlated, indicative of the initial transduction of human multipotent cells with in vivo repopulating activity. Expression of the transduced beta-globin gene in human cells obtained directly from the mice or after their differentiation into erythroid cells in vitro was demonstrated by reverse transcriptase-polymerase chain reaction using specific primers. These experiments represent a significant step toward the realization of a gene therapy approach for human beta-globin gene disorders.

Animals↗

PubMed2002

Overexpression of a dominant negative form of STAT3 selectively impairs hematopoietic stem cell activity.

STAT3 is a key downstream signaling intermediate of gp130, a receptor previously shown to activate hematopoietic stem cell (HSC) self-renewal divisions. These findings prompted us to investigate if the STAT3 pathway is important to HSC activity in vivo. Initial semi-quantitative RT-PCR analyses showed STAT3 to be expressed at slightly higher levels in primitive subsets of both human and murine adult bone marrow cells. To test the effect of abrogating STAT3 activity in HSCs, primitive murine fetal liver cells were transduced at high efficiency with either a bicistronic dominant-negative (dn) or wild-type (wt) STAT3-IRES-GFP retrovirus. Dn STAT3-transduced HSCs showed markedly and permanently reduced in vivo lympho-myeloid reconstituting ability relative to co-transplanted non-transduced HSCs or HSCs transduced with a control (GFP-only) vector. In contrast, the activity of dn STAT3-transduced cells with short term in vivo (CFU-S) or in vitro (CFC) proliferation potential was not affected. Overexpression of wt-STAT3 had very little effect on either HSCs or shorter term progenitors. These findings suggest HSCs express non-limiting levels of STAT3 which, nevertheless, play an important stage-specific and non-redundant role in maintaining the function of HSCs stimulated to divide in adult marrow tissue.

Animals↗

PubMed2002

Detection of the BCR-ABL gene by interphase fluorescence in situ hybridization (iFISH) in chronic myelogenous leukemia patients after hemopoietic stem cell transplantation: the feasibility of iFISH monitoring of therapeutic response in peripheral blood.

The detection of the Philadelphia (Ph) translocation has been accomplished primarily by cytogenetic analysis and reverse transcriptase polymerase chain reaction (RT-PCR). RT-PCR is highly sensitive (1/10(4)-10(6)) but not quantitatively reliable and is thus unsuitable for the monitoring of Ph-positive cells during therapy. Interphase fluorescence in situ hybridization (iFISH) allows analysis of a large number of cells (> 500) in a timely and efficiently quantitative manner. We obtained 118 peripheral blood (PB) and 127 bone marrow (BM) samples from 75 adult chronic myelogenous leukemia (CML) patients undergoing stem cell transplantation. We simultaneously performed nested RT-PCR and iFISH for all samples. False-positive cells were detected in 2.48% +/- 0.93% (mean +/- SD) of PB samples and 2.75% +/- 0.83% of BM samples. The iFISH results for PB and BM ranged from 1.4% to 92.8% and 1.0% to 93.8%, respectively. Correlation analysis of iFISH results for PB versus BM samples showed a strong relation (r = .993). A significant correlation (P < .05) was also found between iFISH and first-round RT-PCR. The sensitivity of BCR-ABL iFISH was similar to that of first-round RT-PCR, and iFISH results for PB and BM were also well correlated. Thus, iFISH analysis of PB and/or BM samples may be more clinically reliable than RT-PCR in the quantitative monitoring of BCR-ABL fusion in CML after transplantation.

Blood Cells↗

PubMed2002

Comprehensive comparison of FISH, RT-PCR, and RQ-PCR for monitoring the BCR-ABL gene after hematopoietic stem cell transplantation in CML.

The reverse transcriptase-polymerase chain reaction (RT-PCR) was compared with fluorescence in situ hybridization (FISH) and real-time quantitative RT-PCR (RQ-PCR) for minimal residual disease (MRD) monitoring in 266 post-transplant bone marrow samples from 78 patients with chronic myelogenous leukemia (CML). The sensitivities of FISH to BCR-ABL positive samples determined by first-round (1st) RT-PCR, second-round (2nd) RT-PCR, and RQ-PCR were 64.2%, 25.8%, and 20.7%, respectively. The BCR-ABL/ABL ratio by RQ-PCR had a mean of 0.000 13 in the 1st RT-PCR-negative samples and 1.42 in the 1st RT-PCR-positive samples (P<0.001), and means of 0.000 39 and 0.51 in the 2nd RT-PCR-negative and -positive samples (P< 0.001). The mean ratios of BCR-ABL/ABL by RQ-PCR were significantly different in N/N (1st/2nd RT-PCR) or N/P and P/P (P<0.001), but not in N/N and N/P, which showed that the discriminative power of RQ-PCR is confined to the 1st RT-PCR level. In this respect, monitoring of the 1st RT-PCR might be useful for estimating normalized BCR-ABL levels after transplantation. Nested RT-PCR was of limited use, as RQ-PCR quantified the BCR-ABL transcripts in 60 (91%) of 66 samples determined to be negative by 2nd RT-PCR. FISH was significantly correlated with RQ-PCR in FISH-positive samples (n=24, r=0.79, P=0.001). An increase of FISH preceded that of RQ-PCR in a few cases with molecular relapse. By analyzing a large number of samples post-transplant, we found that RQ-PCR might be the most useful assay for MRD monitoring; however, FISH and RT-PCR were found to be useful complementary tools.

Bone Marrow↗

PubMed2002

Three-dimensional approach to stem cell therapy.

Recent progress in stem cell research is opening a new hope for cell therapy in regenerative medicine. Two breakthroughs were made in the stem cell era, one, new discoveries in multi-potentiality of adult stem cells beyond the traditionally appreciated extent, and the other, establishment of pluripotent stem cell from human embryo. In addition to the newly identified multi-potentiality of adult stem cells, their ability to be trans-differentiated toward other tissue types (stem cell plasticity) as well as to migrate toward the site of tissue damage make adult stem cells particularly attractive choice for stem cell based therapy. Stem cell therapy for organ regeneration, therefore, could be approached from three distinct dimensions: first, direct differentiation of multi-potent stem cells toward desired tissue types; secondly, regeneration of specific tissues through in vivo stem cell plasticity, and lastly, by tissue-specific stem cells from many types of organs. While each approach in stem cell therapy poses distinctive limitations for their successful clinical applications, understanding regulatory mechanisms of stem cell self-renewal and their in vivo engraftment will mostly extend their medical efficacy of stem cell based therapy.

Animals↗