OCT3/4 regulates transcription of histone deacetylase 4 (Hdac4) in mouse embryonic stem cells.

OCT3/4 is a POU domain transcription factor that is critical for maintenance of pluripotency and self-renewal by embryonic stem (ES) cells and cells of the early mammalian embryo. It has been demonstrated to bind and regulate a number of genes, often in conjunction with the transcription factors SOX2 and NANOG. In an effort to further understand this regulatory network, chromatin immunoprecipitation was used to prepare a library of DNA segments specifically bound by OCT3/4 in undifferentiated mouse ES (mES) cell chromatin. One segment corresponds to a region within the first intron of the gene encoding histone deacetylase 4 (Hdac4), a Class II histone deacetylase. This region acts as a transcriptional repressor and contains at least two functional sites that are specifically bound by OCT3/4. HDAC4 is not expressed in the nuclei of OCT3/4+ mES cells and is upregulated upon differentiation. These findings demonstrate the participation of OCT3/4 in the repression of Hdac4 in ES cells.

Intravascular magnetic resonance/radiofrequency may enhance gene therapy for prevention of in-stent neointimal hyperplasia.

RATIONALE AND OBJECTIVES: We evaluated the potential of using intravascular magnetic resonance (MR)/radiofrequency (RF) to enhance vascular endothelial growth factor (VEGF) gene therapy of in-stent neointimal hyperplasia. MATERIALS AND METHODS: By using a catheter-based approach, VEGF/lentivirus was locally transferred into 10 (five paired) bilateral femoral-iliac arteries of five hypercholesterolemic pigs, whereas the right arteries were heated up to approximately 41 degrees C by using an intravascular MR/RF system. Then, identical stents were placed immediately into the bilateral VEGF-targeted arteries to create in-stent neointimal hyperplasia. At day 60 after gene/stent interventions, the targeted arteries were harvested for histological correlation. RESULTS: X-Ray angiography-detectable in-stent stenoses were found in three of the arteries treated with VEGF genes only, whereas there were no in-stent stenoses in arteries treated by using MR/RF-heated VEGF genes. Correlative histological examination confirmed a 138% reduction in average thickness of neointimal hyperplasia in VEGF/RF-treated arteries compared with VEGF-only-treated arteries (P < .01). CONCLUSION: We report a potential method of using an intravascular MR/RF heating technique to enhance gene therapy of in-stent restenosis.

Functional antigen-presenting leucocytes derived from human embryonic stem cells in vitro.

BACKGROUND: Differentiated cells derived from pluripotent human embryonic stem (hES) cells offer the opportunity for new transplantation therapies. However, hES cells and their differentiated progeny express highly polymorphic MHC molecules that serve as major graft rejection antigens to the immune system of allogeneic hosts. To achieve sustained engraftment of donor cells, strategies must be developed to overcome graft rejection without broadly suppressing host immunity. One approach entails induction of donor-specific immune tolerance by establishing chimeric engraftment in hosts with haemopoietic cells derived from an existing hES cell line. We aimed to develop methods to efficiently differentiate hES cells to haemopoietic cells, including immune-modulating leucocytes, a prerequisite of the tolerance induction strategies applying to hES cell-mediated transplantation. METHODS: We developed a method to generate a broad range of haemopoietic cells from hES-generated embryonic bodies in the absence of murine stromal feeder cells. Embryonic bodies were further cultured in the presence of haemopoietic cytokines. In addition to flow cytometric analyses of haemopoietic cell markers, we analysed the hES cell-derived haemopoietic cells by colony-forming assays (for erythroid and myeloid progenitor cells), cytochemical staining, and mixed leucocyte reactions to determine the functional capacity of the generated antigen-presenting cells. FINDINGS: 12 independent experiments were done. When selected growth factors were added, leucocytes expressing CD45 were generated and released into culture media for 6-7 weeks. Under the condition used, both erythroid and myeloid progenitor cells were generated. About 25% of the generated leucocytes acquired MHC class II and costimulatory molecule expression. These hES-derived, MHC class II+ leucocytes resembled dendritic cells and macrophages, and they functioned as antigen-presenting cells capable of eliciting allogeneic CD4 and CD8 T-cell responses in culture. INTERPRETATION: The hES cell-derived antigen-presenting cells could be used to regulate alloreactive T cells and induce immune tolerance for improvement of the transplant acceptance of hES-cell derivatives.

Detection of dual-gene expression in arteries using an optical imaging method.

We evaluate the in vivo use of an optical imaging method to detect the vascular expression of green fluorescent protein (GFP) or red fluorescent protein (RFP), and to detect the simultaneous expression of GFP and RFP after transduction into arteries by a dual-promoter lentiviral vector driving their concurrent expression. This method involves using a charge-coupled device camera to detect fluorescence, a fiber optic probe to transmit light, and optical filters to distinguish each marker. In animal models, these vectors are locally delivered to target arteries, whereas the gene for a nonfluorescent cell-surface protein is transduced into contralateral arteries as the sham control. The images show distinct areas of bright fluorescence from GFP and RFP along the target arteries on excitation; no exogenous fluorescence is observed in the controls. Measured signal intensities from arteries transduced with the single- and dual-promoter vectors exceed the autofluorescence signal from the controls. Transgene expression of GFP and RFP in vivo is confirmed with confocal microscopy. We demonstrate the use of an optical imaging method to concurrently detect two distinct fluorescent proteins, potentially permitting the expression of multiple transgenes and their localization in the vasculature to be monitored.

Cell-type independent MYC target genes reveal a primordial signature involved in biomass accumulation.

The functions of key oncogenic transcription factors independent of context have not been fully delineated despite our richer understanding of the genetic alterations in human cancers. The MYC oncogene, which produces the Myc transcription factor, is frequently altered in human cancer and is a major regulatory hub for many cancers. In this regard, we sought to unravel the primordial signature of Myc function by using high-throughput genomic approaches to identify the cell-type independent core Myc target gene signature. Using a model of human B lymphoma cells bearing inducible MYC, we identified a stringent set of direct Myc target genes via chromatin immunoprecipitation (ChIP), global nuclear run-on assay, and changes in mRNA levels. We also identified direct Myc targets in human embryonic stem cells (ESCs). We further document that a Myc core signature (MCS) set of target genes is shared in mouse and human ESCs as well as in four other human cancer cell types. Remarkably, the expression of the MCS correlates with MYC expression in a cell-type independent manner across 8,129 microarray samples, which include 312 cell and tissue types. Furthermore, the expression of the MCS is elevated in vivo in Eµ-Myc transgenic murine lymphoma cells as compared with premalignant or normal B lymphocytes. Expression of the MCS in human B cell lymphomas, acute leukemia, lung cancers or Ewing sarcomas has the highest correlation with MYC expression. Annotation of this gene signature reveals Myc's primordial function in RNA processing, ribosome biogenesis and biomass accumulation as its key roles in cancer and stem cells.

Dual transfer of GFP gene and MGd into stem-progenitor cells: toward in vivo MRI of stem cell-mediated gene therapy of atherosclerosis.

RATIONALE AND OBJECTIVES: The aim of this study was to develop a new technique, the use of magnetic resonance (MR) imaging (MRI) to monitor gene/MR-cotransferred stem-progenitor cells (SPCs) recruited to atherosclerosis. MATERIALS AND METHODS: First, a green fluorescent protein (GFP) gene and a T1 MR contrast agent (motexafin gadolinium [MGd]) were cotransferred into neural or bone marrow (BM)-derived SPCs. GFP expression and MGd signal were confirmed by fluorescent microscopy and quantified by flow cytometry. Cell viability and proliferation were then evaluated by trypan blue exclusion and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay, and GFP/MGd-transferred cells were imaged using 1.5-T and 9.4-T MR scanners. For in vivo validation, GFP/MGd-cotransferred beta-galactosidase-BM SPCs were transplanted to apolipoprotein E-knockout mice, and cell migration to atherosclerotic aortas was monitored using 9.4-T micro-MRI with subsequent histologic correlations. RESULTS: Fluorescent microscopy demonstrated simultaneous GFP expression and MGd signals in cotransferred-cells. Quantitative flow cytometry showed GFP-positive cells at 47 +/- 25% and 56 +/- 12% and MGd-positive cells at 96 +/- 6% and 57 +/- 11% for neural stem cells and BM cells, respectively. Cell viability and metabolic rates of cotransferred cells were 86 +/- 4% and 84 +/- 12%, respectively. In vivo MRI revealed high MR signals of the aortic walls in GFP/MGd-transferred mice, which were confirmed by histologic correlations. CONCLUSION: This study has initially proven the new concept of MRI for plaque-specific, cell-mediated gene expression of atherosclerosis.

Cells derived from human umbilical cord blood support the long-term growth of undifferentiated human embryonic stem cells.

Various types of human cells have been tested as feeder cells for the undifferentiated growth of human embryonic stem cells (hESCs) in vitro. We report here the successful culture of two hESC lines (H1 and H9) on human umbilical cord blood (UCB)-derived fibroblast-like cells. These cells permit the long-term continuous growth of undifferentiated and pluripotent hESCs. The cultured hESCs had normal karyotypes, expressed OCT-4, SSEA-4, TRA-1-60, and TRA-1-81, formed cystic embryonic body in vitro and teratomas in vivo after injected into immunodeficient mice. The wide availability of clinical-grade human UCB makes it a promising source of support cells for the growth of hESC for use in cell therapies.

Radiofrequency-enhanced vascular gene transduction and expression for intravascular MR imaging-guided therapy: feasibility study in pigs.

PURPOSE: To evaluate the feasibility of radiofrequency (RF)-enhanced vascular gene transduction and expression by using a magnetic resonance (MR) imaging-heating guidewire as an intravascular heating vehicle during MR imaging-guided therapy. MATERIALS AND METHODS: The institutional committee for animal care and use approved the experimental protocol. The study included in vitro evaluation of the use of RF energy to enhance gene transduction and expression in vascular cells, as well as in vivo validation of the feasibility of intravascular MR imaging-guided RF-enhanced vascular gene transduction and expression in pig arteries. For in vitro experiments, approximately 10(4) vascular smooth muscle cells were seeded in each of four chambers of a cell culture plate. Next, 1 mL of a green fluorescent protein gene (gfp)-bearing lentivirus was added to each chamber. Chamber 4 was heated at approximately 41 degrees C for 15 minutes by using an MR imaging-heating guidewire connected to a custom RF generator. At day 6 after transduction, the four chambers were examined and compared at confocal microscopy to determine the efficiency of gfp transduction and expression. For the in vivo experiments, a lentivirus vector bearing a therapeutic gene, vascular endothelial growth factor 165 (VEGF-165), was transferred by using a gene delivery balloon catheter in 18 femoral-iliac arteries (nine artery pairs) in domestic pigs and Yucatan pigs with atherosclerosis. During gene infusion, one femoral-iliac artery in each pig was heated to approximately 41 degrees C with RF energy transferred via the intravascular MR imaging-heating guidewire, while the contralateral artery was not heated (control condition). At day 6, the 18 arteries were harvested for quantitative Western blot analysis to compare VEGF-165 transduction and expression efficiency between RF-heated and nonheated arterial groups. RESULTS: Confocal microscopy showed gfp expression in chamber 4 that was 293% the level of expression in chamber 1 (49.6% +/- 25.8 vs 16.8% +/- 8.0). Results of Western blot analysis showed VEGF-165 expression for normal arteries in the RF-heated group that was 300% the level of expression in the nonheated group (70.4 arbitrary units [au] +/- 107.1 vs 23.5 au +/- 29.8), and, for atherosclerotic arteries in the RF-heated group, 986% the level in the nonheated group (129.2 au +/- 100.3 vs 13.1 au +/- 4.9). CONCLUSION: Simultaneous monitoring and enhancement of vascular gene delivery and expression is feasible with the MR imaging-heating guidewire.

Human adult marrow cells support prolonged expansion of human embryonic stem cells in culture.

Prolonged propagation of human embryonic stem (hES) cells is currently achieved by coculture with primary mouse embryonic fibroblasts (MEFs) serving as feeder cells. Unlike mouse ES cells, adding growth factors such as leukemia inhibitory factor is insufficient to maintain undifferentiated hES cells without feeder cells. The presence of uncharacterized rodent cells or crude extracts imposes a risk to the clinical applications of hES cells. While others looked for a replacement of MEFs with human fetal cells, we attempted to use easily accessible postnatal human cells such as human marrow stromal cells (hMSCs). Culture-expanded hMSCs from multiple donors were used as feeder cells to support growth of the H1 hES cell line under a serum-free culture condition. Human ES cell colonies cultured on irradiated hMSCs amplified >100-fold during the 30-day continuous culture (in five passages). The longest continuous expansion of hES cells on hMSCs tested to date is 13 passages. The expanded hES cells displayed the unique morphology and molecular markers characteristic of undifferentiated hES cells as observed when they were cultured on MEFs. They expressed the transcription factor Oct-4, a membrane alkaline phosphatase, and the stage-specific embryonic antigen (SSEA)-4, but not the SSEA-1 marker. Expanded hES cells on hMSCs retained unique differentiation potentials in culture and a normal diploid karyotype. The well-studied hMSCs (and this animal cell- and serum-free system) may provide a clinically and ethically feasible method to expand hES cells for novel cell therapies. In addition, this system may help to identify cytokines and adhesion molecules that are required for the self-renewal of hES cells.

Lentiviral vectors with two independent internal promoters transfer high-level expression of multiple transgenes to human hematopoietic stem-progenitor cells.

Lentiviral vectors (LVs) offer several advantages over traditional oncoretroviral vectors. LVs efficiently transduce slowly dividing cells, including hematopoietic stem-progenitor cells (HSCs), resulting in stable gene transfer and expression. Additionally, recently developed self-inactivating (SIN) LVs allow promoter-specific transgene expression. For many gene transfer applications, transduction of more than one gene is needed. We obtained inconsistent results in our attempts to coexpress two transgenes linked by an internal ribosomal entry site (IRES) element in a single bicistronic LV transcript. In more than six bicistronic LVs we constructed containing a gene of interest followed by an IRES and the GFP reporter gene, GFP fluorescence was undetectable in transduced cells. We therefore investigated how to achieve consistent and efficient coexpression of two transgenes by LVs. In a SIN LV containing the elongation factor 1alpha promoter, we included a second promoter from cytomegalovirus, the phosphoglycerate kinase gene, or the HLA-DRalpha gene. Using a single LV containing two constitutive promoters, we achieved strong and sustained expression of both transgenes in transduced engrafting CD34(+) HSCs and their progeny, as well as in other human cell types. Thus, such dual-promoter LVs can coexpress multiple transgenes efficiently in a single target cell and will enable many gene transfer applications.