[Development of a System for Evaluating the In-room-laser Alignment Including the Horizontality and Verticality Integrated the Light/Radiation Field Coincidence Test and the Winston-Lutz Test].

PURPOSE: The in-room laser which is used for patient positioning in radiotherapy is generally projected on the radiation isocenter determined by the Winston-Lutz test and so on. In this study, a couch-mounted verification device was developed that could evaluate all in-room lasers' alignment including the horizontality and verticality at one time. The device has the function to perform the light/radiation field coincidence test and the Winston-Lutz test at the same time. The aim of this report was to introduce the verification procedure for two tests, using the newly developed software and device, and to present the tuning flow of the in-room laser. Moreover, the analysis accuracy of the developed software was evaluated in comparison with commercial software. METHODS: First, the light/radiation field was evaluated by using tungsten markers on the central surface of the device. Next, after aligning the long-carved lines on the front and sides of the device with the in-room lasers, the Winston-Lutz test was carried out by using the tungsten sphere in the center of the device. The acquired images were collectively analyzed using the developed software equipped with the reporting function. Additionally, the result of this Winston-Lutz test was compared with the result from commercial software. RESULTS: A series of the light/radiation field coincidence test and the Winston-Lutz test were analyzed using the developed device and software. The results could be easily confirmed using the reporting function of the software. Regarding the result of the Winston-Lutz test, most of the analysis differences between the developed software and commercially available software were within the pixel size (0.22 mm). DISCUSSIONS: Since the accuracy of the radiation field affects the result of the Winston-Lutz test, the presented procedure of performing the light/radiation coincidence field test in advance facilitates the interpretation of the error of the Winston-Lutz test. Based on the results of the Winston-Lutz test, we were able to demonstrate the tuning flow of all in-room lasers including the horizontality and verticality by using the developed device. CONCLUSIONS: We have developed a couch-mounted verification device and software that can evaluate the light/radiation coincidence field test and the alignment including the horizontality and verticality of the in-room laser used for patient positioning in radiotherapy, and reported its usefulness. The analysis accuracy of the developed software was comparable to that of commercially available software. The use of this device and the developed software would contribute to not only the efficiency of adjusting all in-room lasers' alignment including the horizontality and verticality but also reflect accurately the result of the Winston-Lutz test.

Therapeutic superiority for cartilage repair by CD271-positive marrow stromal cell transplantation.

Recent reports indicated that human isolated CD271+ bone marrow mesenchymal stromal cells (BM-MSCs) have a greater expansion and potential for multipotent differentiation including chondrogenesis than classical plastic adherent (PA) BM-MSCs in vitro. Therefore, we set up a hypothesis that CD271+ MSCs may have a greater chondrogenic potential than PA-MSCs in vitro and in vivo. We investigated the superiority of CD271+ MSCs on chondrogenesis using in vitro expansion and pellet culture system and in vivo rat model of cartilage defect when compared to PA-MSCs. In the in vitro study, CD271+ MSCs showed higher expansion potential and produced larger pellets with higher expressions of chondrogenic genes when compared to the control groups. During the culture, CD271 expression decreased, which resulted in decreased chondrogenesis. In the in vivo study, immunohistochemical staining demonstrated differentiated human chondrocytes identified as double-stained cells with human-specific collagen type 2 and human leukocyte antigen-ABC in CD271+ and PA groups. The number of double-stained cells was significantly higher in the CD271+ group than PA group. Real-time RT-PCR analysis of tissue RNA isolated from the chondral defect site for human-specific chondrogenic markers demonstrated a significantly higher expression in CD271+ group than PA group. Macroscopic examination of chondral defect sites at week 8 revealed glossy white and well-integrated repaired tissues in the CD271+ and PA groups, but not in the PBS group. The average histological score in the CD271+ group was significantly greater than in the other groups. Apoptosis analysis at the cell transplanted site with TUNEL staining showed that the CD271+ group had significantly fewer apoptotic chondrocytes compared with the PA group. These results indicate that CD271+ MSCs have a greater chondrogenic potential than PA-MSCs in both in vitro and in vivo conditions.

Cardiac differentiation of P19CL6 cells by oxytocin.

BACKGROUND: It has been reported that P19 embryonal carcinoma (EC) cells differentiate into beating cardiomyocytes under the action of oxytocin (OT). It has been suggested that dimethylsulfoxide (DMSO) acts via the oxytocin/oxytocin receptor pathway because an oxytocin receptor antagonist not only blocks oxytocin-induced cardiomyocyte differentiation, but also blocks DMSO-induced differentiation. In this study, the differentiation ability of OT was tested using P19CL6 cells. METHODS: P19CL6 cells were cultured as a confluent monolayer and aggregated cells. OT was then added to culture media as an inducing agent. The cells treated with 1% DMSO were used as a positive control group. Differentiated cells were evaluated morphologically and immunocytochemically, as well as by RT-PCR. In addition, a stable line of green fluorescent protein (GFP)-expressing P19CL6 cells were differentiated into beating cardiomyocytes by OT. RESULTS: Aggregated P19CL6 cells could be differentiated into cardiomyocytes, whereas monolayer cells could not differentiate and express specific cardiac muscle marker genes. In the control group, both aggregates and monolayer cells could be differentiated into cardiomyocytes by DMSO. In addition, GFP-expressing P19CL6 cells differentiated efficiently into beating cardiomyocytes when treated with OT. The results of all evaluations confirmed that the differentiated cells were cardiomyocytes. CONCLUSIONS: We concluded that embryoid body formation (cell aggregation) is necessary for the differentiation of P19CL6 cells into cardiomyocytes when using OT as an inducer agent. Furthermore, because of the high rate of differentiation efficiency, GFP-expressing cardiomyocytes derived from P19CL6 cells have the potential to be used for regenerative therapies in experimental models.

Changing modified regions in the genome in hematopoietic stem cell differentiation.

In the process of hematopoietic stem cell (CD133+ cell) differentiation, a drastic change in gene expression occurs which must be regulated by epigenetic mechanisms. One strategy for CD133+ cell differentiation analysis is to identify genomic DNA regions that have been modified in the process of differentiation. However, it is difficult to obtain large amounts of genomic DNA from uniform CD133+ cells. Based on this situation, we screened genomic DNA regions where modifications change during the process of differentiation in human CD133+ cells using differential methylation site scanning (DMSS), which is a method of identifying differentially methylated regions of the genome from a small number of cells. As a result, we cloned three DNA fragments which corresponded to centrosomal protein 68kDA (Cep68), TRIO and F-actin binding protein (TRIOBP), and AMP-activated protein kinase beta (AMPKb).

Functional and gene expression analysis of hTERT overexpressed endothelial cells.

Telomerase dysfunction contributes to cellular senescence. Recent advances indicate the importance of senescence in maintaining vascular cell function in vitro. Human telomerase reverse transcriptase (hTERT) overexpression is thought to lead to resistance to apoptosis and oxidative stress. However, the mechanism in endothelial lineage cells is unclear. We tried to generate an immortal endothelial cell line from human umbilical vein endothelial cells using a no-virus system and examine the functional mechanisms of hTERT overexpressed endothelial cell senescence in vitro. High levels of hTERT genes and endothelial cell-specific markers were expressed during long-term culture. Also, angiogenic responses were observed in hTERT over-expressed endothelial cell. These cells showed a delay in senescence and appeared more resistant to stressed conditions. PI3K/Akt-related gene levels were enhanced in hTERT overexpressed endothelial cells. An up-regulated PI3K/Akt pathway caused by hTERT overexpression might contribute to anti-apoptosis and survival effects in endothelial lineage cells.

Cardiogenic potential of endothelial progenitor cells.

Transplantation of endothelial progenitor cells (EPCs) are one of the promising strategies to recover the capillary flow in ischaemic diseases such as ischaemic heart disease and peripheral artery disease (PAD) in the leg. However, our previous and another group's works suggested the scarcity of the number of EPCs in peripheral blood might cause insufficient effect for the ischaemic diseases. There are several strategies to overcome this issue, such as (1) in vivo EPC expansion; (2) ex vivo EPC expansion; (3) local (not systemic) EPC injection; and (4) modification of EPC by gene transfer. Recent publications from our own and other groups have reported the possibility of cardiogenic potential of EPCs. We would like to focus on the strategies of EPC transplantation and cardiomyogenesis of EPCs in this review.

Gene modified cell transplantation for vascular regeneration.

Cell Transplantation is one of the powerful tools to ameliorate the capillary flow in ischemic condition. EPC (Endothelial Progenitor Cell) was identified in adult peripheral blood and thought to be a suitable candidate for cell transplantation. Also, gene therapy is already promising choice for enhancing angiogenic property. The combination of cell transplantation and gene therapy should be more effective way to regenerate vasculature in ischemic region. Recently, several research reports have come out regarding gene modified cell transplantation. We will mainly focus on the background of EPC, and then gene modified EPC findings in this review.

Simple screening method for differentially methylated regions of the genome using a small number of cells.

Genomic DNA methylation is a major epigenetic mechanism controlling the expression of genetic information. Therefore, identifying regions of the genome that are differentially methylated in different cells is a useful strategy for the study of biological phenomena. To date, several useful screening methods have been established for identifying differentially methylated genomic regions. However, it is impossible to use these methods in fields of study in which it is difficult to obtain a large number of uniform cells, because considerable amounts of genomic DNA are required. Given this situation, we developed a method for preparing large genomic DNA from a small number of cells, and a simple and highly sensitive method for screening for differentially methylated sites. Combined, these two methods comprise a simple screening method, which we named "Differential Methylation Site Scanning" (DMSS), for identifying differentially methylated regions of the genome from a small number of cells. Just 10 cells are sufficient for the method described here.

[Vascular endothelium regeneration therapy].

CD34 positive cells were first defined as endothelial progenitor cells(EPCs) from circulating mononuclear cells in peripheral blood. EPCs have shown to be mobilized from bone marrow by the various factors, incorporate into sites of physiological and pathological neovascularization and differentiate into mature endothelial cells (ECs). Post-natal vasculogenesis has been considered to be involved in neovascularization of adult tissues. Recently, freshly isolated CD34 positive cells transplantation has started as clinical trial for ischemic diseases. In the clinical situation, we should consider the cell number and cell quality derived from the patients who have atherosclerosis background, especially diabetes. Ex vivo expansion or gene modification of EPCs could be the strategies for the next generation cell therapy to overcome these issues.

Therapeutic potential of vasculogenesis and osteogenesis promoted by peripheral blood CD34-positive cells for functional bone healing.

Failures in fracture healing are mainly caused by a lack of vascularization. Adult human circulating CD34+ cells, an endothelial/hematopoietic progenitor-enriched cell population, have been reported to differentiate into osteoblasts in vitro; however, the therapeutic potential of CD34+ cells for fracture healing is still unclear. Therefore, we performed a series of experiments to test our hypothesis that functional fracture healing is supported by vasculogenesis and osteogenesis via regenerative plasticity of CD34+ cells. Peripheral blood CD34+ cells, isolated from total mononuclear cells of adult human volunteers, showed gene expression of osteocalcin in 4 of 20 freshly isolated cells by single cell reverse transcriptase-polymerase chain reaction analysis. Phosphate-buffered saline, mononuclear cells, or CD34+ cells were intravenously transplanted after producing nonhealing femoral fractures in nude rats. Reverse transcriptase-polymerase chain reaction and immunohistochemical staining at the peri-fracture site demonstrated molecular and histological expression of human-specific markers for endothelial cells and osteoblasts at week 2. Functional bone healing assessed by biomechanical as well as radiological and histological examinations was significantly enhanced by CD34+ cell transplantation compared with the other groups. Our data suggest circulating human CD34+ cells have therapeutic potential to promote an environment conducive to neovascularization and osteogenesis in damaged skeletal tissue, allowing the complete healing of fractures.

Dose-dependent contribution of CD34-positive cell transplantation to concurrent vasculogenesis and cardiomyogenesis for functional regenerative recovery after myocardial infarction.

BACKGROUND: Multilineage developmental capacity of the CD34+ cells, especially into cardiomyocytes and smooth muscle cells (SMCs), is still controversial. In the present study we performed a series of experiments to prove our hypothesis that vasculogenesis and cardiomyogenesis after myocardial infarction (MI) may be dose-dependently enhanced after CD34+ cell transplantation. METHODS AND RESULTS: Peripheral blood CD34+ cells were isolated from total mononuclear cells of patients with limb ischemia by apheresis after 5-day administration of granulocyte colony-stimulating factor. PBS and 1x10(3) (low), 1x10(5) (mid), or 5x10(5) (high) CD34+ cells were intramyocardially transplanted after ligation of the left anterior descending coronary artery of nude rats. Functional assessments with the use of echocardiography and a microtip conductance catheter at day 28 revealed dose-dependent preservation of left ventricular function by CD34+ cell transplantation. Necropsy examination disclosed dose-dependent augmentation of capillary density and dose-dependent inhibition of left ventricular fibrosis. Immunohistochemistry for human-specific brain natriuretic peptide demonstrated that human cardiomyocytes were dose-dependently observed in ischemic myocardium at day 28 (high, 2480+/-149; mid, 1860+/-141; low, 423+/-9; PBS, 0+/-0/mm2; P<0.05 for high versus mid and mid versus low). Immunostaining for smooth muscle actin and human leukocyte antigen or Ulex europaeus lectin type 1 also revealed dose-dependent vasculogenesis by endothelial cell and SMC development after CD34+ cell transplantation. Reverse transcriptase-polymerase chain reaction indicated that human-specific gene expression of cardiomyocyte (brain natriuretic peptide, cardiac troponin-I, myosin heavy chain, and Nkx 2.5), SMC (smooth muscle actin and sm22alpha), and endothelial cell (CD31 and KDR) markers were dose-dependently augmented in MI tissue. CONCLUSIONS: Human CD34+ cell transplantation may have significant and dose-dependent potential for vasculogenesis and cardiomyogenesis with functional recovery from MI.

Niche-dependent translineage commitment of endothelial progenitor cells, not cell fusion in general, into myocardial lineage cells.

OBJECTIVE: Previous studies from our laboratory have shown therapeutic potential of ex vivo expanded endothelial progenitor cells (EPCs) for myocardial ischemia. Our purpose was to investigate the mechanisms regulating EPC contribution to myocardial regeneration. METHODS AND RESULTS: To evaluate niche-dependent expression profiles of EPCs in vitro, we performed coculture using cultured EPCs derived from human peripheral blood and rat cardiac myoblast cell line (H9C2). Reverse-transcription polymerase chain reaction (PCR) disclosed the expression of human-specific cardiac markers as well as human-specific smooth muscle markers. Cytoimmunochemistry presented several cocultured cells stained with human specific cardiac antibody. To prove this translineage differentiation in vivo, human cultured EPCs were injected into nude rat myocardial infarction model. Reverse-transcription PCR as well as immunohistochemistry of rat myocardial samples demonstrated the expression of human specific cardiac, vascular smooth muscle, and endothelial markers. We observed the distribution of colors (Qtracker; Quantum Dot Corp) in coculture to detect the fused cells, and the frequency of cell fusion was <1%. CONCLUSIONS: EPCs can contribute to not only vasculogenesis but also myogenesis in the ischemic myocardium in vivo. Transdifferentiation, not cell fusion, is dominant for EPCs commitment to myocardial lineage cells. Ex vivo expanded EPCs transplantation might have enhanced therapeutic potential for myocardial regeneration.

Endothelial progenitor cells for vasculogenesis.

Postnatal vasculogenesis is considered to be involved in neovascularization of adult tissues, because bone marrow-derived endothelial progenitor cells (EPCs) were isolated from circulating mononuclear cells in peripheral blood and were shown to incorporate into sites of physiological and pathological neovascularization and to differentiate into mature endothelial cells. EPCs might have an attractive potential therapeutic application for cardiovascular ischemic diseases as a novel cell-based strategy mainly via a vasculogenesis mechanism.

Stromal cell-derived factor-1 effects on ex vivo expanded endothelial progenitor cell recruitment for ischemic neovascularization.

BACKGROUND: Stromal cell-derived factor-1 (SDF-1) is a chemokine considered to play an important role in the trafficking of hematopoietic stem cells. Given the close relationship between hematopoietic stem cells and endothelial progenitor cells (EPCs), we investigated the effect of SDF-1 on EPC-mediated vasculogenesis. METHODS AND RESULTS: Flow cytometric analysis demonstrated expression of CXCR4, the receptor of SDF-1, by 66+/-3% of EPCs after 7 days in culture. In vitro modified Boyden chamber assay showed a dose-dependent EPC migration toward SDF-1 (control versus 10 ng/mL SDF-1 versus 100 ng/mL SDF-1, 24+/-2 versus 71+/-3 versus 140+/-6 cells/mm2; P<0.0001). SDF-1 attenuated EPC apoptosis (control versus SDF-1, 27+/-1 versus 7+/-1%; P<0.0001). To investigate the effect of SDF-1 in vivo, we locally injected SDF-1 into athymic ischemic hindlimb muscle of nude mice combined with human EPC transplantation to determine whether SDF-1 augmented EPC-induced vasculogenesis. Fluorescence microscopic examination disclosed increased local accumulation of fluorescence-labeled EPCs in ischemic muscle in the SDF-1 treatment group (control versus SDF-1=241+/-25 versus 445+/-24 cells/mm2, P<0.0001). At day 28 after treatment, ischemic tissue perfusion was improved in the SDF-1 group and capillary density was also increased. (control versus SDF-1, 355+/-26 versus 551+/-30 cells/mm2; P<0.0001). CONCLUSION: These findings indicate that locally delivered SDF-1 augments vasculogenesis and subsequently contributes to ischemic neovascularization in vivo by augmenting EPC recruitment in ischemic tissues.

Constitutive human telomerase reverse transcriptase expression enhances regenerative properties of endothelial progenitor cells.

BACKGROUND: The regulatory molecule for cell life span, telomerase, was modified by human telomerase reverse transcriptase (hTERT) gene transfer to investigate its effect on regenerative properties of endothelial progenitor cells (EPCs) in neovascularization. METHODS AND RESULTS: Telomerase activity was enhanced in hTERT-transduced EPCs (Td-TERTs) (1.2-fold versus no transduced EPCs [no-Td] and 1.2-fold versus GFP-transduced EPCs [Td/GFPs] at day 8; 5.2-fold versus no-Td and 4.8-fold versus Td/GFP at day 21, respectively) Mitogenic capacity in Td/TERTs exceeded that in Td/GFPs at day 8 (0.62+/-0.02 versus 0.53+/-0.01, respectively; P<0.01). Vascular endothelial growth factor-induced cell migration in EPCs was markedly enhanced by hTERT overexpression (Td/TERTs versus Td/GFPs, 292+/-12 versus 174+/-6 cells, respectively; P<0.01). hTERT overexpression has rescued EPCs from starvation-induced cell apoptosis, an outcome that was further enhanced in response to vascular endothelial growth factor. The colony appearance of totally differentiated endothelial cells (tdECs) was detected before day 30 only in Td/TERT, whereas no tdEC colonies could be detected in both Td/GFPs and no-Tds. Finally, we investigated in vivo transplantation of heterologous EPCs. Td/TERTs dramatically improved postnatal neovascularization in terms of limb salvage by 4-fold in comparison with that of Td/GFPs; limb perfusion was measured by laser Doppler (0.77+/-0.10 versus 0.47+/-0.06; P=0.02), and capillary density (224+/-78 versus 90+/-40 capillaries/mm2; P<0.01). CONCLUSIONS: These findings provide the novel evidence that telomerase activity contributes to EPC angiogenic properties; mitogenic activity, migratory activity, and cell survival. This enhanced regenerative activity of EPCs by hTERT transfer will provide novel therapeutical strategy for postnatal neovascularization in severe ischemic disease patients.

Endothelial progenitor cell vascular endothelial growth factor gene transfer for vascular regeneration.

BACKGROUND: Previous studies have established that bone marrow-derived endothelial progenitor cells (EPCs) are present in the systemic circulation. In the current study, we investigated the hypothesis that gene transfer can be used to achieve phenotypic modulation of EPCs. METHODS AND RESULTS: In vitro, ex vivo murine vascular endothelial growth factor (VEGF) 164 gene transfer augmented EPC proliferative activity and enhanced adhesion and incorporation of EPCs into quiescent as well as activated endothelial cell monolayers. To determine if such phenotypic modulation may facilitate therapeutic neovascularization, heterologous EPCs transduced with adenovirus encoding VEGF were administered to athymic nude mice with hindlimb ischemia; neovascularization and blood flow recovery were both improved, and limb necrosis/autoamputation were reduced by 63.7% in comparison with control animals. The dose of EPCs used for the in vivo experiments was 30 times less than that required in previous trials of EPC transplantation to improve ischemic limb salvage. Necropsy analysis of animals that received DiI-labeled VEGF-transduced EPCs confirmed that enhanced EPC incorporation demonstrated in vitro contributed to in vivo neovascularization as well. CONCLUSIONS: In vitro, VEGF EPC gene transfer enhances EPC proliferation, adhesion, and incorporation into endothelial cell monolayers. In vivo, gene-modified EPCs facilitate the strategy of cell transplantation to augment naturally impaired neovascularization in an animal model of experimentally induced limb ischemia.