Mechanism of apoptotic resistance of human osteosarcoma cell line,HS-Os-1, against irradiation

In our previous study, we examined reactive oxygen species (ROS) formation in T lymphocytes following 5 Gy of irradiation. Using a CCD camera system, we monitored fluorescence in T lymphocytes loaded with the succinimidyl ester of Dichlorodihydrofluorescein diacetate (H2DCFDA), which is non-fluorescent until oxidized by ROS. We found that ROS formation occurred immediately after irradiation, continued for several hours, and resulted in oxidative DNA damage. Therefore, the origin of the hyper-radiosensitivity of T lymphocytes seemed to be the high production of ROS in the mitochondrial DNA following irradiation. In this study, we examined radiation-induced ROS formation, oxidative DNA damage, early apoptotic changes, and mitochondrial membrane dysfunction in the human osteosarcoma cell line HS-Os-1, which was established from an osteoblastic tumor that arose in the left humerus of an 11-year-old girl and was already morphologically characterized in vitro and in vivo. We found that ROS formation and oxidative DNA damage were actually scarcely seen after irradiation of up to 30 Gy in these cells; that mitochondrial membrane potential was preserved; and that apoptotic changes were not demonstrated despite the relatively high-dose irradiation of 30 Gy. Therefore, the origin of the close similarity of radiosensitivity between adult articular chondrocytes and the human osteosarcoma cell line HS-Os-1, is considered to involve the low degree of ROS formation following irradiation; the similarity possibly results from the strong scavenging ability of these two kinds of cells for free radicals including hydroxyl radicals.     

Immunocytochemical characteristics of human osteosarcoma cell line HS-Os-1: possible implication in apoptotic resistance against irradiation

In our previous study, we examined reactive oxygen species (ROS) formation in T lymphocytes following 5 Gy irradiation. We found that ROS formation occurred immediately after irradiation, continued for several hours, and resulted in oxidative DNA damage. Therefore, the origin of the hyper-radiosensitivity of T lymphocytes seemed to be the high production of ROS in the mitochondrial DNA following irradiation. In the succeeding study, we examined radiation-induced ROS formation, oxidative DNA damage, early apoptotic changes, and mitochondrial membrane dysfunction in the human osteosarcoma cell line HS-Os-1. We found that ROS formation and oxidative DNA damage were actually scarcely seen after irradiation of up to 30 Gy in these cells, that mitochondrial membrane potential was preserved, and that apoptotic changes were not demonstrated despite the relatively high-dose irradiation of 30 Gy. In the present study, we examined the immunocytochemical characteristics of the apoptotic-resistance of the HS-Os-1 cell line against irradiation in order to clarify its possible implications regarding radiosensitivity. The results showed that these cells lack P53 and Bax protein expression, and strong peroxidase activity was confirmed in the nuclei of the cells. Moreover, SODII (manganese superoxide dismutase II) protein expression was gradually increased in spite of irradiation of up to 30 Gy. Therefore, it is concluded that HS-Os-1 cells are originally apoptotic-resistant and that the cells possess a strong ability to scavenge for free radicals. To convert these cells to a state of apoptotic-susceptibility, a powerful oxidant such as hydrogen peroxide might exert such an effect in terms of the production of hydroxyl radicals in lysosomes in the cells as shown in our previous studies. The origin of the radioresistance of the human osteosarcoma cell line HS-Os-1 is considered to to be low degree of ROS formation following irradiation, reflecting the strong scavenging ability of these cells for free radicals including hydroxyl radicals.     

Radiation-induced reactive oxygen species formation prior to oxidative DNA damage in human peripheral T cells

Previously, we demonstrated that human peripheral T lymphocytes revealed early apoptotic changes (annexin V-positive) and late apoptotic changes (propidium iodide-positive), at 13 and 24 h, respectively, after irradiation of 5 Gy. Changes in mitochondrial membrane potential were observed at 10 h after irradiation of 5 Gy. Subsequently, mitochondrial cytochrome c-release was confirmed. In order to elucidate the mechanism which acts prior to the mitochondrial membrane potential changes, we examined in the previous study the radiation dose and the timing of oxidative DNA damage induced in human peripheral T lymphocytes following 10 MV X-ray irradiation. As a result, the production of 8-oxoguanine, i.e., the product of oxidative DNA damage, was clearly identified starting at 10, 6, and 3 h, after 2, 5, and 20 Gy of irradiation, respectively. Therefore, we examined in the present study reactive oxygen species (ROS) formation in T lymphocytes following 5 Gy of irradiation. Using a CCD camera system, we monitored fluorescence in T lymphocytes loaded with the succinimidyl ester of dichlorodihydrofluorescein diacetate (H2DCFDA), which is non-fluorescent until oxidized by ROS. We found that ROS formation occurred immediately after irradiation, continued for several hours, and resulted in oxidative DNA damage. Therefore, the origin of hyper-radiosensitivity of T lymphocytes seemed to be the high production of ROS in the mitochondrial DNA following irradiation.     

Mechanism of hydrogen peroxide-induced apoptosis of the human osteosarcoma cell line HS-Os-1

In our previous study, we examined radiation-induced ROS formation, oxidative DNA damage, early apoptotic changes, and mitochondrial membrane dysfunction in the human osteosarcoma cell line HS-Os-1, which was established from an osteoblastic tumor that arose in the left humerus of an 11-year-old girl and was already morphologically characterized in vitro and in vivo. We found that ROS formation and oxidative DNA damage were scarcely seen after irradiation of up to 30 Gy in these cells; that mitochondrial membrane potential was preserved; and that apoptotic changes were not demonstrated despite the relatively high-dose irradiation of 30 Gy. Based on these results, the radioresistance of the human osteosarcoma cell line HS-Os-1, was considered to arise, at least in part, from the low level of ROS formation following irradiation, which in turn may have resulted from the strong scavenging ability of the cells for free radicals, including hydroxyl radicals. Therefore, in this study, we examined the effect of exogenous hydrogen peroxide, which causes a potent oxidative stress and has been demonstrated to be a potent apoptosis-inducer in many kinds of cells. We found that addition of 1 or 10 mM hydrogen peroxide induced ROS formation, oxidative DNA damage, dysfunction of the mitochondrial membrane potential, and early apoptotic changes in the human osteosarcoma cell line HS-Os-1. We therefore concluded that intracellular ROS formation is involved in the hydrogen peroxide-induced apoptosis of HS-Os-1 cells.     

Apoptotic-resistance of the human osteosarcoma cell line HS-Os-1 to irradiation is converted to apoptotic-susceptibility by hydrogen peroxide: a potent role of hydrogen peroxide as a new radiosensitizer

In our previous study, we demonstrated that the radioresistance of the human osteosarcoma cell line HS-Os-1, was considered to arise, at least in part, from the low level of ROS formation following irradiation, which in turn may have resulted from the strong scavenging ability of the cells for free radicals, including hydroxyl radicals. Following the study, we found that addition of 1 or 10 mM hydrogen peroxide induced ROS formation, oxidative DNA damage, dysfunction of the mitochondrial membrane potential, and early apoptotic changes in the human osteosarcoma cell line HS-Os-1. We therefore speculated that combined use of irradiation and hydrogen peroxide might exert an additive effect for apoptotic-resistant tumors such as the human osteosarcoma cell line HS-Os-1, in terms of preservation of the radiation-induced hydroxyl radical production supported by the intracellular ROS formation that is induced by exogenous hydrogen peroxide addition. Therefore, in this study, we examined the effect of various doses of irradiation on the existence of 0.1 mM hydrogen peroxide in the culture medium. We found that irradiation with 10 or 20 Gy, under the condition of the presence of 0.1 mM hydrogen peroxide, induced ROS formation, oxidative DNA damage, dysfunction of the mitochondrial membrane potential, and early apoptotic changes in the human osteosarcoma cell line HS-Os-1, though ROS formation and oxidative DNA damage were scarcely seen in response to irradiation of up to 30 Gy, as was shown in our previous study. We therefore concluded that the combined modality of irradiation and such a low concentration of hydrogen peroxide (0.1 mM) is potentially applicable in clinical radiotherapy for many kinds of apoptotic-resistant neoplasms in terms of achieving both local control and improving survival benefit of patients.     

Radiation-induced oxidative DNA damage, 8-oxoguanine,in human peripheral T cells

The mechanism leading to the high level of radiosensitivity of T lymphocytes has not yet been fully described. In our previous study, we demonstrated that human peripheral T lymphocytes revealed early apoptotic changes (annexin V-positive) and late apoptotic changes (propidium iodide-positive), at 13 and 24 h after irradiation of 5 Gy, respectively. Changes in mitochondrial membrane potential were observed at 10 h after irradiation of 5 Gy. Subsequently, mitochondrial cytochrome c release was confirmed. In order to elucidate the mechanism which occurs prior to the mitochondrial membrane potential changes, we examined in the present study the radiation dose and the timing of oxidative DNA damage induced in human peripheral T lymphocytes following 10 MV X-ray irradiation. As a result, the production of 8-oxoguanine, i.e., the product of oxidative DNA damage, was clearly identified starting at 10, 6, and 3 h, after 2, 5, and 20 Gy of irradiation, respectively. Therefore, we concluded that it remains necessary to evaluate the extent of radiation-induced oxidative DNA damage. Furthermore, it is important to analyze superoxide radical production and scavenging in terms of the variety of radiosensitivities found among various types of normal tissue cells and neoplastic cells.     

Reactive oxygen species-producing site in radiation-induced apoptosis of human peripheral T cells: involvement of lysosomal membrane destabilization

In our previous studies, we have partly elucidated the mechanism of radiation-induced apoptosis of human peripheral T cells. The exact site of the ROS (reactive oxygen species) formation induced by irradiation has been so far unknown. Therefore, in this study, we investigated the site of ROS formation by utilizing MitoCapture, H2DCFDA (succinimidyl ester of dichlorodihydrofluorescein diacetate), DAPI, and Lysosensor. Our results showed that ROS formation apparently originated in the mitochondria and/or lysosomes instead of in the nuclei of irradiated T cells. Moreover, lysosomal swelling and deformity, possibly revealing lysosomal membrane instability, were observed at 1 h after 5 Gy irradiation of T cells. At 4 h after irradiation of 5 Gy, increase of fluorescence around the lysosomes, possibly revealing lysosomal rupture, was seen. Based on the above results, we concluded the possible existence of a new apoptotic cascade involving early lysosomal membrane destabilization in radiation-induced apoptosis of human peripheral T cells. Therefore, possible involvement of lysosomal protease leakage caused by hydroxyl radical formation in lysosomes (possibly resulting in mitochondrial membrane dysfunction) is considered to play an important role in radiation-induced T cell apoptosis.     

Metabolic effects of X-ray irradiation on adult human articular chondrocytes

There have been few reports regarding the metabolic effects of X-ray irradiation on adult human articular chondrocytes. The purpose of this study was to evaluate whether exposure to X-ray irradiation during tumor surgery can cause impaired metabolism in adult articular cartilage. To achieve this we exposed cultured chondrocytes isolated from normal or degenerated cartilage to varying doses of X-ray irradiation, then measured apoptosis, and the production of chondroitin sulfates (CS), prostaglandin E2 (PGE2) and p38 mitogen-activated protein kinase (MAPK) in these cells. The number of apoptotic cells was not affected by irradiation in chondrocytes from normal or degenerated cartilage. Likewise, the production of C6S and C4S was not altered by irradiation in either group. The concentration of PGE2 in non-degenerated chondrocyte cultures did not change with radiation in a dose-dependent manner. However, the concentration of PGE2 in degenerated chondrocytes increased in a radiation dose-dependent manner. Irradiation at 10 Gy, in degenerated chondrocytes, induced remarkable activation of p38. This suggests that it is important to consider whether there is an osteoarthritic joint in the area that is to receive radiation therapy during tumor surgery.     

The role of apoptotic resistance in irradiated adult articular chondrocytes

There have so far been no studies on the apoptosis of adult articular chondrocytes after X-ray irradiation. The purpose of this study was to assess the apoptotic resistance of articular chondrocytes in X-ray radiation, in order to examine the possibility of irradiated allogenic chondrocyte implantation. Adult human chondrocytes of the non-degenerated cartilage group without X-ray irradiation did not show positive cells of Annexin V and PI staining in a 48 h culture. The Annexin V positive chondrocytes did not increase in a radiation dose dependent manner, and the PI positive cells were slightly increased at 30 Gy irradiation. In the degenerated cartilage group, the PI positive chondrocytes without irradiation were present, and both the Annexin V and PI positive chondrocytes increased in a radiation dose dependent manner. The Annexin V and PI positive staining of chondrocytes in the non-degenerated cartilage group was less than that of the degenerated cartilage group in the same dose of X-ray irradiation exposure. Loss of the mitochondrial membrane potential, revealed in an early stage of apoptosis, did not show in the irradiated chondrocytes of the non-degenerated cartilage, but were demonstrated in those of the degenerated cartilage. These results demonstrated that the non-degenerated chondrocytes of X-ray irradiation were highly resistant for apoptosis, and this knowledge could be applied to allogenic chondrocytes implantation.     

The relative contributions of B and T lymphocytes in the human peripheral blood mutagen test system as determined by cell survival,mitogenic stimulation,and induction of chromosome aberrations by radiation

Ficoll-Hypaque-separated subpopulations of human peripheral blood T and B lymphocytes were exposed to 0, 0.5, 1.0, 2.5, or 5.0 Gy of γ rays. Three parameters were examined: Survival, as measured by trypan blue dye exclusion in unstimulated cultures five days after irradiation; mitotic index, measured in phytohemagglutinin (PHA)-stimulated cultures 48 and 72 hours after irradiation; and chromosome aberration frequency, measured 48 or 60 hours after irradiation. Survival curves of T, B, and null cells are biphasic; the D₀ values for the radiosensitive populations of all three cell types are close to 0.6 Gy but are different for the radioresistant populations: 2.7 Gy for B cells, 4.77 Gy for T cells, and 6.03 Gy for null cells. B cells, as well as T cells, are stimulated to divide by PHA, and B cells comprise at least 10% of the mitotic figures seen in unirradiated cultures at 48 hours. The proportion of B lymphocytes in mitosis at any particular time after PHA stimulation decreases with increasing radiation dose, which reflects a higher mitotic radiosensitivity of B than of T cells. No significant difference, however, in chromosome aberration frequency was found between T and B cells.     

Radioprotection against DNA damage by an extract of Indian green mussel, Perna viridis (L).

This study describes the radioprotective ability of a hydrolysate prepared using an enzyme-acid hydrolysis method from the green mussel Perna viridis in terms of its ability to prevent radiation-induced damage in plasmid DNA, cell death, reactive oxygen species (ROS) formation, and DNA damage in mice lymphocytes. The mussel hydrolysate (MH) present during irradiation showed significant protection from gamma-radiation-induced strand breaks in plasmid DNA as evaluated by gel electrophoresis. Viability studies by trypan blue dye exclusion and MTT assay showed that preincubation of mice splenic lymphocytes with MH protected them from gamma-radiation-mediated killing. Moreover, the presence of MH during irradiation of isolated mice lymphocytes significantly decreased the DNA damage, as measured by comet assay. Measurement of intracellular ROS by dichlorofluorescein fluorescence revealed that the presence of MH effectively reduced the ROS generated in lymphocytes by both chemical method and gamma-irradiation. Prevention of DNA damage both in plasmid and lymphocytes and cell death in lymphocytes appears correlated with reduction of oxidatively generated free radicals. It is concluded that protection against radiation-induced cell death and DNA damage by MH was attributable to reduction of reactive free radical species generated by gamma-radiation.     

Reactive oxygen species-producing site in radiation and hydrogen peroxide-induced apoptosis of human peripheral T cells: Involvement of lysosomal membrane destabilization

In our previous studies, we showed that the apoptotic resistance of the human osteosarcoma cell line HS-Os-1 against irradiation was easily converted to a state of apoptotic-susceptibility by the addition of a relatively low concentration of hydrogen peroxide to the culture medium just prior to irradiation. When we consider the combined use of radiotherapy and hydrogen peroxide in a clinical setting for patients with radioresistant neoplasms, we need to be careful of the possible augmentation of the radiation effect to normal tissues of patients who undergo radiation therapy for their tumor in the presence of a low concentration of hydrogen peroxide in their topical tumor tissue. Therefore, we examined the combined effect of irradiation and hydrogen peroxide compared to that of irradiation alone for human peripheral T cells which were considered to be representative of normal tissue susceptible to apoptosis induced by irradiation. In this study, we compared the morphological changes in human peripheral T cells between both groups by utilizing MitoCapture, H2DCFDA (succinimidyl ester of dichloro-dihydrofluorescein diacetate), DAPI (4',6-diamidino-2-phenylindole), and LysoSensor. Our results showed that ROS formation was apparently augmented in the mitochondria and/or lysosomes instead of in the nuclei of irradiated T cells in the presence of a low concentration of hydrogen peroxide compared to those treated with irradiation alone. Moreover, dysfunction of mitochondrial membrane potential was also more evidently shown in human peripheral T cells irradiated under existence of a low concentration of hydrogen peroxide compared to T cells treated with 5 Gy irradiation alone. Based on these results, we concluded the possible existence of an augmentation effect of irradiation by the existence of a low concentration of hydrogen peroxide for human peripheral T cells. Therefore, we should be alert for the combined effects of radiation therapy and hydrogen peroxide on normal tissues in possible clinical situations when this combination is used for treatment of patients having radioresistant neoplasms such as osteosarcoma, malignant melanoma, and glioblastoma multiforme.     

Reactive oxygen species-producing site in hydrogen peroxide-induced apoptosis of human peripheral T cells: involvement of lysosomal membrane destabilization

In our previous study, we examined the effect of exogenous hydrogen peroxide, which causes a potent oxidative stress and has been demonstrated to be a potent apoptosis-inducer in many kinds of cells. We found that the addition of 1 or 10 mM hydrogen peroxide induced reactive oxygen species (ROS) formation, oxidative DNA damage, dysfunction of the mitochondrial membrane potential, and early apoptotic changes in the human osteosarcoma cell line HS-Os-1. We therefore concluded that intracellular ROS formation was involved in the hydrogen peroxide-induced apoptosis of HS-Os-1 cells. In contrast to the osteosarcoma cell line HS-Os-1, human peripheral T cells are considered to be easily susceptible to oxidative stress, because these cells lack peroxidase activity. Therefore, in this study, we investigated the site of ROS formation by utilizing MitoCapture, H2DCFDA (succinimidyl ester of dichloro-dihydrofluorescein diacetate), DAPI (4',6-diamidino-2-phenylindole), and LysoSensor. Our results showed that ROS formation was apparently diffusely distributed in T cells oxidatively stressed with 0.1 mM hydrogen peroxide. Moreover, lysosomal swelling and deformity, possibly revealing lysosomal membrane destabilization, were observed in these cells. Based on the above results, there exists an apoptotic cascade involving early lysosomal membrane destabilization in the hydrogen peroxide-induced apoptosis of human peripheral T cells. Therefore, the possible involvement of lysosomal protease leakage caused by hydroxyl radical formation in lysosomes (possibly resulting in mitochondrial membrane dysfunction) is considered to play an important role in hydrogen peroxide-induced T cell apoptosis.     

Short-term culture and gammaH2AX flow cytometry determine differences in individual radiosensitivity in human peripheral T lymphocytes

Histone H2AX, a subfamily of histone H2A, is phosphorylated and forms proteinaceous repair foci at the sites of DNA double-strand breaks in response to genotoxic insults, such as ionizing radiation. This process is believed to play a key role in the repair of DNA damage. In this study, we established a flow cytometry (FCM) system for measuring radiation-induced phosphorylated histone H2AX (gammaH2AX) in cultured human T lymphocytes to evaluate individual radiation sensitivity in vitro. Irradiation of short-term ( approximately 7 days) cultured T lymphocytes exhibited significant interindividual, but not interexperimental, differences in the cellular content of gammaH2AX 6 hr after 4 Gy of X-irradiation in three independent experiments using peripheral blood lymphocytes from six healthy donors. However, these differences were not as marked in uncultured lymphocytes, or lymphocytes that were cultured for a prolonged period ( approximately 13 days). The variation of gammaH2AX focus formation in lymphocytes of individuals was reproducible, with differences reaching about 1.5-fold following 7 days of culture. Therefore, the FCM-based gammaH2AX measurement appeared to reflect both the temporal course and the amount of DNA damage within the irradiated lymphocytes. Further, we confirmed that the differences in residual lymphocyte subsets were not involved in individual radiosensitivity. These results suggest that the FCM-based gammaH2AX assay using cultured T lymphocytes might be useful for the rapid and reliable assessment of individual radiation sensitivity involved in DNA damage repair.     

The effect of camphorquinone (CQ) and CQ-related photosensitizers on the generation of reactive oxygen species and the production of oxidative DNA damage

Recent evidence suggests that following visible-light (VL) irradiation, CQ and the CQ-related photosensitizers benzil (BZ), benzophenone (BP), and 9-fluorenone (9-F) generate initiating radicals that may indiscriminately react with molecular oxygen forming reactive oxygen species (ROS). The purpose of this investigation was to determine whether VL-irradiated CQ, BZ, BP, and 9-F cause DNA damage due to the generation of ROS in vitro. ROS formation by CQ and CQ-related photosensitizers+/-dimethyl-p-toluidine (DMT) was investigated in a cell-free system with VL irradiation. DNA damage was determined using PhiX-174 RF I supercoiled double-stranded plasmid DNA and ROS quantified with 4-((9-acridinecarbonyl)amino)-2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPO-9-AC), a fluorogenic ROS-sensitive probe. VL-irradiated CQ, BZ, BP, and 9-F (+/-DMT) produced significant DNA damage at 0.1, 0.5, and 1.0 mM and in a concentration-dependent manner (p<0.05). TEMPO-9-AC revealed that all investigated VL-irradiated photosensitizers produced significant amounts of ROS with BZ in the presence of DMT generating the most ROS after 30, 60, and 90 min. VL-irradiated CQ, BZ, BP, and 9-F +/-DMT continued to generate significant amounts of ROS 90 min after VL irradiation. As a result, future investigations should evaluate the effect of VL-irradiated photosensitizers in cells and possible protective effects provided by antioxidants.     

The immunohistochemical localization of notch receptors and ligands in human articular cartilage, chondroprogenitor culture and ultrastructural characteristics of these progenitor cells

The presence of progenitor/stem cells in human articular cartilage remains controversial. Therefore, we attempted to isolate and culture progenitor/stem cells and to investigate their phenotypic characteristics. Biopsies were obtained (with consent) from patients undergoing arthroscopic surgery. Full depth explants were fixed and cryosectioned or enzymatically digested and the resulting cells cultured and plated on fibronectin-coated dishes. Chondrocytes were cultured until colonies of >32 cells were present. Colonies were trypsinized and expanded in monolayer for pellet culture. Immunolocalization of Notch and its ligands were detected in vivo and in vitro using immunocytochemistry. In vitro studies investigated differences in immunolocalization of Notch and its associated ligands in colony-forming cells and small clusters of non-colony-forming cells. The ultrastructure of the chondroprogenitors was examined by scanning and transmission electron microscopy. Results revealed that the immunolocalization of Notch-1 and its ligand Delta were concentrated in regions closest to the articular surface. Notch-1 was also densely localized in the deeper zone of articular cartilage. Notch-2 immunolabeling was densely localized in all zones of articular cartilage. Jagged-1 was concentrated in the deeper regions of articular cartilage. Notch-1, Delta and Jagged-1 were more abundant in colony-forming cells than non-colony-forming chondrocytes in vitro. Notch-3, Notch-4 and Jagged-2 were absent from all regions of the articular cartilage tissues and cultured cartilage cells in vitro. Ultrastructurally, chondrocytes cultured in monolayer dedifferentiated to fibroblast-like cells with cell surface processes of varying lengths, pellet cultured cells varied in morphology, as flattened and rounded. In conclusion, we propose that adult human articular cartilage may contain cells having progenitor cell features.     

Establishment of Human cell Type-Specific iPS cells with Enhanced Chondrogenic Potential

The propensity of induced pluripotent stem (iPS) cells to differentiate into specific lineages may be influenced by a number of factors, including the selection of the somatic cell type used for reprogramming. Herein we report the generation of new iPS cells, which we derived from human articular chondrocytes and from cord blood mononucleocytes via lentiviral-mediated delivery of Oct4, Klf4, Sox2, and cMyc. Molecular, cytochemical, and cytogenic analyses confirmed the acquisition of hallmark features of pluripotency, as well as the retention of normal karyotypes following reprogramming of both the human articular chondrocytes (AC) and the cord blood (CB) cells. In vitro and in vivo functional analyses formally established the pluripotent differentiation capacity of all cell lines. Chondrogenic differentiation assays comparing iPS cells derived from AC, CB, and a well established dermal fibroblast cell line (HDFa-Yk26) identified enhanced proteoglycan-rich matrix formation and cartilage-associated gene expression from AC-derived iPS cells. These findings suggest that the tissue of origin may impact the fate potential of iPS cells for differentiating into specialized cell types, such as chondrocytes. Thus, we generated new cellular tools for the identification of inherent features driving high chondrogenic potential of reprogrammed cells.     

Autorosette formation in humans: study of the specificity of the T cell receptors for autologous erythrocytes

Spontaneous autorosette formation has been described as being restricted to a subpopulation of the circulating helper/inducer T cell subset. In order to study the specificity of the binding between human lymphocytes and autologous red blood cells (auto-RBC), we have investigated the relationship between autorosette forming cells (auto-RFC) and rosettes formed with allogeneic (allo-) or xenogeneic (xeno-) RBC. Using a mixed rosette assay in which the origin of the erythrocytes was assessed by the FITC labeling of one type of erythrocyte, we have shown that auto-RFC and allo-RFC belong to the same T cell subset, and that the T cells which rosette with auto-RBC can also bind xenogeneic (pig, sheep, rabbit) RBC, although a disparate incidence of rosettes is found depending upon the origin of the erythrocytes. Whether T lymphocytes co-expressed distinct receptors for RBC of different species was then investigated. Preincubation of lymphocytes with monoclonal antibody OKT11A (directed against the T cell receptors for sheep RBC) completely abrogated rosette formation with auto- or allo-RBC, indicating that auto- and allo-RBC interact with the lymphocytes by their receptors for sheep RBC. Therefore, the auto-RFC may represent T lymphocytes having high affinity receptors for sheep RBC.     

Chromosomal radiosensitivity of lymphocytes from Alzheimer''s disease patients.

We have examined chromosome aberrations in gamma irradiated (3 Gy) lymphocytes from five patients with Alzheimer's disease (AD). In each case, the number of dicentrics was significantly higher than the number in irradiated lymphocytes from five age matched normal subjects, the mean value for AD cells being about 25% higher. There was no significant difference in number of acentrics between AD and normal cells. Examination of the number of first, second, and third division metaphases, using fluorescence plus Giemsa staining, indicated that there was no difference in cycling time between AD and normal cells, and that after irradiation both groups showed the same mitotic delay. The similarity of our findings to those of others with irradiated Down's syndrome cells (from adult patients) is discussed.     

Regeneration of hyaline articular cartilage with irradiated transforming growth factor beta1-producing fibroblasts

The regeneration of hyaline articular cartilage by cell-mediated gene therapy using transforming growth factor beta(1) (TGF-beta(1))-producing fibroblasts (NIH 3T3-TGF-beta(1)) has been reported previously. In this study, we investigated whether TGF-beta(1)-producing fibroblasts irradiated with a lethal dose of radiation are still capable of inducing the regeneration of hyaline articular cartilage. NIH 3T3TGF-beta(1) fibroblasts were exposed to doses of 20, 40, or 80 Gy, using a irradiator, and then injected into artificially made partial defects on the femoral condyle of rabbit knee joints. The rabbits were killed 3 or 6 weeks postinjection and hyaline articular cartilage regeneration was evaluated by histological and immunohistochemical staining (n = 5 per each group). Irradiated NIH 3T3-TGFbeta(1) fibroblasts started to die rapidly 3 days after irradiation; moreover, the kinetics of their viability were similar regardless of the radiation intensity. TGF-beta1 expression, measured by ELISA, showed that the TGF-beta(1) protein produced from the irradiated cells peaked 5 days after irradiation and thereafter declined rapidly. Complete filling of the defect with reparative tissue occurred in all the groups, although variations were observed in terms of the nature of the repair tissue. Histological and immunohistochemical staining of the repair tissue showed that the tissue newly formed by irradiated NIH 3T3-TGF-beta(1) fibroblasts after exposure to 20 Gy had hyaline cartilage-like characteristics, as was observed in the nonirradiated controls. On the other hand, the repair tissue formed by NIH 3T3-TGF-beta(1) fibroblasts irradiated with 40 or 80 Gy showed more fibrous cartilage-like tissue. These results suggest that TGF-beta(1)-producing fibroblasts irradiated up to a certain level of lethal dose (i.e., 20 Gy) are able to induce normal-appearing articular cartilage in vivo. Therefore, irradiated heterologous cell-mediated TGF-beta(1) gene therapy may be clinically useful and an efficient method of regenerating hyaline articular cartilage.