Chung-Lan Kao

Identification of CD133-Positive Radioresistant Cells in Atypical Teratoid/ Rhabdoid Tumor

Atypical teratoid/rhabdoid tumor (AT/RT) is an extremely malignant neoplasm in the central nervous system (CNS) which occurs in infancy and childhood. Recent studies suggested that CD133 could be considered a marker for brain cancer stem-like cells (CSCs). However, the role of CD133 in AT/RT has never been investigated. Herein we report the isolation of CD133-positive cells (CD133+), found to have the potential to differentiate into three germ layer tissues, from tissues of nine AT/RT patients. The migration/invasion/malignancy and radioresistant capabilities of CD133+ were significantly augmented when compared to CD133−. The clinical data showed that the amount of CD133+ in AT/RTs correlated positively with the degree of resistance to radiation therapy. Using cDNA microarray analysis, the genotoxic–response profiles of CD133+ and CD133− irradiated with 10 Gy ionizing radiation (IR) were analyzed 0.5, 2, 6, 12 and 24 h post-IR. We then validated these microarray data and showed increased phosphorylation after IR of p-ATM, p-RAD17, and p-CHX2 as well as increased expression of BCL-2 protein in CD133+ compared to CD133−. Furthermore, we found that CD133+ can effectively resist IR with cisplatin- and/or TRAIL-induced apoptosis. Immunohistochemical analysis confirmed the up-regulated expression of p-ATM and BCL-2 proteins in IR-treated CD133+ xenotransgrafts in SCID mice but not in IR-treated CD133−. Importantly, the effect of IR in CD133+ transplanted mice can be significantly improved by a combination of BCL-2 siRNA with debromohymenialdisine, an inhibitor of checkpoint kinases. In sum, this is the first report indicating that CD133+ AT/RT cells demonstrate the characteristics of CSCs. The IR-resistant and anti-apoptotic properties in CD133+ may reflect the clinical refractory malignancy of AT/RTs and thus the activated p-ATM pathway and BCL-2 expression in CD133+ could be possible targets to improve future treatment of deadly diseases like AT/RT.

Resveratrol Promotes Osteogenic Differentiation and Protects Against Dexamethasone Damage in Murine Induced Pluripotent Stem Cells

Resveratrol is a natural polyphenol antioxidant that has been shown to facilitate osteogenic differentiation. A recent breakthrough has demonstrated that ectopic expression of four genes is sufficient to reprogram murine and human fibroblasts into induced pluripotent stem (iPS) cells. However, the roles of resveratrol in the differentiation and cytoprotection of iPS cells have never been studied. In this study, we showed that, in addition to cardiac cells, neuron-like cells, and adipocytes, mouse iPS cells could differentiate into osteocyte-like cells. Using atomic force microscopy that provided nanoscale resolution, we monitored mechanical properties of living iPS cells during osteogenic differentiation. The intensity of mineralization and stiffness in differentiating iPS significantly increased after 14 days of osteogenic induction. Furthermore, resveratrol was found to facilitate osteogenic differentiation in both iPS and embryonic stem cells, as shown by increased mineralization, up-regulation of osteogenic markers, and decreased elastic modulus. Dexamethasone-induced apoptosis in iPS cell-derived osteocyte-like cells was effectively prevented by pretreatment with resveratrol. Furthermore, resveratrol significantly increased manganese superoxide dismutase expression and intracellular glutathione level, thereby efficiently decreasing dexamethasone-induced reactive oxygen species (ROS) production and cytotoxicity. Transplantation experiments using iPS cell-derived osteocyte-like cells further demonstrated that oral intake of resveratrol could up-regulate osteopontin expression and inhibit teratoma formation in vivo. In sum, resveratrol can facilitate differentiation of iPS cells into osteocyte-like cells, protect these iPS cell-derived osteocyte-like cells from glucocorticoid-induced oxidative damage, and decrease tumorigenicity of iPS cells. These findings implicate roles of resveratrol and iPS cells in the stem cell therapy of orthopedic diseases.

A novel in vitro retinal differentiation model by co-culturing adult human bone marrow stem cells with retinal pigmented epithelium cells

Human retinal pigment epithelium (HRPE) cells are important in maintaining the normal physiology within the neurosensory retina and photoreceptors. Recently, transplantation of HRPE has become a possible therapeutic approach for retinal degeneration. By negative immunoselection (CD45 and glycophorin A), in this study, we have isolated and cultivated adult human bone marrow stem cells (BMSCs) with multilineage differentiation potential. After a 2- to 4-week culture under chondrogenic, osteogenic, adipogenic, and hepatogenic induction medium, these BMSCs were found to differentiate into cartilage, bone, adipocyte, and hepatocyte-like cells, respectively. We also showed that these BMSCs could differentiate into neural precursor cells (nestin-positive) and mature neurons (MAP-2 and Tuj1-positive) following treatment of neural selection and induction medium for 1 month. Furthermore, the plasticity of BMSCs was confirmed by initiating their differentiation into retinal cells and photoreceptor lineages by co-culturing with HRPE cells. The latter system provides an ex vivo expansion model of culturing photoreceptors for the treatment of retinal degeneration diseases.

Celecoxib and radioresistant glioblastoma-derived CD133+ cells: improvement in radiotherapeutic effects. Laboratory investigation.

OBJECT Glioblastoma, the most common primary brain tumor, has a poor prognosis, even with aggressive resection and chemoradiotherapy. Recent studies indicate that CD133(+) cells play a key role in radioresistance and recurrence of glioblastoma. Cyclooxygenase-2 (COX-2), which converts arachidonic acid to prostaglandins, is over-expressed in a variety of tumors, including CD133(+) glioblastomas. The COX-2-derived prostaglandins promote neovascularization during tumor development, and conventional radiotherapy increases the proportion of CD133(+) cells rather than eradicating them. The aim of the present study was to investigate the role of celecoxib, a selective COX-2 inhibitor, in enhancing the therapeutic effects of radiation on CD133(+) glioblastomas. METHODS Cells positive for CD133 were isolated from glioblastoma specimens and characterized by flow cytometry, then treated with celecoxib and/or ionizing radiation (IR). Clonogenic assay, cell irradiation, cell cycle analysis, Western blot, and xenotransplantation were used to assess the effects of celecoxib alone, IR alone, and IR with celecoxib on CD133(+) and CD133(-) glioblastoma cells. Three separate xenotransplantation experiments were carried out using 310 severe combined immunodeficient (SCID) mice: 1) an initial tumorigenicity evaluation in which 3 different quantities of untreated CD133(-) cells or untreated or pretreated CD133(+) cells (5 treatment conditions) from 7 different tumors were injected into the striatum of 2 mice (210 mice total); 2) a tumor growth study (50 mice); and 3) a survival study (50 mice). For these last 2 studies the same 5 categories of cells were used as in the tumorigenicity (untreated CD133(-) cells, untreated or pretreated CD133(+) cells, with pretreatment consisting of celecoxib alone, IR alone, or IR and celecoxib), but only 1 cell source (Case 2) and quantity (5 × 10(4) cells) were used. RESULTS High levels of COX-2 protein were detected in the CD133(+) but not the CD133(-) glioblastoma cells. The authors further demonstrated that 30 μM celecoxib was able to effectively enhance the IR effect in inhibiting colony formation and increasing IR-mediated apoptosis in celecoxib-treated CD133(+) glioblastoma cells. Furthermore, reduction in radioresistance was correlated with the induction of G2/M arrest, which was partially mediated through the increase in the level of phosphorylated-cdc2. In vivo xenotransplant analysis further confirmed that CD133(+)-associated tumorigenicity was significantly suppressed by celecoxib treatment. Importantly, pretreatment of CD133(+) glioblastoma cells with a combination of celecoxib and IR before injection into the striatum of SCID mice resulted in a statistically significant reduction in tumor growth and a statistically significant increase in the mean survival rate of the mice. CONCLUSIONS Celecoxib combined with radiation plays a critical role in the suppression of growth of CD133(+) glioblastoma stemlike cells. Celecoxib is therefore a radiosensitizing drug for clinical application in glioblastoma.

Induction of Insulin-Producing Cells Derived from Endometrial Mesenchymal Stem-like Cells

Studies have demonstrated that mesenchymal stem-like cells can be isolated from endometrium. However, the potential of endometrial-derived stem cells to differentiate into insulin-positive cells and functionally secrete insulin remains undetermined. We isolated endometrial mesenchymal stem-like cells (EMSCs) from human endometrial tissue from six donors. The insulin-secreting function of EMSCs was further analyzed in vitro and in transplanted grafts in vivo. We successfully isolated EMSCs from human endometrium, and our results showed that EMSCs expressed high levels of stemness genes (Nanog, Oct-4, Nestin). Under specific induction conditions for 2 weeks, EMSCs formed three-dimensional spheroid bodies (SBs) and secreted C-peptide. The high insulin content of SB-EMSCs was confirmed by enzyme-linked immunosorbent assay, and glucose responsiveness was demonstrated by measuring glucose-dependent insulin secretion. Using cDNA microarrays, we found that the expression profiles of SB-EMSCs are related to those of islet tissues. Insulin and C-peptide production in response to glucose was significantly higher in SB-EMSCs than in undifferentiated EMSC controls. Furthermore, upon differentiation, SB-EMSCs displayed increased mRNA expression levels of NKx2.2, Glut2, insulin, glucagon, and somatostatin. Our results also showed that SB-EMSCs were more resistant to oxidative damage and oxidative damage-induced apoptosis than fibroblasts from the same patient. It is noteworthy that SB-EMSCs xenotransplanted into immunocompromised mice with streptozotocin-induced diabetes restored blood insulin levels to control values and greatly prolonged the survival of graft cells. These data suggest that EMSCs not only play a novel role in the differentiation of pancreatic progenitors, but also can functionally enhance insulin production to restore the regulation of blood glucose levels in an in vivo transplantation model.

Lower limb power rehabilitation (LLPR) using interactive video game for improvement of balance function in older people

Declined balance functions have adverse effects on elderly population. Lower limbs muscle power training is currently an emerging concept in rehabilitation on individuals with decreased balance and mobility. In this prospective, controlled study, we used a human-computer interactive video-game-based rehabilitation device (LLPR) for training of lower limb muscle power in the elderly. Forty (aged >65 years) individuals were recruited from the community. Twenty participants in the exercise group received 30-min training, twice a week, using the LLPR system. The LLPR system allows participants to perform fast speed sit-to-stand (STS) movements. Twenty age-matched participants in the control group performed slow speed STS movements, as well as strengthening and balance exercises, with the same frequency and duration. The results were compared after 12 sessions (6 weeks) of training. The mechanical and time parameters during STS movement were measured using the LLPR system. Modified falls efficacy scale (MFES), Tinetti Performance-Oriented Mobility Assessment (POMA), function reach test, five times sit to stand (FTSS) and Timed Up and Go (TUG) were administered to participants as clinical assessments. Results showed that in the exercise group, all the mechanical and time parameters showed significant improvement. In control group, only the maximal vertical ground reaction force (MVGRF) improved significantly. For clinical assessments (balance, mobility, and self-confidence), exercise group showed significantly better scores. The STS movements in video-game-based training mimic real life situations which may help to transfer the training effects into daily activities. The effectiveness of lower limb muscle training is worthy of further investigation.

Delivery of Oct4 and SirT1 with cationic polyurethanes-short branch PEI to aged retinal pigment epithelium

Cationic polyurethane, a biodegradable non-viral vector, protects DNA from nuclease degradation and helps to deliver genes efficiently. Oct4, a POU-domain transcription factor, is highly expressed in maintaining pluripotency and cellular reprogramming process in stem cells. SirT1, a NAD-dependent histone deacetylase, is an essential mediator of cellular longevity. Herein we demonstrated that both Oct4 and SirT1 (Oct4/SirT1) expression was decreased in an age-dependent manner in retina with aged-related macular degeneration and retinal pigment epithelium cells (RPEs). To investigate the possible rescuing role of Oct4/SirT1, polyurethane-short branch polyethylenimine (PU-PEI) was used to deliver Oct4/SirT1 into aged RPEs (aRPEs) or light-injured rat retinas. Oct4/SirT1 overexpression increased the expression of several progenitor-related genes and the self-renewal ability of aRPEs. Moreover, Oct4/SirT1 overexpression resulted in the demethylation of the Oct4 promoter and enhanced the expression of antioxidant enzymes, which was accompanied by a decrease in intracellular ROS production and hydrogen peroxide-induced oxidative stress. Importantly, PU-PEI-mediated Oct4/SirT1 gene transfer rescued retinal cell loss and improved electroretinographic responses in light-injured rat retinas. In summary, these data suggest that PU-PEI-mediated delivery of Oct4/SirT1 reprograms aRPEs into a more primitive state and results in cytoprotection by regulating the antioxidative capabilities of these cells.

Topical cooling (icing) delays recovery from eccentric exercise-induced muscle damage.

Abstract Tseng, C-Y, Lee, J-P, Tsai, Y-S, Lee, S-D, Kao, C-L, Liu, T-C, Lai, C-S, Harris, MB, and Kuo, C-H. Topical Cooling (Icing) Delays Recovery From Eccentric Exercise–Induced Muscle Damage. J Strength Cond Res 27(5): 1354–1361, 2013—It is generally thought that topical cooling can interfere with blood perfusion and may have positive effects on recovery from a traumatic challenge. This study examined the influence of topical cooling on muscle damage markers and hemodynamic changes during recovery from eccentric exercise. Eleven male subjects (age 20.2 ± 0.3 years) performed 6 sets of elbow extension at 85% maximum voluntary load and randomly assigned to topical cooling or sham groups during recovery in a randomized crossover fashion. Cold packs were applied to exercised muscle for 15 minutes at 0, 3, 24, 48, and 72 hours after exercise. The exercise significantly elevated circulating creatine kinase-MB isoform (CK-MB) and myoglobin levels. Unexpectedly, greater elevations in circulating CK-MB and myoglobin above the control level were noted in the cooling trial during 48–72 hours of the post-exercise recovery period. Subjective fatigue feeling was greater at 72 hours after topical cooling compared with controls. Removal of the cold pack also led to a protracted rebound in muscle hemoglobin concentration compared with controls. Measures of interleukin (IL)-8, IL-10, IL-1&bgr;, and muscle strength during recovery were not influenced by cooling. A peak shift in IL-12p70 was noted during recovery with topical cooling. These data suggest that topical cooling, a commonly used clinical intervention, seems to not improve but rather delay recovery from eccentric exercise–induced muscle damage.

Can sit-to-stand lower limb muscle power predict fall status?

Sit-to-stand (STS) movements are essential for daily activities. Failure to perform STS movements efficiently and smoothly may lead to falls. In this study, we developed a forceplate to analyze vertical ground reaction force (VGRF), STS duration and generated muscle power to investigate which parameters were fall status predictors. A total of 105 participants were included in this study and were grouped into those (1) aged between 20 and 30 years (Young), (2) aged above 65 years without a history of falling (Non-fallers) and (3) aged above 65 with a history of falling in the past 12 months (Fallers). The results indicated a significantly higher maximal lower limb muscle power (MP) for the Young (9.05 ± 3.66 W/kg), followed by Non-fallers (5.50 ± 2.02W/kg) and Fallers (3.66 ± 1.45 W/kg) as well as higher modified falls efficacy scale (MFES) scores for the Young (Young: 9.88 ± 0.10; Non-fallers: 6.27 ± 1.40; Fallers: 4.83 ± 0.89) and shorter times for the five times sit-to-stand test (FSTST) for the young (Young: 6.09 ± 2.20 s; Non-fallers: 15.65 ± 3.30s; Fallers: 19.82 ± 4.46 s). There was a significant difference between the Young group and the Non-fallers in the maximal vertical ground reaction force (VGRF) (138.79 ± 24.20 N/BW in Young, 117.51 ± 8.57 N/BW in old Non-fallers, p < 0.01), and there was a significant difference between the Non-fallers and the Fallers in the duration of the STS movement (2.74 ± 0.87 s for the Non-fallers, 4.27 ± 2.56 s for the Fallers, p < 0.01). The regression analysis results further indicated that only MP and the STS stabilization phase could differentiate individuals who had past fall events. Therefore, the equipment we developed could potentially be useful in the assessment and monitoring of balance and the risk of falling in older people.

Stem cell-based neuroprotective and neurorestorative strategies.

Stem cells, a special subset of cells derived from embryo or adult tissues, are known to present the characteristics of self-renewal, multiple lineages of differentiation, high plastic capability, and long-term maintenance. Recent reports have further suggested that neural stem cells (NSCs) derived from the adult hippocampal and subventricular regions possess the utilizing potential to develop the transplantation strategies and to screen the candidate agents for neurogenesis, neuroprotection, and neuroplasticity in neurodegenerative diseases. In this article, we review the roles of NSCs and other stem cells in neuroprotective and neurorestorative therapies for neurological and psychiatric diseases. We show the evidences that NSCs play the key roles involved in the pathogenesis of several neurodegenerative disorders, including depression, stroke and Parkinson’s disease. Moreover, the potential and possible utilities of induced pluripotent stem cells (iPS), reprogramming from adult fibroblasts with ectopic expression of four embryonic genes, are also reviewed and further discussed. An understanding of the biophysiology of stem cells could help us elucidate the pathogenicity and develop new treatments for neurodegenerative disorders. In contrast to cell transplantation therapies, the application of stem cells can further provide a platform for drug discovery and small molecular testing, including Chinese herbal medicines. In addition, the high-throughput stem cell-based systems can be used to elucidate the mechanisms of neuroprotective candidates in translation medical research for neurodegenerative diseases.