Optimization of Spheroid Colony Culture and Cryopreservation of Nucleus Pulposus Cells for the Development of Intervertebral Disc Regenerative Therapeutics

After the discovery of functionally superior Tie2-positive nucleus pulposus (NP) progenitor cells, new methods were needed to enable mass culture and cryopreservation to maintain these cells in an undifferentiated state with high cell yield. We used six types of EZSPHERE® dishes, which support spheroid-forming colony culture, and examined NP cell spheroid-formation ability, number, proliferation, and mRNA expression of ACAN, COL1A2, COL2A1, and ANGPT1. Six different types of cryopreservation solutions were examined for potential use in clinical cryopreservation by comparing the effects of exposure time during cryopreservation on cell viability, Tie2-positivity, and cell proliferation rates. The spheroid formation rate was 45.1% and the cell proliferation rate was 7.75 times using EZSPHERE® dishes. The mRNA levels for COL2A1 and ANGPT1 were also high. In cryopreservation, CryoStor10 (CS10) produced ≥90% cell viability and a high proliferation rate after thawing. CS10 had a high Tie2-positive rate of 12.6% after culturing for 5 days after thawing. These results suggest that EZSPHERE enabled colony formation in cell culture without the use of hydrogel products and that CS10 is the best cryopreservation medium for retaining the NP progenitor cell phenotype and viability. Together, these data provide useful information of NP cell-based therapeutics to the clinic.

Clinical and functional analysis of SOX9 in colorectal cancer.

519 Background: Colorectal cancer (CRC) is the third leading cause of cancer-related deaths worldwide. CRC develops within the intestinal epithelium, which is constantly self-renewing, developmental genes could promote the initiation and progression of cancer. SOX9 transcription factor is expressed especially in intestinal stem cells and in Paneth cells, thus alterations in expression could greatly promote neoplasia. However, the clinical significance and functional role of SOX9 in CRC remains unclear. The aim of this study is to investigate the association of SOX9 expression with relapse in CRC patients and initiate functional studies by evaluating the effects of silencing SOX9 in stem cells properties such as colonospheres formation in HCT116 CRC cells. Methods: 97 FFPE biopsies from CRC patients in stage I (N = 34) and stage II (N = 63) were analyzed. Immunoreactivity of SOX9 was classified as low and high expression groups based on the percentage of positive nucleus and tissue staining intensity. SOX9 silencing was achieved using a specific siRNA and Lipofectamine-RNAiMax and confirmed by qPCR 24 h postransfection. 40,000 HCT116 and HCT116-siSOX9 cells were seeded under adherent and ultra-low attachment conditions for tumorigenesis assays. After 72 h colonospheres were seeded and quantified. ROC analysis was used to assess the clinical correlation of SOX9 with relapse. Experimental data were expressed as means and differences tested by t-Student. Results: Data showed that 12.3% of patients relapsed. Interestingly, all of them showed lower SOX9 expression (p ≤ 0.05), regardless of their relapse free survival. In functional analysis, SOX9-deficient HCT116 cells formed smaller and less-compacted spheroid when compared to non-transfected HCT116 (p ≤ 0.05). Nonetheless, cell proliferation and migration under adherent condition were similar between the groups. Conclusions: This study did not find association between SOX9 expression and relapse. So far, in vitro assays results suggest that silencing of SOX9 inhibits colonospheres formation in HCT116 cells. Further investigation will be performed in order to evaluate the functional importance of SOX9 in chemotherapy response.

Nuclear factor-kappa B p65 evaluation on the Canadian Prostate Cancer Biomarker Network (CPCBN) TMA-series for biomarker validation.

80 Background: The CPCBN has assembled a TMA-based resource comprising the specimens of 1512 prostate cancer patients treated by radical prostatectomy. This richly annotated and multi-institutional resource is available to researchers who wish to access a large cohort to validate prognostic biomarkers (http://www.tfri.ca/en/research/translational-research/cpcbn.aspx). Over the last decade, our laboratory demonstrated with an independent cohort (Gannon PO, et al. Eur J Cancer. 2013 Jul;49(10):2441-8, Labouba I, et al. PLoS One. 2015 Jul 17;10(7):e0131024), the reproducible association of nuclear factor-kappa B (NF-kB) p65 with patient’s risk of biochemical recurrence. Here, we evaluated the CPCBN TMA-series for p65 expression. Methods: Two independent observers scored the frequency of p65 nuclear localisation on digital images obtained after automated immunohistochemistry analysis of p65. Over the available minimum 3 cores of tumour tissue per patient, an average percentage of positive nucleus frequency was used. Statistical analyses were performed using SPSS software. Results: High nuclear frequency of NF-kB p65 (cut-off at 3%) was associated with an increased risk for patients to experience a biochemical relapse (p < 0.001; Exp(B) = 1.599; 95%CI = 1.321-1.937), develop bone metastasis (p = 0.007; Exp(B) = 2.126; 95%CI = 1.234-3.663)and die from their disease (p = 0.001; Exp(B) = 3.117; 95%CI = 1.55-6.266). In multivariate analyses, p65 also remained independent from clinical parameters (PSA, Gleason score and pTNM): biochemical relapse (p = 0.005; Exp(B) = 1.331; 95%CI = 1.092-1.623), bone metastasis (p = 0.033; Exp(B) = 1.82; 95%CI = 1.04- 3.158), mortality (p = 0.007; Exp(B) = 2.626; 95%CI = 1.298-5.312) Conclusions: Using a large cohort of Canadian men, our study reiterates the previous study linking NF-kB p65 with prostate cancer progression and highlights the suitability of CPCBN TMAs for biomarker validation. Our results also reveal the role of p65 as a predictor of bone metastasis development and prostate cancer-specific mortality.

Evaluating changes in circulating tumor cell count in prostate cancer patients during androgen deprivation plus chemotherapy by using AxonDx enumeration device.

266 Background: Circulating tumor cell (CTC) counts have clinical utility as a prognostic marker in cancer. For prostate cancer (PCa), CTC counts ≥5 CTC/7.5 ml using the FDA-approved CellSearch system convey a negative prognosis with respect to overall survival, but the significance of changes in CTC count during treatment is not fully understood. To determine whether longitudinal CTC counts can be used as a prognostic and/or predictive marker of clinical outcome in PCa patients undergoing androgen deprivation therapy (ADT) plus chemotherapy, we used the AxonDx nCyte non-enrichment-based CTC detection and enumeration system to evaluate CTC counts at baseline, during, and after therapy in 18 patients for up to 11 months. Methods: Whole blood was drawn from PCa patients on clinical trials for ADT plus chemotherapy at PSA evaluation (±radiographic reevaluation) into vacutainers containing K3-EDTA and processed within 6 hr. PBMC containing nucleated cells harvested from 6 ml of whole blood were fixed, permeabilized and blocked with BSA buffer, and stained using a proprietary antibody cocktail containing DAPI, an anti-cytokeratin mixture, the pan-leukocyte marker CD45, and markers selective for granulocytes and lymphocytes. Stained cells were drawn onto 1.2 µm pore filters, washed, placed on counting slides, and analyzed. CTCs are identified as cytokeratin-positive, nucleus-positive and leukocyte marker-negative. Results: CTCs were obtained from 12 patients for 2-9 months at 2-7 time points; from 2 patients for 11 months at 5-7 time points, and from 4 patients for 1 month at 1 time point. We detected time-dependent changes in CTC counts following treatment that did not necessarily correlate with patient’s local prostate specific antigen (PSA) levels but were early indicators of subsequent increase in tumor burden. Conclusions: The AxonDx nCyte system can reliably detect CTCs from PCa patient samples that trend along with other signs of disease progression. CTCs can be used longitudinally for clinical monitoring of patients with PCa prior to signs of progression via tumor markers or scans during ADT plus chemotherapy, providing a reliable biomarker for PCa.

Myricetin Possesses Potential Protective Effects on Diabetic Cardiomyopathy through Inhibiting IκBα/NFκB and Enhancing Nrf2/HO-1

Diabetic cardiomyopathy (DCM) is associated with a greater risk of mortality in patients with diabetes mellitus. Currently, no specific treatment has been suggested for DCM treatment. This study demonstrated that myricetin (M) attenuated DCM-associated cardiac injury in mice subjected to streptozotocin (SZT) and in neonatal rat cardiomyocytes (NRCM) challenged with high glucose.In vivoinvestigation demonstrated 6 months of M treatment (200 mg/kg/d) significantly alleviated cardiac hypertrophy, apoptosis, and interstitial fibrosis. Mechanically, M treatment significantly increased the activity of Nrf2/HO-1 pathway, strengthening antioxidative stress capacity evidenced by reversed activities of GPx and SOD, and decreased MDA production. M treatment also inhibited IκBα/NF-κB pathway, resulting in reduced secretion of inflammation cytokines including IL-1β, TNF-α, and IL-6. Besides, the TGFβ/Smad3 signaling was also blunted in DCM mice treated with M. These beneficial effects of M treatment protected cardiomyocytes from apoptosis as shown by decreased TUNEL-positive nucleus, c-caspase 3, and Bax. Similar effects of M treatment could be reproduced in NRCM treated with high glucose. Furthermore, through silencing Nrf2 in NRCM, we found that the regulation of IκBα/NFκB by M was independent on its function on Nrf2. Thus, we concluded that M possesses potential protective effects on DCM through inhibiting IκBα/NFκB and enhancing Nrf2/HO-1.

Enucleation of Human Erythroblasts Is a Process of Asymmetric Cytokinesis.

Background. During erythroblast enucleation, nuclei surrounded by plasma membrane separate from erythroblast cytoplasm. Enucleation has been thought to be a process similar to cytokinesis. However, more concrete evidence has been difficult to obtain because of a lack of an ex vivo experimental system capable of confirming cytokinesis. Focusing on the mechanism of cell division, we investigated the redistribution of cytoplasmic proteins and integral membrane proteins during enucleation, using ex vivo generation system of mature human blood cells from hematopoietic stem cells. Materials and Methods. The highly purified human CD34+ cells were grown in the presence of interleukin-3, stem cell factor and erythropoietin (EPO) in a liquid phase. After 7 days of culture, the generated cells (day 7 cells) were replaced in a medium with EPO alone. The cells matured and terminally differentiated into reticulocytes during a 13–15-day culture period. We mainly used non-gravity and non-pipetting system to avoid physical stress that may disrupt the connection between the nucleus and reticulocyte. Day 9 cells, predominantly consisted of polychromatophilic erythroblasts and expressed glycophorin A (GPA) at a purity of 97%, were labeled with DNA-staining dye SYTO21 for the direct monitoring of the enucleation process, using differential interference contrast microscopy. We also cultured day 9 cells until day 14, on 4-well culture slides or on the membrane of a cell culture insert system, and removed culture medium by aspiration without centrifugation and pipetting. The day 14 cells on the slide were analyzed using immunohistochemical staining, whereas the cells on the membrane were embedded in O.C.T. compound for confocal microscopy. Results. Approximately a half of erythroblasts enucleated until day 14. The monitoring of the enucleation process at day 13 showed autonomous extrusion of SYTO21 positive nucleus from single erythroblast. Some of the expelled nuclei were still connected with reticulocyte through strings that were positive for antibody against tubulin, actin, GPA, band 3 and glycophorin C (GPC). The expelled nuclei were covered by lamin, a protein specific for nuclear membrane, which were surrounded by a substance positive for GPA, band 3, GPC, p55, 4.1R80, actin, tubulin, b-spectrin, calnexin and cytochrome C, although the distribution of each proteins were asymmetric between nuclei and reticulocytes. An intense area of GPA, GPC, band 3, 4.1R80, actin, tubulin, myosin and b-spectrin was found in the region of the constriction between the extruding nucleus and incipient reticulocyte in enucleating cells. In cells just before enucleation, tubulin and actin formed a radial array around the nucleus. The center of the radial array was positive for centrin and NuMA, indicating that the cenriole formation occurred during an enucleation process. Conclusion. Our investigations show that a part of human erythroblasts enucleate independent of an interaction with accessory cells. The appearance of cenriole and the asymmetric redistribution of cytoplasmic and integral membrane proteins during enucleation strongly suggest that enucleation of human erythroblasts is a process of asymmetric cytokinesis.

Effect of aging on liver regeneration in rats

PURPOSE: Regeneration and/or healing of tissues is believed to be more difficult in elderly people. The liver is one of the most complex organs in the human body, and is involved in a variety of functions. Liver regeneration is the body's protection mechanism against loss of functional liver tissue. The aim of this study is to identify the regenerative capacity of the liver in older animals and to compare it with that of young adult animals. METHODS: Thirty-four Wistar rats were used, of which 17 were 90 days old (young animals) and 17 were 460 days old (old animals). Approximately 70% of the liver was surgically removed. Examinations were carried out after 24 hours and on day 7, using 3 methods: KWON et al.'s formula to identify increase in volume; mitotic figure count in 5 fields; and the percentage of PCNA-positive nuclei in 5 fields. RESULTS: The increase in volume of the remaining liver was greater in the young animals after both 24 hours (p=0.0006) and on day 7 (p=0.0000). Histological cuts showed a greater mitotic figure count in young animals evaluated after 24 hours (p=0.0000). Upon evaluation on day 7, recovery was observed in the old animals. This recovery was similar to that of the young ones (p=0.2851). The PCNA-positive nucleus count was greater in the young animals' liver cuts after 24 hours (p=0.0310), and, while it had decreased in young animals by day 7, recovery was observed in the older animals (p=0.0298). CONCLUSION: The data confirm that age is related to delay in liver regeneration in rats.

Myonuclear Addition to Uninjured Laryngeal Myofibers in Adult Rabbits

Objectives: In normal mature limb skeletal muscle, satellite cells are quiescent and myonuclei do not divide after formation of their associated myofibers in the absence of injury. The possibility of myonuclear addition in uninjured laryngeal myofibers of adult rabbits was investigated in an immunohistochemical pilot study. Methods: Bromodeoxyuridine (brdU), a marker for cell division, was administered by intraperitoneal injection over a 12-hour period in rabbits. The number of brdU-positive myonuclei per myofiber was determined on cross sections through the thyroarytenoid (TA) and posterior cricoarytenoid (PCA) muscles. Results: In the TA muscle, 0.13% ± 0.03% (mean ± SEM) of the myofibers counted had a brdU-positive nucleus. In the PCA muscle, 0.13% ± 0.01% of the myofibers counted had a brdU-positive nucleus. Approximately 0.2% and 0.3% of the myofibers of the TA and PCA muscles, respectively, had brdU-positive satellite cells associated with them. Tibialis anterior and pectoralis major muscle controls were negative for brdU-positive myonuclei. Conclusions: These data support the possibility of continuous remodeling in uninjured adult laryngeal myofibers and accentuate the distinct nature of laryngeal muscle relative to limb skeletal muscle in the rabbit model.

Ab Initio Calculations

Various difficulties of classical physics, including inadequate description of atoms and molecules, led to new ways of visualizing physical realities, ways which are embodied in the methods of quantum mechanics. Quantum mechanics is based on the description of particle motion by a wave function, satisfying the Schrodinger equation, which in its “time-independent” form is: ((−h2/8mπ2)⛛2+V)Ψ=E Ψ or, for short: HΨ = EΨ In this equation, H, the Hamiltonian operator, is defined by H = − ((h2/8mπ2)⛛2+V, where h is Planck’s constant (6.6 10−34 Joules), m is the particle’s mass, ⛛2 is the sum of the partial second derivative with x,y, and z, and V is the potential energy of the system. As such, the Hamiltonian operator is the sum of the kinetic energy operator and the potential energy operator. (Recall that an operator is a mathematical expression which manipulates the function that follows it in a certain way. For example, the operator d/dx placed before a function differentiates that function with respect to x.) E represents the total energy of the system and is a number, not an operator. It contains all the information within the limits of the Heisenberg uncertainty principle, which states that the exact position and velocity of a microscopic particle cannot be determined simultaneously. Therefore, the information provided by Ψ(nit) is in terms of probability: Ψ2(x,t) is the probability of finding the particle between x and x + dx, at time t. The Schrödinger equation applied to atoms will thus describe the motion of each electron in the electrostatic field created by the positive nucleus and by the other electrons. When the equation is applied to molecules, due to the much larger mass of nuclei, their relative motion is considered negligible as compared to that of the electrons (Born-Oppenheimer approximation). Accordingly, the electronic distribution in a molecule depends on the position of the nuclei and not on their motion. The kinetic energy operator for the nuclei is considered to be zero. For a many-electron molecule, the Hamiltonian operator can thus be written as the sum of the electrons’ kinetic energy term, which in turn is the sum of individual electrons’ kinetic energies and the electronic and nuclear potential energy terms.