Development of Bilayered Bone Marrow-derived Cell-Gelatin Grafts for Augmentation Cystoplasty and Reconstruction of Bladder Tissues in Rats

Background : This study attempted to produce a novel graft composed of bone marrow-derived mesenchymal cell (BMC) layer-gelatin sheets for bladder augmentation cystoplasty. Then, we determined if the grafts could reconstruct bladder tissues. Methods : BMCs harvested from the femurs of green fluorescence protein (GFP)-transfected Sprague-Dawley (SD) rats were adherent and proliferating cells on collagen dishes. The cells were then cultured on temperatureresponsive culture dishes. Following this, the BMCs maintaining cell-cell contacts within the monolayer itself were applied to a gelatin sheet. Two BMC layer-gelatin sheets were overlaid together with the cell sides juxtaposed with one another (bilayered BMC-gelatin graft). Bladder top of SD rats were incised and transplanted with the bilayered BMC-gelatin grafts. Similarly, urinary bladders irradiated with 2 Gy once a week for 5 weeks were also conducted. As control, bilayered acellular-gelatin grafts were used. At 4 weeks after transplantation, the bladders were histologically investigated. Results : At 4 weeks after transplantation into either normal or radiation-injured urinary bladders, incised regions closed. The closed regions of bladder top had reconstructed tissues that were formed with urothelium, and smooth muscle layers. Within the reconstructed tissues, the thickness of the smooth muscle layers in the bilayered BMC-gelatin graft-transplanted bladders were larger compared to controls. The GFP-positive transplanted BMCs were detected. Some of the cells were simultaneously positive for smooth muscle or nerve cell markers. Conclusion : This study showed that the bilayered BMC-gelatin grafts that were experimentally produced could reconstruct bladder tissues. The grafts would be developed as grafts for bladder augmentation cystoplasty.Article信州医学雑誌 71(3) : 167-177, (2023)journal articl

Hepatic stem cells with self-renewal and liver repopulation potential are harbored in CDCP1-positive subpopulations of human fetal liver cells

BackgroundMature human hepatocytes are critical in preclinical research and therapy for liver disease, but are difficult to manipulate and expand in vitro. Hepatic stem cells (HpSCs) may be an alternative source of functional hepatocytes for cell therapy and disease modeling. Since these cells play an import role in regenerative medicine, the precise characterization that determines specific markers used to isolate these cells as well as whether they contribute to liver regeneration still remain to be shown.MethodIn this study, human HpSCs were isolated from human primary fetal liver cells (FLCs) by flow cytometry using CDCP1, CD90, and CD66 antibodies. The isolated CDCP1+CD90+CD66– HpSCs were cultured on dishes coated with type IV collagen in DMEM nutrient mixture F-12 Ham supplemented with FBS, human γ-insulin, nicotinamide, dexamethasone, and l-glutamine for at least 2 weeks, and were characterized by transcriptomic profiling, quantitative real-time PCR, immunocytochemistry, and in-vivo transplantation.ResultsThe purified CDCP1+CD90+CD66– subpopulation exhibited clonal expansion and self-renewal capability, and bipotential capacity was further identified in single cell-derived colonies containing distinct hepatocytes and cholangiocytes. Moreover, in-vivo liver repopulation assays demonstrated that human CDCP1+CD90+CD66– HpSCs repopulated over 90% of the mouse liver and differentiated into functional hepatocytes with drug metabolism activity.ConclusionsWe identified a human hepatic stem/progenitor population in the CDCP1+CD90+CD66– subpopulation in human FLCs, indicating CDCP1 marker could potentially be utilized to identify and isolate HpSCs for further cytotherapy of liver disease

spERt Technology: A novel strategy to improve productivity through enhanced polyribosome assembly on the endoplasmic reticulum in CHO cells

In cell line development process, it is frequently observed that increased mRNA levels do not always correlate with protein expression levels in CHO cells. In line with this gap, the endoplasmic reticulum (ER) in CHO cells is much less proliferated as compared with that in terminally differentiated (i.e., professional) secretory cells, suggesting that there is still room to improve their specific productivity if translational efficiency on the ER can be up-regulated. Here we present a novel engineering approach (spERt Technology) to improve specific production rates by mimicking the ER translational apparatus of professional secretory cells. In spERt Technology, we exploit the unique factors that are required for translationally active polyribosome formation on the ER to directly enhance the translational efficiency (1, 2). A high antibody (Ab) producing clone generated by a novel screen using flow cytometry (3) was used as a model cell line. The factors were introduced into the high producer and a series of the spERt Technology - introduced cell lines were generated Among these cell lines, we selected one of the best clones (spERt-f9) having stable and high productivity. Polyribosome analysis of these cell lines revealed that enhanced assembly of the ER polyribosomes as expected (1). Consistent with the highly developed polyribosomes, the spERt-introduced cell lines produced higher levels of Ab than that of parental cells, and showed prominent increase of specific production rates. Further optimization of feeding process resulted in remarkable increase of productivity in spERt-f9 cells: Ab titers of 7.6 g/L and 9.5 g/L on day 14 and 17, respectively, were achieved in shake flask fed-batch cultures by using chemically defined media. Importantly, high cell viabilities were maintained in spERt-f9 cells throughout the culture periods. In addition, lower glucose consumption and reduced accumulation of ammonia were observed. Product quality in these cells were analyzed and compared with that in the parental cells. In conclusion, spERt Technology enables to improve productivity of high Ab producers, associated with reduced accumulation of waste metabolites and high cell viabilities

The Effect of Cranial Change on Oropharyngeal Airway and Breathing During Sleep

Mandibular micrognathia is one of the characteristics of obstructive sleep apnea syndrome. The purpose of this study was to assess the effects of bimaxillary surgery without maxillary advancement on the upper airway using computational fluid dynamics (CFD) results of comparing pre- and post-operative finite element model. Seven female patients with jaw deformity, who underwent two-jaw surgery (Le Fort1 osteotomy and bilateral sagittal split ramus osteotomy; BSSRO) were enrolled. Maxillary was moved for correcting occlusal plane and mandibular was moved to advancement. Pharyngeal airway space and breathing during sleep were evaluated, comparing the periods of 2 days before and 6 months after the operation. The cross-sectional area of the level of the hard palate (HP) and the level of the tip of the uvula (TU), and airway volume of total, HP-TU, and TP- the level of the base of the epiglottis (BE) were increased. AI and AHI in 2 days before and 6 months after were decreased. As the result of nasal ventilation condition, velocity of HP and TU in 2 days before and 6 months after were decreased. We think that it was revealed that movement of the maxilla without advancement did not affect to the morphology and function of airway

TIGIT/CD155 axis mediates resistance to immunotherapy in patients with melanoma with the inflamed tumor microenvironment

Background Patients with cancer benefit from treatment with immune checkpoint inhibitors (ICIs), and those with an inflamed tumor microenvironment (TME) and/or high tumor mutation burden (TMB), particularly, tend to respond to ICIs; however, some patients fail, whereas others acquire resistance after initial response despite the inflamed TME and/or high TMB. We assessed the detailed biological mechanisms of resistance to ICIs such as programmed death 1 and/or cytotoxic T-lymphocyte-associated protein 4 blockade therapies using clinical samples. Methods We established four pairs of autologous tumor cell lines and tumor-infiltrating lymphocytes (TILs) from patients with melanoma treated with ICIs. These tumor cell lines and TILs were subjected to comprehensive analyses and in vitro functional assays. We assessed tumor volume and TILs in vivo mouse models to validate identified mechanism. Furthermore, we analyzed additional clinical samples from another large melanoma cohort. Results Two patients were super-responders, and the others acquired resistance: the first patient had a non-inflamed TME and acquired resistance due to the loss of the beta-2 microglobulin gene, and the other acquired resistance despite having inflamed TME and extremely high TMB which are reportedly predictive biomarkers. Tumor cell line and paired TIL analyses showed high CD155, TIGIT ligand, and TIGIT expression in the tumor cell line and tumor-infiltrating T cells, respectively. TIGIT blockade or CD155-deletion activated T cells in a functional assay using an autologous cell line and paired TILs from this patient. CD155 expression increased in surviving tumor cells after coculturing with TILs from a responder, which suppressed TIGIT+ T-cell activation. Consistently, TIGIT blockade or CD155-deletion could aid in overcoming resistance to ICIs in vivo mouse models. In clinical samples, CD155 was related to resistance to ICIs in patients with melanoma with an inflamed TME, including both primary and acquired resistance. Conclusions The TIGIT/CD155 axis mediates resistance to ICIs in patients with melanoma with an inflamed TME, promoting the development of TIGIT blockade therapies in such patients with cancer