YAP Promotes Cell Proliferation and Stemness Maintenance of Porcine Muscle Stem Cells under High-Density Condition

Muscle stem cells (MuSCs) isolated ex vivo are essential original cells to produce cultured meat. Currently, one of the main obstacles for cultured meat production derives from the limited capacity of large-scale amplification of MuSCs, especially under high-density culture condition. Here, we show that at higher cell densities, proliferation and differentiation capacities of porcine MuSCs are impaired. We investigate the roles of Hippo-YAP signaling, which is important regulators in response to cell contact inhibition. Interestingly, abundant but not functional YAP proteins are accumulated in MuSCs seeded at high density. When treated with lysophosphatidic acid (LPA), the activator of YAP, porcine MuSCs exhibit increased proliferation and elevated differentiation potential compared with control cells. Moreover, constitutively active YAP with deactivated phosphorylation sites, but not intact YAP, promotes cell proliferation and stemness maintenance of MuSCs. Together, we reveal a potential molecular target that enables massive MuSCs expansion for large-scale cultured meat production under high-density condition.

Effects of Epibiotic Diatoms on the Productivity of the Calanoid Copepod Acartia tonsa (Dana) in Intensive Aquaculture Systems

We evaluated here the effects of the epibiotic diatom Tabularia sp. on the productivity of the calanoid copepod Acartia tonsa (Dana) for assessing their risk on copepod intensive aquaculture industry for the provision of live feed. In the first experiment, uninfested and intensively infested females were cultivated individually for the assessment of egg production. Intensively infested females appeared to have a significantly lower egg production (5.0–9.0 eggs/female/d) than uninfested females (22.0–26.0 eggs/female/d) during 5 consecutive days. In the second experiment, effects of culture densities on diatom epibiosis were investigated in 9 L cultures at three different densities (200, 400, and 600 ind. L–1). Another culture at higher volume (250 L) and lowest density (200 ind. L–1) was also carried out to test the effect of culture volume on diatom epibiosis. The infestation rate (%), infestation intensity (ratio of surface diatom coverage levels, classified as levels 0–3) and daily egg harvest rate (number of harvested eggs per day per liter) were evaluated among the four culture populations. The copepods had higher infestation rate (53.69–60.14%) and intensity rate (high ratios at level 2 and 3) when the densities were increased from 200 ind./L to 400 and 600 ind./L. Although egg harvest increased with increasing culture density, it seemed that the diatom-infested A. tonsa population reach a saturated egg production when the density was higher than 400 ind./L. Nevertheless, the differences of culture volumes (250 and 9 L) appeared to be not to have any effect when the copepods were cultivated at the same density (200 ind./L). This study reveals for the first time that the epibiosis of the diatom Tabularia sp. reduces the individual egg production, and egg harvest rate in high-density culture of the copepod A. tonsa. Our findings implicate that diatom epibiosis should be avoid in copepod intensive culture systems.

High-density hiPSCs expansion supported by growth factors accumulation in a simple dialysis-culture platform

Three-dimensional aggregate-suspension culture can produce large numbers of human induced pluripotent stem cells (hiPSCs); however, use of expensive growth factors and method-induced mechanical stress potentially result in inefficient production costs and difficulties in preserving pluripotency. Here, we developed a simple, miniaturized, dual-compartment dialysis-culture device based on a conventional membrane-culture insert with deep well plates. The device allowed growth-factor accumulation and improved cell expansion up to ~ 32 × 106 cells/mL, and reduction of excessive shear stress and agglomeration following addition of the functional polymer FP003 supported high-density expansion. The results revealed accumulation of several growth factors, including fibroblast growth factor 2 and insulin, along with endogenous NODAL, which acts as a substitute for depleted transforming growth factor-β1 in maintaining pluripotency. Because we used the same growth-factor formulation per volume in the upper culture compartment, cost reduction increased significantly in proportional manner with cell density. We showed that growth-factor-accumulation dynamics in a low-shear-stress environment successfully improved hiPSC proliferation, pluripotency, and differentiation potential. This miniaturised dialysis-culture system demonstrated the feasibility and cost-effective mass production of hiPSCs in high-density culture.

Growth Performances and Proximate Composition of Mystus Cavasius (Hamilton, 1822) Cultured in Recirculating Aquaculture System Under Different Stocking Densities

Experiment was conducted to evaluate the high density culture of Mystus cavasius and its effects on growth performances, survival rate and proximate composition of the fish in recirculating aquaculture system. Fishes were cultured at 571, 714, 857 and 1000 fries/m3 as the initial stocking density. No significant differences (p > 0.05) were found in specific growth rate, average daily gain, food conversation ratio, protein efficiency ratio, condition factor (k) and survival rate (%) of fish under the culture period of 120 days among the different stocking densities. The average survival rate was ≥ 99% among the treatments at the end of the culture period. The moisture contents were between 77.10 and 77.75%, ash content was 2.58 and 2.61%, crude protein was 15.86 and 16.07%, crude lipid was 5.45 and 5.68% with no significant differences (p > 0.05) among the treatments. There were no significant variations in DO, TAN, NO3-N, NO2-N and pH among the treatments during culture. This study showed that stocking density of at least 1000 fries/m3 was the best option as the total gain was highest compared to other lower densities under similar facilities. Dhaka Univ. J. Biol. Sci. 29(2): 137-145, 2020 (July)

Clinically compatible differentiation protocol for human pluripotent stem cell-derived dopaminergic progenitor cells

Cell replacement therapy with human pluripotent stem cells has the potential to be a new therapy for Parkinson’s disease (PD). This protocol induces human induced pluripotent stem cells (iPSCs) to dopaminergic progenitor cells (DAPs) as clinically compatible donor cells in 30 days. The protocol includes starting with high density culture, cell sorting by using a cell surface marker for floor plate, and a maturation culture to form floating aggregates. The DAPs differentiated with this protocol were used in a pre-clinical tumorigenicity and efficacy study aiming for approval to start a clinical trial in Japan.

Chondrogenic Potential of Pellet Culture Compared to High-Density Culture on a Bacterial Cellulose Hydrogel

Cell-based approaches of cartilage lesions use different culture systems to obtain optimal cell quality. Pellet cultures with high cellular density (HD) are the gold standard to keep chondrocytes in a differentiated stage. Bacterial cellulose (BC) hydrogel is discussed to prevent cellular aging and dedifferentiation. The hypothesis of this study was that HD culture on BC hydrogel (HD hydrogel) might reach the chondrogenic potential of pellet culture (pellet). Human articular osteoarthritic (OA) and non-osteoarthritic (non-OA) chondrocytes were cultured for seven days within pellets and compared to HD hydrogel and HD polystyrene. Gene expression analysis and histological assessment were performed. We observed no significant change of COL2A1 expression by the culture system (pellet, HD hydrogel and HD polystyrene) but a significant change of COL2A1/COL1A1-ratio, with the highest ratio in pellets. Chondrocytes on HD hydrogel showed an elevated expression of MMP13 and on polystyrene an increased expression of COL1A1 and MMP13. The patterns of gene expression changes observed in OA and non-OA chondrocytes in reaction to the different culture systems were similar in those two cell groups. Pellet cultures moreover formed a histomorphologically superior neocartilage. Concluding, human chondrocytes kept the potential to express COL2A1 in all HD culture systems. However, pellets excelled in a higher COL2A1/COL1A1-ratio, a higher extracellular matrix deposit and in not developing degeneration and dedifferentiation markers. This underlines the superiority of pellet culture in maintaining the chondrogenic potential of human chondrocytes in vitro.