Proliferation of mammalian cells with Chlorococcum littorale algal compounds without serum support.

In recent years, serum-free medium for mammalian cell cultivation has attracted a lot of attention, considering the high cost of production and environmental load involved in developing the conventional animal sera. The use of alternative growth-promoting products in mammalian cell cultivation such as extracts from microalgae has proven to be quite beneficial and environmental-friendly. This research aims to cultivate mammalian cells with growth-promoting factors derived from Chlorococcum littorale. We have established a simple extraction using the ultrasonication method and applied the extract in place of serum on mammalian C2C12 cell lines, 3T3 cell lines, and CHO cell lines to compare and analyze the effectiveness of the extract. Cell passage was conducted in a suspended culture condition with the addition of the extract. The results indicate that the extract from microalgae shows a high proliferation rate in all cell lines without fetal bovine serum. Moreover, it is eco-friendly and has huge potential to replace the traditional cell culture system. It could be applied in the fields of regenerative medicine, gene/cell therapies, as well as cultured meat production.

Development of serum-free and grain-derived-nutrient-free medium using microalga-derived nutrients and mammalian cell-secreted growth factors for sustainable cultured meat production.

Considering the amount of global resources and energy consumed, and animal welfare issues associated with traditional meat production, cultured meat production has been proposed as a solution to these problems and is attracting worldwide attention. Cultured meat is produced by culturing/proliferating animal muscle cells in vitro. This process requires significant amounts of culture medium, which accounts to a major portion of the production cost. Furthermore, it is composed of nutrients derived from grains and heterotrophic microorganisms and fetal bovine serum (FBS), which will impact the sustainability of cultured meat in future. Here, we developed a novel medium containing nutrients extracted from microalga and cell-secreted growth factors. First, rat liver epithelial RL34 cells were cultured by adding Chlorella vulgaris extract (CVE) to inorganic salt solution. The supernatant, containing the RL34 cell-secreted growth factors, was used as the conditioned medium (CM). This CM, with CVE added as a nutrient source, was applied to primary bovine myoblast cultures. This serum-free and grain-derived-nutrient-free medium promoted the proliferation of bovine myoblasts, the main cell source for cultured beef. Our findings will allow us to take a major step toward reducing production costs and environmental impacts, leading to an expansion of the cultured meat market.

Harvest of quality-controlled bovine myogenic cells and biomimetic bovine muscle tissue engineering for sustainable meat production.

Alternative technology for meat production holds the potential to alleviate ethical, environmental, and public health concerns associated with conventional meat production. Cultured meat produced using cell culture technology promises to become a viable alternative to animal-raised meat for the future of the food industry. In this study, biomimetic bovine muscle tissue was artificially fabricated from myogenic cells extracted from bovine meat. Our primary culture method relies on three key factors; a sequential digesting process, enzymatic treatment with pronase, and coating with laminin fragment on culture dishes. This method allows the efficient collection of large numbers of primary cells from bovine cheek meat, purifies the myogenic cells from the cell mixture, and then continuously grows the myogenic cells in vitro. In addition, using our "quality control" methods, we were able to determine the "cell quality", including the proliferative and differentiation capability in each step of the primary culture. Furthermore, to mimic native bovine meat, the quality-controlled bovine myogenic cells were cultured on a micropatterned thermoresponsive substrate stimulating a native-like aligned structure of cells, which were then transferred onto a fibrin-based gel. This gel-based culture environment promoted structural and functional maturation of the myogenic cells, resulting in the production of bovine muscle tissues with sarcomere structures, native-like membrane structures, and contractile ability. We believe that these biomimetic features of "tissue-engineered meat" are important for the production of future cultured meat, which will need native-like nutrients, texture and taste. Therefore, our meat production approach will provide a new platform to produce more native biomimetic tissue-engineered meat in the near future.

Proliferation and differentiation of primary bovine myoblasts using Chlorella vulgaris extract for sustainable production of cultured meat.

Recently, cultured meat obtained from livestock-derived cells is being considered as a sustainable food source that reduces the use of natural resources. This study aimed to show that nutrients extracted from Chlorella vulgaris were beneficial in the culture of primary bovine myoblasts (PBMs), a major cell source for cultured meat production. Nutrients (glucose, amino acids, and vitamins) present in the animal-cell culture media were effectively recovered from C. vulgaris using acid hydrolysis treatment. On culture in nutrient-free inorganic salt solution, cell death was induced in most PBMs after 6 days of cultivation. However, the addition of C. vulgaris extract (CVE) significantly improved PBM viability, which was comparable to the viability in conventional culture medium (Dulbecco's modified Eagle's medium). Furthermore, by adding horse serum to induce differentiation, the formation of myotubes was confirmed when CVE were used. Together, the results showed that CVE could be used as an alternative to the conventional culture medium for PBMs. These findings will not only lower the environmental risks associated with the establishment of this eco-friendly cell culture system, but also highlight microalgae as a potent nutrient source that can replace conventional grain-dependent nutrient sources.

Bioengineering of a scaffold-less three-dimensional tissue using net mould.

Tissue engineering has attracted attention worldwide because of its application in regenerative medicine, drug screening, and cultured meat. Numerous biofabrication techniques for producing tissues have been developed, including various scaffold and printing methods. Here, we have proposed a novel tissue engineering method using a net metal mould without the use of a scaffold. Briefly, normal human dermal fibroblasts seeded on a dimple plate were subjected to static culture technique for several days to form spheroids. Spheroids of diameter ⩾200µm were poured into a net-shaped mould of gap ⩽100µm and subjected to shake-cultivation for several weeks, facilitating their fusion to form a three-dimensional (3D) tissue. Through this study, we successfully constructed a scaffold-free 3D tissue having strength that can be easily manipulated, which was difficult to construct using conventional tissue engineering methods. We also investigated the viability of the 3D tissue and found that the condition of the tissues was completely different depending on the culture media used. Collectively, this method allows scaffold-free culture of 3D tissues of unprecedented thickness, and may contribute largely to next-generation tissue engineering products.

Exposure to valproic acid during middle to late-stage corticogenesis induces learning and social behavioral abnormalities with attention deficit/hyperactivity in adult mice.

Single prenatal exposure to valproic acid (VPA) in rodents is a widely used preclinical model of autism spectrum disorder (ASD). Continuous prenatal VPA exposure has been recently proposed as a new ASD model that closely captures the neuropathological features of ASD, including increases in cerebral cortex volume and the number of cortical upper layer neurons. We investigated the influence of prenatal VPA exposure on the behavior of adult offspring of pregnant dams that received intraperitoneal injections of VPA twice on one day during the genesis of cortical upper layer neurons. Mice exposed to VPA at E14 (E14-VPA) showed typical behavior abnormalities including reduced social interaction, hyperactivity, and poor maze learning due to attention deficit/impulsivity relative to healthy controls. Histological analysis revealed that E14-VPA mice had significantly increased neuronal density and impaired neural activity in the prefrontal cortex, but not the somatosensory area, which is likely linked to the observed abnormalities in social behavior. These results suggest that this VPA exposure method is a good model for gaining new insights into the underlying neuropathology of ASD.

Novel RNA- and FMRP-binding protein TRF2-S regulates axonal mRNA transport and presynaptic plasticity.

Despite considerable evidence that RNA-binding proteins (RBPs) regulate mRNA transport and local translation in dendrites, roles for axonal RBPs are poorly understood. Here we demonstrate that a non-telomeric isoform of telomere repeat-binding factor 2 (TRF2-S) is a novel RBP that regulates axonal plasticity. TRF2-S interacts directly with target mRNAs to facilitate their axonal delivery. The process is antagonized by fragile X mental retardation protein (FMRP). Distinct from the current RNA-binding model of FMRP, we show that FMRP occupies the GAR domain of TRF2-S protein to block the assembly of TRF2-S-mRNA complexes. Overexpressing TRF2-S and silencing FMRP promotes mRNA entry to axons and enhances axonal outgrowth and neurotransmitter release from presynaptic terminals. Our findings suggest a pivotal role for TRF2-S in an axonal mRNA localization pathway that enhances axon outgrowth and neurotransmitter release.

Activation of TRPML1 clears intraneuronal Aß in preclinical models of HIV infection.

Antiretroviral therapy extends the lifespan of human immunodeficiency virus (HIV)-infected patients, but many survivors develop premature impairments in cognition. These residual cognitive impairments may involve aberrant deposition of amyloid ß-peptides (Aß). By unknown mechanisms, Aß accumulates in the lysosomal and autophagic compartments of neurons in the HIV-infected brain. Here we identify the molecular events evoked by the HIV coat protein gp120 that facilitate the intraneuronal accumulation of Aß. We created a triple transgenic gp120/APP/PS1 mouse that recapitulates intraneuronal deposition of Aß in a manner reminiscent of the HIV-infected brain. In cultured neurons, we found that the HIV coat protein gp120 increased the transcriptional expression of BACE1 through repression of PPARγ, and increased APP expression by promoting interaction of the translation-activating RBP heterogeneous nuclear ribonucleoprotein C with APP mRNA. APP and BACE1 were colocalized into stabilized membrane microdomains, where the ß-cleavage of APP and Aß formation were enhanced. Aß-peptides became localized to lysosomes that were engorged with sphingomyelin and calcium. Stimulating calcium efflux from lysosomes with a TRPM1 agonist promoted calcium efflux, luminal acidification, and cleared both sphingomyelin and Aß from lysosomes. These findings suggest that therapeutics targeted to reduce lysosomal pH in neurodegenerative conditions may protect neurons by facilitating the clearance of accumulated sphingolipids and Aß-peptides.

Scaffold function of long non-coding RNA HOTAIR in protein ubiquitination.

Although mammalian long non-coding (lnc)RNAs are best known for modulating transcription, their post-transcriptional influence on mRNA splicing, stability and translation is emerging. Here we report a post-translational function for the lncRNA HOTAIR as an inducer of ubiquitin-mediated proteolysis. HOTAIR associates with E3 ubiquitin ligases bearing RNA-binding domains, Dzip3 and Mex3b, as well as with their respective ubiquitination substrates, Ataxin-1 and Snurportin-1. In this manner, HOTAIR facilitates the ubiquitination of Ataxin-1 by Dzip3 and Snurportin-1 by Mex3b in cells and in vitro, and accelerates their degradation. HOTAIR levels are highly upregulated in senescent cells, causing rapid decay of targets Ataxin-1 and Snurportin-1, and preventing premature senescence. These results uncover a role for a lncRNA, HOTAIR, as a platform for protein ubiquitination.

NF90 coordinately represses the senescence-associated secretory phenotype.

A hallmark trait of cellular senescence is the acquisition of a senescence-associated secretory phenotype (SASP). SASP factors include cytokines and their receptors (IL-6, IL-8, osteoprotegerin, GM-CSF), chemokines and their ligands (MCP-1, HCC4), and oncogenes (Gro1 and Gro2), many of them encoded by mRNAs whose stability and translation are tightly regulated. Using two models of human fibroblast senescence (WI-38 and IDH4 cells), we report the identification of RNA-binding protein NF90 as a post-transcriptional repressor of several SASP factors. In 'young', proliferating fibroblasts, NF90 was highly abundant, associated with numerous SASP mRNAs, and inhibited their expression. By contrast, senescent cells expressed low levels of NF90, thus allowing SASP factor expression to increase. NF90 elicited these effects mainly by repressing the translation of target SASP mRNAs, since silencing NF90 did not increase the steady-state levels of SASP mRNAs but elevated key SASP factors including MCP-1, GROa, IL-6, and IL-8. Our findings indicate that NF90 contributes to maintaining low levels of SASP factors in non-senescent cells, while NF90 reduction in senescent cells allows SASP factor expression to rise.

RNA-binding protein AUF1 represses Dicer expression.

MicroRNA (miRNA) biogenesis is tightly regulated by numerous proteins. Among them, Dicer is required for the processing of the precursor (pre-)miRNAs into the mature miRNA. Despite its critical function, the mechanisms that regulate Dicer expression are not well understood. Here we report that the RNA-binding protein (RBP) AUF1 (AU-binding factor 1) associates with the endogenous DICER1 mRNA and can interact with several segments of DICER1 mRNA within the coding region (CR) and the 3'-untranslated region (UTR). Through these interactions, AUF1 lowered DICER1 mRNA stability, since silencing AUF1 lengthened DICER1 mRNA half-life and increased Dicer expression, while overexpressing AUF1 lowered DICER1 mRNA and Dicer protein levels. Given that Dicer is necessary for the synthesis of mature miRNAs, the lowering of Dicer levels by AUF1 diminished the levels of miRNAs tested, but not the levels of the corresponding pre-miRNAs. In summary, AUF1 suppresses miRNA production by reducing Dicer production.

Human VAPA and the yeast VAP Scs2p with an altered proline distribution can phenocopy amyotrophic lateral sclerosis-associated VAPB(P56S).

A human isoform of the vesicle-associated membrane protein-associated proteins (VAPs), VAPB, causes amyotrophic lateral sclerosis eight due to the missense mutation of Pro-56, whereas human VAPA and the yeast VAP Scs2p proteins are not significantly affected by similar mutations. We have found that VAPA and Scs2p have three prolines present in a conserved region however VAPB has only two prolines in this region. Consequently, this mutation in VAPB (VAPB(P56S)) leaves a single proline in this region whereas other VAPs can retain two proline residues even if the proline equivalent to the Pro-56 is substituted. When Scs2p and VAPA were mutated to be equivalent to VAPB(P56S) in terms of the distribution of proline residues in this region, Scs2p became inactive and aggregated, and VAPA localize to membranous aggregates indistinguishable from those induced by VAPB(P56S). This suggests that the appropriate distribution of three conserved prolines, not the existence of a particular proline, confers VAPA and Scs2p resistance to the Pro-56 mutation and, therefore, is critical for VAP activities.