Aerobic glycolysis and its by-product lactate accumulation are usually associated with adverse culture phenotypes such as poor cell viability and productivity. Due to the lack of knowledge on underlying mechanisms and accompanying biological processes, the regulation of aerobic glycolysis has been an ongoing challenge in culture process development for therapeutic protein productivity. NAD+ , a coenzyme and co-substrate in energy metabolism, promotes the conversion of inefficient glycolysis into an efficient oxidative phosphorylation pathway. However, the effect of NAD+ on CHO cells for biopharmaceutical production has not been reported yet. In this work, we aimed to elucidate the influence of NAD+ on cell culture performance by examining metabolic shifts and mAb productivity. The supplementation of NAD+ increased the intracellular concentration of NAD+ and promoted SIRT3 expression. Antibody titer and the specific productivity in the growth phase were improved by up to 1.82 and 1.88-folds, respectively, with marginal restrictions on cell growth. NAD+ significantly reduced the accumulation of ROS and the degree of glycolysis, determined by lactate accumulation versus glucose consumption. In contrast, oxidative phosphorylation capacity and amino acid consumption rate increased substantially. Collectively, these results suggest that NAD+ contributes to improving therapeutic protein productivity in bioprocessing via inducing an energy metabolic shift. This article is protected by copyright. All rights reserved.

Human Adipose-Derived Mesenchymal Stem/Stromal Cells (hAD-MSCs) have great potential for tissue regeneration. Since transplanted hAD-MSCs are likely to be placed in a hypoxic environment, culturing the cells under hypoxic conditions might improve their post-transplantation survival and regenerative performance. The combination of hAD-MSCs and PCL-nHA nanofibers synergically improves the contribution of both components for osteoblast differentiation. In this work, we hypothesized that this biomaterial constitutes a hypoxic environment for hAD-MSCs. We studied the cellular re-arrangement and the subcellular ultrastructure by Transmission Electron Microscopy (TEM) of hAD-MSCs grown into PCL-nHA nanofibers, and we compared them with the same cells grown in two-dimensional cultures, over tissue culture-treated plastic, or glass coverslips. Among the most evident changes, PCL-nHA grown cells showed enlarged mitochondria, and accumulation of glycogen granules, consistent with a hypoxic environment. We observed a 3.5 upregulation (p = 0.0379) of Hypoxia Inducible Factor (HIF)-1A gene expression in PCL-nHA grown cells. This work evidences for the first time intra-cellular changes in three-dimensional compared to two-dimensional cultures, which are adaptive responses of the cells to an environment more closely resembling that of the in vivo niche after transplantation, thus PCL-nHA nanofibers are adequate for hAD-MSCs pre-conditioning.

N6 -methyladenosine (m6A) is the most abundant internal modification on eukaryotic mRNA and has been implicated in a wide range of fundamental cellular processes. This modification is regulated and interpreted by a set of writer, eraser, and reader proteins. To date, there have been no reports on the potential of mRNA epigenetic regulators to influence recombinant protein expression in mammalian cells. In this study, the potential of manipulating the expression of the m6A YTH domain-containing readers, YTHDF1, 2 and 3 to improve recombinant protein yield based on their role in regulating mRNA stability and promoting translation were evaluated. Using siRNA-mediated gene depletion, cDNA over-expression, and methylation-specific RNA immunoprecipitation, it is demonstrated that (i) knock-down of YTHDF2 enhances (~2-fold) the levels of recombinant protein derived from GFP and EPO transgenes in CHO cells; (ii) the effects of YTHDF2 depletion on transgene expression is m6A-mediated; and (iii) YTHDF2 depletion, or over-expression of YTHDF1 increases viral protein expression and yield of infectious lentiviral (LV) particles (~2-3-fold) in HEK293 cells. We conclude that various transgenes can be subjected to regulation by m6A regulators in mammalian cell lines and that these findings demonstrate the utility of epitranscriptomic-based approaches to host cell line engineering for improved recombinant protein and viral vector production.

Auxotrophic marker genes have been widely used for genetic engineering in yeast. However, the effects of amino acids or nucleotides deficiency in auxotrophic strains on cell growth and product synthesis were rarely reported. In this study, a total of eight auxotrophic strains of Saccharomyces cerevisiae with single knockout of selection markers were obtained. Cell growth and free fatty acid (FFA) production of these auxotrophic strains were evaluated with supplementation of different concentrations of amino acids or nucleotides. Generally, except ade2Δ mutants, most auxotrophic strains showed decreased cell growth and FFA production, which could be recovered by adding higher concentrations of supplements. LEU2 deletion (leu2Δ) damaged both cell growth and FFA production even with supplementation of 1000 mg L-1 leucine. This study shows that growth and product biosynthesis of auxotrophs could be limited by insufficient supplementation of amino acids or nucleotides, and provides guidance on supplementation of these nutrients during fermentation to maximize cell growth and product biosynthesis.

Bone morphogenetic protein-2 (BMP-2) and transforming growth factor-β1 (TGF-β1) reportedly induce the osteogenic and tenogenic differentiation of anterior cruciate ligament (ACL)-derived stem cells (LDSCs), respectively. However, few studies have investigated the effect of BMP-2/TGF-β1 on the differentiation of LDSC. We developed a BMP-2/TGF-β1 gene insertion into an LDSC cell sheet that promotes tendon-bone healing in a mouse ACL reconstruction (ACLR) model. CD34+ LDSCs were isolated from human ACL stump tissues, virally transduced to express BMP-2 or TGF-β1, and then embedded within cell sheets. All mice underwent ACLR using an autograft wrapped with a cell sheet and were randomly divided into three groups: BMP-2-, TGF-β1-, and BMP-2/TGF-β1-transduced. At 4 and 8 weeks, tendon-bone healing was evaluated by micro-CT, biomechanical test, and histological analysis. BMP-2 and TGF-β1 promoted the osteogenic and tenogenic differentiation of LDSC in vitro. BMP-2/TGF-β1-transduced LDSC sheet application contributed to early improvement in mean failure load and graft stiffness, accelerated maturation of the tendon-bone junction, and inhibited bone tunnel widening. Furthermore, reduced M1 macrophage infiltration and a higher M2 macrophage percentage were observed in the BMP-2/TGF-β1-transduced LDSC group. This work demonstrated that BMP-2 and TGF-β1 promoted CD34+ LDSCs osteogenic and tenogenic differentiation in vitro and in vivo, which accelerated the tendon-bone healing after ACLR using autografts wrapped with cell sheets in a mouse model.