Flowering and Seed Production across the Lemnaceae

Plants in the family Lemnaceae are aquatic monocots and the smallest, simplest, and fastest growing angiosperms. Their small size, the smallest family member is 0.5 mm and the largest is 2.0 cm, as well as their diverse morphologies make these plants ideal for laboratory studies. Their rapid growth rate is partially due to the family’s neotenous lifestyle, where instead of maturing and producing flowers, the plants remain in a juvenile state and continuously bud asexually. Maturation and flowering in the wild are rare in most family members. To promote further research on these unique plants, we have optimized laboratory flowering protocols for 3 of the 5 genera: Spirodela; Lemna; and Wolffia in the Lemnaceae. Duckweeds were widely used in the past for research on flowering, hormone and amino acid biosynthesis, the photosynthetic apparatus, and phytoremediation due to their aqueous lifestyle and ease of aseptic culture. There is a recent renaissance in interest in growing these plants as non-lignified biomass sources for fuel production, and as a resource-efficient complete protein source. The genome sequences of several Lemnaceae family members have become available, providing a foundation for genetic improvement of these plants as crops. The protocols for maximizing flowering described herein are based on screens testing daylength, a variety of media, supplementation with salicylic acid or ethylenediamine-N,N′-bis(2-hydroxyphenylacetic acid) (EDDHA), as well as various culture vessels for effects on flowering of verified Lemnaceae strains available from the Rutgers Duckweed Stock Cooperative.

Effect of dexamethasone as osteogenic supplementation in in vitro osteogenic differentiation of stem cells from human exfoliated deciduous teeth

In in vitro culture systems, dexamethasone (DEX) has been applied with ascorbic acid (ASC) and β-glycerophosphate (βGLY) as culture media supplementation to induce osteogenic differentiation of mesenchymal stem cells. However, there are some inconsistencies regarding the role of DEX as osteogenic media supplementation. Therefore, this study verified the influence of DEX culture media supplementation on the osteogenic differentiation, especially the capacity to mineralize the extracellular matrix of stem cells from human exfoliated deciduous teeth (SHED). Five groups were established: G1—SHED + Dulbecco’s Modified Eagles’ Medium (DMEM) + fetal bovine serum (FBS); G2—SHED + DMEM + FBS + DEX; G3—SHED + DMEM + FBS + ASC + βGLY; G4—SHED + DMEM + FBS + ASC + βGLY + DEX; G5—MC3T3-E1 + α Minimal Essential Medium (MEM) + FBS + ASC + βGLY. DNA content, alkaline phosphatase (ALP) activity, free calcium quantification in the extracellular medium, and extracellular matrix mineralization quantification through staining with von Kossa, alizarin red, and tetracycline were performed on days 7 and 21. Osteogenic media supplemented with ASC and β-GLY demonstrated similar effects on SHED in the presence or absence of DEX for DNA content (day 21) and capacity to mineralize the extracellular matrix according to alizarin red and tetracycline quantifications (day 21). In addition, the presence of DEX in the osteogenic medium promoted less ALP activity (day 7) and extracellular matrix mineralization according to the von Kossa assay (day 21), and more free calcium quantification at extracellular medium (day 21). In summary, the presence of DEX in the osteogenic media supplementation did not interfere with SHED commitment into mineral matrix depositor cells. We suggest that DEX may be omitted from culture media supplementation for SHED osteogenic differentiation in vitro studies.

Use of Multi-Omics Approach to Produce a Robust Serum-Free Media for the Expansion of T Cells

Chimeric antigen receptor (CAR) T-cell therapy represents a major advancement in personalized cancer treatment. Generating robust and well characterized manufacturing processes for cell therapy has become crucial. Traditional manufacturing processes activate and expand T cells in media containing human serum, which supports cell growth and viability. However, serum batches vary significantly from lot to lot and require frequent screening for contaminants that can be detrimental to patients. T cell manufacturing processes independent of human serum will render adoptive T cell therapy less expensive, more consistent, and safer for patients. In order to create a best-in class serum-free medium that is even more robust than serum-containing media, we need to gain a better understanding of the metabolic requirements of T cells during expansion. For this purpose, we have utilized a multi-omics approach to fully characterize the proteomic and metabolomic signatures of T cells expanded in a serum free, xeno free medium at different phases of growth. We sampled cells at days 3, 5, and 7 for label-free proteomics and untargeted extra- and intracellular metabolomics analysis in order to identify metabolic patterns that could be corrected with media supplementation. We identified over 6,043 proteins and 900 metabolites from 4 donors and detected 1,200 significantly different proteins and 312 metabolites. Using an in-house bioinformatics strategy to analyze the multi-omics data, we focused on several metabolic and signaling pathways that were significantly different at days 5 and 7 compared to day 3. The "corrective" media supplementation that we added based on these results demonstrated a tremendous increase in cell growth and viability yielding a 2.6- and 3.2-fold increase in cell growth on days 5 and 7, respectively, and an increase in viability of 15-20% and 11-15% on days 5 and 7, respectively, compared to the un-supplemented prototype. In addition, cells maintained high viability throughout the whole culture and phenotype was not affected. These data demonstrate that multi-omics is a powerful tool for understanding T cell metabolism and identifying components to develop robust and reproducible serum-free media that produces high quality T cells. Disclosures No relevant conflicts of interest to declare.