The following student awards were presented at the 2020 World Congress on In Vitro Biology Virtual Pre-recorded Meeting held from June 6-10, 2020. Information on additional awardees at the 2020 World Congress was presented in the previous issue of the In Vitro Report. Information related to the available specific student awards can be found on the SIVB website or by contacting the SIVB Business Office.


Flower Power: A Rapid In Vitro Regeneration Protocol from In Vitro Cannabis sativa Inflorescences

The legalization of recreational cannabis (Cannabis sativa L.) in North America has driven the need for large-scale propagation of disease free, chemically defined clones. Currently, cannabis is propagated using stem cuttings from mother plants, which can occupy up to 15% of commercial production space and are susceptible to pests and diseases. In vitro growth of cannabis allows for rapid clonal propagation of axenic plants for research and germplasm storage, but published methods are few and multiplication rates can be low.  A recent study from our lab explored the regenerative potential of cannabis inflorescences, using greenhouse grown flowers.  Subsequent studies have shown that morphologically normal plants can regenerate from in vitro florets in the absence of plant growth regulators (PGRs).  Since cannabis inflorescences are comprised of compact internodes, they represent a meristem-dense region with a high multiplication potential.  We hypothesized that micropropagation using inflorescences can enhance multiplication rates in C. sativa over existing vegetative methods while providing an alternative approach to culturing day neutral (autoflowering) plants and some photoperiod sensitive genotypes.  We explored the use of PGRs (BAP and TDZ) to enhance the multiplication rate of explants regenerated from individual or groups of florets and assessed the incorporation of inflorescences into alginate-based synthetic seeds. This study provides a framework for the rapid multiplication of clonal explants using C. sativa inflorescences.  Our findings have the potential to facilitate the automation of cannabis micropropagation while providing a roadmap to overcoming.

Adrian S. Monthony, University of Guelph, Guelph, Ontario, CANADA. In Vitro Cellular and Developmental Biology, 56:S25, 2020


Anti-inflammatory Mechanism of the Prenylated Stilbenoid Arachidin-3: A Natural Product Derived from Peanut Hairy Roots

Stilbenoids are a non-flavonoid class of polyphenols that are important for their potential medicinal applications. Resveratrol is one of the most well-studied stilbenoid and several studies have described its anti-inflammatory, antioxidant, cardio-protective and anticancer activities. Prenylated stilbenoids, which include arachidin-1 and arachidin-3, are produced to counteract biotic and abiotic stresses in peanut (Arachis hypogaea). Despite their importance to plant and human health, the molecular mechanisms underlying the bioactivities of prenylated stilbenoids are still poorly understood. To address this issue, we are using lipopolysaccharide (LPS)-treated RAW 264.7 mouse macrophages to understand the anti-inflammatory molecular mechanism of arachidin-3 in vitro. This prenylated stilbenoid was produced in elicitor-treated hairy root cultures of peanut and purified by semi-preparative HPLC. Our preliminary results suggest that arachidin-3 is not toxic to the cells at low micromolar concentrations. Arachdin-3 pretreatment also reduces the IL-6 production in LPS-treated RAW 264.7 cells.  Furthermore, cell imaging assays showed that arachidin-3 acts by inhibiting LPS-induced NF-κB activation and attenuating high mobility group box 1 (HMGB1) protein signaling in the RAW 264.7 cells. These studies will increase our understanding of the molecular mechanism mediated by arachidin-3 and carry important translational implications for the application of prenylated stilbenoids as anti-inflammatory compounds. 

Md Rokib Hasan, Arkansas State University, Jonesboro, AR. In Vitro Cellular and Developmental Biology, 56:S36-37, 2020


High Yields Secretion of Human Erythropoietin from Tobacco Cell for Ex Vivo Production of Red Blood Cells

Human blood transfusion is crucial in healthcare, which entirely depends on the human donors. Ex vivo generation of clinically available red blood cells (RBCs) from hematopoietic stem cells (HSCs) offers a promising solution to overcome the challenges associated with current use of donor blood, including shortage of blood supply and risk of transfusion-transmitted infections. However, ex vivo expansion and differentiation of HSCs into RBCs requires large quantities of hematopoietic growth factors/cytokines, particularly erythropoietin (EPO) that is a key cytokine responsible for effective erythropoiesis. High-quality functional EPO is increasingly demanded for fundamental research and clinical applications. Plant cell culture is an emerging alternative bioproduction platform for therapeutic proteins, as it offers advantages in safety, scalability and cost over other eukaryotic and prokaryotic systems. However, low protein productivity and secretion is a common bottleneck preventing the commercialization of this platform. To overcome these bottlenecks, we expressed EPO with a designer peptide tag, termed (SP)20 consisting of 20 tandem repeats of a “Ser-Pro” motif.  This de novo designed tag is expected to direct extensive Oglycosylation on each Pro residue in plant cells and function as a molecular carrier in boosting extracellular secretion and stability of EPO. Tobacco codon-optimized EPO gene with the (SP)20 tag attached at either N-terminal or C-terminal region was stably expressed in tobacco BY-2 cells.  BY-2 cell secreted EPO products were purified with hydrophobic interaction chromatography and Ni+ affinity chromatography for their function assay. The yield of (SP)20-tagged EPO was significantly higher than the EPO without the tag. The in vitro expansion and differentiation of hematopoietic stem cell (CD34+ cells) was established to test the plant cell-produced EPO products. This research develops a new plant cell-based platform for high yield production of EPO, facilitating manufacturing of HSCs-derived RBCs at large scale for clinical applications.

Uddhab Karki, Arkansas State University, Jonesboro, AR. In Vitro Cellular and Developmental Biology, 56:S26-273, 2020


Optimization of a Polarized 2D Differentiated Human Colonoid Transwell Model

The colonic epithelial barrier plays a crucial role in preventing permeation of toxins and microbiota while allowing absorption of water and nutrients. Colonic epithelial polarity, consisting of distinct apical and basolateral membrane domains, is crucial for regulating these functions. To develop a model of the human colon in vitro, we aimed to optimize a 2D transwell system of colonic monolayers by assessing media components contributing to proliferation, differentiation and polarization. Colonoids were established from donor resections or biopsies and cultured in Matrigel in media containing Advanced DMEM/F-12, 2mM GlutaMax, 10mM Hepes, N2 supplement, B27 supplement minus vitamin A, 1mM N-Acetyl-L-cysteine, 500nM A8301, 10μM SB202190, 10μM Y27632, 100ng/mL EGF and 50% LWRN conditioned media including 10% FBS. Colonoids were trypsin-dissociated and 200,000 cell aggregates were plated per collagen-IV coated 0.33cm2 transwell insert. Medium was replaced after 24hrs with differentiation medium minus stem cell support factors LWRN, SB202190, and with added 10nM gastrin, 50ng/mL EGF, 50ng/mL noggin and experimental concentrations of A8301 and Y27632. Trans-epithelial electrical resistance was recorded to assess monolayer confluence and barrier function while tight junction protein expression was visualized by immunofluorescence. Optimized methods for generating a polarized and differentiated monolayer included components that support initial cell proliferation: adding Rho Kinase inhibitor 2.5μM Y27632 for the first 48hrs while removing TGFβ inhibitor A8301, and pre-treating colonoids with LWRN selected for high Wnt activity to drive a cystic morphology. These conditions were optimal for creating a reproducibly uniform monolayer that models the colonic epithelial barrier.

Caroline L. McCarthy, University of Michigan Medical School, Ann Arbor, MI. In Vitro Cellular and Developmental Biology, 56:S63-64, 2020


Tomato (Solanum lycopersicum L.) Class II Glutaredoxin Mutants Generated Via CRISPR/Cas9 System Are Susceptible to Multiple Abiotic Stresses

Global environmental change and rapid population growth make transgenic technologies necessary for crop improvement.  CRISPR/Cas9 system can be precisely designed to generate multiplex genome editing, providing a powerful tool for studying functions of gene families in plants.  Glutaredoxins (GRXs) are low molecular weight oxidoreductases that are involved in oxidative stress responses; however, despite their importance, their function in plants has not been well understood.  In this study, we successfully designed and applied pYLCRISPR/Cas9 multiplex vector system to edit Solanum lycopersicum class II glutaredoxins (SlGRXS14, S15, S16, and S17) and used mutant plants to study physiological functions of knock-out genes.  Our genotyping data showed highly efficient gene editing in T0 plants that were genetically inherited to T1 and T2 generations. Transgene-free single and multiple null mutants in T3 were evaluated under control, heat, drought, chilling, cadmium and short photoperiod stress conditions.  In standard conditions, single and multiple mutants did not show any phenotypic differences compared to wild-type plants.  Unsuccessful attempts to find any single, double, triple or quadruple mutant lines containing S15 null mutants suggests that complete loss of function of S15 is embryonic lethal for tomato.  However, upon exposure to different abiotic stresses, wild-type and mutant plants showed significant phenotypic differences that were easily distinguishable.  Phenotyping data suggested that function of GRX gene family is critical for plant’s survival under abiotic stress conditions, making them attractive targets for crop improvement. 

Tayebeh Kakeshpour, Kansas State University, Manhattan, KS. In Vitro Cellular and Developmental Biology, 56:S25-26, 2020


Sulforaphane Inhibits Colon Adenoma Organoid Formation and Induces Differentiation in a Dose-dependent Manner

Colorectal cancer ranks as the 2nd leading cause of cancer mortality in the United States. Preventive strategies for individuals with familial colon cancer syndromes remain limited. Sulforaphane (SFN), an isothiocyanate in cruciferous vegetables and potent activator of the Nrf2 transcription factor, has chemopreventive effects in vitro and in vivo, including inducing growth arrest and/or apoptosis. We have previously demonstrated that SFN inhibits breast cancer stem cells, the hypothesized cells of origin for carcinogenesis. Whether SFN can act as a cancer preventive agent through targeting colon stem cells is unknown. To investigate this, we treated APC-mutant patient-derived human colon adenoma organoids with 4 doses of SFN (1.5, 3.1, 6.25, and 12.5 µM) or DMSO-control for 6 days and quantified the number and size of organoids formed. Additionally, we performed RNAseq on each treatment group and calculated benchmark doses for pathway alterations using BMDExpress2. Organoid formation decreased with SFN treatment in a dose-dependent manner and the highest dose decreased organoid formation by 80% compared to control. The number of differentially expressed genes (DEG) also increased in a dose-dependent manner. Organoids treated with the lowest dose had 254 DEG (83 down-regulated and 171 up-regulated) compared to control while the highest dose had 7099 DEGs (3478 down and 3621 up). Increasing doses of SFN increased transcription of many Nrf2 target genes, including NQO1, HMOX1, and ALDH3A1. Increasing doses of SFN also increased the expression of colon differentiation genes, including KRT20, AQP3, and ALPP, and decreased the expression of colon stem cell genes LGR5, OLFM4, and EPHB2. BMDExpress2 analysis estimated the median benchmark dose for the gene ontology “stem cell proliferation” pathway at less than 1µM.  Ongoing work is characterizing the stem cell specific effects of SFN in a mouse model of colorectal carcinogenesis. These results provide evidence that SFN induces a differentiation-associated phenotype in colon adenoma cells at physiologically relevant doses.

Evan Michael Hill, University of Michigan Medical School, Ann Arbor, MI. In Vitro Cellular and Developmental Biology, 56:S20-21, 2020

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