IMPLEMENTING ORGANOID MODELS FOR STUDYING MICROBE-HOST INTERACTION IN LIVESTOCK

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: EVANS-ALLEN
• Reporting Frequency: Annual
• Accession No.: 1025145
• Project Start Date: Nov 3, 2020
• Project End Date: Sep 30, 2023
• Program Code: [(N/A)]- (N/A)

Recipient Organization
TENNESSEE STATE UNIVERSITY
3500 JOHN A. MERRITT BLVD
NASHVILLE,TN 37209

Performing Department
Agricultural and Environmental Sciences

Non Technical Summary
Organoids, also known as "mini-organs", are three-dimensional (3D) constructs that differentiate from stem cells to recapitulate the cellular architecture and functionality of native organs. Multiple types of organoids have been cultured primarily from human and mouse stem cells, which have profoundly revolutionized the research and clinic practice in human medicine. Compared with the traditional 2D cell culture, organoid culture is outstanding per its genetic stability, physiological resemblance and potency in modeling organogenesis and microbe-host interactions; meanwhile, fully addressing the concerns of animal welfare.We hypothesize that the principle and procedures for generating organoid from human and mouse stem cells are generally applicable in livestock species, but predict species-specific optimization and discoveries in livestock species according to their phylogenic difference. Our long-term goal intends to establish organoid cultures for most if not all organs as validated using human and mouse stem cells. However, because the research focus of the PI's projects is relevant primarily to animal respiratory and digestive systems, the lung and intestinal organoids are therefore concentrated in this proposal, and will be realized with the following Specific Objectives (Obj.): Obj. 1: Optimize the protocols for porcine intestinal and lung organoids culture and apply to promote current research projects in animal immunogenetics and host-pathogen interactionObj. 2: Develop organoid cultures in cattle and chickens to establish livestock organoid stocks, and use them collaboratively for new projects.

Animal Health Component

30%

Research Effort Categories

Basic

40%

Applied

30%

Developmental

30%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	3599	1050	50%
305	3399	1030	25%
304	3530	1090	25%

Knowledge Area
311 - Animal Diseases; 305 - Animal Physiological Processes; 304 - Animal Genome;

Subject Of Investigation
3530 - Other swine products; 3599 - Swine, general/other; 3399 - Beef cattle, general/other;

Field Of Science
1090 - Immunology; 1030 - Cellular biology; 1050 - Developmental biology;

Keywords

organoids

livestock

animal health

swine

bovine

Goals / Objectives
We hypothesize that the principle and procedures for generating organoid from human and mouse stem cells are generally applicable in livestock species [1-9,15-17], but predict species-specific optimization and discoveries in livestock species according to their phylogenic difference. Our long-term goal intends to establish organoid cultures for most if not all organs as validated using human and mouse stem cells [25-31]. However, because the research focus of the PI's projects is relevant primarily to animal respiratory and digestive systems [18-24], the lung and intestinal organoids are therefore concentrated in this proposal, and will be realized with the following Specific Objectives (Obj.):Obj. 1: Optimize the protocols for porcine intestinal and lung organoids culture and apply to promote current research projects in animal immunogenetics and host-pathogen interactionObj. 2: Develop organoid cultures in cattle and chickens to establish livestock organoid stocks, and use them collaboratively for new projects.

Project Methods
Materials and methods:?Bovine gastrointestinal organoid culture: Procedures will be generated from several recent studies [15-17,27-31]. Sections of intestine from outbred fetuses or cattle at 10-20 month-old will be obtained from reliable resources (Animal Technologies, TX). Intestine segments will be cut and opened longitudinally, placed and transported in HBSS. Further process of intestinal crypt wash, enrichment, and culture will be generally conducted as for pigs but will be optimized for centrifuge forces, medium volumes and culture time for organoid differentiation.Chicken gastrointestinal organoid culture: Avian enteroids will be produced as described by Acharya et al 2020 with modification. Briefly, intestinal segments between the pancreatic loop and ileocecal junction will be aseptically removed and placed in HBSS. The mucosal tissues will be extruded into fresh complete medium by draining intestine segments of 5-6 chicks longitudinally with the help of tweezers. The pooled tissues will be dispersed by trituration and centrifuged at 200 g for 10 min to remove the supernatant. This process will be repeated once after which the tissue pellets will be resuspended in DMEM:F12 medium [containing 10% FBS, 1 x insulin transferrin selenite (ITS), 1 × polyamine (www.sigmaaldrich.com), and 1× bovine pituitary extract (Cell Applications Inc., San Diego, CA) as per the manufacturer's recommendation, henceforth referred to as"culture medium']. After overnight incubation in hydrophobic suspension cell culture plates (Sarstedt, Germany), this will generate the sheared ends of the villi to self-repair and form spherical enteroids. The cultures containing cell debris and the enteroids will be gently triturated to remove the attached cells then strained through 40 μm Falcon cell strainers (www.vwr.com) by three successive transfers and washes with excess volumes of complete medium. The enteroids will be then transferred from the strainer set in fresh medium and centrifuged at 200 × g for 10 min, reconstituted in fresh culture medium. The enteroids will be cultured in hydrophobic suspension culture wells. The villus crypts and the enteroids will be examined and photographed as described above [27-31].Other types of organoid culture from livestock: If other types of organoids needed for the laboratory and collaborative uses, we are prepared to develop and stock other organoids from other livestock species using the strategy as for generation of porcine lung organoid culture [27-31]. 2D monolayer enteroid culture: Expanded 3D enteroids will be recovered from Matrigel after 7 to 14 days of growth by the addition of ice-cold DMEM-F-12 medium, transferred into 15-ml tubes, and centrifuged at 250 × g at 4°C for 5 min. The enteroid pellet will be incubated in 0.25% trypsin-EDTA (Gibco) for 5 min at 37°C and dissociated by repeated pipetting to obtain a single-cell suspension. DMEM-F-12 medium with 10% (vol/vol) FBS will be added to the single-cell suspension, and the mixture was centrifuged at 800 × g for 5 min. The cell pellets will be resuspended in complete IntestiCult organoid growth medium at room temperature (RT) and seeded at 50 enteroid cells per well in a Matrigel-precoated 96-well plate. After 3 days of differentiation, the planner monolayer 2D enteroids were ready for experiments [15].Organoid passage: Organoids, which will be cultured for up to ~10 days and appeared to be 'crowded' in the Matrigel, will be deemed for passaging. Medium is removed and 500-μl warmed DPBS added to the wells. Organoid-containing Matrigel will be resuspended in the DPBS and transferred to a centrifuge tube. All of the organoids suspended in the Matrigel will be fragmented to separate the crypts/villi by gentle pipetting in the tube before centrifugation. The organoid fragment suspension will be then centrifuged at 50 × g for 3 min at 4 °C (bovine) or 300 × g for 10 min at 4 °C. Supernatant is aspirated, taking care to avoid the organoid pellet that is then resuspended in the 70% Matrigel, 30% IntestiCult/bovine medium mix described previously. Passaged organoids in Matrigel will be plated onto pre-warmed coverslips at 30 μl per well. Following a polymerisation period of 30 min, 200 μl of IntestiCult/bovine medium at RT will be added to the wells [15-17].Cryopreservation and resuscitation of organoids: At day 6 or 7 of culture, medium is removed from wells and 500-μl warmed DPBS added. Organoid-containing Matrigel is resuspended in the DPBS and transferred to a centrifuge tube. The organoids will be fragmented by gentle pipetting in the tube before centrifugation. The organoid fragment suspension will be centrifuged at 50 × g for 3 min at 4 °C (bovine) or 300 × g for 10 min at 4 °C (porcine). Supernatant will be removed and organoid pellets resuspended in CryoStor® (Stem Cell Technologies) using a volume of 1-ml CryoStor® per three wells organoids. Vials are frozen at - 80 °C for 24 h in a Nalgene® Mr Frosty freezing container (Thermo Fisher Scientific, Cheshire, UK) before being transferred to liquid nitrogen for long-term storage [15-17].For resuscitation, vials are removed from liquid nitrogen and defrosted in a waterbath at 37 °C until the freezing medium became liquid but not warm. The slurry in the vial will be transferred to a 15-ml tube with 5-ml IntestiCult added. Suspensions will be centrifuged at 50 × g for 3 min at 4 °C (bovine) or 300 × g for 10 min at 4 °C (porcine), or other species-specific conditions. Organoid pellets will be then resuspended in Matrigel and covered with medium as per the passage process.RNA isolation and qRT-PCR: Total cellular mRNA will be extracted using a total RNA extraction kit (Biotek, Canada) according to the manufacturer's instructions. Total mRNA (1 μg) will be reverse transcribed to cDNA and relative gene expression levels are quantified by one-stem qRT-PCR using SYBR green PCR mix (Life Technologies) based on the cycle threshold (ΔΔCt) method (42). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as the internal control [18-24].Immunofluorescence assay. Organoids or organoid monolayers will be fixed with 4% paraformaldehyde (PFA) for 30 min at RT. After incubation with a permeabilizing buffer (Life Technologies), the cells will be blocked with blocking buffer (PBS with 5% FBS and 5% skim milk), incubated with the primary antibodies for 2 h at 37°C, and stained with the secondary antibodies for 1 h at 37°C. The surface differentiation markers of will be detected using various validated antibodies. For examples, porcine intestinal epithelial cells will be detected using primary anti-Ki-67 (1 μg/ml; Abcam, USA) for proliferative cells, anti-Lgr5 (1:50; Novus Biologicals, USA) for stem cells, anti-mucin 2 (1:50; Abcam, USA) for goblet cells, antivillin (1:100; Abcam, USA) and anti-chromogranin A (1:100; Santa Cruz, USA) for enteroendocrine cells, and antilysozyme (1:50; Santa Cruz, USA) for Paneth cells. The cells are then labeled with secondary antibody conjugated to Alexa Fluor 488 donkey polyclonal antibody against rabbit IgG (1:1,000; Thermo Fisher Scientific, USA) or Alexa Fluor 546 goat anti-mouse IgG antibody (1:500; Thermo Fisher Scientific, USA). DAPI (4′,6-diamidino-2-phenylindole) (1:100; Sigma, USA) will be used to counter-stain cellular nuclei. The stained cells will be visualized and micrographs will be collected using an Evos FL Auto2 fluorescence microscope [18-24].Statistical analysis: All the results in the figures are presented, wherever appropriate, as the means ± the standard errors of the mean (SEMs) from three independent experiments or replicates and are analyzed using a Sigmaplot or a GraphPad Prism (GraphPad Software, Inc.) software. Differences are considered significant if the P value was <0.05.

Progress 11/03/20 to 09/30/21

Outputs
Target Audience:Target Audience Group I: Industry and academic professionals including swine immunologists, virologists, nutritionists, consultants and other researcher workers, as well as producers, veterinarians and veterinary researchers. Efforts: 1) Conference presentation at scientific meetings such as the Annual Conference of The American Association of Immunologists (AAI), International PRRS Symposium, and the Conference of Research Workers in Animal Diseases (CRWAD); 2) TSU's day at the capital city of Tennessee.; 3) Invited seminars and lectures at national and international academic institutes; 4) publications in book chapters, and peer-reviewed and open-assessed scientific journals including Front Microbiology, Viruses, Front Immunol. Group II: Graduate and undergraduate students. Efforts: 1) In addition to the first and third efforts above, the data will also be presented specifically at the University annual Research Day/Forum; and 2) other in-campus and inter-campus student research forums; 3) exchange and publication of students' research theses; 4) incorporation and introduction in lectures/labs of animal health/disease/immunology courses; 5) the department and inter-department seminar presentation, topic lectures or lab research training Group III: other interested public audiences. Efforts: Through the activity of TSU agricultural extension system, other localnews media and online broadcasting (YouTube, etc.) Changes/Problems:No major changes/problem expected so far. What opportunities for training and professional development has the project provided?In part, this grant has provided opportunity to train: Undergraduate research assistant: (1) Ms. Autumn Peterson, is a TSU Biology TLSAMP Traineeship student, she joined our research program and helped in this project during May, 2020 to May, 2021. She has co-authored one publication and an abstract to TLSAMP 2020 (2) Ms. Kendall McCoy, is a TSU Biology TLSAMP Traineeship student, she joined our research program and helped in this project during May, 2020 to May, 2021. She has co-authored one publication and an abstract to TLSAMP 2020 (3) Ms. LeAnn Lopez, is a TSU Agriculture Dean Scholarship student, she joined our research program and helped in this project during May, 2020 to May, 2021. She has co-authored one publication and an abstract to TLSAMP 2020 Graduate students and postdoctoral research associate: (1) Mr. Jiuyi Li, a Master Graduate student, has started his graduate program in Animal Science and been primarily working in this project since Jan 2021. He has co-authored two publications. (2) Ms. Yun Tian, a Ph.D. Graduate student, has started his graduate program in Animal Science and been partially working in this project since September 2020. She has been a primary author for nine relevant publications. (3) Dr. Collins Khwatenge, a postdoctoral research associate supported by another project, has been partially working in this project since September 2020. He has co-authored three publications. How have the results been disseminated to communities of interest?We have disseminated the project results to communities through the following pathways: (1) Publish our results mostly in peer-reviewed and open-access journals including Genes, Viruses, Front Immunol, and Front Microbiology (2) Present our results in on-campus seminars and class teaching in particular for DVM and graduate students in Animal Science (3) Broadcast our discoveries through both classical (animal workers' gathers such as in Swine Days) and online medias (such as http://www.tnstate.edu/research/documents/ResearchHorizons2019.pdf http://veterinarynews.dvm360.com/kansas-state-researchers-investigate-threat-influenza-amphibians https://www.ars.usda.gov/research/publications/publication/?seqNo115=327085 http://phys.org/news/2016-07-veterinary-team-wild-amphibians-zoonotic.html https://www.youtube.com/watch?v=Du26POi4DcI https://www.youtube.com/watch?v=08NIdnTWKkU) (4) Actively present our results in scientific conferences and symposiums, or invited seminars. What do you plan to do during the next reporting period to accomplish the goals?Further development of animal organoids 1) in different organ systems, 2) other livestock species, and 3) further application in modeling viral infections and functional genomic characterization per virus-host interaction.

Impacts
What was accomplished under these goals? For Obj. 1 and 2, 1) We have developed the protocols for culturing porcine and bovine intestinal organoids through collaboration with Drs. Rahul Nelli and Luis Gabriel Giménez-Lirola at Iowa State University. 2) The organoids have been used to validate the viral infection using SARS-CoV2 and other animal viruses. 3) The virus-infected organoids and primary animal samples, are further processed for functional genomics analysis by Dr. Laura C. Miller at USDA ARS, IA.

Publications

• Type: Journal Articles Status: Published Year Published: 2021 Citation: Sang Y, Miller LC, Nelli RK, Gimenez-Lirola LG (2021) Harness Organoid Models for Virological Studies in Animals: A Cross-Species Perspective. Front Microbiology, 16 September 2021 | https://doi.org/10.3389/fmicb.2021.725074
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Tian Y, DeJesus FA, Khwatenge C, Li J, Robert J, Sang Y. (2021) Virus-Targeted Transcriptomic and Functional Analyses Implicate Ranaviral Interaction with Host Interferon Response in Frog Virus 3-infected Frog Tissues. Viruses 2021, 13(7), 132.
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Sang ER, Tian Y, Miller LC, Sang Y. (2020) Epigenetic evolution of ACE2 and IL-6 genes as non-canonical interferon-stimulated genes correlates to COVID-19 susceptibility in vertebrates. Genes (Basel). 2021 Jan 25;12(2):154.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Lopez L, Sang PC, Pate M, Tian Y, Sang Y. (2020) Dysregulated Interferon Response Underlying Severe COVID19. Viruses, 2020, 12(12), 1433.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Fleming D, Miller LC, Tian Y, Li Y, Ma W, Sang Y. (2020) Impact of Porcine Reproductive and Respiratory Syndrome Virus, Influenza B, and Their Coinfection on Antiviral Response in the Porcine Lung. Pathogens, 2020, 9, 934.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: McCoy K, Peterson A., Tian Y, Sang Y. (2020) Immunogenetic Association Underlying Severe COVID19. Vaccines, 2020, 8(4), 700.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Sang ER, Tian Y, Gong Y, Miller LC, Sang Y. (2020) Integrate structural analysis, isoform diversity, and interferon-inductive propensity of ACE2 to refine SARS-COV2 susceptibility prediction in vertebrates. Heliyon. 2020 Sep;6(9):e04818.
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Tian Y, Khwatenge C, Li J, DeJesus FA, Robert J, Sang Y. (2021) Virus-Targeted Transcriptomic Analyses in Frog Virus 3-infected Frog Tissues Reveal Non-Coding Regulatory Elements in Intergenic Regions of Ranaviral Genome and Their Molecular Interaction with Host Immune Response. Front Immunol. 2021 Jun 17;12:705253.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Dowson H, Sang Y, Lunney J. Porcine cytokines, chemokines and growth factors: 2019 Update. Res. Vet. Sci. 2020 Aug;131:266-300.