Establishment And Characterization Of Mouse Intestinal Crypt Cell Culture System And Its Utilization In Tumorigenesis Research

BackgroundCancer stem cells are considered as the source for tumor recurrence and cause ofconventional treatment’s failure to cure tumors. However, the origin of cancer stem cells hasbeen controversial while it has been postulated that cancer stem cells may be derived fromnormal adult stem cells in the tissues. In recent years, studies of intestinal adenomas and cryptstem cells provide fresh evidence for the cancer stem cell hypothesis. Understanding hownormal adult stem cells are transformed to cancer stem cells will help us not only tounderstand the mechanisms underlying tumorigenesis but also to identify new and moreeffective cancer therapeutic targets.Adult stem cells exist in many tissues in the body to maintain tissue homeostasis andself-renewal. Intestinal epithelial stem cells are located at the bottom of crypt. After leavingthe bottom niche, stem cells enter a rapid proliferation phage, and migrate along the basementmembrane, then lose proliferative capacity (cell cycle arrest) on the way to the top of theintestinal epithelial villi. At the peak of the villi the cells will shed into the intestine lumen.The well-understood kinetics of intestinal epithelial proliferation and differentiation, andsimple micro-anatomical structure make iSCs (intestinal stem cells) an ideal model for adultstem cell research.The lack of successful expansion and culturing adult stem cells plagued stem cellresearch and clinic therapy for many years. Recently, using three-dimensional culturetechnique, researchers from the Netherlands have successfully cultivated long-term survivalself-sustainable organoids, and micro-intestine-like structure (or “mini-gut”). Villus-Cryptorganoid structures simulate normal intestinal epithelium armature epithelial cells lined onsurface of the cavity enclosed the represpective lumen of the intestine, and projecting budsrepresent the crypt. The organoids also show that stem cells and Paneth cells locate at the bottom of the crypt. Other types of intestinal epithelial cells (such as goblet cells) can also bedetected in organoid structures. Thus, the organoids provide a valuable platform andconvenience for studying stem cell biology. Whether single intestinal epithelial cells can forma complete organoid has gradually been regarded as an important criterion to evaluate themultipotency of stem cells. However, this method has its limitations: in addition to its highcost, delivery of the genes of interest into cultured organoids (in particular stem cells) is ratherdifficult.There are two general purposes for delivering genes into cells: transient expression andstable expression. For transient expression, transfection of expression plasmids is an option,but more commonly used approach is the replication-defective adenoviruses. The stableexpression can be regularly achieved by using recombinant retrovirus or lentivirus. Althoughthey are replication-defective, the virus contained DNA sequences are integrated into thegenome. However, operating staff may be exposed to the risks of infection and safety concern.Furthermore, packaging retroviruses are laborious and the infection efficiency is low, makingit an inconvenient and inefficient strategy. In recent years, some of the newly discoveredtransposon systems from lower organisms can efficiently be used to transduce mouse andhuman cells, such as piggyBac, compared with retrovirus (and lentivirus), piggyBac systemoffers numerous advantages: safe, large DNA cargo sizes, time and labor-saving, andmultiple copies integrations into genome.ObjectiveTo optimize and create intestinal crypt stem cells culture system in a two-dimensionaland three-dimensional manner; To establish a new tool and research approach for simulatingand/or reconstructing malignant transformation of intestinal stem cells in vitro.Materials and methods1. Use high-fidelity PCR, DNA precipitation, restriction enzyme digestion, DNAligations/cloning, electroporation, regular PCR screening, agarose gel electrophoresis, DNAsequencing and other related experiments to complete the construction and development ofpiggyBac plasmid vector, as well as identification and sequencing. Use the establishedpiggyBac system to build a variety of cancer-related genes (e.g., N-terminal deleted Beta-Catenin, and mutant Kras) expression vector and fluorescence labeled constructs (RFP, GFP,and FGL---Firefly Luciferase-GFP-LacZ). 2. Isolate mouse intestinal crypt primary cells and culture them in an optimized Matrigelbased three-dimensional culture system; infect cultured organoids with adenovirus and recordthe efficiency and effectiveness by fluorescence microscopy. Time-Lapse recording positionchanges of live cell’s fluorescence.3. Using isolated primary cells and two-dimensional culture, transfect withimmortalization vector pMPH86(piggyBac based), infect with piggyBac-transposase-expressing adenovirus, followed by drug selection. The established mouse intestinal cryptcells, designated as iMICs. Single iMIC’s ability to form organoids was evaluated in thethree-dimensional cell culture system.4. Identify, analyze and characterize the iMICs and the formed organoids in3D systemusing alkaline phosphatase staining, alcian blue, Schiff staining, immunofluorescence staining(whole mount staining). Subcutaneous injection in nude mice was set up to test iMICs’ability to grow in vivo, as well as other stable cell lines derived from iMICs. Histologicevaluation was carried out to analyze the retrieved masses.Results1. Using standard process of cloning, I constructed piggyBac transposon-basedexpression vectors, including pMPBLox, pMPH, pMPN, pMPH86, pMPN86etc, many ofwhich were used as basic tools in my research and my lab mates’ experiment. The process ofestablishing stable cell line by these constructs is proven convenient and accessible.Characters of the cell line are stable and consistent.2. I successfully established three-dimensional culture system for intestinal crypts cells,organoids grow healthily maintain long-term survival and continuous passage. Adenovirusexpressing fluorescent protein efficiently infect three-dimensional culture system organoids,infection efficiency rises as a virus dose dependent way. Two methods of infection (mix andseed, incubate and seed) be proven effectively deliver genes into organoids; too high viralload will be toxic for organoids. Fluorescence can be detected the presence for at least than10days, verify BMPs’ inhibition effect on crypt information.3. I successfully isolated and cultured intestinal primary crypt cells and establishedimmortalized intestinal crypt cell line, iMICs (using piggyBac based vector: pMPH86), whichcan only grow healthily in organoid medium. The iMIC cells exhibit typical epithelial cellmorphology even after passaged over20generations. For2D cultured iMICs, alkaline phosphatase staining, Schiff staining, alcian blue staining showed only a small amount of cellperformed positive staining in, but Villin, Beta-Catenin and PCNA was found widelyexpressed by immunofluorescence staining; after the cells grew to completely confluence,tight junction marker ZO-1was readily detected. Small percentage of cells express marker ofintestinal Paneth cells: Lysozyme. For iMIC formed organoids, combined a whole mountprocess with specific chemical staining and immunofluorescence staining, we find positivestaining of alkaline phosphatase staining, Schiff staining, Alcian blue staining are readilydetected, as well as PCNA, Beta-Catenin, Villin, E-cadherin, lysozyme, Musin-2.4. More than70%of iMIC cells can form vacuolar organoid structures which aredifferent from widetpye organoid which featured with small branches. Nevertheless, iMIC-Noggin cells get floated in two-dimensional culture system and form structures similar to wildtype organoid structures with buds.5.To simulate tumorigenesis process, we also established the iMIC based cell lineswhich overexpress colon cancer related genes, iMIC-NTD Beta-Catenin (or oncogenic Beta-catenin)(abbreviated as iMIC-NTDBC), iMIC-mutKras and iMIC-NTDBC/mutKras.Different with iMIC, the three overexpress cell lines can grow in multi-layer manner undertwo-dimensional culture situation, and in three-dimensional culture system, iMIC-NTDBCform same vacuolar organoids like iMIC, but some iMIC-mutKras and iMIC-NTDBC/mutKras cells formed disorganized cell mass.5. In vivo, we found iMIC cells will survive and form glandular and crypt-like structureunder the support of Matrigel subcutaneously, but not with regular subcutaneous injectionprocess. Conversely, iMIC derived cells, iMIC-NTDBC and iMIC-mutKras, posess highgrowth activity independence of Matrigel. They formed solid mass without Villus-Crypt likestructure.Conclusions1. I developed a series of piggyBac based expression plasmids, which have been provento be more convenient and efficient than recombination retrovirus (and lentivirus), and couldbe widely used for lab research and potential clinical therapy.2. Adenovirus mediated genes delivery is an efficient way for intestinal organoids in3-dimensional culture system.3. iMICs, a conditional immortalized cell line we firstly established, retain the characterstics of the original intestinal stem cells, and should be recommended as a powerfultool for intestinal tumorigenesis.