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Generation of Porcine Intestinal Enteroids and Transducible Spheroids
Hassan Khalil*1, Nan Ye Lei1, Garrett Brinkley1, Upendra K. Kar1, Michael Lewis2, Martin G. Martin3, James Dunn1, Matthias G. Stelzner1,4
1Surgery, University of California, Los Angeles, Los Angeles, CA; 2Pathology, VA Greater Los Angeles Medical Center, Los Angeles, CA; 3Pediatrics, University of California, Los Angeles, Los Angeles, CA; 4Surgery, VA Greater Los Angeles Medical Center, Los Angeles, CA
Background: Porcine models are important tools for preclinical studies of stem cell transplantation. However, methods for the long-term culture and genetic manipulation of small intestinal crypt stem cells from adult pigs are lacking.
Methods: Two-cm segments of jejunum were procured from 10-14 week-old Yorkshire pigs (n=17). Crypts were isolated by EDTA chelation, suspended in Matrigel and grown in various culture media. We first used murine media containing only epidermal growth factor, noggin, and R-spondin 1 ("ENR medium") and then tested supplementation of Wnt3a, nicotinamide and small molecule inhibitors of GSK3 (CHIR99021), p160ROCK (Y27632), p38 MAP kinase (SB202190), and TGFβ receptor (LY2157299). We also isolated intestinal subepithelial myofibroblasts (ISEMFs), cultured them in DMEM with 10% FBS and added ISEMF or Wnt3a-conditioned medium (CM) to crypt cultures in a 1:1 ratio. We evaluated all media for enterosphere-forming efficiency and assessed cell lineage differentiation by immunohistology and fluorescence microscopy (FM). After we found that enteroids could be grown long-term in ENR medium with nicotinamide, Wnt3a-CM, SB202190 and LY2157299, we passaged enteroids while adding various compounds, e.g., PGE2 and the Notch ligand Jagged1, and tested media combinations to produce transducible cells. When crypts were grown in ENR medium with ISEMF-CM, CHIR99021 and Y27632, spheroids formed in culture that could be transduced using a lentiviral vector encoding a GFP reporter. Spheroids were maintained in this medium for 2 days and reporter activity was assessed by FM.
Results: ENR medium did not sustain adult porcine enterospheres beyond 5 days in culture. Addition of nicotinamide and small molecule inhibitors drastically improved 2-5 day survival of enterospheres but did not support long-term culture. Crypts grown in presence of porcine ISEMF-CM or co-cultured with porcine ISEMFs formed large, thin-walled spheroids. In contrast, Wnt3a-CM resulted in complex enteroids with budding extensions. Passaged enteroids continued to proliferate well for up to 5 weeks in culture when PGE2 was added at time of passage to enhance growth whereas Jagged1 had no discernible effect. Successful lentiviral transfection of spheroids was demonstrated with reporter activity present in >90% of transduced spheroids compared to controls.
Discussion: We describe a novel method to maintain adult porcine crypt cells in culture over several weeks. Porcine crypts can be induced by specific supplements to form enteroids which can be repeatedly passaged in vitro. Porcine ISEMFs produce factors that induce formation of spheroids which can be successfully transduced with >90% efficiency using lentiviral vectors.
Acknowledgment: This work was conducted by the Intestinal Stem Cell Consortium. The ISCC is supported by the NIDDK and NIAID (grant #DK085535).
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