Organoids are 3D organotypic cultures derived from tissue stem cells, embryonic cells and induced pluripotent stem cells (iPSCs), that mimic the structure and function of organs ranging from the colon1 to the brain2. They are amenable to essentially all cell biological and molecular technologies that are commonly used for cell lines, including genome editing with CRISPR-Cas technology3. Organoids are proving to be an enabling tool for applications ranging from fundamental biology to personalized or regenerative medicine (Method of Year 2017, Nat. Methods). Organoids grown from adult stem cells (obtained from tissue biopsies of almost any organ and animal species) provide a potentially powerful research platform e.g. for ex vivo phenotyping in livestock breeding programs and investigating genotype-to-phenotype relationships. Organoids derived from pluripotent stem cells are increasingly used in human biology and for designing new therapies; they provide controlled genetic backgrounds and the ability to drive their differentiation into virtually any cell type. Here, NI13, a line of porcine induced pluripotent-like cells (iPSLCs) generated through a non-integrative reprogramming strategy4 was used for in vitro differentiation towards the anterior (AFE) and posterior (PE) domains of the primitive gut in the presence of Activin-A (Activin/Nodal/TGF-β pathway) and CHIR99021 (WNT pathway) respectively5. Cells were subsequently embedded in matrigel to test their ability to organize in 3D structures. Incubation of PE-differentiated cells in medium optimized for porcine gut organoids (van der Hee et al., 2018, Stem Cell Research submitted) generated immature organoid structures which could be maintained in culture for up to two months as previously described for human PSCs5. Most of them had a defined lumen with traces of mucus but without clear evidence of presence of Goblet cells and multiple cell types of the gut epithelium; at day8 of 3D culture, the CDX2 and HOXC5 mid-hindgut markers were found highly enriched by qPCR. Incubation of the AFE-differentiated cells in Pneumacult ALI medium (StemCell Technologies) generated short-lived spheroids resembling bronchiospheres; a subsequent modification of this protocol using a defined medium generated larger and aggregating 3D structures in which the main markers of lung progenitors (NKW2.1 and FOXP2) could be readily detected. These preliminary results underline the potential of iPSCs to produce organoids for livestock research and point to the importance to achieve fully pluripotent cell lines for the major farm animal species. Acknowledgements: This work was supported by a WIAS fellowship of Wageningen University to E. Giuffra (Sept.-Dec. 2017). References 1 Date, S. & Sato, T. Mini-gut organoids: reconstitution of the stem cell niche. Annu Rev Cell Dev Biol 31, 269-289, doi:10.1146/annurev-cellbio-100814-125218 (2015). 2 Di Lullo, E. & Kriegstein, A. R. The use of brain organoids to investigate neural development and disease. Nat Rev Neurosci 18, 573-584, doi:10.1038/nrn.2017.107 (2017). 3 Driehuis, E. & Clevers, H. CRISPR/Cas 9 genome editing and its applications in organoids. Am J Physiol Gastrointest Liver Physiol 312, G257-G265, doi:10.1152/ajpgi.00410.2016 (2017). 4 Congras, A. et al. Non integrative strategy decreases chromosome instability and improves endogenous pluripotency genes reactivation in porcine induced pluripotent-like stem cells. Sci Rep 6, 27059, doi:10.1038/srep27059 (2016). 5 Hannan, N. R. et al. Generation of multipotent foregut stem cells from human pluripotent stem cells. Stem Cell Reports 1, 293-306, doi:10.1016/j.stemcr.2013.09.003 (2013).
|Number of pages||2|
|Publication status||Published - 26 Mar 2018|
|Event||11. Symposium of the French Domestic Animal Immunology Network - Tours, Tours, France|
Duration: 26 Mar 2018 → 27 Mar 2018
|Conference||11. Symposium of the French Domestic Animal Immunology Network|
|Period||26/03/18 → 27/03/18|