A novel culture system for adult porcine intestinal crypts
| Autor: | Jiafang Wang, Garrett J. Brinkley, Martin G. Martin, Andrew Scott, Michael T. Lewis, James C.Y. Dunn, Ziyad Jabaji, Nan Ye Lei, Matthias Stelzner, Hassan A. Khalil, Upendra K. Kar |
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| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Aging Cellular differentiation Sus scrofa Medical Physiology Regenerative Medicine Tissue Culture Techniques Mice 0302 clinical medicine Conditioned Transduction Genetic Epidermal growth factor Stem Cell Research - Nonembryonic - Human Transduction of intestinal epithelium Intestinal Mucosa Myofibroblasts Temperature Immunohistochemistry Intestines medicine.anatomical_structure 030220 oncology & carcinogenesis Intestinal subepithelial myofibroblast Stem Cell Research - Nonembryonic - Non-Human Stem cell Development of treatments and therapeutic interventions Biotechnology Histology Crypt Biology Article Pathology and Forensic Medicine Viral vector 03 medical and health sciences Transduction Genetic medicine Animals Enteroid Cryopreservation Matrigel Neurology & Neurosurgery 5.2 Cellular and gene therapies Porcine intestinal culture Cell Biology Stem Cell Research Molecular biology Small intestine Culture Media Transplantation 030104 developmental biology Culture Media Conditioned Digestive Diseases Intestinal spheroid |
| Zdroj: | Cell and tissue research, vol 365, iss 1 |
| Popis: | Porcine models are useful for investigating therapeutic approaches to short bowel syndrome and potentially to intestinal stem cell (ISC) transplantation. Whereas techniques for the culture and genetic manipulation of ISCs from mice and humans are well established, similar methods for porcine stem cells have not been reported. Jejunal crypts were isolated from murine, human, and juvenile and adult porcine small intestine, suspended in Matrigel, and co-cultured with syngeneic intestinal subepithelial myofibroblasts (ISEMFs) or cultured without feeder cells in various culture media. Media containing epidermal growth factor, noggin, and R-spondin 1 (ENR medium) were supplemented with various combinations of Wnt3a- or ISEMF-conditioned medium (CM) and with glycogen synthase kinase 3 inhibitor (GSK3i), and their effects were studied on cultured crypts. Cell lineage differentiation was assessed by immunohistochemistry and quantitative polymerase chain reaction. Cultured porcine cells were serially passaged and transduced with a lentiviral vector. Whereas ENR medium supported murine enteroid growth, it did not sustain porcine crypts beyond 5days. Supplementation of Wnt3a-CM and GSK3i resulted in the formation of complex porcine enteroids with budding extensions. These enteroids contained a mixture of stem and differentiated cells and were successfully passaged in the presence of GSK3i. Crypts grown in media supplemented with porcine ISEMF-CM formed spheroids that were less well differentiated than enteroids. Enteroids and spheroids were transfected with a lentivirus with high efficiency. Thus, our method maintains juvenile and adult porcine crypt cells long-term in culture. Porcine enteroids and spheroids can be successfully passaged and transduced by using lentiviral vectors. |
| Databáze: | OpenAIRE |
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