| Autor: |
Duleba, Marcin, Yamamoto, Yusuke, Neupane, Rahul, Rao, Wei, Xie, Jingzhong, Qi, Yutao, Liew, Audrey-Ann, Niroula, Suchan, Zhang, Yanting, Mahalingam, Rajasekaran, Wang, Shan, Goller, Kristina, Ajani, Jaffer A., Vincent, Matthew, Ho, Khek Yu, Hou, Jason K., Hyams, Jeffrey S., Sylvester, Francisco A., Crum, Christopher P., McKeon, Frank, Xian, Wa |
| Zdroj: |
Nature Protocols; 20240101, Issue: Preprints p1-16, 16p |
| Abstrakt: |
‘Adult’ or ‘somatic’ stem cells harbor an intrinsic ability to regenerate tissues. Heterogeneity of such stem cells along the gastrointestinal tract yields the known segmental specificity of this organ and may contribute to the pathology of certain enteric conditions. Here we detail technology for the generation of ‘libraries’ of clonogenic cells from 1-mm-diamter endoscopic biopsy samples from the human gastrointestinal tract. Each of the 150–300 independent clones in a typical stem cell library can be clonally expanded to billions of cells in a few weeks while maintaining genomic stability and the ability to undergo multipotent differentiation to the specific epithelia from which the sample originated. The key to this methodology is the intrinsic immortality of normal intestinal stem cells (ISCs) and culture systems that maintain them as highly immature, ground-state ISCs marked by a single-cell clonogenicity of 70% and a corresponding 250-fold proliferative advantage over spheroid technologies. Clonal approaches such as this enhance the resolution of molecular genetics, make genome editing easier, and may be useful in regenerative medicine, unravelling heterogeneity in disease, and facilitating drug discovery. |
| Databáze: |
Supplemental Index |
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