A 3D system to model human pancreas development and its reference single-cell transcriptome atlas identify signaling pathways required for progenitor expansion.
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ABSTRACT:
SUBMITTER: Goncalves CA
PROVIDER: S-EPMC8149728 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Although the cell of origin for pancreatic cancer remains unknown, prior studies have suggested that pancreatic neoplasia may be initiated in progenitor-like cells. To examine the effects of oncogene activation within the pancreatic progenitor pool, we devised a system for real-time visualization of both normal and oncogenic KRAS-expressing pancreatic progenitor cells in living zebrafish embryos.By using BAC transgenes under the regulation of ptf1a regulatory elements, we expressed either extended green fluorescent protein (eGFP) alone or eGFP fused to oncogenic KRAS in developing zebrafish pancreas.After their initial specification, normal eGFP-labeled pancreatic progenitor cells were observed to actively migrate away from the forming endodermal gut tube, and subsequently underwent characteristic exocrine differentiation. In contrast, pancreatic progenitor cells expressing oncogenic KRAS underwent normal specification and migration, but failed to differentiate. This block in differentiation resulted in the abnormal persistence of an undifferentiated progenitor pool, and was associated with the subsequent formation of invasive pancreatic cancer. These tumors showed several features in common with the human disease, including evidence of abnormal Hedgehog pathway activation.These results provide a unique view of the tumor-initiating effects of oncogenic KRAS in a living vertebrate organism, and suggest that zebrafish models of pancreatic cancer may prove useful in advancing our understanding of the human disease.
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Project description:BackgroundTransplantation of insulin-producing cells is considered an important diabetes therapy. Many research studies have shown that insulin-producing cells can be derived from the in-vitro cultured pancreatic colonies with self-renewal ability and multilineage potential. Even though these progenitor-like colonies have been prepared from adult pancreas cells, the efficient culture method is hardly established and regulation of the colonies is rarely known. We confirmed previously that single cells acquired from adult mouse pancreas could form cyst-like colonies in a 3D semi-solid system containing Matrigel and methylcellulose. These colonies could be passaged continuously without losing progenitor-like capacity. In the previous culturing system, however, conditioned medium from murine embryonic-stem-cell-derived pancreatic-like cells was used. This unregulated ingredient may reduce repeatability and affect following study. Thus, a new culturing system with certain components needs to be developed.MethodsSingle cell suspension was acquired from adult mouse pancreas and cultured in a Matrigel-based 3D system with epidermal growth factor, Nicotinamide, B27, and Noggin to form ring colonies. Serial-passage assay was performed to evaluate self-renewal ability. Real-time polymerase chain reaction and immunostaining were used to detect the expression of progenitor-related genes. A 2D differentiation method was used to testify the multilineage potency of the colonies. High-throughput sequencing (HTS) of the colonies was performed to profile the differentially expressed genes.ResultsWe developed a 3D culturing system deprived of conditioned medium to propagate those colonies with high proliferative efficiency. HTS of the transcriptome of mRNAs, microRNAs (miRNAs) and long noncoding RNAs (lncRNAs) showed differentially expressed genes compared to the whole pancreas (as control). In mRNAs, several surface marker genes were identified in the colonies. Moreover in noncoding RNAs, miR-21a, miR-31 and miR-155 were upregulated and miR-217, miR-802 and miR-375 were downregulated in colonies along with a number of other miRNAs and lncRNAs.ConclusionsOur results offer an efficient culture system for pancreatic progenitor-like colonies and HTS of the colonies serves as a target resource for following study of in-vitro cultured pancreatic progenitors. These findings should also contribute to our understanding of the transcriptional regulation of these progenitor-like colonies and the mechanisms behind their functions.
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