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Background: Transplantation 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., Methods: Single 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., Results: We 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., Conclusions: Our 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.